Methods and compositions for enhancing efficacy and reducing toxicity of non-transplant CD8-depleted allogeneic donor lymphocyte infusions - Patent Application 20070123333

Abstract

Provided herein are methods and compositions for enhancing efficacy and reducing toxicity of non-transplant CD8-depleted allogeneic donor lymphocyte infusion. The compositions include isolated leukocytes obtained from a donor subject that (i) is mismatched with the recipient subject for at least one human leukocyte antigen (HLA) class II allele mismatch in the donor-to-recipient (graft-versus-host) direction to the recipient subject, or (ii) is mismatched with the recipient for at least one human leukocyte antigen (HLA) class II allele mismatch in the donor-to-recipient (graft-versus-host) direction to the recipient subject and matches with the recipient for at least one human leukocyte antigen (HLA) class II allele, and has CD4+ T cell immunity to an antigen present in the recipient subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial Nos. 63 / 250,061, filed September 29, 2021; 63 / 287,890, filed December 9, 2021; and 63 / 332,616, filed April 19, 2022. The disclosures of the prior applications are deemed to be part of the disclosure of this application in their entireties and are incorporated herein by reference. [Technical field]

[0002] The present disclosure relates generally to allogeneic lymphocyte compositions, and more specifically to the use of such compositions in methods of increasing the efficacy and reducing the toxicity of non-transplant CD8-depleted allogeneic donor lymphocyte infusions. [Background technology]

[0003] Cancer immunotherapy has emerged as a promising cancer treatment. The goal of cancer immunotherapy is to harness the immune system to selectively destroy cancer cells while sparing normal tissue.

[0004] Cytotoxic CD8+ T cells are a major population of immune cells that control and eliminate tumor cells. Zhang et al., (2020), Front. Cell Dev. Biol., 8:17. However, T cells are not as effective against cancer as expected. Due to immune tolerance and immune suppression mechanisms, CD8+ T cells are often suboptimally primed and for these and other reasons become ineffective or enter a dysfunctional state known as T cell exhaustion. T cell exhaustion reduces the ability of cytotoxic CD8+ T cells to kill cancer cells, leading to attenuated effector functions that lead to tumor progression.

[0005] There has been considerable interest in developing methods to revive or replace exhausted T cells to restore anti-tumor immune responses, but developing effective methods to revive exhausted T cells remains a challenge.

[0006] Allogeneic T-cell therapy is a form of cancer immunotherapy in which lymphocytes are harvested from the peripheral blood or bone marrow of healthy donors and infused into cancer patients. This treatment is usually performed in the form of allogeneic stem cell transplantation, where patients undergo extensive immunosuppressive conditioning and are then infused with a stem cell graft containing an unselected population of mature T cells. The aim of allogeneic stem cell transplantation is to obtain sustained engraftment of donor cells, but it is associated with significant toxicities, including graft-versus-host disease (GVHD), an immunological attack against normal tissues, and this therapy has not proven effective in treating solid tumor malignancies. International Application No. PCT / US2013 / 032129 describes an approach called non-transplant donor lymphocyte infusion, in which patients are treated with lymphodepleting chemotherapy followed by infusion of CD8+ cell-depleted peripheral blood cells from healthy, partially or completely human leukocyte antigen (HLA)-mismatched donors. The proposed therapeutic mechanism of mismatched CD8-depleted donor lymphocyte infusion (DLI) is that alloreactive donor CD4+ T cells secrete cytokines and signal through recipient antigen-presenting cells (APCs) to reverse the exhaustion of recipient-derived tumor-specific CD8+ T cells. Although this therapy can induce antitumor responses against hematological malignancies, its efficacy against solid tumors is limited, possibly by reversion of tumor-specific CD8+ T cells to an exhausted state.

[0007] Thus, there remains an unmet need for methods to efficiently and persistently awaken exhausted T cells and induce tumor regression while reducing the risk of persistent engraftment and graft-versus-host disease. Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure relates to allogeneic lymphocyte compositions and methods for increasing the efficacy and reducing the toxicity of non-transplant CD8-depleted allogeneic lymphocyte infusions.

[0009] The present disclosure provides allogeneic lymphocyte compositions that contain CD4+ T cells that have been modified to deplete CD8+ T cells and enhance in vivo anti-tumor activity. Without being committed to a particular mechanism, the modifications include depleting CD4+ T cells from type 1, i.e., T, which secrete interferon gamma (IFNγ) and provide optimal help to the recipient's CD8+ T cells. h 1 It can increase the frequency of CD4+ T cells or make CD4+ T cells more resistant to exhaustion or suppression of activity. The allogeneic lymphocyte composition can provide a source of CD4+ T cell help to reverse T cell exhaustion in the subject, while depletion of donor CD8+ T cells reduces the risk of persistent engraftment and graft-versus-host disease. In addition to helping restore exhausted CD8+ T cells, it can also help restore donor T h 1 cells can reprogram the tumor microenvironment to be immunostimulatory and reverse CD8+ T cell exhaustion while suppressing graft-versus-host disease toxicity.

[0010] The present disclosure relates to a pharmaceutical composition comprising a plurality of isolated leukocytes mismatched with a recipient subject for at least one human leukocyte antigen (HLA) class II allele in a donor-to-recipient (graft-to-host) orientation to the recipient subject. The isolated leukocytes can be obtained from a donor subject. The isolated leukocytes can also be obtained from other sources, such as cell lines or umbilical cord blood. The leukocytes are depleted of CD8+ T cells by about 10-fold or more compared to undepleted leukocytes. The leukocytes can be further modified to suppress Bruton's tyrosine kinase (BTK), interleukin-2-inducible T cell kinase (ITK), delta isoform of phosphoinositide 3 kinase (PI3Kδ), helios, Blimp1, SOCS1, GATA3, IL-10, STAT3, TGF-β receptor II, LAG-3, PD-1, TNF-α, TOX, CD25, foxp3, Ezh2, or a combination thereof.

[0011] The present disclosure also relates to a pharmaceutical composition comprising a plurality of isolated leukocytes obtained from an allogeneic donor subject. The donor's CD4+ T cells are stimulated in vivo or ex vivo with an antigen present in the recipient subject, and the donor's lymphocytes contain at least one HLA class II allele match to the recipient. Alternatively, the donor's CD4+ T cells are stimulated in vivo or ex vivo with an antigen not present in the recipient, and the donor's lymphocytes contain at least one HLA class II allele match to the recipient, and both the antigen and the donor's CD4+ T cells are introduced into the recipient. The plurality of isolated leukocytes can also be obtained from other sources, such as cell lines or umbilical cord blood. The leukocytes are depleted of CD8+ T cells by about 10-fold or more compared to undepleted leukocytes. The leukocytes are engineered to inhibit Bruton's tyrosine kinase (BTK), interleukin 2-inducible T cell kinase (ITK), delta isoform of phosphoinositide 3 kinase (PI3Kδ), helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or combinations thereof.

[0012] The present disclosure also relates to a pharmaceutical composition comprising a plurality of isolated leukocytes obtained from a donor subject, (i) mismatched with the recipient subject for at least one HLA class II allele in a donor-to-recipient (graft-versus-host) direction to the recipient subject, (ii) the donor's CD4+ T cells have been stimulated in vivo or ex vivo with an antigen present in the recipient subject, the donor subject comprising at least one human leukocyte HLA class II allele match to the recipient. Alternatively, the donor's CD4+ T cells (i) mismatched with the recipient subject for at least one HLA class II allele mismatch in a donor-to-recipient (graft-versus-host) direction to the recipient subject, (ii) the donor's CD4+ T cells have been stimulated in vivo or ex vivo with an antigen not present in the recipient subject, the donor subject comprising at least one human leukocyte HLA class II allele match to the recipient, and (iii) both the antigen and the donor's CD4+ T cells are introduced into the recipient. The plurality of isolated leukocytes may also be obtained from other sources, such as cell lines or umbilical cord blood. The leukocytes are depleted of CD8+ T cells by about 10-fold or more compared to undepleted leukocytes. At least a portion of the CD4+ T cells are modified to inhibit the activity of Bruton's tyrosine kinase (BTK), interleukin-2-inducible T cell kinase (ITK), delta isoform of phosphoinositide 3-kinase (PI3Kδ), helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof.

[0013] The present disclosure also relates to a method of producing an allogeneic lymphocyte composition comprising obtaining a peripheral blood cell composition that is (i) mismatched with a recipient subject for at least one HLA class II allele in a donor-to-recipient (graft-versus-host) direction to the recipient subject, and (ii) has CD4+ T cell immunity to an antigen present in the recipient subject, the donor subject comprising at least one HLA class II allele match to the recipient. Alternatively, the donor's CD4+ T cells are (i) mismatched with the recipient subject for at least one HLA class II allele mismatch in a donor-to-recipient (graft-versus-host) direction to the recipient subject, (ii) the donor's CD4+ T cells have been stimulated in vivo or ex vivo with an antigen not present in the recipient subject, the donor subject comprising at least one human leukocyte HLA class II allele match to the recipient, and (iii) both the antigen and the donor's CD4+ T cells are introduced into the recipient. The method further includes isolating leukocytes from the peripheral blood cell composition and depleting the number of CD8+ T cells in the leukocytes at least 10-fold greater than in undepleted leukocytes. The method then includes inhibiting Bruton's tyrosine kinase (BTK), interleukin-2-inducible T cell kinase (ITK), delta isoform of phosphoinositide 3-kinase (PI3Kδ), helios, blimp1, SOCSI, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof. The method further includes inhibiting T of at least a portion of the T cells. h 1 Treatment to promote differentiation into CD4+T cells, CD4+T h The method may further include culturing the leukocytes in vitro. The method may further include culturing the leukocytes ex vivo.

[0014] The method of producing an allogeneic lymphocyte composition further comprises adding one or more cytokines. The one or more cytokines are IL-2, IL-7, IL-12, IL-15, IL-18, IFNγ, or IL-21. The method may further comprise adding one or more antibodies. The one or more antibodies may be an anti-IL3 antibody, an anti-IL-4 antibody, an anti-CD3 antibody, an anti-CD200 antibody, or an anti-CD28 antibody.

[0015] A portion of the T cells in the pharmaceutical composition is h 1 CD4+ T cells can be differentiated into T cells by inhibiting BTK. h 1 CD4+ T cell differentiation can be biased. T cells can be biased toward T h 1. Biased towards CD4+ T cell differentiation.

[0016] T cells are able to upregulate T h 1. Biasing CD4+T cell differentiation, T h Inhibition of Foxp3 can maintain T cells in a differentiated state or render them resistant to exhaustion or functional inhibition. h 1. Biasing CD4+T cell differentiation, T h Inhibition of GATA3 can maintain T cells in a differentiated state or render them resistant to exhaustion or functional inhibition. h 1. Biasing CD4+T cell differentiation, T h STAT3 inhibition can maintain T cells in a differentiated state or render them resistant to exhaustion or functional inhibition. h 1. Biasing CD4+T cell differentiation, T h T cells can be maintained in a differentiated state or made resistant to exhaustion or functional inhibition by inhibition of CD25. h 1. Biasing CD4+T cell differentiation, T h Inhibition of Ezh2 can maintain T cells in a differentiated state or render them resistant to exhaustion or functional inhibition. h 1. Biasing CD4+T cell differentiation, T hInhibition of BTK and ITK can maintain T cells in a differentiated state or render them resistant to exhaustion or functional inhibition. h 1. Biasing CD4+T cell differentiation, T h Inhibition of both BTK and PI3Kδ can maintain T cells in a differentiated state or render them resistant to exhaustion or functional inhibition. h 1. CD4+ T cell differentiation can be skewed toward regulatory T cell differentiation. T cells can be skewed toward regulatory T cell differentiation by inhibition of both ITK and PI3Kδ. h 1. CD4+ T cell differentiation can be skewed toward regulatory T cell differentiation. T cells can be skewed toward regulatory T cell differentiation by inhibiting BTK, ITK, and PI3Kδ. h 1. CD4+ T cell differentiation can be skewed toward regulatory T cell differentiation. T cells can be skewed toward regulatory T cell differentiation by blocking TGF-β receptor II. h 1. Biased towards CD4+ T cell differentiation, T h LAG-3 inhibition can maintain T cells in a differentiated state or render them resistant to exhaustion or functional suppression. h 1. Biased towards CD4+ T cell differentiation, T h Blockade of PD-1 can maintain T cells in a differentiated state or render them resistant to exhaustion or functional suppression. h 1. Biased towards CD4+ T cell differentiation, T h It is possible to maintain the cells in a differentiated state or to render them resistant to exhaustion or functional inhibition.

[0017] BTK, ITK and PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, TGF-β receptor II, LAG-3, PD-1, TNF-α, or Ezh2 can be inhibited by inhibitors or genetic modification. The BTK inhibitor can be acalabrutinib, zanubrutinib, LFM-A13, dasatinib, or AVL-292. In an embodiment, the BTK inhibitor is not ibrutinib. The ITK inhibitor can be aminothiazole, aminobenzimidazole, indole, pyridine, or prn694. The PI3Kδ inhibitor can be idelalisib, copanlisib, duvelisib, umbralisib, ME-4401, RP6503, perifosine, buparlisib, or dactolisib.

[0018] The ITK inhibitor, BTK inhibitor, PI3Kδ inhibitor, Helios inhibitor, Blimp1 inhibitor, SOCS1 inhibitor, TGF-β receptor II inhibitor, LAG-3 inhibitor, PD-1 inhibitor, TNF-α inhibitor, Foxp3 inhibitor, GATA3 inhibitor, IL-10 inhibitor, STAT3 inhibitor, CD25 inhibitor, Ezh2 inhibitor, or TOX inhibitor can be a small molecule, small interfering RNA (siRNA) or short hairpin RNA (shRNA).

[0019] BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, Ezh2, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, or TOX can be inhibited by deleting the BTK gene, ITK gene, PI3Kδ gene, Helios gene, Blimp1 gene, SOCS1 gene, Foxp3 gene, GATA3 gene, IL-10 gene, STAT3 gene, Ezh2 gene, CD25 gene, TGF-β receptor II gene, LAG-3 gene, PD-1 gene, TNF-α gene, or TOX gene from the genome. The BTK gene, ITK gene, PI3Kδ gene, Helios gene, Blimp1 gene, SOCS1 gene, Foxp3 gene, GATA3 gene, IL-10 gene, STAT3 gene, Ezh2 gene, CD25 gene, TGF-β receptor II gene, LAG-3 gene, PD-1 gene, TNF-α gene, or TOX gene can be deleted from the genome using CRISPR or TALEN.

[0020] The differentiation of T cells into T regulatory cells can be attenuated by inhibitors or genetic modification. The differentiation of T cells into regulatory T cells can be attenuated by the inhibition of PI3Kδ, Foxp3, CD25, TGF-β receptor II, LAG-3, PD-1, or Ezh2. PI3Kδ, Foxp3, CD25, TGF-β receptor II, LAG-3, PD-1, or Ezh2 can be attenuated by using PI3Kδ inhibitors, Foxp3 inhibitors, CD25 inhibitors, TGF-β receptor II inhibitors, LAG-3 inhibitors, PD-1 inhibitors, or Ezh2 inhibitors, or by genetic modification. The PI3Kδ inhibitor can be idelalisib, copanlisib, duvelisib, umbralisib, ME-4401, RP6503, perifosine, buparlisib, or dactolisib. The genetic modification may include a deletion of the PI3Kδ gene, the Foxp3 gene, the CD25 gene, or the Ezh2 gene.

[0021] The differentiation of T cells into regulatory T cells or the function of regulatory T cells can be attenuated using small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA) against BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, Ezh2, CD25, TGF-β receptor II, LAG-3, PD-1 or TOX. The differentiation of T cells into regulatory T cells or the function of regulatory T cells can be attenuated by modifying isolated leukocytes from a donor subject to express a dominant negative transforming growth factor βRII receptor.

[0022] The differentiation of T cells into Th2 cells or their function as Th2 cells can be attenuated by inhibitors or genetic modification. The differentiation of T cells into Th2 cells or their function as Th2 cells can be attenuated by inhibition of GATA3. The differentiation of T cells into Th17 cells or their function as Th17 cells can be attenuated by inhibition of STAT3.

[0023] The HLA class II match can be an HLA-DRB1 allele, an HLA-DQB1 allele, or an HLA-DPB1 allele.

[0024] The activation of myeloid cells in the pharmaceutical composition can be inhibited. The activation of myeloid cells can be inhibited by inhibiting BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, TGF-β receptor II, LAG-3, PD-1, TNF-α, or TOX. BTK can be inhibited by at least about 50% in CD4+ T cells compared to the basal activity of BTK. ITK can be inhibited by at least about 50% in CD4+ T cells compared to the basal activity of ITK. PI3Kδ can be inhibited by at least about 50% compared to the basal activity of PI3Kδ. The amount of inhibited ITK, BTK, or PI3Kδ can be measured by Western blot.

[0025] Expression of IFNγ or IL-12 is increased by at least about 10-fold compared to leukocytes that have not been modified to downregulate BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or TOX. Expression of TGFβ or IL-10 is decreased by at least about 10-fold compared to leukocytes that have not been modified to downregulate BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, CD25, Exh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or TOX.

[0026] The amount of IFNγ, IL-12, TGFβ or IL-10 can be measured by intracellular cytokine staining or ELISA.

[0027] Expression of IFNγ or IL-12 may be increased by at least about 10-fold compared to leukocytes that have not been modified to inhibit BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, TOX, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof. Expression of TGFβ or IL-10 may be decreased by at least about 10-fold compared to leukocytes that have not been modified to inhibit BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, TOX, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof.

[0028] The differentiation of T cells into Th2 cells or the function of Th2 cells can be attenuated by inhibitors or genetic modification. The differentiation of T cells into Th2 cells or the function of T cells as Th2 cells can be attenuated by inhibition of GATA3.

[0029] Inhibition of STAT3 can attenuate T cell differentiation or function as Th17 cells. Inhibition of Foxp3 can bias T cells toward Th1 CD4+ T cell differentiation or function. Inhibition of GATA3 can bias T cells toward Th1 CD4+ T cell differentiation or function. Inhibition of STAT3 can bias T cells toward Th1 CD4+ T cell differentiation or function. Inhibition of CD25 can bias T cells toward Th1 CD4+ T cell differentiation or function. Inhibition of Ezh2 can bias T cells toward Th1 CD4+ T cell differentiation or function.

[0030] The present disclosure further relates to a method of treating cancer, comprising administering to a subject in need thereof an effective amount of (i) a lymphocyte depleting agent and (ii) a pharmaceutical composition disclosed herein.

[0031] Disclosed herein is a method for treating cancer, comprising administering to a subject in need thereof effective amounts of (i) a lymphocyte depleting agent, (ii) an inhibitor of NLR family pyrin domain containing 3 (NLRP3), and (iii) a pharmaceutical composition disclosed herein.

[0032] Disclosed herein is a method for treating cancer, comprising administering to a subject in need thereof effective amounts of (i) a lymphocyte depleting agent, (ii) an agent that inhibits the differentiation of T cells into regulatory T cells or an agent that inhibits the function of regulatory T cells, and (iii) a pharmaceutical composition disclosed herein.

[0033] Disclosed herein is a method of enhancing anti-tumor immunity in a subject having cancer, comprising administering to a subject in need thereof an effective amount of (i) a lymphocyte depleting agent and (ii) a pharmaceutical composition disclosed herein.

[0034] The lymphocyte depleting agent can be a cytoreductive agent. The cytoreductive agent can be an alkylating agent, an alkylsulfonate, a nitrosourea, a triazene, an antimetabolite, a pyrimidine analog, a purine analog, a vinca alkaloid, an epipodophyllotoxin, an antibiotic, dibromomannitol, deoxyspergualin, dimethylmyleran, or thiotepa. The alkylating agent can be cyclophosphamide. The purine analog can be fluarabin, cladribine, or pentostatin. The cancer can be a blood cancer. The cancer can be a solid cancer. The blood cancer can be a leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome, or myeloproliferative disease.

[0035] The leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome or myeloproliferative disorder can be non-Hodgkin's lymphoma, chronic lymphocytic leukemia, small lymphocytic lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, acute myeloid leukemia, hairy cell leukemia, AIDS-related lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, mycosis fungoides, primary central nervous system lymphoma, Sézary syndrome, T-cell lymphoma, Waldenström's macroglobulinemia, chronic myelogenous leukemia, chronic myelomonocytic leukemia, polycythemia vera, essential thrombocythemia or idiopathic myelofibrosis.

[0036] The solid tumor can be a sarcoma, carcinoma, neurofibroma, colon cancer, lung cancer, ovarian cancer, pancreatic cancer or breast cancer.

[0037] The methods disclosed herein may further comprise administering to a subject in need thereof an additional therapeutic agent, which is a chemotherapeutic agent, a radiation therapy, an immunotherapy agent, a T cell agonist cytokine, a CAR-T, a CAR-NK, a natural killer cell, a gamma delta T cell, an antibody drug conjugate, an antibody, a bispecific or trispecific T cell or NK cell engager, an immune checkpoint inhibitor, a small molecule inhibitor, or an oncolytic virus therapy or vaccine.

[0038] The antibody can be rituximab, obinutuzumab, ofatumumab, cetuximab, trastuzumab, pertuzumab, brentuximab vedotin, gemtuzumab, trastuzumab emtansine, inotuzumab ozogamicin, glembatumumab vedotin, lorvotuzumab mertansine, cantotuzumab mertansine, or milatuzumab doxorubicin.

[0039] The immune checkpoint inhibitor can be an inhibitor or antibody of PD-L1, PD-1, CTLA-4, LAG-3, TIGIT or TIM-3.

[0040] The small molecule inhibitor can be dasatinib, nilotinib, ponatinib, imatinib, bosutinib, asciminib, lapatinib or vismodegib.

[0041] The pharmaceutical composition may be administered after the lymphocyte depleting agent. [Brief description of the drawings]

[0042] [Figure 1] FIG. 1 is a schematic diagram showing the test protocol MHC haploidentical donor vaccinated against tumor antigen CD4+ T cells carrying a gene deletion. [Diagram 2]FIG. 2A is a graph showing tumor-free survival in MHC haploidentical C57BL / 6xC3H (B6C3 F1) mice several days after tumor inoculation. The graph shows tumor-free survival in TC1-luci-bearing recipients of CD4+ T cells from unvaccinated donors (squares) versus vaccinated donors (circles). FIG. 2B is a graph showing tumor-free survival in MHC haploidentical C57BL / 6xC3H (B6C3 F1) mice several days after tumor inoculation. The graph shows tumor-free survival in pBI-11 vaccinated donor cells nucleofected with Cas9 reagent (squares) versus pBI-11 vaccinated donor cells without Cas9 nucleofection (circles). FIG. 2C is a graph showing tumor-free survival in MHC haploidentical C57BL / 6xC3H (B6C3 F1) mice several days after tumor inoculation. Tumor-free survival rates of CD4+ T cells from unvaccinated donors (triangles), CD4+ T cells from pBI-11 vaccinated and Cas9 nucleofected donors (squares), and pBI-11 vaccinated donor cells with ITK gene deletion (circles) are shown. Figure 2D is a graph showing tumor-free survival rates several days after tumor inoculation in MHC haploidentical C57BL / 6xC3H (B6C3 F1) mice. Tumor-free survival rates of pBI-11 vaccinated donor cells with Foxp3 gene deletion (open squares), transforming growth factor beta receptor type 2 (TGFBR2) gene deletion (open circles), SOCS1 gene deletion (circles), ITK gene deletion (squares), or PD1 gene deletion (triangles) are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] The present disclosure relates to allogeneic lymphocyte compositions and methods for increasing the efficacy and reducing the toxicity of non-transplant CD8-depleted allogeneic lymphocyte infusions.

[0044] The present inventors have developed an allogeneic lymphocyte composition enriched for CD4+Th1 cells and depleted of CD8+T cells. Without being bound by theory or mechanism, the present inventors believe that this allogeneic lymphocyte composition can provide a source of CD4+T cells that can reduce immune suppression and / or increase immune system activation and / or provide signals that reverse CD8+T cell exhaustion upon infusion, restoring endogenous anti-tumor responses while minimizing the risk of sustained engraftment and graft-versus-host disease.

[0045] The allogeneic lymphocyte composition disclosed herein includes a plurality of isolated white blood cells, which can be obtained from a donor subject or another source, such as umbilical cord blood or cell line. The white blood cells can be mismatched with the recipient for at least one human leukocyte antigen (HLA) class II allele in the donor-to-recipient (graft-to-host) direction to the recipient subject. Alternatively, the donor can include at least one HLA class II allele mismatch with the recipient and at least one HLA class II allele match with the recipient in the donor-to-recipient (graft-to-host) direction. The mismatch or match of the HLA class II allele can be found in HLA-DRB1, HLA-DQB1, or HLA-DPB1. For the purpose of the present treatment, low-expressed HLA class II molecules, such as HLA-DPA1, HLA-DQA1, and HLA-DRB3, -DRB4, and -DRB5, may not be considered. In some examples, the allogeneic compositions disclosed herein may include a plurality of isolated white blood cells obtained from a cell line or umbilical cord blood, modified or unmodified to target a specific target. The cell line may be HLA class II allele matched, partially matched, or mismatched to the subject.

[0046] The leukocytes may be depleted of CD8+ T cells by about 10-fold or more compared to undepleted leukocytes. The leukocytes may be further modified to inhibit Bruton's tyrosine kinase (BTK), interleukin 2-inducible T cell kinase (ITK), phosphatidylinositol 3 kinase delta isoform (PI3Kδ), helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof. Without wishing to be bound by theory or mechanism, the inventors believe that inhibition of BTK, ITK, PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof, can promote differentiation of naive CD4+ T cells into a state favorable for helping effector cells of antitumor or antiviral immunity, such as type 1 (Th1) CD4+ T cells, or prevent post-naive CD4+ T cells from converting into cells with suboptimal helper activity for antitumor or antiviral immunity. For example, a portion of T cells can be preferentially differentiated into a subtype of CD4+ T cells (e.g., Th1). Inhibition of BTK, ITK, PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or combinations thereof, can also prevent naive CD4+ T cells from differentiating into states that are not optimal for promoting anti-tumor or anti-viral immunity, such as Th2, Th17, or regulatory T cells, or can prevent CD4+ T cells from becoming exhausted or being suppressed by other cells from mediating anti-tumor or anti-viral activity.

[0047] Certain exemplary and preferred embodiments are described in further detail herein, and the embodiments within this specification should not be construed as limiting the scope of the present disclosure.

[0048] A. Composition of allogeneic lymphocytes The present disclosure relates to an allogeneic lymphocyte composition. The allogeneic lymphocyte composition includes isolated leukocytes obtained from a donor. The donor may include at least one HLA class II allele mismatch to the recipient in the donor-to-recipient (graft-versus-host, or GVH) direction. The HLA class II allele mismatch may be found at HLA-DRB1, HLA-DQB1, or HLA-DPB1.

[0049] Donors who have not been vaccinated against one or more tumor-specific antigens, including neoantigens, generally have a low frequency of tumor-specific CD4+ T cells. When using donors who have not been vaccinated against one or more tumor-specific antigens, the ability of the immune system to restore endogenous antitumor immunity is based on the activity of alloreactive CD4+ T cells, so HLA class II matching between donor and recipient is not required.

[0050] When vaccinating donors against tumor-specific antigens or expanding tumor-specific CD4+ T cells ex vivo, some degree of HLA class II allele matching is necessary because the expanded tumor-specific CD4+ T cells are restricted to the donor's HLA class II molecules and are predicted to be ineffective in providing help in the recipient unless the recipient expresses at least one HLA class II molecule shared by the donor. Among HLA class II molecules, the preferred molecules for sharing are the highly expressed molecules HLA-DRB1>HLA-DPB1>HLA-DQB1>>>HLADRB3,4,5=HLA-DQA1.

[0051] The donor may be partially or completely mismatched in HLA class II alleles, such as HLA-DRB1, HLA-DQB1 and HLA-DPB1, in the donor's anti-recipient (GVH) direction. The donor may be partially or completely mismatched in HLA class II alleles, such as HLA-DRB1, HLA-DQB1 and HLA-DPB1, and completely matched for class I alleles. The donor may be completely mismatched with the unshared HLA of the first-degree relative of the recipient who is a candidate for allogeneic stem cell transplantation.

[0052] The donor's leukocytes may be stimulated in vivo or ex vivo to increase the frequency of CD4+ T cells that proliferate and / or secrete IFNγ in response to tumor or viral antigens compared to unstimulated leukocytes. This stimulation may consist of deliberate in vivo vaccination of the donor against tumor or viral antigens. Alternatively, or in addition, the donor's leukocytes, including CD4+ T cells, may be stimulated ex vivo with antigen-presenting cells (APCs), such as dendritic cells, pulsed with tumor or viral antigens in the presence or absence of cytokines that polarize the CD4+ T cells. The tumor or viral antigens may be present in the recipient. If the donor is immunized or donor cells are stimulated ex vivo with antigen-pulsed APCs, the donor must contain at least one HLA class II allele match to the recipient. The HLA class II allele match may be found in HLA-DRB1, HLA-DQB1, or HLA-DPB1. In embodiments, the immunized donor may have at least one HLA class II allele mismatch to the recipient in the donor-to-recipient (graft-versus-host) direction and at least one HLA class II allele match to the recipient. If the donor's leukocytes have not been stimulated to increase the frequency of tumor-specific or virus-specific CD4+ T cells, the donor's HLA class II molecules HLA-DRB1, HLA-DQB1, and HLA-DPB1 may be completely mismatched to the recipient in the donor's anti-recipient (GVH) direction.

[0053] In embodiments, the allogeneic lymphocyte composition may include leukocytes obtained from a cell line or umbilical cord blood.

[0054] Donor samples can be obtained from cord blood banks.When donor samples are obtained from cord blood banks, desirable samples may contain infrequent and / or rare HLA alleles, because subjects are unlikely to contain serum antibodies against infrequent and / or rare HLA allele types.Exemplary rare alleles include, but are not limited to, A*24:41, B*07:02:28, B*35:03:03, B*39:40N, DRB1*13:23, DRB1*14:111, B*44:16 and DRB1*01:31, C*06:49N, B*37:03N, A*24:312N and A*30:76N.

[0055] In an embodiment, the recipient may not have detectable antibodies that are reactive to the HLA of the donor.Detectable antibodies can be determined using conventional methods known to those skilled in the art.For example, the recipient must not have antibodies that are detectable by complement-dependent cytotoxicity against the HLA molecules of the donor in flow cytometry cross-match assay, or a mean fluorescence intensity (MFI) of 3000 or more in solid-phase immunoassay, because positive results are undesirable.

[0056] The number of natural killer cells in the allogeneic composition may be less than or equal to the number of natural killer cells in the peripheral blood composition.

[0057] In an embodiment, the CD4+ T cells present in the composition are not activated ex vivo.

[0058] The white blood cells present in the allogeneic composition are depleted of CD8+ T cells. The CD8+ T cells can be depleted by any known method. For example, the CD8+ T cells can be depleted using a magnetic bead cell sorter or flow cytometry. The depletion of CD8+ T cells can involve the use of anti-CD8+ antibodies bound to magnetic particles, or anti-CD8+ antibodies plus complement.

[0059] The white blood cells are approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65 about one in, about one in 70, about one in 75, about one in 80, about one in 85, about one in 90, about one in 95, about one in 100, about one in 200, about one in 300, about one in 400, about one in 500, about one in 600, about one in 700, about one in 800, about one in 900, about one in 1,000 or more CD8+ T cells are depleted.

[0060] The leukocytes of the allogeneic lymphocyte compositions disclosed herein are further modified to inhibit the activity of BTK, ITK, or PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof. Without wishing to be bound by theory or mechanism, the inventors believe that suppression of BTK, ITK, PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof, can promote differentiation of naive CD4+ T cells into a state favorable for helping effector cells of antitumor or antiviral immunity, such as type 1 (Th1) CD4+ T cells, or prevent post-naive CD4+ T cells from being converted into cells with suboptimal helper activity for antitumor or antiviral immunity. For example, a portion of T cells can be preferentially differentiated into CD4+ T cell subtypes, such as Th1. Alternatively, differentiation of T cells into another CD4+ T cell subtype (e.g., Th2 or Treg) can be suppressed. Without wishing to be bound by theory or mechanism, the inventors also believe that inhibition of BTK, ITK, PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or combinations thereof in leukocytes (e.g., donor leukocytes) may enhance the efficacy and reduce the toxicity of non-transplant allogeneic donor lymphocyte infusions.

[0061] CD4+ T cells are T lymphocytes that express a T cell receptor that recognizes peptide antigens presented in the context of class II major histocompatibility complex (MHCII) molecules. Tay et. al. (2021), Cancer Gene Therapy, 28:5-17. CD4+ T cells can differentiate into any of several diverse functional subtypes in response to context-dependent signals, and thus provide "help" to appropriate effector immune cells in their primary role as central coordinators of the immune response. CD4+ T cells mediate antitumor immunity primarily by providing support to CD8+ T cell and antibody responses, inducing the tumoricidal capacity of macrophages, secreting effector cytokines such as IFNγ and tumor necrosis factor alpha (TNF-α), and, under certain circumstances, direct cytotoxicity against tumor cells.

[0062] CD4+ T cells can differentiate into Th1 cells expressing IFNγ and TNF-α, Th2 cells expressing IL-4, IL-5 and IL-13; Th9 cells expressing IL-9 and IL-21; Th17 cells expressing IL-17; TFH cells expressing IL-6 and IL-21; and Treg cells expressing TGFβ and IL-10. Tay et. al. (2021), Cancer Gene Therapy, 28:5-17.

[0063] The leukocyte composition disclosed herein may include tumor infiltrating lymphocytes ("TILs"), chimeric receptor T cells ("CAR-Ts"), or T cell receptor ("TCR")-transduced T cells. For example, the leukocyte composition may include TILs. For example, the leukocyte composition may include CAR-T cells. For example, the leukocyte composition may include TCR-transduced T cells.

[0064] As disclosed above, the leukocytes of the allogeneic lymphocyte composition disclosed herein are modified to inhibit BTK, ITK, or PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof. The BTK pathway can be inhibited alone or together with ITK, PI3Kδ, or a combination thereof. The ITK pathway can be inhibited alone or together with BTK, PI3Kδ, or a combination thereof. The PI3Kδ pathway can be inhibited alone or together with BTK, ITK, or a combination thereof. In some examples, each of the BTK, ITK, or PI3Kδ pathways is inhibited. Helios may be inhibited alone or in combination with BTK, ITK or PI3Kδ, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX. Blimp1 may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Helios, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, SOCS1, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX. SOCS1 may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Blimp1, Helios, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX. TGF-β receptor II may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Blimp1, Helios, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, SOCS1, LAG-3, PD-1, TNF-α, or TOX. LAG-3 may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Blimp1, Helios, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, TGF-β receptor II, SOCS1, PD-1, TNF-α, or TOX.PD-1 may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Blimp1, Helios, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, TGF-β receptor II, LAG-3, SOCS1, TNF-α, or TOX. TNF-α may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Blimp1, Helios, Foxp3, GATA3, IL-10, STAT3, CD25, Ezh2, TGF-β receptor II, LAG-3, PD-1, SOCS1, or TOX. Foxp3 may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, STAT3, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, Ezh2, or TOX. GATA3 may be inhibited alone or in combination with BTK, ITK or PI3Kδ, Helios, Blimp1, SOCS1, GATA3, IL-10, STAT3, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, Ezh2 or TOX. GATA3 may be inhibited alone or in combination with BTK, ITK or PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, IL-10, STAT3, CD25, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX. IL-10 may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, TGF-β receptor II, LAG-3, PD-1, TNF-α, STAT3 or TOX. STAT3 may be inhibited alone or in combination with BTK, ITK or PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, Ezh2 or TOX. TOX may be inhibited alone or with BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, IL-10, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, Ezh2 or STAT3.CD25 may be inhibited alone or in combination with BTK, ITK or PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, IL-10, STAT3, GATA3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX. IL-10 may be inhibited alone or in combination with BTK, ITK or PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, STAT3, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX. Ezh2 may be inhibited alone or in combination with BTK, ITK, PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, IL-10, STAT3, CD25, GATA3, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX. IL-10 may be inhibited alone or together with BTK, ITK or PI3Kδ, Helios, Blimp1, SOCS1, Foxp3, GATA3, STAT3, TGF-β receptor II, LAG-3, PD-1, TNF-α or TOX.

[0065] The allogeneic lymphocyte composition may be modified to inhibit BTK. The allogeneic lymphocyte composition may be modified to inhibit ITK. The allogeneic lymphocyte composition may be modified to inhibit PI3Kδ. The allogeneic lymphocyte composition may be modified to inhibit BTK and PI3Kδ. The allogeneic lymphocyte composition may be modified to inhibit BTK and ITK. The allogeneic lymphocyte composition may be modified to inhibit ITK and PI3Kδ. The allogeneic lymphocyte composition may be modified to inhibit BTK, ITK and PI3Kδ. The allogeneic lymphocyte composition may be modified to inhibit helios. The allogeneic lymphocyte composition may be modified to inhibit blimp1. The allogeneic lymphocyte composition may be modified to inhibit SOCS1. The allogeneic lymphocyte composition may be modified to inhibit TGF-β receptor II. The allogeneic lymphocyte composition may be modified to inhibit LAG-3. The allogeneic lymphocyte composition may be modified to inhibit PD-1. The allogeneic lymphocyte composition may be modified to inhibit TNF-α. The allogeneic lymphocyte composition may be modified to inhibit GATA3. The allogeneic lymphocyte composition may be modified to inhibit IL-10. The allogeneic lymphocyte composition may be modified to inhibit STAT3. The allogeneic lymphocyte composition may be modified to inhibit TOX. The allogeneic lymphocyte composition may be modified to inhibit CD25. The allogeneic lymphocyte composition may be modified to inhibit foxp3. The allogeneic lymphocyte composition may be modified to inhibit Ezh2.

[0066] The allogeneic lymphocyte composition can be modified to suppress the differentiation of CD4+ T cells into T regulatory (Treg) cells or inhibit CD4+ Treg function. Inhibition of PI3Kδ can attenuate the differentiation into Treg cells or Treg function. Inhibition of Foxp3 can suppress the differentiation into Treg cells. Inhibition of CD25 can suppress the differentiation into Treg cells. Inhibition of Ezh2 can suppress the differentiation into Treg cells.

[0067] T cell differentiation into Treg cells can be suppressed by engineering isolated leukocytes to express a dominant-negative transforming growth factor-βRII receptor. Liu et al., Nature, 2020, 587(7832):115-120.

[0068] BTK, ITK or PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, foxp3, Ezh2, or a combination thereof can be suppressed (e.g., inhibited) using a pharmacological agent. Alternatively, BTK, ITK or PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, foxp3, Ezh2, or a combination thereof can be inhibited by genetic modification.

[0069] Th1-mediated events, through their effects on innate immune cells such as macrophages and neutrophils, may contribute to immunotherapy toxicity, including cytokine release syndrome (Imus et al., Biol Blood Marrow Transplant, (2019), 25(12):2431-2437) or liver toxicity (Guan et al., Cell Death Dis., (2021), 12(5):431.). Without wishing to be bound by theory or mechanism, inhibition of BTK, ITK, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α or combinations thereof may also reduce or prevent cytokine release syndrome. Inhibition of BTK, ITK, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof can reduce or prevent cytokine release syndrome through inhibition of myeloid cell activation.

[0070] CD4+ T cells in white blood cells can be measured using conventional methodologies known to those skilled in the art, such as flow cytometry. The expression levels of cytokines expressed by CD4+ T cells can be measured by those skilled in the art using conventional methodologies, such as ELISA or intracellular cytokine staining followed by cell surface staining and flow cytometry.

[0071] i.BTK BTK is a member of the Tec family of non-receptor tyrosine kinases, which consists of PH, TH, SH3, SH2 and catalytic domains. BTK is involved in the signal transduction of multiple receptors, including growth factor receptors, cytokine receptors, G protein-coupled receptors, antigen receptors and integrins. BTK then activates many key downstream signaling pathways that control cell migration, adhesion, survival and proliferation.

[0072] BTK can be suppressed (i.e., inhibited) by pharmacological agents or genetic recombination. The pharmacological agent can be an inhibitor of BTK. Suitable inhibitors of BTK include, but are not limited to, acalabrutinib, zanubrutinib, tirabrutinib, evobrutinib, trebrutinib, rilzabrutinib, remibrutinib, tirabrutinib, branebrutinib, orelabrutinib, BIIB091, AC0058, PRN473LFM-A13, dasatinib, GD-4059, or AVL-292. The inhibitor can be a small molecule, a small interfering RNA (siRNA), or a short hairpin RNA (shRNA). In an embodiment, the BTK inhibitor used herein is not ibrutinib. The BTK inhibitor may have a half inhibitory concentration of less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, or less than about 100 nM.

[0073] TK can be inhibited by deleting or "knocking out" the BTK gene from the genome. Techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing, and mutational knockout. Gene silencing can be achieved, for example, using RNA interference, siRNA, or shRNA. Conditional knockout methods can be used to inactivate the BTK gene. Loss-of-function mutations can help suppress gene function by creating mutations in the BTK gene. Gene editing techniques that can be employed to inhibit BTK include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to inhibit BTK. Mutations can be created in one or more protein domains. For example, mutations can be made in the pleckstrin homology domain, the proline-rich TEC homology (TH) domain or the SRC homology domain (SH2 or SH3).

[0074] Inhibition of BTK can decrease the number or frequency of Th2 polarized T cells in white blood cells. Inhibition of BTK can increase the number or frequency of Th1 polarized T cells in white blood cells. Inhibition of BTK can promote the differentiation of T cells into Th1. Inhibition of BTK can decrease the expression of IL-10, IL-4, or IL-13 in white blood cells. Inhibition of BTK can increase the expression of IFN-γ in white blood cells. Inhibition of BTK can increase the expression of IL-12 in white blood cells.

[0075] Inhibition of BTK can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which BTK is not inhibited. Inhibition of BTK can reduce the Th2 population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which BTK is not inhibited.

[0076] Inhibition of BTK can increase the expression of one or more Th1 cell-associated markers. Inhibition of BTK can increase the expression of one or more Th1 cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which BTK is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, inhibition of BTK can increase the expression of IFN-γ. For example, inhibition of BTK can increase IL-2. For example, inhibition of BTK can increase the expression of IL-12.

[0077] Inhibition of BTK can reduce expression of one or more Th2 cell-associated markers. Inhibition of BTK can reduce expression of one or more Th2 cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which BTK is not inhibited. The one or more Th2-associated markers may include CCR3, CCR4, CCR7, CCR8, CD4, CD30, CD81, CD184, CD278, c-maf, CRTH2, Gata-3, GM-CSF, IFN yR, IgD, IL-1R, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, ST2L / T1, Tim-1, or any combination thereof. In particular, the one or more Th2-associated markers may include IL-4, IL-5, IL-6, IL-10, IL-13, IL-15, or any combination thereof. For example, inhibition of BTK can reduce the expression of IL-4. For example, inhibition of BTK can reduce the expression of IL-5. For example, inhibition of BTK can reduce the expression of IL-6. For example, inhibition of BTK can reduce the expression of IL-10. For example, inhibition of BTK can reduce the expression of IL-13, For example, inhibition of BTK can reduce the expression of IL-15.

[0078] Inhibition of BTK can increase the ratio of Th1 T cells to Th2 T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0079] Inhibition of BTK reduces the ratio of Th2 T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be by a factor of 0, about a factor of 150, about a factor of 200, about a factor of 250, about a factor of 300, about a factor of 350, about a factor of 400, about a factor of 450, about a factor of 500, about a factor of 550, about a factor of 600, about a factor of 650, about a factor of 700, about a factor of 750, about a factor of 800, about a factor of 850, about a factor of 900, about a factor of 950, about a factor of 1000 or more.

[0080] Cytokine release syndrome is a known complication of the treatment of hematological malignancies with chimeric antigen receptor-modified (CAR) T cells or with T cell-replete, HLA haploidentical blood or bone marrow transplants. In embodiments, inhibition of BTK can reduce cytokine release syndrome following non-transplanted CD8-depleted lymphocyte infusions.

[0081] Cytokine release syndrome is rated on a scale of 0 to 5. Inhibition of BTK can reduce cytokine release syndrome scores to 0, 1, 2, 3, or 4.

[0082] Inhibition of BTK can reduce IL-1β expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which BTK is not inhibited.

[0083] Inhibition of BTK can reduce the percentage of leukocytes undergoing pyroptosis among total leukocytes by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which BTK is not inhibited.

[0084] The activity of BTK, as measured, for example, by phosphorylation of one of its substrates (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2; PLC-γ2), can be inhibited by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0085] ii.ITK Interleukin-2 (IL-2)-inducible T cell kinase (ITK) is a non-receptor tyrosine kinase highly expressed in T cell lineages and regulates various aspects of T cell development and function, primarily through its functions downstream of the T cell receptor.

[0086] ITK can be suppressed (i.e., inhibited or attenuated) by pharmacological agents or genetic recombination. The pharmacological agent can be an inhibitor of ITK. Suitable inhibitors of ITK include, but are not limited to, aminothiazole, aminobenzimidazole, indole, pyridine or prn694. The inhibitor can be a small molecule, a small interfering RNA (siRNA) or a short hairpin RNA (shRNA). The ITK inhibitor can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0087] ITK can be inhibited by knocking out the ITK gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and mutational knockout. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the ITK gene. Loss-of-function mutations can help suppress gene function by creating mutations in the ITK gene. Gene editing techniques that can be employed to inhibit ITK include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to inhibit ITK. Mutations can be created in one or more protein domains of ITK.

[0088] Inhibition of ITK can reduce the number of Th2 polarized T cells in white blood cells. Inhibition of ITK can increase the number of Th1 polarized T cells in white blood cells. Inhibition of ITK can promote the differentiation of T cells into Th1. Inhibition of ITK can reduce the expression of IL-10, IL-4, or IL-13 in white blood cells. Inhibition of ITK can increase the expression of IFN-γ in white blood cells. Inhibition of ITK can increase the expression of IL-12 in white blood cells.

[0089] Inhibition of ITK can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which ITK is not inhibited. Inhibition of ITK can reduce the Th2 population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which ITK is not inhibited.

[0090] Inhibition of ITK can increase the expression of one or more Th1 cell-associated markers. Inhibition of ITK can increase the expression of one or more Th1 cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more, compared to leukocytes in which ITK is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-a, IFN-gamma, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-gamma, IL-2, IL-12, or any combination thereof. For example, inhibition of ITK may increase the expression of IFN-gamma. For example, inhibition of ITK may increase the expression of IL-2. For example, inhibition of ITK may increase the expression of IL-12.

[0091] Inhibition of ITK can reduce the expression of one or more Th2 cell-associated markers. Inhibition of ITK can reduce the expression of one or more Th2 cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which ITK is not inhibited. The one or more Th2-associated markers may include CCR3, CCR4, CCR7, CCR8, CD4, CD30, CD81, CD184, CD278, c-maf, CRTH2, Gata-3, GM-CSF, IFN yR, IgD, IL-1R, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, ST2L / T1, Tim-1, or any combination thereof. In particular, the one or more Th2-associated markers may include IL-4, IL-6, IL-10, IL-13, IL-15, or any combination thereof. For example, inhibition of ITK can reduce the expression of IL-4. For example, inhibition of ITK can reduce the expression of IL-5. For example, inhibition of ITK can reduce the expression of IL-6. For example, inhibition of ITK can reduce the expression of IL-10. For example, inhibition of ITK can reduce the expression of IL-13. For example, inhibition of ITK can reduce the expression of IL-15.

[0092] Inhibition of ITK can increase the ratio of Th1 T cells to Th2 T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0093] By suppressing ITK, the ratio of Th2 T cells to Th1 T cells can be reduced to about 1 / 1, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 10, about 1 / 15, about 1 / 20, about 1 / 25, about 1 / 30, about 1 / 35, about 1 / 40, about 1 / 45, about 1 / 50, about 1 / 55, about 1 / 60, about 1 / 65, about 1 / 70, about 1 / 75, about 1 / 80, about 1 / 85, about 1 / 90, about 1 / 95, about 1 / 10 The reduction can be by a factor of 0, about a factor of 150, about a factor of 200, about a factor of 250, about a factor of 300, about a factor of 350, about a factor of 400, about a factor of 450, about a factor of 500, about a factor of 550, about a factor of 600, about a factor of 650, about a factor of 700, about a factor of 750, about a factor of 800, about a factor of 850, about a factor of 900, about a factor of 950, about a factor of 1000 or more.

[0094] The activity of ITK can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0095] iii.PI3Kδ PI3Kδ can be suppressed (i.e., inhibited or attenuated) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of PI3Kδ. Suitable inhibitors of PI3Kδ include, but are not limited to, idelalisib, copanlisib, duvelisib, umbralisib, ME-4401, RP6503, perifosine, buparlisib or dactolisib. The inhibitor can be a small molecule, a small interfering RNA (siRNA) or a short hairpin RNA (shRNA). The PI3Kδ inhibitor can have a half-maximal inhibitory concentration of less than about 1000 nM, less than about 900 nM, less than about 800 nM, less than about 700 nM, less than about 600 nM, less than about 500 nM, less than about 400 nM, less than about 300 nM, less than about 200 nM, or less than about 100 nM.

[0096] PI3Kδ can be suppressed by knocking out the PI3Kδ gene from the genome. The techniques of knocking out genes are known to those skilled in the art. Known gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. For example, the RNA interference, siRNA or shRNA can be against CD25, foxp3 or Ezh2. Conditional knockout methods can be used to inactivate the PI3Kδ gene. Loss-of-function mutations can help suppress gene function by creating mutations in the PI3Kδ gene. Gene editing techniques that can be employed to suppress PI3Kδ include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can be used to inhibit PI3Kδ, or mutations can be made in one or more protein domains.

[0097] Genetic modification of PI3Kδ can include deleting the genes for CD25, foxp3, or Ezh2.

[0098] Inhibition of PI3Kδ suppresses the expression of CD4+CD25+foxp3+ regulatory T cells (T reg Inhibition of PI3Kδ can reduce the number or function of T h Inhibition of PI3Kδ increases the number of T1-polarized T cells. h 1. Inhibition of PI3Kδ can reduce the expression of TGFβ and IL-10 in leukocytes. Inhibition of PI3Kδ can increase the expression of IFN-γ in leukocytes. Inhibition of PI3Kδ can increase the expression of IL-12 in leukocytes.

[0099] Inhibition of PI3Kδ increased T h Inhibition of PI3Kδ can increase the population of T.sup.1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which PI3Kδ is not inhibited. reg The population of cells can be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more.

[0100] Inhibition of PI3Kδ inhibits one or more T h Inhibition of PI3Kδ can increase the expression of one or more T cell-associated markers compared to leukocytes in which PI3Kδ is not inhibited. hThe expression of one or more T cell-associated markers may be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more. h 1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-α, TRANCE, sCD40L, or any combination thereof. In particular, one or more T h 1-related markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, inhibition of PI3Kδ can increase the expression of IFN-γ. For example, inhibition of PI3Kδ can increase the expression of IL-2. For example, inhibition of PI3Kδ can increase the expression of IL-12.

[0101] Inhibition of PI3Kδ inhibits one or more T reg Inhibition of PI3Kδ can reduce the expression of T cell-associated markers. reg Compared with unsuppressed leukocytes, one or more T reg The expression of cell-associated markers may be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more. reg Related markers may include TGFβ or IL-10 or a combination thereof.For example, suppression of PI3Kδ can reduce the expression of TGFβ.For example, suppression of PI3Kδ can reduce the expression of IL-10.

[0102] Inhibition of PI3Kδ inhibits T reg The ratio of Th1 T cells to T cells can be increased by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0103] Inhibition of PI3Kδ inhibits T vs Th1 T cells reg The ratio of T cells is set to about 1 / 1, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 10, about 1 / 15, about 1 / 20, about 1 / 25, about 1 / 30, about 1 / 35, about 1 / 40, about 1 / 45, about 1 / 50, about 1 / 55, about 1 / 60, about 1 / 65, about 1 / 70, about 1 / 75, about 1 / 80, about 1 / 85, about 1 / 90, about 1 / 95, about 1 / 10 ... The reduction can be by a factor of 0, about a factor of 150, about a factor of 200, about a factor of 250, about a factor of 300, about a factor of 350, about a factor of 400, about a factor of 450, about a factor of 500, about a factor of 550, about a factor of 600, about a factor of 650, about a factor of 700, about a factor of 750, about a factor of 800, about a factor of 850, about a factor of 900, about a factor of 950, about a factor of 1000 or more.

[0104] The activity of PI3Kδ can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0105] iv.GATA3 GATA3 can be suppressed (i.e., inhibited or attenuated) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of GATA3. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The GATA3 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0106] GATA3 can be suppressed by knocking out the GATA3 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the GATA3 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the GATA3 gene. Gene editing techniques that can be employed to suppress GATA3 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress GATA3. Mutations can be created in one or more protein domains.

[0107] Inhibition of GATA3 reduces T h Inhibition of GATA3 increases the number of T1-polarized T cells. hInhibition of GATA3 can reduce the expression of IL-10, IL-4, or IL-13 in leukocytes. Inhibition of GATA3 can increase the expression of IFN-γ in leukocytes. Inhibition of GATA3 can increase the expression of IL-12 in leukocytes. Inhibition of GATA3 can promote the differentiation of T h The number of bipolarized T cells can be reduced.

[0108] Inhibition of GATA3 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which GATA3 is not inhibited. Inhibition of GATA3 can reduce the Th2 population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which GATA3 is not inhibited.

[0109] Inhibition of GATA3 can increase expression of one or more relevant Th1 cell associated markers. Inhibition of GATA3 can increase expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which GATA3 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, inhibition of GATA3 may increase the expression of IFN-γ. For example, inhibition of GATA3 may increase IL-2. For example, inhibition of GATA3 may increase the expression of IL-12.

[0110] Inhibition of GATA3 can reduce expression of one or more relevant Th2 cell associated markers. Inhibition of GATA3 can reduce expression of one or more Th2 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which GATA3 is not inhibited. The one or more Th2-associated markers may include CCR3, CCR4, CCR7, CCR8, CD4, CD30, CD81, CD184, CD278, c-maf, CRTH2, Gata-3, GM-CSF, IFN yR, IgD, IL-1R, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, ST2L / T1, Tim-1, or any combination thereof. In particular, the one or more Th2-associated markers may include IL-4, IL-5, IL-6, IL-10, IL-13, IL-15, or any combination thereof. For example, inhibition of GATA3 can reduce the expression of IL-4. For example, inhibition of GATA3 can reduce the expression of IL-5. For example, inhibition of GATA3 can reduce the expression of IL-6. For example, inhibition of GATA3 can reduce the expression of IL-10. For example, inhibition of GATA3 can reduce the expression of IL-13, For example, inhibition of GATA3 can reduce the expression of IL-15.

[0111] Inhibition of GATA3 can increase the ratio of Th1 T cells to Th2 T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0112] Inhibition of GATA3 reduces the ratio of Th2 T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0113] Cytokine release syndrome is a known complication of the treatment of hematological malignancies with chimeric antigen receptor modified (CAR) T cells or with T cell repletion, HLA haploidentical blood or bone marrow transplantation. In embodiments, inhibition of GATA3 can reduce cytokine release syndrome after non-transplant CD8-depleted donor lymphocyte transfusion. Inhibition of GATA3 can reduce cytokine release syndrome by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more compared to the activity without inhibition of GATA3.

[0114] Inhibition of GATA3 can reduce IL-1β expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which GATA3 is not inhibited.

[0115] Inhibition of GATA3 can reduce the occurrence of pyroptosis by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which GATA3 is not inhibited.

[0116] The activity of GATA3 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0117] v.STAT3 STAT3 can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be STAT3 inhibitors. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The STAT3 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0118] STAT3 can be suppressed by knocking out the STAT3 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the STAT3 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the STAT3 gene. Gene editing techniques that can be employed to suppress STAT3 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress STAT3. Mutations can be created in one or more protein domains.

[0119] Inhibition of STAT3 can increase the number of Th1 polarized T cells in leukocytes. Inhibition of STAT3 can promote the differentiation of T cells into Th1. Inhibition of STAT3 can decrease the expression of IL-10, IL-4, or IL-13 in leukocytes. Inhibition of STAT3 can increase the expression of IFN-γ in leukocytes. Inhibition of STAT3 can increase the expression of IL-12 in leukocytes. Inhibition of STAT3 can decrease the number of Th17 polarized T cells or Tfh polarized T cells in leukocytes.

[0120] Inhibition of STAT3 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which STAT3 is not inhibited. Inhibition of STAT3 can reduce the population of Th17 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which STAT3 is not inhibited. Inhibition of STAT3 can reduce the population of Tfh cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which STAT3 is not inhibited.

[0121] Inhibition of STAT3 can increase expression of one or more relevant Th1 cell associated markers Inhibition of STAT3 can increase expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which STAT3 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, inhibition of STAT3 may increase the expression of IFN-γ. For example, inhibition of STAT3 may increase the expression of IL-2. For example, inhibition of STAT3 may increase the expression of IL-12.

[0122] Inhibition of STAT3 can reduce the expression of one or more Treg cell-associated markers. Inhibition of STAT3 can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which Treg is not suppressed. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, inhibition of STAT3 can reduce TGFβ expression. For example, inhibition of STAT3 can reduce IL-10 expression.

[0123] Inhibition of STAT3 can reduce the expression of one or more Tfh cell-associated markers. Inhibition of STAT3 can reduce the expression of one or more Tfh cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which Tfh is not inhibited. The one or more Tfh-associated markers can include IL-21, IL-4, or any combination thereof. For example, inhibition of STAT3 can reduce IL-21 expression. For example, inhibition of STAT3 can reduce IL-4 expression.

[0124] Inhibition of STAT3 inhibits T regThe ratio of Th1 T cells to T cells can be increased by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0125] Inhibition of STAT3 inhibits T cell proliferation against Th1 T cells. reg Set the ratio of T cells to about 1 / 1, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 10, about 1 / 15, about 1 / 20, about 1 / 25, about 1 / 30, about 1 / 35, about 1 / 40, about 1 / 45, about 1 / 50, about 1 / 55, about 1 / 60, about 1 / 65, about 1 / 70, about 1 / 75, about 1 / 80, about 1 / 85, about 1 / 90, about 1 / 95, about 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0126] Inhibition of STAT3 can increase the ratio of Th1 T cells to Tfh T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0127] Inhibition of STAT3 reduces the ratio of Tfh T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be by a factor of 0, about a factor of 150, about a factor of 200, about a factor of 250, about a factor of 300, about a factor of 350, about a factor of 400, about a factor of 450, about a factor of 500, about a factor of 550, about a factor of 600, about a factor of 650, about a factor of 700, about a factor of 750, about a factor of 800, about a factor of 850, about a factor of 900, about a factor of 950, about a factor of 1000 or more.

[0128] STAT3 activity can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0129] vi.fox3 foxp3 can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of foxp3. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The foxp3 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0130] foxp3 can be suppressed by knocking out the FOXP3 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the FOXP3 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the FOXP3 gene. Gene editing techniques that can be employed to suppress foxp3 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress foxp3. Mutations can be created in one or more protein domains.

[0131] Inhibition of foxp3 can increase the number of Th1 polarized T cells in white blood cells. Inhibition of foxp3 can promote the differentiation of T cells into Th1. Inhibition of foxp3 can decrease the expression of IL-10, IL-4, or IL-13 in white blood cells. Inhibition of foxp3 can increase the expression of IFN-γ in white blood cells. Inhibition of foxp3 can increase the expression of IL-12 in white blood cells. Inhibition of foxp3 can decrease the number of Treg polarized T cells in white blood cells.

[0132] Inhibition of foxp3 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which foxp3 is not inhibited. Inhibition of foxp3 can reduce the population of Treg cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which foxp3 is not inhibited.

[0133] Inhibition of foxp3 can increase the expression of one or more relevant Th1 cell associated markers. Inhibition of foxp3 can increase the expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which foxp3 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of foxp3 can increase the expression of IFN-γ. For example, suppression of foxp3 can increase IL-2. For example, suppression of foxp3 can increase the expression of IL-12.

[0134] Inhibition of foxp3 can reduce the expression of one or more relevant Treg cell-associated markers. Inhibition of foxp3 can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which foxp3 is not inhibited. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, inhibition of foxp3 can reduce the expression of TGFβ. For example, inhibition of foxp3 can reduce the expression of IL-10.

[0135] Inhibition of foxp3 can increase the ratio of Th1 T cells to Treg T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0136] Inhibition of foxp3 reduces the ratio of Treg T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0137] The activity of foxp3 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0138] vii.CD25 CD25 can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of CD25. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The CD25 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0139] CD25 can be suppressed by knocking out the CD25 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the CD25 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the CD25 gene. Gene editing techniques that can be employed to suppress CD25 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress CD25. Mutations can be created in one or more protein domains.

[0140] Suppression of CD25 inhibits T h CD25 inhibition increases the number of T cells polarized by T h Inhibition of CD25 can reduce the expression of IL-10, IL-4, or IL-13 in leukocytes. Inhibition of CD25 can increase the expression of IFN-γ in leukocytes. Inhibition of CD25 can increase the expression of IL-12 in leukocytes. Inhibition of CD25 can promote the differentiation of T reg The number of polarized T cells can be reduced.

[0141] CD25 inhibition increased T cell proliferation compared with non-CD25-inhibited leukocytes. hThe population of T 1 cells can be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more, as compared to leukocytes in which CD25 is not suppressed. reg The population of cells can be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more.

[0142] Inhibition of CD25 inhibits one or more of the relevant T h CD25 inhibition can increase the expression of one or more T-cell associated markers compared to non-CD25-inhibited leukocytes. h The expression of one or more T cell-associated markers may be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more. hThe Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of CD25 can increase the expression of IFN-γ. For example, suppression of CD25 can increase IL-2. For example, suppression of CD25 can increase the expression of IL-12.

[0143] Inhibition of CD25 inhibits one or more of the relevant T reg CD25 inhibition can reduce the expression of one or more T cell-associated markers compared to non-CD25-suppressed leukocytes. reg The expression of cell-associated markers may be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more. reg Related markers can include TGFβ or IL-10 or any combination thereof.For example, suppression of CD25 can reduce the expression of TGFβ.For example, suppression of CD25 can reduce the expression of IL-10.

[0144] CD25 inhibition inhibits T reg T for T cells hThe proportion of 1 T cells can be increased by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0145] CD25 inhibition inhibits T h 1 T against T cells reg The ratio of T cells is set to about 1 / 1, about 1 / 2, about 1 / 3, about 1 / 4, about 1 / 5, about 1 / 10, about 1 / 15, about 1 / 20, about 1 / 25, about 1 / 30, about 1 / 35, about 1 / 40, about 1 / 45, about 1 / 50, about 1 / 55, about 1 / 60, about 1 / 65, about 1 / 70, about 1 / 75, about 1 / 80, about 1 / 85, about 1 / 90, about 1 / 95, about 1 / 10 ... The reduction can be by a factor of 0, about a factor of 150, about a factor of 200, about a factor of 250, about a factor of 300, about a factor of 350, about a factor of 400, about a factor of 450, about a factor of 500, about a factor of 550, about a factor of 600, about a factor of 650, about a factor of 700, about a factor of 750, about a factor of 800, about a factor of 850, about a factor of 900, about a factor of 950, about a factor of 1000 or more.

[0146] The activity of CD25 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0147] viii.Ezh2 Ezh2 can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of Ezh2. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The Ezh2 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0148] Ezh2 can be suppressed by knocking out the Ezh2 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing, and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA, or shRNA. Conditional knockout methods can be used to inactivate the Ezh2 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the Ezh2 gene. Gene editing techniques that can be employed to suppress Ezh2 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress Ezh2. Mutations can be created in one or more protein domains.

[0149] Inhibition of Ezh2 reduces T expression in leukocytes h The number of T1-polarized T cells can be increased. Inhibition of Ezh2 increases the number of T1-polarized T cells. hInhibition of Ezh2 can reduce the expression of IL-10, IL-4, and IL-13 in leukocytes. Inhibition of Ezh2 can increase the expression of IFN-γ in leukocytes. Inhibition of Ezh2 can increase the expression of IL-12 in leukocytes. Inhibition of Ezh2 can promote the differentiation of T reg The number of polarized T cells can be reduced.

[0150] Inhibition of Ezh2 reduced T h Inhibition of Ezh2 can increase the population of T-cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Ezh2 is not inhibited. reg The population of cells can be reduced by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more.

[0151] Suppression of Ezh2 inhibits one or more of the associated T h Inhibition of Ezh2 can increase the expression of one or more T cell-associated markers compared to leukocytes in which Ezh2 is not inhibited. hExpression of one cell-associated markers can be increased by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of Ezh2 may increase the expression of IFN-γ. For example, suppression of Ezh2 may increase IL-2. For example, suppression of Ezh2 may increase the expression of Ezh2.

[0152] Suppression of Ezh2 can reduce the expression of one or more relevant Treg cell-associated markers. Suppression of Ezh2 can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Ezh2 is not suppressed. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, suppression of Ezh2 can reduce the expression of TGFβ. For example, suppression of Ezh2 can reduce the expression of IL-10.

[0153] Suppression of Ezh2 inhibits T regThe ratio of Th1 T cells to T cells can be increased by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0154] Inhibition of Ezh2 reduces the ratio of Treg T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0155] The activity of Ezh2 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0156] ix. Helios Helios can be suppressed (i.e., inhibited) by pharmacological agents or genetic recombination. The pharmacological agents can be inhibitors of Helios. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The inhibitors of Helios can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0157] Helios can be suppressed by knocking out the IKZF2 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the IKZF2 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the IKZF2 gene. Gene editing techniques that can be employed to suppress Helios include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress Helios. Mutations can be created in one or more protein domains.

[0158] Inhibition of Helios reduces T h Helios suppression increases the number of T cells polarized in the T hInhibition of Helios can reduce the expression of IL-10, IL-4, or IL-13 in leukocytes. Inhibition of Helios can increase the expression of IFN-γ in leukocytes. Inhibition of Helios can increase the expression of IL-12 in leukocytes. Inhibition of Helios can promote the differentiation of T reg The number of polarized T cells can be reduced.

[0159] Inhibition of Helios can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which Helios is not inhibited. Inhibition of Helios can reduce the Treg population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Helios is not inhibited.

[0160] Inhibition of Helios can increase expression of one or more relevant Th1 cell associated markers. Inhibition of Helios can increase expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Helios is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, Granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, inhibition of Helios may increase the expression of IFN-γ. For example, inhibition of Helios may increase IL-2. For example, inhibition of Helios may increase the expression of IL-12.

[0161] Inhibition of Helios can reduce the expression of one or more relevant Treg cell-associated markers. Inhibition of Helios can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which Helios is not inhibited. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, inhibition of Helios can reduce the expression of TGFβ. For example, inhibition of Helios can reduce the expression of IL-10.

[0162] Inhibition of Helios can increase the ratio of Th1 T cells to Treg T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0163] Inhibition of Helios reduces the ratio of Treg T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0164] Inhibition of Helios can reduce CD8+ T cell exhaustion. Inhibition of Helios can reduce CD8+ T cell exhaustion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more compared to activity without inhibition of Helios.

[0165] Inhibition of Helios can increase the population of CD8+ T cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Helios is not inhibited.

[0166] The activity of Helios can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0167] x.Blimp1 Blimp1 can be suppressed (i.e. inhibited) by a pharmacological agent or genetic modification. The pharmacological agent can be an inhibitor of Blimp1. The Blimp1 inhibitor can be a small molecule, a small interfering RNA (siRNA) or a short hairpin RNA (shRNA). The Blimp1 inhibitor can have a half maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0168] Blimp1 can be suppressed by knocking out the PRDM1 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and mutational knockout. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the PRDM1 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the PRDM1 gene. Gene editing techniques that can be employed to suppress Blimp1 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress Blimp1. Mutations can be created in one or more protein domains.

[0169] Inhibition of Blimp1 can increase Th1 polarized T cells in leukocytes. Inhibition of Blimp1 can promote differentiation of T cells into Th1. Inhibition of Blimp1 can decrease the expression of IL-10, IL-4, and IL-13 in leukocytes. Inhibition of Blimp1 can increase the expression of IFN-γ in leukocytes. Inhibition of Blimp1 can increase the expression of IL-12 in leukocytes. Inhibition of Blimp1 can decrease the number of Treg polarized T cells in leukocytes.

[0170] Inhibition of Blimp1 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Blimp1 is not inhibited. Inhibition of Blimp1 can reduce the Treg population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Blimp1 is not inhibited.

[0171] Inhibition of Blimp1 can increase the expression of one or more relevant Th1 cell associated markers. Inhibition of Blimp1 can increase the expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Blimp1 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, Granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, inhibition of Blimp1 may increase the expression of IFN-γ. For example, inhibition of Blimp1 may increase IL-2. For example, inhibition of Blimp1 may increase the expression of IL-12.

[0172] Inhibition of Blimp1 can reduce the expression of one or more relevant Treg cell-associated markers. Inhibition of Blimp1 can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which Blimp1 is not inhibited. The one or more Treg-associated markers can include TGFβ or IL-10 or a combination thereof. For example, inhibition of Blimp1 can reduce the expression of TGFβ. For example, inhibition of Blimp1 can reduce the expression of IL-10.

[0173] Inhibition of Blimp1 can increase the ratio of Th1 T cells to Treg T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0174] Inhibition of Blimp1 reduces the ratio of Treg T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0175] Inhibition of Blimp1 can reduce CD8+ T cell exhaustion. Inhibition of Blimp1 can reduce CD8+ T cell exhaustion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more compared to activity in the absence of Blimp1 inhibition.

[0176] Inhibition of Blimp1 can increase the population of CD8+ T cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which Blimp is not inhibited.

[0177] The activity of Blimp1 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0178] xi.TOX TOX (Thymocyte selection-related HMG BOX) can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of TOX. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The inhibitors of TOX can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0179] TOX can be suppressed by knocking out the TOX gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the TOX gene. Loss-of-function mutations can help suppress gene function by creating mutations in the TOX gene. Gene editing techniques that can be employed to suppress TOX include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress TOX. Mutations can be created in one or more protein domains.

[0180] Inhibition of TOX can reduce CD8+ T cell exhaustion. Inhibition of TOX can reduce CD8+ T cell exhaustion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more compared to activity without inhibition of TOX.

[0181] Inhibition of TOX can increase the population of CD8+ T cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TOX is not inhibited.

[0182] xii.IL-10 IL-10 can be suppressed (i.e., inhibited) by pharmacological agents or genetic recombination. The pharmacological agents can be inhibitors of IL-10. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The inhibitors of IL-10 can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0183] IL-10 can be suppressed by knocking out the IL-10 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the IL-10 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the IL-10 gene. Gene editing techniques that can be employed to suppress IL-10 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress IL-10. Mutations can be created in one or more of the protein domains.

[0184] Suppression of IL-10 can decrease the number of Th2 polarized T cells in white blood cells. Suppression of IL-10 can increase the number of Th1 polarized T cells in white blood cells. Suppression of IL-10 can promote the differentiation of T cells into Th1. Suppression of IL-10 can decrease the expression of IL-10, IL-4, or IL-13 in white blood cells. Suppression of IL-10 can increase the expression of IFN-γ in white blood cells. Suppression of IL-10 can increase the expression of IL-12 in white blood cells.

[0185] Inhibition of IL-10 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited. Inhibition of IL-10 can reduce the Th2 population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0186] Inhibition of IL-10 can increase expression of one or more relevant Th1 cell associated markers. Inhibition of IL-10 can increase expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of IL-10 may increase the expression of IFN-γ. For example, suppression of IL-10 may increase IL-2. For example, suppression of IL-10 may increase the expression of IL-12.

[0187] Inhibition of IL-10 can reduce expression of one or more relevant Th2 cell associated markers. Inhibition of IL-10 can reduce expression of one or more Th2 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited. The one or more Th2-associated markers may include CCR3, CCR4, CCR7, CCR8, CD4, CD30, CD81, CD184, CD278, c-maf, CRTH2, Gata-3, GM-CSF, IFN yR, IgD, IL-1R, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-15, ST2L / T1, Tim-1, or any combination thereof. In particular, the one or more Th2-associated markers may include IL-4, IL-6, IL-10, IL-13, IL-15, or any combination thereof. For example, suppression of IL-10 can reduce the expression of IL-4. For example, suppression of IL-10 can reduce the expression of IL-5. For example, suppression of IL-10 can reduce the expression of IL-6. For example, suppression of IL-10 can reduce the expression of IL-10. For example, suppression of IL-10 can reduce the expression of IL-13. For example, suppression of IL-10 can reduce the expression of IL-15.

[0188] Inhibition of IL-10 can increase the ratio of Th1 T cells to Th2 T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold, or more.

[0189] Suppression of IL-10 inhibits Th2 T cells against Th1 T cells The proportion of T cells can be increased by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0190] The activity of IL-10 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0191] Suppression of IL-10 can promote inflammation and the production of inflammatory cytokines in other T cells, and can increase the expression of IL-1, IL-12, IL-18, TNF-α, IFN-γ, or GM-CSF.

[0192] Inhibition of IL-10 can increase IL-1 expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0193] Inhibition of IL-10 can increase IL-12 expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0194] Inhibition of IL-10 can increase IL-18 expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0195] Inhibition of IL-10 can increase TFN-α expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0196] Inhibition of IL-10 can increase expression of IL-1 by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0197] Inhibition of IL-10 can increase IL-18 expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0198] Inhibition of IL-10 can increase expression of GM-CFS by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which IL-10 is not inhibited.

[0199] xiii.SOCS1 SOCS1 can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of SOCS1. The SOCS1 inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The SOCS1 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, or less than about 100nM.

[0200] SOCS1 can be suppressed by knocking out the SOCS1 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the SOCS1 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the SOCS1 gene. Gene editing techniques that can be employed to suppress SOCS1 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress SOCS1. Mutations can be created in one or more protein domains.

[0201] Inhibition of SOCS1 can increase the number of Th1 polarized T cells in white blood cells. Inhibition of SOCS1 can promote the differentiation of T cells into Th1. Inhibition of SOCS1 can decrease the expression of IL-10, IL-4, or IL-13 in white blood cells. Inhibition of SOCS1 can increase the expression of IFN-γ in white blood cells. Inhibition of SOCS1 can increase the expression of IL-12 in white blood cells. Inhibition of SOCS1 can decrease the number of Treg polarized T cells in white blood cells.

[0202] Inhibition of SOCS1 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which SOCS1 is not inhibited. Inhibition of SOCS1 can reduce the Treg population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which SOCS1 is not inhibited.

[0203] Inhibition of SOCS1 can increase the expression of one or more relevant Th1 cell-associated markers. Inhibition of SOCS1 can increase the expression of one or more Th1 cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which SOCS1 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, inhibition of SOCS1 may increase the expression of IFN-γ. For example, inhibition of SOCS1 may increase IL-2. For example, inhibition of SOCS1 may increase the expression of IL-12.

[0204] Suppression of SOCS1 can reduce the expression of one or more relevant Treg cell-associated markers. Suppression of SOCS1 can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which SOCS1 is not suppressed. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, suppression of SOCS1 can reduce TGFβ expression. For example, suppression of SOCS1 can reduce IL-10 expression.

[0205] Inhibition of SOCS1 can increase the ratio of Th1 T cells to Treg T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0206] Inhibition of SOCS1 reduces the ratio of Treg T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0207] Inhibition of SOCS1 can reduce CD8+ T cell exhaustion. Inhibition of SOCS1 can reduce CD8+ T cell exhaustion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more, compared to activity when SOCS1 is not inhibited.

[0208] Inhibition of SOCS1 can increase the population of CD8+ T cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which SOCS1 is not inhibited.

[0209] The activity of SOCS1 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0210] xiv.PD-1 PD-1 can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of PD-1. The PD-1 inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The PD-1 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0211] PD-1 can be suppressed by knocking out the PD-1 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the PD-1 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the PD-1 gene. Gene editing techniques that can be employed to suppress PD-1 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress PD-1. Mutations can be created in one or more protein domains.

[0212] Suppression of PD-1 can increase the number of Th1 polarized T cells in white blood cells. Suppression of PD-1 can promote the differentiation of T cells into Th1. Suppression of PD-1 can decrease the expression of IL-10, IL-4 or IL-13 in white blood cells. Suppression of PD-1 can increase the expression of IFN-γ in white blood cells. Suppression of PD-1 can increase the expression of IL-12 in white blood cells. Suppression of PD-1 can decrease the number of Treg polarized T cells in white blood cells.

[0213] Inhibition of PD-1 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which PD-1 is not inhibited. Inhibition of PD-1 can reduce the Treg population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which PD-1 is not inhibited.

[0214] Inhibition of PD-1 can increase expression of one or more relevant Th1 cell associated markers. Inhibition of PD-1 can increase expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which PD-1 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of PD-1 may increase the expression of IFN-γ. For example, suppression of PD-1 may increase IL-2. For example, suppression of PD-1 may increase the expression of IL-12.

[0215] Inhibition of PD-1 can reduce the expression of one or more relevant Treg cell-associated markers. Inhibition of PD-1 can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which PD-1 is not inhibited. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, inhibition of PD-1 can reduce the expression of TGFβ. For example, inhibition of PD-1 can reduce the expression of IL-10.

[0216] Inhibition of PD-1 can increase the ratio of Th1 T cells to Treg T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0217] Inhibition of PD-1 reduces the ratio of Treg T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0218] Inhibition of PD-1 can reduce CD8+ T cell exhaustion. Inhibition of PD-1 can reduce CD8+ T cell exhaustion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more compared to activity without inhibition of PD-1.

[0219] Inhibition of PD-1 can increase the population of CD8+ T cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which PD-1 is not inhibited.

[0220] The activity of PD-1 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0221] xv.LAG-3 LAG-3 can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of LAG-3. The inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The LAG-3 inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0222] LAG-3 can be suppressed by knocking out the LAG-3 gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA or shRNA. Conditional knockout methods can be used to inactivate the LAG-3 gene. Loss-of-function mutations can help suppress gene function by creating mutations in the LAG-3 gene. Gene editing techniques that can be employed to suppress LAG-3 include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress LAG-3. Mutations can be created in one or more protein domains.

[0223] Suppression of LAG-3 can increase the number of Th1 polarized T cells in leukocytes. Suppression of LAG-3 can promote the differentiation of T cells into Th1. Suppression of LAG-3 can decrease the expression of IL-10, IL-4, or IL-13 in leukocytes. Suppression of LAG-3 can increase the expression of IFN-γ in leukocytes. Suppression of LAG-3 can increase the expression of IL-12 in leukocytes. Suppression of LAG-3 can decrease the number of Treg polarized T cells in leukocytes.

[0224] Inhibition of LAG-3 can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which LAG-3 is not inhibited. Inhibition of LAG-3 can reduce the population of Treg cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which LAG-3 is not inhibited.

[0225] Inhibition of LAG-3 can increase expression of one or more relevant Th1 cell associated markers. Inhibition of LAG-3 can increase expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which LAG-3 is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of LAG-3 may increase the expression of IFN-γ. For example, suppression of LAG-3 may increase IL-2. For example, suppression of LAG-3 may increase the expression of IL-12.

[0226] Suppression of LAG-3 can reduce the expression of one or more relevant Treg cell-associated markers. Suppression of LAG-3 can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which LAG-3 is not suppressed. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, suppression of LAG-3 can reduce the expression of TGFβ. For example, suppression of LAG-3 can reduce the expression of IL-10.

[0227] Inhibition of LAG-3 can increase the ratio of Th1 T cells to Treg T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0228] Inhibition of LAG-3 reduces the ratio of Treg T cells to Th1 T cells to approximately 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be about 1 / 2, about 150 / 2, about 200 / 2, about 250 / 3, about 300 / 350 / 4, about 400 / 450 / 5, about 500 / 5, about 550 / 6, about 650 / 7, about 750 / 8, about 800 / 8, about 850 / 9, about 900 / 950 / 1, about 1000 / 1 or more.

[0229] The activity of LAG-3 can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0230] xvi. TNF-α TNF-α can be suppressed (i.e., inhibited) by pharmacological agents or genetic modification. The pharmacological agents can be inhibitors of TNF-α. The TNF-α inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The TNF-α inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM.

[0231] TNF-α can be suppressed by knocking out the TNF-α gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing, and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA, or shRNA. Conditional knockout methods can be used to inactivate the TNF-α gene. Loss-of-function mutations can help suppress gene function by creating mutations in the TNF-α gene. Gene editing techniques that can be employed to suppress TNF-α include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress TNF-α. Mutations can be created in one or more protein domains.

[0232] Suppression of TNF-α can increase the number of Th1 polarized T cells in white blood cells. Suppression of TNF-α can promote the differentiation of T cells into Th1. Suppression of TNF-α can decrease the expression of IL-10, IL-4, or IL-13 in white blood cells. Suppression of TNF-α can increase the expression of IFN-γ in white blood cells. Suppression of TNF-α can increase the expression of IL-12 in white blood cells. Suppression of TNF-α can decrease the number of Treg polarized T cells in white blood cells.

[0233] Inhibition of TNF-α can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TNF-α is not inhibited. Inhibition of TNF-α can reduce the Treg population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TNF-α is not inhibited.

[0234] Inhibition of TNF-α can increase expression of one or more relevant Th1 cell associated markers. Inhibition of TNF-α can increase expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TNF-α is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of TNF-α can increase the expression of IFN-γ. For example, suppression of TNF-α can increase IL-2. For example, suppression of TNF-α can increase the expression of IL-12.

[0235] Suppression of TNF-α can reduce the expression of one or more relevant Treg cell-associated markers. Suppression of TNF-α can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which TNF-α is not suppressed. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, suppression of TNF-α can reduce the expression of TGFβ. For example, suppression of TNF-α can reduce the expression of IL-10.

[0236] Inhibition of TNF-α leads to reg The ratio of Th1 T cells to T cells can be increased by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0237] By suppressing TNF-α, the ratio of Treg T cells to Th1 T cells can be reduced to about 1 / 2, 1 / 3, 1 / 4, 1 / 5, 1 / 10, 1 / 15, 1 / 20, 1 / 25, 1 / 30, 1 / 35, 1 / 40, 1 / 45, 1 / 50, 1 / 55, 1 / 60, 1 / 65, 1 / 70, 1 / 75, 1 / 80, 1 / 85, 1 / 90, 1 / 95, 1 / 10 ... The reduction can be by a factor of 0, about a factor of 150, about a factor of 200, about a factor of 250, about a factor of 300, about a factor of 350, about a factor of 400, about a factor of 450, about a factor of 500, about a factor of 550, about a factor of 600, about a factor of 650, about a factor of 700, about a factor of 750, about a factor of 800, about a factor of 850, about a factor of 900, about a factor of 950, about a factor of 1000 or more.

[0238] Inhibition of TNF-α can reduce CD8+ T cell exhaustion. Inhibition of TNF-α can reduce CD8+ T cell exhaustion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more compared to activity without inhibition of TNF-α.

[0239] Inhibition of TNF-α can increase the population of CD8+ T cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TNF-α is not inhibited.

[0240] The activity of TNF-α can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0241] Cytokine release syndrome is a known complication of the treatment of hematological malignancies with chimeric antigen receptor-modified (CAR) T cells or with T cell-replete, HLA haploidentical blood or bone marrow transplants. In embodiments, inhibition of TNF-α can reduce cytokine release syndrome following non-transplant CD8-depleted donor lymphocyte infusion.

[0242] Cytokine release syndrome is rated on a scale of 0 to 5. Inhibition of TNF-α can reduce the cytokine release syndrome score to 0, 1, 2, 3, or 4.

[0243] Inhibition of TNF-α can reduce IL-1β expression by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TNF-α is not inhibited.

[0244] Inhibition of TNF-α can reduce the percentage of leukocytes undergoing pyroptosis among total leukocytes by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TNF-α is not inhibited.

[0245] The activity of TNF-α, as measured, for example, by phosphorylation of its substrate (e.g., 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase gamma-2; PLC-γ2), can be inhibited by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0246] xvii. TGF-β receptor II TGF-β receptor II can be suppressed (i.e., inhibited) by pharmacological agents or genetic recombination. The pharmacological agents can be inhibitors of TGF-β receptor II. The TGF-β receptor II inhibitors can be small molecules, small interfering RNA (siRNA) or short hairpin RNA (shRNA). The TGF-β receptor II inhibitors can have a half-maximal inhibitory concentration of less than about 1000nM, less than about 900nM, less than about 800mM, less than about 700nM, less than about 600nM, less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, or less than about 100nM.

[0247] TGF-β receptor II can be suppressed by knocking out the TGF-β receptor II gene from the genome. The techniques for knocking out genes are known to those skilled in the art. Gene knockout methods in the art include, but are not limited to, gene silencing, conditional knockout, homologous recombination, gene editing, and knockout by mutation. Gene silencing can be achieved, for example, using RNA interference, siRNA, or shRNA. Conditional knockout methods can be used to inactivate the TGF-β receptor II gene. Loss-of-function mutations can help suppress gene function by creating mutations in the TGF-β receptor II gene. Gene editing techniques that can be employed to suppress TGF-β receptor II include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and CRISPR-based systems (e.g., CRISPR-Cas9). Commercially available kits can also be used to suppress TGF-β receptor II. Mutations can be created in one or more protein domains.

[0248] Inhibition of TGF-β receptor II can increase the number of Th1 polarized T cells in white blood cells. Inhibition of TGF-β receptor II can promote the differentiation of T cells into Th1. Inhibition of TGF-β receptor II can decrease the expression of IL-10, IL-4 or IL-13 in white blood cells. Inhibition of TGF-β receptor II can increase the expression of IFN-γ in white blood cells. Inhibition of TGF-β receptor II can increase the expression of IL-12 in white blood cells. Inhibition of TGF-β receptor II can decrease the number of Treg polarized T cells in white blood cells.

[0249] Inhibition of TGF-β receptor II can increase the population of Th1 cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TGF-β receptor II is not inhibited. Inhibition of TGF-β receptor II can reduce the Treg population by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TGF-β receptor II is not inhibited.

[0250] Inhibition of TGF-β receptor II can increase the expression of one or more relevant Th1 cell associated markers. Inhibition of TGF-β receptor II can increase the expression of one or more Th1 cell associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TGF-β receptor II is not inhibited. The one or more Th1-associated markers may include CCR1, CD4, CD26, CD94, CD119, CD183, CD195, CD212, GM-CSF, granzyme B, IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-18R, IL-23, IL-27, IL-27R, lymphotoxin, perforin, t-bet, Tim-3, TNF-a, TRANCE, sCD40L, or any combination thereof. In particular, the one or more Th1-associated markers may include IFN-γ, IL-2, IL-12, or any combination thereof. For example, suppression of TGF-β receptor II can increase the expression of IFN-γ. For example, suppression of TGF-β receptor II can increase IL-2. For example, suppression of TGF-β receptor II can increase the expression of IL-12.

[0251] Inhibition of TGF-β receptor II can reduce the expression of one or more relevant Treg cell-associated markers. Inhibition of TGF-β receptor II can reduce the expression of one or more Treg cell-associated markers by about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to white blood cells in which TGF-β receptor II is not inhibited. The one or more Treg-associated markers can include TGFβ or IL-10 or any combination thereof. For example, inhibition of TGF-β receptor II can reduce the expression of TGFβ. For example, inhibition of TGF-β receptor II can reduce the expression of IL-10.

[0252] Inhibition of TGF-β receptor II can increase the ratio of Th1 T cells to Treg T cells by about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 55-fold, about 60-fold, about 65-fold, about 70-fold, about 75-fold, about 80-fold, about 85-fold, about 90-fold, about 95-fold, about 100-fold, about 150-fold, about 200-fold, about 250-fold, about 300-fold, about 350-fold, about 400-fold, about 450-fold, about 500-fold, about 550-fold, about 600-fold, about 650-fold, about 700-fold, about 750-fold, about 800-fold, about 850-fold, about 900-fold, about 950-fold, about 1000-fold or more.

[0253] Inhibition of TGF-β receptor II reduces the ratio of Treg T cells to Th1 T cells to approximately one-half, one-half, one-third, one-quarter, one-fifth, one-tenth, one-fifteenth, one-twentieth, one-twenty-fifth, one-thirtieth, one-thirty-fifth, one-fortieth ... The reduction can be by a factor of 0, about a factor of 150, about a factor of 200, about a factor of 250, about a factor of 300, about a factor of 350, about a factor of 400, about a factor of 450, about a factor of 500, about a factor of 550, about a factor of 600, about a factor of 650, about a factor of 700, about a factor of 750, about a factor of 800, about a factor of 850, about a factor of 900, about a factor of 950, about a factor of 1000 or more.

[0254] Inhibition of TGF-β receptor II can reduce CD8+ T cell exhaustion. Inhibition of TGF-β receptor II can reduce CD8+ T cell exhaustion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or more compared to activity without inhibition of TGF-β receptor II.

[0255] Inhibition of TGF-β receptor II can increase the population of CD8+ T cells by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to leukocytes in which TGF-β receptor II is not inhibited.

[0256] The activity of TGF-β receptor II can be suppressed (i.e., inhibited) by about 1%, about 2%, about 3%, about 4%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or more compared to basal activity.

[0257] Therapeutic Applications The present disclosure also relates to methods for treating a disease or condition, such as cancer, comprising administering to a subject in need thereof a lymphocyte depleting and / or immunostimulating agent and administering an allogeneic lymphocyte composition as described herein.

[0258] The lymphocyte depleting agent can be a cellular reducing agent. Exemplary cellular reducing agents include, but are not limited to, alkylating agents, alkylsulfonates, nitrosoureas, triazenes, antimetabolites, pyrimidine analogs, purine analogs, vinca alkaloids, epiodophyllotoxins, antibiotics, dibromomannitol, deoxyspergualin, dimethylmyleran, and thiotepa.

[0259] The lymphocyte depleting agent can be a chemotherapeutic agent or a biologic agent. Exemplary chemotherapeutic and / or biologic agents include antibodies, B cell receptor pathway inhibitors, T cell receptor inhibitors, PI3K inhibitors, IAP inhibitors, mTOR inhibitors, radioimmunotherapeutic agents, DNA damaging agents, histone deacetylase inhibitors, protein kinase inhibitors, hedgehog inhibitors, Hsp90 inhibitors, telomerase inhibitors, Jakl / 2 inhibitors, protease inhibitors, IRAK inhibitors, PKC inhibitors, PARP inhibitors, CYP3 A4 inhibitors, AKT inhibitors, Erk inhibitors, proteosome inhibitors, alkylating agents, antimetabolites, plant alkaloids, terpenoids, cytotoxins, topoisomerase inhibitors, CD79A inhibitors, CD79B inhibitors, CD19 inhibitors, Lyn inhibitors, Syk inhibitors, PI3K inhibitors, Blnk inhibitors, PLCy inhibitors, PKCP inhibitors, CD22 inhibitors, Bcl-2 inhibitors, IRAK inhibitors, and / or other agents. 1 / 4 inhibitors, JAK inhibitors (e.g., ruxolitinib, baricitinib, CYT387, lestauritinib, pacritinib, TG101348, SAR302503, tofacitinib (Xeljanz), etanercept (Enbrel), GLPG0634, R256), microtubule inhibitors, TopoII inhibitors, anti-TWEAK antibodies, anti-IL17 bispecific antibodies, CK2 inhibitors, anaplastic lymphoma kinase (ALK) and c-Met inhibitors, demethylase inhibitors (such as demethylase, HDM, LSDI and KDM), fatty acid synthase inhibitors (such as spirocyclic piperidine derivatives), glucocorticosteroid receptor agonists, fused anti-CD19-cytotoxic drug conjugates, alternative antagonists, p70S6K inhibitors, immunomodulators, AKT / PKB inhibitors, procaspase-3 activators PAC-1, BRAF inhibitors, lactate dehydrogenase A (LDH-A) inhibitors, CCR2 inhibitors, CXCR4 inhibitors, chemokine receptor antagonists, DNA double-strand break repair inhibitors, NOR202, GA-101, TLR2 inhibitors, muromonab-CD3, rituximab (Rituxan), carfilzomib, fludarabine, cyclophosphamide, vincristine, chlorambucil, ifosfamide, doxorubicin, mesalazine, thalidomide, revlimid, lenalidomide, temsirolimus, everolimus, fostamatinib, paclitaxel, docetaxel, ofatumumab,Dexamethasone, bendamustine, CAL-101, ibritumomab, tositumomab, bortezomib, pentostatin, endostatin, ritonavir, ketoconazole, anti-VEGF antibodies, herceptin, cetuximab, cisplatin, carboplatin, docetaxel, erlotinib, etoposide, 5-fluorouracil, gemcitabine, ifosfamide, imatinib mesylate (Gleevec), gefitinib, erlotinib, procarbazine, irinotecan, leucovorin, mesylate, cefotaxime ... Chlorethamine, methotrexate, oxaliplatin, paclitaxel, sorafenib, sunitinib, topotecan, vinblastine, GA-1101, dasatinib, sipuleucel-T, disulfiram, epigallocatechin-3-gallate, salinosporamide A, ONX0912, CEP-18770, MLN9708, R-406, lenarinomide, spirocyclic piperidine derivatives, quinazoline carboxamide azetidine compounds, thiotepa, DWA2114R, NK121, IS 3 295, 254-S, alkylsulfonates (such as busulfan, improsulfan, and piposulfan), aziridines (such as benzodepa, carboquone, meturedepa, and uredepa), ethylenimines, methylmelamines (such as altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine), chlornaphazine, estramustine, ifosfamide, mechlorethamine, oxide hydrochloride, novobiocin, phenesterine, prednimustine, trofosfamide, uracil mustard, nitrosoureas (carmustine, chlorozotocin, fotemustine, romosulfan ... sucrine, nimustine, ranimustine, etc.), antibiotics (aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carubicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, rodorubicin, streptonigrin,streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, etc.), antimetabolites (methotrexate and 5-fluorouracil (5-FU) etc.), folic acid analogues (denopterin, methotrexate, pteropterin, trimetrexate etc.), purine analogues (fludarabine, 6-mercaptopurine, thiamiprine, thioguanine etc.), pyrimidine analogues (ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.), androgens (calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone, etc.), adrenal inhibitors (aminoglutethimide, mitotane, trilostane, etc.), folic acid supplements (folinic acid, etc.), aceglatone, aldophosphamide glycoside, aminolevulinic acid, amsacrine, bestravcil, bisantrene, edatrexate, dephosphamide, demecolcine, diazicon, etazolidine, Flunitine, elliptinium acetate, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidamine, mitoguazone, mitoxantrone, mopidamol, nitracrine, pentostatin, phenameth, pirarubicin, podophyllic acid, 2-ethylhydrazide, procarbazine, polysaccharide-K, razoxane, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2''-trichlorotriethylamine, urethane, vindesine, dacarbazine, mannan Nomustine, mitobronitol, mitolactol, pipobroman, gactosine, cytosine arabinoside, taxoids such as paclitaxel and docetaxel, 6-thioguanine, mercaptopurine, methotrexate, platinum analogues, platinum, etoposide (VP-16), ifosfamide, mitomycin C, mitoxantrone, vincristine, vinorelbine, navelbine, novantrone, teniposide, daunomycin, aminopterin, xeloda, ibandronate, CPT1 1, topoisomerase inhibitors RFS2000, difluoromethylornithine (DMFO), retinoic acid, esperamicin, capecitabine and pharma- ceutically acceptable salts, acids or derivatives thereof, antihormonal agents such as antiestrogens (e.g. tamoxifen, raloxifene,Aromatase inhibitors 4(5)-imidazoles (such as 4-hydroxytamoxifen, trioxyphene, ketoxifene, LY117018, onapristone, and toremifene (Fareston)), antiandrogens (such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin), ACK inhibitors (such as AVL-263 (Avila Therapeutics / Celgene Corporation), AVL-292 (Avila Therapeutics / Celgene Corporation), AVL-291 (Avila Therapeutics / Celgene Corporation), BMS-488516 (Bristol-Myers Squibb), BMS-509744 (Bristol-Myers Squibb), CGI-1746 (CGI Pharma / Gilead Sciences), CTA-056, GDC-0834 (Genentech), HY-11066 (also CTK4I7891, HMS3265G21, HMS3265G22, HMS3265H21, HMS3265H22, 439574-61-5, AG-F-54930), ONO-4059 (Ono Pharmaceutical Co., Ltd.), ONO-WG37 (Ono Pharmaceutical Co., Ltd.), PLS-123 (Peking University), RN486 (Hoffmann-La Roche), HM71224 (Hanmi Pharmaceutical Company Limited), or combinations thereof.

[0260] The compositions and methods disclosed herein can be used with any suitable cancer, including, but not limited to, bladder cancer, brain cancer, breast cancer, colorectal cancer, cervical cancer, gastrointestinal cancer, genitourinary cancer, head and neck cancer, lung cancer, ovarian cancer, prostate cancer, renal cancer, skin cancer, testicular cancer, cardiac cancer (e.g., sarcomas, e.g., angiosarcoma, fibrosarcoma, rhabdomyosarcoma, and liposarcoma), myxoma, rhabdomyosarcoma, fibroma, lipoma, teratoma, lung cancer (e.g., bronchogenic carcinoma, e.g., squamous cell, small undifferentiated cell carcinoma, large undifferentiated cell, and adenocarcinoma, alveolar and mesothelioma), gastrointestinal cancers (e.g., cancer of the esophagus, e.g., squamous cell carcinoma, adenocarcinoma, leiomyosarcoma and lymphoma), stomach cancer (e.g., carcinoma, lymphoma and leiomyosarcoma), pancreatic cancer (e.g., tubular adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor and bipoma), small intestine cancer (e.g., adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma and fibroma), large intestine cancer (e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma and leiomyoma), genitourinary cancer Cancer of the kidney (e.g. adenocarcinoma, Wilm's tumor (nephroblastoma), lymphoma and leukemia), cancer of the bladder and urethra (e.g. squamous cell carcinoma, transitional cell carcinoma and adenocarcinoma), cancer of the prostate (e.g. adenocarcinoma and sarcoma), cancer of the testes (e.g. seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous tumor and marginal tumor), cancer of the liver (e.g. hepatocellular carcinoma, e.g. hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), bone cancer (e.g. osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma (reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochondroma (osteochondroma), benign chondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor), cancer of the nervous system (e.g. cancer of the skull, e.g. osteoma, hemangioma, granuloma, xanthomas, and osteitis dissecans), cancer of the meninges (e.g. meningioma, meningeal sarcoma and gliomatosis), cancer of the brain (e.g. astrocytoma, medulloblastoma, glioma, ependymoma, embryonal tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma and congenital tumors),Cancer of the spinal cord (e.g. neurofibroma, meningioma, glioma and sarcoma), gynaecological cancer (e.g. cancer of the uterus, e.g. endometrial cancer, cancer of the cervix, e.g. cervical carcinoma and preneoplastic cervical dysplasia), cancer of the ovary, e.g. ovarian cancer including serous cystadenocarcinoma, mucinous cystadenocarcinoma, unclassified carcinoma, granulosa theca cell tumor, Sertoli-Leydig cell tumor, dysplastic cell tumor and malignant teratoma, cancer of the vulva, e.g. squamous cell carcinoma, carcinoma in situ, adenocarcinoma, fibrosarcoma and melanoma, cancer of the vagina, e.g. clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma and embryonal striated carcinoma myosarcoma, and fallopian tube cancer, e.g., myeloma), blood cancer (e.g., blood cancer, e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma and myelodysplastic syndrome, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and Waldenstrom's macroglobulinemia), skin cancer (e.g., malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, lenticular dysplastic nevus, lipoma, hemangioma, dermatofibroma, keloid, psoriasis), and adrenal cancer (including, e.g., neuroblastoma). In certain embodiments, when the disease is cancer, it can include, for example, lung cancer tumor, breast cancer tumor, prostate cancer tumor, brain cancer tumor, or skin cancer tumor.

[0261] The subject may have a solid tumor. In some embodiments, the subject may have a sarcoma, carcinoma, or neurofibroma. In some embodiments, the subject may have colon cancer. In some embodiments, the subject may have lung cancer. In some embodiments, the subject may have ovarian cancer. In some embodiments, the subject may have pancreatic cancer. In some embodiments, the subject may have prostate cancer. In some embodiments, the subject may have proximal or distal bile duct cancer. In some embodiments, the subject may have breast cancer. In some embodiments, the subject may have HER2-positive breast cancer. In some embodiments, the subject may have HER2-negative breast cancer. In some embodiments, the subject has been treated for a solid tumor, and the method is applied to treat the subject as an adjuvant therapy, i.e., the method is applied to the subject when the cancer is in complete remission to prevent recurrence of the cancer.

[0262] The subject may have a hematological cancer. In some embodiments, the cancer is a leukemia, lymphoma, myeloma, myelodysplastic syndrome, or myeloproliferative neoplasm. In some embodiments, the cancer is a non-Hodgkin's lymphoma. In some embodiments, the cancer is a Hodgkin's lymphoma. In some embodiments, the cancer is a B-cell malignancy. In some embodiments, the B-cell malignancy is selected from the group consisting of chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL), germinal center diffuse large B-cell lymphoma (GCBCL), and leukemia-associated lymphoma (LEM). In some embodiments, the cancer is a T-cell malignancy. In some embodiments, the T cell malignancy is peripheral T cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large cell lymphoma, angioimmunoblastic lymphoma, cutaneous T cell lymphoma, adult T cell leukemia / lymphoma (ATLL), blastic NK cell lymphoma, enteropathy-type T cell lymphoma, hemocytosplenomegaly gamma delta T cell lymphoma, lymphoblastic lymphoma, nasal NK / T cell lymphoma, or therapy-related T cell lymphoma. In some embodiments, the subject may have multiple myeloma.

[0263] The subject may have recurrent or refractory cancer.

[0264] The methods disclosed herein may further involve administering one or more additional agents for treating cancer, such as, for example, chemotherapeutic agents (e.g., adriamycin, cervidin, bleomycin, alkeran, velban, oncovin, fluorouracil, thiotepa, methotrexate, bisantrene, noanthrone, tiguanine, citalibine, procarabidine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cell therapy (e.g., CAR-T, T cell therapy, natural killer cell therapy, gamma delta T cell therapy), and oncolytic viruses.

[0265] Non-limiting examples of additional agents for treating cancer include acivicin, aclarubicin, acodazole hydrochloride, acronine, adzelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, visnafide dimesylate, bizeresin, bleomycin sulfate, brequinar sodium, bropirimine. , busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, cilormycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexorumaplatin, dezaguanine, dezaguanine mesylate, diazicon, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate salt, dromostanolone propionate, duazomycin, edatrexate, efurnithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropizine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etoprine, fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, flurocitabine, foskidone, fostriecin sodium, gemcitabine , gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosine, interleukin II (including recombinant interleukin II, rIL2), interferon α-2a, interferon α-2b, interferon α-nl, interferon α-n3, interferon β-Ia, interferon γ-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, liraozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride,Masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium, metoprine, metholedepa, mitindomide, mitocalcin, mitochromine, mitogillin, mitomarcine, mitomycin, mitospar, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogamicin, ormaplatin, oxy Slane, paclitaxel, pegaspagase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pivobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin, puromycin hydrochloride, pyrazofurin, ribopurine, logretimide, safingol, safingol hydrochloride, semustine, simtrazene , Sparfosate sodium, Sparsomycin, Spirogermanium hydrochloride, Spiromustine, Spiroplatin, Streptonigrin, Streptozocin, Surofenur, Tallysomycin, Tecogalan sodium, Tegafur, Teroxantrone hydrochloride, Temoporfin, Teniposide, Teroxylon, Testolactone, Thiamiprine, Thioguanine, Thiotepa, Tiazofurin, Tirapazamine, Toremifene citrate, Trestrone acetate, Triciribine phosphate salt, trimetrexate, trimetrexate glucuronate, triptorelin, tuburozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinylcinate sulfate, benzurozole sulfate, vinorelbine tartrate, vinzolidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride.

[0266] The methods disclosed herein can further include administration of an anti-tumor antibody / drug conjugate, including, but not limited to, rituximab, cetuximab, trastuzumab, and pertuzumab, brentuximab vedotin, gemtuzumab ozogamicin, trastuzumab emtansine, inotuzumab ozogamicin, glenbatumumab vedotin, lorvotuzumab mertansine, cantuzumab mertansine, or milatuzumab doxorubicin.

[0267] The methods disclosed herein may further include administering an antiviral agent. Exemplary antiviral agents include acyclovir, famciclovir, ganciclovir, penciclovir, valacyclovir, valganciclovir, idoxuridine, trifluridine, brivudine, cidofovir, docosanol, fomivirsen, foscarnet, tromantadine, imiquimod, podophyllotoxin, entecavir, lamivudine, telbivudine, clevudine, adefovir, tenofovir, boceprevir, telaprevir, pleconaril, arbidol, amantadine, rimantadine, oseltamivir, zanamivir, peramivir, inosine, interferons (e.g., interferon alpha-2b, pegylated interferon alpha-2a), ribavirin, and the like. These include, but are not limited to, valproate / talibavirin, abacavir, emtricitabine, lamivudine, didanosine, zidovudine, apricitabine, stamidine, elvucitabine, rasivir, amdoxovir, stavudine, zalcitabine, tenofovir, efavirenz, nevirapine, etravirine, rilpivirine, loviride, delavirdine, atazanavir, fosamprenavir, lopinavir, darunavir, nerfumavir, ritonavir, saquinavir, tipranavir, amprenavir, indinavir, enfuvirtide, maraviroc, vicriviroc, PRO140, ibalizumab, raltegravir, elvitegravir, bevirimat and vibecon.

[0268] The compositions disclosed herein are typically administered systemically, for example, by intravenous injection or infusion.Other routes of administration, such as oral, parenteral, intravenous, intraarticular, intraperitoneal, intramuscular, subcutaneous, intracavitary, transdermal, intrahepatic, intracranial, nebulization / inhalation, bronchoscopic placement, or intratumoral administration, may be used.

[0269] The dosage is determined by the attending physician and other clinical factors. The dosage for a single patient varies depending on many factors, such as the patient's size, body surface area, age, sex, the specific compound administered, administration time and route, type of treatment, general health status, and other drugs administered at the same time. "Effective amount" refers to the amount of active ingredient that is sufficient to affect the course and severity of the disease and to reduce or ameliorate such pathology, and can be determined using known methods.

[0270] C. Preparation method The present disclosure also relates to a method of preparing an allogeneic lymphocyte composition as disclosed herein. The method includes obtaining a peripheral blood cell composition from a donor subject that is allogeneic to a recipient subject, or from a cell line or umbilical cord blood. The peripheral blood cell composition can be a whole blood product or an apheresis product. The peripheral blood cell composition can be obtained using means known in the art, such as, for example, venipuncture. The peripheral blood cell composition includes both CD8+ and CD4+ T cells. The peripheral blood cell composition can be obtained from a human or non-human subject. Preferentially, the peripheral blood cell composition is obtained from a human.

[0271] The white blood cells from the donor subject may be mismatched with the recipient subject for at least one HLA class II allele mismatch in the donor-to-recipient (graft-to-host) direction to the recipient subject. Alternatively, the donor may have at least one HLA class II allele mismatch with the recipient in the donor-to-recipient (graft-to-host) direction and at least one HLA class II allele match with the recipient. The HLA class II allele mismatch or match may be HLA-DRB1, HLA-DQB1 or HLA-DPB1.

[0272] When the donor and the recipient are ABO blood type incompatible and the allogeneic white blood cell composition contains a large number of red blood cells, making the allogeneic lymphocyte composition may further include reducing the number of red blood cells. As used herein, "ABO blood type incompatibility" refers to when the recipient has a major ABO red blood cell incompatibility with the donor, such as when the recipient has blood type O and the donor has blood type A, B or AB, when the recipient has blood type A and the donor has blood type B or AB, or when the recipient has blood type B and the donor has blood type A or AB. The number of red blood cells may comprise a packed volume of about 50 mL or less, for example, a packed volume of about 50 mL or less, preferably a packed volume of about 30 mL or less, and further, "packed volume" should be defined, for example, centrifugation of the lymphocyte composition provides a packed volume of 50 mL or less of red blood cells, and a measured volume sample of the lymphocyte composition may also be screened to provide a proportionally representative volume of packed blood cells.

[0273] Mononuclear cells are then isolated from the peripheral blood cell composition, for example, by Ficoll-Hypaque gradient separation. The number of CD8+ cells in the white blood cells can then be reduced. The number of CD8+ cells in the white blood cells can be reduced by about 1 / 2, about 3 / 4, about 5 / 6, about 7 / 8, about 9 / 10, about 15 / 10, about 20 / 25, about 30 / 35, about 40 / 45, about 50 / 55, about 6 / 10, about 8 / 15, about 9 / 10, about 11 / 15, about 12 / 10, about 13 / 10, about 14 / 15, about 15 / 10, about 16 / 10, about 17 / 10, about 18 / 10, about 19 / 11, about 20 / 25, about 25 / 30, about 26 / 10, about 27 / 10, about 28 / 10, about 29 / 10, about 30 / 35, about 31 / 32, about 32 / 36, about 33 / 37, about 34 / 38, about 35 / 39, about 36 / 40, about 37 / 45, about 38 / 46, about 39 / 47, about 39 / 48, about 39 / 49, about 40 / 50, about 40 / 5 ...0, about 40 / 50, about 40 The amount of removal can be about 1 in 0, about 1 in 65, about 1 in 70, about 1 in 75, about 1 in 80, about 1 in 85, about 1 in 90, about 1 in 95, about 1 in 100, about 1 in 200, about 1 in 300, about 1 in 400, about 1 in 500, about 1 in 600, about 1 in 700, about 1 in 800, about 1 in 900, about 1,000 or more.

[0274] The leukocytes are further engineered to inhibit BTK, ITK, or PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or combinations thereof. Without wishing to be bound by theory or mechanism, the inventors believe that inhibition of BTK, ITK, PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof, can promote differentiation of naive CD4+ T cells into a state favorable for helping effector cells of antitumor or antiviral immunity, such as type 1 (Th1) CD4+ T cells, or prevent post-naive CD4+ T cells from converting into cells with suboptimal helper activity for antitumor or antiviral immunity. For example, a portion of T cells can preferentially differentiate into CD4+ T cell subtypes, particularly Th1.

[0275] The method can include promoting differentiation of at least a portion of the T cells into Th1 CD4+ T cells. The differentiation of the portion of the T cells into Th1 CD4+ T cells can be promoted by inhibiting BTK, ITK, PI3Kδ, helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof.

[0276] The method involves culturing leukocytes in vitro.

[0277] The method of producing an allogeneic composition may further comprise stimulating antigen-specific lymphocytes in the composition with antigen-presenting cells pulsed with an antigenic peptide.

[0278] The method of producing an allogeneic composition may further include adding one or more additional agents, such as a cytokine or an antibody.

[0279] The additional agent can be a cytokine. Exemplary cytokines that can be added include IL-2, IL-7, IL-12, IL-15, IL-18, IFNγ, IL-21, CCDC134, GM-CSF, or LYG1.

[0280] The additional agent can be an antibody. Exemplary antibodies include an anti-IL3 antibody, an anti-IL-4 antibody, an anti-CD3 antibody, an anti-CD200 antibody, or an anti-CD28 antibody.

[0281] The additional agent can be an inhibitor. Exemplary inhibitors include inhibitors of MEK1 / 2, ERK, p38, Cox-2, Pi13k, c5l2, setdb1 or Got1.

[0282] Other exemplary agents include, but are not limited to, receptor agonists (e.g., RARα or TLR), transcription factors (e.g., T-bet and Tbx21), lipoarabinomannan or lipomannan derived from BCG cell bodies, and the like.

[0283] D. Definition Throughout the specification and claims, various terms related to the aspects of the specification are used. Such terms should be given their normal meaning in the art unless otherwise indicated. Other specifically defined terms should be interpreted in a manner consistent with the definitions provided herein. The techniques and procedures described or referenced herein are generally well understood and commonly employed by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions, unless otherwise indicated.

[0284] As used herein, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise. Terms such as "including," "such as," and the like are intended to include without limitation, unless otherwise specified.

[0285] Unless otherwise specified, "at least," "less than," and "about," or similar terms preceding a series or range, should be understood to refer to every element in the series or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0286] The term "cancer" refers to a physiological condition in mammals in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or specific morphological features. In many cases, cancer may take the form of a tumor or mass, but may also exist alone in a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The term cancer includes all types of cancer and metastases, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancer include, but are not limited to, carcinomas, lymphomas, embryonal tumors, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer (e.g., triple-negative breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial cancer (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and various types of head and neck cancer. Triple-negative breast cancer refers to breast cancer in which the expression of the estrogen receptor (ER), progesterone receptor (PR), and Her2 / neu genes is negative.

[0287] As used herein, the term "T cell exhaustion" refers to the progressive loss of effector function (loss of IL-2, TNF-α and IFN-γ production, or inability to kill cells expressing the T cell's cognate antigen) and sustained expression of inhibitory receptors that have transcriptional programs distinct from functional effector or memory T cells, such as PD-1, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3), CTLA-4, lymphocyte activation gene 3 (LAG-3) and CD160.

[0288] As used herein, the term "subject" refers to any animal, such as any mammal, including, but not limited to, humans, non-human primates, and rodents. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0289] As used herein, the term "therapeutically effective amount" refers to an amount of a compound (i.e., an allogeneic lymphocyte composition) described herein that is sufficient to achieve the desired pharmacological or physiological effect under the conditions of administration. For example, a "therapeutically effective amount" may be an amount sufficient to alleviate the signs or symptoms of a disease or condition (e.g., a tumor). Those skilled in the art will understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject. The therapeutically effective amount of a pharmaceutical composition may vary depending on factors such as the disease state, age, sex, and weight of the individual, as well as the ability of the pharmaceutical composition to elicit the desired response in the individual. Based on these and other considerations, an ordinary skilled clinician can determine the appropriate amount to administer to achieve the desired therapeutic benefit.

[0290] Equivalent It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the present invention described herein will be apparent and may be made using suitable equivalents without departing from the scope of the present disclosure or embodiments. Although specific compounds and methods have been described in detail above, the same will be more clearly understood by reference to the following examples, which are provided for illustrative purposes and are not intended to be limiting.

[0291] This invention is further illustrated by the following examples, which are not intended to be limiting in any way. EXAMPLES

[0292] Example 1. Donor E7 priming, ex vivo expansion, and T cell proliferation in E7-expressing head and neck cancer mouse models hVerification of antitumor effect of 1-polarization

[0293] The experimental goal was to test the efficacy of exogenous CD4+ T cell help against endogenous antitumor effectors in a mouse model of HPV-associated head and neck cancer. Table 1 shows the drugs and treatment protocol for this study.

[0294] [Table 1]

[0295] CD8- refers to the depletion of CD8+ T cells. CD8+ depletion is performed using Miltenyi CD8 depletion columns.

[0296] For ex vivo expansion, mouse dendritic cells (DCs) are enriched using a mouse pan-dendritic cell isolation kit (Miltenyi Biotech). DCs are pulsed with 1 μg / mL each of HPV16 E6 and E7 peptides (JPT Peptide Technologies) for 2 h in RPMI1640 (complete medium) containing 2 mmol / L L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% heat-inactivated fetal bovine serum. E6 / E7-pulsed DCs are then transferred to flasks containing mouse splenocytes (treated with 1 μM ITK inhibitor for 1 h before mixing) in a 1:20 ratio with complete medium containing 25 U / mL recombinant mouse IL-2 and 20 ng / mL recombinant mouse GMCSF and cultured for 5 days. Cells are then washed and rested for 2 days in complete medium + IL-2 and GM-CSF before restimulation.

[0297] ITK samples (i.e., groups 9 and 10) are exposed ex vivo to 1 μM ITK inhibitor for 1 h before incubation with peptide-pulsed dendritic cells and cytokines.

[0298] ICB refers to immune checkpoint blockade. ICB is performed on day 2 where indicated using RMP1-14 (anti-PD1; BioXCell) 100 mg IP in 100 mL HBSS and 10F.9G2 (anti-PD-L1; BioXCell) 100 mg IP in 100 mL HBSS.

[0299] Example 2. T of CD4+ T cells h Characterization of the fate and efficacy of ex vivo expanded E7-primed donor T cells under conditions designed to favor one phenotype We previously showed that in a syngenic cell transplantation model, primed whole spleens were curative, whereas primed CD8-depleted spleens conferred no benefit. + After infusion, T cells are T h 2 or T reg Such alternatives (i.e. non-T h 1) CD4 + We hypothesize that the T h 1. Ex vivo expansion of E7-specific T cells and in vivo T h We hypothesized that deletion of ITK as a means to promote retention of the CD4+ phenotype would enhance the antitumor efficacy of infusions in a syngenic model. + Among T cells, T h These manipulations promote the CD4.1 phenotype. + T cell T reg We hypothesized that this would prevent the conversion to

[0300] The goal of this study was to characterize the fate and efficacy of E7-primed donor T cells expanded ex vivo under conditions designed to favor a Th1 phenotype in CD4+ T cells. Table 2 lists the agents and treatment protocol for this study.

[0301] [Table 2]

[0302] Three mice per group will be sacrificed at 1, 2 and 3 weeks after DLI. Spleen cells will be stimulated with dendritic cells pulsed with the overlapping pentadecapeptide of E7 (JPT peptide). E7-specific donor CD4+ T cell status will be characterized by intracellular staining for IFN-γ, IL-4, IL-17A or foxp3, and extracellular staining for CD4 and CD45.1, followed by flow cytometry.

[0303] It is believed that primed E7-specific CD4+ T cells become exhausted or convert to alternative CD4 phenotypes (e.g., Th2, Th17, Treg). In the syngenic model, donor cells can be tracked and a portion of mice can be sacrificed at intervals after infusion to examine the number and function of E7-specific T cells.

[0304] The same experiment is repeated using CB6 F1 donors and B6C3 F1 recipients.

[0305] Example 3. Comparison of different E7 vaccines as immunogens for non-transplant donor lymphocyte infusions The objective of the study was to determine the optimal vaccine to induce E6 / E7-specific Th1 CD4+ T cells and enhance the antitumor effect of CD8-depleted donor lymphocyte infusion.

[0306] First, we characterize the effect of one, two, and three doses of the vaccine on CD4 + T cell responses to human papillomavirus type 16 (HPV16) E6 / E7 antigens.

[0307] Nine BALB / cx B6 mice in each group are vaccinated once, twice or three times weekly with either the RNA-LPX mRNA vaccine, PDS0101 or PapiVax DNA vaccine. One week after each vaccination, three mice from each group are sacrificed. Either splenocytes or vaccine-draining lymph node cells are stimulated with either HPV16 E6 overlapping peptides (JPT Peptides, Germany) or E7 overlapping peptides. Six hours after stimulation, extracellular CD4 and CD8 and intracellular interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-a) are stained and analyzed by flow cytometry. Table 3 shows the drugs and treatment protocol of this study.

[0308] [Table 3]

[0309] Next, we will evaluate the antitumor effect of non-transplant cell therapy using donors vaccinated with either RNA-LPX, PDS0101 or PapiVax vaccine. Table 4 shows the drugs and treatment protocol of this study.

[0310] [Table 4]

[0311] Mice are photographed weekly to follow their survival.

[0312] Once the optimal vaccine is identified, a decision will be made whether to add recipient vaccination to donor vaccination. Table 5 lists the drugs and treatment protocol for this study.

[0313] [Table 5]

[0314] Example 4. CRISPR-mediated excision of selected genes in donor CD4+ T cells The aim of this study is to prevent the conversion of T cells to regulatory T cells in vivo. h The goal of this study is to test CRISPR-mediated ablation of selected genes in donor CD4+ T cells as a means to polarize the tumor-specific CD4+ T cells toward the 1 pathway and enhance the antitumor efficacy of nontransplant donor lymphocyte infusions. Table 6 shows the drugs and treatment protocol of this study.

[0315] [Table 6]

[0316] For each of groups 2-7, BTK and ITK activity after inhibition will be assayed by measuring the amount of phospho-BTK or phospho-ITK protein after CD3 / CD28 stimulation (see J. Dubovsky et al. Blood 122: 2539, 2013). The frequency of IL-4 vs. IFN-γ producing CD4+ T cells will be measured by intracellular cytokine staining and flow cytometry.

[0317] Example 5. E7-specific CD4 + To compare in vivo priming against E7 with or without ex vivo restimulation to pure ex vivo priming and restimulation to generate and expand Th1 cells and enhance the antitumor effect of nontransplant donor lymphocyte infusion The aim of this study is to determine whether donor vaccination can be eliminated from the protocol.

[0318] A. Donor Cell Preparation CB6 F1 donor mice or splenocytes are treated as shown in Table 7 below.

[0319] [Table 7]

[0320] For in vivo priming or boosting, 25 μg of pcDNA3-CRT / E7 is administered intramuscularly.

[0321] B. Ex vivo priming or boosting Murine dendritic cells (DCs) are enriched using a mouse pan-dendritic cell isolation kit (Miltenyi Biotech). DCs are pulsed with 1 μg / mL E7 peptide (JPT Peptide Technologies) for 2 h in RPMI1640 (complete medium) containing 2 mmol / L L-glutamine, 100 U / mL penicillin, 100 μg / mL streptomycin, and 10% heat-inactivated fetal bovine serum. E7-pulsed DCs are then transferred to flasks containing mouse splenocytes (treated with 1 μM ITK inhibitor for 1 h before mixing) in a 1:20 ratio with complete medium containing 25 U / mL recombinant mouse IL-2 and 20 ng / mL recombinant mouse GMCSF and cultured for 5 days. Cells are then washed and rested for 2 days in complete medium + IL-2 and GM-CSF before restimulation. Table 8 shows the drugs and treatment protocol for this study.

[0322] [Table 8]

[0323] On day 15, the frequency of E7-specific Th1 cells among all donor cells in groups 2-7 is determined by intracellular cytokine staining and flow cytometry. Mouse splenocytes are stimulated for 5 hours in complete medium with 1 μg / mL E7 peptide and with or without 5 μg / mL Brefeldin A. Cells are then washed and stained for IFN-γ and TNF-α along with CD4 and CD8.

[0324] Example 6. Anti-lymphoma effect of CD8-depleted donor lymphocyte infusion. + Effect of ITK deletion alone or in combination with BTK deletion in T cells The purpose of this study was to identify donor strains and their T h 1 (e.g., C57BL / 6) or T hThe objective of this study is to verify whether the bias towards BALB / c (e.g., BALB / c) affects the antitumor immunity of CD8-depleted DLI and its enhancement by BTK or ITK deletion in donor CD4+ T cells. Table 9 shows the tumor types, lineages of origin, and donor lineages in this study.

[0325] [Table 9]

[0326] The experimental design was the same for all three tumor types, with only differences in tumor dose and donor and recipient strains. Table 10 shows the treatment protocol for this study.

[0327] [Table 10]

[0328] Example 7. Efficacy of non-transplant donor lymphocyte infusion for common malignant tumors (lung, breast, ovarian) The aim of this study is to test whether non-transplant DLI can be incorporated into the treatment of common malignancies and be synergistic with checkpoint blockade. The cancers being studied are lung, breast and ovarian cancers.

[0329] A. Lung cancer

[0330] Tables 11 and 12 show the treatment protocols for lung cancer studies.

[0331] [Table 11]

[0332] [Table 12]

[0333] B. Breast cancer Table 13 shows the treatment protocols for breast cancer studies.

[0334] [Table 13]

[0335] C. Ovarian cancer Table 14 shows the treatment protocols for breast cancer studies.

[0336] [Table 14]

[0337] Example 8. Effect of donor neoantigen vaccination on the antitumor efficacy of CD8-depleted non-transplanted DLI against neoantigen-expressing tumors The aim of this study was to test whether a donor vaccine strategy could be employed for the treatment of sporadic tumors through vaccination against tumor neoantigens.

[0338] In this experiment, we test two methods to increase the frequency of neoAg-specific CD4+ T cells in CD8-depleted NEDLI: 1) in vivo vaccination with CD4+ T cell neoepitopes (with or without subsequent neopeptide stimulation); or 2) in vitro "priming" with sequential stimulation of CD4+ T cells with neopeptide+DCs. The experimental design is shown in Table 15 below.

[0339] [Table 15]

[0340] B16-F10 melanomas derived from C57BL / 6 (B6; H-2b) are grown in F1 hybrids; immunogenic CD4+ neoepitopes have been identified (see reference 4). 1) B6 x C3H (B6C3; H-2bxk) F1 or MHC-haploidentical BALB / cx B6 (CB6; H-2bxd) F1 mice are vaccinated with either the mutant neoepitope M30 (groups 2, 6) encoded by the kinesin family member 18b gene (Kif18b) or the corresponding wild-type peptide (groups 1, 5) by injecting a vaccine containing 100 μg of synthetic peptide and 50 μg of poly(I:C) in a volume of 200 μL phosphate-buffered saline into the flank. Vaccination efficacy and responder cell phenotype (CD4 vs. CD8) will be examined by flow cytometry and intracellular cytokine staining (ICS) for interferon gamma (IFNγ) or tumor necrosis factor alpha (TNF-α) as described in reference 5. Two weeks after vaccination, CD8+ cells will be removed from the spleen of euthanized donor mice and infused into B16-F10 bearing B6C3 mice treated with Cy the day before infusion.

[0341] Alternatively, CD8-depleted cells from immunized donors are cultured for 5 days with M30-pulsed donor DCs 5 days prior to infusion (groups 3, 7); 2) spleen cells from naive B6C3 or CB6 F1 mice are stimulated twice weekly with M30-pulsed autologous dendritic cells + 20 U / mL IL-2 (groups 4, 8). The frequency of M30-specific IFNγ+CD4+T cells is measured by ICS before and after ex vivo stimulation. NeoAg-specific CD4+T cells can be purified using an IFNγ capture assay (Miltenyi Biotec) and further expanded using anti-CD3 and anti-CD28 coated beads.

[0342] References 1. Williams R, Lee DW, Elzey BD, Anderson ME, Hostager BS, Lee JH. Preclinical models of HPV+ and HPV- HNSCC in mice: an immune clearance of HPV+ HNSCC. Head & Neck: Journal for the Sciences and Specialties of the Head and Neck. 2009;31(7):911-918. 2. Ahrends T, Babala N, Xiao Y, Yagita H, van Eenennaam H, Borst J. CD27 Agonism Plus PD-1 Blockade Recapitulates CD4+ T-cell Help in Therapeutic Anticancer Vaccination. Cancer Res. 2016;76(10):2921-2931. 3. Minard-Colin V, Xiu Y, Poe JC, et al. Lymphoma depletion during CD20 immunotherapy in mice is mediated by macrophage FcγRI, FcγRIII, and FcγRIV. Blood. 2008;112(4):1205-1213. 4. Castle JC, Kreiter S, Diekmann J, Lower M, Roemer N, Graaf J. Exploiting the mutanome for tumor vaccination. Cancer Res. 2012;72. 5. Kreiter S, Vormehr M, van de Roemer N, et al. Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature. 2015;520(7549):692-696.

[0343] Example 9. Depletion of CD8+ T cells CD8+ T cells are removed from human blood products using the CliniMACS system with CliniMACS® CD8 Reagent. CD8+ T cells are labeled with monoclonal antibodies bound to superparamagnetic particles and removed from the blood product by passing them through the CliniMACS system, which incorporates a separation column with a strong permanent magnet and a ferromagnetic matrix to remove the labeled cells.

[0344] Example 10. Differential effects of single gene deletions on the antitumor efficacy of CD4+ T cells from MHC haploidentical donors vaccinated against tumor antigens Previous experiments demonstrated that prior vaccination of healthy donors against a tumor antigen (E7 of human papillomavirus type 16 [HPV16]) enhanced the antitumor efficacy of donor CD8-depleted lymphocytes infused after cyclophosphamide treatment of MHC haploidentical recipients with advanced E7-expressing tumors. CD4+ T cells from vaccinated donors slightly extended the survival of tumor-bearing recipients, but all recipients eventually succumbed to progressive tumors. See US, Publication No: 2022 / 0163997.

[0345] It has been hypothesized that growing tumors eventually polarize infused CD4+ T cells to render them ineffective at providing antitumor immune help, but deleting key genes involved in CD4+ T cell differentiation / polarization may prevent this loss of CD4+ T cell help and thereby sustain antitumor immune responses.

[0346] To test this hypothesis, BALB / cx C57BL / 6 (CB6 F1) donors were vaccinated three times a week with 25 micrograms of pBI-11, a DNA vaccine encoding the E6 and E7 antigens of HPV16, intramuscularly. One week after the third vaccination, CD4+ T cells were enriched to near purity using immunomagnetic beads and either left untreated, transfected with Cas9 nucleoprotein, or transfected with Cas9 nucleoprotein and guide RNA to inactivate single genes: interleukin-2-inducible T cell kinase (ITK), forkhead box p3 (FOXP3), transforming growth factor beta receptor type II (TGFBR2), suppressor of cytokine signaling-1 (SOCS1), or programmed death molecule-1 (PDCD1). After resting the cells overnight, 25 million unvaccinated, vaccinated but non-nucleofected, vaccinated and Cas9-nucleofected, or single gene-deficient CD4+ T cells were co-injected with 5 million syngenic (CB6 F1) CD3-depleted splenocytes into MHC haploidentical C57BL / 6 x C3H (B6C3 F1) mice that had received 50,000 TC-1 tumor cells (expressing HPV16 E6 and E7) 14 days prior to infusion and 200 mg / kg cyclophosphamide intraperitoneally the day prior to infusion. See Figure 1.

[0347] Donor vaccination against HPV E7 enhanced the antitumor effect of cyclophosphamide CD8-depleted, MHC haploidentical donor lymphocyte infusion, with the difference in tumor-free survival between TC1-luci-bearing recipients of unvaccinated and TC1-luci-bearing recipients of vaccinated donor cells approaching statistical significance by day 42 post-tumor inoculation (p=0.096; Figure 2A). Nucleofection with Cas9 reagent, but not gene knockout, did not significantly affect tumor-free survival in donor lymphocyte infusion recipients (Figure 2B; p=0.99). In contrast, CRISPR-mediated deletion of the ITK gene in donor CD4+ T cells further increased tumor-free survival compared to recipients of vaccinated CD4+ T cells without ITK deletion (p=0.17) or recipients of CD4+ T cells from unvaccinated donors (p~0.07; Figure 2C). Different single gene deletions in CD4+ T cells of E6 / E7 vaccinated donors had different effects on DLI-induced antitumor immunity (Figure 2D). Interestingly, tumor-bearing recipients of vaccinated CD4+ T cells containing deletions of either PD-1 or TGFβR2 had slightly worse tumor-free survival than recipients of genetically unmodified CD4+ T cells and significantly worse overall survival than recipients of cells with deletions of either ITK or foxp3 (Figure 2D and Table 16).

[0348] [Table 16]

[0349] These results indicate that CRISPR-mediated deletion of genes involved in CD4+ T cell differentiation and function may affect the antitumor efficacy of cell therapy from healthy donors vaccinated against tumor antigens.-Deletion of ITK or FOXP3 appears to be beneficial, whereas deletion of PDCD1 or TGFBR2 results in poorer tumor-free survival compared to genetically unmodified CD4+ T cells.

[0350] Example 11. Additional gene deletions for antitumor efficacy of CD4+ T cells from MHC haploidentical donors vaccinated against tumor antigens BALB / cx C57BL / 6 (CB6 F1) donors are vaccinated three times a week with intramuscular injections of 25 micrograms of pBI-11, a DNA vaccine encoding the E6 and E7 antigens of HPV16. One week after the third vaccination, CD4+ T cells are enriched to near purity using immunomagnetic beads and either left untreated, transfected with Cas9 nucleoprotein, or transfected with Cas9 nucleoprotein and guide RNAs to inactivate single genes: Bruton's tyrosine kinase (BTK), delta isoform of phosphoinositide 3 kinase (PI3Kδ), helios, blimp1, GATA3, IL-10, STAT3, TOX, CD25, Ezh2, LAG-3, TNF-α, or combinations thereof. After resting the cells overnight, 3 million unvaccinated, vaccinated but non-nucleofected, vaccinated and Cas9-nucleofected CD4+ T cells, or single gene-deficient CD4+ T cells are co-injected with 5 million syngenic (CB6 F1) CD3-deleted splenocytes into MHC haploidentical C57BL / 6 x C3H (B6C3 F1) mice that had been administered 50,000 TC-1 tumor cells (expressing HPV16 E6 and E7) 14 days prior to infusion and 200 mg / kg cyclophosphamide intraperitoneally the day prior to infusion.

[0351] Example 12. Combination of gene deletions on the antitumor effect of CD4+ T cells from MHC haploidentical donors vaccinated against tumor antigens

[0352] BALB / cx C57BL / 6 (CB6 F1) donors are vaccinated three times a week with intramuscular injections of 25 micrograms of pBI-11, a DNA vaccine encoding the E6 and E7 antigens of HPV16. One week after the third vaccination, CD4+ T cells are enriched to near purity using immunomagnetic beads and either left untreated, transfected with Cas9 nuclear protein, or transfected with Cas9 nuclear protein and guide RNA to inactivate a combination of two or more genes: Bruton's tyrosine kinase (BTK), interleukin-2-inducible T cell kinase (ITK), phosphoinositide 3 kinase delta isoform (PI3Kδ), helios, blimp1, SOCS1, GATA3, IL-10, STAT3, TGF-β receptor II, LAG-3, PD-1, TNF-α, TOX, CD25, foxp3, and Ezh2. After resting the cells overnight, 3 million unvaccinated, vaccinated but non-nucleofected, vaccinated and Cas9-nucleofected, or single gene-deficient CD4+ T cells are infused together with 5 million syngenic (CB6 F1) CD3-deleted splenocytes into MHC haploidentical C57BL / 6 x C3H (B6C3 F1) mice that received 50,000 TC-1 tumor cells (expressing HPV16 E6 and E7) 14 days prior to infusion and 200 mg / kg cyclophosphamide intraperitoneally the day before infusion.

[0353] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims.

Claims

1. 1. A pharmaceutical composition comprising a plurality of isolated leukocytes obtained from a donor subject and mismatched with the recipient subject for at least one human leukocyte antigen (HLA) class II allele in a donor-to-recipient (graft-versus-host) orientation to the recipient subject, The leukocytes have CD8+ T cells depleted at about 10 times or more compared to undepleted leukocytes, The leukocytes have been modified to inhibit Bruton's tyrosine kinase (BTK), interleukin-2-inducible T-cell kinase (ITK), the delta isoform of phosphoinositide 3 kinase (PI3Kδ), helios, blimpl, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof.

2. A pharmaceutical composition comprising a plurality of isolated leukocytes obtained from an allogeneic donor subject, wherein the donor's CD4+ T cells have been stimulated in vivo or ex vivo with an antigen present in a recipient subject, the donor subject comprising at least one HLA class II allele match to the recipient; The leukocytes have CD8+ T cells depleted at about 10 times or more compared to undepleted leukocytes, The leukocytes have been modified to inhibit Bruton's tyrosine kinase (BTK), interleukin-2-inducible T-cell kinase (ITK), the delta isoform of phosphoinositide 3 kinase (PI3Kδ), helios, blimpl, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, foxp3, Ezh2, TGF-β receptor II, LAG-3, PD-1, TNF-α, or a combination thereof.

3. 1. A pharmaceutical composition comprising a plurality of isolated leukocytes obtained from a donor subject, (i) mismatched with the recipient subject for at least one HLA class II allele mismatch in a donor-to-recipient (graft-versus-host) direction to the recipient subject, and (ii) wherein the donor's CD4+ T cells have been stimulated in vivo or ex vivo with an antigen present in the recipient subject, the donor subject comprising at least one human leukocyte HLA class II allele match to the recipient, The leukocytes have CD8+ T cells depleted at about 10 times or more compared to undepleted leukocytes, 10. The method of claim 1, wherein at least a portion of the CD4+ T cells have been modified to inhibit the activity of Bruton's tyrosine kinase (BTK), interleukin-2-inducible T-cell kinase (ITK), the delta isoform of phosphoinositide 3-kinase (PI3Kδ), helios, blimpl, SOCS1, GATA3, IL-10, STAT3, TGF-β receptor II, LAG-3, PD-1, TNF-α, TOX, CD25, foxp3, Ezh2, or a combination thereof.

4. At least a portion of the T cells are T ｈ 1. The pharmaceutical composition according to any one of claims 1 to 3, wherein the cells are differentiated into CD4+ T cells.

5. the T cells are transformed into T cells by inhibition of BTK, ITK, PI3Kδ, Foxp3, GATA3, STAT3, CD25, or Ezh2. ｈ 1. The pharmaceutical composition according to any one of claims 1 to 3, which is biased towards CD4+ T cell differentiation. (i) the T cells are biased toward T h 1 CD4+ T cell differentiation by inhibition of both BTK and ITK; (ii) the T cells are biased toward T h 1 CD4+ T cell differentiation and opposed to regulatory T cell differentiation by inhibition of BTK and PI3Kδ; or (iii) the T cells are biased towards T h 1 CD4+ T cell differentiation and against regulatory T cell differentiation by inhibition of both ITK and PI3Kδ; The pharmaceutical composition according to any one of claims 1 to 3.

7. The T cells are inhibited by BTK, ITK, and PI3Kδ. ｈ 1. The pharmaceutical composition of claim 3, which is biased towards CD4+ T cell differentiation and opposed to regulatory T cell differentiation.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein BTK, ITK and PI3Kδ, helios, blimpl, SOCS1, GATA3, IL-10, STAT3, TOX, CD25, TGF-β receptor II, LAG-3, PD-1, TNF-α, foxp3, or Ezh2 is inhibited by an inhibitor or genetic modification.

9. The pharmaceutical composition of any one of claims 1 to 3, wherein the BTK inhibitor is acalabrutinib, zanubrutinib, LFM-A13, dasatinib, or AVL-292.

10. 9. The pharmaceutical composition of claim 8, wherein the inhibitor of BTK is not ibrutinib.

11. 9. The pharmaceutical composition of claim 8, wherein the inhibitor of ITK is an aminothiazole, aminobenzimidazole, indole, pyridine, or prn694.

12. 9. The pharmaceutical composition of claim 8, wherein the inhibitor of PI3Kδ is idelalisib, copanlisib, duvelisib, umbralisib, ME-4401, RP6503, perifosine, bupallisib, or dactolisib.

13. The method of claim 12, wherein the differentiation of the T cells into regulatory T cells is attenuated by inhibition of PI3Kδ, Foxp3, CD25, or Ezh2; 4. The pharmaceutical composition of claim 1, wherein PI3Kδ, Foxp3, CD25, or Ezh2 is inhibited by a PI3Kδ inhibitor, a Foxp3 inhibitor, a CD25 inhibitor, or an Ezh2 inhibitor, or by genetic modification.

14. 14. The pharmaceutical composition of claim 13, wherein the PI3Kδ inhibitor is idelalisib, copanlisib, duvelisib, umbralisib, ME-4401, RP6503, perifosine, bupallisib, or dactolisib.

15. 4. The pharmaceutical composition of claim 2 or 3, wherein the HLA class II match is an HLA-DRB1 allele, an HLA-DQB1 allele, or an HLA-DPB1 allele.

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