Cell Therapy Methods

Abstract

The present invention is in the field of cell therapy and provides compositions and methods for treating cancer and / or viral infection in patients.The present invention provides lymphocytes comprising a synthetic polynucleotide encoding at least one iron-regulating protein and, optionally, a chimeric antigen receptor.The present invention also provides methods for producing these lymphocytes and administering them to patients.For example, the lymphocytes can be T cells or natural killer cells.

Description

[Technical Field]

[0001] summary The present invention is in the field of cell therapy and provides compositions and methods for treating cancer and / or viral infections in patients. The present invention provides lymphocytes comprising a synthetic polynucleotide encoding at least one iron-regulatory protein and, optionally, a chimeric antigen receptor. The present invention further provides methods for producing these lymphocytes and administering them to patients. [Background technology]

[0002] Introduction Over the past few decades, the efficacy of the immune system in cancer development and treatment has become a major focus of research. While targeted therapy and immunotherapy using immune checkpoint blockade have significantly improved survival for many cancer patients, a large proportion of patients still experience disease progression despite these treatments. Adoptive cell therapy (ACT) can offer an additional treatment option for these patients and involves the intravenous transfer of either tumor-resident or peripheral blood-modified immune cells into cancer patients to mediate antitumor function. Currently, ACT can be categorized into three distinct types, each with its own unique mechanism of action: ACT using tumor-infiltrating lymphocytes (TILs), ACT using T cell receptor (TCR) gene therapy, and ACT using chimeric antigen receptor (CAR)-modified T cells. The use of other immune cell types, such as natural killer cells, as the basis for cell therapy is also within the realm of current research.

[0003] The first studies using TILs were conducted by Rosenberg and colleagues at the Surgery Branch of the National Institutes of Health (SB, NIH, Bethesda, Maryland, USA), who grew TILs from various mouse tumors and demonstrated in vivo antitumor activity. Current TIL therapy involves ex vivo expansion of TILs from resected tumor material, adoptive transfer into patients after a lymphocyte-depleting conditioning regimen, and subsequent support with interleukin-2 (IL-2). Using this regimen, several phase I / II clinical trials have achieved remarkable objective tumor responses of approximately 50% in patients with metastatic melanoma. Following the success of TILs in melanoma patients, the generation of TILs from other solid tumor types has also been tested. To date, TILs have been successfully grown from nonmelanoma tumor types, such as cervical cancer, renal cell carcinoma, breast cancer, and non-small cell lung cancer, with varying rates of tumor reactivity.

[0004] Following the use of naturally occurring TILs in tumors and the treatment options based on them, peripheral blood T cells can be isolated and genetically modified in vitro to express TCRs targeting specific tumor antigens for use in ACT. Using this method, large pools of tumor-specific T cells can be generated, and potent antitumor activity and objective clinical responses have been observed in up to 30% of treated patients. Antigen presentation via major histocompatibility complex (MHC) is required for recognition by the modified TCR. However, it is well known that many cancer types can escape T cell-mediated immune responses by downregulation or loss of MHC expression. To circumvent the need for MHC on tumor cells for recognition by tumor-specific T cells, artificial receptors such as CAR molecules have been developed. ACT using CAR-modified T cells is similar to TCR-modified T cells, but retains the ability for effector function regardless of MHC expression. In addition to the use of protein antigens, other antigens, such as carbohydrate or glycolipid antigens, have also been explored. Impressive clinical responses have already been observed using CD19-specific CAR T cells for hematological malignancies, which has led to the exploration of using CAR therapy for solid tumors as well.

[0005] Hematopoietic stem cell transplantation (HSCT) offers a chance for cure for many patients with high-risk cancers or primary immunodeficiency syndromes, but transplant recipients remain susceptible to infectious complications due to persistent and severe immunosuppression. These risks are modified by the conditioning regimen, graft type, and duration of myelosuppression. With advances in conditioning regimens and improvements in posttransplant management, an increasing number of patients are eligible to receive mismatched, unrelated, or haploidentical donor HSCT. While outcomes for patients with severe or otherwise untreatable disease have improved significantly, immunosuppression required for engraftment and, when indicated, for the treatment of graft-versus-host disease (GVHD) creates the potential for infection. Viral infections, in particular, cause significant morbidity and mortality, and the risk increases with delayed T-cell immune reconstitution. The relationship between the effects of immunosuppression, immune reconstitution, and GVHD and infection is complex and intertwined. Pharmacological treatment and prophylactic options for viral infections remain limited and often ineffective, with significant morbidity associated with acute kidney injury and bone marrow suppression. Treatments can develop resistance, do not confer long-term protection, and patients remain at risk for viral reactivation. Given the correlation between delayed T cell immune recovery and viral disease, adoptive cell therapy is a logical alternative to pharmacological treatment. Unmanipulated lymphocyte infusions from seropositive donors have been infused into patients with life-threatening diseases, such as EBV-associated lymphoma, with demonstrated clinical efficacy, primarily associated with risks associated with GVHD. This strategy has evolved over the past two decades, and donor lymphocyte products have been successful in reconstituting viral immunity in the host as a treatment for viral disease (reactivation, new exposure, and lymphoma) and as a prophylaxis. Following these initial studies, virus-specific T cell (VST) selection and / or expansion have been refined to maximize viral cytotoxicity and minimize alloreactivity, reducing and nearly eliminating the risk of GVHD. Current studies have demonstrated that VSTs provide targeted therapy and have demonstrated a very favorable safety profile to date.

[0006] Natural killer cells (NK cells) have been studied to a lesser extent than T cells for their ACT potential. However, several properties of NK cells make them ideal candidates for adoptive cell therapy. In addition to being highly cytotoxic effectors, NK cells are not restricted by antigen specificity and rapidly produce pro-inflammatory cytokines that enhance the adaptive immune response.

[0007] NK cells from cancer patients are often dysfunctional, exhibiting reduced proliferation rates, diminished responses to cytokine stimulation, and impaired effector function. Therefore, early immunotherapeutic strategies aimed to enhance or restore the function of endogenous NK cells. These strategies involved IL-2-induced activation of autologous NK cells ex vivo, followed by reinfusion into the patient with combined IL-2 treatment during the course of treatment. Unfortunately, IL-2-activated NK cells did not affect tumor growth, and this treatment regimen was associated with severe side effects. The use of allogeneic NK cells for the treatment of cancer patients is more promising because they are fully functional compared to patient-specific NK cells. Furthermore, allogeneic NK cells have a graft-versus-leukemia / tumor (GvL / GvT) effect and do not induce graft-versus-host disease (GvHD), thus causing less immunopathology.

[0008] Clinically meaningful responses have been achieved, particularly for the treatment of hematologic malignancies such as acute myeloid lymphoma (AML) and non-Hodgkin's lymphoma (NHL). However, NK cell activity alone is often insufficient to adequately control tumor growth, and the treatment of solid tumors is particularly challenging due to the restrictive tumor microenvironment. Therefore, strategies to enhance NK cell function have been extensively investigated. One approach to enhancing the antitumor activity of NK cells is the use of cytokines. Various cytokines (IL-2, IL-12, IL-15, IL-18, IL-21, and type I interferons) have been used to expand and activate NK cells in vitro before adoptive transfer.

[0009] One promising cytokine combination for maximizing NK cell function is the combinatorial use of IL-12, IL-18, and IL-15. Stimulation with this combination induces a population of NK cells with "memory-like" features, such as long-term survival and enhanced effector function. Preclinical studies have demonstrated that cytokine-enhanced (CE) NK cells have substantial potential as anti-leukemia cell therapy. In in vivo tumor models of lymphoma or melanoma, CE NK cells possessed enhanced effector function (IFN-γ production and cytotoxicity). Furthermore, after adoptive transfer into immunodeficient NOD-SCID-γc- / - mice (NSG), IL-2-enhanced CE NK cells persisted longer than control NK cells. Finally, in AML xenografted NSG mice, CE NK cells substantially reduced AML burden and improved overall survival.

[0010] The molecular mechanisms driving the increased effector function of CE NK cells are currently unknown. Regarding T cells, it is well established that the function of certain subsets, e.g., naive versus memory CD8+ T cells, is linked to distinct metabolic regulatory mechanisms. Therefore, altering metabolic patterns in CE NK cells may support enhanced function. Elucidating the molecular mechanisms underlying the differentiation of CE NK cells and their superior effector functionality is an important prerequisite for improving clinical efficacy.

[0011] In general, highly proliferating cells strictly depend on iron to support fundamental processes such as energy metabolism / respiration, DNA synthesis and repair, and cell cycle control. The large amounts of iron required by proliferating cells, including lymphocytes, are supplied by transferrin, which is taken up via the cell surface receptor CD71. CD71 is commonly used as a lymphocyte activation marker and is expressed on activated NK cells. To date, only a few studies have investigated the importance of iron metabolism for lymphocyte function. A mutation in the CD71 receptor (TFRCY20H / Y20H) has recently been shown to impair T and B cell function due to impaired proliferation.

[0012] Decreased iron levels impair NK cell cytotoxicity, and in this context, it has been proposed that dysfunctional NK cells may contribute to cancer development in rats. Furthermore, low serum ferritin levels have been associated with reduced NK cell activity in humans. However, the specific effects of iron on NK cell-mediated immunity remain elusive.

[0013] Intracellular iron homeostasis is a tightly regulated process involving the coordination of iron uptake, utilization, and storage. Intracellular iron homeostasis is primarily regulated at the post-transcriptional level by the iron regulatory protein / iron-responsive element (IRP / IRE) regulatory system. IRP1 and IRP2 are RNA-binding proteins that recognize IREs in distinct mRNAs, thereby controlling their stability and translation into proteins. The activities of IRP1 and IRP2 are regulated in response to intracellular iron levels. Canonical IREs are present in the 5' or 3' UTRs of mRNAs encoding iron acquisition, iron storage, iron utilization, ATP production, and iron transport.

[0014] Under iron-deficient conditions, IRP activity is high, and IRPs bind to IREs within the 5' or 3' UTR of the corresponding mRNA. IRPs can differentially affect protein expression depending on the location of the IRE. Translation of mRNAs with IREs in the 5' UTR (e.g., FTH1 mRNA, ferritin light chain 1) is inhibited by IRP binding. In contrast, binding of IRPs to 3' UTR IREs (e.g., TFRC mRNA, CD71) stabilizes the mRNA, resulting in enhanced translation. Thus, the IRP / IRE regulatory network coordinates with intracellular iron homeostasis by selectively regulating the translation of certain mRNAs in response to intracellular iron status. Summary of the Invention [Problem to be solved by the invention]

[0015] Despite the recent success of adoptive cell therapy, there is still a need for improvements to make these therapies available to more patients.One of the general requirements for the success of adoptive cell therapy is to ensure that immune cells expand robustly in patients after being infused.This leads to, on the one hand, a reduction in the number of infusions, and, on the other hand, higher therapeutic efficacy.Therefore, there is a need in the art for improved means and methods related to cell therapy.More specifically, there is a need for immune cells that expand robustly in vivo after being administered to a subject. [Means for solving the problem]

[0016] The technical problem is solved by the embodiments presented in the claims, namely, the present invention relates to the following items:

[0017] 1. A lymphocyte comprising a synthetic polynucleotide encoding at least one iron-regulatory protein.

[0018] 2. The lymphocyte according to item 1, wherein the lymphocyte is a T cell or a natural killer cell.

[0019] 3. The lymphocyte of any one of items 1 or 2, wherein at least one iron-regulatory protein is constitutively expressed.

[0020] 4. The lymphocyte of any one of items 1 to 3, wherein at least one iron-regulatory protein is IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NOs: 2-6).

[0021] 5. The lymphocyte of any one of items 1 to 4, further comprising a chimeric antigen receptor.

[0022] 6. The lymphocyte according to item 5, wherein the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain.

[0023] 7. The lymphocyte according to item 6, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof, in particular, the antigen-binding fragment is a Fab or scFv.

[0024] 8. The lymphocyte of any one of items 6 or 7, wherein the antigen-binding domain specifically binds to a tumor antigen.

[0025] 9. The lymphocyte according to item 8, wherein the tumor antigen is present on the surface of cells of a target cell population or tissue.

[0026] 10. A pharmaceutical composition comprising lymphocytes according to any one of items 1 to 9 and a pharmaceutically acceptable carrier.

[0027] 11. A lymphocyte according to any one of items 1 to 9 or a pharmaceutical composition according to item 10 for use in therapy.

[0028] 12. A lymphocyte according to any one of items 1 to 9 or a pharmaceutical composition according to item 10 for use in the treatment of cancer.

[0029] 13. The lymphocyte or pharmaceutical composition for use according to item 12, wherein the cancer is a blood cancer or a solid tumor, in particular, the blood cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia or multiple myeloma, and the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

[0030] 14. A lymphocyte according to any one of items 1 to 9 or a pharmaceutical composition according to item 10 for use in the prevention and / or treatment of a viral infection.

[0031] 15. The lymphocyte or pharmaceutical composition for use according to item 14, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpesvirus, in particular the human herpesvirus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpesvirus 8 (HHV8).

[0032] 16. A method for treating a subject with cancer or for preventing and / or treating a viral infection in a subject, comprising administering to the subject a therapeutically effective amount of a lymphocyte according to any one of items 1 to 7 or a pharmaceutical composition according to item 10.

[0033] 17. The method according to item 16, wherein the cancer is a blood cancer or a solid tumor, in particular, the blood cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia or multiple myeloma, and the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

[0034] 18. The method according to item 16, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus, or human herpesvirus, and in particular, the human herpesvirus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV), or human herpesvirus 8 (HHV8).

[0035] 19. A method for producing lymphocytes according to any one of items 1 to 9, comprising: a) providing lymphocytes obtained from a subject; b) introducing into lymphocytes a synthetic polynucleotide encoding at least one iron-regulatory protein; c) expressing the gene(s) encoded by the synthetic polynucleotide; A method comprising:

[0036] 20. The method according to item 19, wherein in step (b), a second synthetic polynucleotide encoding a chimeric antigen receptor is introduced.

[0037] 21. The method according to item 20, wherein a synthetic polynucleotide encoding a chimeric antigen receptor is combined with a synthetic polynucleotide encoding at least one iron-regulatory protein.

[0038] 22. The method according to any one of items 19 to 21, wherein the lymphocytes are activated before or after introducing at least one synthetic polynucleotide into the lymphocytes.

[0039] 23. The method according to any one of items 19 to 22, wherein at least one synthetic polynucleotide is introduced into the lymphocytes by viral transduction, in particular by retroviral transduction. DETAILED DESCRIPTION OF THE INVENTION

[0040] Thus, in one embodiment, the present invention relates to a lymphocyte comprising a synthetic polynucleotide encoding at least one iron-regulatory protein.

[0041] Thus, the present invention is based on the surprising finding that CD71-mediated iron uptake is a critical metabolic checkpoint for activated NK cells, acting as a go / no-go gatekeeper for cell proliferation. In cytokine-enhanced (CE) NK cells, excess levels of iron-regulatory proteins unexpectedly create a pseudo-iron-deficient state, which selectively enhances CD71 translation and therefore increases cell proliferation.

[0042] The molecular mechanisms underlying the enhanced effector function of CE NK cells remain unclear. Example 2 specifically demonstrates that CE NK cells express significantly higher levels of CD71 than naive (NV) NK cells in response to stimulation with IL-12 and IL-18 and tumor target cells (Figures 2B, 2C, and 2D). Example 5 further demonstrates that cytokine activation induces transcription of the TFRC gene, which encodes CD71, but to a greater extent in CE NK cells (Figure 5B). Higher TFRC mRNA expression in CE NK cells compared with NV NK cells directly translates into increased protein expression (Figures 2B and C). Accordingly, Example 6 demonstrates that the expression levels of the iron regulatory proteins (IRPs), IRP1 and IRP2, are higher in CE NK cells compared with NV NK cells (Figure 6A). Because IRPs are known to regulate TFRC mRNA translation by stabilizing it, we can conclude that higher amounts of IRPs result in higher amounts of CD71 protein in CE NK cells compared with NV NK cells. These findings are surprising because IRP expression is known to be regulated in response to intracellular iron levels. However, in CE NK cells, IRP expression is upregulated despite the presence of abundant iron in the surrounding medium, thereby creating a pseudo-iron-deficient state. Upon stimulation, this pseudo-iron-deficient state allows for increased stabilization of CD71 mRNA, resulting in increased CD71 protein expression and, therefore, proliferation.

[0043] Based on these findings, the inventors concluded that inducing a pseudo-iron-deficient state in lymphocytes, such as NK cells or T cells, leads to increased proliferation after administration to a subject, thus resulting in a subsequent larger effector population. Necessary treatment of lymphocytes with cytokines and / or feeder cell systems is often difficult or even impossible to control in in vivo applications, and the present invention instead provides a solution for inducing a pseudo-iron-deficient state in lymphocytes by overexpressing at least one IRP in said lymphocytes. Thus, pseudo-iron-deficient lymphocytes overexpressing an IRP according to the present invention have enhanced function, particularly enhanced proliferation, compared to lymphocytes that do not overexpress an IRP. Thus, in an alternative embodiment, the present invention relates to lymphocytes overexpressing at least one iron-regulatory protein.

[0044] We have demonstrated that forced IRP expression results in increased proliferation of different types of lymphocytes. For example, we showed that lentiviral overexpression of IRP2 (SEQ ID NO: 2; NCBI RefSeq: NM_004136.4) resulted in increased expression of CD71 and, more importantly, increased proliferation of Jurkat T cells (Figure 8F). Furthermore, lentiviral overexpression increased the expression of CD4 + T cells and CD8 + We showed that lentiviral overexpression of IRP2 in CAR T cells resulted in increased proliferation of CAR T cells upon antigen stimulation, whereas IRP2 overexpression had no effect on unstimulated CAR T cell proliferation (Figure 8K).

[0045] Thus, we convincingly demonstrated that the regulatory effect of IRPs on CD71 expression and the correlation between CD71 expression and cell proliferation are well conserved among different types of lymphocytes, particularly between T cells and NK cells. Given these findings, we conclude that overexpression of IRPs in lymphocytes results in increased proliferation of said lymphocytes. Therefore, it is plausible that forced IRP overexpression is an attractive strategy for achieving robust in vivo proliferation of lymphocytes infused into patients during lymphocyte-based therapy.

[0046] IRPs, also known as iron-responsive element-binding proteins, are proteins that bind to iron-responsive elements (IREs) and thereby regulate human iron metabolism. Two distinct IRPs, designated IRP1 and IRP2, have been described in humans. The activities of IRP1 and IRP2 are regulated differently. IRP1 contains an iron-sulfur cluster and functions as a cytosolic aconitase under iron-rich conditions. Iron deficiency results in the loss of iron from the iron-sulfur cluster, altering the conformation of IRP1 and allowing it to bind to the IRE of mRNA. In contrast, IRP2 is rapidly degraded by the ubiquitin proteasome system under relative iron excess. Under iron-depletion conditions, the adaptor protein FBXL5 is degraded, resulting in increased levels of IRP2. Thus, ubiquitin ligases function as iron sensors and regulators of iron homeostasis. Tissue-specific variations in IRP1 and IRP2 activity have been described, and IRP1 and IRP2 knockout mice have distinct phenotypes.

[0047] The term "polynucleotide," as used herein, refers to a sequence of nucleotides joined by phosphodiester bonds. Polynucleotides of the present invention can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules in either single-stranded or double-stranded form. Nucleotide bases are represented herein by the single-letter code: adenine (A), guanine (G), thymine (T), cytosine (C), inosine (I), and uracil (U). Polynucleotides of the present invention can be prepared using standard techniques well known to those skilled in the art.

[0048] As used herein, a "synthetic polynucleotide" refers to a polynucleotide of non-natural origin that has been incorporated into a lymphocyte. Synthetic polynucleotides can be produced by recombinant techniques, including polymerase chain reaction, or by chemical synthesis. Methods for producing synthetic polynucleotides having specific polynucleotide sequences are known to those skilled in the art. Furthermore, methods for incorporating synthetic polynucleotides into lymphocytes are known to those skilled in the art. Within the scope of the present invention, a synthetic polynucleotide preferably comprises at least one gene or coding sequence encoding an IRP, wherein the at least one gene or coding sequence is operably linked to at least one regulatory element, e.g., a promoter not naturally associated with the endogenous gene encoding the at least one IRP. Such synthetic polynucleotides can be obtained by a number of strategies. For example, a polynucleotide comprising a gene or coding sequence encoding an IRP under the control of a promoter or another regulatory element not naturally associated with the endogenous gene encoding the IRP can be incorporated into a lymphocyte. Alternatively, a polynucleotide encoding an IRP can be integrated into the genome of a lymphocyte and thus operably linked to a regulatory element not naturally associated with the endogenous gene encoding the IRP. Furthermore, the synthetic polynucleotides of the present invention can be obtained by incorporating into lymphocytes a polynucleotide containing a regulatory element that is not naturally associated with the endogenous gene encoding an IRP, thereby operably linking the regulatory element to the endogenous gene encoding an IRP in the lymphocyte. Alternatively, the synthetic polynucleotides of the present invention can be obtained by modifying the endogenous regulatory element of the gene encoding an IRP by genetic engineering or genome editing methods, thereby altering the expression of the endogenous IRP gene in the lymphocyte. For example, a regulatory element, such as a promoter of the endogenous gene encoding an IRP, can be modified so that the gene encoding an IRP is constitutively expressed in the lymphocyte.Methods of genetic engineering are well known in the art and include the use of CRISPR / Cas9 or engineered nucleases such as meganucleases, zinc finger nucleases or TALENs.

[0049] The term "operably linked" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the heterologous nucleic acid sequence. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to link two protein-coding regions, in the same reading frame. The term "promoter," as used herein, is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0050] Cells, for example lymphocytes, can be said to contain synthetic polynucleotides when synthetic polynucleotides are present inside the cell, that is, when they are enclosed by the cytoplasmic membrane of the cell.Synthetic polynucleotides can be delivered to cells in any form and by any method known in the art.For example, synthetic polynucleotides can be present inside the cell as a part of circular DNA vectors, such as plasmids, in the form or part of linear DNA, or in the form or part of mRNA.However, it is preferred that synthetic polynucleotides are integrated into lymphocyte genome as DNA.

[0051] The term "encoding" refers to the inherent property of a particular sequence of nucleotides within a polynucleotide, such as a coding sequence, gene, cDNA, or RNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence and the biological properties resulting therefrom. Thus, a coding sequence or gene encodes a protein when the protein is produced in a cell or other biological system by transcription of the coding sequence or gene into mRNA and translation of the mRNA corresponding to that coding sequence or gene. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and typically presented in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of the coding sequence, gene, or cDNA.

[0052] The term "coding sequence," as used herein, refers to a nucleic acid sequence that, when placed under the control of appropriate regulatory or expression control sequences, is transcribed and translated into a polypeptide. The term "gene," as used herein, refers to a DNA sequence, including, but not limited to, a DNA sequence that can be transcribed into mRNA, which can be translated into a polypeptide chain, or that can be transcribed into rRNA or tRNA, or that serves as a recognition site for enzymes and other proteins involved in DNA replication, transcription, and regulation. When used in its endogenous context, the term "gene" is generally understood to include all introns and other DNA sequences spliced ​​from an mRNA transcript, along with variants resulting from alternative splicing sites. However, when the term "gene" is used in reference to a synthetic polynucleotide, the term is broadly understood to further include coding sequences corresponding in sequence to spliced ​​mRNA variants of the gene or cDNA derived from the spliced ​​mRNA of the gene.

[0053] The term "lymphocyte" as used herein refers to any mononuclear non-phagocytic leukocyte derived from lymphoid stem cells found in blood, lymph, and lymphoid tissues; lymphocytes include natural killer cells (NK cells; function in cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity). Preferably, the lymphocytes of the present invention are NK cells or T cells. Thus, in a preferred embodiment, the present invention relates to lymphocytes according to the present invention that are T cells or natural killer cells.

[0054] T cells are a type of lymphocyte that develop in the thymus and play a central role in immune responses. T cells can be distinguished from other lymphocytes by the presence of T cell receptors on the cell surface. These immune cells arise as progenitor cells, originating from the bone marrow, and develop into several distinct types of T cells upon migration to the thymus. T cell differentiation continues after leaving the thymus. The lymphocytes of the present invention can be any T cell, e.g., helper CD4 + T cells, cytotoxic CD8 + T cells, memory T cells, regulatory CD4 + T cells may be CD4 T cells, natural killer T cells, or gamma delta T cells. In certain embodiments, T cells according to the present invention are CD4 + T cells or CD8 + T cells, and optionally, CD4 + T cells or CD8 + The T cells contain a chimeric antigen receptor.

[0055] Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte essential to the innate immune system. The role of NK cells is similar to that of cytotoxic T cells in the adaptive immune response of vertebrates. NK cells provide a rapid response to virus-infected cells, acting approximately three days after infection, and also respond to tumorigenesis. NK cells were named "natural killers" due to their initial concept of ability to lyse tumor cells without prior sensitization. NK cell inhibitory receptors primarily associate with major histocompatibility class I (MHC class I) molecules, which are ubiquitously expressed on the surface of nucleated cells. Healthy cells expressing high levels of MHC class I maintain self-tolerance and are protected from killing by NK cells. In contrast, viral infection or malignant transformation induces NK cell activation by removing inhibitory signals. Activating NK cell receptors recognize stress-induced ligands on virus-infected or malignant cells. Expression of these stimulatory ligands on target cells can overcome the constitutive inhibition delivered by inhibitory receptors and thus activate NK cells.

[0056] Within the scope of the present invention, the lymphocytes may be any lymphocytes. Preferably, the lymphocytes of the present invention are T cells or NK cells. Thus, in one embodiment, the present invention relates to lymphocytes according to the present invention that are T cells or NK cells. In another embodiment, the present invention relates to cytotoxic CD8 + T cells, helper CD4 + In a further embodiment, the present invention relates to a lymphocyte according to the present invention, which is a T cell or a NK cell. + In a further embodiment, the present invention relates to a lymphocyte according to the present invention, which is a T cell or a NK cell. + In another embodiment, the present invention relates to a lymphocyte according to the present invention, which is a helper CD4 T cell. + The lymphocytes according to the invention are T cells.

[0057] In one embodiment, the invention relates to a lymphocyte according to the invention which is a tumor-infiltrating lymphocyte, a T cell comprising a modified TCR or a virus-specific T cell.

[0058] That is, the lymphocytes are lymphocytes suitable for cell therapy applications, such as TILs, T cells comprising a modified TCR, or virus-specific T cells. Preferably, the TILs, T cells comprising a modified TCR, or virus-specific T cells comprise a synthetic polynucleotide encoding at least one iron-regulatory protein, preferably, the iron-regulatory protein is IRP1 and / or IRP2, more preferably, the iron-regulatory protein is IRP2, and even more preferably, the iron-regulatory protein is IRP2 set forth in SEQ ID NO:2.

[0059] In certain embodiments, the lymphocytes of the present invention can be tumor-infiltrating lymphocytes (TILs). TILs are white blood cells that leave the bloodstream and migrate toward tumors. TILs include T cells and B cells and are part of a larger category of "tumor-infiltrating immune cells," which consist of various proportions of both mononuclear and polymorphonuclear immune cells (e.g., T cells, B cells, natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, basophils, etc.). The abundance of TILs varies depending on tumor type and stage and, in some cases, is related to disease prognosis. TILs can be used in cell therapy, in which they are isolated from a patient's tumor and expanded ex vivo. The expanded TILs can then be assayed for specific tumor recognition, and the tumor-specific TILs can then be reinfused into the patient, if necessary, after an additional expansion step.

[0060] Within the scope of the present invention, synthetic polynucleotides encoding at least one iron-regulatory protein can be introduced ex vivo into TILs obtained from a patient, particularly from the patient's tumor. The IRP-overexpressing TILs can then be infused into the patient during the course of cancer treatment, preferably after one or more additional expansion and / or selection steps.

[0061] In certain embodiments of the present invention, the lymphocytes of the present invention may be T cells comprising a modified T cell receptor (TCR). The term "T cells comprising a modified T cell receptor" or "TCR-modified T cells" refers to T cells that have been genetically modified to express a particular TCR. TCR-modified T cells can be generated by obtaining a population of T cells from a subject and introducing a genetic element encoding a T cell receptor into the population of T cells. The TCR may be a naturally occurring TCR or an engineered TCR.

[0062] TCR-modified T cells can be used in cell therapy to enhance a patient's immune response to a specific antigen, for example, an antigen identified as being produced by a tumor in the patient. Overexpression of iron-regulatory proteins, preferably IPR1 and / or IPR2, more preferably IPR2, in a population of TCR-modified T cells can result in more robust in vivo proliferation of these T cells when infused into a patient, and thus can elicit a stronger immune response in the patient against the antigen recognized by the TCR. The TCR-modified T cells of the present invention can be any type of T cell. The TCR-modified T cells of the present invention can be CD4 + T cells or CD8 + Preferably, it is a T cell.

[0063] In a particular embodiment of the present invention, the lymphocytes according to the present invention are virus-specific T cells. "Virus-specific T cells" are T cells stimulated by a viral antigen, e.g., CD4 + T cells or CD8 + The virus-specific T cells are T cells.When administered to patients, virus-specific T cells can be used to treat viral infection in patients.The overexpression of iron-regulatory protein, preferably IRP1 and / or IRP2, more preferably IRP2, in virus-specific T cell population can lead to the more robust in vivo proliferation of these T cells when infused into patients, and thus can elicit a stronger immune response against the antigen recognized by TCR in the patient.

[0064] In another embodiment, the present invention relates to a lymphocyte according to the invention, in which at least one iron-regulatory protein is constitutively expressed.

[0065] The at least one IRP encoded by the synthetic polynucleotide and contained in the cells of the present invention can be operably linked to any promoter or regulatory element known in the art. Thus, the at least one IRP can be constitutively expressed, i.e., expressed in most cell types in most cases, or inducibly expressed, i.e., expressed only under certain physiological conditions and / or in response to specific signals and / or inducer molecules. However, within the scope of the present invention, constitutive expression of the at least one IRP is preferred. Alternatively, the at least one IRP can be inducibly expressed under conditions frequently encountered in cell therapy applications, e.g., by signals, molecules, and / or processes associated with lymphocyte activation.

[0066] The term "expression," as used herein, refers to the production of a desired end-product molecule in a target cell. The end-product molecule may include, for example, an RNA molecule, a peptide, a protein, or a combination thereof. Within the scope of the present invention, the end-product is preferably an iron-regulating protein.

[0067] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell under most or all physiological conditions of the cell.

[0068] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is the elongation growth factor-1a (EF-1a) promoter. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter.

[0069] Thus, in one particular embodiment, the invention relates to a lymphocyte according to the invention, in which the synthetic polynucleotide encoding at least one iron-regulatory protein is under the control of a constitutive promoter.

[0070] A polynucleotide encoding a protein or polypeptide is "under the control of a constitutive promoter" if the constitutive promoter is responsible for initiating transcription of the polynucleotide encoding said protein or polypeptide. Methods for placing a polynucleotide encoding a protein or polypeptide under the control of a promoter, for example, by methods of molecular cloning, are known to those skilled in the art.

[0071] The constitutive promoter can be any constitutive promoter known in the art, preferably a constitutive promoter that initiates transcription in mammalian cells, more preferably human cells. For example, the constitutive promoter can be any one of the constitutive promoters listed above.

[0072] In one particular embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein the constitutive promoter is the EF-1α promoter.

[0073] Human elongation factor-1 alpha (EF-1 alpha) is a constitutive promoter of human origin that can be used to drive ectopic gene expression in a variety of in vitro and in vivo situations. Without being bound by theory, EF-1 alpha is often useful in situations where the activity of other promoters (such as CMV) is attenuated or silenced (such as in embryonic stem cells).

[0074] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0075] In yet another embodiment, the invention relates to a lymphocyte according to the invention, wherein at least one iron-regulatory protein is IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NOs: 2-6).

[0076] That is, the at least one IRP encoded by the synthetic polynucleotide contained in the lymphocytes of the present invention can be any IRP known in the art. However, the IRP is preferably human IRP1 and / or human IRP2. In humans, four different isoforms of IRP2 have been described, with two different sequences for isoform 3 (SEQ ID NOS: 2-6). Thus, in certain embodiments of the present invention, lymphocytes of the present invention can comprise a synthetic polynucleotide encoding a protein having the amino acid sequence of SEQ ID NOS: 1. In other embodiments of the present invention, lymphocytes of the present invention can comprise one or more synthetic polynucleotides encoding a protein having the amino acid sequence of SEQ ID NOS: 2-6. In further embodiments of the present invention, lymphocytes of the present invention can comprise a synthetic polynucleotide encoding a protein having one or more of the amino acid sequences of SEQ ID NOS: 1 and 2-6. Lymphocytes of the present invention can also comprise two or more synthetic polynucleotides, where a first synthetic polynucleotide encodes a protein having the amino acid sequence of SEQ ID NOS: 1 and a second or any further synthetic polynucleotide encodes a protein having the amino acid sequence of SEQ ID NOS: 2-6.

[0077] In one embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP1 (SEQ ID NO: 1). In another embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP2 (SEQ ID NO: 2). In yet another embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP2 (SEQ ID NO: 3). In yet another embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP2 (SEQ ID NO: 4). In yet another embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP2 (SEQ ID NO: 5). In yet another embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP2 (SEQ ID NO: 6). In another embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP1 (SEQ ID NO: 1) and IRP2 (SEQ ID NO: 2). In a preferred embodiment of the invention, the invention relates to a lymphocyte according to the invention, wherein at least one iron regulatory protein is IRP2 (SEQ ID NO: 2).

[0078] We demonstrated that overexpression of IRP2 in lymphocytes resulted in more robust proliferation of these lymphocytes. We further demonstrated that silencing IRP1 in lymphocytes had a similar effect to silencing IRP2, but the effect was less significant than that of IRP2 (Figures 8C and 8D). However, it should be noted that silencing IRP1 was less efficient than silencing IRP2 (Figure 8A). Therefore, it is plausible that overexpression of IRP1 can also result in increased proliferation of lymphocytes. Furthermore, it is plausible that simultaneous overexpression of IRP1 and IRP2 can result in increased proliferation of lymphocytes.

[0079] In one embodiment, the invention relates to a lymphocyte according to the invention, further comprising a chimeric antigen receptor.

[0080] The term "chimeric antigen receptor" or "CAR" or "CARs," as used herein, refers to an engineered receptor in which antigen specificity has been transferred to lymphocytes, such as T cells and NK cells. The CAR of the present invention can be any CAR known in the art. The CAR of the present invention preferably comprises at least one extracellular antigen-binding domain, a transmembrane domain, one or more costimulatory signaling regions, and an intracellular signaling domain. In certain embodiments of the present invention, the CAR can be a bispecific CAR specific for two different antigens or epitopes. When the antigen-binding domain specifically binds to the target antigen, the signaling domain activates intracellular signaling. For example, the signaling domain utilizes the antigen-binding properties of antibodies to redirect T cell specificity and reactivity to a selected target in an MHC-unrestricted manner. MHC-unrestricted antigen recognition allows CAR-expressing T cells to recognize antigens independently of antigen processing, thus circumventing a major mechanism of tumor escape. Furthermore, when expressed in T cells, the CAR advantageously does not dimerize with the endogenous T cell receptor (TCR) alpha and beta chains. In the case of NK cells, expression of a CAR can facilitate directing NK cells to target antigens. However, in contrast to CAR T cells, CAR NK cells can retain expression of their activating and inhibitory receptors. Thus, unlike CAR-T cells, CAR-NK cells can still exert their "native" functions even when the antigen targeted by the CAR is downregulated.

[0081] Within the scope of the present invention, a lymphocyte is said to contain a chimeric antigen receptor if it contains coding sequences encoding a CAR and expresses these coding sequences, such that the CAR is anchored to the lymphocyte's membrane. The coding sequences encoding the CAR components can be located on one or more synthetic polynucleotides. In certain embodiments of the present invention, the coding sequences encoding the CAR components and one or more coding sequences encoding IRPs can be located on a single synthetic polynucleotide. Alternatively, the coding sequences encoding the CAR components and one or more coding sequences encoding IRPs can be located on two or more separate polynucleotides. For example, a polynucleotide encoding a CAR and a polynucleotide encoding one or more IRPs can be introduced into a cell by two independent viral transduction events and thus integrated into different parts of the genome. One or more synthetic polynucleotides comprising a CAR coding sequence, and optionally, an IRP coding sequence(s), can be included in the lymphocyte in any form and can be introduced into the lymphocyte by any method known in the art. For example, the synthetic polynucleotide encoding CAR and / or one or more iron-regulating proteins can be present inside the cell as a circular DNA vector, such as a plasmid, in the form of or as a part of linear DNA, or in the form of or as a part of mRNA. However, it is preferred that one or more synthetic polynucleotides comprising a CAR coding sequence and optionally an IRP coding sequence(s) are integrated into the genome of lymphocytes as DNA. Methods for introducing DNA into the genome of lymphocytes are known to those skilled in the art. Synthetic polynucleotides encoding IRP1 and / or IRP2, and optionally CAR, can be introduced into the genome by any method known in the art. In certain embodiments, synthetic polynucleotides encoding IRP1 and / or IRP2, and optionally CAR, can be introduced into the genome of lymphocytes by viral transduction. However, other methods for introducing synthetic DNA into the genome of lymphocytes, such as CRISPR / Cas9, are also encompassed by the present invention.

[0082] In another embodiment, the invention relates to a lymphocyte according to the invention, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region and a signaling domain.

[0083] The lymphocytes according to the present invention may comprise a chimeric antigen receptor (CAR) comprising an extracellular domain and an intracellular domain. The extracellular domain may comprise one or more target-specific binding elements, otherwise referred to as antigen-binding moieties. The intracellular domain, or alternatively, the cytoplasmic domain, may comprise one or more costimulatory signaling regions and signaling domains. The costimulatory signaling region refers to the part of the CAR that comprises the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to antigens.

[0084] A spacer domain can be incorporated between the extracellular and transmembrane domains of a CAR, or between the cytoplasmic and transmembrane domains of a CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain with either the extracellular or cytoplasmic domain within a polypeptide chain. A spacer domain can contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0085] The CAR of the present invention can comprise one or more target-specific binding elements, which are also referred to as antigen-binding moieties.The selection of the moiety depends on the type and number of ligands that define the surface of target cells.For example, the antigen-binding domain can be selected to recognize the ligand that acts as a cell surface marker on target cells that is associated with specific pathological conditions.Therefore, examples of cell surface markers that can serve as ligands for the antigen moiety domain of the CAR of the present invention include those associated with viral infection, bacterial infection and parasitic infection, autoimmune disease and cancer cells.

[0086] Depending on the desired antigen to be targeted, the CAR of the present invention can be engineered to include an appropriate antigen-binding moiety that is specific for the desired target antigen. For example, if the desired antigen to be targeted is CD19, an antibody against CD19 can be used as the antigen-binding moiety for incorporation into the CAR of the present invention.

[0087] Regarding transmembrane domain, CAR can be designed to include a transmembrane domain fused with the extracellular domain of CAR.In certain embodiments, the transmembrane domain naturally associated with the extracellular domain or cytoplasmic domain of CAR can be used.In some cases, transmembrane domain can be selected or modified by amino acid substitution so as to avoid such domain from combining with the transmembrane domain of the same or different surface membrane protein, in order to minimize interaction with other members of receptor complex.

[0088] The transmembrane domain can be derived from either a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present invention can be derived from the alpha, beta, or zeta chains of the T cell receptor, CD28, CD8, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154 (i.e., at least the transmembrane region(s) thereof). Alternatively, the transmembrane domain can be synthetic, in which case the transmembrane domain predominantly contains hydrophobic residues such as leucine and valine. Preferably, a phenylalanine, tryptophan, and valine triplet is found at each end of the synthetic transmembrane domain.

[0089] If desired, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane and cytoplasmic signaling domains of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0090] The cytoplasmic domain or alternatively the intracellular signaling domain of the CAR of the present invention is responsible for activating at least one of the normal effector functions of the lymphocyte in which the CAR is placed. The term "effector function" refers to the specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, so long as the truncated portion transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal. Preferred examples of intracellular signaling domains for use in the CARs of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same functional capability.

[0091] It is known that signals generated by the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act independently of antigen to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0092] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs (ITAMs).

[0093] Examples of ITAMs containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule of the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[0094] The cytoplasmic domain of the CAR can be designed to include a CD3-zeta signaling domain alone or in combination with any other desired cytoplasmic domain(s) useful for the CAR of the present invention. For example, the cytoplasmic domain of the CAR can include a CD3 zeta chain portion and a costimulatory signaling region. The costimulatory signaling region refers to the part of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for efficient lymphocyte response to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0095] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be linked to each other in random order or in a specified order. If necessary, the linkage can be formed by a short oligopeptide or polypeptide linker, preferably a linker of 2 to 10 amino acids in length. A glycine-serine doublet provides a particularly suitable linker.

[0096] In certain embodiments, the cytoplasmic domain can be designed to include the signaling domain of CD3 zeta and the signaling domains of CD28 and / or 4-1BB.

[0097] In yet another embodiment, the invention relates to a lymphocyte according to the invention, wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof, in particular, wherein the antigen-binding fragment is a Fab or scFv.

[0098] That is, the antigen-binding domain of the CAR can be any domain known in the art that can specifically bind to a particular antigen, but preferably the antigen-binding domain of the CAR is an antibody or an antigen-binding fragment of an antibody.

[0099] The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. An antibody can be an intact immunoglobulin derived from natural or recombinant sources, or an immunoreactive portion of an intact immunoglobulin. An antibody is generally a tetramer of an immunoglobulin molecule. The antibody of the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab, and F(ab)2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA; 85: 5879-5883; Bird et al., 1988, Science; 242: 423-426).

[0100] The term "immunoglobulin" or "Ig" as used herein is defined as a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCRs (B cell receptors) or antigen receptors. Five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal fluids, and respiratory and genitourinary mucus. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects and is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, making it important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function but can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity triggered by the release of mediators from mast cells and basophils upon allergen exposure.

[0101] The term "antibody fragment" refers to a portion of an intact antibody and to the variable region that determines the antigenicity of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies formed from antibody fragments, scFv antibodies, and multispecific antibodies.

[0102] The term "Fab," as used herein, refers to a region of an antibody (monovalent antigen-binding fragment) that consists of one constant domain and one variable domain of each of the heavy and light chains, but in which the heavy chain is truncated and therefore lacks the CH2 and CH3 domains, and may also lack part or all of the hinge region. Fab fragments can be produced by digesting whole antibodies with the enzyme papain. Fab can refer to this region in isolation or in the context of a full-length antibody, immunoglobulin construct, or Fab fusion protein.

[0103] "scFv" refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present as a single polypeptide chain. See, e.g., U.S. Patent Nos. 4,946,778, 5,260,203, 5,455,030, and 5,856,456. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFvs, see Pluckthun (1994) The Pharmacology of Monoclonal Antibodies, vol. 113, ed. Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315. The complex between the VH and VL domains of an Fv fragment can also be stabilized by disulfide bonds (U.S. Patent No. 5,747,654).

[0104] "Antibody heavy chain," as used herein, refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation. "Antibody light chain," as used herein, refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformation, with λ light chains referring to the two major antibody light chain isotypes.

[0105] The term "synthetic antibody," as used herein, refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage. The term should also be construed to refer to an antibody produced by synthesis of a DNA molecule encoding the antibody, where the DNA molecule expresses the antibody protein, or an amino acid sequence that specifies the antibody, and where the DNA or amino acid sequence is obtained using synthetic DNA or amino acid sequencing techniques that are available and well known in the art.

[0106] Those skilled in the art are aware of methods for generating CARs with various antigen-binding domains, transmembrane domains, costimulatory signaling regions, and / or signaling domains, and methods for introducing such CARs into lymphocytes, such as T cells or NK cells.

[0107] In one embodiment, the invention relates to a lymphocyte according to the invention, wherein the antigen-binding domain specifically binds to a tumor antigen.

[0108] That is, the antigen-binding domain of a CAR can bind to any antigen known in the art. However, it is preferred that the antigen-binding domain of a CAR specifically bind to a tumor antigen. The term "antigen" or "Ag," as used herein, is defined as a molecule that elicits an immune response. This immune response can involve either antibody production or activation of specific immune-competent cells, or both. Those skilled in the art will understand that any macromolecule, including virtually any protein or peptide, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence that encodes a protein that elicits an immune response, and thus encodes an "antigen," can be used in the present invention. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that antigens can be generated, synthesized, or derived from biological samples, including, but not limited to, tissue samples, tumor samples, cells, or biological fluids.

[0109] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen-binding portion of the CAR depends on the specific type of cancer being treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0110] Tumor antigens may contain one or more antigenic cancer epitopes associated with malignant tumors. Malignant tumors express numerous proteins that can serve as target antigens for immune attack. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens are oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidate target antigens in B-cell lymphomas. Several of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies, with limited success.

[0111] The type of tumor antigen referred to in the present invention may be tumor-specific antigen (TSA) or tumor-associated antigen (TAA).TSA is specific to tumor cells and is not found in other cells in the body.TAA-associated antigens are not specific to tumor cells, but are instead expressed in normal cells under conditions that cannot induce a state of immune tolerance to the antigen.The expression of antigens in tumors can occur under conditions that allow the immune system to respond to the antigen.TAA can be an antigen that is expressed in normal cells during fetal development, when the immune system is immature and unable to respond, or TAA can be an antigen that is usually present at very low levels in normal cells, but is expressed at much higher levels in tumor cells.

[0112] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erb-B3, c-met, nm23_H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4(791Tgp72), alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\I, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB / 70K, NY-CO-1, RCAS-1, SDCCAG16, TA-90\Mac-2 binding protein, cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0113] The antigen-binding portion of the CAR can be any of, but not limited to, CD19, CD20, CD22, CD30, CD123, CD171, CS-1, ROR1, mesothelin, CD33, IL3Ra, c-Met, PSMA, glycolipid F77, EGFRvIII, GD-2, CD7, NY-ESO-1 TCR, MAGE-A3 TCR, CLL-1, GD3, BCMA, Tn Ag, PSMA, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-lRa, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, folate receptor alpha, ErbB2 (Her2 / neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, Globo H, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, LAGE-la, legumain, HPV E6,E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RUL, RU2, intestinal carboxylesterase, mut Additional antigens that may be targeted include hsp70-2, CD79a, CD79b, CD72, LAIRl, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1.

[0114] In certain embodiments, the lymphocytes of the invention comprising a CAR can be used to treat hematological cancers, particularly acute lymphoblastic leukemia and / or diffuse large B-cell lymphoma. In these embodiments, the antigen-binding portion of the CAR can specifically target CD19.

[0115] In certain embodiments, the lymphocytes of the invention comprising a CAR can be used to treat hematological cancers, particularly refractory Hodgkin's lymphoma. In these embodiments, the antigen-binding portion of the CAR can specifically target CD30.

[0116] In certain embodiments, the lymphocytes of the invention comprising a CAR can be used to treat hematological cancers, particularly acute myeloid leukemia. In these embodiments, the antigen-binding portion of the CAR can specifically target CD33, CD123, or FLT3.

[0117] In certain embodiments, the lymphocytes of the invention comprising a CAR can be used to treat hematological cancers, particularly multiple myeloma. In these embodiments, the antigen-binding portion of the CAR can specifically target BCMA.

[0118] In certain embodiments, the CAR contained in the lymphocyte of the present invention can bind to two antigens, hi certain embodiments, the bispecific CAR can bind to CD19 and CD22 or CD19 and CD20.

[0119] Generally, CAR has the advantage that it does not depend on the presentation of tumor antigens by MHC molecules on the surface of target cells.Instead, CAR can theoretically bind to any molecule that CAR can contact on the surface of tumor cells, provided that the antigen binding domain of CAR specifically binds to the antigen.Therefore, tumor antigens are preferably antigens present on the surface of tumor or malignant cells.More preferably, tumor antigens are antigens that are more abundant on the surface of tumor or malignant cells than on the surface of healthy or non-tumor cells.Even more preferably, tumor antigens are antigens that are present on the surface of tumor cells or malignant cells, but not on the surface of healthy or non-tumor cells.

[0120] The term "specifically binds," as used herein with respect to an antigen-binding domain or antibody, refers to an antigen-binding domain or antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antigen-binding domain or antibody that specifically binds to an antigen from one species will also bind to that antigen from one or more other species. However, such cross-species reactivity does not in itself change the classification of the antigen-binding domain or antibody as specific. In another example, an antigen-binding domain or antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not in itself change the classification of the antibody as specific. In some cases, the terms "specific binding" or "specifically binding" can be used with respect to the interaction of an antigen-binding domain, antibody, protein, or peptide with a second chemical species to mean that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, an antigen-binding domain or antibody recognizes and binds to a specific protein structure rather than recognizing and binding to proteins in general. If an antigen-binding domain or antibody is specific for epitope "A," the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antigen-binding domain or antibody will reduce the amount of labeled A that binds to the antigen-binding domain or antibody.

[0121] In another embodiment, the invention relates to a lymphocyte according to the invention, wherein the tumor antigen is present on the surface of the cells of the target cell population or tissue.

[0122] The lymphocytes according to the present invention that contain CAR can bind to tumor antigens present on the cell surface of target cells.Target cells can be part of a cell population or tissue.Generally, when tumor antigens are exposed by target cells and therefore can be contacted with the antigen binding domain of CAR, the tumor antigen can be said to be "present on the cell surface".

[0123] The tumor antigen can be any protein produced by a tumor cell, or more preferably, any part of a protein produced by a tumor cell and expressed on the cell surface of said tumor cell. Preferably, the tumor antigen is part of the extracellular domain of a membrane-anchored protein that is accessible to the antigen-binding domain of the CAR.

[0124] However, the present invention also encompasses tumor antigens that are presented on the surface of target cells by other molecules, particularly MHC molecules.In this case, tumor antigens are preferably peptides derived from proteins.Tumor antigens can be derived from proteins produced by tumor cells, for example.Alternatively, tumor antigens can be derived from extracellular proteins that have previously been taken up by tumor cells, for example, by endocytosis.In either case, proteins can be processed by target cells into peptides, which can then be presented on the surface of target cells, for example, by MHC molecules.

[0125] In one embodiment, the invention relates to a lymphocyte according to the invention, wherein the antigen binding domain specifically binds to a viral antigen.

[0126] That is, the lymphocytes of the present invention can be used to treat viral infections in subjects. The CAR contained in the lymphocytes of the present invention can contain an antigen-binding domain that specifically binds to a viral antigen. The viral antigen can be any component of a viral particle that can contact the CAR, such as an antigen that forms part of the surface and / or protein coat of the virus. The viral antigen recognized by the CAR is an antigen derived from human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus, or human herpesvirus. In particular, the human herpesvirus is preferably cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV), or human herpesvirus 8 (HHV8).

[0127] In one embodiment, the invention relates to a lymphocyte according to the invention, wherein the CAR is encoded by a polynucleotide, and wherein the polynucleotide encoding the CAR is transcriptionally linked to a synthetic polynucleotide encoding IRP1 and / or IRP2.

[0128] Within the scope of the present invention, it is preferred that the CAR is encoded by a polynucleotide that is integrated into the genome of a lymphocyte. It is more preferred that the polynucleotide encoding the CAR and the polynucleotide encoding IRP1 and / or IRP2 are integrated into the same locus of the genome of the lymphocyte. It is more preferred that the polynucleotide encoding the CAR and the polynucleotide encoding IRP1 and / or IRP2 are integrated into the same locus of the genome of the lymphocyte, thus transcriptionally linking two or more polynucleotides. Two or more polynucleotides are said to be transcriptionally linked when the transcription of the coding sequences contained in two or more polynucleotides is driven by a single promoter, thus resulting in a single transcript encoding two or more polypeptides. It is preferred that the promoter is located upstream (5') of the coding sequence or polynucleotide that is transcriptionally linked. That is, the coding sequence encoding the CAR and the coding sequence(s) encoding IRP1 and / or IRP2 can be transcribed by a single promoter.

[0129] To allow synthesis of a functional protein, the coding sequence encoding the CAR and the coding sequence(s) encoding IRP1 and / or IRP2 can be separated by an internal ribosome entry site (IRES) or can be connected by a polynucleotide encoding a self-cleaving peptide.

[0130] When the coding sequence encoding CAR and the coding sequence(s) encoding IRP1 and / or IRP2 are separated by an IRES, each coding sequence in the transcript is translated independently. However, when the coding sequence encoding CAR and the coding sequence(s) encoding IRP1 and / or IRP2 are connected by a self-cleaving peptide, the entire transcript is translated into a polyprotein that is then cleaved into a single protein by self-cleavage during or after translation.

[0131] In one embodiment, the invention relates to a lymphocyte according to the invention, wherein the polynucleotide encoding the CAR and the synthetic polynucleotide encoding IRP1 and / or IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

[0132] The term "self-cleaving peptide," as used herein, refers to a peptide sequence with cleavage activity occurring between two amino acid residues within the peptide sequence itself. For example, in a 2A / 2B peptide or a 2A / 2B-like peptide, cleavage occurs between the glycine residue of the 2A peptide and the proline residue of the 2B peptide. This occurs through a "ribosomal skipping mechanism" in which normal peptide bond formation between the 2A glycine residue and the 2B proline residue of the 2A / 2B peptide during translation is impaired, while translation of the remainder of the 2B peptide is unaffected. Such ribosomal skipping mechanisms are well known in the art and are known to be used by several viruses to express several proteins encoded by a single messenger RNA.

[0133] Thus, in one embodiment, the invention relates to a lymphocyte according to the invention, wherein the self-cleaving peptide is the 2A self-cleaving peptide.

[0134] In a preferred embodiment, the invention relates to a lymphocyte according to the invention, wherein the self-cleaving peptide is T2A. T2A is a self-cleaving peptide comprising the peptide sequence EGRGSLLTCGDVEENPGP (SEQ ID NO: 7).

[0135] It should be understood that when two coding sequences are linked by a polynucleotide encoding a self-cleaving peptide, the coding sequence encoding the first polypeptide, the coding sequence encoding the self-cleaving peptide, and the coding sequence encoding the second polypeptide are encoded in the same reading frame.

[0136] Within the scope of the present invention, it is preferred that the polynucleotide encoding the CAR and the polynucleotide(s) encoding IRP1 and / or IRP2 are encoded on the same synthetic polynucleotide. In certain embodiments, the synthetic polynucleotide encoding the CAR, IRP1, and / or IRP2 is integrated into the genome of a lymphocyte by viral transduction. In certain embodiments, the polynucleotide encoding the CAR and the polynucleotide encoding IRP1 contained in the synthetic polynucleotide are separated by an IRES. In another embodiment, the polynucleotide encoding the CAR and the polynucleotide encoding IRP2 contained in the synthetic polynucleotide are separated by an IRES. In other embodiments, the polynucleotide encoding the CAR and the polynucleotide encoding IRP1 contained in the synthetic polynucleotide are connected by a polynucleotide encoding a self-cleaving peptide, particularly a 2A self-cleaving peptide, in particular T2A. In other embodiments, the polynucleotide encoding the CAR and the polynucleotide encoding IRP2 contained in the synthetic polynucleotide are connected by a polynucleotide encoding a self-cleaving peptide, particularly a 2A self-cleaving peptide, in particular T2A.

[0137] In certain embodiments, the synthetic polynucleotide encoding CAR, IRP1, and / or IRP2 is under the control of a constitutive promoter. In certain embodiments, the promoter is part of the synthetic polynucleotide. In certain embodiments, the constitutive promoter is an EF-1α promoter. However, it should be understood that those skilled in the art know a wide range of promoters that can be used instead of the EF-1α promoter. Furthermore, it should be understood that Example 10 is merely a proof-of-concept, and that by optimizing the expression of CAR and / or IRP1 / 2 in lymphocytes, more efficient in vivo proliferation of lymphocytes can be achieved.

[0138] In certain embodiments, the synthetic polynucleotide has the structure: 5'-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-CAR-T2A-IRP1-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-CAR-T2A-IRP2-3'.

[0139] In certain embodiments, the synthetic polynucleotide has the structure: 5'-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP1-T2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP2-T2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP1-P2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP2-P2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP1-E2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP2-E2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP1-F2A-CAR-3'. In other embodiments, the synthetic polynucleotide has the structure: 5'-constitutive promoter-IRP2-F2A-CAR-3'.

[0140] However, it should be understood that the present invention also encompasses lymphocytes in which a first polynucleotide encoding IRP1 and / or IRP2 and a second polynucleotide encoding CAR are integrated into the lymphocyte's genome at different locations and expressed independently. Preferably, the polynucleotide encoding IRP1 and / or IRP2 and the polynucleotide encoding CAR are integrated into the lymphocyte's genome by two independent viral transduction events. The two independent viral transduction events may occur simultaneously or in stages.

[0141] In another embodiment, the present invention relates to a viral vector comprising at least one polynucleotide encoding IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NOs: 2 to 6).

[0142] That is, the present invention further relates to a viral vector that can be used to incorporate iron regulatory proteins into cells, preferably lymphocytes.The viral vector can be any viral vector suitable for incorporating polynucleotides into cells, preferably lymphocytes.Therefore, in a specific embodiment, the present invention relates to a viral vector according to the present invention that is derived from lentivirus, adeno-associated virus (AAV), adenovirus, herpes simplex virus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus or poxvirus.In a preferred embodiment, the present invention relates to a viral vector according to the present invention that is derived from lentivirus or adeno-associated virus (AAV).In a more preferred embodiment, the present invention relates to a viral vector according to the present invention that is derived from lentivirus.

[0143] A viral vector may contain one or more transgenes. As used herein, the term "transgene" refers to a specific nucleic acid sequence encoding a polypeptide or a portion of a polypeptide that is expressed in a cell into which the nucleic acid sequence is inserted. The term transgene encompasses (1) a nucleic acid sequence not naturally found in a cell (i.e., a heterologous nucleic acid sequence, such as a nucleic acid encoding a CAR); (2) a nucleic acid sequence that is a mutant version of a nucleic acid sequence naturally found in the cell into which it is introduced; (3) a nucleic acid sequence that functions to add additional copies of the same (i.e., homologous) or similar nucleic acid sequence (e.g., IRP1 and / or IRP2) naturally occurring in the cell into which it is introduced; or (4) a naturally occurring or homologous silent nucleic acid sequence whose expression is induced in the cell into which it is introduced. A mutant version refers to a nucleic acid sequence that contains one or more nucleotides that differ from the wild-type or naturally occurring sequence; i.e., a mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, a transgene may also include a sequence encoding a leader peptide or signal sequence, thereby allowing the transgene product to be secreted from the cell.

[0144] The synthetic polynucleotides encoding IRP1 and / or IRP2 contained in the lymphocytes of the invention, and optionally the promoter and / or CAR, can be introduced into the lymphocytes, preferably by viral transduction. It should therefore be understood that the synthetic polynucleotides disclosed in relation to the lymphocytes of the invention can be contained in a viral vector of the invention.

[0145] In certain embodiments, the viral vector comprises a single transgene. For example, in certain embodiments, the viral vector comprises a polynucleotide encoding IRP1 (SEQ ID NO: 1). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 2). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 3). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 4). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 5). In other embodiments, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 6). Preferably, the viral vector comprises a polynucleotide encoding IRP2 (SEQ ID NO: 2).

[0146] In certain embodiments, a viral vector may contain more than one transgene. For example, a viral vector may contain two or more polynucleotides encoding IRP1 (SEQ ID NO: 1) and one or more isotypes of IRP2 (SEQ ID NOs: 2-6). In another embodiment, a viral vector may contain two or more polynucleotides encoding two or more isotypes of IRP2 (SEQ ID NOs: 2-6).

[0147] Furthermore, the viral vector may comprise one or more polynucleotides encoding IRP1 and / or IRP2 and an additional polynucleotide encoding a CAR. Thus, in one embodiment, the present invention relates to a viral vector according to the present invention comprising an additional polynucleotide encoding a CAR. Thus, the viral vector can be used to simultaneously incorporate a polynucleotide encoding a CAR and at least one polynucleotide encoding IRP1 and / or IRP2 into a cell, preferably a lymphocyte.

[0148] In certain embodiments, the present invention relates to a viral vector according to the present invention, in which a polynucleotide encoding a CAR is transcriptionally linked to a polynucleotide(s) encoding IRP1 and / or IRP2. The polynucleotide encoding a CAR and the polynucleotide(s) encoding IRP1 and / or IRP2 can be transcriptionally linked as described above. That is, the polynucleotide encoding a CAR and the polynucleotide(s) encoding IRP1 and / or IRP2 can be under the control of a common promoter.

[0149] The polynucleotide encoding the CAR and the one or more polynucleotides encoding IRP1 and / or IRP2 can be separated by one or more IRESes and can be connected by one or more polynucleotides encoding a self-cleaving peptide as described herein.

[0150] In certain embodiments, the present invention relates to a viral vector according to the present invention, wherein the polynucleotide encoding the CAR and the polynucleotide(s) encoding IRP1 and / or IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

[0151] In certain embodiments, the present invention relates to a viral vector according to the invention, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

[0152] In certain embodiments, the present invention relates to a viral vector according to the invention, wherein the self-cleaving peptide is T2A.

[0153] In another embodiment, the viral vector may further comprise a promoter controlling the expression of one or more polynucleotides encoding CAR and IRP1 and / or IRP2. Thus, in another embodiment, the present invention relates to a viral vector according to the present invention, in which at least one polynucleotide encoding IRP1 and / or IRP2, and optionally CAR, are under the control of a promoter. The promoter may be a constitutive promoter or an inducible promoter, for example, one of the constitutive promoters or inducible promoters specified elsewhere herein. Preferably, the promoter is a constitutive promoter, such as the EF-1α promoter. Thus, in a specific embodiment, the present invention relates to a viral vector according to the present invention, in which the constitutive promoter is the EF-1α promoter.

[0154] In certain embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP1-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-self-cleaving peptide-IRP2-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-T2A-IRP1-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-T2A-IRP2-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-P2A-IRP1-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-P2A-IRP2-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-E2A-IRP1-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-E2A-IRP2-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-F2A-IRP1-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-CAR-F2A-IRP2-3'.

[0155] In certain embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP1-self-cleaving peptide-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP2-self-cleaving peptide-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP1-T2A-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP2-T2A-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP1-P2A-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP2-P2A-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP1-E2A-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP2-E2A-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP1-F2A-CAR-3'. In other embodiments, a viral vector may comprise a polynucleotide having the structure: 5'-constitutive promoter-IRP2-F2A-CAR-3'.

[0156] Molecular biology methods for introducing transgenes and / or regulatory elements such as promoters into viral vectors are known to those skilled in the art.

[0157] In one embodiment, the present invention relates to a pharmaceutical composition comprising lymphocytes according to the present invention and a pharmaceutically acceptable carrier.

[0158] The lymphocytes of the present invention can be administered alone or as a pharmaceutical composition comprising the lymphocytes of the present invention. Briefly, pharmaceutical compositions of the present invention can comprise the lymphocytes or lymphocyte populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions can include buffers, such as neutral buffered saline, phosphate buffered saline, carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Pharmaceutical compositions according to the present invention can be administered with diluents and / or in combination with other components, such as IL-2 or other cytokines or cell populations. The compositions of the present invention are preferably formulated for intravenous administration.

[0159] In another embodiment, the present invention relates to a pharmaceutical composition comprising a viral vector according to the present invention and a pharmaceutically acceptable carrier.

[0160] In certain embodiments, the direct treatment of the subject is intended by directly introducing the vector.The viral vector composition can be formulated for delivery by any available route, including but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, rectal, and vaginal.Commonly used delivery routes include inhalation, parenteral, and transmucosal.

[0161] In certain embodiments, a pharmaceutical composition according to the invention may comprise a viral vector according to the invention comprising a polynucleotide encoding at least one iron-regulatory protein and a second viral vector comprising a polynucleotide encoding a CAR, i.e., the polynucleotide(s) encoding the one or more iron-regulatory proteins and the polynucleotide encoding the CAR may be located on two separate viral vectors but may be included in the same pharmaceutical composition.

[0162] In various embodiments, pharmaceutical compositions can include a viral vector in combination with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition.

[0163] In some embodiments, active agents, i.e., viral vectors described herein and / or other agents administered with the vectors, are prepared with carriers that protect the compounds from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Methods for preparing such compositions will be apparent to those skilled in the art. Suitable materials can be purchased from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomes can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. In some embodiments, the compositions are targeted to specific cell types or virally infected cells. For example, the compositions can be targeted to cell surface markers, such as endogenous markers or viral antigens expressed on the surface of infected cells, using monoclonal antibodies.

[0164] For ease of administration and uniformity of dosage, it is advantageous to prepare the composition as unit dosage form.Unit dosage form as used herein refers to a physically separate unit that is suitable as a unit dosage for the subject to be treated; each unit comprises a predetermined amount of viral vector, which is calculated to bring about desired therapeutic effect together with pharmaceutical carrier.

[0165] The unit dose need not be administered as a single injection, but may include continuous infusion over a period of time. The unit dose of the viral vectors described herein can be conveniently described in terms of transducing units (TU) of the viral vector, which are defined by titrating the vector on a cell line such as HeLa or 293. In certain embodiments, the unit dose is 10 3 TU, 10 4 TU, 10 5 TU, 10 6 TU, 10 7 TU, 10 8 TU, 10 9 TU, 10 10 TU, 10 11 TU, 10 12 TU, 10 13 It can range from TU and higher.

[0166] The pharmaceutical composition can be administered at various intervals as needed for different periods, for example, once a week for about 1 week to about 10 weeks; about 2 weeks to about 8 weeks; about 3 weeks to about 7 weeks; about 4 weeks; about 5 weeks; about 6 weeks, etc. It may be necessary to administer the therapeutic composition indefinitely. Those skilled in the art will understand that certain factors, including but not limited to, the severity of the disease or disorder, previous treatment, the overall health and / or age of the subject, and other existing diseases, can affect the dosage and timing required to effectively treat the subject. Treatment of a subject with a viral vector can include a single treatment, or in many cases, can include a series of treatments.

[0167] The exemplary dose for administering viral vector and the method for determining the appropriate dose are known in the art.It is further understood that the appropriate dose of viral vector can depend on specific recipient and administration mode.The appropriate dose level for any specific subject can depend on various factors, including subject's age, weight, overall health, sex and diet, administration time, administration route, excretion rate, other administered therapeutic agents, etc.

[0168] In certain embodiments, viral vectors can be delivered to a subject, for example, by intravenous injection, topical administration, or stereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA, 91: 3054). In certain embodiments, vectors can be delivered orally or by inhalation and can be encapsulated or otherwise manipulated to protect them from degradation, enhance uptake into tissues, cells, etc. Pharmaceutical preparations can include the viral vector in an acceptable diluent or can comprise a slow-release matrix in which the viral vector is embedded. Alternatively or additionally, where the vector can be produced intact from recombinant cells, as in the case of retroviral or lentiviral vectors, the pharmaceutical preparation can include one or more cells that produce the vector. Pharmaceutical compositions comprising the viral vectors described herein can be included in a container, pack, or dispenser, optionally along with instructions for administration.

[0169] The foregoing compositions, methods and uses are illustrative and not limiting, and using the teachings provided herein, other variations of the compositions, methods and uses will be readily available to those skilled in the art.

[0170] In another embodiment, the present invention relates to a lymphocyte according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention for use in therapy.

[0171] That is, the lymphocytes according to the present invention, the viral vectors according to the present invention, or pharmaceutical compositions comprising the lymphocytes and / or viral vectors according to the present invention can be used for treatment.

[0172] In certain embodiments, the present invention relates to a lymphocyte according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention for use in the treatment of cancer.

[0173] The present invention provides the use of CAR as defined in the present invention to redirect the specificity of lymphocytes, such as T cells or NK cells, to tumor antigens.Disclosed herein is a type of cell therapy, in which lymphocytes are genetically modified to express at least one IRP and CAR, and the resulting cells are injected into recipients who need them.The injected cells can kill tumor cells in recipients.Unlike antibody therapy, lymphocytes according to the present invention can replicate in vivo, resulting in long-term persistence, thereby providing sustained tumor control.

[0174] Due to the overexpression of at least one IRP, the lymphocytes described herein can undergo robust in vivo expansion and can persist for a long period of time. Without being bound by any particular theory, the anti-tumor immune response elicited by the lymphocytes of the present invention can be an active immune response or a passive immune response. Furthermore, CAR-mediated immune response can be part of an adoptive immunotherapy approach, in which CAR-modified lymphocytes induce an immune response specific to the antigen-binding portion of CAR.

[0175] Although lymphocytes that express at least one IRP and CAR are preferred for cancer treatment, this can also be assumed by lymphocytes that only express at least one IRP and do not express CAR.In this case, the synthetic polynucleotide that encodes at least one IRP can be introduced into lymphocytes ex vivo, and then the genetically engineered lymphocytes can be administered to cancer patients.If necessary, lymphocytes can be stimulated ex vivo with tumor antigen to increase the specificity for a certain type of cancer or tumor.In certain embodiments, the genetically engineered lymphocytes that express at least one IRP can be directly injected into tumor.

[0176] However, it should be understood that the present invention also encompasses the use of lymphocytes that overexpress IRP1 and / or IRP2, but do not express CAR, in the treatment of cancer.

[0177] For example, IRP1 and / or IRP2 can be overexpressed in TIL or TCR modified T cells, and then used for cell therapy.The inventors have demonstrated that the overexpression of IRP in lymphocytes results in more robust lymphocyte proliferation.Therefore, it is reasonable to say that the overexpression of IRP in TIL or TCR modified T cells results in more effective cell therapy.

[0178] Furthermore, NK cells can be made to overexpress IRP1 and / or IRP2, which are then used in cell therapy. In certain embodiments, the NK cells are allogeneic NK cells.

[0179] To "treat" a disease, as that term is used herein, means to reduce the frequency or severity of at least one sign or symptom of the disease or disorder in a subject.

[0180] A "disease" is a state of health in an animal (including a human) in which the animal is unable to maintain homeostasis and the animal's health will continue to deteriorate unless the disease is reversed. In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but the animal's health is less favorable than it would be if the disorder were not present. A disorder, if left untreated, does not necessarily cause a further deterioration in the animal's health.

[0181] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof amenable to the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0182] The term "cancer," as used herein, is defined as a disease characterized by the rapid, uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0183] Cancers that can be treated using lymphocytes according to the present invention include non-vascularized tumors or tumors that are not yet substantially vascularized, as well as vascularized tumors.Cancers can include non-solid tumors (e.g., hematological tumors, such as leukemia and lymphoma) and solid tumors.Cancers that can be treated using lymphocytes according to the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphatic tumors, benign and malignant tumors, and malignant lesions, such as sarcomas, carcinomas, and melanomas.Adult tumors / cancers and pediatric tumors / cancers are also included.

[0184] In one embodiment, the present invention relates to a lymphocyte, a viral vector, or a pharmaceutical composition for use according to the present invention, wherein the cancer is a blood cancer or a solid tumor. In a specific embodiment, the blood cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia, or multiple myeloma, and the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma, or sarcoma.

[0185] Blood cancer is cancer of blood or bone marrow.Examples of blood (or hematopoietic) cancer include leukemia, including acute leukemia (for example, acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemia (for example, chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0186] A solid tumor is an abnormal mass of tissue, usually containing no cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, gallbladder carcin ... These include ductal carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (e.g., brain stem glioma and mixed glioma), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0187] The lymphocytes of the present invention can be designed to target CD19 and can be used to treat cancers and disorders including, but not limited to, pre-B ALL (pediatric indication), adult ALL, mantle cell lymphoma, diffuse large B-cell lymphoma, salvage after allogeneic bone marrow transplantation, and the like.

[0188] The CAR-modified lymphocytes described herein can also function as a type of vaccine for ex vivo immunization and / or in vivo therapy in a subject, preferably a human.

[0189] For ex vivo immunization, at least one of the following is performed in vitro before administering lymphocytes to a subject: i) expanding the cells, ii) introducing at least one synthetic nucleotide encoding at least one IRP and / or CAR into the cells, and / or iii) cryopreserving the cells.

[0190] Ex vivo procedures are well known in the art. Briefly, lymphocytes are isolated from a subject (preferably human) and genetically modified (i.e., transduced or transfected in vitro) with at least one vector expressing at least one IRP and / or CAR disclosed herein. CAR-modified cells expressing at least one IRP can be administered to a recipient to provide therapeutic benefit. The recipient can be human, and modified lymphocytes can be autologous to the recipient. Alternatively, lymphocytes can be allogeneic, syngeneic, or xenogeneic to the recipient.

[0191] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells described in U.S. Patent No. 5,199,942 can be applied to the cells of the present invention. Other suitable methods are known in the art, and therefore the present invention is not limited to any particular method of ex vivo cell expansion. Briefly, ex vivo culture and expansion of lymphocytes involves (1) harvesting CD34+ hematopoietic stem and progenitor cells from a mammal, either from peripheral blood collection or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cell growth factors described in U.S. Patent No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can be used for cell culture and expansion.

[0192] In addition to using cell-based vaccines for ex vivo immunization, the present disclosure also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0193] Generally, lymphocytes activated and expanded as described herein can be used to treat and prevent diseases occurring in immunocompromised individuals. In particular, CAR-modified lymphocytes described herein can be used to treat chronic lymphocytic leukemia (CCL). In certain embodiments, lymphocytes described herein can be used to treat patients at risk of developing CCL. Therefore, the present disclosure provides a method for treating or preventing CCL, comprising administering a therapeutically effective amount of lymphocytes of the present invention to a subject in need thereof.

[0194] Alternatively, lymphocytes for use in treating cancer can be NK cells, TILs, or TCR-modified lymphocytes. NK cells, TILs, or TCR-modified T cells can be modified using the methods of the present invention to overexpress IRP1 and / or IRP2, thereby resulting in more efficient expansion of the NK cells, TILs, or TCR-modified T cells in vivo.

[0195] NK cells used in cancer therapy may be allogeneic NK cells, as they have a graft-versus-leukemia / tumor (GvL / GvT) effect and do not cause graft-versus-host disease (GvHD), and therefore cause less immunopathology.

[0196] TILs for use in cancer therapy can be obtained as described in WO2018 / 182817 and can be further modified by introducing at least one polynucleotide encoding at least one IRP.

[0197] In one embodiment, the present invention relates to a lymphocyte according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention for use in the prevention and / or treatment of a viral infection.

[0198] That is, lymphocytes according to the present invention can also be used to prevent and treat viral infection.For example, it is known that virus-specific T cells can be used to prevent or treat viral infection in subjects who have undergone hematopoietic stem cell transplantation, but this is not limited thereto.Virus-specific T cells can be generated by stimulating and expanding T cells with viral antigens, such as antigen-presenting cells that present viral antigenic peptides, whole virus particles, viral lysates, whole virus proteins, or viral vectors.Alternatively, virus-specific T cells can be generated by expressing natural or engineered T cell receptors that are known to bind to specific viral antigens.The obtained virus-specific T cells can then be administered to subjects suffering from or at risk of viral infection.

[0199] By expressing at least one IRP in virus-specific T cells, thereby creating a pseudo-iron-deficient state, virus-specific T cells can be more robustly proliferated after administration to a subject. As a result, virus-specific T cells that have been genetically engineered to express at least one IRP can be more effective in preventing or treating viral infections than virus-specific T cells that have not been genetically engineered. The synthetic polynucleotide encoding at least one IRP can be introduced into T cells before, during, or after stimulation of T cells with viral antigens.

[0200] The prevention and treatment of above-mentioned viral infection do not necessarily require the presence of CAR.However, by using the lymphocyte of the present invention that further comprises CAR, at least in some cases, lymphocyte recognition of virus can be improved.In this case, CAR can preferably comprise the antigen binding domain that specifically binds to viral antigen.

[0201] In one embodiment, the invention relates to a lymphocyte, a viral vector or a pharmaceutical composition for use according to the invention, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpesvirus, in particular wherein the human herpesvirus is caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpesvirus 8 (HHV8).

[0202] Human cytomegalovirus (CMV) is a widespread β-herpesvirus with a population prevalence of 50–100%. CMV can manifest as a mild, self-limited disease in immunocompetent hosts, but can cause severe, life-threatening disease in immunocompromised hosts. Because CMV persists in a latent form after acute infection, CMV-specific CD4+ and CD8+ T cells are required to maintain viral quiescence. In post-HSCT patients, in the absence of donor immunity and other immunocompromised conditions, CMV may reactivate in the form of retinitis, pneumonitis, hepatitis, or enteritis. Adoptive transfer of CMV-specific T cells is a logical strategy for treating and preventing CMV reactivation in such individuals, and numerous clinical trials have demonstrated excellent overall efficacy of virus-specific T cells. CMV-specific VSTs generated from naive T cells in umbilical cord blood (UCB) have also proven effective. These VSTs exhibit specificity for atypical epitopes while maintaining functionality.

[0203] EBV is a ubiquitous, highly immunogenic gamma-herpesvirus that can cause unique complications after transplantation. More than 90% of the population is infected and remains seropositive for life. The manifestation of primary EBV infection varies widely, from asymptomatic infection to debilitating viral illness. In most cases, EBV then remains latent for life in B cell and mucosal epithelial reservoirs under continuous T cell immune surveillance. In these healthy individuals, up to 2% of circulating T cells are EBV-specific. During the period of immunodeficiency after HSCT, EBV reactivation can lead to viremia and life-threatening post-transplant lymphoproliferative disorder (PTLD). The monoclonal antibody rituximab successfully treats severe EBV disease in many patients by eliminating EBV-harboring B cells, but it also results in a prolonged decline in antibody production and is not always successful in controlling PTLD.

[0204] Adenovirus infection can range from mild upper respiratory tract infection to a range of life-threatening pneumonia, gastrointestinal, hepatic, renal, and neurological complications. Following infection, latency is maintained in lymphoid tissues, but the virus can reactivate during the prolonged absence of T cell immunity. Adenoviruses cause potentially fatal viral complications in recipients after HSCT. Antiviral drugs such as ribavirin are largely ineffective. However, adenovirus-specific T cells generated from healthy donors have proven effective in treating even advanced disease. For this reason, adenovirus antigens are often incorporated into the generation of multiple virus-specific T cell products.

[0205] The BK and JC polyomaviruses are normally latent in healthy tissues of the majority of adult individuals but reactivate after HSCT and in immunocompromised individuals. The BK virus can manifest as nephropathy and life-threatening hemorrhagic cystitis (HC). Rarely, the closely related JC virus causes fatal brain damage from progressive multifocal leukoencephalopathy. Polyoma-specific VSTs are under development to combat these viruses. A single case report describes the successful use of BK VST, which subsequently resulted in complete resolution of the patient's HC without bystander organ toxicity, GVHD, or graft rejection. It is clear that the platform developed for ex vivo selection and expansion of VSTs can be readily adapted to many other viruses associated with immunodeficiency, and future developments include developing VSTs targeting a range of viruses, including VZV, HHV, and even HIV or influenza.

[0206] In one embodiment, the present invention relates to a method for treating a subject with cancer or for preventing and / or treating a viral infection in a subject, the method comprising administering to the subject a therapeutically effective amount of a lymphocyte according to the invention, a viral vector according to the invention or a pharmaceutical composition according to the invention.

[0207] The lymphocytes or pharmaceutical compositions described herein can be administered in a manner appropriate to the disease to be treated (or prevented). The number and frequency of administration will depend on factors such as the condition of the patient and the type and severity of the patient's disease, but appropriate dosages can be determined by clinical trials.

[0208] As used herein, "effective amount" refers to an amount that provides a therapeutic or preventative benefit. The term "therapeutically effective amount" refers to the amount of the subject compound that elicits the biological or medical response of a tissue, system, or subject that is sought by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of compound that, when administered, is sufficient to prevent or alleviate to some extent one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount varies depending on the compound, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0209] When an "immunologically effective amount," "antitumor effective amount," "tumor inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of lymphocytes or compositions of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, type of viral infection, severity of viral infection, and / or condition of the patient (subject). Pharmaceutical compositions comprising lymphocytes described herein can be administered in amounts of 10 cells / kg body weight, including all integer values ​​within the range. 4 ~10 9 cells, preferably 10 cells per kg of body weight 5 ~10 6 It can generally be shown that the lymphocyte composition can be administered in a single dose. The lymphocyte composition can also be administered multiple times at these doses. Lymphocyte administration can be carried out using injection techniques commonly known for immunotherapy (Rosenberg et al., 1988, New Eng. J. of Med; 319: 1676.). The optimal dosage and treatment regimen for a particular patient can be easily determined by those skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[0210] It may be desirable to administer activated lymphocytes to a subject, then subsequently draw blood again (or perform apheresis), activate lymphocytes therefrom according to the present invention, and reinfuse these activated and expanded lymphocytes into the patient. This process can be performed multiple times every few weeks. Lymphocytes can be activated from blood draws ranging from 10 mL to 400 mL; for example, lymphocytes can be activated from blood draws of 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 mL, 80 mL, 90 mL, or 100 mL. Without being bound by theory, the use of this multiple blood draw / multiple reinfusion protocol can aid in the selection of certain lymphocyte populations.

[0211] Administration of the lymphocytes or compositions can be carried out in any convenient manner, including by aerosol inhalation, injection, oral ingestion, transfusion, implantation, or transplantation. The lymphocytes or compositions described herein can be administered to a subject subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally. For example, the lymphocytes or compositions described herein can be administered to a patient by intradermal or subcutaneous injection. In another example, the lymphocytes or compositions described herein can preferably be administered by iv injection. The lymphocytes or compositions can be injected directly into a tumor, lymph node, or site of infection.

[0212] In certain cases, lymphocytes activated and expanded using the methods described herein or other methods known in the art for expanding lymphocytes to therapeutic levels are administered to a patient in conjunction with (e.g., before, simultaneously with, or after) any number of related treatment modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin 2, ribavirin, rituximab, cytarabine (also known as ARA-C), or natalizumab for MS patients, or efalizumab for psoriasis patients, or other treatments for PML patients. It is also disclosed herein that the lymphocytes of the present invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents, e.g., CAMPATH, anti-CD3 antibody or other antibody therapy, cytoxin, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (Liu et al., 1991, Cell; 66: 807-815; Henderson et al., 1991, Immun; 73: 316-321; Bierer et al., 1993, Curr. Opin. Immun; 5: 763-773). It is also disclosed herein that the lymphocytes or compositions of the invention can be administered to patients in conjunction with (e.g., before, simultaneously with, or after) T-cell depletion therapy using bone marrow transplantation, chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. It is also described herein that the lymphocytes or compositions of the invention can be administered after B cell depleting therapy, such as an agent that reacts with CD20, eg, Rituxan.For example, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In certain cases, the subject may receive an infusion of the expanded immune cells of the present invention after the transplant, or the expanded cells may be administered before or after surgery.

[0213] The dosages of the above treatments administered to a subject will vary depending on the exact nature of the condition being treated and the recipient of the treatment. Scaling of dosages for human administration can be performed according to art-accepted practices.

[0214] In one embodiment, the invention relates to a method according to the invention, wherein the cancer is a hematological cancer or a solid tumor, in particular wherein the hematological cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia and multiple myeloma, or wherein the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma and sarcoma.

[0215] In one embodiment, the invention relates to a method according to the invention, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpesvirus, in particular the human herpesvirus is caused by cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpesvirus 8 (HHV8).

[0216] In one embodiment, the present invention relates to a method for producing lymphocytes according to the present invention, the method comprising the steps of: a) providing lymphocytes obtained from a subject; b) introducing a synthetic polynucleotide encoding at least one iron-regulatory protein into the lymphocytes of step (a), wherein the iron-regulatory protein is IRP1 (SEQ ID NO: 1) and / or IRP2 (SEQ ID NOs: 2-6); and c) expressing the at least one iron-regulatory protein encoded by the synthetic polynucleotide introduced into the lymphocytes in step (b). It should be understood that the lymphocytes can be any lymphocyte disclosed herein.

[0217] Optionally, the present invention further relates to a method according to the invention, wherein in step (b) a second synthetic polynucleotide encoding a chimeric antigen receptor (CAR) is introduced into the lymphocytes.

[0218] That is, the method according to the present invention can be used to generate lymphocytes that overexpress IRP1 and / or IRP2. Furthermore, the method according to the present invention can be used to generate lymphocytes containing two synthetic polynucleotides: a first synthetic polynucleotide encoding IRP1 and / or IRP2 and a second synthetic polynucleotide encoding a CAR. In the latter case, it should be understood that the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polynucleotide encoding a CAR can be fused to each other as described herein. Alternatively, the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polynucleotide encoding a CAR can be unrelated. That is, the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polypeptide encoding a CAR can be introduced into lymphocytes independently. Preferably, the synthetic polynucleotides are introduced into lymphocytes by viral transduction and integrated into the lymphocyte genome. Thus, the first synthetic polynucleotide encoding IRP1 and / or IRP2 and the second synthetic polypeptide encoding a CAR can be contained in different viral vectors. A first viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2 and a second viral vector comprising a synthetic polynucleotide encoding a CAR can be introduced into lymphocytes in a single transduction experiment. Alternatively, lymphocytes can be transduced stepwise with a first viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2 and a second viral vector comprising a synthetic polynucleotide encoding a CAR. For example, lymphocytes according to the present invention can be first transduced with a viral vector comprising a synthetic polynucleotide encoding a CAR to generate, but not limited to, CAR T cells or CAR NK cells, and then in a second step, transduced with a viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2.Alternatively, lymphocytes can be first transduced with a viral vector comprising a synthetic polynucleotide encoding IRP1 and / or IRP2, and in a second step transduced with a viral vector comprising a synthetic polynucleotide encoding a CAR.

[0219] Before carrying out the genetic modification of lymphocytes of the present invention, lymphocyte sources can be obtained from subjects.Lymphocytes can be obtained from multiple sources, including peripheral blood, mononuclear cells, bone marrow, lymph node tissue, blood, thymus tissue, tissue from infected sites, ascites, pleural effusion, spleen tissue and tumor.Within the scope of the present invention, any type of lymphocytes available in the art can be used.Generally, those skilled in the art are familiar with the methods for isolating a specific type of lymphocyte from a suitable source.

[0220] Certain types of lymphocytes, specifically peripheral blood mononuclear cells (PBMCs), can be obtained from a unit of blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. Additionally, cells derived from an individual's circulating blood can be obtained by apheresis. Apheresis products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. Cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps. For example, cells can be washed with phosphate-buffered saline (PBS). Alternatively, the wash solution can be calcium-free and magnesium-free, or it can be free of many, but not all, divalent cations. As will be readily understood by those skilled in the art, the washing step can be accomplished by methods known to those skilled in the art, such as, for example, by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 29 cell processor, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca+-free, Mg+-free PBS, PlasmaLyte A, or other saline solutions, with or without buffers. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium. Alternatively, specific types of lymphocytes can be isolated by, for example, centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation to lyse red blood cells and deplete monocytes.

[0221] Specific lymphocyte subpopulations, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, CD16+ and CD56+ NK cells, or CD3+, CD56+, and CD161+ NKT cells, can be further isolated by positive or negative selection techniques. It is known in the art that each type of lymphocyte has a surface antigen. Therefore, those skilled in the art can select positive or negative selection conditions that allow for the enrichment or isolation of specific types of lymphocytes. Furthermore, commercially available kits for enriching and / or isolating specific types of lymphocytes are known to those skilled in the art.

[0222] In certain embodiments, T cells can be isolated by incubation with anti-CD3 / anti-CD28-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient for positive selection of the desired T cells. The period can range from 30 minutes to 36 hours or longer, including all integer values ​​in between. In certain embodiments, the period is at least 0.5, 1, 2, 3, 4, 5, or 6 hours. Alternatively, the period can be 10 to 24 hours. For the isolation of T cells from leukemia patients, longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times can be used for the isolation of T cells in any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Furthermore, longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply allowing T cells to bind to CD3 / CD28 beads for a shorter or longer period of time, and / or by increasing or decreasing the ratio of beads to T cells, subpopulations of T cells can be preferentially selected for or relative to the start of culture or other time points during the process; and by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or relative to the start of culture or other desired time points. Those skilled in the art will appreciate that multiple rounds of selection can also be used in connection with the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use the "unselected" cells for the activation and expansion process. The "unselected" cells can also be subjected to additional rounds of selection.

[0223] Enrichment of lymphocyte populations by negative selection can be achieved using a combination of antibodies directed against surface markers specific to negatively selected cells. One method is negative magnetic immunoadhesion or flow cytometry cell sorting and / or selection using a cocktail of monoclonal antibodies directed against cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail generally comprises antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select regulatory T cells, which generally express CD4+CD25+, CD62L, GITR+, and FoxP3+. Alternatively, in certain embodiments, regulatory T cells can be depleted by anti-CD25-conjugated beads or other similar selection methods.

[0224] In certain embodiments, NK cells from healthy donors or patients can be enriched from PBMCs or directly from blood by incubation with magnetic beads using a human NK cell negative selection isolation kit (Miltenyi Biotec or STEMCELL Technologies) according to the manufacturer's instructions. For example, when using Miltenyi Biotec's isolation kit, unwanted cells (i.e., T cells, B cells, macrophages, and monocytes) can be removed using a cocktail of biotin-conjugated monoclonal anti-human antibodies against antigens not expressed by NK cells at a concentration of 2.5 billion cells per mL in PBMCs. The biotin-conjugated antibody-labeled unwanted cells can then be magnetically labeled using an NK cell microbead cocktail and removed using a MACS column. For example, when using STEMCELL Technologies' isolation kit, unwanted cells (i.e., T cells, B cells, macrophages, and monocytes) can be removed using a tetrameric anti-human antibody complex against an antigen not expressed by NK cells at a concentration of 50 million cells per mL in PBMCs. The unwanted cells labeled with the tetrameric antibody complex can then be magnetically labeled with dextran-coated magnetic particles and removed using a magnet.

[0225] To isolate a desired population of lymphocytes by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed (i.e., increase the concentration of cells) to ensure maximum contact between the cells and beads. For example, in one embodiment, a concentration of 2 billion cells per mL can be used. In one embodiment, a concentration of 1 billion cells per mL can be used. In further embodiments, greater than 100 million cells per mL can be used. In further embodiments, concentrations of 0, 15, 20, 25, 30, 35, 40, 45, or 50 million cells per mL can be used. In yet another embodiment, a cell concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per mL can be used. In a further embodiment, a concentration of 125 million or 150 million cells per mL can be used. The use of a high concentration can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of a high cell concentration can enable more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells, or more efficient capture from samples containing many tumor cells (i.e., leukemic blood, tumor tissue, etc.). Such cell populations have therapeutic value and are desirable to obtain. For example, the use of a high cell concentration can enable more efficient selection of CD8+ T cells, which typically have weak CD28 expression.

[0226] In related embodiments, it may be desirable to use low concentrations of cells. By significantly diluting the mixture of cells and surfaces (e.g., particles such as beads), particle-cell interactions can be minimized. This can select for cells that express high amounts of the desired antigen to be bound to the particles. For example, CD4+ T cells may express high levels of CD28 and, at dilute concentrations, can be captured more efficiently than CD8+ T cells.

[0227] Lymphocytes can be incubated on a rotator at varying speeds for various lengths of time at either 2-10°C or room temperature. Cells for stimulation can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing step can result in a more uniform product by removing granulocytes and, to some extent, monocytes within the cell population. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this regard, one method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or culture medium containing 1.25% Plasmalyte-A, 3.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte-A. The cells can then be frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods can also be used, or uncontrolled freezing can be performed immediately at -20°C or in liquid nitrogen.

[0228] Cryopreserved cells can be thawed, washed as described herein, allowed to stand at room temperature for 1 hour, and then activated using the methods of the present invention.

[0229] It is also contemplated for the present invention that a blood sample or apheresis product may be collected from a subject at a time prior to the need for cells expanded as described herein. In this manner, a source of cells to be expanded can be collected at any time necessary, and desired cells, such as lymphocytes, can be isolated and frozen for later use in cell therapy for any number of diseases or conditions that would benefit from cell therapy, such as those described herein. The blood sample or apheresis can be obtained from a generally healthy subject. In certain embodiments, a blood sample or apheresis can be obtained from a generally healthy subject who is at risk for developing a disease but has not yet developed the disease, and the desired cells can be isolated and frozen for later use. In certain embodiments, the cells can be expanded, frozen, and used at a later time. In certain embodiments, a sample can be collected from a patient shortly after diagnosis of a particular disease described herein, but prior to any treatment. Additionally, cells can be isolated from blood samples or apheresis from subjects prior to any number of relevant treatment modalities, including, but not limited to, treatment with natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or p70S6 kinase (rapamycin), which is important for growth factor-induced signal transduction (99-101). In certain embodiments, cells can be isolated from a patient and frozen for later use in conjunction with (e.g., before, simultaneously with, or after) bone marrow or stem cell transplantation, chemotherapy such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or T cell depletion therapy using antibodies to OKT3 or CAMPATH.In certain embodiments, cells can be pre-isolated and frozen for later use in treatment after B cell depleting therapy, such as with an agent reactive with CD20, e.g., Rituxan.

[0230] In certain embodiments of the present invention, lymphocytes can be obtained from patients immediately after treatment. In this regard, it has been observed that after certain cancer treatments, particularly those using drugs that damage the immune system, the quality of lymphocytes obtained immediately after treatment, during the period when patients are typically recovering from the treatment, can be optimal or improved in terms of their ability to expand ex vivo. Similarly, after ex vivo manipulation using the methods described herein, these cells can be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, within the context of the present invention, it is contemplated to harvest blood cells, including lymphocytes, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create a subject state in which repopulation, recirculation, regeneration, and / or expansion of specific cell types is advantageous, particularly during a defined time frame after treatment. Exemplary cell types include T cells, NK cells, B cells, dendritic cells, and other cells of the immune system.

[0231] The present invention encompasses one or more synthetic polynucleotides, comprising one or more IRPs and optionally CAR coding polynucleotide sequences.The synthetic polynucleotides encoding desired molecules can be obtained by using recombinant methods known in the art, such as by screening libraries from cells that express the gene, by extracting the gene from a vector that is known to contain the gene, or by directly isolating the gene from cells and tissues that contain the gene using standard techniques.Alternatively, the gene of interest can be produced synthetically instead of by cloning.

[0232] The present invention also provides a vector into which the synthetic polynucleotide of the present invention can be inserted.Vector derived from retroviruses such as lentiviruses allows long-term stable integration of transgenes and their transmission in daughter cells, making it a suitable tool for achieving long-term gene transfer.Compared with vectors derived from oncoretroviruses such as murine leukemia viruses, lentiviral vectors have the additional advantage of being able to transduce non-proliferating cells such as hepatocytes.Lentiviral vectors also have the additional advantage of being less immunogenic.

[0233] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid into a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. This term should also be construed to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0234] Briefly, the expression of natural or synthetic polynucleotides encoding IRP and optionally CAR is generally achieved by operably linking the polynucleotide encoding IRP or optionally CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector.The vector may be suitable for replication and / or integration in eukaryotes.Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of desired polynucleotides.

[0235] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0236] The expression constructs of the present invention can also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art.

[0237] IRP and optionally the synthetic polynucleotide encoding CAR can be cloned into many types of vectors.For example, polynucleotide can be cloned into vectors including but not limited to plasmid, phagemid, phage derivative, animal virus and cosmid.Particularly interesting vectors include expression vector, replication vector, probe generation vector and sequencing vector.

[0238] Furthermore, expression vectors can be provided to cells in the form of viral vectors.Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.Generally, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0239] Numerous virus-based systems have been developed for gene transfer into mammalian cells.For example, retrovirus provides a convenient platform for gene delivery systems.Using techniques known in the art, a selected gene can be inserted into a vector and packaged into retroviral particles.The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo.Many retroviral systems are known in the art.Within the scope of the present invention, it is preferred to use adenoviral vector or lentiviral vector.

[0240] Additional promoter elements, such as enhancers, can be used to modulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is frequently flexible, and thus promoter function can be preserved even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. It is clear that, depending on the promoter, individual elements can function cooperatively or independently to activate transcription.

[0241] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any operably linked polynucleotide sequence. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α) promoter. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters; inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0242] To evaluate the expression of IRP or, if necessary, CAR polypeptide or a portion thereof, the expression vector introduced into cells can also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a group of cells to be transfected or infected via a viral vector.In other embodiments, the selection marker can be carried on a separate piece of DNA and used in a co-transfection procedure.Both the selection marker and the reporter gene can be adjacent to an appropriate regulatory sequence to allow expression in host cells.Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0243] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes encoding polypeptides whose expression is manifested by some easily detectable property, such as enzymatic activity, that is not present or expressed in recipient organisms or tissues. The expression of the reporter gene can be assayed at an appropriate time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (Ui-Tei et al., 2000, FEBS Letters; 479: 79-82). Suitable expression systems are well known and can be prepared using known techniques or commercially available.

[0244] Methods for introducing and expressing genes into cells are known in the art.As for expression vectors, the vector can be easily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art.For example, the expression vector can be transferred into host cells by physical means, chemical means, or biological means.

[0245] Physical means for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous polynucleotides are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0246] The biological method for introducing target polynucleotide into host cell includes the use of DNA and RNA vector.Virus vector, especially retrovirus vector, has become the most widely used method for inserting gene into mammalian, for example, human cell.Other virus vectors can be derived from lentivirus, poxvirus, herpes simplex virus I, adenovirus and adeno-associated virus, etc.See, for example, U.S. Patent No. 5,350,674 and U.S. Patent No. 5,585,362.

[0247] Chemical means for introducing polynucleotides into host cells include polymer complexes, nanocapsules, microspheres, beads, and colloidal dispersion systems, such as lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). When a non-viral delivery system is used, an exemplary delivery vehicle can be a liposome. The use of lipid formulations for introducing polynucleotides into host cells is contemplated (in vitro, ex vivo, or in vivo). In another embodiment, the polynucleotide can be associated with a lipid. A lipid-associated polynucleotide may be encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule associated with both the liposome and the polynucleotide, entrapped within a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained in or complexed with a micelle, or otherwise associated with a lipid. Compositions involving lipids, lipid / DNA, or lipid / expression vectors are not limited to any particular structure in solution. For example, the composition may exist as a micelle or in a bilayer structure with a "collapsed" structure. The composition may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that occur naturally within the cytoplasm as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0248] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol and can therefore be used as the sole solvent. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as vesicular structures with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement to form a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991, Glycobiology; 5; 505-10). However, compositions with structures in solution that differ from the usual vesicular structure are also encompassed. For example, lipids may assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0249] The terms "transfected" or "transformed" or "transduced," as used herein, refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0250] Regardless of the method used to introduce an exogenous synthetic polynucleotide into a host cell, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of a particular peptide by, for example, immunological means (ELISA, Western blot, flow cytometry) or by the assays described herein to identify agents within the scope of the present invention.

[0251] In another embodiment, the present invention relates to a method according to the invention, in which a synthetic polynucleotide encoding a chimeric antigen receptor (CAR) is combined with a synthetic polynucleotide encoding at least one iron-regulatory protein, in particular wherein the at least one iron-regulatory protein is IRP1 and / or IRP2.

[0252] The chimeric antigen receptor and at least one IRP can be encoded by separate synthetic polynucleotides that are not contiguous with each other and are not directly connected.In this case, the synthetic polynucleotide encoding at least one IRP and the synthetic polynucleotide encoding CAR can be introduced into cells separately using the same or different methods.Alternatively, the gene(s) encoding at least one IRP and the gene encoding CAR can be combined in a single synthetic polynucleotide.Two synthetic polynucleotides can be said to be combined when the combined synthetic polynucleotide contains all the genes encoded by two separate synthetic polypeptides.

[0253] For example, if it is planned to introduce genes encoding at least one IRP and a CAR into lymphocytes by viral transduction, the gene(s) encoding at least one IRP and the gene encoding the CAR can be included in separate viral vectors or can be combined into a single viral vector.

[0254] In yet another embodiment, the present invention relates to a method according to the present invention, wherein the lymphocytes are activated before or after introducing one or more synthetic polynucleotides into the lymphocytes.

[0255] Whether before or after the lymphocytes have been genetically modified to express at least one IRP, or optionally a desired CAR, the lymphocytes can be activated and expanded prior to administration to the subject. Those skilled in the art will recognize that specific conditions are required to activate different types of lymphocytes.

[0256] Methods for activating NK cells are known to those skilled in the art. For example, the NK cells described herein can be activated by culturing them in an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza), CellGro Medium (Cellgenix), IMDM (Gibco)) supplemented with IL-15 and / or IL-12 and / or IL-18, which may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum, human serum, or horse serum). NK cell activation can also be achieved by supplementing the medium with IL-2. NK cell activation can be improved by adding a feeder cell line to the culture. Suitable feeder cell lines for NK cell activation are cancer cell lines, genetically modified K562 cells, or EBV-transformed lymphoblastoid cell lines or autologous peripheral blood mononuclear cells (irradiated).

[0257] Generally, the T cells described herein can be activated by contact with a surface bearing an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, a population of T cells can be stimulated by contacting them with an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD2 antibody immobilized on the surface, or by contacting them with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For costimulation of accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule can be used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. Anti-CD3 antibody and anti-CD28 antibody can be used to stimulate the proliferation of either CD4+ or CD8+ T cells. Anti-CD28 antibodies 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used, as can other methods known in the art (Berg et al., 1998, Transplant Proc; 30(8): 3975-3977; Haanen et al., 1999, J. Exp. Med; 190: 13191328; Garland et al., 1999, J. Immunol Meth. 227: 53-63).

[0258] The primary and costimulatory signals for T cells can be provided by different protocols. For example, the agents providing each signal can be in solution or coupled to a surface. If coupled to a surface, the agents can be coupled to the same surface (i.e., a "cis" configuration) or to separate surfaces (i.e., a "trans" configuration). Alternatively, one agent can be coupled to a surface and the other in solution. For example, the agent providing the costimulatory signal can be attached to a surface, and the agent providing the primary activation signal can be in solution or coupled to a surface, or both agents can be in solution. Alternatively, the agents can be in soluble form and then cross-linked to a surface, such as a cell expressing an Fc receptor or antibody or other binding agent that binds the agent. In this regard, see, for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810, which relate to artificial antigen-presenting cells (aAPCs) intended for use in T cell activation and expansion.

[0259] The two agents are immobilized on beads either on the same bead, i.e., "cis," or on separate beads, i.e., "trans." By way of example, the agent providing the primary activation signal can be an anti-CD3 antibody or its antigen-binding fragment, and the agent providing the costimulatory signal can be an anti-CD28 antibody or its antigen-binding fragment, with both agents co-immobilized on the same bead at equivalent molecular weights. For example, a 1:1 ratio of each antibody bound to beads for CD4+ T cell expansion and T cell growth can be used. In certain cases, a ratio of anti-CD3:CD28 antibodies bound to beads can be used such that increased T cell expansion is observed compared to that observed using a 1:1 ratio. An increase of about 1-fold to about 3-fold compared to that observed using a 1:1 ratio can be observed. The ratio of CD3:CD28 antibodies bound to beads can range from 100:1 to 1:100, and all integer values ​​therebetween. In some cases, more anti-CD28 antibody can be bound to the particles than anti-CD3 antibody, i.e., the CD3:CD28 ratio can be less than 1. In certain cases, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to the beads can be greater than 2:1. For example, antibodies bound to beads at a CD3:CD28 ratio of 1:100 can be used, antibodies bound to beads at a CD3:CD28 ratio of 1:75 can be used, antibodies bound to beads at a CD3:CD28 ratio of 1:50 can be used, antibodies bound to beads at a CD3:CD28 ratio of 1:30 can be used, antibodies bound to beads at a CD3:CD28 ratio of 1:10 can be used, or antibodies bound to beads at a CD3:CD28 ratio of 1:3 can be used. Alternatively, antibodies bound to beads at a CD3:CD28 ratio of 3:1 can be used.

[0260] To stimulate T cells or other target cells, particle-to-cell ratios ranging from 1:500 to 500:1, and all integer values ​​therebetween, can be used. As will be readily understood by those skilled in the art, the particle-to-cell ratio can depend on the particle size relative to the target cells. For example, small beads can only bind to a small number of cells, while larger beads can bind to a large number of cells. The ratio of anti-CD3-coupled particles and anti-CD28-coupled particles to T cells that results in T cell stimulation can vary as described above, but certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, and 15:1, with one preferred ratio being at least 1:1 particles per T cell. Alternatively, a particle-to-cell ratio of 1:1 or less can be used. A preferred particle:cell ratio can be 1:5. The particle-to-cell ratio can vary depending on the stimulation day. For example, the particle to cell ratio can be 1:1 to 10:1 on the first day, and then additional particles can be added to the cells every day or every other day for up to 10 days, resulting in a final ratio of 1:1 to 1:10 (depending on the cell count on the day of addition). Alternatively, the particle to cell ratio can be 1:1 on the first day of stimulation and adjusted to 1:5 on the third and fifth days of stimulation. In another case, particles can be added every day or every other day to achieve a final ratio of 1:1 on the first day and 1:5 on the third and fifth days of stimulation. In another case, the particle to cell ratio can be 2:1 on the first day of stimulation and adjusted to 1:10 on the third and fifth days of stimulation. In another case, particles can be added every day or every other day to achieve a final ratio of 1:1 on the first day and 1:10 on the third and fifth days of stimulation. Those skilled in the art will appreciate that various other ratios may be suitable for use in the present invention. In particular, the ratio varies depending on particle size and cell size and type.

[0261] T cells can be combined with drug-coated beads, then bead and cell can be separated, and then cell can be cultured.Alternatively, drug-coated beads and cell can be not separated before culture, but can be cultured together.In another case, bead and cell can be concentrated first by applying force such as magnetic force, which results in increased ligation of cell surface marker, thereby inducing cell stimulation.

[0262] cells (e.g., 10 4 ~10 9T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads, 1:1 ratio) can be combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Again, one of skill in the art will readily appreciate any cell concentration that can be used. To ensure maximum contact between the cells and particles, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration). For example, a concentration of approximately 2 billion cells per mL can be used. In other cases, concentrations greater than 100 million cells per mL can be used. In further cases, concentrations of 10 million, 15 million, 20 million, 25 million, 30 million, 35 million, 40 million, 45 million, or 50 million cells per mL can be used. In yet another case, a concentration of 75 million, 80 million, 85 million, 90 million, 95 million, or 100 million cells per mL can be used. In yet another case, a concentration of 125 million or 150 million cells per mL can be used. The use of a high concentration can result in increased cell yield, cell activation, and cell expansion. Furthermore, the use of a high cell concentration can enable more efficient capture of cells that may weakly express the target antigen of interest, such as CD28-negative T cells. In certain embodiments, such a cell population has therapeutic value and is desirable to obtain. For example, the use of a high cell concentration can enable more efficient selection of CD8+ T cells, which usually have weak CD28 expression.

[0263] The mixture can be cultured for a few hours (approximately 3 hours) up to about 14 days, or any hourly integer value in between. The mixture can be cultured for 21 days. In some cases, the beads and T cells can be cultured together for about 8 days. In other cases, the beads and T cells can be cultured together for 2-3 days. Several cycles of stimulation may also be desirable, and therefore, the T cell culture time may be 60 days or longer.

[0264] T cells exposed to various stimulation times may exhibit different characteristics. For example, a typical blood or apheresis peripheral blood mononuclear cell product contains a higher percentage of helper T cells (TH, CD4+) than cytotoxic or suppressor T cells (TC, CD8+). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors results in a T cell population that is predominantly TH cells before about 8-9 days, whereas after about 8-9 days, the T cell population contains increasingly more TC cells. Thus, depending on the goal of treatment, it may be advantageous to infuse a T cell population that is predominantly TH cells into a subject. Similarly, if an antigen-specific subset of TC cells is isolated, it may be beneficial to expand this subset to a greater extent.

[0265] Furthermore, in addition to CD4 and CD8 markers, other phenotypic markers also vary significantly but are largely reproducible during the course of the cell expansion process, thus allowing activated T cell products to be tailored for specific purposes.

[0266] Suitable conditions for lymphocyte culture include an appropriate medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo 15 (Lonza), CellGro Medium (Cellgenix), IMDM (Gibco)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine serum, human serum, or horse serum), interleukin 2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, IL-18, TGF-β, and TNF-α, or any other additives for cell growth known to those of skill in the art. Other additives for cell growth may include, but are not limited to, surfactants, plasmanate, and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture media can include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15, X-Vivo 20, IMDM, and CellGro, Optimizer, which are supplemented with amino acids, sodium pyruvate, and vitamins, and are either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones and / or cytokine(s) sufficient for the growth and expansion of NK cells and T cells. Antibiotics, such as penicillin and streptomycin, can only be included in experimental cultures and cannot be included in cultures of cells to be infused into subjects. Lymphocytes can be maintained under conditions necessary to support growth, for example, under an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air + 5% CO2).

[0267] In a further embodiment, the present invention relates to a method according to the invention, wherein at least one synthetic polynucleotide is introduced into lymphocytes by viral transduction, in particular by lentiviral transduction.

[0268] As described above, the synthetic polynucleotide(s) encoding at least one IRP and optionally a CAR can be introduced into lymphocytes by any method known in the art. However, it is preferred to introduce the synthetic polynucleotide(s) into lymphocytes by the above-mentioned viral transduction. More preferably, the viral vector used to introduce the synthetic polynucleotide into lymphocytes is a lentiviral vector. Because viral vectors are usually integrated into the genome of host cells at random locations, it is preferred that the synthetic polynucleotide comprises a gene encoding at least one IRP and the regulatory elements necessary for expressing the gene encoding at least one IRP in host cells.

[0269] " Lentivirus " as used herein refers to the genus of Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells.Lentivirus can deliver significant amount of genetic information to the DNA of host cell, and therefore is one of the most efficient methods of gene delivery vector.HIV, SIV and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means for achieving significant level of gene transfer in vivo.

[0270] In one embodiment, the invention relates to a method according to the invention, wherein a synthetic polynucleotide encoding a CAR is transcriptionally linked to a synthetic polynucleotide encoding IRP1 and / or IRP2.

[0271] In one embodiment, the invention relates to a method according to the invention, wherein a synthetic polynucleotide encoding a CAR and a polynucleotide encoding IRP1 and / or IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

[0272] In one embodiment, the present invention relates to a method according to the present invention, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

[0273] In one embodiment, the invention relates to a method according to the invention, wherein the self-cleaving peptide is T2A.

[0274] In one embodiment, the present invention relates to a method according to the invention, wherein one or more synthetic polynucleotides are introduced into lymphocytes by viral transduction.

[0275] In one embodiment, the present invention relates to a method according to the present invention, wherein viral transduction is performed using a viral vector according to any of the embodiments presented herein.

[0276] Examples of synthetic polynucleotides encoding IRP1 and / or IRP2, and optionally a CAR, promoters and / or further regulatory elements (e.g., a polynucleotide encoding an IRES or a self-cleaving peptide) are disclosed elsewhere herein and apply mutatis mutandis to the claimed methods. Viral vectors according to the invention are preferably used in methods according to the invention. [Brief explanation of the drawings]

[0277] [Figure 1A-C]Figure 1: Naive and cytokine-enhanced NK cells similarly rely on glycolysis for IFN-γ production. (A) Schematic of the experiment used to generate CE NK cells. (B) IFN-γ production by unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 18 donors). (C) GMFI of CD69 expression in unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 13 donors). (D) PCA of transcriptome data showing group relationships in unstimulated NV NK cells and CE NK cells (or IL-12 / IL-18-stimulated NV NK cells and CE NK cells). The proportion of variance components is expressed as a percentage (n = 5 donors). (E) Heatmap of relative expression levels of mRNAs encoding glycolytic genes from unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (n = 5 donors) of transcriptome data. (F) Upper panel: Representative mitochondrial perturbation assay of unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (G) Upper panel: Representative histograms of NBDG uptake in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of NBDG uptake in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 15 donors). (H) IFNG mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (I) Upper panel: IFN-γ production by unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG (mean ± SEM, n = 6 donors). Lower panel: IFN-γ production by unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 under 10 mM glucose and under 2 mM glucose (mean ± SEM, n = 5 donors). Statistical significance was assessed by paired two-tailed Student's t-test (C, F, H, I) or linear regression analysis (B, G, H, I). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 1D-E]Figure 1: Naive and cytokine-enhanced NK cells similarly rely on glycolysis for IFN-γ production. (A) Schematic of the experiment used to generate CE NK cells. (B) IFN-γ production by unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 18 donors). (C) GMFI of CD69 expression in unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 13 donors). (D) PCA of transcriptome data showing group relationships in unstimulated NV NK cells and CE NK cells (or IL-12 / IL-18-stimulated NV NK cells and CE NK cells). The proportion of variance components is expressed as a percentage (n = 5 donors). (E) Heatmap of relative expression levels of mRNAs encoding glycolytic genes from unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (n = 5 donors) of transcriptome data. (F) Upper panel: Representative mitochondrial perturbation assay of unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (G) Upper panel: Representative histograms of NBDG uptake in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of NBDG uptake in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 15 donors). (H) IFNG mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (I) Upper panel: IFN-γ production by unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG (mean ± SEM, n = 6 donors). Lower panel: IFN-γ production by unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 under 10 mM glucose and under 2 mM glucose (mean ± SEM, n = 5 donors). Statistical significance was assessed by paired two-tailed Student's t-test (C, F, H, I) or linear regression analysis (B, G, H, I). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 1F]Figure 1: Naive and cytokine-enhanced NK cells similarly rely on glycolysis for IFN-γ production. (A) Schematic of the experiment used to generate CE NK cells. (B) IFN-γ production by unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 18 donors). (C) GMFI of CD69 expression in unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 13 donors). (D) PCA of transcriptome data showing group relationships in unstimulated NV NK cells and CE NK cells (or IL-12 / IL-18-stimulated NV NK cells and CE NK cells). The proportion of variance components is expressed as a percentage (n = 5 donors). (E) Heatmap of relative expression levels of mRNAs encoding glycolytic genes from unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (n = 5 donors) of transcriptome data. (F) Upper panel: Representative mitochondrial perturbation assay of unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (G) Upper panel: Representative histograms of NBDG uptake in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of NBDG uptake in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 15 donors). (H) IFNG mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (I) Upper panel: IFN-γ production by unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG (mean ± SEM, n = 6 donors). Lower panel: IFN-γ production by unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 under 10 mM glucose and under 2 mM glucose (mean ± SEM, n = 5 donors). Statistical significance was assessed by paired two-tailed Student's t-test (C, F, H, I) or linear regression analysis (B, G, H, I). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 1G]Figure 1: Naive and cytokine-enhanced NK cells similarly rely on glycolysis for IFN-γ production. (A) Schematic of the experiment used to generate CE NK cells. (B) IFN-γ production by unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 18 donors). (C) GMFI of CD69 expression in unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 13 donors). (D) PCA of transcriptome data showing group relationships in unstimulated NV NK cells and CE NK cells (or IL-12 / IL-18-stimulated NV NK cells and CE NK cells). The proportion of variance components is expressed as a percentage (n = 5 donors). (E) Heatmap of relative expression levels of mRNAs encoding glycolytic genes from unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (n = 5 donors) of transcriptome data. (F) Upper panel: Representative mitochondrial perturbation assay of unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (G) Upper panel: Representative histograms of NBDG uptake in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of NBDG uptake in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 15 donors). (H) IFNG mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (I) Upper panel: IFN-γ production by unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG (mean ± SEM, n = 6 donors). Lower panel: IFN-γ production by unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 under 10 mM glucose and under 2 mM glucose (mean ± SEM, n = 5 donors). Statistical significance was assessed by paired two-tailed Student's t-test (C, F, H, I) or linear regression analysis (B, G, H, I). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 1H-I]Figure 1: Naive and cytokine-enhanced NK cells similarly rely on glycolysis for IFN-γ production. (A) Schematic of the experiment used to generate CE NK cells. (B) IFN-γ production by unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 18 donors). (C) GMFI of CD69 expression in unstimulated (no stim) NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (mean ± SEM, n = 13 donors). (D) PCA of transcriptome data showing group relationships in unstimulated NV NK cells and CE NK cells (or IL-12 / IL-18-stimulated NV NK cells and CE NK cells). The proportion of variance components is expressed as a percentage (n = 5 donors). (E) Heatmap of relative expression levels of mRNAs encoding glycolytic genes from unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells (n = 5 donors) of transcriptome data. (F) Upper panel: Representative mitochondrial perturbation assay of unstimulated NV NK cells and CE NK cells or IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (G) Upper panel: Representative histograms of NBDG uptake in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of NBDG uptake in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 15 donors). (H) IFNG mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (I) Upper panel: IFN-γ production by unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2-DG (mean ± SEM, n = 6 donors). Lower panel: IFN-γ production by unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 under 10 mM glucose and under 2 mM glucose (mean ± SEM, n = 5 donors). Statistical significance was assessed by paired two-tailed Student's t-test (C, F, H, I) or linear regression analysis (B, G, H, I). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

[0278] [Figure 2A]Figure 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation. (A) Top panel: Representative histograms of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Bottom panel: MFI (mean ± SEM, n = 8 donors) of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (B) Top panel: Representative histograms of CD71 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Lower panel: GMFI and percentage of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 14 donors). (C) Left panel: Representative Western blot of total CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Right panel: Total CD71 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 13 donors). (D) Left panel: GMFI of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6). Right panel: Percentage of CD71+ NK cells in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (E) Top panel: Representative histogram of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 10 donors). (F) Upper panel: Schematic of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle panel: Representative histogram of CFSE dilution in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Percentage of proliferation of unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells analyzed by CFSE dilution (mean ± SEM, n = 13 donors). (G) Heatmap of relative expression levels of mRNA encoding cell cycle genes (GO:0006098) in unstimulated (unstimulated) or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (H) Percentage of proliferation of unstimulated (no stimulation) NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18+BIP (1 μM, 10 μM, and 50 μM) analyzed by CFSE dilution (mean ± SEM, n = 11 donors for no stimulation, stimulation with IL-12 / IL-18, and IL-12 / IL-18+BIP 10 μM, n = 8 donors for stimulation with IL-12 / IL-18+BIP 1 μM, and n = 3 donors for stimulation with IL-12 / IL-18+BIP 50 μM). (I) GMFI (mean ± SEM, n = 5 donors) of CD69 expression in unstimulated (unstimulated) NV NK cells and CE NK cells or NV NK cells and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP 100 μM.(J) Upper panel: Heatmap of relative expression levels of mRNA encoding the PPP gene (GO:0006098) in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (n = 5 donors). Lower panel: Percentage of proliferated cells analyzed by CFSE dilution in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 6AN 50 μM (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear regression analysis (A, B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 2B-C]Figure 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation. (A) Top panel: Representative histograms of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Bottom panel: MFI (mean ± SEM, n = 8 donors) of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (B) Top panel: Representative histograms of CD71 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Lower panel: GMFI and percentage of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 14 donors). (C) Left panel: Representative Western blot of total CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Right panel: Total CD71 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 13 donors). (D) Left panel: GMFI of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6). Right panel: Percentage of CD71+ NK cells in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (E) Top panel: Representative histogram of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 10 donors). (F) Upper panel: Schematic of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle panel: Representative histogram of CFSE dilution in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Percentage of proliferation of unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells analyzed by CFSE dilution (mean ± SEM, n = 13 donors). (G) Heatmap of relative expression levels of mRNA encoding cell cycle genes (GO:0006098) in unstimulated (unstimulated) or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (H) Percentage of proliferation of unstimulated (no stimulation) NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18+BIP (1 μM, 10 μM, and 50 μM) analyzed by CFSE dilution (mean ± SEM, n = 11 donors for no stimulation, stimulation with IL-12 / IL-18, and IL-12 / IL-18+BIP 10 μM, n = 8 donors for stimulation with IL-12 / IL-18+BIP 1 μM, and n = 3 donors for stimulation with IL-12 / IL-18+BIP 50 μM). (I) GMFI (mean ± SEM, n = 5 donors) of CD69 expression in unstimulated (unstimulated) NV NK cells and CE NK cells or NV NK cells and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP 100 μM.(J) Upper panel: Heatmap of relative expression levels of mRNA encoding the PPP gene (GO:0006098) in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (n = 5 donors). Lower panel: Percentage of proliferated cells analyzed by CFSE dilution in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 6AN 50 μM (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear regression analysis (A, B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 2D-E]Figure 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation. (A) Top panel: Representative histograms of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Bottom panel: MFI (mean ± SEM, n = 8 donors) of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (B) Top panel: Representative histograms of CD71 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Lower panel: GMFI and percentage of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 14 donors). (C) Left panel: Representative Western blot of total CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Right panel: Total CD71 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 13 donors). (D) Left panel: GMFI of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6). Right panel: Percentage of CD71+ NK cells in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (E) Top panel: Representative histogram of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 10 donors). (F) Upper panel: Schematic of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle panel: Representative histogram of CFSE dilution in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Percentage of proliferation of unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells analyzed by CFSE dilution (mean ± SEM, n = 13 donors). (G) Heatmap of relative expression levels of mRNA encoding cell cycle genes (GO:0006098) in unstimulated (unstimulated) or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (H) Percentage of proliferation of unstimulated (no stimulation) NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18+BIP (1 μM, 10 μM, and 50 μM) analyzed by CFSE dilution (mean ± SEM, n = 11 donors for no stimulation, stimulation with IL-12 / IL-18, and IL-12 / IL-18+BIP 10 μM, n = 8 donors for stimulation with IL-12 / IL-18+BIP 1 μM, and n = 3 donors for stimulation with IL-12 / IL-18+BIP 50 μM). (I) GMFI (mean ± SEM, n = 5 donors) of CD69 expression in unstimulated (unstimulated) NV NK cells and CE NK cells or NV NK cells and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP 100 μM.(J) Upper panel: Heatmap of relative expression levels of mRNA encoding the PPP gene (GO:0006098) in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (n = 5 donors). Lower panel: Percentage of proliferated cells analyzed by CFSE dilution in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 6AN 50 μM (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear regression analysis (A, B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 2F]Figure 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation. (A) Top panel: Representative histograms of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Bottom panel: MFI (mean ± SEM, n = 8 donors) of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (B) Top panel: Representative histograms of CD71 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Lower panel: GMFI and percentage of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 14 donors). (C) Left panel: Representative Western blot of total CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Right panel: Total CD71 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 13 donors). (D) Left panel: GMFI of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6). Right panel: Percentage of CD71+ NK cells in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (E) Top panel: Representative histogram of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 10 donors). (F) Upper panel: Schematic of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle panel: Representative histogram of CFSE dilution in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Percentage of proliferation of unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells analyzed by CFSE dilution (mean ± SEM, n = 13 donors). (G) Heatmap of relative expression levels of mRNA encoding cell cycle genes (GO:0006098) in unstimulated (unstimulated) or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (H) Percentage of proliferation of unstimulated (no stimulation) NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18+BIP (1 μM, 10 μM, and 50 μM) analyzed by CFSE dilution (mean ± SEM, n = 11 donors for no stimulation, stimulation with IL-12 / IL-18, and IL-12 / IL-18+BIP 10 μM, n = 8 donors for stimulation with IL-12 / IL-18+BIP 1 μM, and n = 3 donors for stimulation with IL-12 / IL-18+BIP 50 μM). (I) GMFI (mean ± SEM, n = 5 donors) of CD69 expression in unstimulated (unstimulated) NV NK cells and CE NK cells or NV NK cells and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP 100 μM.(J) Upper panel: Heatmap of relative expression levels of mRNA encoding the PPP gene (GO:0006098) in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (n = 5 donors). Lower panel: Percentage of proliferated cells analyzed by CFSE dilution in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 6AN 50 μM (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear regression analysis (A, B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 2G-H]Figure 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation. (A) Top panel: Representative histograms of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Bottom panel: MFI (mean ± SEM, n = 8 donors) of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (B) Top panel: Representative histograms of CD71 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Lower panel: GMFI and percentage of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 14 donors). (C) Left panel: Representative Western blot of total CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Right panel: Total CD71 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 13 donors). (D) Left panel: GMFI of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6). Right panel: Percentage of CD71+ NK cells in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (E) Top panel: Representative histogram of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 10 donors). (F) Upper panel: Schematic of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle panel: Representative histogram of CFSE dilution in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Percentage of proliferation of unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells analyzed by CFSE dilution (mean ± SEM, n = 13 donors). (G) Heatmap of relative expression levels of mRNA encoding cell cycle genes (GO:0006098) in unstimulated (unstimulated) or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (H) Percentage of proliferation of unstimulated (no stimulation) NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18+BIP (1 μM, 10 μM, and 50 μM) analyzed by CFSE dilution (mean ± SEM, n = 11 donors for no stimulation, stimulation with IL-12 / IL-18, and IL-12 / IL-18+BIP 10 μM, n = 8 donors for stimulation with IL-12 / IL-18+BIP 1 μM, and n = 3 donors for stimulation with IL-12 / IL-18+BIP 50 μM). (I) GMFI (mean ± SEM, n = 5 donors) of CD69 expression in unstimulated (unstimulated) NV NK cells and CE NK cells or NV NK cells and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP 100 μM.(J) Upper panel: Heatmap of relative expression levels of mRNA encoding the PPP gene (GO:0006098) in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (n = 5 donors). Lower panel: Percentage of proliferated cells analyzed by CFSE dilution in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 6AN 50 μM (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear regression analysis (A, B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 2I-J]Figure 2: Activated CE NK cells are characterized by high levels of cell surface CD71 and rapid cell proliferation. (A) Top panel: Representative histograms of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Bottom panel: MFI (mean ± SEM, n = 8 donors) of CD98 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. (B) Top panel: Representative histograms of CD71 expression on unstimulated (unstimulated) NV NK cells and CE NK cells or on IL-12 / IL-18-stimulated NV NK cells and CE NK cells. Lower panel: GMFI and percentage of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 14 donors). (C) Left panel: Representative Western blot of total CD71 expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Right panel: Total CD71 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 13 donors). (D) Left panel: GMFI of CD71 expression in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6). Right panel: Percentage of CD71+ NK cells in unstimulated (unstimulated) NV and CE NK cells or K562-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (E) Top panel: Representative histogram of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells.Lower panel: GMFI of Tf-488 uptake in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 10 donors). (F) Upper panel: Schematic of the experiment used to analyze CFSE dilution in NV and CE NK cells. Middle panel: Representative histogram of CFSE dilution in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Percentage of proliferation of unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells analyzed by CFSE dilution (mean ± SEM, n = 13 donors). (G) Heatmap of relative expression levels of mRNA encoding cell cycle genes (GO:0006098) in unstimulated (unstimulated) or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (H) Percentage of proliferation of unstimulated (no stimulation) NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18+BIP (1 μM, 10 μM, and 50 μM) analyzed by CFSE dilution (mean ± SEM, n = 11 donors for no stimulation, stimulation with IL-12 / IL-18, and IL-12 / IL-18+BIP 10 μM, n = 8 donors for stimulation with IL-12 / IL-18+BIP 1 μM, and n = 3 donors for stimulation with IL-12 / IL-18+BIP 50 μM). (I) GMFI (mean ± SEM, n = 5 donors) of CD69 expression in unstimulated (unstimulated) NV NK cells and CE NK cells or NV NK cells and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + BIP 100 μM.(J) Upper panel: Heatmap of relative expression levels of mRNA encoding the PPP gene (GO:0006098) in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (n = 5 donors). Lower panel: Percentage of proliferated cells analyzed by CFSE dilution in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 6AN 50 μM (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (F, H, I, J) or linear regression analysis (A, B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

[0279] [Figure 3A]Figure 3: CD71-mediated iron uptake and dietary iron availability affect NK cell function. (A) Top panel: Schematic of the experiment used to analyze CFSE dilution in spleen-derived WT and TfrcY20H / Y20H NK cells. Bottom left panel: Representative histogram of CFSE dilution in spleen-derived WT and TfrcY20H / Y20H NK1.1+ NK cells stimulated with IL-12 / IL-18. Bottom right panel: Percentage of proliferation of spleen-derived WT and TfrcY20H / Y20H NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 5 for WT NK cells, n = 6 for TfrcY20H / Y20H NK cells). (B) Upper panel: Schematic of MCMV infection experiments in mice fed a + / - iron diet for 6 weeks. Lower panel: Serum levels of iron, ferritin, UIBC, TIBC, and hematocrit in mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 8–18 for iron, ferritin, UIBC, and TIBC; n = 3 for hematocrit). (C) Left panel: Percentage of NK1.1+ NK cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right panel: Percentage of CD8+ cells, CD4+ cells, and CD19+ cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (D) Left panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right panel: Percentages of KLRG1+ and CD62L+ NK1.1+ NK cells in the spleens of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (E) Left panel: Viral titers in the liver and spleen of mice infected with WT MCMV and fed a + / - iron diet for 6 weeks at 3 dpi (each dot represents data from cells isolated from one mouse; data are presented as fold-change differences normalized to mice fed a + iron diet; horizontal lines indicate median values; n = 10).Right panel: Viral titers in the liver and spleen of MCMV-infected Δm157 mice fed a + / - iron diet for 6 weeks at 3 dpi (each dot represents data from cells isolated from one mouse; data are presented as the difference in fold change normalized to that of mice fed a + iron diet; horizontal lines indicate the median; n = 9–10). (F) Percentage of IFN-γ+ in NK1.1+ NK cells in the liver and spleen of WT MCMV-infected mice fed a + / - feed for 6 weeks at 1.5 dpi (mean ± SEM, n = 4–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant). [Figure 3B-C]Figure 3: CD71-mediated iron uptake and dietary iron availability affect NK cell function. (A) Top panel: Schematic of the experiment used to analyze CFSE dilution in spleen-derived WT and TfrcY20H / Y20H NK cells. Bottom left panel: Representative histogram of CFSE dilution in spleen-derived WT and TfrcY20H / Y20H NK1.1+ NK cells stimulated with IL-12 / IL-18. Bottom right panel: Percentage of proliferation of spleen-derived WT and TfrcY20H / Y20H NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 5 for WT NK cells, n = 6 for TfrcY20H / Y20H NK cells). (B) Upper panel: Schematic of MCMV infection experiments in mice fed a + / - iron diet for 6 weeks. Lower panel: Serum levels of iron, ferritin, UIBC, TIBC, and hematocrit in mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 8–18 for iron, ferritin, UIBC, and TIBC; n = 3 for hematocrit). (C) Left panel: Percentage of NK1.1+ NK cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right panel: Percentage of CD8+ cells, CD4+ cells, and CD19+ cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (D) Left panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right panel: Percentages of KLRG1+ and CD62L+ NK1.1+ NK cells in the spleens of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (E) Left panel: Viral titers in the liver and spleen of mice infected with WT MCMV and fed a + / - iron diet for 6 weeks at 3 dpi (each dot represents data from cells isolated from one mouse; data are presented as fold-change differences normalized to mice fed a + iron diet; horizontal lines indicate median values; n = 10).Right panel: Viral titers in the liver and spleen of MCMV-infected Δm157 mice fed a + / - iron diet for 6 weeks at 3 dpi (each dot represents data from cells isolated from one mouse; data are presented as the difference in fold change normalized to that of mice fed a + iron diet; horizontal lines indicate the median; n = 9–10). (F) Percentage of IFN-γ+ in NK1.1+ NK cells in the liver and spleen of WT MCMV-infected mice fed a + / - feed for 6 weeks at 1.5 dpi (mean ± SEM, n = 4–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant). [Figure 3D-F]Figure 3: CD71-mediated iron uptake and dietary iron availability affect NK cell function. (A) Top panel: Schematic of the experiment used to analyze CFSE dilution in spleen-derived WT and TfrcY20H / Y20H NK cells. Bottom left panel: Representative histogram of CFSE dilution in spleen-derived WT and TfrcY20H / Y20H NK1.1+ NK cells stimulated with IL-12 / IL-18. Bottom right panel: Percentage of proliferation of spleen-derived WT and TfrcY20H / Y20H NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 5 for WT NK cells, n = 6 for TfrcY20H / Y20H NK cells). (B) Upper panel: Schematic of MCMV infection experiments in mice fed a + / - iron diet for 6 weeks. Lower panel: Serum levels of iron, ferritin, UIBC, TIBC, and hematocrit in mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 8–18 for iron, ferritin, UIBC, and TIBC; n = 3 for hematocrit). (C) Left panel: Percentage of NK1.1+ NK cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right panel: Percentage of CD8+ cells, CD4+ cells, and CD19+ cells in the spleen of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (D) Left panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). Right panel: Percentages of KLRG1+ and CD62L+ NK1.1+ NK cells in the spleens of mice fed a + / - iron diet for 6 weeks (mean ± SEM, n = 5). (E) Left panel: Viral titers in the liver and spleen of mice infected with WT MCMV and fed a + / - iron diet for 6 weeks at 3 dpi (each dot represents data from cells isolated from one mouse; data are presented as fold-change differences normalized to mice fed a + iron diet; horizontal lines indicate median values; n = 10).Right panel: Viral titers in the liver and spleen of MCMV-infected Δm157 mice fed a + / - iron diet for 6 weeks at 3 dpi (each dot represents data from cells isolated from one mouse; data are presented as the difference in fold change normalized to that of mice fed a + iron diet; horizontal lines indicate the median; n = 9–10). (F) Percentage of IFN-γ+ in NK1.1+ NK cells in the liver and spleen of WT MCMV-infected mice fed a + / - feed for 6 weeks at 1.5 dpi (mean ± SEM, n = 4–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant).

[0280] [Figure 4A-B]Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection. (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17–22). (B) Top left panel: Percentage and absolute number of CD8+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Top right panel: Percentage and absolute number of CD4+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9–19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11–22). Lower panel: Percentage and absolute number of CD19+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12–22). (D) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6).(E) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Lower panel: Percentages of KLRG1+, CD62L+, and Ly6C+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). (F) Percentages of Ly49H+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5–6). (G) Upper left panel: Schematic of adoptive transfer experiments into Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells upon MCMV infection. Upper right panel: Representative flow plots gated on adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of a recipient infected with WT MCMV at 7 dpi. Lower panel: Percentages of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of recipients infected with WT MCMV at 7 and 30 dpi. (Each dot represents data from cells isolated from one mouse, and bars indicate ±SEM. Two independent experiments were performed: n = 5 for 7 dpi and n = 3–5 for 30 dpi; n = 4 for 7 dpi and n = 2 for 30 dpi.) (H) Left panel: Schematic of adoptive transfer experiments into Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells.Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood at 6 dpi (each dot represents data from cells isolated from one mouse, and bars indicate ±SEM, n = 3–4). (I) Top left panel: Schematic of adoptive transfer experiment into Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells upon WT MCMV infection. Top right panel: Representative histogram of CFSE dilution in adoptively transferred WT NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of recipients infected with WT MCMV, 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of recipients infected with WT MCMV at 3.5 dpi (mean ± SEM, two independent experiments; n = 5 in the first experiment, n = 4 in the second experiment). (J) Percentage of proliferation of spleen-derived Tfrcfl / fl NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 3–4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 4–6). Bottom panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 3–6).(L) Viral titers in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 dpi (n = 5). (Each dot represents data from cells isolated from one mouse; the horizontal line indicates the median.) (M) Percentage of IFN-γ+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Right panels: Percentage of KLRG1+ among NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. [Figure 4C-D]Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection. (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17–22). (B) Top left panel: Percentage and absolute number of CD8+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Top right panel: Percentage and absolute number of CD4+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9–19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11–22). Lower panel: Percentage and absolute number of CD19+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12–22). (D) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6).(E) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Lower panel: Percentages of KLRG1+, CD62L+, and Ly6C+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). (F) Percentages of Ly49H+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5–6). (G) Upper left panel: Schematic of adoptive transfer experiments into Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells upon MCMV infection. Upper right panel: Representative flow plots gated on adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of a recipient infected with WT MCMV at 7 dpi. Lower panel: Percentages of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of recipients infected with WT MCMV at 7 and 30 dpi. (Each dot represents data from cells isolated from one mouse, and bars indicate ±SEM. Two independent experiments were performed: n = 5 for 7 dpi and n = 3–5 for 30 dpi; n = 4 for 7 dpi and n = 2 for 30 dpi.) (H) Left panel: Schematic of adoptive transfer experiments into Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells.Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood at 6 dpi (each dot represents data from cells isolated from one mouse, and bars indicate ±SEM, n = 3–4). (I) Top left panel: Schematic of adoptive transfer experiment into Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells upon WT MCMV infection. Top right panel: Representative histogram of CFSE dilution in adoptively transferred WT NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of recipients infected with WT MCMV, 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of recipients infected with WT MCMV at 3.5 dpi (mean ± SEM, two independent experiments; n = 5 in the first experiment, n = 4 in the second experiment). (J) Percentage of proliferation of spleen-derived Tfrcfl / fl NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 3–4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 4–6). Bottom panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 3–6).(L) Viral titers in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 dpi (n = 5). (Each dot represents data from cells isolated from one mouse; the horizontal line indicates the median.) (M) Percentage of IFN-γ+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Right panels: Percentage of KLRG1+ among NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. [Figure 4E-F]Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection. (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17–22). (B) Top left panel: Percentage and absolute number of CD8+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Top right panel: Percentage and absolute number of CD4+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9–19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11–22). Lower panel: Percentage and absolute number of CD19+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12–22). (D) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6).(E) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Lower panel: Percentages of KLRG1+, CD62L+, and Ly6C+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). (F) Percentages of Ly49H+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5–6). (G) Upper left panel: Schematic of adoptive transfer experiments into Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells upon MCMV infection. Upper right panel: Representative flow plots gated on adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of a recipient infected with WT MCMV at 7 dpi. Lower panel: Percentages of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of recipients infected with WT MCMV at 7 and 30 dpi. (Each dot represents data from cells isolated from one mouse, and bars indicate ±SEM. Two independent experiments were performed: n = 5 for 7 dpi and n = 3–5 for 30 dpi; n = 4 for 7 dpi and n = 2 for 30 dpi.) (H) Left panel: Schematic of adoptive transfer experiments into Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells.Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood at 6 dpi (each dot represents data from cells isolated from one mouse, and bars indicate ±SEM, n = 3–4). (I) Top left panel: Schematic of adoptive transfer experiment into Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells upon WT MCMV infection. Top right panel: Representative histogram of CFSE dilution in adoptively transferred WT NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of recipients infected with WT MCMV, 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of recipients infected with WT MCMV at 3.5 dpi (mean ± SEM, two independent experiments; n = 5 in the first experiment, n = 4 in the second experiment). (J) Percentage of proliferation of spleen-derived Tfrcfl / fl NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 3–4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 4–6). Bottom panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 3–6).(L) Viral titers in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 dpi (n = 5). (Each dot represents data from cells isolated from one mouse; the horizontal line indicates the median.) (M) Percentage of IFN-γ+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Right panels: Percentage of KLRG1+ among NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. [Figure 4G-H]Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection. (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17–22). (B) Top left panel: Percentage and absolute number of CD8+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Top right panel: Percentage and absolute number of CD4+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9–19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11–22). Lower panel: Percentage and absolute number of CD19+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12–22). (D) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6).(E) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Lower panel: Percentages of KLRG1+, CD62L+, and Ly6C+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). (F) Percentages of Ly49H+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5–6). (G) Upper left panel: Schematic of adoptive transfer experiments into Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells upon MCMV infection. Upper right panel: Representative flow plots gated on adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of a recipient infected with WT MCMV at 7 dpi. Lower panel: Percentages of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of recipients infected with WT MCMV at 7 and 30 dpi. (Each dot represents data from cells isolated from one mouse, and bars indicate ±SEM. Two independent experiments were performed: n = 5 for 7 dpi and n = 3–5 for 30 dpi; n = 4 for 7 dpi and n = 2 for 30 dpi.) (H) Left panel: Schematic of adoptive transfer experiments into Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells.Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood at 6 dpi (each dot represents data from cells isolated from one mouse, and bars indicate ±SEM, n = 3–4). (I) Top left panel: Schematic of adoptive transfer experiment into Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells upon WT MCMV infection. Top right panel: Representative histogram of CFSE dilution in adoptively transferred WT NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of recipients infected with WT MCMV, 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of recipients infected with WT MCMV at 3.5 dpi (mean ± SEM, two independent experiments; n = 5 in the first experiment, n = 4 in the second experiment). (J) Percentage of proliferation of spleen-derived Tfrcfl / fl NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 3–4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 4–6). Bottom panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 3–6).(L) Viral titers in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 dpi (n = 5). (Each dot represents data from cells isolated from one mouse; the horizontal line indicates the median.) (M) Percentage of IFN-γ+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Right panels: Percentage of KLRG1+ among NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. [Figure 4I-J]Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection. (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17–22). (B) Top left panel: Percentage and absolute number of CD8+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Top right panel: Percentage and absolute number of CD4+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9–19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11–22). Lower panel: Percentage and absolute number of CD19+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12–22). (D) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6).(E) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Lower panel: Percentages of KLRG1+, CD62L+, and Ly6C+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). (F) Percentages of Ly49H+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5–6). (G) Upper left panel: Schematic of adoptive transfer experiments into Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells upon MCMV infection. Upper right panel: Representative flow plots gated on adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of a recipient infected with WT MCMV at 7 dpi. Lower panel: Percentages of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of recipients infected with WT MCMV at 7 and 30 dpi. (Each dot represents data from cells isolated from one mouse, and bars indicate ±SEM. Two independent experiments were performed: n = 5 for 7 dpi and n = 3–5 for 30 dpi; n = 4 for 7 dpi and n = 2 for 30 dpi.) (H) Left panel: Schematic of adoptive transfer experiments into Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells.Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood at 6 dpi (each dot represents data from cells isolated from one mouse, and bars indicate ±SEM, n = 3–4). (I) Top left panel: Schematic of adoptive transfer experiment into Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells upon WT MCMV infection. Top right panel: Representative histogram of CFSE dilution in adoptively transferred WT NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of recipients infected with WT MCMV, 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of recipients infected with WT MCMV at 3.5 dpi (mean ± SEM, two independent experiments; n = 5 in the first experiment, n = 4 in the second experiment). (J) Percentage of proliferation of spleen-derived Tfrcfl / fl NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 3–4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 4–6). Bottom panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 3–6).(L) Viral titers in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 dpi (n = 5). (Each dot represents data from cells isolated from one mouse; the horizontal line indicates the median.) (M) Percentage of IFN-γ+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Right panels: Percentage of KLRG1+ among NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. [Figure 4K]Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection. (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17–22). (B) Top left panel: Percentage and absolute number of CD8+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Top right panel: Percentage and absolute number of CD4+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9–19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11–22). Lower panel: Percentage and absolute number of CD19+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12–22). (D) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6).(E) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Lower panel: Percentages of KLRG1+, CD62L+, and Ly6C+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). (F) Percentages of Ly49H+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5–6). (G) Upper left panel: Schematic of adoptive transfer experiments into Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells upon MCMV infection. Upper right panel: Representative flow plots gated on adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of a recipient infected with WT MCMV at 7 dpi. Lower panel: Percentages of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of recipients infected with WT MCMV at 7 and 30 dpi. (Each dot represents data from cells isolated from one mouse, and bars indicate ±SEM. Two independent experiments were performed: n = 5 for 7 dpi and n = 3–5 for 30 dpi; n = 4 for 7 dpi and n = 2 for 30 dpi.) (H) Left panel: Schematic of adoptive transfer experiments into Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells.Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood at 6 dpi (each dot represents data from cells isolated from one mouse, and bars indicate ±SEM, n = 3–4). (I) Top left panel: Schematic of adoptive transfer experiment into Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells upon WT MCMV infection. Top right panel: Representative histogram of CFSE dilution in adoptively transferred WT NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of recipients infected with WT MCMV, 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of recipients infected with WT MCMV at 3.5 dpi (mean ± SEM, two independent experiments; n = 5 in the first experiment, n = 4 in the second experiment). (J) Percentage of proliferation of spleen-derived Tfrcfl / fl NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 3–4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 4–6). Bottom panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 3–6).(L) Viral titers in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 dpi (n = 5). (Each dot represents data from cells isolated from one mouse; the horizontal line indicates the median.) (M) Percentage of IFN-γ+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Right panels: Percentage of KLRG1+ among NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant. [Figure 4L-N]Figure 4: CD71 supports NK cell proliferation and optimal effector function during viral infection. (A) Left panel: Percentage and absolute number of NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Right panel: Percentage and absolute number of NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 17–22). (B) Top left panel: Percentage and absolute number of CD8+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Top right panel: Percentage and absolute number of CD4+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 4). Lower panel: Percentage and absolute number of CD19+ cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). (C) Upper left panel: Percentage and absolute number of CD8+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 9–19). Upper right panel: Percentage and absolute number of CD4+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 11–22). Lower panel: Percentage and absolute number of CD19+ cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 12–22). (D) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 10). Lower panel: Percentages of CD62L+ and Ly6C+ NK1.1+ NK cells in the livers of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 6).(E) Upper panel: Percentages of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). Lower panel: Percentages of KLRG1+, CD62L+, and Ly6C+ NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5). (F) Percentages of Ly49H+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice (mean ± SEM, n = 5–6). (G) Upper left panel: Schematic of adoptive transfer experiments into Klra8- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells upon MCMV infection. Upper right panel: Representative flow plots gated on adoptively transferred CD45.1+ and CD45.2+ (Ly49H+NK1.1+) NK cells in the liver of a recipient infected with WT MCMV at 7 dpi. Lower panel: Percentages of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood of recipients infected with WT MCMV at 7 and 30 dpi. (Each dot represents data from cells isolated from one mouse, and bars indicate ±SEM. Two independent experiments were performed: n = 5 for 7 dpi and n = 3–5 for 30 dpi; n = 4 for 7 dpi and n = 2 for 30 dpi.) (H) Left panel: Schematic of adoptive transfer experiments into Rag2- / -IL2rg- / - recipients to track the expansion of WT and Tfrcfl / fl NK cells.Right panel: Percentage of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver, spleen, lung, and blood at 6 dpi (each dot represents data from cells isolated from one mouse, and bars indicate ±SEM, n = 3–4). (I) Top left panel: Schematic of adoptive transfer experiment into Klra8- / - recipients to analyze CFSE dilution in WT and Tfrcfl / fl NK cells upon WT MCMV infection. Top right panel: Representative histogram of CFSE dilution in adoptively transferred WT NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells in the liver of recipients infected with WT MCMV, 3.5 dpi. Lower panel: CFSE GMFI of adoptively transferred WT (Ly49H+NK1.1+CD45.1+) NK cells and Tfrcfl / flNcr1Cre (Ly49H+NK1.1+CD45.2+) NK cells in the liver and spleen of recipients infected with WT MCMV at 3.5 dpi (mean ± SEM, two independent experiments; n = 5 in the first experiment, n = 4 in the second experiment). (J) Percentage of proliferation of spleen-derived Tfrcfl / fl NK1.1+ NK cells and Tfrcfl / flNcr1Cre NK1.1+ NK cells stimulated with IL-15 LD or IL-12 / IL-18, analyzed by CFSE dilution (mean ± SEM, n = 3–4). (K) Upper panel: Schematic diagram of MCMV infection experiments in Tfrcfl / fl and Tfrcfl / flNcr1cre mice. Middle panel: Percentage and absolute number of NK1.1+ NK cells in the liver of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 4–6). Bottom panel: Percentage and absolute number of NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 and 5.5 dpi (mean ± SEM, n = 3–6).(L) Viral titers in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 3.5 dpi (n = 5). (Each dot represents data from cells isolated from one mouse; the horizontal line indicates the median.) (M) Percentage of IFN-γ+ NK1.1+ NK cells in the liver and spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 1.5 dpi (mean ± SEM, n = 4). (N) Left panel: Percentage of CD27+CD11b-, CD27+CD11b+, and CD27-CD11b+ NK1.1+ NK cells in the spleen of Tfrcfl / fl and Tfrcfl / flNcr1cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Right panels: Percentage of KLRG1+ among NK1.1+ NK cells in the spleens of Tfrcfl / fl and Tfrcfl / flNcr1Cre mice infected with WT MCMV at 5.5 dpi (mean ± SEM, n = 3–5). Statistical significance was assessed by unpaired, two-tailed Student's t-test (A, B, C, D, E, F, I, J, K, L, M, N). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; ns, not significant.

[0281] [Figure 5A-B]Figure 5: Glycolysis is required for the induction of CD71 in activated NK cells. (A) Top panel: Representative Western blots of total CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + ActD (1 μM and 10 μM), and IL-12 / IL-18 + CHX (10 μg / ml and 100 μg / ml). Bottom left panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + ActD 10 μM (mean ± SEM, n = 3 donors). Lower right panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + CHX 100 μg / ml (mean ± SEM, n = 2 donors). (B) TFRC mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (C) Upper left panel: GMFI (mean ± SEM, n = 6 donors) of CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG. Upper right panel: Representative Western blot of total CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG.Lower panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells and NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2DG (mean ± SEM, n = 5 donors). (D) GMFI of CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18 under 10 mM glucose and 2 mM glucose (mean ± SEM, n = 5 donors). (E) GMFI of Tf-488 uptake in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2DG (mean ± SEM, n = 5 donors). (F) Upper panel: Representative Western blot of total c-Myc expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Total c-Myc expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, B, C, D, E, F) or linear regression analysis (B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 5C-D]Figure 5: Glycolysis is required for the induction of CD71 in activated NK cells. (A) Top panel: Representative Western blots of total CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + ActD (1 μM and 10 μM), and IL-12 / IL-18 + CHX (10 μg / ml and 100 μg / ml). Bottom left panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + ActD 10 μM (mean ± SEM, n = 3 donors). Lower right panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + CHX 100 μg / ml (mean ± SEM, n = 2 donors). (B) TFRC mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (C) Upper left panel: GMFI (mean ± SEM, n = 6 donors) of CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG. Upper right panel: Representative Western blot of total CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG.Lower panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells and NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2DG (mean ± SEM, n = 5 donors). (D) GMFI of CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18 under 10 mM glucose and 2 mM glucose (mean ± SEM, n = 5 donors). (E) GMFI of Tf-488 uptake in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2DG (mean ± SEM, n = 5 donors). (F) Upper panel: Representative Western blot of total c-Myc expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Total c-Myc expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, B, C, D, E, F) or linear regression analysis (B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant. [Figure 5E-F]Figure 5: Glycolysis is required for the induction of CD71 in activated NK cells. (A) Top panel: Representative Western blots of total CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + ActD (1 μM and 10 μM), and IL-12 / IL-18 + CHX (10 μg / ml and 100 μg / ml). Bottom left panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + ActD 10 μM (mean ± SEM, n = 3 donors). Lower right panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + CHX 100 μg / ml (mean ± SEM, n = 2 donors). (B) TFRC mRNA expression in unstimulated NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG. Transcription levels relative to 18S mRNA levels were determined and normalized to unstimulated NV NK cells (mean ± SEM, n = 6 donors). (C) Upper left panel: GMFI (mean ± SEM, n = 6 donors) of CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG. Upper right panel: Representative Western blot of total CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18, IL-12 / IL-18 + 2-DG.Lower panel: Total CD71 expression normalized to actin in unstimulated NV and CE NK cells and NV and CE NK cells stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2DG (mean ± SEM, n = 5 donors). (D) GMFI of CD71 expression in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18 under 10 mM glucose and 2 mM glucose (mean ± SEM, n = 5 donors). (E) GMFI of Tf-488 uptake in unstimulated NV and CE NK cells or stimulated with IL-12 / IL-18 or IL-12 / IL-18 + 2DG (mean ± SEM, n = 5 donors). (F) Upper panel: Representative Western blot of total c-Myc expression in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Total c-Myc expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, B, C, D, E, F) or linear regression analysis (B, C, E). *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

[0282] [Figure 6A]Figure 6: Cytokine pre-stimulation induces the IRP / IRE regulatory system. (A) Top panel: Transcriptome data for ACO1 and IREB2 mRNA expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). Middle panel: Representative Western blots for total IRP1 and IRP2 expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Bottom panel: Total IRP1 and IRP2 expression normalized to actin in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 7 donors for IRP1, n = 6 donors for IRP2). (B) Heatmap of relative expression levels of mRNAs encoding genes with IREs in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (C) Upper left panel: EIF4E mRNA expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors) from transcriptome data. Upper right panel: Representative Western blot of total eIF4E expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Total eIF4E expression normalized to actin in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (D) Upper panel: Representative histograms of HPG incorporation in unstimulated (unstimulated) or IL-12 / IL-18 stimulated NV and CE NK cells.Lower panel: GMFI of HPG incorporation in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 5 donors). (E) Left panel: Representative Western blot of total ferritin heavy chain 1 expression in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18. Right panel: Total ferritin heavy chain 1 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 4 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, C, E) or linear regression analysis (D). *p < 0.05, **p < 0.01; ns, not significant. [Figure 6B-C]Figure 6: Cytokine pre-stimulation induces the IRP / IRE regulatory system. (A) Top panel: Transcriptome data for ACO1 and IREB2 mRNA expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). Middle panel: Representative Western blots for total IRP1 and IRP2 expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Bottom panel: Total IRP1 and IRP2 expression normalized to actin in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 7 donors for IRP1, n = 6 donors for IRP2). (B) Heatmap of relative expression levels of mRNAs encoding genes with IREs in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (C) Upper left panel: EIF4E mRNA expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors) from transcriptome data. Upper right panel: Representative Western blot of total eIF4E expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Total eIF4E expression normalized to actin in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (D) Upper panel: Representative histograms of HPG incorporation in unstimulated (unstimulated) or IL-12 / IL-18 stimulated NV and CE NK cells.Lower panel: GMFI of HPG incorporation in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 5 donors). (E) Left panel: Representative Western blot of total ferritin heavy chain 1 expression in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18. Right panel: Total ferritin heavy chain 1 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 4 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, C, E) or linear regression analysis (D). *p < 0.05, **p < 0.01; ns, not significant. [Figure 6D-E]Figure 6: Cytokine pre-stimulation induces the IRP / IRE regulatory system. (A) Top panel: Transcriptome data for ACO1 and IREB2 mRNA expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). Middle panel: Representative Western blots for total IRP1 and IRP2 expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Bottom panel: Total IRP1 and IRP2 expression normalized to actin in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 7 donors for IRP1, n = 6 donors for IRP2). (B) Heatmap of relative expression levels of mRNAs encoding genes with IREs in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors). (C) Upper left panel: EIF4E mRNA expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (n = 5 donors) from transcriptome data. Upper right panel: Representative Western blot of total eIF4E expression in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells. Lower panel: Total eIF4E expression normalized to actin in unstimulated NV and CE NK cells or IL-12 / IL-18-stimulated NV and CE NK cells (mean ± SEM, n = 6 donors). (D) Upper panel: Representative histograms of HPG incorporation in unstimulated (unstimulated) or IL-12 / IL-18 stimulated NV and CE NK cells.Lower panel: GMFI of HPG incorporation in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 5 donors). (E) Left panel: Representative Western blot of total ferritin heavy chain 1 expression in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18. Right panel: Total ferritin heavy chain 1 expression normalized to actin in unstimulated (unstimulated) NV and CE NK cells or NV and CE NK cells stimulated with IL-12 / IL-18 (mean ± SEM, n = 4 donors). Statistical significance was assessed by paired two-tailed Student's t-test (A, C, E) or linear regression analysis (D). *p < 0.05, **p < 0.01; ns, not significant.

[0283] [Figure 7A-C]Figure 7: The IRP / IRE regulatory system regulates CD71 expression in NK cells. (A) TFRC mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, data from transcriptome data (n=5). (B) FTH1 mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, transcriptome data (n=5). (C) Representative Western blot of total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA. Total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA (n=7). (D) Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA. Total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA (n=6). (E) Left panel: Representative histogram of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA (n=4-5). (F) Left panel: Representative Western blot of total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA. Right panel: Total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA (n=5). (G) Left panel: Representative Western blot of total IRP1 expression in NKL cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in NKL cells transfected with control or Aco1 siRNA (n=6). (H) Left panel: Representative Western blot of total IRP2 expression in NKL cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in NKL cells transfected with control or IREB2 siRNA (n=7). (I) Left panel: Representative histogram of CD71 expression in NKL cells transfected with control, ACO1, or IREB2 siRNA.Right panel: GMFI of CD71 expression in NKL cells transfected with control, Aco1, or IREB2 siRNA (n=5). (J) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and Aco1 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and Aco1 siRNA (n=6). (K) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and IREB2 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and IREB2 siRNA (n=5). All averaged data are presented as mean ± sem and were analyzed using an unpaired, two-tailed Student's t-test (a,b,d,e) or ANOWA (c). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. (L) Left panel: Representative Western blot of total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells. Right panel: Total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells (n=3). (M) Left panel: GMFI of CD71 expression in control or IREB2 sgRNA-transfected NK92 cells (n=5). Right panel: Number of control or IREB2 sgRNA-transfected NK92 cells (n=4). All averaged data are presented as mean ± sem and were analyzed using a two-tailed Student's t-test (A-B), an unpaired two-tailed Student's t-test (C, D, G, H, J, K, L, M), or ANOWA (E, F, I). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ***p<0.001, ns, not significant. [Figure 7D-F]Figure 7: The IRP / IRE regulatory system regulates CD71 expression in NK cells. (A) TFRC mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, data from transcriptome data (n=5). (B) FTH1 mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, transcriptome data (n=5). (C) Representative Western blot of total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA. Total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA (n=7). (D) Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA. Total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA (n=6). (E) Left panel: Representative histogram of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA (n=4-5). (F) Left panel: Representative Western blot of total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA. Right panel: Total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA (n=5). (G) Left panel: Representative Western blot of total IRP1 expression in NKL cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in NKL cells transfected with control or Aco1 siRNA (n=6). (H) Left panel: Representative Western blot of total IRP2 expression in NKL cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in NKL cells transfected with control or IREB2 siRNA (n=7). (I) Left panel: Representative histogram of CD71 expression in NKL cells transfected with control, ACO1, or IREB2 siRNA.Right panel: GMFI of CD71 expression in NKL cells transfected with control, Aco1, or IREB2 siRNA (n=5). (J) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and Aco1 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and Aco1 siRNA (n=6). (K) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and IREB2 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and IREB2 siRNA (n=5). All averaged data are presented as mean ± sem and were analyzed using an unpaired, two-tailed Student's t-test (a,b,d,e) or ANOWA (c). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. (L) Left panel: Representative Western blot of total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells. Right panel: Total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells (n=3). (M) Left panel: GMFI of CD71 expression in control or IREB2 sgRNA-transfected NK92 cells (n=5). Right panel: Number of control or IREB2 sgRNA-transfected NK92 cells (n=4). All averaged data are presented as mean ± sem and were analyzed using a two-tailed Student's t-test (A-B), an unpaired two-tailed Student's t-test (C, D, G, H, J, K, L, M), or ANOWA (E, F, I). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ***p<0.001, ns, not significant. [Figure 7G-I]Figure 7: The IRP / IRE regulatory system regulates CD71 expression in NK cells. (A) TFRC mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, data from transcriptome data (n=5). (B) FTH1 mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, transcriptome data (n=5). (C) Representative Western blot of total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA. Total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA (n=7). (D) Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA. Total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA (n=6). (E) Left panel: Representative histogram of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA (n=4-5). (F) Left panel: Representative Western blot of total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA. Right panel: Total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA (n=5). (G) Left panel: Representative Western blot of total IRP1 expression in NKL cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in NKL cells transfected with control or Aco1 siRNA (n=6). (H) Left panel: Representative Western blot of total IRP2 expression in NKL cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in NKL cells transfected with control or IREB2 siRNA (n=7). (I) Left panel: Representative histogram of CD71 expression in NKL cells transfected with control, ACO1, or IREB2 siRNA.Right panel: GMFI of CD71 expression in NKL cells transfected with control, Aco1, or IREB2 siRNA (n=5). (J) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and Aco1 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and Aco1 siRNA (n=6). (K) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and IREB2 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and IREB2 siRNA (n=5). All averaged data are presented as mean ± sem and were analyzed using an unpaired, two-tailed Student's t-test (a,b,d,e) or ANOWA (c). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. (L) Left panel: Representative Western blot of total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells. Right panel: Total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells (n=3). (M) Left panel: GMFI of CD71 expression in control or IREB2 sgRNA-transfected NK92 cells (n=5). Right panel: Number of control or IREB2 sgRNA-transfected NK92 cells (n=4). All averaged data are presented as mean ± sem and were analyzed using a two-tailed Student's t-test (A-B), an unpaired two-tailed Student's t-test (C, D, G, H, J, K, L, M), or ANOWA (E, F, I). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ***p<0.001, ns, not significant. [Figure 7J-L]Figure 7: The IRP / IRE regulatory system regulates CD71 expression in NK cells. (A) TFRC mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, data from transcriptome data (n=5). (B) FTH1 mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, transcriptome data (n=5). (C) Representative Western blot of total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA. Total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA (n=7). (D) Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA. Total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA (n=6). (E) Left panel: Representative histogram of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA (n=4-5). (F) Left panel: Representative Western blot of total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA. Right panel: Total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA (n=5). (G) Left panel: Representative Western blot of total IRP1 expression in NKL cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in NKL cells transfected with control or Aco1 siRNA (n=6). (H) Left panel: Representative Western blot of total IRP2 expression in NKL cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in NKL cells transfected with control or IREB2 siRNA (n=7). (I) Left panel: Representative histogram of CD71 expression in NKL cells transfected with control, ACO1, or IREB2 siRNA.Right panel: GMFI of CD71 expression in NKL cells transfected with control, Aco1, or IREB2 siRNA (n=5). (J) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and Aco1 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and Aco1 siRNA (n=6). (K) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and IREB2 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and IREB2 siRNA (n=5). All averaged data are presented as mean ± sem and were analyzed using an unpaired, two-tailed Student's t-test (a,b,d,e) or ANOWA (c). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. (L) Left panel: Representative Western blot of total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells. Right panel: Total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells (n=3). (M) Left panel: GMFI of CD71 expression in control or IREB2 sgRNA-transfected NK92 cells (n=5). Right panel: Number of control or IREB2 sgRNA-transfected NK92 cells (n=4). All averaged data are presented as mean ± sem and were analyzed using a two-tailed Student's t-test (A-B), an unpaired two-tailed Student's t-test (C, D, G, H, J, K, L, M), or ANOWA (E, F, I). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ***p<0.001, ns, not significant. [Figure 7M]Figure 7: The IRP / IRE regulatory system regulates CD71 expression in NK cells. (A) TFRC mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, data from transcriptome data (n=5). (B) FTH1 mRNA expression in NV and CE NK cells without stimulation versus IL-12 / IL-18, transcriptome data (n=5). (C) Representative Western blot of total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA. Total IRP1 expression in NK92 cells transfected with control or Aco1 siRNA (n=7). (D) Representative Western blot of total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA. Total IRP2 expression in NK92 cells transfected with control or IREB2 siRNA (n=6). (E) Left panel: Representative histogram of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in NK92 cells transfected with control, Aco1, or IREB2 siRNA (n=4-5). (F) Left panel: Representative Western blot of total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA. Right panel: Total FTH1 expression in NK92 cells transfected with control, Aco1, and IREB2 siRNA (n=5). (G) Left panel: Representative Western blot of total IRP1 expression in NKL cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in NKL cells transfected with control or Aco1 siRNA (n=6). (H) Left panel: Representative Western blot of total IRP2 expression in NKL cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in NKL cells transfected with control or IREB2 siRNA (n=7). (I) Left panel: Representative histogram of CD71 expression in NKL cells transfected with control, ACO1, or IREB2 siRNA.Right panel: GMFI of CD71 expression in NKL cells transfected with control, Aco1, or IREB2 siRNA (n=5). (J) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and Aco1 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and Aco1 siRNA (n=6). (K) Left panel: Representative Western blot of total FTH1 expression in NKL cells transfected with control and IREB2 siRNA. Right panel: Total FTH1 expression in NKL cells transfected with control and IREB2 siRNA (n=5). All averaged data are presented as mean ± sem and were analyzed using an unpaired, two-tailed Student's t-test (a,b,d,e) or ANOWA (c). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. (L) Left panel: Representative Western blot of total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells. Right panel: Total IRP2 expression in control or IREB2 sgRNA-transfected NK92 cells (n=3). (M) Left panel: GMFI of CD71 expression in control or IREB2 sgRNA-transfected NK92 cells (n=5). Right panel: Number of control or IREB2 sgRNA-transfected NK92 cells (n=4). All averaged data are presented as mean ± sem and were analyzed using a two-tailed Student's t-test (A-B), an unpaired two-tailed Student's t-test (C, D, G, H, J, K, L, M), or ANOWA (E, F, I). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ***p<0.001, ns, not significant.

[0284] [Figure 8A-B]Figure 8: Enforced IRP expression is a molecular module that similarly supports T cell proliferation. (A) Left panel: Representative Western blot of total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA (n=5). (B) Left panel: Representative Western blot of total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA (n=4). (C) Left panel: Representative histogram of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA (n=7). (D) Left panel: Representative Western blot of total FTH1 expression in Jurkat cells transfected with control, Aco1, or IREB2 siRNA. Right panel: Total FTH1 expression in Jurkat cells transfected with control, Aco1, and IREB2 siRNA (n=5). (E) Representative Western blot of total IRP2 expression in IRP2 knockout (ko) Jurkat cells transfected with a control vector encoding mCherry (LV-mCherry) or IREB2 (LV-IREB2). (F) Top panel: Representative histogram of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry or LV-IREB2. Bottom left panel: GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry versus GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-IREB2 (n=4). Bottom right panel: Number of IRP2 ko Jurkat cells transduced with LV-mCherry versus number of IRP2 ko Jurkat cells transduced with LV-IREB2 (n=3).(G) Left panel: Representative histogram of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: GMFI of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2 (n=2). (H) Left panel: Representative histogram of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: MFI of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus IREB2 (LV-IREB2) (n=2). (I) Representative Western blot of total IRP2 expression in untransduced CD4+ T cells (UTD), PSMA-specific CAR CD4+ T cells (CAR), and PSMA-specific CAR CD4+ T cells co-expressing IRP2 (CAR-IREB2). (J) Top panel: Representative histogram of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). Bottom panel: Representative histogram of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). (K) Upper panel: Percentage of unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3). Lower panel: Percentage of Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3).All averaged data are presented as mean ± sem and were analyzed using an unpaired two-tailed Student's t-test (a, b, f) or ANOWA (c, d). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. [Figure 8C-D]Figure 8: Enforced IRP expression is a molecular module that similarly supports T cell proliferation. (A) Left panel: Representative Western blot of total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA (n=5). (B) Left panel: Representative Western blot of total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA (n=4). (C) Left panel: Representative histogram of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA (n=7). (D) Left panel: Representative Western blot of total FTH1 expression in Jurkat cells transfected with control, Aco1, or IREB2 siRNA. Right panel: Total FTH1 expression in Jurkat cells transfected with control, Aco1, and IREB2 siRNA (n=5). (E) Representative Western blot of total IRP2 expression in IRP2 knockout (ko) Jurkat cells transfected with a control vector encoding mCherry (LV-mCherry) or IREB2 (LV-IREB2). (F) Top panel: Representative histogram of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry or LV-IREB2. Bottom left panel: GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry versus GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-IREB2 (n=4). Bottom right panel: Number of IRP2 ko Jurkat cells transduced with LV-mCherry versus number of IRP2 ko Jurkat cells transduced with LV-IREB2 (n=3).(G) Left panel: Representative histogram of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: GMFI of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2 (n=2). (H) Left panel: Representative histogram of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: MFI of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus IREB2 (LV-IREB2) (n=2). (I) Representative Western blot of total IRP2 expression in untransduced CD4+ T cells (UTD), PSMA-specific CAR CD4+ T cells (CAR), and PSMA-specific CAR CD4+ T cells co-expressing IRP2 (CAR-IREB2). (J) Top panel: Representative histogram of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). Bottom panel: Representative histogram of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). (K) Upper panel: Percentage of unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3). Lower panel: Percentage of Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3).All averaged data are presented as mean ± sem and were analyzed using an unpaired two-tailed Student's t-test (a, b, f) or ANOWA (c, d). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. [Figure 8E-F]Figure 8: Enforced IRP expression is a molecular module that similarly supports T cell proliferation. (A) Left panel: Representative Western blot of total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA (n=5). (B) Left panel: Representative Western blot of total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA (n=4). (C) Left panel: Representative histogram of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA (n=7). (D) Left panel: Representative Western blot of total FTH1 expression in Jurkat cells transfected with control, Aco1, or IREB2 siRNA. Right panel: Total FTH1 expression in Jurkat cells transfected with control, Aco1, and IREB2 siRNA (n=5). (E) Representative Western blot of total IRP2 expression in IRP2 knockout (ko) Jurkat cells transfected with a control vector encoding mCherry (LV-mCherry) or IREB2 (LV-IREB2). (F) Top panel: Representative histogram of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry or LV-IREB2. Bottom left panel: GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry versus GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-IREB2 (n=4). Bottom right panel: Number of IRP2 ko Jurkat cells transduced with LV-mCherry versus number of IRP2 ko Jurkat cells transduced with LV-IREB2 (n=3).(G) Left panel: Representative histogram of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: GMFI of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2 (n=2). (H) Left panel: Representative histogram of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: MFI of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus IREB2 (LV-IREB2) (n=2). (I) Representative Western blot of total IRP2 expression in untransduced CD4+ T cells (UTD), PSMA-specific CAR CD4+ T cells (CAR), and PSMA-specific CAR CD4+ T cells co-expressing IRP2 (CAR-IREB2). (J) Top panel: Representative histogram of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). Bottom panel: Representative histogram of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). (K) Upper panel: Percentage of unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3). Lower panel: Percentage of Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3).All averaged data are presented as mean ± sem and were analyzed using an unpaired two-tailed Student's t-test (a, b, f) or ANOWA (c, d). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. [Figure 8G-I]Figure 8: Enforced IRP expression is a molecular module that similarly supports T cell proliferation. (A) Left panel: Representative Western blot of total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA (n=5). (B) Left panel: Representative Western blot of total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA. Right panel: Total IRP2 expression in Jurkat cells transfected with control or IREB2 siRNA (n=4). (C) Left panel: Representative histogram of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA. Right panel: GMFI of CD71 expression in Jurkat cells transfected with control, ACO1, or IREB2 siRNA (n=7). (D) Left panel: Representative Western blot of total FTH1 expression in Jurkat cells transfected with control, Aco1, or IREB2 siRNA. Right panel: Total FTH1 expression in Jurkat cells transfected with control, Aco1, and IREB2 siRNA (n=5). (E) Representative Western blot of total IRP2 expression in IRP2 knockout (ko) Jurkat cells transfected with a control vector encoding mCherry (LV-mCherry) or IREB2 (LV-IREB2). (F) Top panel: Representative histogram of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry or LV-IREB2. Bottom left panel: GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-mCherry versus GMFI of CD71 expression in IRP2 ko Jurkat cells transduced with LV-IREB2 (n=4). Bottom right panel: Number of IRP2 ko Jurkat cells transduced with LV-mCherry versus number of IRP2 ko Jurkat cells transduced with LV-IREB2 (n=3).(G) Left panel: Representative histogram of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: GMFI of CD71 expression in primary CD4+ T cells transduced with LV-mCherry versus LV-IREB2 (n=2). (H) Left panel: Representative histogram of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus LV-IREB2. Right panel: MFI of CD71 expression in primary CD8+ T cells transduced with LV-mCherry versus IREB2 (LV-IREB2) (n=2). (I) Representative Western blot of total IRP2 expression in untransduced CD4+ T cells (UTD), PSMA-specific CAR CD4+ T cells (CAR), and PSMA-specific CAR CD4+ T cells co-expressing IRP2 (CAR-IREB2). (J) Top panel: Representative histogram of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). Bottom panel: Representative histogram of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells. MFI of CD71 expression in Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells (n=3). (K) Upper panel: Percentage of unstimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3). Lower panel: Percentage of Fab-stimulated UTD-, CAR-, and CAR-IREB2-transduced CD4+ T cells that entered 0, 1, and 2 cycles of cell expansion (n=3).All averaged data are presented as mean ± sem and were analyzed using an unpaired two-tailed Student's t-test (a, b, f) or ANOWA (c, d). Asterisks indicate significance between groups. *p<0.05, **p<0.01, ns, not significant. [Figure 8J]Figure 8: Enforced IRP expression is a molecular module that similarly supports T cell proliferation. (A) Left panel: Representative Western blot of total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA. Right panel: Total IRP1 expression in Jurkat cells transfected with control or Aco1 siRNA (n=5). (B) Left panel: Re...

Claims

1. A lymphocyte comprising a synthetic polynucleotide encoding at least one iron regulatory protein (IRP), wherein said at least one iron regulatory protein is IRP2 (SEQ ID NO: 2), and wherein said at least one iron regulatory protein is constitutively expressed.

2. The lymphocyte of claim 1 , which is a T cell or a natural killer (NK) cell.

3. The lymphocyte of claim 2, which is a tumor-infiltrating lymphocyte, a modified T cell, or a virus-specific T cell.

4. 4. The lymphocyte of claim 1, wherein the synthetic polynucleotide encoding the at least one iron-regulatory protein is under the control of a constitutive promoter.

5. The lymphocyte of claim 4, wherein the constitutive promoter is an EF-1α promoter.

6. The lymphocyte of claim 1 , further comprising a chimeric antigen receptor (CAR).

7. The lymphocyte of claim 6, wherein the CAR comprises an antigen-binding domain, a transmembrane domain, a costimulatory signaling region, and a signaling domain.

8. The lymphocyte of claim 7 , wherein the antigen-binding domain is an antibody or an antigen-binding fragment thereof.

9. The lymphocyte of claim 8, wherein the antigen-binding fragment is a Fab or scFv.

10. The lymphocyte of claim 7 , wherein the antigen-binding domain specifically binds to a tumor antigen or a viral antigen.

11. The lymphocyte of claim 10, wherein the tumor antigen is present on the surface of a cell of a target cell population or tissue.

12. 12. The lymphocyte of any one of claims 6 to 11, wherein the CAR is encoded by a polynucleotide, and the polynucleotide encoding the CAR is transcriptionally linked to the synthetic polynucleotide encoding IRP2.

13. 13. The lymphocyte of claim 12, wherein the polynucleotide encoding the CAR and the synthetic polynucleotide encoding IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

14. The lymphocyte of claim 13, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

15. The lymphocyte of claim 13 or 14, wherein the self-cleaving peptide is T2A.

16. 16. A pharmaceutical composition comprising lymphocytes according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

17. A composition comprising lymphocytes according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 for use in therapy.

18. 17. A composition comprising lymphocytes according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 for use in the treatment of cancer.

19. 19. The composition or pharmaceutical composition for use according to claim 18, wherein the cancer is a blood cancer or a solid tumor.

20. 20. The composition or pharmaceutical composition for use according to claim 19, wherein the hematological cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia or multiple myeloma, and the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

21. A composition comprising a lymphocyte according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 for use in the prevention and / or treatment of a viral infection.

22. 22. The composition or pharmaceutical composition for use according to claim 21, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpesvirus, in particular, the human herpesvirus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpesvirus 8 (HHV8).

23. 17. A composition comprising lymphocytes according to any one of claims 1 to 15 or a pharmaceutical composition according to claim 16 for treating a subject with cancer or for preventing and / or treating a viral infection in a subject, characterized in that a therapeutically effective amount of the lymphocytes or the pharmaceutical composition is administered to the subject.

24. 24. The composition or pharmaceutical composition of claim 23, wherein the cancer is a blood cancer or a solid tumor, in particular, the blood cancer is acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Hodgkin's lymphoma, acute myeloid leukemia or multiple myeloma, and the solid tumor is colon cancer, breast cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, lung cancer, neuroblastoma, glioblastoma or sarcoma.

25. 24. The composition or pharmaceutical composition of claim 23, wherein the viral infection is caused by human immunodeficiency virus (HIV), adenovirus, polyomavirus, influenza virus or human herpesvirus, in particular, the human herpesvirus is cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), varicella-zoster virus (VZV) or human herpesvirus 8 (HHV8).

26. 16. An in vitro or ex vivo method for producing lymphocytes according to any one of claims 1 to 15, comprising: a) providing lymphocytes obtained from a subject; b) introducing into the lymphocytes of step (a) a synthetic polynucleotide encoding at least one iron-regulatory protein, wherein the iron-regulatory protein is IRP2 (SEQ ID NO: 2); c) expressing the at least one iron-regulatory protein encoded by the synthetic polynucleotide introduced into the lymphocytes in step (b); A method comprising:

27. 27. The in vitro or ex vivo method of claim 26, wherein a second synthetic polynucleotide encoding a chimeric antigen receptor (CAR) is introduced into the lymphocytes in step (b).

28. 28. The in vitro or ex vivo method of claim 27, wherein the synthetic polynucleotide encoding the CAR is combined with the synthetic polynucleotide encoding IRP2.

29. 29. The in vitro or ex vivo method of claim 27 or 28, wherein the synthetic polynucleotide encoding the CAR is transcriptionally linked to the synthetic polynucleotide encoding IRP2.

30. 30. The in vitro or ex vivo method of any one of claims 27 to 29, wherein the synthetic polynucleotide encoding the CAR and the polynucleotide encoding IRP2 are linked by a polynucleotide encoding a self-cleaving peptide.

31. 31. The in vitro or ex vivo method of claim 30, wherein the self-cleaving peptide is a 2A self-cleaving peptide.

32. 32. The in vitro or ex vivo method of claim 30 or 31, wherein the self-cleaving peptide is T2A.

33. 33. An in vitro or ex vivo method according to any one of claims 26 to 32, wherein the one or more synthetic polynucleotides are introduced into the lymphocytes by viral transduction.

34. 34. The in vitro or ex vivo method of any one of claims 26 to 33, wherein the lymphocytes are activated before or after introducing the one or more synthetic polynucleotides into the lymphocytes.

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