Method of enhancing immune response activity of cell, cell having enhanced immune response activity, method for treating tumor, and cell composition for cell therapy

Abstract

A method of enhancing immune response activity of a cell is provided. The method includes modification of a plasma membrane damage repair mechanism of the cell.

Description

METHOD OF ENHANCING IMMUNE RESPONSE ACTIVITY OF CELL, CELL HAVING ENHANCED IMMUNE RESPONSE ACTIVITY, METHOD FOR TREATING TUMOR, AND CELL COMPOSITION FOR CELL THERAPY

[0001] The present disclosure relates to a method of enhancing immune response activity of a cell, a cell having enhanced immune response activity, a method for treating tumor in a subject, and a cell composition for cell therapy including the cell.Background

[0002] Recently, immunotherapy utilizing the immune response of a cell has been largely studied. Among cells, immune cells have been thoroughly examined and studied for their immune functions. During the course of the immune response, immune cells are regularly exposed to various substances that can compromise the integrity of their plasma membrane, such as bacterial pore-forming toxins, reactive oxygen species, and perforin. Substantial damage to the plasma membrane has the potential to lead to cell death. Thus, the ability of immune cells to endure these membrane assaults may be important for mounting an effective immune response. However, the role and mechanisms of immune cell adaptation to plasma membrane damage remain largely unknown.

[0003] Among the immune cells, T cells have been thoroughly studied for their unique features. For example, the CAR (Chimeric Antigen Receptor)-T cell is attracting attention and has been studied recently. PTL 1, for example, suggests a method for treating mantle cell lymphoma (MCL) or B cell ALL in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a T cell product comprising autologous T cells expressing an anti-CD 19 chimeric antigen receptor.

[0004] PTL 1: WO2021092290A1Summary

[0005] The conventional approach for immunotherapy is open for innovation. We have studied the role and mechanisms of immune cell (e.g., T cell) adaptation to plasma membrane damage and found that modification of plasma membrane damage repair mechanism provides a new aspect for immunotherapy.

[0006] The present disclosure relates to the following.

[0007] [1] A method of enhancing immune response activity of a cell comprising: modifying a plasma membrane damage repair mechanism of the cell.

[0008] [2] The method according to [1], wherein the cell is an immune cell.

[0009] [3] The method according to [2], wherein the immune cell is derived from a stem cell.

[0010] [4] The method according to [3], wherein the stem cell is an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, a lymphoid stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0011] [5] The method according to any one of [1] to [4], wherein the cell is selected from a group including CD8 T cells, CD4 T cells, and NKT (natural killer T) cells.

[0012] [6] The method according to any one of [1] to [5], wherein modifying the plasma membrane damage repair mechanism of the cell includes controlling membrane trafficking.

[0013] [7] The method according to any one of [1] to [6], wherein modifying the plasma membrane damage repair mechanism of the cell includes controlling an expression of one or more genes related to the plasma membrane damage repair mechanism of the cell and / or inhibiting a function of one or more proteins encoded by the one or more genes related to the plasma membrane damage repair mechanism of the cell.

[0014] [8] The method according to [7], wherein the one or more genes related to the plasma membrane damage repair mechanism of the cell includes at least one gene selected from a group including genes encoding components of ESCRT (Endosomal Sorting Complex Required for Transport) complex, genes encoding proteins involved in membrane traffic, and genes encoding extracellular ATP receptors.

[0015] [9] The method according to [7] or [8], wherein the one or more genes related to the plasma membrane damage repair mechanism of the cell includes at least one gene selected from a group including genes encoding components of ESCRT-0 including HRS, STAM1, and STAM2; genes encoding components of ESCRT-1 including VPS23, VPS28, VPS37A, VPS37B, VPS37C, and VPS37D; genes encoding components of ATPase complex including VPS4A, VPS4B, and LIP5; genes encoding components of ESCRT-II including VPS22, VPS25, and VPS36; genes encoding components of ESCRT-III including CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHMP1A, CHMP1B, CHMP5, and CHMP7; genes encoding regulators of membrane trafficking including Rab GTPases, GEF, GAP, GDI, and RAMP3; and genes encoding extracellular ATP receptors including P2X1,P2X2,P2X3,P2X4,P2X5,P2X6,P2X7,P2Y1,P2Y2,P2Y3,P2Y4,P2Y5,P2Y6,P2Y7,P2Y8,P2Y9,P2Y10,P2Y11,P2Y12,P2Y13,and P2Y14; and a gene encoding acid sphingomyelinase (ASM).

[0016]

[0010] The method according to any one of [7] to [9], wherein controlling an expression of one or more genes related to the plasma membrane damage repair mechanism of the cell includes suppressing the one or more genes related to the plasma membrane damage repair mechanism of the cell.

[0017]

[0011] The method according to

[0010] , wherein suppressing is performed by a technique selected from a group consisting of RNA interference (RNAi), short interfering RNS (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) and DNA-directed RNA interference (ddRNAi).

[0018]

[0012] The method according to

[0010] or

[0011] , wherein suppressing is performed by modifying a genome of the cell using CRISPR, ZFN, TALEN, retroviral transduction or lentiviral transduction.

[0019]

[0013] The method according to any one of [1] to [9], wherein modifying a plasma membrane damage repair mechanism of the cell includes overexpressing genes related to the plasma membrane damage repair mechanism of the cell.

[0020]

[0014] The method according [1] to [9], wherein modifying a plasma membrane damage repair mechanism of the cell includes inhibiting one or more genes or one or more proteins related to the plasma membrane damage repair mechanism of the cell.

[0021]

[0015] The method according to any one of [1] to

[0014] , wherein the immune response activity is at least one of anti-tumor activity, anti-inflammatory activity, anti-bacterial activity, or anti-viral activity.

[0022]

[0016] A method for treating tumor in a subject in need thereof, comprising: producing a cell having enhanced immune response activity according to the method of any one of [1] to

[0015] ; and administering an effective amount of the cell into the subject.

[0023]

[0017] A cell having enhanced immune response activity and comprising a modified plasma membrane damage repair mechanism.

[0024]

[0018] The cell according to

[0017] , wherein the cell is an immune cell.

[0025]

[0019] The cell according to

[0017] , wherein the immune cell is derived from a stem cell.

[0026]

[0020] The cell according to

[0019] , wherein the stem cell is an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, a lymphoid stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.

[0027]

[0021] The cell according to any one of

[0017] to

[0020] , wherein the cell is a selected from a group including CD8 T cells, CD4 T cells, and NKT (natural killer T) cells.

[0028]

[0022] The cell according to any one of

[0017] to

[0021] , wherein the cell has controlled expression of one or more genes related to the plasma membrane damage repair mechanism.

[0029]

[0023] The cell according to

[0022] , wherein the one or more genes related to the plasma membrane damage repair mechanism includes at least one gene selected from a group including genes encoding components of ESCRT (Endosomal Sorting Complex Required for Transport) complex, genes encoding proteins involved in membrane traffic, and genes encoding extracellular ATP receptors.

[0030]

[0024] The cell according to

[0022] or

[0023] , wherein the one or more genes related to the plasma membrane damage repair mechanism includes at least one of genes encoding components of ESCRT-0 including HRS, STAM1, and STAM2; genes encoding components of ESCRT-1 including VPS23, VPS28, VPS37A, VPS37B, VPS37C, and VPS37D; genes encoding components of ATPase complex including VPS4A, VPS4B, and LIP5; genes encoding components of ESCRT-II including VPS22, VPS25, and VPS36; genes encoding components of ESCRT-III including CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHMP1A, CHMP1B, CHMP5, and CHMP7; genes encoding regulators of membrane trafficking including Rab GTPases, GEF, GAP, GDI, and RAMP3; and genes encoding extracellular ATP receptors including P2X1,P2X2,P2X3,P2X4,P2X5,P2X6,P2X7,P2Y1,P2Y2,P2Y3,P2Y4,P2Y5,P2Y6,P2Y7,P2Y8,P2Y9,P2Y10,P2Y11,P2Y12,P2Y13,and P2Y14; and a gene encoding acid sphingomyelinase (ASM).

[0031]

[0025] The cell according to any one of

[0022] to

[0024] , wherein the cell has suppressed expression of one or more genes related to the plasma membrane damage repair mechanism.

[0032]

[0026] The cell according to any one of

[0022] to

[0024] , wherein the cell has overexpression of one or more genes related to the plasma membrane damage repair mechanism.

[0033]

[0027] The cell according to any one of

[0022] to

[0024] , wherein the cell has inhibition of one or more genes or one or more proteins related to the plasma membrane damage repair mechanism.

[0034]

[0028] The cell according to any one of

[0017] to

[0027] , wherein the immune response activity is at least one of anti-tumor activity, anti-inflammatory activity, anti-bacterial activity, or anti-viral activity.

[0035]

[0029] The cell according to any one of

[0017] to

[0028] , wherein the cell expresses CAR (Chimeric Antigen Receptor) or TCR (T Cell Receptor).

[0036]

[0030] A cell composition for cell therapy including the cell according to any one of

[0017] to

[0029] .

[0037]

[0031] A pharmaceutical composition comprising the cell composition according to

[0030] and pharmaceutically acceptable excipients.

[0038] In the accompanying drawings: FIG. 1 is a schematic illustration of an exemplary plasma membrane repair mechanism of a CD8+T cell. FIG. 2 is a diagram representing an experimental result showing that plasma membrane damage facilitates adaptation of CD8+T cells to subsequent similar damage. FIG. 3A is a Venn diagram showing commonly upregulated genes in each condition. FIG. 3B is a diagram representing ranking of differentially expressed genes based on log2 fold change. FIG. 4A is a diagram representing results obtained by nucleofection with CRISPR RNPs for observing effects of CRISPR knock out of Vps37b or Ramp3. FIG. 4B is a diagram representing results obtained by overexpression of Vps37b and / or Ramp3 by using retroviral transduction. FIG. 5A is a diagram representing the relative intensity of FM1-43 signals in control cells, VPS37B knocked out cells and RAMP3 knocked out cells. FIG. 5B is a diagram representing the time for FM1-43 signals to reach maximum intensity in control cells, VPS37B knocked out cells and RAMP3 knocked out cells. FIG. 5C is a diagram showing that MCF-7 cells were transduced with a vector carrying human VPS37B or RAMP3 fused with mCherry fluorescent protein and irradiated with laser. FIG. 6A is a diagram representing experimental results suggesting that VPS37B is involved in plasma membrane repair by promoting the localization of other ESCRT proteins. FIG. 6B is a diagram representing experimental results showing that RAMP 3 is associated with various intracellular vesicles, including endosomes and lysosomes. FIG. 7A is a diagram showing that a plasma membrane damage adaptation process induces upregulation of Vps37b and Ramp3. FIG. 7B is a diagram showing that upregulation of Vps37b and Ramp3 is induced in not only damaged cells but also neighboring cells. FIG. 7C is a diagram showing that the expression of Vps37b and Ramp3 increased in cells cultured in the supernatant from electroporated cells. FIGs 8A-D are diagrams showing experimental results that support the idea that an extracellular ATP increases the expression of Vps37b and Ramp3 through cAMP signaling. FIGs 9A-B are diagrams showing the analysis result of RNA-seq data obtained in Experiment 2. FIG. 9C is a diagram that suggests that induction of Vps37b and Ramp3 was partially inhibited by the deletion of P2ry10 (also known as P2y10) FIGs 10A-B are diagrams that show the results indicating that LM infection increases plasma membrane damage in CD8+T cells. FIGs 11A-C are diagrams that show results indicating that loss of Vps37b or Ramp3 in CD8+T cells impairs their clonal expansion. FIGs 12A-B are diagrams that show results indicating that Vps37b or Ramp3 deficiency enhances plasma membrane damage in CD8+T cells during anti-tumor responses. FIGs 13A-E are diagrams that show results indicating that plasma membrane damage before TCR-stimulation upregulates expression of perforin and IFN-g, in T cells after activation, which might be due to enhanced multiple signaling pathways, including the UPR pathway. FIGs 14A-D are diagrams showing results indicating that loss of Vps37b or Ramp3 enhances anti-tumor responses of CD8+T cells.DETAILED DESCRIPTION

[0039] The following provides a detailed description of embodiments of the present disclosure.

[0040] In the present disclosure, the term “immune response activity” as used herein encompasses any known activity enhanced because of immune response. The activity includes, for example, anti-tumor activity, anti-inflammatory activity, anti-bacterial activity, and anti-viral activity. The term “anti-tumor activity” means a reduction in the rate of proliferation, viability, or metastatic activity of tumor cells. For example, anti-tumor activity can be shown by a decline in growth rate of tumor cells or tumor size stability or reduction, or longer survival due to therapy as compared to control without therapy. Such activity can be assessed using in vitro or in vivo tumor models, including but not limited to xenograft models, allograft models, and other known models known in the art to investigate anti-tumor activity. The term “enhancing immune response activity of a cell” as used herein means that the immune response by the cells is enhanced as compared to control cell (e.g., a untreated cell, a cell before treatment, or cell received mock treatment). The term “plasma membrane damage repair mechanism” encompasses adaptation process of a cell against plasma membrane damage. The term “modifying plasma membrane damage repair mechanism” means inducing at least one irregular reaction in a plasma membrane damage repair mechanism. In one example, the modification includes controlling membrane trafficking. In other example, the modification includes controlling an expression of one or more genes related to the plasma membrane damage repair mechanism of the cell. In another example, the modification includes inhibiting one or more genes or one or more proteins related to the plasma membrane damage repair mechanism of the cell. As use herein, "inhibiting one or more genes related to the plasma membrane damage repair mechanism of the cell" includes inhibition of the expression of the genes, inhibition of the translation of the genes, degradation of mRNA of the genes, alternative splicing of the genes, or a combination thereof as compared to control cell (e.g., an untreated cell, a cell before treatment, or cell received mock treatment). As use herein, "inhibiting one or more proteins related to the plasma membrane damage repair mechanism of the cell" includes inhibition of the function of the proteins, inhibition of the folding of the proteins, degradation of the proteins, inhibition of production of the proteins or a combination thereof as compared to control cell (e.g., an untreated cell, a cell before treatment, or cell received mock treatment). In another example, the modification includes inhibiting signaling mediated by extracellular ATP. Examples that can be used in such an inhibition may be a compound (e.g., synthesized or native), a protein, an antibody, a peptide, a small molecule, a nucleic acid, or an aptamer. The term “membrane trafficking” as used herein encompasses variety of processes that go into the movement of cargo, including, for example, proteins and other macromolecules, using membrane bound transport vesicles. The membrane trafficking may occur in a process of membrane damage repair mechanism. The term “immune cell” as used herein encompasses any known cells related to immune system. In one example, an immune cell is part of the immune system and helps the body fight infections and other diseases. The term “T cell” as used herein encompasses any known T cell. The term T cell encompasses, for example, CD8 T cell, CD4 T cell, and NKT cell. The term “ESCRT” is an abbreviation of Endosomal Sorting Complex Required for Transport. Such ESCRT complex plays a pivotal role in extraction of minor injuries on a cell surface (Reference 1). Vps37b, as explained below, is a component, i.e., subunit, of ESCRT. The phrase “suppressing the one or more genes related to the plasma membrane damage repair mechanism” as used herein encompasses reducing the one or more genes by any means as compared to control cell (e.g., a untreated cell, a cell before treatment, or cell received mock treatment). The reducing can be any level. In fact, 100% reduction to 1% reduction can be covered by the term “suppressing.” In one example the reduction level of the one or more genes can be 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, and 20% or less, compared to the normal level of gene expression. The reduction level of the one or more genes can be evaluated through reverse transcription quantitative PCR (RT-qPCR), immunoblot, or flow cytometry analyses. Among these RT-qPCR is optimal in the light of feasibility. Suppressing the one or more genes related to the plasma membrane damage repair mechanism may be introduced into CAR-T cells, TCR-T cells or other cells used in cellular therapies. CAR-T cells have genetic modification to target tumor tissue, killing tumor cells that carry cell surface markers targeted by the expressed chimeric antigen receptors (CAR). TCR-T cells are cells that are genetically engineered to express the T cell receptor (TCR) specific to the target antigen. The term “overexpression” of gene(s) as used herein encompasses excessive expression of the gene(s) as compared to control cell (e.g., a untreated cell, a cell before treatment, or cell received mock treatment). In one example, the level of gene over expression can be 101% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, and 170% or more, compared to the normal level of gene expression. The level of gene overexpression can be evaluated through reverse transcription quantitative PCR (RT-qPCR), immunoblot, or flow cytometry analyses. Among these RT-qPCR is optimal in the light of feasibility. The term “proteins related to the plasma membrane damage repair mechanism of the cell” as used herein encompasses any proteins involved in the plasma membrane damage repair mechanism of the cell. Examples for such proteins are, EEA1, RAB4A, RAB5A, RAB7, RAB8, RAB11, LAMP1, components of ESCRT-0 (e.g. HRS (also known as HGS), STAM1, and STAM2), ESCRT-1 (e.g. VPS23 (also known as TSG101), VPS28, VPS37A, VPS37B, VPS37C, and VPS37D), ATPase complex (e.g. VPS4A, VPS4B, and LIP5 (also known as VTA1)), ESCRT-II (VPS22 (also known as EAP30), VPS25 (also known as EAP20), and VPS36 (also known as EAP45)), ESCRT-III (e.g. CHMP2A (also known as VPS2A), CHMP2B (also known as VPS2B), CHMP3, CHMP4A (also known as SNF7F), CHMP4B (also known as SNF7B), CHMP4C (also known as SNF7C), CHMP6 (also known as VPS20), CHMP1A (also known as DID2A), CHMP1B (also known as DID2B), CHMP5 (also known as VPS60), and CHMP7), , regulators of membrane trafficking (e.g. Rab GTPases, GEF, GAP, GDI, and RAMP3), and extracellular ATP receptors (P2X1,P2X2,P2X3,P2X4,P2X5,P2X6,P2X7,P2Y1,P2Y2,P2Y3,P2Y4,P2Y5,P2Y6,P2Y7,P2Y8,P2Y9,P2Y10,P2Y11,P2Y12,P2Y13,and P2Y14), and acid sphingomyelinase (ASM). Although these proteins may be referred to by aliases, proteins referred to by aliases are also included in proteins related to the plasma membrane damage repair mechanism of the cell herein. Furthermore, although these proteins are examples of human proteins, homolog of non-human animals are also included in proteins related to the plasma membrane damage repair mechanism of the cell herein. Similarly, although genes may be referred to by aliases, genes referred to by aliases are also included in genes related to the plasma membrane damage repair mechanism of the cell herein. Furthermore, although genes are examples of human genes, homolog of non-human animals are also included in genes related to the plasma membrane damage repair mechanism of the cell herein. As will be understood by a skilled person, “proteins related to the plasma membrane damage repair mechanism of the cell” are encoded by “genes related to the plasma membrane damage repair mechanism of the cell”. The reference to a gene can be made by reference to the protein encoded by the gene. The reference to a protein can be made by reference to the gene encoding the protein. The term “treatment (treating)” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent or the presently disclosed cells to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. The term “subject” may encompass any human or nonhuman animal. The term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms “cancer”, “cancerous”, “cell proliferative disorder”, “proliferative disorder”, and “tumor” are not mutually exclusive as referred to herein.

[0041] In the specification, the technical features described in different embodiments can be combined with each other unless otherwise stated.

[0042] (Method of enhancing immune response activity of a cell) The presently disclosed method of enhancing immune response activity of a cell includes a step of modifying a plasma membrane damage repair mechanism of the cell. The plasma membrane damage can be induced by membrane damaging substances. Examples of membrane damaging substances include pore-forming toxins (PFTs), reactive oxygen species, deoxycholic acid, and perforin. In one example, the cell can be selected from immune cells. In more detail, the cell can be selected from a group including, CD4 and CD8 T cells and NKT cells. Once these cells are exposed to any of the membrane damaging substances, plasma membrane of each cell may be damaged.

[0043] The conventional studies revealed several kinds of plasma membrane repair mechanisms. For example, in vitro studies reveal that nanometer-scale damage in the plasma membrane is spontaneously resealed (References 2, 3), whereas the repair of more extensive damage is hindered by membrane tension, generated by the combination of intracellular cytoskeltal tension and osmotic pressure (Reference 4). To facilitate the closure of these larger pores, cells employ active repair mechanisms. Damage to the plasma membrane triggers a rapid influx of Ca2+, resulting in localized and temporary increases in cytosolic Ca2+levels. This Ca2+influx activates the plasma membrane repair mechanisms (Reference 5)

[0044] ESCRT mediated plasma membrane repair The ESCRT complex plays a pivotal role in the extrusion of minor injuries on the cell surface (Reference 6). The mechanism by which ESCRT facilitates membrane repair involves ESCRT-III (Reference 7), mainly composed of the charged MVB protein (CHMP) family (Reference 8). ESCRT-III components form spiral structures on the membrane that induce membrane scission in cooperation with the vacuolar protein sorting associated protein 4 (VPS4) (References 9, 10). The recruitment of ESCRT-III to the membrane wound site can be induced through various pathways, including direct engagement with the ESCRT I subunit TSG101 and the ESCRT-I-associated protein ALIX (also known as AIP1) (Reference 10).

[0045] Endocytosis mediated plasma membrane repair While the ESCRT pathway promotes the detachment of damaged membranes outward from the cell. The process of inward endocytosis represents another mechanism by which mammalian cells can excise minor damaged membrane sections or transmembrane openings induced by PFTs from the plasma membrane. This removal involves the initiation of clathrin and caveolin-mediated endocytosis, leading to endosome formation, subsequently directed towards degradation (References 11, 12). Furthermore, the endosome assembly can be induced via the lysosomal acid sphingomyelinase (ASM) axis. Plasma membrane damage causes a Ca2+ influx at the damage site, promoting the exocytosis of secretory lysosomes containing ASM (Reference 13). Following its release, ASM catalyzes the hydrolysis of sphingomyelin present on the plasma membrane’s outer layer, yielding ceramide (Reference 13). Owing to its high affinity for cholesterol and various lipids, ceramide facilitates the assembly of lipid rafts proximal to the damage locus (Reference 14). These rafts then promote localized membrane bending, enhancing endocytosis around the damaged areas to excise stable transmembrane pores (Reference 14). The internalized segments of the damaged membrane are then processed through the endo-lysosomal system for degradation within multivesicular bodies (Reference 14).

[0046] Exocytosis mediated plasma membrane repair Two models have been proposed for how exocytosis is involved in plasma membrane repair. The first model, known as the patch model, suggests the creation of an extensive, uniform membrane under the injury site through the homotypic fusion of internal compartments, which subsequently integrates with the plasma membrane to mend the impaired area (References 15, 16). The alternative model proposed that the decrease in plasma membrane tension, due to several exocytic events near a wound, promotes the natural resealing of the plasma membrane (Reference 17).

[0047] In the process of seeking the presently disclosed method, the following plasma membrane repair mechanism workable in a CD8+T cell has been studied. FIG. 1 represents a schematic illustration of an exemplary plasma membrane repair mechanism of a CD8+T cell.

[0048] The experiments that support this finding will be explained in the EXAMPLES section of the present specification. When a plasma membrane of a CD8+T cell is damaged, release of adenosine triphosphate (ATP) is induced. This extracellular ATP binds to the P2ry10 receptor existing in the plasma membrane in a paracrine or autocrine manner. The engagement of ATP with the P2ry10 receptor leads to the synthesis of cyclic adenosine monophosphate (cAMP), which subsequently induces the expression of at least one gene related to the plasma membrane damage repair mechanism. Such gene is selected from the group including genes encoding the protein Vps37b (Gene ID: 79720) and Ramp3 (Gene ID: 10268). The NCBI (National Center for Biotechnology Information) Gene ID of the one encoding the protein Vps37b is 79720 and the Gene ID of the one encoding the protein Ramp3 is 10268. Of course, candidates for the genes to be manipulated are not limited to these two. In the specification, the Gene IDs are shown as examples only, the same proteins may be referred to by other Gene IDs.

[0049] Such candidates can be selected from a group of genes or molecules related to Endosomal Sorting Complex Required for Transport (ESCRT) molecule, a group of genes encoding protein involved in membrane traffic, and the like, as mentioned above.

[0050] The upregulation of genes related to the plasma membrane damage, for example genes encoding components of ESCRT-0 (e.g. HRS (also known as HGS), STAM1, and STAM2), genes encoding components of ESCRT-1 (e.g. VPS23 (also known as TSG101), VPS28, VPS37A, VPS37B, VPS37C, and VPS37D), genes encoding components of ATPase complex (e.g. VPS4A, VPS4B, and LIP5 (also known as VTA1)), genes encoding components of ESCRT-II (VPS22 (also known as EAP30), VPS25 (also known as EAP20), and VPS36 (also known as EAP45)), genes encoding components of ESCRT-III (e.g. CHMP2A (also known as VPS2A), CHMP2B (also known as VPS2B), CHMP3, CHMP4A (also known as SNF7F), CHMP4B (also known as SNF7B), CHMP4C (also known as SNF7C), CHMP6 (also known as VPS20), CHMP1A (also known as DID2A), CHMP1B (also known as DID2B), CHMP5 (also known as VPS60), and CHMP7), genes encoding components of regulators of membrane trafficking (e.g. Rab GTPases, GEF, GAP, GDI, and RAMP3), and genes encoding components of extracellular ATP receptors (P2X1,P2X2,P2X3,P2X4,P2X5,P2X6,P2X7,P2Y1,P2Y2,P2Y3,P2Y4,P2Y5,P2Y6,P2Y7,P2Y8,P2Y9,P2Y10,P2Y11,P2Y12,P2Y13,and P2Y14), gene encoding acid sphingomyelinase (ASM), enhances membrane repair and adaptation to plasma membrane damage.

[0051] Experimental data described in the EXAMPLES section of the present specification suggests that the level of immune response activity can be controlled by modifying a plasma membrane damage repair mechanism of the cell, especially by promoting or suppressing the expression of at least one of these genes. In fact, by suppressing one or more genes related to the plasma membrane damage repair mechanism of the cell, immune response activity is enhanced. Among the immune response activity, as shown in the data described in the EXPMPLES section of the present specification, anti-tumor activity is enhanced by suppressing Vps37b or Ramp3.

[0052] Specifically, experimental data described in the EXAMPLES section of the present specification indicates that enhanced anti-tumor activity acquired by modifying a plasma membrane damage repair mechanism of the cell, especially by the Vps37b or Ramp3 suppression is effective for melanoma treatment. Such result may indicate that the presently disclosed method is also effective for solid cancer treatment.

[0053] Also, the immune response activity is enhanced by modifying a plasma membrane damage repair mechanism of the cell, especially by inhibiting the one or more genes or one or more proteins related to the plasma membrane damage repair mechanism of the cell. Similar to the situation where the expression of the one or more genes is suppressed, the inhibition of the one or more genes or one or the more proteins related to the plasma membrane damage repair mechanism of the cell leads to enhancement of immune response activity.

[0054] Further, the immune response activity is enhanced by modifying a plasma membrane damage repair mechanism of the cell, especially by overexpressing genes related to the plasma membrane damage repair mechanism of the cell. Experimental data described in the EXAMPLES section of the present specification suggests that by inducing overexpression of at least one of the above-mentioned genes, immune response activity can be enhanced. In more detail, by overexpressing one or more genes related to the plasma membrane damage repair mechanism of the cell, anti-bacterial activity is enhanced.

[0055] The gene suppression can be performed by a technique selected from the group consisting of RNA interference (RNAi), short interfering RNS (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) and DNA-directed RNA interference (ddRNAi).

[0056] Alternatively, gene suppression can be performed by modifying a genome of the cell using CRISPR, ZFN, or TALEN.

[0057] Moreover, inhibition of the one or more genes or one or more proteins related to the plasma membrane damage repair mechanism of the cell can be performed by dosing an appropriate inhibitor.

[0058] Further, gene overexpression can be performed by retroviral or lentiviral transduction.

[0059] Experimental data described in the EXAMPLES section of the present specification suggests that the extracellular ATP increases the expression of one or more genes related to the plasma membrane damage repair mechanism of the cell. In fact, the expression levels of Vps37b or Ramp3 are controlled by means of differing dose of ATP and thereby the plasma membrane damage repair mechanism of the cell can be modified. Similarly, the expression levels of Vps37b or Ramp3 may be controlled by suppressing the expression of extracellular ATP receptors and / or inhibiting the function of extracellular ATP receptors.

[0060] (Method for treating tumor in a subject) The presently disclosed method for treating a tumor in a subject includes steps of producing a cell having enhanced immune response activity according to the method as described above and administering effective amount of the cell into the subject. The method may optionally include any additional steps as needed. For example, the method can include lymphocyte depletion therapy, as needed.

[0061] In the step of producing a cell having enhanced immune response activity, cells as a material can be taken out from a subject. The cells, for example, immune cells (e.g., T cells) taken out from the subject are subjected to several manipulations under the above-mentioned method so as to produce cells having enhanced immune response activity.

[0062] The immune cell (e.g., T cell) may be extracted from blood (e.g., peripheral blood mononuclear cells (PBMC)). Alternatively, the immune cell (e.g., T cell) may be derived from a stem cell, e.g. by in vitro differentiation. The stem cell can be an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, a lymphoid stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell. The immune cell (e.g., T cell) may be obtained from the subject, or from any source, such as a donor, a blood bank, or a cell bank (e.g., a stem cell bank).

[0063] The presently disclosed method for treating a tumor in a subject may be used in combination with other methods of treating tumors for example by chemotherapy, irradiation therapy, tumor-targeted therapy, adjuvant therapy, immunotherapy, hormonal therapy, gene therapy or surgery. Tumors for which presently disclosed methods for treating a tumor are useful include all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0064] Once cells having enhanced immune response activity are prepared, the cells are administered to the subject with an effective amount of dose. The effective amount can be determined appropriately based on the result of medical diagnosis.

[0065] Specifically, experimental data described in the EXAMPLES section of the present specification indicates that cells having enhanced anti-tumor activity caused by Vps37b or Ramp3 suppression are effective for melanoma treatment. Hence, the presently method for treating tumor in a subject is also effective for solid cancer treatment.

[0066] (Cell having enhanced immune response activity) The presently disclosed cell having enhanced immune response activity includes modified plasma membrane damage repair mechanism. The preferable attribute and preparation method for the cell is as described above. In one example, the cell can be selected from immune cells. In more detail, the cell can be selected from a group including CD4 and CD8 T cells and NKT cells. As described above, the cell preferably has controlled, or in more detail, suppressed or enhanced expression of one or more genes related to the plasma membrane damage repair mechanism. The candidates for the one or more genes related to the plasma membrane damage repair mechanism are as explained above. In one example, the cell may have inhibition of one or more genes or one or more proteins related to the plasma membrane damage repair mechanism. The presently disclosed enhanced immune response activity may be introduced into CAR-T cells, TCR-T cells, or other cells used in cellular therapies.

[0067] (Cell composition for cell therapy) The presently disclosed cell composition for cell therapy includes the presently disclosed cell. The cell composition may include solvent and additives, such as pH adjuster. The effective number of cells is 103to 1010cells per kg body weight of a subject. The concentration of the cells in the cell composition can be determined based on the effective number of cells. The presently disclosed cell composition may be pharmaceutical composition, further comprising suitable pharmaceutically acceptable excipients, as needed. Suitable pharmaceutically acceptable excipients may include binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavouring agents, flavour masking agents, colouring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents. The pharamaceutical composition may be used in combination with one or more additional pharmaceutical agents such as, for example, chemotherapeutics, anti-inflammatory agents, steroids, immunosuppressants, check point inhibitor, therapeutic antibody, metabolic enzyme inhibitors, chemokine receptor inhibitors, and phosphatase inhibitors, as well as targeted therapies.Examples

[0068] Materials and methods The materials and methods used in the following examples are explained as below.

[0069] Mice B6SJL and OT-I mice were obtained from the Jackson Laboratory and were kept under specific pathogen-free conditions. Sex-matched, 6-12-week-old mice were used for experiments. All of the protocols for animal experiments were approved by the Animal Care and Use Committee at the Okinawa Institute of Science and Technology Graduate University.

[0070] Cell culture Naive CD8+T cells were isolated using the Mojosort mouse naive CD8+T cell isolation kit (BioLegend). Naive CD8+T cells purified from B6SJL or OT-I mice were cultured in 48-well non-treated culture plates (1 × 106cells per well) coated with 5 μg / mL anti-CD3ε antibody (BioLegend). The cells were maintained in complete RPMI (Invitrogen) media supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin (Sigma), 55 μMβ-mercaptoethanol (Invitrogen), 10 mM HEPES (Invitrogen), 1% non-essential amino acids (Invitrogen), and 1 mM sodium pyruvate (Invitrogen). Additionally, 1 μg / mL anti CD28 antibody (BioLegend), 10 ng / mL IL-2 (BioLegend) were added. For co-culture experiments with electroporated CD8+T cells or culture with supernatant from electroporated CD8+T cells, CD45.2+CD8+T cells were electroporated using the 4D-Nucleofector system (4D-Nucleofector X Unit: AAF-1002X; Lonza) with program code DS-137. Either the electroporated cells or the supernatant was added to the CD45.1+ CD8+T cell culture. ATP (Sigma-Aldrich) and dbcAMP (Santa Cruz Biotechnology) were used at concentrations of 25-100 μM. Apyrase (Sigma-Aldrich) was used at 40 U / ml. Rp-8-Br-cAMP (Santa Cruz Biotechnology) was used at 50 μM. Plat-E cells were grown in DMEM (FUJIFILM Wako) supplemented with 10% FBS, 1% penicillin / streptomycin, 1 μg / mL puromycin, and 10 μg / mL blasticidin. HeLa and MCF-7 cells were obtained from RIKEN BRC. Hela cells were cultured in DMEM supplemented with 1% penicillin-streptomycin, 1% glutamine, and heat-inactivated 10% FBS. MCF-7 cells were seeded onto 0.1% gelatin coated dish and grown in DMEM-low glucose (FUJIFILM Wako). The culture conditions were maintained in a humidified incubator at 37°C with 5% CO2.

[0071] Plasma membrane damage For RNA-sequencing (RNA-seq), cells were treated with deoxycholic acid (400 μM), sodium dodecyl sulfate (0.008%), or streptolysin O (SLO; 200 ng / mL) in complete media for 1 hour at 37°C, or they were electroporated using program code DS-137 followed by a 2-h culture. To analyze the effect of plasma membrane damage on CD8+T cell activation, naive CD8+T cells were treated with SLO (3 μg / mL) or deoxycholic acid (600 mM) for one hour, subsequently washed, and cultured in fresh media for an additional hour. Following these treatments, cells were activated with anti-CD3 and anti-CD28 antibodies, along with IL-2, for 72 hours. For the membrane permeability assay, SLO and propidium iodide (PI; 1 μg / mL, Sigma) were added to the complete medium, and cellular uptake of these dyes was evaluated using flow cytometry (FACS).

[0072] RNA-seq CD8+Tcells were subjected to RNA isolation using the RNAdvance Cell V2 kit (Beckman Coulter), following the manufacturer’s protocols. The quantification of RNA was carried out using an RNA HS Assay Kit (Thermo Fisher) and a Qubit Flex Fluorometer (Thermo Fisher). The library preparation was performed using the QuantSeq 3 mRNA-Seq Library Prep Kit FWDforIllumina (Lexogen), following the manufacturer’s procedure. The libraries were measured using the Qubit 1x dsDNA HS Assay Kit (Thermo Fisher) and a Qubit Flex Fluorometer (Thermo Fisher), and their quality was evaluated using D1000 ScreenTape and High Sensitivity D5000 ScreenTape on a Tapestation 2200 (Agilent). The pooled libraries underwent sequencing using an Illumina NovaSeq 6000 equipment, with 1 × 100-bp reads.

[0073] RNA-seq data analysis In order to evaluate the quality of the data, FastQC (v0.11.9). Trimmomatic (Reference 18) (v0.39) was used to eliminate adapter and low-quality sequences. The following parameters were applied: SLIDINGWINDOW:4:20, LEADING:20, TRAILING:20, MINLEN:20, and HEADCROP:12. The sequencing reads were mapped to the mm10 reference genome using HISAT2 (Reference 19) (v2.2). The feature Counts function from the Subread (Reference 20) package (v2.0.1) was used to count the number of overlapping genes in the GENCODE (v30) reference transcriptome annotations. The flags-s 1 and-t gene were used. Statistical analysis using the Wald test in DESeq2 (v1.34.0) (Reference 21) was used to identify differentially expressed genes. The clusterProfiler package (v4.2.2) (Reference 22) was used to perform gene set enrichment analysis. Motif enrichment analysis was performed using Rcis Target (v1.23.1). In short, Rcis Target identified transcription factor binding motifs enriched in differentially expressed genes. We set the cut-off for the normalized enrichment score (NES) at 3.5. The motif rankings version used was mm10refseq-r80, covering 500 bp upstream and 100 bp downstream of the transcription start site.

[0074] qPCR Total RNA was extracted using Isogen II (Nippon Gene). RNA quantity was measured with a Nanodrop 2000 spectrophotometer (Thermo). cDNA synthesis was carried out using the ReverTra Ace qPCR Kit (FSQ-101; Toyobo). The synthesized cDNA served as the template for qPCR, which was performed using KAPA SYBR FAST (NIPPON Genetics) on a StepOnePlus thermal cycler (Thermo). qPCR primers used in this study are listed in Table 1. In Table 1, the sequences of primers and ID numbers of their target genes are shown. The primers named “Hprt Fow” and “Hprt Rev” are corresponding to the target gene Hprt1 having Gene ID of 15452. The primers named “Vps37b Fow” and “Vps37b Rev” are corresponding to the target gene Vps37b having Gene ID of 330192. The primers named “Ramp3 Fow” and “Ramp3 Rev” are corresponding to the target gene Ramp3 having Gene ID of 56089.

[0075] Table 1

[0076] Nucleofection To prepare the crRNA / tracrRNA duplex, the Alt-R crRNA and Alt-R tracrRNA (IDT) were each reconstituted to 100 μM using Nuclease-Free Duplex Buffer (IDT). The oligos were mixed together at equal concentrations, and then heated to 95℃for 5 minutes in a PCR thermocycler to allow them to anneal. After that, they were cooled down to room temperature over 10 minutes. Two crRNA-tracrRNA duplexes (2 μL each, 100 pmol each, total 4 μL) were combined with 2 μL (60 pmol) of TrueCut Cas9 Protein v2 (Thermo Fisher Scientific) in a PCR strip. The mixture was gently mixed by pipetting and then incubated at room temperature for a minimum of 10 minutes. Naive CD8+T cells were cultured in a complete medium supplemented with 10 ng / mL of recombinant mouse IL-7 (BioLegend) for 24 hours before transfection. Preactivated CD8+T cells were grown in complete media at a density of 1 × 106cells / mL with 5 μg / mL plate-bound anti-CD3 (BioLegend), 1 μg / mL anti-CD28 (BioLegend), and 10 ng / ml of recombinant IL-2 (BioLegend). Prior to nucleofection, T cells were subjected to a 3-day preactivation. Around 5-10 million T cells were suspended in 20 μL of primary cell nucleofection solution (P4 Primary Cell 4D-Nucleofector X kit S; 32 RCT, V4XP-4032; Lonza), with the addition of 4 μM Alt-R Cas9 Electroporation Enhancer (IDT). The cells were subsequently mixed with the ribonucleoproteins (RNPs) and incubated at room temperature for a duration of 2 minutes. The mixture of cells and ribonucleoproteins (RNPs) was moved to strips of cuvettes designed for nucleofection (4D-Nucleofector X kit S; Lonza). Electroporation was conducted using a 4D Nucleofector machine, utilizing program code DS-189 for naive T cells and CM-189 for preactivated T cells. After nucleofection, the cells were moved to 24-well containing pre-warmed T cell media supplemented with IL-7 (BioLegend; 10 ng / mL). The cells were cultured for at least overnight and then used for future examination. The crRNAs utilized in this investigation are documented in Table 2. Unless otherwise specified, Vps37b #1 and Ramp3 #1 were employed for the purpose of deleting Vps37b and Ramp3. In Table 2, the crRNA sets named “Vps37b #1” and “Vps37b #2” are corresponding to the target gene Vps37b having Gene ID of 330192. The crRNA sets named “Ramp3 #1” and “Ramp3 #2” are corresponding to the target gene Ramp3 having Gene ID of 56089. The crRNA set named “P2ry10” is corresponding to the target gene P2ry10 having Gene ID of 78826.

[0077] Table 2

[0078] Overexpression and RNAi in human cells For transfection of vectors overexpressing VPS37B or RAMP3, MCF-7 cells were incubated with a mixture of 4 μg / mL polyethyleneimine (PEI) and 400 ng / mL plasmid DNA for 30 min. Cells were then washed and cultured with fresh medium. Laser damage assays were conducted at 24-48 hours post-transfection. For imaging, cells were resuspended in Fluorobrite DMEM (Invitrogen) supplemented with 1% penicillin-streptomycin, 1% glutamine, and heat-inactivated 10% FBS. For transfection of vectors overexpressing CHMP4B, Ramp3 fused with mCherry, and intracellular vesicle markers, Hela cells were incubated with a mixture of 1 mg / mL polyethyleneimine (PEI) and 1.2 μg / mL plasmid DNA for 1 hour. Cells were then washed and cultured with fresh medium. Cells were observed under confocal microscope at 24 h post-transfection. The plasmid vectors used are listed below: pCMV-TagGFP2-G4S3-CHMP4B pCMV-mCherry-G4S3-VPS37B pCMV-RAMP3ss-mCherry-G4S3-RAMP3(C) pFX-EGFP-EEA1 EGFP-Rab4a-7 pLVX-EF1a-EGFP-RAB5A-IRES-Puromycin EGFP-Rab7A EGFP-Rab8a-wt pLVX-EF1a-EGFP-RAB11B-IRES-Puromycin LAMP1-mGFP For RNAi experiments, HeLa cells were incubated with a mixture of 3 nM siRNA (IDT) and 2 μL / mL RNAiMAX transfection reagent (Thermo Fisher Scientific) for 12 h. Cells were then washed and cultured with fresh medium. Laser damage assays were conducted 24-72h post-transfection. The siRNA sequences used are listed in Table 3.

[0079] Table 3

[0080] Laser damage assay The laser damage assay was performed using a Nikon laser scanning confocal microscope (A1, NIS-Elements 6.0) with a 60x objective lens. A region approximately 0.5 μm in diameter on the plasma membrane was selected and irradiated with a 405 nm laser for approximately 1 second. The 405 nm laser was set to 100% power.

[0081] Retroviral transduction Full-length mouse Vps37b and Ramp3 were inserted into MIGR1 IRES-GFP or MIGR1 IRES-dsRed vectors. To prepare virus particles, Plat-E cells were seeded into 10-cm dishes and allowed to grow overnight. The next day, the plasmids and the packaging plasmid pCL Eco were mixed with PEI at a 5:1 PEI ratio in 3 ml of PBS and added to the Plat-E cells, incubating overnight. The following day, the medium was changed, and the viral supernatant was collected four days after transduction. For retroviral transduction, T cells were plated in 48-well plates pre-coated with retronectin (Takara). The viruses were immobilized onto the retronectin-coated plates by centrifuging the virus-containing supernatant at 2000 xg for 120 min. After removing the supernatant, wells were washed once with PBS, and anti CD3 / CD28-stimulated CD8+T cells were added at a concentration of 1 × 106cells per well, along with IL-2 (10 ng / mL; BioLegend). The plates were then centrifuged at 600 g for 30 min to ensure contact between the T cells and retronectin-bound viruses.

[0082] OT-I adoptive T cell transfer For listeria infection model, naive CD8+T cells were isolated from CD45.2+CD45.1+OT I mice. After isolation, cells were subjected to nucleofection with RNPs targeting either Vps37b or Ramp3, along with non-targeting RNPs. Nucleofected cells were then incubated overnight at 37℃. Subsequently, 1 × 104cells per mouse were intravenously administered into congenic recipient CD45.1+B6SJL mice. For tumor experiments, 5-10 × 106in vitro activated OT-I T cells were nucleofected for gene deletion and expanded in CD8+T cell culture media supplemented with IL-7 and IL-15. Subsequently, 1.5 × 106cells were injected intravenously in 100 μL PBS on day 8.

[0083] Listeria monocytogenes infection LM-OVA, which is erythromycin resistant, was grown in Brain-Heart Infusion (BHI) broth containing erythromycin (5 μg / ml) at 37℃ with shaking at 220 rpm until LM-OVA was grown to OD600 at about 0.1, then washed with sterile PBS. One day after the adoptive T cell transfer, mice were intravenously infected with 5×103colony-forming units (cfu) of LM OVA. For evaluation of the proliferated OVA specific CD8+T cells, spleen was harvested and analyzed by flowcytometry.

[0084] Flow cytometry Before the antibody staining, the cells were incubated with an anti-Fc receptor-blocking antibody (anti-CD16 / CD32; BioLegend). Subsequently, dead cells were stained with Zombie Nir (BioLegend). For cell surface molecule analysis, cells were stained with fluorochrome conjugated antibodies in PBS containing 2% FBS for 20 minutes on ice. For intracellular molecule analysis, cells were stained with fluorochrome-conjugated antibodies using the Foxp3 Staining Buffer Set (eBioscience) following the manufacturer’s instructions. To analyze intracellular cytokines, cells were re-stimulated with phorbol 12-myristate 13-acetate (PMA; Sigma; 50 ng / mL) and ionomycin (Sigma; 500 ng / mL) in the presence of brefeldin A (BioLegend; 5 μg / mL). The following antibodies were used at a 1:200 dilution: anti CD16 / 32 (BioLegend), anti-CD3 (BioLegend), anti-CD8 (BioLegend), anti-CD45.1 (BioLegend), anti-CD45.2 (BioLegend), anti-IFNγ (BioLegend), anti-Bim (CST), anti-perforin (BioLegend), and anti-Ki67 (BioLegend). To quantify plasma membrane damage in LM OVA-infected mice, blood samples were collected, mixed with FITC-dextran (1.25 mg / mL; Sigma), and incubated for 1 hour on ice. The uptake of the fluorescent dye was then evaluated using flow cytometry (FACS).

[0085] B16-OVA model Mice were intradermally challenged with 2 × 105B16-Ova cells in 50 μL PBS. Tumor size was measured every two days. Tumor volume was calculated as 0.5 × longer diameter × shorter diameter2.

[0086] Coculture with B16-OVA cells For co-culture of CD8+T cells with tumor cells, B16-OVA cells were seeded at 7 × 104cells per well in a 96 well plate. Following nucleofection, 3.5 × 105nucleofected CD8+T cells were transferred to wells containing B16-OVA cells. After 20 hours, FITC-dextran was added and cells were incubated for an additional 1 hour to evaluate fluorescent dye uptake. To distinguish dead cells from live damaged cells, CD8+T cells were collected and then stained with Zombie-NIR.

[0087] scRNA-seq CD8+T cells isolated from tumor tissues were used for scRNA-seq analysis. Cells from individual mice were stained separately with different oligonucleotide-tagged antibodies (TotalSeq-B hashtag antibodies) following the manufacturer’s protocol and pooled in equal proportions. Approximately 20,000 cells were placed onto the 10X Genomics Chromium Controller as single-cell suspensions. The library preparation was conducted using the Chromium Next GEM Single Cell 5 v2 (Dual Index) Reagent Kit, following the instructions provided by the manufacturer. The reverse transcription reaction was conducted using a Bio-Rad T100 Thermal Cycler (Bio-Rad). All libraries were evaluated using a TapeStation and quantified using a Qubit Fluorometer. The libraries were combined and processed for sequencing using an Illumina NovaSeq platform (Illumina). The sequencing parameters used were as follows: read 1-26 cycles, i7-10 cycles, i5-10 cycles, and read 2-90 cycles. The aim was to obtain 20,000 reads per cell RNA library

[0088] scRNA-seq data analysis The CellRanger Single-Cell Software Suite (v.6.0.0; 10x Genomics) (Reference 23) was employed to process barcodes and count transcripts. The alignment to the GRCm39 reference genome was performed using default parameters. The scRNA-seq data was subjected to additional analysis using the Seurat R package (v.4.1.0) (Reference 24). The subset function was used to eliminate cells that had mitochondrial gene counts exceeding 5% or fewer than 500 genes. The datasets were processed with the NormalizeData and FindVariableFeatures functions, followed by the data scaling using the ScaleData function. Each dataset underwent unsupervised clustering using the following procedure: (i) The top variable genes discovered by FindVariableFeatures were utilized for principal component analysis (PCA). (ii) The RunTSNE function was used to do t-SNE dimensional reduction on the top 15 principal components. The FindClusters function was then used to produce unsupervised clustering. For annotating single cells, we used the ProjecTILs (Reference 25) dataset as a reference and the default settings of Seurat v4’s FindTransferAnchors(), TransferData(), and AddMetaData() functions.

[0089] Statistical analysis Statistical differences were analyzed using Student’s t-test for two-group comparisons, and analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test or Tukey’s HSD test for multiple group comparisons. These statistical analyses were conducted using R version 4.3.2

[0090] (Experiment 1) Adaptation of CD8+T cells to plasma membrane damage To assess the adaptability of CD8+T cells to plasma membrane damage, we first examined whether transient sub-lethal plasma membrane damage would enhance resistance of CD8+T cells to subsequent similar damage. We added primary membrane damage to CD8+T cells using conventional electroporation that makes transient pores on plasma membrane. Three days later, we treated cells with streptolysin o (SLO), a bacterial pore-forming toxin, and measured membrane damage levels by tracking the uptake of propidium iodide (PI), a membrane-impermeable fluorescent dye, commonly used to detect dead cells in a population. This revealed that pre-electroporated cells exhibited reduced PI uptake during SLO treatment compared to non-electroporated cells (FIG. 2). This indicates that CD8+T cells have an adaptive response to plasma membrane damage. To obtain the data shown in FIG. 2, CD8+T cells were treated with or without electroporation and cultured for 3 days. Subsequently, cells were treated with SLO (1500 ng / mL) for 1 hour in the presence of PI. Percentages of PI-positive cells were analyzed using flow cytometry. In the bar graph on the righthand, abbreviation “EP” stands for “Electroporation.” The p-value was calculated by a two-tailed unpaired Student’s t-test (** p < 0.01). Error bars represent mean ± standard deviation (SD).

[0091] (Experiment 2) Plasma membrane damage induces transcriptional change in CD8+T cells We hypothesized that genes induced by plasma membrane damage are involved in the adaptation response to such damage. To identify genes commonly induced under various plasma membrane-damaging conditions, we subjected naive CD8+T cells to electroporation or transiently exposed them to membrane-damaging agents, SLO, sodium dodecyl sulfate (SDS), or deoxycholic acid (DCA). After a 2-hour culture, we analyzed these cells using RNA sequencing (RNA-seq). This revealed that 153 genes were upregulated across all conditions (FIG. 3A), including Vps37b and Ramp3 (FIG.3B). Vps37b is a component of the ESCRT complex (Reference 26), while Ramp3 plays a role in membrane trafficking (References 27, 28). Both ESCRT and membrane trafficking are crucial for plasma membrane repair (References 29, 30). Therefore, we decided to focus on these two proteins as candidates for key factors in T cell adaptation to plasma membrane damage. To obtain the result shown in FIG. 3A and FIG. 3B, Naive CD8+T cells were subjected to electroporation or treated with either SLO (200 ng / mL), SDS (0.008%), or DCA (400 mM) for 1 hour, followed by a 2-hour culture. Cells were then used for RNA-seq analysis. Genes whose expression was significantly altered in each condition compared to untreated controls (adjusted p-value < 0.05, |log2 fold change| >1) were identified as differentially expressed genes. FIG. 3A represents Venn diagram showing commonly upregulated genes in each condition. The abbreviation “EP” stands for electroporation. FIG. 3B represents ranking of differentially expressed genes based on log2 fold change.

[0092] (Experiment 3) Vps37b and Ramp3 are involved in the adaptation to plasma membrane damage To test whether Vps37b and Ramp3 are involved in resistance of CD8+T cells to plasma membrane damage, we deleted Vps37b and Ramp3 in CD8+T cells using the CRISPR / Cas9 system. Following nucleofection with Cas9-gRNA complexes targeting Vps37b or Ramp3, we exposed cells to SLO in the presence of PI dye. The results revealed that CRISPR knock out (KO) of Vps37b or Ramp3 significantly decreased resistance of CD8+T cells to plasma membrane damage induced by SLO, as indicated by increased PI uptake (FIG. 4A). Additionally, we investigated whether overexpression of Vps37b or Ramp3 enhances resistance of CD8+T cells to plasma membrane damage. We found that overexpression of Ramp3, but not Vps37b, mitigated plasma membrane damage (FIG. 4B). Moreover, simultaneous overexpression of Vps37b and Ramp3 enhanced resistance to plasma membrane damage more than Ramp3 alone (FIG. 4B). These observations suggest that Ramp3 increases T cell resistance to plasma membrane damage, and Vps37b further supports Ramp3 function. Collectively, our data suggest that upregulation of Vps37b and Ramp3 is required for adaptation of CD8+T cells to plasma membrane damage. To obtain the result shown in FIG. 4A and FIG. 4B, CD8+T cells were activated and nucleofected with CRISPR RNPs targeting either Vps37b or Ramp3 alongside non-targeting RNPs (FIG. 4A) or retrovirally transduced with pMIG-GFP (empty vector), pMIG Vps37b-GFP (Vps37b), or pMIG-Ramp3-GFP (Ramp3) (FIG. 4B). Then, cells were treated with SLO (500 ng / mL and 750 ng / ml, respectively.) in the presence of PI, and PI uptake was assessed by FACS. The p-value was calculated by Dunnett’s test. Error bars represent mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001.

[0093] (Experiment 4) VPS37B and RAMP3 are involved in plasma membrane repair Next, we investigated how VPS37B and RAMP3 are involved in plasma membrane damage tolerance. To this end, we knocked down VPS37B or RAMP3 in a human breast cancer cell line, MCF-7 cells, using siRNA specific to these genes. Subsequently, we irradiated the plasma membranes of these cells with a laser, and the extent of plasma membrane damage and repair was assessed by influx of a membrane impermeable dye, FM1-43. Knockdown of VPS37B or RAMP3 not only augmented the FM1-43 influx but also prolonged the time until the FM1-43 signal reached its peak (FIG. 5A, B). This suggests that VPS37B and RAMP3 may facilitate closure of plasma membrane wound. To further investigate this possibility, we ectopically expressed fluorescent-tagged VPS37B or RAMP3 in MCF-7 cells and monitored their localization after laser irradiation. VPS37B and RAMP3 accumulated at the damaged site within 15 min and 40 min of plasma membrane damage, respectively (FIG. 5C). These findings suggest that VPS37B and RAMP3 are directly involved in plasma membrane repair. To obtain the result shown in FIG. 5A and FIG. 5B, VPS37B or RAMP3 was knocked down by siRNA in Hela cells. These cells were irradiated by laser, and the influx of FM1-43 dye was assessed under a confocal microscope. FIG. 5A shows the relative intensity of FM1-43 signals. FIG. 5B shows time for FM1-43 signals to reach maximum intensity. FIG. 5C shows MCF-7 cells were transduced with a vector carrying human VPS37B or RAMP3 fused with mCherry fluorescent protein and irradiated with laser. Localization of VPS37B and RAMP3 was visualized under a confocal microscope. Laser-irradiated sites are marked by arrow heads. Areas indicated with white boxes are enlarged on the lower right corner of each panel. Scale bars indicate 10 μm.

[0094] (Experiment 5) Vps37b and Ramp3 promote plasma membrane repair by distinct mechanisms Since VPS37B is a subunit of the ESCRT complex, VPS37B may contribute to the repair of plasma membrane damage by regulating other ESCRT protein functions. To test this possibility, we expressed fluorescent-tagged charged multivesicular body protein 4B (CHMP4B), a subunit of the ESCRT complex (Reference 7), in MCF-7 cells and knocked down VPS37B. Analysis of CHMP4B localization after laser irradiation revealed that the knockdown of VPS37B, but not RAMP3, inhibited the recruitment of CHMP4B to the site of damage (FIG. 6A). This suggests that VPS37B is involved in plasma membrane repair by promoting the localization of other ESCRT proteins. RAMP3 is known to localize to intracellular vesicles and regulate vesicle trafficking (References 27, 28). Given that the fusion of intracellular vesicles with the plasma membrane contributes to plasma membrane repair (Reference 30), RAMP3 may influence this process by controlling the localization of these vesicles. Specifically, RAMP3 has been reported to localize to Rab4 positive early endosomes and recycling vesicles (Reference 27). To further investigate the localization of RAMP3, we employed various markers of intracellular vesicles, including EEA1 (early endosome), RAB4B (early and recycling endosome), RAB5A (early endosome and phago some), RAB7A (late endosome, lysosome, and autophagosome), RAB8A (recycling endo some), RAB11 (recycling endosome and trans-Golgi network), and LAMP1 (lysosome). This revealed that RAMP3 co-localizes with vesicles containing EEA1, RAB5A, RAB7A, RAB8A, and LAMP1 (FIG. 6B) suggesting that RAMP3 is associated with various intra cellular vesicles, including endosomes and lysosomes. Given membrane repair is promoted by endosome and lysosome fusion with the plasma membrane (References 31-33), RAMP3 may facilitate plasma membrane repair by regulating the trafficking of these intracellular vesicles. In summary, VPS37B and RAMP3 appear to regulate plasma membrane repair through distinct mechanisms: VPS37B by modulating ESCRT proteins, and RAMP3 potentially by controlling the localization and trafficking of endosomes and lysosomes in response to plasma membrane damage. To obtain the result shown in FIG. 6A, Hela cells were transfected with a vector expressing CHMP4B fused with TagGFP2, followed by RNAi knockdown of VPS37B or RAMP3, and then subjected to laser irradiation. On the left hand of FIG. 6A, timelapse con focal microscopy images showing CHMP4B localization after laser irradiation are shown. In these timelapse images, arrow heads indicate laser-irradiated sites. A Graph on the left hand of FIG. 6A, indicates the kinetics of signal intensity of TagGFP2, which reflects CHMP4B accumulation at laser-irradiated sites. As to FIG. 6B, Hela cells were cotransfected with a vector expressing RAMP3 fused with mCherry red fluorescent protein and vectors expressing intracellular vesicle markers, EEA1, RAB4A, RAB5A, RAB7, RAB8, RAB11, or LAMP1, each fused with EGFP. Co-localization of RAMP3 with vesicle markers was assessed using confocal microscopy. The scale bars, shown in FIGs 6A and 6B represents 10 μm.

[0095] (Experiment 6) Plasma membrane damage adaptation is induced in both damaged cells and neighboring cells Plasma membrane repair activity is enhanced not only in cells with plasma membrane damage but also in the surrounding undamaged cells (Reference 34). Therefore, we examined expression of Vps37b and Ramp3 in CD8+T cells with plasma membrane damage and those surrounding them without such damage. For this purpose, naive CD8+T cells that were exposed to plasma membrane damage through electroporation were co-cultured with those not subjected to electroporation, and expression of Vps37b and Ramp3 mRNA was analyzed by qPCR. We observed an increase in Vps37b and Ramp3 expression not only in electroporated cells but also in non-electroporated cells co-cultured with them (FIG. 7A, B). We reasoned that substances secreted from damaged cells increase expression of Vps37b and Ramp3 in surrounding undamaged cells. To test this, we collected the conditioned medium of electroporated CD8+T cells and used them to culture naive CD8+T cells. This revealed that the expression of Vps37b and Ramp3 increased in naive CD8+T cells cultured in the supernatant from electroporated CD8+T cells (FIG. 7C). These results suggest that substances secreted by cells with plasma membrane damage induce the expression of Vps37b and Ramp3 in a paracrine manner. To obtain the result shown in FIG. 7A, CD8+T cells were electroporated, cultured for 2 hours, and the expression levels of Vps37b and Ramp3 were assessed by qPCR. To obtain the result shown in FIG. 7B, CD8+T cells were co-cultured with electroporated or non-electroporated cells for 2 hours and the expression levels of Vps37b and Ramp3 were assessed by qPCR. To obtain the result shown in FIG. 7 C, CD8+T cells were cultured in the conditioned media of electroporated CD8+T cells (cell wash supernatant immediately after electroporation) or control cells for 2 hours and the expression levels of Vps37b and Ramp3 were assessed by qPCR. The p-value was calculated by a Student’s t-test. Error bars represent mean±standard deviation(SD). *p<0.05, **p<0.01, ***p<0.001.

[0096] (Experiment 7) ATP is a key molecule to induce Vps37b and Ramp3 A previous report suggests that ATP, released from damaged cells, enhances plasma membrane repair in neighboring cells through cAMP signaling (Reference 34). Therefore, we treated naive CD8+T cells with or without ATP for 4 hours and assessed the expression of Vps37b and Ramp3 mRNA. The results revealed that ATP treatment of naive CD8+T cells increased the expression of Vps37b and Ramp3 (FIG. 8A). It is well-established that cAMP signaling plays a crucial role in the ATP-induced promotion of plasma membrane repair (References 34, 35). Consistently, treatment of naive CD8+T cells with the membrane-permeable cAMP analogue, dbcAMP, increased the expression of Vps37b and Ramp3 mRNA (FIG. 8B). Next, to assess whether ATP is crucial for the upregulation of Vps37b and Ramp3 in cells with plasma membrane damage, we added the ATP-degrading enzyme apyrase or cAMP inhibitor Rp-8-Br-cAMP during the electroporation of naive CD8+T cells. Apyrase and Rp-8-Br-cAMP partially inhibited the increased expression of Vps37b and Ramp3 in electroporated CD8+T cells (FIG. 8C). Moreover, the conditioned media of electroporated CD8+T cells in the presence of apyrase lost the paracrine effect to induce Vps37B and Ramp3 (FIG. 8D). These results indicate that ATP released from damaged cells contributes to the increased expression of Vps37b and Ramp3 through cAMP synthesis in both damaged and neighboring non-damaged cells via autocrine and paracrine signaling. To obtain the result shown in FIGs 8A to 8D, ATP signaling is stimulated or suppressed in CD8+T cells, and expression levels of Vps37b and Ramp3 were assessed by qPCR. To obtain the result shown in FIG. 8A, CD8+T cells were treated with ATP, at the indicated concentration for 4 hours. To obtain the result shown in FIG. 8B CD8+T cells were treated with the membrane permeable cAMP analog dbcAMP, at the indicated concentration for 4 hours. To obtain the result shown in FIG. 8C, CD8+T cells were electroporated in the presence of the ATP-degrading enzyme apyrase or the cAMP inhibitor Rp-8-Br-cAMP and were cultured for 2 hours. To obtain the result shown in FIG. 8D, the conditioned media of electroporated or nonelectroporated CD8+T cells (cell wash supernatant immediately after electroporation) in the presence or absence of apyrase for 2 hours. In FIG. 8D, “nonEPcond.” refers to conditioned media of non-electroporated cells. “EPcond.” refers to conditioned media of electroporated cells. The p-value was calculated by a Dunnet’s test or Tukey’s HSD test. Error bars represent mean ± standard deviation (SD). **p<0.01,***p< 0.001.

[0097] (Experiment 8) P2ry10 is involved in the ATP-mediated induction of Vps37b and Ramp3 Engagement of some P2ry receptors by ATP leads to the production of cAMP through the activation of adenylate cyclase (Reference 36). Therefore, we hypothesized that ATP induces the expression of Vps37b and Ramp3 via P2ry receptors. To investigate this hypothesis, we first analyzed the expression of the P2ry receptors in CD8+T cells using our RNAseq dataset obtained in Fig. 3.2. In naive CD8+T cells, P2ry10 expression was the highest, and it increased upon plasma membrane damage (FIGs 9A, B). Next, to investigate the role of P2ry10 in the induction of Vps37b and Ramp3, we deleted P2ry10 in CD8+T cells using the CRISPR / Cas9 system and exposed them to a membrane-damaging environment through electroporation. The induction of Vps37b and Ramp3 was partially inhibited by the deletion of P2ry10 (FIG. 9 C), suggesting that P2ry10 is involved in the ATP-dependent increase in expression of Vps37b and Ramp3. In summary, ATP released from cells with plasma membrane damage stimulates the P2ry10 receptor- cAMP pathway to induce expression of Vps37b and Ramp3 in both damaged and surrounding non-damaged CD8+T cells, thereby promoting adaptation of these cells to plasma membrane damage. To obtain the result shown in FIGs 9A, B, RNA-seq data obtained in experiment 2 were analyzed. As to the data shown in FIG. 9A, expression levels of each P2ry receptor in naive CD8+T cells are measured. FIG. 9B shows volcano plots showing expression changes upon plasma membrane damage treatment. To obtain the results shown in FIG. 9C, CD8+T cells were nucleofected with CRISPR RNP targeting P2ry10 and were cultured for 3 days. Subsequently, cells were electroporated, and 2 hours later, expression levels of Vps37b and Ramp3 were assessed by qPCR. In FIG. 9C, “EP” represents electroporation. The p-value was calculated by a Student’s t-test. Error bars represent mean ± standard deviation (SD). **p<0.01.

[0098] (Experiment 9) Listeria monocytogenes infection increases plasma membrane damage in CD8+T cells in vivo To understand the role of adaptation of CD8+T cells to plasma membrane damage in vivo, we first evaluated levels of plasma membrane damage in CD8+T cells during bacterial infection. Listeria monocytogenes (LM) is a pathogenic bacterium that produces listeriolysin O (LLO), one of the CDCs. Thus, it can cause plasma membrane damage in CD8+T cells during infection. We intravenously injected mice with LM and collected CD8+T cells from the blood 4 days post-infection. Then, to assess the severity of plasma membrane damage, we incubated these cells with membrane impermeable FITC-dextran and assessed its incorporation into cells. The results showed that percentages of cells took up FITC-dextran was higher in CD8+T cells isolated from LM-infected mice compared to those from uninfected mice (FIG. 10A). We also analyzed expression of Vps37b and Ramp3 in CD8+T cells isolated from spleens and found that the expression of these genes was elevated in CD8+T cells isolated from LM-infected mice compared to those from uninfected mice (FIG. 10B). These results suggest that LM infection increases plasma membrane damage in CD8+T cells. To obtain the result shown in FIGs 10A-B, mice were intravenously injected with LM or PBS. Four days later, blood and splenocytes were collected. To obtain the result shown in FIG. 10A blood was collected and incubated with FITC-dextran for 1 hour. The uptake of FITC dextran in CD44+activated CD8+T cells was assessed by FACS. To obtain the result shown in FIG. 10B, splenic CD44+CD8+T cells were sorted and the expression levels of Vps37b and Ramp3 mRNA were assessed by qPCR. The p-value was calculated by a Student’s t-test. Error bars represent mean ± standard deviation (SD). * p <0.05, ** p < 0.01.

[0099] (Experiment 10) Plasma membrane damage adaptation is crucial for CD8+T cell responses during LM infection To clarify the importance of adaptation of CD8+T cells to plasma membrane damage during LM infection, we evaluated the effect of deficiency of Vps37b or Ramp3 in CD8+T cell responses. For this, we adoptively transferred naive OVA-specific OT-I CD8+ T cells, transduced with CRISPR RNPs targeting Vps37b or Ramp3, into mice. We then infected mice with LM expressing OVA (LM-OVA) and analyzed responses of the transferred cells. The results showed that, 7 days after infection, CRISPR KO of Vps37b or Ramp3 significantly reduced the accumulation of transferred OT-I T cells compared to control cells (FIG. 11A). Furthermore, the expression of Bim, an apoptosis marker, was elevated in cells lacking Vps37b or Ramp3 compared to control cells (FIG. 11B). The expression of Ki67, a marker of cell proliferation, was decreased in Vps37b- or Ramp3-deficient cells (FIG. 11C). These results indicate that Vps37b and Ramp3 are essential for clonal expansion of CD8+T cells in response to LM infection. Furthermore, the observation implies that CD8+T cells need to adapt to LM-induced plasma membrane damage using Vps37b and Ramp3 to avoid apoptosis and cell cycle arrest. To obtain the result shown in FIGs A-C, CD45.1+CD45.2+naive OT-I T cells were nucleofected with RNPs targeting Vps37b or Ramp3, or non-targeting control RNPs. These cells (1x104) were transferred into CD45.1+congenic recipient mice, followed by LM-OVA infection. Cells were collected from spleens on day 7 post-infection and analyzed by flowcytometry. Representative dot plot and statistical analysis of the percentage of the transferred OT-I T cells (FIG. 11A) and expression of the apoptosis marker Bim (FIG. 11B) and the cell proliferation marker Ki67 (FIG. 11C). The p-value was calculated by a Dunnett’s test. Error bars in FIGs 11A-C represent mean ± standard deviation (SD). **p<0.01,***p<0.001.

[0100] (Experiment 11) Increased plasma membrane damage in CD8+T cells during anti-tumor response We hypothesized that CD8+T cells may experience increased plasma membrane damage not only in infection with pathogens producing pore-forming toxins but also in prolonged inflammatory environment, such as in tissues with cancer or chronic infection, due to increased exposure to membrane-damaging molecules, including self-producing perforin. To address this possibility, we investigated levels of plasma membrane damage in CD8+ T cells in response to tumor cells and the role of Vps37b and Ramp3 in repairing such damage. We co-cultured OT-I T cells with B16-OVA, a melanoma cell line expressing OVA, and plasma membrane damage levels in OT-I T cells was assessed by FITC-dextran uptake. We found that there was a significant increase in uptake of FITC-dextran in CD8+T cells co-cultured with B16-OVA (FIG. 12A). Moreover, this effect was further increased by CRISPR KO of Vps37b or Ramp3, although the increase by Vps37b KO was to a lesser extent and did not reach statistical significance (FIG. 12A). In contrast, CRISPR KO of Vps37b or Ramp3 did not affect the percentage of dead cells in CD8+ T cells cultured with B16-OVA (FIG. 12B). These findings suggest that plasma membrane damage increases in CD8+ T cells during anti-tumor responses and that Vps37b and Ramp3 play a role in repairing this damage. To obtain the result shown in FIGs 12A, B, OT-I T cells activated with anti-CD3 and anti-CD28 antibodies and IL-2, were nucleofected with RNPs targeting Vps37b or Ramp3, or non-targeting control and cultured for 10 days. Cells were then co-cultured with B16-OVA cells at a ratio of 1:5 for 20 h. To obtain the result shown in FIG. 12A, cells were then incubated with FITC-dextran for 1 hour, and its uptake was analyzed by flow cytometry. To obtain the result shown in FIG. 12B, dead cells were stained with Zombie dye and analyzed by flow cytometry. The p-value was calculated by a Dunnet’s test. Error bars shown in FIGs 11A-B represent mean ± standard deviation (SD). *p < 0.05.

[0101] (Experiment 12) Sub-lethal plasma membrane damage upregulates IFN-γ expression Recent reports suggest that sublethal plasma membrane damage increases expression of several inflammatory cytokines and chemokines in cell lines (References 37, 38). To determine if plasma membrane damage induces similar effects on CD8+T cells, we exposed naive CD8+T cells to plasma membrane-damaging conditions through electroporation or treatment with DCA or SLO. Subsequently, we activated these cells with TCR (T-cell Receptor) stimulation for 72 hours and analyzed the expression of anti-tumor effector proteins, perforin and IFN-γ. The results showed that preexposure to these membrane-damaging conditions enhanced expression of perforin and IFN-γ after activation of CD8+T cells (FIG. 13A). To further investigate the impact of plasma membrane damage on CD8+T cell functions, we performed RNA-seq on CD8+T cells that were subjected to electroporation followed by TCR stimulation for 72 hours. This revealed that there were 508 upregulated genes, including Ifng, and 453 downregulated genes in CD8+T cells activated after electroporation compared to controls without electroporation (FIGs 13B-C). Gene set enrichment analysis revealed that MYC target genes, oxidative phosphorylation genes, and genes involved in the unfolded protein response (UPR) were enriched in genes upregulated in CD8+T cells activated after electroporation (FIG. 13D). Additionally, using RcisTarget, we identified several transcription factor (TF)-binding motifs overrepresented in the differentially expressed genes in CD8+T cells activated after electroporation , including binding motifs for ATF3, which is induced by the UPR pathway (Reference 39), at loci including Ifng (FIG. 13E). Taken together, these results suggest that plasma membrane damage before TCR-stimulation upregulates expression of perforin and IFN-γ, in T cells after activation, which might be due to enhanced multiple signaling pathways, including the UPR pathway. To obtain the result shown in FIG. 13A, naive CD8+T cells were treated with SLO (3μg / mL) or DCA (600mM) for 1 hour, washed, and then cultured in fresh media without membrane-damaging agents for 1 hour. Alternatively, cells were subjected to electroporation and then cultured for 2 hours. Subsequently, these cells were activated with anti-CD3, anti-CD28 antibodies, and IL-2 for 72 hours. Cells were then assessed for expression of IFN-γ and perforin using FACS. To obtain the result shown in FIGs 13B-E, naive CD8+T cells were electroporated and then cultured for 2 hours. Cells were then activated with anti-CD3, anti-CD28 antibodies, and IL-2 for 72 hours, and gene expression profiles were analyzed by RNA-seq. FIG. 13B is a volcano plot showing differentially expressed genes. FIG. 13C shows expression of Ifng. FIG. 13D shows the result of gene set enrichment analysis. The abbreviation “NES# stands for normalized enrichment score. FIG. 13E shows the result of Motifenrichment analysis done by using Rcistarget. As to FIG. 13A, the p-value was calculated by a Dunnet’s test. Error bars on the above figures represent mean ± standard deviation (SD). *p<0.05,**p<0.01,*** p<0.001.

[0102] (Experiment 13) Loss of Vps37b or Ramp3 enhances anti-tumor responses of CD8+ T cells Our findings suggested that plasma membrane damage increases in CD8+T cells during anti-tumor responses, and that there is a link between such damage and the enhanced expression of effector molecules. Considering these, we next evaluated the effect of loss of Vps37b or Ramp3 on in vivo anti-tumor responses of CD8+T cells. We transplanted B16 OVA melanoma cells to mice followed by adoptive transfer of OT-I T cells transduced with CRISPR RNPs targeting Vps37b or Ramp3. Notably, CRISPR KO of Vps37b or Ramp3 significantly enhanced activity of OT-I T cells to suppress B16-OVA growth (FIG. 14A). Furthermore, we found that CRISPR KO of Ramp3 increased the percentage of IFN-γ expressing OT-I T cells in tumor tissues (FIG. 14B). To further characterize the effect of Ramp3 deficiency on anti-tumor CD8+ T cell responses, we performed scRNA-seq analysis of tumor-infiltrating OT-I T cells with or without Ramp3 CRISPR KO. Among 6 clusters identified in Seurat prediction (FIG. 14C), Ramp3 KO decreased frequency of CD8+T cells predicted as terminally exhausted cells (Tex), while increasing that of cells predicted as pre cursor exhausted T (Tpex) cells (FIG. 14D). In summary, these results suggest that increased plasma membrane damage due to deficiency of Vps37b or Ramp3 leads to enhanced IFN-γ expression and decreased exhaustion in CD8+T cells, thereby enhancing their anti-tumor activity. To obtain the result shown in FIGs 14A-D, OT-I T cells activated with anti-CD3 and anti-CD28 antibodies and IL-2 were nucleofected with RNPs targeting Vps37b, Ramp3, or control RNPs and expanded with IL-7 and IL-15 invitro. C57BL / 6mice were inoculated with B16-OVAcells (2×105cells, intradermally). Eight days later, mice were injected with OT-I T cells (1.5 × 106cells, intravenously). To obtain the result shown in FIG. 14A, tumor volume was measured every two days. To obtain the result shown in FIG. 14B, twenty days after tumor implantation, IFN-g expression in tumor-infiltrating OT-I T cells was analyzed by FACS. To obtain the result shown in FIG. 14C, twenty days after tumor implantation, tumor-infiltrating OT-I T cells with CRISPR KO of Ramp3 or control were isolated and analyzed by scRNA-seq. To obtain the result shown in FIG. 14C, uniform Manifold Approximation and Projection (UMAP) visualization of scRNA-seq data. CD8+ T cell populations were annotated based on the TIL atlas data set. FIG. 14D shows percentages of cells predicted as terminally exhausted cells (Tex) or precursor exhausted T(Tpex) cells. As to FIG. 14A, the p-value was calculated by a Dunnet’s test. As to FIG. 14B, the p-value was calculated by Student’s t-test. Error bars in the above figures represent mean ± standard deviation (SD). *p<0.05, **p<0.01.

[0103] An innovative method of enhancing immune response activity of a cell is provided.REFERENCES

[0104] 1. Sarah J. Routledge, Graham Ladds, and David R. Poyner. The effects of RAMPs upon cell signalling. Molecular and Cellular Endocrinology, 449:12-20, 2017. 2. David E Golan, M Robert Alecio, William R Veatch, and Robert R Rando. Lateral mobility of phospholipid and cholesterol in the human erythrocyte membrane: effects of protein-lipid interactions. Biochemistry, 23(2):332-339, 1984. 3. Nancy L Thompson and Daniel Axelrod. Reduced lateral mobility of a fluorescent lipid probe in cholesterol-depleted erythrocyte membrane. Biochimica et Biophysica Acta (BBA)-Biomembranes, 597(1):155-165, 1980. 4. Donald E Ingber, Ning Wang, and Dimitrije Stamenovi? Tensegrity, cellular bio physics, and the mechanics of living systems. Reports on Progress in Physics, 77(4):046603, 2014. 5. Adam Horn and Jyoti K Jaiswal. Cellular mechanisms and signals that coordinate plasma membrane repair. Cellular and Molecular Life Sciences, 75(20):3751-3770, 2018. 6. Caroline Barisch, Joost C. M. Holthuis, and Katia Cosentino. Membrane damage and repair: a thin line between life and death. Biological Chemistry, 404(5):467-490, 2023. 7. Ana Joaquina Jimenez, Paolo Maiuri, Julie Lafaurie-Janvore, Severine Divoux, Matthieu Piel, and Franck Perez. ESCRT Machinery Is Required for Plasma Mem brane Repair. Science, 343(6174):1247136, 2014. 8. Suman Lata, Manfred Roessle, Julianna Solomons, Marc Jamin, Heinrich G. Gottlinger, Dmitri I. Svergun, and Winfried Weissenhorn. Structural Basis for Autoinhibition of ESCRT-III CHMP3. Journal of Molecular Biology, 378(4):818-827, 2008. 9. Anna-Katharina Pfitzner, Vincent Mercier, Xiuyun Jiang, Joachim Moser von Filseck, Buzz Baum, Andela Saric, and Aurelien Roux. An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission. Cell, 182(5):1140-1155.e18, 2020. 10. Marina Vietri, Maja Radulovic, and Harald Stenmark. The many functions of ES CRTs. Nature Reviews Molecular Cell Biology, 21(1):25-42, 2020. 11. Ana Joaquina Jimenez and Franck Perez. Plasma membrane repair: the adaptable cell life-insurance. Current Opinion in Cell Biology, 47:99-107, 2017 12. Matthias Corrotte, Patricia E Almeida, Christina Tam, Thiago Castro-Gomes, Maria Cecilia Fernandes, Bryan A Millis, Mauro Cortez, Heather Miller, Wenxia Song, Timothy K Maugel, and Norma W Andrews. Caveolae internalization repairs wounded cells and muscle fibers. eLife, 2:e00926, 2013. 13. Christina Tam, Vincent Idone, Cecilia Devlin, Maria Cecilia Fernandes, Andrew Flannery, Xingxuan He, Edward Schuchman, Ira Tabas, and Norma W. Andrews. Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair. Journal of Cell Biology, 189(6):1027-1038, 2010. 14. Shiqi Xu, Tyler J. Yang, Suhong Xu, and Yi-Nan Gong. Plasma membrane repair empowers the necrotic survivors as innate immune modulators. Seminars in Cell & Developmental Biology, 156:93-106, 2024. 15. Paul L. McNeil, Steven S. Vogel, Katsuya Miyake, and Mark Terasaki. Patching plasma membrane disruptions with cytoplasmic membrane. Journal of Cell Science, 113(11):1891-1902, 2000. 16. Nicholas R. Davenport, Kevin J. Sonnemann, Kevin W. Eliceiri, and William M. Be ment. Membrane dynamics during cellular wound repair. Molecular Biology of the Cell, 27(14):2272-2285, 2016. 17. Norma W. Andrews and Matthias Corrotte. Plasma membrane repair. Current Biology, 28(8):R392-R397, 2018. 18. Anthony M. Bolger, Marc Lohse, and Bjoern Usadel. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 30(15):2114-2120, 2014. 19. Daehwan Kim, Joseph M Paggi, Chanhee Park, Christopher Bennett, and Steven L Salzberg. Graph-based genome alignment and genotyping with HISAT2 and HISAT genotype. Nature biotechnology, 37(8):907-915, 2019. 20. Yang Liao, Gordon K. Smyth, and Wei Shi. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30(7):923-930, 2014. 21. Michael I Love, Wolfgang Huber, and Simon Anders. Moderated estimation of fold change and dispersion for rna-seq data with deseq2. Genome biology, 15:1-21, 2014. 22. Guangchuang Yu, Li-Gen Wang, Yanyan Han, and Qing-Yu He. clusterProfiler: an R Package for Comparing Biological Themes AmongGeneClusters. OMICS:AJournal of Integrative Biology, 16(5):284-287, 2012. 23. Grace XY Zheng, Jessica M Terry, Phillip Belgrader, Paul Ryvkin, Zachary W Bent, Ryan Wilson, Solongo B Ziraldo, Tobias D Wheeler, Geoff P McDermott, Junjie Zhu, et al. Massively parallel digital transcriptional profiling of single cells. Nature communications, 8(1):14049, 2017. 24. Yuhan Hao, Stephanie Hao, Erica Andersen-Nissen, William M. Mauck III, Shiwei Zheng, AndrewButler, MaddieJ.Lee, AaronJ.Wilk, Charlotte Darby, MichaelZagar, Paul Hoffman, Marlon Stoeckius, Efthymia Papalexi, Eleni P. Mimitou, Jaison Jain, Avi Srivastava, Tim Stuart, Lamar B. Fleming, Bertrand Yeung, Angela J. Rogers, Juliana M. McElrath, Catherine A. Blish, Raphael Gottardo, Peter Smibert, and Rahul Satija. Integrated analysis of multimodal single-cell data. Cell, 2021. 25. Massimo Andreatta, Jesus Corria-Osorio, Soren Muller, Rafael Cubas, George Coukos, and Santiago J. Carmona. Interpretation of T cell states from single-cell transcriptomics data using reference atlases. Nature Communications, 12(1):2965, 2021. 26. Melissa D. Stuchell, Jennifer E. Garrus, Barbara Muller, Kirsten M. Stray, Sanaz Ghaffarian, Rena McKinnon, Hans-Georg Krausslich, Scott G. Morham, and Wes ley I. Sundquist. The Human Endosomal Sorting Complex Required for Trans port (ESCRT-I) and Its Role in HIV-1 Budding*. Journal of Biological Chemistry, 279(34):36059-36071, 2004. 27. Duncan I. Mackie, Natalie R. Nielsen, Matthew Harris, Smriti Singh, Reema B. Davis, Danica Dy, Graham Ladds, and Kathleen M. Caron. RAMP3 determines rapid recycling of atypical chemokine receptor-3 for guided angiogenesis. Proceedings of the National Academy of Sciences, 116(48):24093-24099, 2019. 28. Jennifer M. Bomberger, Narayanan Parameswaran, Carolyn S. Hall, Nambi Aiyar, and William S. Spielman. Novel Function for Receptor Activity-modifying Proteins (RAMPs) in Post-endocytic Receptor Trafficking* . Journal of Biological Chemistry, 280(10):9297-9307, 2005. 29. Dustin A. Ammendolia, William M.Bement, and John H. Brumell. Plasma membrane integrity: implications for health and disease. BMC Biology, 19(1):71, 2021. 30. Catarina Dias and Jesper Nylandsted. Plasma membrane integrity in health and disease: significance and therapeutic potential. Cell Discovery, 7(1):4, 2021. 31. Anita Reddy, Elisabet V Caler, and Norma W Andrews. Plasma membrane repair is mediated by ca2+-regulated exocytosis of lysosomes. Cell, 106(2):157-169, 2001. 32. Jyoti K Jaiswal, Norma W Andrews, and Sanford M Simon. Membrane proximal lysosomes are the major vesicles responsible for calcium-dependent exocytosis in nonsecretory cells. The Journal of cell biology, 159(4):625-635, 2002. 33. Nikita Raj, Lilo Greune, Martin Kahms, Karina Mildner, Rico Franzkoch, Olympia Ekaterini Psathaki, Thomas Zobel, Dagmar Zeuschner, Jurgen Klingauf, and Volker Gerke. Early endosomes act as local exocytosis hubs to repair endothelial membrane damage. Advanced Science, 10(13):2300244, 2023. 34. Tatsuru Togo. Cell membrane disruption stimulates cAMP and Ca2+ signaling to potentiate cell membrane resealing in neighboring cells. Biology Open, 6(12):1814-1819, 2017. 35. Tatsuru Togo. Autocrine purinergic signaling stimulated by cell membrane disruption is involved in both cell membrane repair and adaptive response in mdck cells. Biochemical and biophysical research communications, 511(1):161-164, 2019. 36. Mariana Alves, Edward Beamer, and Tobias Engel. The metabotropic purinergic p2y receptor family as novel drug target in epilepsy. Frontiers in pharmacology, 9:322927, 2018. 37. Kojiro Suda, Yohsuke Moriyama, Nurhanani Razali, Yatzu Chiu, Yumiko Masuk agami, Koutarou Nishimura, Hunter Barbee, Hiroshi Takase, Shinju Sugiyama, Yuta Yamazaki, Yoshikatsu Sato, Tetsuya Higashiyama, Yoshikazu Johmura, Makoto Nakanishi, and Keiko Kono. Plasma membrane damage limits replicative lifespan in yeast and induces premature senescence in human fibroblasts. Nature Aging, 4(3):319-335, 2024. 38. Weihong Wang, Joshua S. Prokopec, Yixin Zhang, Maria Sukhoplyasova, Himaly Shinglot, Man-Tzu Wang, Andreas Linkermann, Jacob Stewart-Ornstein, and Yi-Nan Gong. Sensing plasma membrane pore formation induces chemokine production in survivors of regulated necrosis. Developmental Cell, 57(2):228-245.e6, 2022. 39. Jaclyn N Hayner, Jixiu Shan, and Michael S Kilberg. Regulation of the atf3 gene by a single promoter in response to amino acid availability and endoplasmic reticulum stress in human primary hepatocytes and hepatoma cells. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 1861(2):72-79, 2018.

Claims

A method of enhancing immune response activity of a cell comprising:modifying a plasma membrane damage repair mechanism of the cell.The method according to claim 1, wherein the cell is an immune cell.The method according to claim 2, wherein the immune cell is derived from a stem cell.The method according to claim 3, wherein the stem cell is an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, a lymphoid stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.The method according to any one of claims 1 to 4, wherein the cell is selected from a group including CD4 T cells, CD8 T cells, and NKT (natural killer T) cells.The method according to any one of claims 1 to 5, wherein modifying the plasma membrane damage repair mechanism of the cell includes controlling membrane trafficking.The method according to any one of claims 1 to 6, wherein modifying the plasma membrane damage repair mechanism of the cell includes controlling an expression of one or more genes related to the plasma membrane damage repair mechanism of the cell and / or inhibiting a function of one or more proteins encoded by the one or more genes related to the plasma membrane damage repair mechanism of the cell.The method according to claim 7, wherein the one or more genes related to the plasma membrane damage repair mechanism of the cell includes at least one gene selected from a group including genes encoding components of ESCRT (Endosomal Sorting Complex Required for Transport) complex, genes encoding proteins involved in membrane traffic, and genes encoding extracellular ATP receptors.The method according to claim 7 or 8, wherein the one or more genes related to the plasma membrane damage repair mechanism of the cell includes at least one gene selected from a group including genes encoding components of ESCRT-0 including HRS, STAM1, and STAM2; genes encoding components of ESCRT-1 including VPS23, VPS28, VPS37A, VPS37B, VPS37C, and VPS37D; genes encoding components of ATPase complex including VPS4A, VPS4B, and LIP5; genes encoding components of ESCRT-II including VPS22, VPS25, and VPS36; genes encoding components of ESCRT-III including CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHMP1A, CHMP1B, CHMP5, and CHMP7; genes encoding regulators of membrane trafficking including Rab GTPases, GEF, GAP, GDI, and RAMP3; and genes encoding extracellular ATP receptors including P2X1,P2X2,P2X3,P2X4,P2X5,P2X6,P2X7,P2Y1,P2Y2,P2Y3,P2Y4,P2Y5,P2Y6,P2Y7,P2Y8,P2Y9,P2Y10,P2Y11,P2Y12,P2Y13,and P2Y14; and a gene encoding acid sphingomyelinase (ASM).The method according to any one of claims 7 to 9, wherein controlling an expression of one or more genes related to the plasma membrane damage repair mechanism of the cell includes suppressing the one or more genes related to the plasma membrane damage repair mechanism of the cell.The method according to claim 10, wherein suppressing is performed by a technique selected from a group consisting of RNA interference (RNAi), short interfering RNS (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) and DNA-directed RNA interference (ddRNAi).The method according to claim 10 or 11, wherein suppressing is performed by modifying a genome of the cell using CRISPR, ZFN, TALEN, retroviral transduction or lentiviral transduction.The method according to any one of claims 1 to 9, wherein modifying a plasma membrane damage repair mechanism of the cell includes overexpressing genes related to the plasma membrane damage repair mechanism of the cell.The method according to any one of claims 1 to 9, wherein modifying a plasma membrane damage repair mechanism of the cell includes inhibiting one or more genes or one or more proteins related to the plasma membrane damage repair mechanism of the cell.The method according to any one of claims 1 to 14, wherein the immune response activity is at least one of anti-tumor activity, anti-inflammatory activity, anti-bacterial activity, or anti-viral activity.A method for treating tumor in a subject comprising:producing a cell having enhanced immune response activity according to the method of any one of claims 1 to 15; andadministering an effective amount of the cell into the subject.A cell having enhanced immune response activity and comprising a modified plasma membrane damage repair mechanism.The cell according to claim 17, wherein the cell is an immune cell.The cell according to claim 18, wherein the immune cell is derived from a stem cell.The cell according to claim 19, wherein the stem cell is an adult stem cell, an embryonic stem cell, a cord blood stem cell, a progenitor cell, a bone marrow stem cell, a lymphoid stem cell, an induced pluripotent stem cell, or a hematopoietic stem cell.The cell according to any one of claims 17 to 20, wherein the cell is selected from a group including CD8 T cells, CD4 T cells, and NKT (Natural Killer T) cellsThe cell according to any one of claims 17 to 21, wherein the cell has controlled expression of one or more genes related to the plasma membrane damage repair mechanism.The cell according to any one of claim 22, wherein the one or more genes related to the plasma membrane damage repair mechanism includes at least one of a gene selected from a group including genes encoding components of ESCRT (Endosomal Sorting Complex Required for Transport) complex, genes encoding proteins involved in membrane traffic, and genes encoding extracellular ATP receptors.The cell according to claim 22 or 23, wherein the one or more genes related to the plasma membrane damage repair mechanism includes at least one gene selected from a group including genes encoding components of ESCRT-0 including HRS, STAM1, and STAM2; genes encoding components of ESCRT-1 including VPS23, VPS28, VPS37A, VPS37B, VPS37C, and VPS37D; genes encoding components of ATPase complex including VPS4A, VPS4B, and LIP5; genes encoding components of ESCRT-II including VPS22, VPS25, and VPS36; genes encoding components of ESCRT-III including CHMP2A, CHMP2B, CHMP3, CHMP4A, CHMP4B, CHMP4C, CHMP6, CHMP1A, CHMP1B, CHMP5, and CHMP7; genes encoding regulators of membrane trafficking including Rab GTPases, GEF, GAP, GDI, and RAMP3; and genes encoding extracellular ATP receptors including P2X1,P2X2,P2X3,P2X4,P2X5,P2X6,P2X7,P2Y1,P2Y2,P2Y3,P2Y4,P2Y5,P2Y6,P2Y7,P2Y8,P2Y9,P2Y10,P2Y11,P2Y12,P2Y13,and P2Y14; and a gene encoding acid sphingomyelinase (ASM).The cell according to any one of claims 22 to 24, wherein the cell has suppressed expression of one or more genes related to the plasma membrane damage repair mechanism.The cell according to any one of claims 22 to 24, wherein the cell has overexpression of one or more genes related to the plasma membrane damage repair mechanism.The cell according to any one of claims 22 to 24, wherein the cell has inhibition of one or more genes or one or more proteins related to the plasma membrane damage repair mechanism.The cell according to any one of claims 17 to 27, wherein the immune response activity is at least one of anti-tumor activity, anti-inflammatory activity, anti-bacterial activity, or anti-viral activity.The cell according to any one of claims 17 to 28, wherein the cell expresses CAR (Chimeric Antigen Receptor) or TCR (T Cell Receptor).A cell composition for cell therapy including the cell according to any one of claims 17 to 29.A pharmaceutical composition comprising the cell composition according to claim 30 and pharmaceutically acceptable excipients.

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