Expansion of gamma delta t cells, compositions, and use thereof

Abstract

To provide methods of expanding γδ T cells from a non-haematopoietic tissue as a cell source, and further to provide compositions of expanded γδ T cells and methods of using the expanded γδ T cells, for example as part of an adoptive T cell therapy.SOLUTION: A method of expanding γδ T cells comprises the steps of: (i) providing a population of γδ T cells obtained from a non-haematopoietic tissue; and (ii) culturing the γδ T cells for at least 5 days in the presence of effective amounts of (a) IL-2 or IL-9, (b) IL-15, and (c) IL-21 to produce an expanded population of γδ T cells.SELECTED DRAWING: None

Description

[Background technology]

[0001] Growing interest in T cell immunotherapy for cancer has focused on the apparent ability of CD8+ and CD4+αβ T cell subsets to recognize cancer cells and mediate host-protective functional potential, particularly when disinhibited by clinically mediated antagonism of inhibitory pathways exerted by PD1, CTLA4, and other receptors. Nevertheless, many questions remain. For example, there appear to be numerous key clinical situations in which such treatments are unlikely to be effective. Serious adverse events often occur, the ability to predict efficacy or adverse events is extremely limited, and there is little explanation for the interactions that enable the host to sense tumor cells ("immunogenicity"), which must precede activation of conventional antigen-specific CD8+ and CD4+αβ T cell responses.

[0002] Gamma delta T cells (γδ T cells) are a subset of T cells that express a distinct γδ T cell receptor (TCR) on their surface. This TCR consists of one gamma (γ) chain and one delta (δ) chain. Human γδ T cells are broadly classified into one or two subtypes: peripheral blood-resident γδ T cells and non-hematopoietic tissue-resident γδ T cells. While most blood-resident γδ T cells express the Vδ2 TCR, this is uncommon in tissue-resident γδ TCR cells, which typically use Vδ1 and / or other Vδ chains. Because obtaining large numbers of non-hematopoietic tissue-resident γδ T cells is challenging and there are no standard protocols for their isolation and expansion, they have not been well characterized or studied for therapeutic applications. Therefore, there remains a need to identify and expand sufficient numbers of non-hematopoietic tissue-resident γδ T cells for study and potential therapeutic applications, such as adoptive T cell therapy. Summary of the Invention

[0003] The present invention provides methods for expanding γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) from a non-hematopoietic tissue source (e.g., non-hematopoietic tissue-derived γδ T cells, e.g., non-hematopoietic tissue-derived V51 T cells). The expansion method comprises culturing γδ T cells (e.g., γδ T cells isolated from stromal cells of a non-hematopoietic tissue) in the substantial absence of TCR stimulation and / or in the presence of IL-4, IL-15, IL-21, and / or IL-2. Further provided are the expanded γδ T cells (e.g., non-hematopoietic tissue-derived γδ T cells, e.g., non-hematopoietic tissue-derived V51 T cells) and methods of using the γδ T cells (e.g., as part of adoptive T cell therapy, e.g., for cancer treatment).

[0004] In one embodiment, the invention features a method for expanding γδ T cells by: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; and (ii) culturing the γδ T cells in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) for at least 5 days to generate an expanded population of γδ T cells. For example, the γδ T cells can be cultured in the presence of IL-2, IL-15, and IL-4; IL-2, IL-15 and IL-21; or IL-2, IL-15, IL-4, and IL-21.

[0005] In another aspect, the present invention provides a method for expanding γδ T cells by the steps of: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; and (ii) culturing the γδ T cells in the presence of effective amounts of IL-2, IL-4, IL-15, and IL-21 for at least 5 days to generate an expanded population of γδ T cells.

[0006] In some embodiments of both of the foregoing aspects, the γδ T cells are simultaneously exposed to IL-2, IL-4, IL-15, and IL-21 for at least 5 days. In some embodiments, step (ii) comprises culturing the γδ T cells in the absence of an exogenous TCR pathway agonist. In some embodiments, the method further comprises, after step (i), separating the γδ T cells from non-hematopoietic cells to produce a separated population of γδ T cells, and step (ii) comprises: (a) culturing the γδ T cells substantially in the absence of contact with stromal cells; (b) culturing the γδ T cells substantially in the absence of contact with tumor cells; and / or (c) culturing the γδ T cells substantially in the absence of contact with support cells.

[0007] In another aspect, the method for expanding γδ T cells comprises the steps of: (i) providing a non-hematopoietic tissue, a tissue containing non-hematopoietic cells, and γδ T cells; (ii) separating the γδ T cells from the non-hematopoietic cells to obtain an isolated population of γδ T cells; and (iii) culturing in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1 for at least 5 days to generate an expanded population of γδ T cells. In some embodiments, step (ii) comprises culturing the γδ T cells in the presence of IL-2, IL-15, IL-4, and / or IL-21 (e.g., IL-2, IL-15, and IL-4; IL-2, IL-15 and IL-21; or IL-2, IL-15, IL-4, and IL-21). In some embodiments, the γδ T cells are exposed to IL-2, IL-15, IL-4, and / or IL-21 simultaneously. In some embodiments, step (iii) comprises culturing the γδ T cells in the absence of substantial contact of the γδ T cells with stromal cells. In some embodiments, step (iii) comprises culturing the γδ T cells in the absence of an exogenous TCR pathway agonist.

[0008] In another aspect, the invention features a method for expanding γδ T cells, including the steps of: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; and (ii) culturing the γδ T cells in the presence of effective amounts of IL-2, IL-4, IL-15, and IL-21 for at least 5 days to generate an expanded population of γδ T cells. The γδ T cells may be exposed to IL-2, IL-4, IL-15, and IL-21 simultaneously, e.g., for at least 5 days, or may be exposed to one or more factors prior to exposure to the other factors. In some embodiments, step (ii) comprises culturing the γδ T cells in the presence of effective amounts of human recombinant IL-2, human recombinant IL-4, human recombinant IL-15, and human recombinant IL-21 for at least 5 days to generate an expanded population of γδ T cells. In some embodiments, step (ii) comprises culturing the γδ TCR cells in the absence of an exogenous TCR pathway agonist (e.g., anti-CD3), e.g., in the absence of substantial TCR pathway activation. In some embodiments, step (ii) comprises culturing the γδ T cells in the presence of IL-21 at a concentration of 1 ng / mL to 1,000 ng / mL (e.g., about 10 ng / mL, or about 100 ng / mL). Step (ii) may comprise culturing the γδ T cells in the presence of one or more factors selected from the group consisting of IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1).

[0009] In another aspect, a method for expanding γδ T cells is provided by: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; and (ii) culturing the γδ T cells for at least 5 days in the presence of effective amounts of IL-2 and IL-15 to generate an expanded population of γδ T cells. In some embodiments of this aspect, the γδ T cells are simultaneously exposed to IL-2 and IL-15. Step (ii) may comprise culturing the γδ T cells in the absence of an exogenous TCR pathway agonist or in the substantial absence of TCR pathway activation. In some embodiments, step (ii) comprises culturing the γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. In some embodiments, the γδ T cells are cultured in the presence of IL-4 and / or IL-21. In some embodiments, step (ii) comprises culturing γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21, where the concentration of IL-21 can be between 1 ng / mL and 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0010] In yet another aspect, the invention features a method for expanding γδ T cells by the following steps: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; and (ii) culturing the γδ T cells for at least 5 days under conditions substantially free of TCR pathway activation to generate an expanded population of γδ T cells. In some embodiments, step (ii) comprises culturing the γδ T cells in the presence of IL-2 and IL-15. In some examples, the γδ T cells are simultaneously exposed to IL-4 and IL-15. In some embodiments, step (ii) comprises culturing the γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, the γδ T cells may be cultured in the presence of IL-4, IL-21, or both. In some embodiments, γδ T cells are cultured in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be between 1 ng / mL and 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0011] In some embodiments of any of the foregoing aspects, the method of the invention further comprises, after step (i), separating the γδ T cells from non-hematopoietic cells to produce a separated population of γδ T cells, and step (ii) comprises culturing the γδ T cells substantially in the absence of contact with stromal cells, substantially in the absence of contact with tumor cells, and / or substantially in the absence of contact with support cells (e.g., irradiated support cells, B cells, or antigen-presenting cells).

[0012] In another aspect, featured herein is a method for expanding γδ T cells by the following steps: (i) providing non-hematopoietic cells and non-hematopoietic tissue containing γδ T cells; (ii) separating the γδ T cells from the non-hematopoietic cells to obtain an isolated population of γδ T cells; and (iii) culturing the γδ T cells for at least 5 days under conditions substantially free of contact between stromal cells and the γδ T cells to obtain an expanded population of γδ T cells. In some embodiments, step (iii) comprises culturing the γδ T cells in the absence of an exogenous TCR pathway agonist and / or in the substantial absence of TCR pathway activation. In some embodiments, step (iii) comprises culturing the γδ T cells in the presence of IL-2 and IL-15. For example, the γδ T cells may be exposed to IL-2 and IL-15 simultaneously. In some embodiments, step (iii) comprises culturing γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, in some examples, step (iii) comprises culturing γδ T cells in the presence of IL-4, IL-21, or both. In some embodiments, step (iii) comprises culturing γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0013] In another aspect, the present invention provides a method for expanding γδ T cells by the following steps: (i) providing a non-hematopoietic tissue, wherein the tissue contains non-hematopoietic cells and γδ T cells; (ii) separating the γδ T cells from the non-hematopoietic cells to obtain an isolated population of γδ T cells; and (iii) culturing the γδ T cells in the presence of IL-2 and IL-15 for at least 5 days to obtain an expanded population of γδ T cells. In some embodiments, step (iii) comprises culturing the γδ T cells in the absence of substantial contact between stromal cells and the γδ T cells. In some embodiments, step (iii) comprises culturing the γδ T cells in the absence of an exogenous TCR pathway agonist or in the absence of substantial TCR pathway activation. In some embodiments, step (iii) comprises culturing the γδ T cells in the presence of IL-2 and IL-15. The γδ T cells may be exposed to IL-2 and IL-15 simultaneously. In some examples, step (iii) comprises culturing γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, γδ T cells may be cultured in the presence of IL-2 and / or IL-21. In some embodiments, step (iii) comprises culturing γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0014] In yet another aspect, featured herein is a method for expanding γδ T cells by the following steps: (i) providing a non-hematopoietic tissue, wherein the tissue contains non-hematopoietic cells and γδ T cells; (ii) separating the γδ T cells from the non-hematopoietic cells to obtain an isolated population of γδ T cells; and (iii) culturing the γδ T cells for at least 5 days under conditions substantially free of TCR pathway activation to obtain an expanded population of γδ T cells. In some examples, step (iii) comprises culturing the γδ T cells in the absence of an exogenous TCR pathway agonist and / or in the absence of substantial contact between stromal cells and the γδ T cells. In some embodiments, step (iii) comprises culturing the γδ T cells in the presence of IL-2 and IL-15. For example, the γδ T cells may be exposed to IL-2 and IL-15 simultaneously. In some embodiments, step (iii) comprises culturing γδ T cells in the presence of one or more factors selected from the group consisting of IL-4, IL-21, IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, HPL, and SDF-1. For example, γδ T cells may be cultured in the presence of IL-2 and / or IL-21. In some embodiments, step (iii) comprises culturing γδ T cells in the presence of IL-2, IL-4, IL-15, and IL-21. The concentration of IL-21 can be 1 ng / mL to 1,000 ng / mL (e.g., 10 ng / mL or 100 ng / mL).

[0015] In some embodiments of any of the aforementioned methods, the step of separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells on a synthetic scaffold constructed to release cells from non-hematopoietic tissue. In some examples, separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells with non-hematopoietic cells in the presence of IL-2, IL-15, or both. In some embodiments, the separated population of lymphocytes comprises a separated population of γδ T cells, and the separated population of γδ T cells comprises a separated population of Vδ1 T cells and / or double-negative (DN) cells. In some embodiments, prior to the expansion step, 1-10% of the separated population of lymphocytes are γδ T cells. In some embodiments, prior to the expansion step, 1-10% of the separated population of lymphocytes are Vδ1 T cells. Prior to the expansion step, at least 80% of the separated population of γδ T cells may be Vδ1 T cells and / or less than 10% of the separated γδ T cells may be Vδ2 T cells. In some embodiments, αβ T cells and / or NK cells are removed from the isolated population of γδ T cells (eg, before the expansion step).

[0016] In some embodiments, prior to the expansion step, the isolated population of γδ T cells contains at least 10% CCR3 + cells, at least 10% CCR4 + cells, at least 10% CCR7 + cells, at least 10% CCR8 + cells, or at least 10% CD103 + In some embodiments, prior to the expansion step, the isolated population of γδ T cells comprises a higher number of CCR3 T cells compared to a reference population (e.g., a reference population of blood-resident V52 T cells). + cells, CCR4 + cells, CCR7 + cells, and / or CCR8 +In some embodiments, prior to the expansion step, the isolated population of V51 T cells comprises a higher number of NKG2D T cells compared to a reference population (e.g., a reference population of blood-resident V52 T cells). + cells, CD56 + cells, CD69 + Cells and / or TIM3 + Contains cells.

[0017] In some embodiments of any of the foregoing aspects, after 14 days or less of culture during the expansion step, the expanded population of γδ T cells comprises at least 20-fold more γδ T cells than the population of γδ T cells isolated prior to the expansion step. Additionally or alternatively, after 21 days or less of culture during the expansion step, the expanded population of γδ T cells may comprise at least 50-fold more γδ T cells than the population of γδ T cells isolated prior to expansion. The expanded population of γδ T cells comprises an expanded population of V51 T cells. In some embodiments, after 14 days or less of culture during the expansion step, the expanded population of V51 T cells comprises at least 20-fold more V51 T cells than the population of V51 T cells isolated prior to expansion. Additionally or alternatively, after 21 days or less of culture during the expansion step, the expanded population of V51 T cells comprises at least 50-fold more V51 T cells than the population of V51 T cells isolated prior to expansion.

[0018] In some embodiments of any of the foregoing aspects, the expanded population of γδ T cells expresses CD27. For example, the expanded population of γδ T cells may exhibit a higher median CD27 surface expression than the isolated population of γδ T cells. In some examples, the expanded population of γδ T cells exhibits at least two-fold higher median CD27 surface expression than the isolated population of γδ T cells. Additionally or alternatively, the expanded population of γδ T cells may exhibit a higher number of CD27 than the isolated population of γδ T cells. + For example, the expanded population of γδ T cells may have at least 5% more CD27 T cells than the isolated population of γδ T cells.+ In some embodiments, the expanded population of V51 T cells express CD27. In some embodiments, the expanded population of V51 T cells exhibits a higher median CD27 surface expression than the isolated population of V51 T cells. For example, the expanded population of V51 T cells may exhibit at least two-fold higher median CD27 surface expression than the isolated population of V51 T cells. Additionally or alternatively, the expanded population of V51 T cells may exhibit a higher frequency of CD27 than the isolated population of V51 T cells. + For example, the expanded population of V51 T cells may have at least a 5% higher frequency of CD27 T cells compared to the isolated population of V51 T cells. + The cell may be a cellular component.

[0019] In some embodiments of any of the foregoing aspects, the expanded population of γδ T cells exhibits a lower median surface expression of TIGIT than the isolated population of γδ T cells. For example, the expanded population of γδ T cells may exhibit a median surface expression of TIGIT that is at least 50% lower than the isolated population of γδ T cells. Additionally or alternatively, the expanded population of γδ T cells may exhibit a lower frequency of TIGIT than the isolated population of γδ T cells. + For example, the expanded population of γδ T cells may have at least a 20% lower frequency of TIGIT T cells than the isolated population of γδ T cells. + In some embodiments, the expanded population of V51 T cells exhibits a lower median surface expression of TIGIT than the isolated population of V51 T cells. For example, the expanded population of V51 T cells may exhibit a median surface expression of TIGIT that is at least 50% lower than the isolated population of V51 T cells. Additionally or alternatively, the expanded population of V51 T cells may have a lower frequency of TIGIT+ cells than the isolated population of V51 T cells. For example, the expanded population of V51 T cells may have a frequency of TIGIT+ cells that is at least 20% lower than the isolated population of V51 T cells.

[0020] In some embodiments of any of the foregoing aspects, the expanded population of γδ T cells exhibits increased surface expression of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to a reference population (e.g., compared to the isolated population of γδ T cells, e.g., compared to the isolated population of γδ T cells prior to expansion). Additionally or alternatively, the number of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 may be increased in the expanded population of γδ T cells compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before expansion). In some embodiments, the expanded population of γδ T cells exhibits reduced surface expression of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before expansion). Additionally or alternatively, the expanded population of γδ T cells may have a lower frequency of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64 compared to a reference population (e.g., compared to the isolated population of γδ T cells, e.g., compared to the isolated population of γδ T cells prior to expansion).

[0021] In some embodiments, the expanded population of V51 T cells exhibits increased surface expression of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells prior to expansion). In some embodiments, the expanded population of V51 T cells exhibits a higher frequency of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells prior to expansion). In some embodiments, the expanded population of γδ T cells exhibits a lower surface expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before expansion). In other embodiments, the expanded population of γδ T cells exhibits a lower frequency of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to a reference population (e.g., compared to a population of isolated γδ T cells, e.g., compared to a population of isolated γδ T cells before expansion).

[0022] In some embodiments of any of the preceding aspects, step (iii) comprises culturing the γδ T cells substantially in contact with stromal cells, substantially in contact with support cells, and / or substantially in contact with tumor cells. In some embodiments, the non-hematopoietic tissue is not tumor cells.

[0023] In some embodiments of any of the preceding aspects, the non-hematopoietic tissue is skin (e.g., human skin, e.g., skin obtained by punch biopsy). In other embodiments, the non-hematopoietic tissue is gastrointestinal tissue.

[0024] In any of the foregoing aspects and embodiments, the method of expanding γδ T cells may be carried out in vitro.

[0025] In any of the foregoing aspects and embodiments, the step of providing non-hematopoietic tissue may be the step of providing non-hematopoietic cells obtained from a subject (e.g., a human or non-human animal subject).

[0026] In another aspect, the invention features expanded γδ T cells obtained by the method of any one of the preceding aspects.

[0027] In other aspects, the invention provides pharmaceutical compositions comprising the expanded γδ T cells of the above aspects. In some embodiments, the pharmaceutical composition further comprises an additional therapeutic agent selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and a combination of two or more agents. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent (e.g., IL-2, e.g., low dose IL-2, e.g., 0.3×10 per day). 6 ~3.0×10 6 IU of IL-2, e.g., 1.0 x 10 per day 6 IU of IL-2).

[0028] In another aspect, the invention features a pharmaceutical composition of the previous aspect for use in a method of treating a subject by adoptive T cell therapy.

[0029] In another aspect, the invention features the expanded γδ T cells of any of the preceding aspects for use in a method of treating a subject by adoptive T cell therapy.

[0030] In yet another aspect, the invention provides use of the expanded γδ T cells of any of the preceding aspects, or a pharmaceutical composition thereof, in the manufacture of a medicament for treating cancer (e.g., a solid cancer), an infectious disease (e.g., a cytomegalovirus (CMV) infection), or an immune disorder in a subject.

[0031] In another aspect, the invention provides the expanded γδ T cell of any of the preceding aspects, or a pharmaceutical composition thereof, for use in a method of treating cancer (e.g., a solid cancer), an infectious disease (e.g., a cytomegalovirus (CMV) infection), or an immune disease in a subject.

[0032] In another aspect, the invention provides a method of treating a subject by adoptive T cell therapy, comprising administering to a subject in need thereof a therapeutically effective amount of expanded γδ T cells obtained by the method of any of the preceding embodiments. In some embodiments, the therapeutically effective amount of expanded γδ T cells is 10×10 per administration. 12 Less than 10 x 10 cells throughout the course of treatment 12 In some embodiments, the method further comprises administering one or more additional therapeutic agents to the subject in need of treatment. The additional therapeutic agent may be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, and a combination of two or more of these agents. The additional therapeutic agent may be administered simultaneously with the expanded γδ T cells, prior to administration of the expanded γδ T cells, or after administration of the expanded γδ T cells. In some embodiments, the additional therapeutic agent is an immunotherapeutic agent. In one embodiment, the immunotherapeutic agent is IL-2 (e.g., a low dose of IL-2, e.g., 0.3×10 per day). 6 ~3.0×10 6 IU of IL-2, e.g., 1.0 x 10 per day6 IU of IL-2). These embodiments apply to any of the aspects described above and below relating to the use of expanded γδ T cells obtained by the methods described herein (or a pharmaceutical composition comprising these γδ T cells) in a method of treating a subject by adoptive T cell therapy.

[0033] In another aspect, the invention features a method of treating a subject with adoptive T cell therapy, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of any of the previous aspects.

[0034] In some embodiments of any of the foregoing aspects, the subject is a human (e.g., a human cancer patient (e.g., a human cancer patient being treated for a solid cancer), or a human cancer patient being treated for an infection (e.g., a viral infection such as CMV)).

[0035] In another aspect, the invention features a method of expanding γδ T cells, including the steps of: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; (ii) culturing the γδ T cells in the presence of effective amounts of (a) IL-2 or IL-9; (b) IL-15; and (c) IL-21 for at least 5 days to generate an expanded population of γδ T cells. In some embodiments, the γδ T cells in step (ii) are further cultured in the presence of IL-4.

[0036] In yet another aspect, the invention features a method for expanding γδ T cells by the following steps: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; and (ii) culturing the γδ T cells in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-21, stromal cell-derived factor (SDF, e.g., SDF-1), IL-1β, IL-12, IL-18, and IL-33 for at least 5 days to generate an expanded population of γδ T cells. In some embodiments, step (ii) comprises culturing the γδ T cells in the absence of an exogenous TCR pathway agonist. In some embodiments, step (ii) comprises culturing the γδ T cells in serum-free medium. In some embodiments, after step (i), the γδ T cells are separated from non-hematopoietic cells to generate a population of separated γδ T cells. Additionally, step (ii) comprises culturing the γδ T cells in the substantial absence of stromal cell contact, the substantial absence of tumor cell contact, and / or the substantial absence of supportive cell contact.

[0037] In another aspect, the invention features a method for expanding γδ T cells through the steps of: (i) providing non-hematopoietic tissue, wherein the tissue contains non-hematopoietic cells and γδ T cells; and (iii) culturing the tissue in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-21, SDF, IL-1β, IL-12, IL-18, and IL-33 for at least 5 days to generate an expanded population of γδ T cells. The γδ T cells may be cultured in the presence of IL-2, IL-15, and IL-21. Additionally or alternatively, the γδ T cells may be cultured in serum-free medium.

[0038] In yet another aspect, the invention features an isolated population of γδ T cells having the phenotype of any of the expanded γδ T cell populations described above. For example, in some embodiments, at least 50% of the isolated γδ T cells express CD27 and are substantially free of TIGIT. In some embodiments, at least 50% of the isolated γδ T cells express Vδ1.

[0039] In other embodiments, the invention includes pharmaceutical compositions of the isolated γδ T cells of the previous embodiments.

[0040] In another aspect, there is provided a use of the pharmaceutical composition described herein.

[0041] In another aspect, the invention features a method of treating a subject via adoptive T cell therapy, comprising administering to a subject in need of treatment a therapeutically effective amount of the expanded γδ T cells, the isolated population, or the pharmaceutical composition.

[0042] Within the scope of each aspect of the present invention, any embodiment described herein can be combined with other described embodiments. [Brief explanation of the drawings]

[0043] [Figure 1A]Human skin contains a distinct population of resident γδ T cells. Figure 1A: Skin-resident lymphocytes were isolated using the organotypic cell culture "Clark protocol" published by Clark et al. (Clark et al., Journal of Investigational Dermatology, 2006, 126(5):1059-70). Among CD45+ cells, anti-CD3 was used to stain T cells, and anti-CD56 antibodies were used to identify NK cells (CD3- CD56+). Among CD3+ cells, antibodies against the pan-γδ T cell receptor were used to identify skin-resident γδ T cells, and anti-CD8α was used to identify the proportion of conventional CD4 and CD8-positive αβ T cells within the CD3+ pan-γδ TCR gate. [Figure 1B] This shows that human skin contains a distinct population of resident γδ T cells. Figure 1B shows an overview of these experiments using the Clark protocol for 7–10 donors. Using this protocol, lymphocytes within human skin remained in contact with dermal fibroblasts, and either no cytokines were added, or interleukin-2 (IL-2), interleukin-15 (IL-15), or IL-2 and IL-15 were added. This demonstrated that the use of cytokines did not alter the composition of skin-resident lymphocytes, except for a slightly larger population of γδ T cells when IL-15 or IL-2 and IL-15 were added to the cultures, demonstrating the effectiveness of the Clark et al. protocol. The lymphocyte composition after 3 weeks of organotypic skin culture is shown as an overview for four donors, using the cytokines indicated. [Figure 1C]Figure 1C: Human skin contains distinct populations of resident γδ T cells. Figure 1C: Skin-resident γδ cells comprise primarily Vδ1-expressing γδ T cells (76.24% ± 17.3), a small population of Vδ2-expressing T cells (3.06% ± 6.1), and a population of pan-γδ TCR-positive cells (also referred to herein as double-negative (DN) γδ T cells) that stain negative for Vδ1 and Vδ2 (20.7% ± 13.97). Control staining of blood from healthy volunteers shows a strong localization of human γδ T cells, as the predominant population of γδ T cells in blood expressed the Vδ2 TCR chain. [Figure 1D] Figure 1D: Human skin contains a distinct population of resident γδ T cells. Although skin-resident γδ T cells display markers previously associated with chronically activated T cells, these markers do not necessarily reflect chronic activation and are characteristic indicators of tissue residency. Histograms show staining of the indicated markers for γδ T cells (filled histograms) and appropriate isotype controls for each antibody (open histograms). [Figure 2A]These figures show that skin-resident γδ T cells derived directly from human skin via the Clark protocol exhibit so-called TH1-biased responses upon activation by conventional means for activating T cells, as well as upon activation with NKG2D ligands alone. Figure 2A: Skin-resident γδ T cells exhibit strong expression of the activatory and NK cell-associated receptor NKG2D (filled histogram; compared with the isotype represented by the open histogram). Upon activation with plate-bound recombinant MICA (one of the known ligands for the NKG2D receptor), skin γδ T cells respond without any other stimuli and independently of TCR ligation, as the response is abolished in the presence of a blocking NKG2D antibody. Cells were stimulated for 6 hours in the presence of brefeldin A and 100 units of IL-2 / mL, followed by analysis of degranulation by staining for CD107a. TNFα and IFN-γ production was analyzed by permeabilization after surface staining and subsequent staining for intracellular cytokines. Phorbol 12-myristate 13-acetate (P) in combination with ionomycin (I) was used as a positive control for T cell activation. [Figure 2B] Figure 2B shows that skin-resident γδ T cells derived directly from human skin via the Clark protocol exhibit so-called TH1-biased responses upon activation by conventional means for activating T cells, as well as upon activation with NKG2D ligands alone. (Figure 2B) Skin-resident γδ T cells exhibit TH1-biased responses. γδ T cells were harvested using the Clark protocol, stimulated with PMA and ionomycin for 6 hours in the presence of brefeldin A, and stained for intracellular cytokines. Freshly isolated γδ T cells from human skin produce TNFα and IFN-γ upon stimulation, but produce low or undetectable amounts of cytokines associated with Th2 or Th-17 cells (e.g., IL-4, IL-17A, IL-13, IL-22), whereas conventional CD4+ αβ T cells exhibit a much more diverse range of cytokine production. [Figure 2C] Figure 2C shows that skin-resident γδ T cells derived directly from human skin via the Clark protocol exhibit so-called TH1-biased responses upon activation by conventional means for activating T cells, as well as upon activation with NKG2D ligand alone. Figure 2C: Among lymphocytes derived directly from human skin, varying levels of NKG2D receptors are expressed by γδ T cells, CD8a+ conventional αβ T cells, and NK cells. Among these cells, NK cells respond to exposure to NKG2D ligand alone, whereas among T cells, only the γδ T cell population exhibits a cytokine response upon stimulation with NKG2D ligand in the absence of any TCR stimulation (see the upper row of the flow cytometry dot plots). The response can be blocked using a soluble blocking anti-NKG2D antibody, indicating that the response is exclusively mediated through the NKG2D receptor. [Figure 2D] (Figure 2D) Skin-resident γδ T cells derived directly from human skin via the Clark protocol exhibit so-called TH1-biased responses upon activation by conventional means for activating T cells, as well as upon activation with NKG2D ligands alone. (Figure 2D) Of skin-resident γδ T cells, only Vδ1 γδ T cells and DN γδ T cells exhibit an innate-like ability to be activated by recombinant MICA alone (represented by *). Vδ2-expressing T cells, found in low numbers in the skin, do not exhibit such a response. [Figure 3A]This figure shows that only skin-resident γδ T cells respond to isolation from the dermal stroma with robust activation and expansion. (Figure 3A) Skin-resident lymphocytes were isolated using the Clark protocol. After 3 weeks of organotypic culture, skin lymphocytes were harvested, separated from any remaining skin cells, including fibroblasts, and transferred to tissue culture wells at a density of 1 million lymphocytes / mL, supplemented with 100 U / mL of IL-2. After an additional 3 weeks, resident γδ T cells were significantly expanded and enriched in skin lymphocyte cultures. This significant expansion was restricted to skin-resident γδ T cells, as evidenced by the fact that the majority of Vδ1+ T cells expanded 127.18-fold over the 3-week period, whereas conventional αβ T cells expanded only 5.21-fold (more than 20-fold lower). [Figure 3B] Figure 3B: Only skin-resident γδ T cells respond to separation from the dermal stroma with robust activation and expansion. (Figure 3B) Skin-resident Vδ1+ T cells respond to tissue removal by robustly upregulating the marker Ki-67 (indicating cell cycle) over 14 days (isotype control is represented by the dashed white histogram; Ki-67 expression at day 0 is represented by the white histogram; Ki-67 expression at day 7 is represented by the light gray histogram; Ki-67 expression at day 14 is represented by the dark gray histogram). Furthermore, skin-resident Vδ1 T cells, which are largely negative for IL-2 receptor alpha (CD25) when in contact with the dermal stroma, upregulate CD25 after separation from the tissue (isotype control: dashed histogram; day 0 staining: light gray histogram; day 7 staining: dark gray histogram). [Figure 3C] Only skin-resident γδ T cells respond to isolation from the dermal stroma with robust activation and expansion. (Fig. 3C) A rapid cell cycle, indicated by Ki-67 median fluorescence intensity (MFI), is observed only in skin-resident γδ T cells, represented by Vδ1+ T cells, but not in conventional αβ T cells or NK cells, and the MFI actually decreases in these cells over the course of 14 days. [Figure 3D] Figure 3D: Only skin-resident γδ T cells respond to isolation from the dermal stroma with robust activation and expansion. Skin lymphocytes isolated from stromal cells exhibit a significantly enriched population of resident γδ T cells after 3 weeks of culture. This population of γδ T cells contains a large number of Vδ1-positive cells (77.49% ± 17.04) and pan-γδ TCR-positive DN T cells (21.46% ± 16.92). The initial small population of Vδ2 T cells seen in freshly collected skin lymphocytes using the Clark protocol declines and is almost lost after 3 weeks of expansion of tissue γδ T cells (0.6% ± 1.204). [Figure 4A] This figure shows that skin-resident γδ T cells respond to tissue removal and are suppressed by skin stromal cells, particularly fibroblasts, via a contact-dependent mechanism. Figure 4A: Mixed dermal lymphocytes were cultured in organotypic culture according to the Clark protocol and harvested after 3 weeks. Mixed lymphocytes were then seeded onto a confluent layer of autologous dermal fibroblasts and into transwells to control for the presence of soluble inhibitory factors produced by fibroblasts. After 14 days, fold expansion, calculated via the absolute number of cells present, was measured for γδ T cells and conventional αβ T cells. Skin-resident γδ T cells showed significant expansion when isolated from tissue and in the presence of fibroblasts, but only in the absence of direct cell contact with autologous fibroblasts. Conventional αβ T cells did not show such a response under any of the conditions tested. [Figure 4B]Skin-resident γδ T cells respond to tissue removal and are suppressed by skin stromal cells, particularly fibroblasts, via a contact-dependent mechanism. (Figure 4B) Mixed lymphocytes obtained from organotypic cultures were seeded on a monolayer of autologous fibroblasts (light gray histogram) or into empty wells (dark gray histogram) and cultured for 7 days with IL-2. Skin-resident Vδ1 T cells (left panel) and pan-γδ TCR DN T cells (right panel) remained quiescent in the direct presence of fibroblasts but showed robust activation when isolated from skin organotypic cultures and in the absence of fibroblasts, as indicated by upregulated expression (MFI) of CD25, the Th-1-associated transcription factor T-bet, and the cell cycle marker Ki-67 (dashed white histogram represents the corresponding isotype control). [Figure 5A] Figure 5 shows that expanding skin γδ T cells show signs of disinhibition and acquisition of potent cytotoxicity. Figure 5A: Skin-resident γδ T cells were expanded for 14 days after isolation from organotypic cell culture. γδ T cells were then negatively sorted by flow cytometry by excluding all conventional T cells stained with a pan-αβ TCR monoclonal antibody. 150,000 sorted γδ T cells were then seeded in duplicate into 96-well flat-bottom culture plates and cultured for 24 hours without added cytokines or any activating ligands. Supernatants were collected and analyzed for produced cytokines using an Affymetrix LUMINEX®-based cytokine array. [Figure 5B]This figure shows that expanding cutaneous γδ T cells exhibit signs of disinhibition and acquisition of potent cytotoxicity. (Figure 5B) Negatively sorted γδ T cells were also seeded on cancer cell lines seeded 1 day earlier at a concentration of 10,000 cells / well. As a control, negatively sorted conventional cutaneous αβ T cells were used. T cells were seeded at the indicated effector:target ratios in the presence of 100 U / mL IL-2 and in the presence or absence of blocking NKG2D antibodies. Skin-resident γδ T cells demonstrated superior killing of malignant cell lines compared with conventional αβ T cells, as indicated by caspase-cleaved epithelial-specific cytokeratin 18 (CK18) release (measured via ELISA). Cytotoxicity was at least partially mediated by the NKG2D receptor, as indicated by its reduction in cultures containing antibodies blocking the NKG2D receptor. [Figure 6A] Analysis of tissue-resident γδ T cells in the human gastrointestinal tract. Figure 6A: An adaptation of the Clark protocol enabled the isolation of gut-resident lymphocytes. Mixed gut lymphocytes typically contain primarily Vδ1 T cells, but also a large population of tissue-resident γδ T cells containing Vδ2 and double-negative γδ T cells. [Figure 6B] Analysis of tissue-resident γδ T cells within the human gut (Figure 6B): γδ T cells isolated from gut organotypic cultures show similar responses to skin-derived γδ T cells, as they upregulate Ki-67 over time if separated from the gut stroma. [Figure 6C] Analysis of tissue-resident γδ T cells within the human gut (Figure 6C): Gut-derived γδ T cells respond to innate-like stimuli such as recombinant MICA by producing IFN-γ, as measured by CD107a upregulation, and by degranulation. [Figure 6D]Analysis of tissue-resident γδ T cells within the human gut (Fig. 6D): γδ T cells isolated from gut organotypic cultures show similar responses to skin-derived γδ T cells and expand over time in cell culture, as evidenced by their overall enrichment in lymphocyte cultures without contact with gut stroma. [Figure 7A] Figure 7 shows the tissue phenotype of expanded skin-derived γδ T cells: Figure 7A: Skin-derived γδ T cells stain positive for the skin homing chemokine receptors CCR4 and CCR8. [Figure 7B] Figure 7B shows the tissue phenotype of expanded skin-derived γδ T cells.B: Different expression levels for expanded γδ T cells derived from skin or blood, respectively. [Figure 8] This figure shows that disinhibition of skin-derived γδ T cells without any TCR stimulation results in spontaneous Th1 cytokine production and, interestingly, production of the atopic cytokine IL-13, in contrast to freshly TCR-activated γδ T cells. Consistent with freshly induced γδ T cells, disinhibited and expanding γδ T cells produce negligible amounts of Th2-associated cytokines (e.g., IL-4 and IL-5). Skin-derived γδ T cells were expanded for 14 days and negatively sorted by depleting conventional αβ T cells. 150,000 mixed γδ T cells were cultured in duplicate in 96-well flat-bottom plates from four donors at a density of 1 million cells / mL without any stimulation or cytokine addition. Supernatants were collected after 24 hours and analyzed using an Affymetrix LUMINEX®-based cytokine array. [Figure 9A] FIG. 1 shows that expanded and negatively sorted skin-derived γδ T cells display potent cytotoxicity against various human tumor cell lines co-cultured with them, as measured by the release of caspase-cleaved cytokeratin 18 from target cells using ELISA. [Figure 9B]FIG. 1 shows that expanded and negatively sorted skin-derived γδ T cells display potent cytotoxicity against various human tumor cell lines co-cultured with them, as measured by the release of caspase-cleaved cytokeratin 18 from target cells using ELISA. [Figure 9C] FIG. 1 shows that expanded and negatively sorted skin-derived γδ T cells display potent cytotoxicity against various human tumor cell lines co-cultured with them, as measured by the release of caspase-cleaved cytokeratin 18 from target cells using ELISA. [Figure 10A] Figure 10 shows that fresh, unexpanded skin-derived V51 T cells display markers of previous T cell activation. Figure 10A: Skin-derived V51 T cells highly express CD69 and TIM3, and lowly express CD28. Furthermore, they display high expression of the activation marker NKG2D. This phenotype is maintained by skin-derived V51 T cells during in vitro expansion. In contrast, V51 T cells derived from human blood do not have these signs of activation and do not express CD69 or TIM3. Compared to skin-derived V51 T cells, blood-derived V51 T cells have much lower NKG2D expression, whereas blood-derived V51 T cells express the costimulatory molecule CD28. [Figure 10B] Fresh, unexpanded skin-derived V51 T cells display markers of prior T cell activation. (Fig. 10B) Only skin-derived V51 T cells are reactive to NKG2D ligands, such as recombinant MICA, in the absence of any other stimuli, such as ligands for the T cell receptor. Blood-derived V51 or V52 T cells did not display such responsiveness to innate-like stimuli. Cells were seeded in 96-well plates with recombinant MICA or anti-CD3 antibody, or both, as described. Cells were cultured for 6 hours, with the final 4 hours in IL-2 100 U / mL and BFA, followed by surface antigen staining, permeabilization, and intracellular staining for IFN-γ. [Figure 11]Figure 1 shows that skin-derived V51 T cells express minimal levels of CD16 but show substantial surface expression of the high-affinity IgG receptor CD64. Thus, in addition to direct cytotoxic activity, tissue-derived V51 T cells could also be used to increase the efficacy of monoclonal antibody therapies, such as CD20 or Her2 therapy, as they will be directed by antibodies to sites of malignancies and metastases, recognize opsonized tumor cells, and kill them via antibody-dependent cellular cytotoxicity (ADCC). Results shown are from one representative donor (out of four). [Figure 12] Figure 1 shows the expansion of V51 T cells in IL-2 (left panel), IL-15 (middle panel), and IL-2 + IL-15 (right panel). Freshly isolated skin-derived lymphocytes were cultured in 96-well flat-bottom plates in RPMI medium containing 10% FCS and 1% Pen / Strep, and either IL-2, IL-15, or IL-2 + IL-15 were added for 7 days. Both IL-2 and IL-15, as well as the combination of the two cytokines, induced the expansion of V51 T cells, as indicated by a shift in Ki-67 staining compared to isotype (true negative) staining in the absence of stromal cells. Ki-67 specifically stains cells that have left the G0 phase of the cell cycle and is commonly associated with expansion. [Figure 13] Flow cytometry results showing expression of CD9, CCR3 and CD39 on the surface of expanded V51 T cells on day 21. Expanded skin-derived V51 T cells maintained high levels of the cell surface markers CCR3, CD39 and CD9 as shown by (dark histograms) compared to the corresponding isotype staining (true negative, open histograms). [Figure 14]Figure 1 shows mRNA expression of CCR3 and CD9 in skin-derived V51 T cells (dark bars) and blood-derived V51 T cells (light bars). Skin-derived V51 T cells were expanded as described herein, and blood-derived V51 T cells were expanded using a plate-bound antibody against the V5 T cell receptor (20 μg / mL). After expansion, V51 T cells were isolated using fluorescence-activated cell sorting (FACS), and RNA was isolated from three donors for both groups (blood = gray, skin = black). Total mRNA was sequenced, and expression levels of the indicated mRNAs were normalized and log2-transformed. All expression levels are shown as direct comparisons and as ratios to GAPDH, a common housekeeping gene expressed at high levels in most human cells. [Figure 15] Figure 1 shows IL-13 mRNA expression in skin-derived V51 T cells (dark bars) and blood-derived V51 T cells (light bars). Skin-derived V51 T cells were expanded as disclosed herein, and blood-derived V51 T cells were expanded using a high dose of plate-bound antibody against the V5 T cell receptor (20 μg / mL). After expansion, V51 T cells were isolated using FACS, and RNA was isolated from three donors for both groups (blood = gray, skin = black). Total mRNA was sequenced, and mRNA expression levels to IL-13 were normalized and log2 transformed. Expression levels are shown as a direct comparison and as a ratio to GAPDH. [Figure 16A] Figure 16 shows cytokine production in skin-derived V51 T cells after TCR stimulation with PMA / ionomycin (Figure 16A) or anti-CD3 (Figure 16B). Following isolation and expansion, skin-derived V51 T cells were purified using fluorescence-activated cell sorting (FACS). 150,000 V51 T cells were seeded into 96-well flat-bottom plates in duplicate for three donors and stimulated for 24 hours with either plate-bound CD3 (5 μg / mL) or PMA / ionomycin. Supernatants were analyzed for absolute amounts of the indicated cytokines using a LUMINEX® platform. [Figure 16B]Figure 16 shows cytokine production in skin-derived V51 T cells after TCR stimulation with PMA / ionomycin (Figure 16A) or anti-CD3 (Figure 16B). Following isolation and expansion, skin-derived V51 T cells were purified using fluorescence-activated cell sorting (FACS). 150,000 V51 T cells were seeded into 96-well flat-bottom plates in duplicate for three donors and stimulated for 24 hours with either plate-bound CD3 (5 μg / mL) or PMA / ionomycin. Supernatants were analyzed for absolute amounts of the indicated cytokines using a LUMINEX® platform. [Figure 17A] Figure 17 shows the results for each amplification condition. Representative flow cytometry plots and gating schemes are shown for isolated lymphocytes (after 21 days of culture) (Figure 17A) and lymphocytes expanded for 20 days in the presence of IL-2, IL-4, IL-15, and IL-21 (Figure 17B). [Figure 17B] Figure 17 shows the results for each amplification condition. Representative flow cytometry plots and gating schemes are shown for isolated lymphocytes (after 21 days of culture) (Figure 17A) and lymphocytes expanded for 20 days in the presence of IL-2, IL-4, IL-15, and IL-21 (Figure 17B). [Figure 17C] Fig. 17C shows the results for each expansion condition, with the results for IL-2 (100 U / mL) and IL-15 (10 ng / mL) as the standard, and shows the corrected expansion fold of V51 T cells under various conditions. [Figure 17D] Figure 17D shows the results for each amplification condition: fold amplification as a result of treatment with IL-2+IL-15, IL2+IL-15+IL-4, IL-2+IL-15+IL-21, and IL-2+IL-15+IL-4+IL-21 is shown relative to the separated population. [Figure 17E]Figure 17E shows the results for each amplification condition. Figure 17E shows the total number of Vδ1 cells on day 21 (before amplification) and day 42 (after amplification). Amplification was performed under the following conditions: 100 U / mL IL-2, 100 U / mL IL-2 + 10 ng / mL IL-15, or 100 U / mL IL-2 + 5 ng / mL IL-4 + 10 ng / mL IL-15 + 100 ng / mL IL-21, as indicated (n = 8-17). [Figure 17F] Figure 17 shows the results for each amplification condition. Figure 17F: The mean number (plus standard error of the mean (SEM)) of V51+ T cells in each condition at both time points is shown. **p=0.001. Student's two-arm unpaired t-test (n=8-17). [Figure 17G] Figure 17G shows the results for each expansion condition. Expansion of V51+ T cells using different concentrations of IL-21 is shown, normalized to expansion using 100 U / mL IL-2, 5 ng / mL IL-4, and 10 ng / mL IL-15 alone (n=3). [Figure 17H] Figure 17H ​​shows the results for each expansion condition. Expansion of Vδ1 T cells using 100 U / mL IL-2 + 5 ng / mL IL-4 + 10 ng / mL IL-15 + 10 ng / mL IL-21 is shown relative to expansion with IL-2 alone. [Figure 18A] Figure 18 shows the characteristics of CD27 expression by expanded V51 T cells: Figure 18A: Corrected CD27 expression (mean fluorescence intensity (MFI)) normalized to CD27 expression as a result of IL-2 (100 U / mL) and IL-15 (10 ng / mL). [Figure 18B] Figure 18B: Characterization of CD27 expression by expanded V51 T cells. Figure 18B:I MFI of CD27 is shown relative to the isolated population as a result of IL-2+IL-15, IL2+IL-15+IL-4, IL-2+IL-15+IL-21 and IL-2+IL-15+IL-4+IL-21 treatment. [Figure 18C]Figure 18C: Characterization of CD27 expression by expanded V51 T cells. Figure 18C: V51+ CD27 expression assessed by flow cytometry with different concentrations of IL-21, as compared to expression with 100 U / mL IL-2, 5 ng / mL IL-4 and 10 ng / mL IL-15 alone (n=3). [Figure 18D] Figure 18D: Characterization of CD27 expression by expanded V51 T cells. Figure 18D: V51+ CD27 expression using 100 U / mL IL-2 + 5 ng / ml IL-4 + 10 ng / ml IL-15 + 10 ng / ml IL-21 assessed by flow cytometry, normalized to expansion with IL-2 alone (n=4). [Figure 19] Fig. 1 shows the characteristics of TIGIT surface expression by expanded V51 T cells, showing the fold-corrected expression values ​​(MFI) of TIGIT under various conditions, with TIGIT expression by IL-2 and IL-15 treatment as the standard. [Figure 20] FIG. 1 shows a plot of surface expression of TIGIT on individual cells as a function of CD27 expression. [Figure 21] Figure 1 shows the cytokine-supported expansion and enrichment of tissue-derived γδ T cells in the presence or absence of blood-derived serum or plasma fractions. Tissue-isolated mixed lymphocyte populations containing 2% γδ T cells were expanded in medium containing IL-2, IL-4, IL-15, and IL-21 with or without 10% human AB serum. The data show comparable successful expansion (432-fold) and enrichment (2% to 77%) in the human serum-free condition compared to the results in the human serum-containing condition (295-fold expansion and enrichment from 2% to 75%). [Figure 22]Figures 22A-22D show an example of enrichment of isolated tissue-derived γδ cells from a mixed lymphocyte population. Cells were isolated from a single sample of human tissue and replicated and subsequently expanded in TexMACS medium containing IL-2, IL-4, IL-15, and IL-21, with 10% serum (left-hand column) or 5% Cell Therapy System Serum (CTS™) (right-hand column). Profiles of the isolated and expanded cell cultures are as indicated in the figure. Figure 22A shows that the initial isolated culture contains relatively low numbers (<10%) of the desired tissue-derived γδ T cells. Conversely, Figures 22B-22D show that after expansion, the resulting cell populations are highly enriched for tissue-derived γδ T cells. [Figure 23] Figure 10 shows constitutive TIGIT expression on gut-resident V51 cells. Data were generated using V51 cells isolated from the gut epithelium using a standard isolation protocol for the release of lymphocytes within colonic epithelial cells. [Figure 24A] Figure 24A shows that the poliovirus receptor (PVR) specifically inhibits TCR signaling, as measured by IFNγ (Figure 24A) and TNFα (Figure 24B). Cells were cultured with IL-2 and IL-15 and activated with anti-CD3 antibody. [Figure 24B] Figure 24A shows that the poliovirus receptor (PVR) specifically inhibits TCR signaling, as measured by IFNγ (Figure 24A) and TNFα (Figure 24B). Cells were cultured with IL-2 and IL-15 and activated with anti-CD3 antibody. [Figure 25A] Figure 25B shows that the inhibitory effect of PVR is lost on TIGIT-negative V51+ / V53+ cells, as measured by IFNγ (Figure 25B) and TNFα (Figure 25B) expression. Cells were cultured with IL-2, IL-15, IL-4 and IL-21 and activated with anti-CD3 antibody. [Figure 25B]Figure 25 shows that the inhibitory effect of PVR is lost on TIGIT-negative V51 / V53 cells, as measured by IFNγ (Figure 25A) and TNFα (Figure 25B) expression. Cells were cultured with IL-2, IL-15, IL-4, and IL-21 and activated with anti-CD3 antibody. [Figure 26A] Figure 26 shows graphs demonstrating that IL-9 can replace the function of IL-2 in expanding skin-derived γδ T cells. Skin tissue from three donors (TS052, TS056, and SK073) was plated on 9 mm grids and cultured in medium supplemented with IL-2 and IL-15 for 3 weeks. Isolated lymphocytes were then expanded in medium supplemented with IL-2, IL-4, IL-15, and IL-21 (left bars) or IL-4, IL-9, IL-15, and IL-21 (right bars). After 3 weeks of expansion, the final numbers of γδ T cells / grid (Figure 26A) and Vδ1 cells / grid (Figure 26B) were calculated. Replacing IL-2 with IL-9 in expanding skin-derived γδ T cells resulted in comparable expansion effects. Histograms show mean + / - SEM. [Figure 26B] Figure 26 shows graphs demonstrating that IL-9 can replace the function of IL-2 in expanding skin-derived γδ T cells. Skin tissue from three donors (TS052, TS056, and SK073) was plated on 9 mm grids and cultured in medium supplemented with IL-2 and IL-15 for 3 weeks. Isolated lymphocytes were then expanded in medium supplemented with IL-2, IL-4, IL-15, and IL-21 (left bars) or IL-4, IL-9, IL-15, and IL-21 (right bars). After 3 weeks of expansion, the final numbers of γδ T cells / grid (Figure 26A) and Vδ1 cells / grid (Figure 26B) were calculated. Replacing IL-2 with IL-9 in expanding skin-derived γδ T cells resulted in comparable expansion effects. Histograms show mean + / - SEM. [Figure 27A]Figures 27A-C show that IL-9 can substitute for IL-2 in expanding skin-derived γδ T cells, as measured by fold change in expansion (Figure 27A), % of γδ TCR+ T cells (Figure 27B), and % of Vδ1+ T cells (Figure 27C). Skin tissue was derived from six donors (SK073, SK075, SK077, TS052, TS053, and TS056). 2CK = 2 cytokines (IL-2 + IL-15); 4CK = 4 cytokines (IL-2 + IL-15 + IL-21 + IL-4). [Figure 27B] Figures 27A-C show that IL-9 can substitute for IL-2 in expanding skin-derived γδ T cells, as measured by fold change in expansion (Figure 27A), % of γδ TCR+ T cells (Figure 27B), and % of Vδ1+ T cells (Figure 27C). Skin tissue was derived from six donors (SK073, SK075, SK077, TS052, TS053, and TS056). 2CK = 2 cytokines (IL-2 + IL-15); 4CK = 4 cytokines (IL-2 + IL-15 + IL-21 + IL-4). [Figure 27C] Figures 27A-B show that IL-9 can substitute for IL-2 in expanding skin-derived γδ T cells, as measured by fold change in expansion (Figure 27A), % of γδ TCR+ T cells (Figure 27B), and % of Vδ1+ T cells (Figure). Skin tissue was derived from six donors (SK073, SK075, SK077, TS052, TS053, and TS056). 2CK = 2 cytokines (IL-2 + IL-15); 4CK = 4 cytokines (IL-2 + IL-15 + IL-21 + IL-4). DETAILED DESCRIPTION OF THE INVENTION

[0044] I. Introduction Provided herein are methods for expanding γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells, and / or double-negative T cells) using a non-hematopoietic tissue as a cell source (e.g., non-hematopoietic tissue-derived γδ T cells, e.g., non-hematopoietic tissue-derived Vδ1 T cells). The expansion method comprises culturing γδ T cells (e.g., γδ T cells isolated from stromal cells of a non-hematopoietic tissue) in the substantial absence of TCR stimulation and / or in the presence of interleukin-4 (IL-4), interleukin-15 (IL-15), interleukin-21 (IL-21), and / or interleukin-2 (IL-2). Additionally provided are compositions of expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells, and / or DN T cells), and methods of using the expanded γδ T cells (e.g., adoptive T cell therapy, e.g., for the treatment of cancer).

[0045] II. Definition Aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments. As used herein, the singular forms "a," "an," and "the" include plural application unless specifically indicated otherwise.

[0046] As used herein, the term "about" refers to a typical error range for each value, which is readily known to those skilled in the art. Reference herein to "about" a value or parameter includes (and discloses) embodiments directed to the value or parameter itself. In some examples, "about" includes variations of +20%, in some examples +10%, in some examples +5%, in some examples +1%, or in some examples +0.1% from the specified value, where such variations are suitable for practicing the disclosed methods.

[0047] As used herein, the terms "substantial" and "substantially" refer to qualitative terms indicating the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the art of biology will understand that biological and chemical phenomena rarely reach and / or progress toward completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential inherent lack of completeness in many biological and chemical phenomena. When describing a physical scenario, such as a receptor / ligand interaction or cell-cell contact, such a scenario is substantial if its functional outcome is detectable by conventional means available to the practitioner of the method. For example, "substantial TCR activation" refers to a detectable level of TCR activation in a population of cells (e.g., a statistically significant degree of TCR activation). In some embodiments, the TCR is activated by ECs on each cell population. 50 Exposure of up to 0.1%, 0.5%, 1%, 5%, 10%, 20%, 30%, or 40% of the expanded cells to a TCR pathway agonist (e.g., an antibody, e.g., anti-CD3, or a lectin) results in substantial activation. Similarly, "substantial cell contact" (e.g., substantial support cell contact, substantial stromal cell contact, or substantial tumor cell contact) refers to the degree of cell-to-cell contact that can result in a detectable change in the expanded cells (e.g., reduced expansion). In some instances, substantial cell contact occurs when a contaminating cell type (e.g., support cells, stromal cells, or tumor cells) is present in the culture at a concentration of up to 0.1%, 0.5%, 1%, 5%, 10%, or 20% relative to the population of expanded cells. A "substantial number" of cells or a "substantial amount" of an agent similarly refers to the number or amount necessary to produce a substantial effect, as defined above.

[0048] As used herein, "non-hematopoietic cells" includes stromal cells and epithelial cells. Stromal cells are non-hematopoietic connective tissue cells of any organ and support the function of the organ's parenchymal cells. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinocytes, endothelial cells, and non-hematopoietic tumor cells. Epithelial cells are non-hematopoietic cells that line the cavities and surfaces of blood vessels and organs throughout the body. They are usually flat, cylindrical, or cuboid in shape and can be arranged as a single cell layer or two or more cell layers.

[0049] As used herein, "non-hematopoietic tissue-resident γδ T cells," "non-hematopoietic tissue-derived," and "native non-hematopoietic tissue γδ T cells" refer to γδ T cells present in the non-hematopoietic tissue at the time the tissue was removed. Non-hematopoietic tissue-resident γδ T cells can be obtained from any suitable non-hematopoietic tissue of a human or non-human animal. A non-hematopoietic tissue is a tissue other than blood or bone marrow. In some embodiments, the γδ T cells are not obtained from a sample of a specific type of biological fluid, such as blood or synovial fluid. Suitable examples of non-hematopoietic tissues of a human or non-human animal include skin or a portion thereof (e.g., dermis or epidermis), gastrointestinal tract (e.g., gastrointestinal epithelium, colon, small intestine, stomach, appendix, cecum, or rectum), mammary gland tissue, lung (preferably not tissue obtained by bronchoalveolar lavage), prostate, liver, and pancreas. In some embodiments, non-hematopoietic tissue-resident γδ T cells can be derived from lymphoid tissue, such as the thymus, spleen, or tonsils. γδ T cells may be resident in human cancer tissues, such as, for example, breast and prostate. In some embodiments, the γδ T cells are not obtained from human cancer tissue. Samples of non-hematopoietic tissues may be obtained by standard techniques, such as, for example, explantation (e.g., biopsy). Non-hematopoietic tissue-resident γδ T cells include, for example, non-V52 T cells, such as V51 T cells, double-negative (DN) T cells, V53 T cells, and V55 T cells.

[0050] As used herein, "IL-2" refers to native or recombinant IL-2 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-2 receptor (IL-2R) subunits. These agents can support the growth of the IL-2-dependent cell line CTLL-2 (33; American Type Culture Collection (ATCC®) TIB 214). Mature human IL-2 occurs as a 133-amino acid sequence (lacking an additional N-terminal 20-amino acid signal peptide) as described by Fujita, et al. Cell 1986. 46.3:401-407. IL-2 muteins are polypeptides with specific substitutions made to the interleukin-2 protein while retaining the ability to bind to IL-2Rβ, as described in U.S. Patent Application Publication No. 2014 / 0046026. IL-2 muteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more positions or at other residues in the native IL-2 polypeptide chain. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications results in an IL-2 mutein that retains IL-2Rβ binding activity. Examples of muteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0051] Nucleic acids encoding human IL-2 can be obtained by conventional procedures, such as polymerase chain reaction (PCR). The amino acid sequence of human IL-2 (Gene ID 3558) can be found in Genbank under accession locator NP_000577.2 GI:28178861. The amino acid sequence of mouse (Mus musculus) IL-2 (Gene ID 16183) can be found in Genbank under accession locator NP_032392.1 GI:7110653.

[0052] IL-2 can refer to IL-2 derived from various mammalian species, including, for example, human, monkey, bovine, porcine, equine, and murine. Variants may include conservative substitutions in the sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as between Ile, Val, Leu, or Ala, or the substitution of one polar residue for another, such as between Lys and Arg, between Glu and Asp, or between Gln and Asn. Other examples of such conservative substitutions include the substitution of entire regions with similar hydrophobic properties. Naturally occurring IL-2 variants are also included in the present invention. Examples of such variants include proteins generated by alternative mRNA splicing events or proteolytic cleavage of the IL-2 protein, which retain the binding properties of IL-2. Alternative splicing of mRNA can result in cleaved IL-2 proteins that retain biological activity. Changes due to proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to the proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-2 protein. In some embodiments, the termini or interior of the protein can be modified with chemical groups, such as polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the termini or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0053] As used herein, "IL-15" refers to native or recombinant IL-15 or its variants (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics) that act as agonists for one or more IL-15 receptor (IL-15R) subunits. IL-15 is a known T cell growth factor that, like IL-2, can support the proliferation of the IL-2-dependent cell line CTLL-2. IL-15 was first reported by Grabstein et al. as a 114-amino acid mature protein (Grabstein, et al. Science 1994. 264.5161: 965-969). As used herein, the term "IL-15" refers to native or recombinant IL-15 and its muteins, analogs, subunits, or complexes (e.g., receptor complexes, e.g., sushi peptides, as described in WO 2007 / 046006), each of which can stimulate the proliferation of CTLL-2 cells. In an assay for CTLL-2 proliferation, cell supernatants transfected with recombinantly expressed in-frame fusions of precursor and mature IL-15 can induce the proliferation of CTLL-2 cells.

[0054] Human IL-15 can be obtained by the procedure described in Grabstein et al. (Grabstein, et al. Science 1994. 264.5161: 965-969) or by conventional procedures such as polymerase chain reaction (PCR). A human IL-15 cDNA was deposited with ATCC® on February 19, 1993, and assigned accession number 69245.

[0055] The amino acid sequence of human IL-15 (Gene ID 3600) can be found in GenBank under the accession locators NP000576.1 GI: 10835153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2). The amino acid sequence of mouse (Mus musculus) IL-15 (Gene ID 16168) can be found in GenBank under the accession locator NP_001241676.1 GI: 363000984.

[0056] IL-15 can also refer to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. As used herein, an IL-15 "mutein" or "variant" is a polypeptide having an amino acid sequence that is substantially homologous to the sequence of native mammalian IL-15 but differs from that of native mammalian IL-15 due to amino acid deletions, insertions, and substitutions. A variant may include conservative substitutions in the sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue with another aliphatic residue, such as between Ile, Val, Leu, or Ala, or the substitution of one polar residue with another polar residue, such as between Lys and Arg, between Glu and Asp, or between Gln and Asn. Other examples of such conservative substitutions include the well-known substitution of entire regions with similar hydrophobic properties. Naturally occurring IL-15 variants are also encompassed by the present invention. Examples of such variants include proteins generated by alternative mRNA splicing events or proteolytic cleavage of the IL-15 protein, which retain the binding properties of IL-15. Alternative splicing of mRNA can result in cleaved IL-15 proteins that retain biological activity. Changes due to proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to the proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-15 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the termini or interior of the protein can be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0057] As used herein, "IL-4" refers to native or recombinant IL-4 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists at one or more IL-4 receptor (IL-4R) subunits. Such agents support the differentiation of naive helper T cells (Th0 cells) into Th2 cells. Mature human IL-4 occurs as a 129-amino acid sequence (shortened by an additional N-terminal 24-amino acid signal peptide). IL-4 muteins are polypeptides with specific substitutions in the interleukin-4 protein that retain the ability to bind to IL-4Rα, as described in U.S. Patent No. 6,313,272. IL-4 muteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more positions or at other residues in the native IL-4 polypeptide chain. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications will produce IL-4 muteins that retain IL-2Rα binding activity. Exemplary muteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0058] Nucleic acids encoding human IL-4 can be obtained by conventional procedures, such as polymerase chain reaction (PCR). The amino acid sequence of human IL-4 (Gene ID 3565) can be found in Genbank under accession locator NG_023252. The amino acid sequence of mouse (Mus musculus) IL-4 (Gene ID 16189) can be found in Genbank under accession locator NC_000077.6.

[0059] IL-4 can also refer to IL-4 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may contain conservative substitutions in the sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as between Ile, Val, Leu, or Ala, or the substitution of one polar residue for another, such as between Lys and Arg, between Glu and Asp, or between Gln and Asn. Other examples of such conservative substitutions include the substitution of entire regions with similar hydrophobic properties. Naturally occurring IL-4 variants are also included in the present invention. Examples of such variants include proteins generated by alternative mRNA splicing events or proteolytic cleavage of the IL-4 protein, which retain the binding properties of IL-4. Alternative splicing of mRNA can result in cleaved IL-4 proteins that retain biological activity. Changes due to proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to the proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-4 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the termini or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0060] As used herein, "IL-21" refers to native or recombinant IL-21 or variants thereof that act as agonists at one or more IL-21 receptor (IL-21R) subunits (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). Such agents are capable of inhibiting natural killer (NK) and cytotoxic (CD8) responses. + (IL-21) can support the proliferation of T cells. Mature human IL-21 occurs as a 133 amino acid sequence (lacking a signal peptide consisting of an additional 22 amino acids at the N-terminus). IL-21 muteins are polypeptides containing specific substitutions in the interleukin-21 protein while retaining the ability to bind to IL-21Rα, as described in U.S. Patent No. 9,388,241. IL-21 muteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more positions in the native IL-21 polypeptide chain or at other residues. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications will generate IL-21 muteins that retain IL-21Rα binding activity. Examples of muteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0061] Nucleic acids encoding human IL-21 can be obtained by conventional procedures, such as polymerase chain reaction (PCR). The amino acid sequence of human IL-21 (Gene ID 59067) can be found in Genbank under accession locator NC_000004.12. The amino acid sequence of mouse (Mus musculus) IL-21 (Gene ID 60505) can be found in Genbank under accession locator NC_000069.6.

[0062] IL-21 can also refer to IL-21 derived from various mammalian species, including, for example, humans, monkeys, cows, pigs, horses, and mice. Variants may contain conservative substitutions in the sequence, meaning that a given amino acid residue is replaced by a residue with similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as between Ile, Val, Leu, or Ala, or the substitution of one polar residue for another, such as between Lys and Arg, between Glu and Asp, or between Gln and Asn. Other examples of such conservative substitutions include the substitution of entire regions with similar hydrophobic properties. Naturally occurring IL-21 variants are also included in the present invention. Examples of such variants include proteins generated by alternative mRNA splicing events or proteolytic cleavage of the IL-21 protein, which retain the binding properties of IL-21. Alternative splicing of mRNA can result in cleaved IL-21 proteins that retain biological activity. Changes due to proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to the proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-21 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the termini or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0063] As used herein, "IL-9" refers to native or recombinant IL-9 or variants thereof (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) that act as agonists for one or more IL-9 receptor (IL-9R) subunits. Mature human IL-9 occurs as a 144-amino acid sequence. IL-9 muteins are polypeptides with specific substitutions made to the interleukin-9 protein while retaining the ability to bind to IL-9R. IL-9 muteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more positions or at other residues in the native IL-9 polypeptide chain. In accordance with the present disclosure, any of the above insertions, deletions, substitutions, and modifications will generate IL-9 muteins that retain IL-9R binding activity. Examples of muteins include those containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acid substitutions.

[0064] Nucleic acids encoding human IL-9 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-9 is provided by UniProtKB P15248.

[0065] IL-9 can also refer to IL-9 derived from various mammalian species, including, for example, humans, monkeys, cattle, pigs, horses, and mice. Variants may include conservative substitutions in the sequence, meaning that a given amino acid residue is replaced with a residue having similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as between Ile, Val, Leu, or Ala, or the substitution of one polar residue for another, such as between Lys and Arg, between Glu and Asp, or between Gln and Asn. Other examples of such conservative substitutions include the substitution of entire regions with similar hydrophobic properties. Naturally occurring IL-9 variants are also included in the present invention. Examples of such variants include proteins generated by alternative mRNA splicing events or proteolytic cleavage of the IL-9 protein, which retain the binding properties of IL-9. Alternative splicing of mRNA can result in cleaved IL-9 proteins that retain biological activity. Changes due to proteolysis include, for example, differences in the N- or C-terminus upon expression in different types of host cells due to the proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-9 protein. In some embodiments, the termini of the protein can be modified with chemical groups, such as polyethylene glycol, to alter its physical properties (Yang, et al. Cancer 1995. 76:687-694). In some embodiments, the termini or interior of the protein may be modified with additional amino acids (Clark-Lewis, et al. PNAS 1993. 90:3574-3577).

[0066] One or more of the above agents can be incorporated into an expansion protocol in an amount sufficient to generate expanded γδ T cells. As used herein, the phrase "in an amount effective to" refers to an amount that induces a detectable result (e.g., a statistically significant increase in the number of cells compared to the starting population, e.g., p<0.05). When multiple agents are present at once, an effective amount refers to the combined effect of all agents (e.g., the combined effect of IL-2 and IL-15, or the combined effect of IL-2 or IL-9, IL-4, IL-15, and IL-21).

[0067] "T cell receptor (TCR) pathway agonists" or "agents that activate the TCR pathway" refer to compounds that induce proliferation or other activation of T cells, such as αβ T cells and / or blood-resident γδ T cells, through TCR signaling. T cell signaling modulators function by sequential activation of the Src-related protein tyrosine kinases (PTKs), Lck and Fyn, and the 70 kDa zeta chain (TCR)-associated protein kinase (ZAP70). These PTKs phosphorylate polypeptides, including linker-activator of T cells (LAT), which leads to downstream stimulation via extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor of activated T cells (NFAT). Costimulation, for example, via CD28 and CD45, promotes phosphorylation and enhances the TCR signaling pathway. Therefore, substances that target the TCR or parts of the costimulatory pathway can activate T cell signaling. TCR pathway agonists include antibodies (e.g., monoclonal antibodies, e.g., anti-TCRVδ1, anti-TCRδTCS-1, anti-TCRPANγδ, and anti-CD3 antibodies), lectins (e.g., plant lectins, e.g., concanavalin A; lectins from Phaseolus vulgaris (PHA-P), Phytolacca Americana, Triticum vulgaris, Lens culinaris, Glycine max, Maackia amurensis, Pisum sativum, and Sambucus nigra), synthetic phosphoantigens (e.g., BrHPP (bromohydrin pyrophosphate), 2M3B1PP (2-methyl-3-butenyl-1-pyrophosphate), HMBPP ((E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate), or IPP (isopentenyl pyrophosphate)), and N-bisphosphonates (e.g., zoledronate).TCR pathway agonists include coreceptor agonists and include antibodies (e.g., monoclonal antibodies, e.g., anti-CD2, anti-CD6, anti-CD9, anti-CD28, anti-CD43, anti-CD94, anti-CD160, anti-SLAM, anti-NKGD2, anti-2B4, anti-HLA-A, anti-HLA-b, anti-HLA-C, and anti-ICAM-3 antibodies) and proteins (e.g., recombinant proteins, e.g., recombinant human proteins, e.g., CD7L, CD26, CD27L, CD30L, CD40L, OX40L, 4-1BBL, ICAM-1, fibronectin, hydrocortisone, and variants thereof, e.g., Fc fusion proteins, e.g., CD27L-Fc). TCR pathway agonists can be soluble or membrane-bound and can be presented on cells, such as artificial antigen-presenting cells (aAPCs), as in the case of, for example, MHC or HLA complexes. Suitable aAPCs for activating T cell signaling are known in the art. Suitable methods for activating T cells by exogenously adding TCR pathway agonists are well known in the art and are summarized in Figure 1 of Deniger et al. (Deniger, et al. Frontiers in Immunology, 2014. 5(636):1-10).

[0068] "Exogenous TCR pathway agonist" refers to a TCR pathway agonist that is not derived from non-hematopoietic tissue or its donor (i.e., exogenously added). Thus, in some embodiments of the present invention, it will be understood that TCR pathway agonist (e.g., soluble fibronectin or cell-bound ICAM-1) may be present in the culture as residual material from the non-hematopoietic tissue. In some embodiments, the residual TCR pathway agonist is at negligible concentrations and does not substantially activate T cells.

[0069] As used herein, the terms "synthetic scaffold," "scaffold," and "grid" are used interchangeably to refer to a non-native, three-dimensional structure suitable for supporting cell growth. Explants may be attached to the synthetic scaffold to promote lymphocyte migration from the explant to the scaffold. Synthetic scaffolds are constructed of natural and synthetic materials, such as polymers (e.g., natural or synthetic polymers, e.g., polyvinylpyrrolidone, polymethylmethacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (tantalum, titanium, platinum, and metals of the same element group as platinum, niobium, hafnium, tungsten, and combinations of these alloys). Biological factors (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, antibodies and fragments thereof, cytokines (e.g., IL-2 or IL-15, and combinations thereof) can be coated onto the scaffold surface or encapsulated within the material of the scaffold to enhance cell adhesion, migration, survival, or proliferation, according to methods known in the art. This and other methods can be used to isolate lymphocytes from many other types of non-hematopoietic tissues, such as, for example, the gastrointestinal tract, prostate, and breast. Exemplary synthetic scaffolds contemplated for use as part of the present invention include those used in the Clark protocol.

[0070] As used herein, the terms "separation," "separated," or "separating" refer to the disruption or inhibition of physical contact between different cell populations (e.g., the separation of hematopoietic cells (e.g., lymphocytes) from non-hematopoietic cells). Separation can be achieved, for example, by pipetting the mixed cell population to forcibly disrupt intermembrane junctions, or by inducing the cell population to "crawl out" from the tissue matrix, for example, by culturing the cell population with chemokines or cytokines, as described in Carrasco et al. (Carrasco A. et al. Journal of Immunological Methods 2013. 389(1-2):29-37). Separation can also be maintained by using a transwell culture system or similar culture method that inhibits physical contact between different cell populations.

[0071] As used herein, an "isolated population of γδ cells" refers to a population of hematopoietic cells comprising γδ cells that have been separated from their non-hematopoietic tissue of origin (e.g., according to a separation protocol described herein) so as to be substantially free of contact with non-hematopoietic cells. Similarly, an "isolated population of V51 T cells" refers to a population of hematopoietic cells comprising V51 T cells that have been separated from their non-hematopoietic tissue of origin (e.g., according to a separation protocol described herein) so as to be substantially free of contact with non-hematopoietic cells. Thus, in these examples, separation refers to the separation of hematopoietic cells (e.g., lymphocytes) from non-hematopoietic cells (e.g., stromal and / or epithelial cells).

[0072] The term "antibody" is used in the broadest sense and covers, inter alia, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they perform the desired biological activity.

[0073] As used herein, an "expansion step" refers to a stage of culture after isolation, in which a certain number of γδ T cells are increased by cell division. It will be understood that cell division can occur during the isolation stage while the γδ T cells are in contact with stromal cells, but the expansion step does not begin until isolation is complete. Thus, when a population of isolated cells is characterized as being at a time point "before the expansion step," this means a time point after isolation culture and before expansion culture.

[0074] As used herein, an "expanded population of γδ cells" refers to a population of hematopoietic cells comprising γδ T cells that have been continuously cultured under conditions that induce γδ cell expansion, i.e., an increase in the number of γδ cells. Similarly, as used herein, an "expanded population of V51 T cells" refers to a population of hematopoietic cells comprising V51 T cells that have been continuously cultured under conditions that induce V51 T cell expansion, i.e., an increase in the number of V51 cells.

[0075] As used herein, "feeder cells" refers to exogenous cells added to a culture to provide cell-to-cell surface contact for non-hematopoietic tissue-derived cells. Feeder cells may be primary cells (e.g., tissue-derived) or cells derived from a cell line. Feeder cells may be live or irradiated cells, and include tumor cells, fibroblasts, B cells, and other antigen-presenting cells.

[0076] As used herein, the term "marker" refers to a DNA, RNA, protein, carbohydrate, glycolipid, or cell-based molecular marker, the expression or presence of which in a patient sample can be detected by standard methods (or methods disclosed herein).

[0077] A single cell or population of cells "expresses" a marker of interest when the mRNA encoding the protein, or the protein itself, including a fragment thereof, is determined to be present in the cell or population. Marker expression can be detected in a variety of ways. For example, in some embodiments, marker expression is indicated by the surface density of the marker on the cells. For example, mean fluorescence intensity (MFI), used as a readout in flow cytometry, represents the density of the marker on a population of cells. Those skilled in the art will understand that the value of MFI depends on staining parameters (e.g., concentration, duration, and temperature) and the composition of the fluorescent dye. However, MFI can be quantitative when examined in the context of appropriate controls. For example, if the MFI of an antibody against a marker is significantly higher than the MFI of an appropriate isotype control antibody on the same population of cells stained under comparable conditions, the population of cells is said to express that marker. Additionally or alternatively, a population of cells can be analyzed using positive and negative gates (e.g., gates set for isotype or "fluorescence minus one" (FMO) controls) to detect marker expression on a cell-by-cell basis according to conventional flow cytometry analysis methods. By this metric, a population can be said to "express" a marker if the number of cells detected as positive for the marker is significantly greater than background (e.g., by gating on an isotype control).

[0078] As used herein, the expression of a population is described as the proportion (percentage) of positive cells, and when this proportion is compared with the corresponding proportion of positive cells in a reference population, the difference in proportion indicates the proportion of the parent population of each population. For example, if a marker is expressed in 10% of cells in population A, and the same marker is expressed in 1% of cells in population B, then population A can be said to have a 9% higher frequency of marker-positive cells (i.e., 10%-1%, not 10%÷1%). Multiplying the frequency by the number of cells in the parent population calculates the difference in absolute number of cells. In the above example, if population A has 100 cells and population B has 10 cells, then population A has 100 times as many cells as population B; i.e., (10%×100)÷(1%×10).

[0079] The expression level may be a nucleic acid expression level (e.g., a DNA expression level or an RNA expression level, e.g., an mRNA expression level). Any suitable method for measuring nucleic acid expression may be used. In some embodiments, the nucleic acid expression level is measured using qPCR, rtPCR, RNA-seq, multiplex qPCR or RT-qPCR, microarray analysis, serial analysis of gene expression (SAGE), MassARRAY technology, in situ hybridization (e.g., FISH), or a combination thereof.

[0080] As used herein, a "reference population" of cells is a population of cells corresponding to cells of interest, and the phenotype of the cells of interest is measured. For example, the expression level of a marker on an isolated population of γδ cells derived from a non-hematopoietic tissue is compared to that of γδ T cells derived from a hematopoietic tissue (e.g., blood-resident γδ cells, e.g., blood-resident γδ cells from the same donor or another donor), or to that of γδ T cells derived from a non-hematopoietic tissue expanded under different conditions (e.g., with substantial TCR activation, in the presence of an exogenous TCR activator (e.g., anti-CD3), or with substantial contact with stromal cells (e.g., fibroblasts)). Comparison can also be made to an earlier state of the same population. For example, the reference population can be a population of isolated cells prior to expansion. In this case, the expanded population is compared to the composition of the same population prior to the expansion step; that is, in this case, the previous composition is the reference population.

[0081] "Cancer" refers to the abnormal proliferation of malignant cancer cells, and includes leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL), lymphomas such as Hodgkin's lymphoma, non-Hodgkin's lymphoma, and multiple myeloma, as well as solid cancers such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder cancer and biliary tract cancer, thyroid cancer, thymus cancer, bone cancer, and brain cancer.

[0082] Cancer cells in a cancer patient can be immunologically distinguishable from the individual's normal somatic cells (e.g., cancerous tumors can be immunogenic). For example, cancer cells can induce a systemic immune response in the cancer patient's body against one or more antigens expressed by the cancer cells. The antigens that can induce an immune response can be tumor antigens or can be shared with normal cells. Patients with cancer can exhibit at least one recognizable sign, symptom, or laboratory finding sufficient to diagnose cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks such as Harrison's Principles of Internal Medicine (Longo DL, Fauci AS, Kasper DL, Hauser SL, Jameson J, Loscalzo J. eds. 18e. New York, NY: McGraw-Hill; 2012). For example, diagnosing an individual's cancer can include identifying a characteristic cell type (e.g., cancer cells) in a sample of bodily fluid or tissue obtained from the individual.

[0083] As used herein, a "solid cancer" is a cancer of any body tissue other than blood, bone marrow, or lymphatic system. Solid cancers can be further divided into cancers of epithelial cell origin and cancers of non-epithelial cell origin. Examples of solid tumors of epithelial cells include tumors of the digestive tract, colon, breast, prostate, lung, kidney, liver, pancreas, ovary, head and neck, oral cavity, stomach, duodenum, small intestine, large intestine, anus, gallbladder, labia, nasopharynx, skin, uterus, male reproductive organs, urinary tract, bladder, and skin. Examples of solid tumors of non-epithelial cells include sarcomas, brain tumors, and bone tumors.

[0084] The patient, subject, or individual suitable for the above treatment may be a mammal such as a rodent (e.g., guinea pig, hamster, rat, mouse), murine (e.g., mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, simian (e.g., monkey or ape), monkey (e.g., marmoset or baboon), ape (e.g., gorilla, chimpanzee, orangutan or gibbon), or human.

[0085] In some embodiments, the patient, subject, or individual is a human. In other preferred embodiments, non-human mammals can be used, particularly mammals traditionally used as models for demonstrating therapeutic efficacy in humans.

[0086] As used herein, "treatment" (and grammatical variations such as "treat" or "treating") refers to a clinical intervention in a human or animal (e.g., in veterinary applications) to achieve a desired therapeutic effect, such as inhibiting or slowing the progression of a disease state, including slowing the rate of progression, halting the rate of progression, ameliorating a disease state, curing or ameliorating (partially or totally) a disease state, preventing, delaying, alleviating or arresting one or more symptoms and / or signs of a condition, or prolonging the survival of a subject or patient beyond that expected in the absence of treatment.

[0087] Treatment as a preventative measure (e.g., prevention) is also included. For example, a patient, subject, or individual who is susceptible to or at risk of developing or recurring cancer can be treated as described herein. Such treatment can prevent or delay the development or recurrence of cancer in the patient, subject, or individual.

[0088] In particular, treatment includes the suppression of cancer growth, including complete remission of cancer and / or the suppression of cancer metastasis. Cancer growth generally manifests itself in any one of several indicators, indicating a change to a more advanced form within the cancer. Thus, indicators for measuring the suppression of cancer growth include a decrease in the viability of cancer cells, a decrease in tumor size or morphology (e.g., determined using computed tomography (CT), ultrasound, or other imaging methods), a delay in tumor growth, destruction of tumor blood vessels, an improvement in delayed-type hypersensitivity skin test performance, an increase in the activity of cytotoxic T lymphocytes, and a decrease in the level of tumor-specific antigens. Reducing immunosuppression in an individual's cancerous tumor can improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or reduce the individual's tendency to develop cancer.

[0089] In some embodiments, expanded γδ T cells (e.g., γδ T cells from non-hematopoietic tissue, e.g., Vδ1 T cells from non-hematopoietic tissue) are administered to delay disease development or slow the progression of a disease or disorder.

[0090] As used herein, "administering" means providing a patient with a dose of a therapy (e.g., adoptive T cell therapy, including, for example, γδ T cells derived from non-hematopoietic tissue) or composition (e.g., a pharmaceutical composition, e.g., a pharmaceutical composition comprising γδ T cells derived from non-hematopoietic cells). Compositions utilized in the methods described herein can be administered, for example, intramuscularly, intravenously, intradermally, transcutaneously, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, intraprostatically, intrathoracically, intratracheally, subarachnoidally, intranasally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctivally, intravesically, mucosally, intrapericardially, intraumbilically, intraocularly, intraorbitally, intravitreally (e.g., via intravitreal injection), ophthalmically, orally, topically, transdermally, by inhalation, injection, implantation, infusion, continuous infusion, localized perfusion directly bathing target cells, catheter, lavage, cream, or lipid composition. The compositions utilized in the methods described herein may be administered systemically or locally, and the method of administration may vary depending on a variety of factors, such as the therapeutic agent or composition being administered and the severity of the condition, disease, or disorder being treated.

[0091] A "therapeutically effective amount" refers to that amount of a therapeutic agent to treat or inhibit a disease or disorder in a mammal. In the case of cancer, a therapeutically effective amount of a therapeutic agent (e.g., non-hematopoietic tissue-derived γδ T) may reduce the number of cancer cells, reduce the size of the primary tumor, inhibit (i.e., slow to some extent, and preferably stop) cancer cell invasion into peripheral tissues, inhibit (i.e., slow to some extent, and preferably stop) tumor metastasis, inhibit tumor growth for some period of time, and / or alleviate to some extent one or more symptoms associated with the disorder. To the extent a drug can prevent the growth of cancer cells and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. In the case of cancer treatment, in vivo efficacy can be measured, for example, by assessing survival time, time to disease progression (TTP), response rate (e.g., complete response (CR) and partial response (PR)), duration of response, and / or quality of life.

[0092] The term "concurrently," as used herein, refers to the administration of two or more therapeutic agents that at least partially overlap in time. Thus, concurrent administration includes dosing regimens in which administration of one or more agents continues after the administration of one or more other agents is discontinued. For example, in some embodiments, non-hematopoietic tissue-derived γδ T cells and IL-2 may be administered simultaneously.

[0093] The term "pharmaceutical composition" refers to a formulation that is in a form that effectively utilizes the biological activity of one or more active ingredients contained therein and that does not contain additional ingredients that are unacceptably toxic to the patient receiving the formulation.

[0094] III. Methods for Isolating and Expanding γδ T Cells The present invention provides methods for isolating and expanding γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) from non-hematopoietic tissue of a human or non-human animal that can be or has been removed from a patient. In some embodiments, the non-hematopoietic tissue from which γδ T cells are isolated and expanded is skin (e.g., human skin) and is obtained by methods known in the art. In some embodiments, the skin is obtained by punch biopsy. Alternatively, the methods for isolating and expanding γδ T cells provided herein can be applied to the gastrointestinal tract (e.g., colon), breast, lung, prostate, liver, spleen, or pancreas. γδ T cells may be resident in human cancer tissue, e.g., breast or prostate tumors. In some embodiments, γδ T cells may be derived from human cancer tissue (e.g., solid tumor tissue). In other embodiments, γδ T cells may be from non-hematopoietic tissue other than human cancer tissue (e.g., tissue that does not have a substantial number of tumor cells). For example, the γδ T cells may be derived from an area of ​​skin (eg, healthy skin) away from the periphery of or adjacent to the cancer.

[0095] While γδ T cells in the blood account for the majority of γδ T cells, they are primarily Vδ2 T cells. In non-hematopoietic tissues, the majority of γδ T cells are primarily Vδ1 T cells, accounting for 70–80% of the non-hematopoietic tissue-resident γδ T cell population. However, some Vδ2 T cells are also found in non-hematopoietic tissues, such as the gastrointestinal tract, where they account for 10–20% of γδ T cells (Figure 6). Some γδ T cells resident in non-hematopoietic tissues express neither Vδ1 nor Vδ2 TCRs. Such cells are termed double-negative (DN) TCRs herein. DN γδ T cells are likely to be predominantly Vδ3-expressing T cells and predominantly Vδ5-expressing T cells. Therefore, γδ T cells resident in non-hematopoietic tissues and expanded by the methods of the present invention are preferably not Vδ2 T cells but, for example, Vδ1 T cells with a small number of DN γδ T cells.

[0096] Those skilled in the art will appreciate that certain non-hematopoietic tissues may be highly vascularized, and therefore, in practice, non-hematopoietic tissue samples may be susceptible to contamination with peripheral blood-resident cells. To avoid or minimize such contamination, care can be taken to remove peripheral blood from isolation and expansion cultures according to methods known in the art, such as by thoroughly washing the tissue with an appropriate buffer to remove cells. For example, in some embodiments, the population of γδ T cells isolated from lung tissue is not obtained by bronchoalveolar lavage.

[0097] Isolation of non-hematopoietic tissue-resident γδ T cells from non-hematopoietic tissues In some embodiments, a key step is the targeted separation of non-hematopoietic tissue-resident T cells (e.g., within a mixed lymphocyte population that includes αβ cells, natural killer (NK) cells, B cells, and γδ2 T cells, as well as non-γδ2 T cells, etc.) from non-hematopoietic cells of the tissue from which the T cells were obtained (e.g., stromal cells, particularly fibroblasts), e.g., after days or weeks of culture. This allows for the preferential and rapid expansion of non-hematopoietic tissue-derived Vδ1 T cells and DN γδ T cells over days and weeks.

[0098] The present invention provides methods comprising the isolation of γδ T cells (e.g., non-V52 T cells, e.g., V51 T cells and / or DN T cells) from non-hematopoietic tissue (e.g., skin, e.g., obtained by punch biopsy). In certain embodiments, the isolation of γδ T cells from non-hematopoietic cells comprises culturing γδ T cells with non-hematopoietic cells on a synthetic scaffold configured to promote the release of cells from the non-hematopoietic tissue. Any scaffold suitable for the isolation of lymphocytes from solid tissue can be used. The synthetic scaffold can be composed of natural and / or synthetic materials (e.g., natural or natural polymers, e.g., polyvinylpyrrolidone, polymethylmethacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (tantalum, titanium, platinum, and metals of the same element group as platinum, niobium, hafnium, tungsten, and combinations of alloys thereof). Biological factors (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, antibodies and fragments thereof, cytokines (e.g., IL-2 or IL-15), and combinations thereof), chemokines, and / or chemoattractants can be coated onto the scaffold surface or encapsulated within the scaffold material to promote cell adhesion, migration, survival, or proliferation according to methods known in the art. In some embodiments, the synthetic scaffold is a Cellfoam scaffold as described in the Clerk protocol. Alternatively, other methods, e.g., enzyme-based degradation of extracellular matrix components (e.g., collagenase), can be used to separate lymphocytes from many other non-hematopoietic tissue types.

[0099] The isolation culture can be carried out for anywhere from 1 hour (e.g., for simple digestion) to 42 days (e.g., for culture on a scaffold). For example, if the isolation step is carried out on a scaffold, the culture can be carried out for at least 5 days (e.g., at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 18 days, at least 20 days, at least 21 days, at least 24 days, at least 28 days, at least 30 days, at least 35 days, or at least 40 days, e.g., between 7 and 14 days, between 14 and 21 days, or between 21 and 35 days, e.g., about 14 days or about 21 days).

[0100] During isolation of γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells), the non-hematopoietic tissue and cells derived therefrom may be cultured in the presence of a biological factor to promote egress from the tissue or to promote survival of one or more cell subpopulations. In some embodiments, the isolation culture is in the presence of IL-2, e.g., IL-2 at a concentration of at least 10 IU / mL (e.g., 10 IU / mL to 1,000 IU / mL, 20 IU / mL to 800 IU / mL, 25 IU / mL to 750 IU / mL, 30 IU / mL to 700 IU / mL, 40 IU / mL to 600 IU / mL, 50 IU / mL to 500 IU / mL, 75 IU / mL to 250 IU / mL, or 100 IU / mL). In some embodiments, the isolation culture contains about 100 IU / mL of IL-2. Additionally or alternatively, the isolation culture may contain IL-15, e.g., at least 0.1 ng / mL of IL-15 (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, or 100 ng / mL to In some embodiments, the isolation culture contains about 20 ng / mL of IL-15.

[0101] In some embodiments, isolation of γδ T cells from non-hematopoietic tissue involves culturing in the presence of both IL-2 and IL-15 (each at any of the concentrations listed above), in some cases at a concentration of about 100 IU / mL of IL-2 and 20 ng / mL of IL-15.

[0102] γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) may be cultured under conditions in which IL-6, IL-23 and IL-1β are absent or these cytokines are present at low concentrations (e.g., less than 20 ng / mL), as the addition of this cytokine combination may act to reduce proliferation of non-hematopoietic derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells).

[0103] In isolation from non-hematopoietic tissues (e.g., skin), γδ T cells are typically part of a larger population of lymphocytes that includes, for example, αβ T cells, B cells, and natural killer (NK) cells. In some embodiments, 1% to 10% of the isolated population of lymphocytes are pre-expanded γδ T cells (e.g., 1% to 10% of an isolated population of skin-derived lymphocytes are pre-expanded γδ T cells). In most cases, the population of γδ T cells (e.g., a population of skin-derived γδ T cells) includes a large population of Vδ1 T cells. In some embodiments, 1% to 10% of the isolated population of lymphocytes (e.g., skin-derived lymphocytes) are pre-expanded Vδ1 T cells (e.g., within an isolated population of pre-expanded γδ T cells, Vδ1 T cells may represent more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the population). In some instances, less than 10% of an isolated population of γδ T cells are pre-expanded V52 T cells (e.g., less than 10% of an isolated population of skin-derived γδ T cells are pre-expanded V52 T cells).

[0104] Non-V51 T cells or non-DN T cells, such as V52 T cells, αβ T cells, B cells or NK cells, can be removed from the isolated population of γδ T cells (e.g., before, during or after expansion).

[0105] Prior to expansion, isolated γδ T cells (e.g., γδ T cells isolated from the skin, e.g., V51 T cells isolated from the skin) have a phenotype that is distinct from corresponding cells derived from a hematopoietic tissue (e.g., γδ T cells derived from the blood, e.g., V52 T cells derived from the blood). For example, an isolated population of γδ T cells may express higher levels of CCR3, CCR4, CCR7, CCR8, or CD103 than a reference population, e.g., a population of non-hematopoietic tissue-resident, TCR-activated γδ T cells, or a corresponding population of cells derived from a hematopoietic tissue (e.g., γδ T cells derived from the blood, e.g., V52 T cells derived from the blood). In some embodiments, an isolated population of γδ T cells expresses at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR3 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR4 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR7 + Cells; 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR8 + cells; and / or 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD103 + The isolated population of γδ T cells may express one or more, two or more, three or more, four or more, five or more, or all six of CCR3, CCR4, CCR7, CCR8, or CD103.

[0106] In some embodiments, the isolated population of γδ T cells expresses higher levels of NKGD2, CD56, CD69, and / or TIM3 than a reference population, e.g., a population of non-hematopoietic tissue-resident TCR-activated γδ T cells, or a corresponding hematopoietic tissue-derived cell population (e.g., blood-derived γδ T cells, e.g., blood-derived V52 T cells). In some embodiments, the isolated population of γδ T cells expresses at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more NKGD2. + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD56 + Cells, at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD69 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more TIM3 + The isolated population of γδ T cells may express one or more, two or more, three or more, four or more, or all five of NKGD2, CD56, CD69, and / or TIM3.

[0107] Isolated populations of non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells, e.g., skin-derived Vδ1 T cells) can be characterized by their function. Functional assays known in the art and exemplified in Example 3 can measure functional differences between non-hematopoietic tissue-derived cells of the invention (e.g., an isolated population of γδ T cells, e.g., a population of skin-derived Vδ1 T cells, or an expanded population of γδ T cells, e.g., skin-derived Vδ1 T cells) and reference cells (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells, or a corresponding population of cells from a hematopoietic tissue, e.g., blood-derived γδ T cells, e.g., blood-derived V52 T cells). In some embodiments, an isolated population of non-hematopoietic tissue-derived γδ T cells (e.g., a population of γδ T cells isolated substantially without TCR pathway activation contact) secretes higher levels of IL-13 than a reference population (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells, e.g., a population of anti-CD3-activated non-hematopoietic tissue-derived γδ T cells). For example, an isolated population of non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, such as V51 T cells and / or DN T cells) may secrete IL-13 at 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1,000-fold or higher concentrations than a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells, e.g., a population of anti-CD3-activated non-hematopoietic tissue-resident γδ T cells). Similarly, the number or frequency of IL-13-secreting non-hematopoietic tissue-derived γδ T cells in a population of isolated cells can be greater than that of a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells, e.g., a population of anti-CD3-activated non-hematopoietic tissue-resident γδ T cells).For example, the frequency of IL-13-secreting cells in an isolated population of γδ T cells (e.g., the frequency of IL-13-secreting cells in an isolated population of Vδ1 T cells) may be higher than a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells, e.g., a population of anti-CD3-activated non-hematopoietic tissue-resident γδ T cells). In some embodiments, the frequency of IL-13-secreting cells in an isolated population of γδ T cells (e.g., the frequency of IL-13-secreting cells in an isolated population of DN T cells or Vδ1 T cells of the invention) is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater than a reference population of cells (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells, such as a population of anti-CD3-activated non-hematopoietic tissue-resident γδ T cells).

[0108] Expansion of non-hematopoietic tissue-resident γδ T cells The present invention features methods for expanding non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells). These methods can be performed in vitro. In some embodiments, non-hematopoietic tissue-resident γδ T cells are expanded from a population of γδ T cells isolated from non-hematopoietic tissues by the methods described above. Generally, non-hematopoietic tissue-resident γδ T cells can be expanded automatically if they are removed from physical contact with stromal cells (e.g., skin fibroblasts). Thus, the scaffold-based culture methods described above can be used to induce such separation and release the suppression of γδ T cells, leading to their expansion. Thus, in some embodiments, the expansion step is substantially free of TCR pathway activation (e.g., exogenous TCR pathway activators are not included in the culture medium). Furthermore, the present invention provides methods for expanding non-hematopoietic-derived resident γδ TCR cells that do not involve contact with supportive cells, tumor cells, and / or antigen-presenting cells.

[0109] The present inventors have developed an expansion protocol comprising culturing non-hematopoietic tissue-resident γδ T cells in the presence of a cocktail of biological factors effective to support efficient expansion of γδ T cells. In one embodiment, the present invention provides a method for expanding γδ T cells by providing a population of γδ T cells obtained from a non-hematopoietic tissue (e.g., an isolated population of non-hematopoietic tissue-derived γδ T cells, e.g., a population of non-hematopoietic tissue-derived γδ T cells isolated by a method described herein) and culturing the γδ T cells in the presence of IL-2, IL-4, IL-15, and / or IL-21. These cytokines or analogs thereof can be cultured with the cells for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days or longer, e.g., 5-40 days, 7-35 days, 14-28 days, or about 21 days) in an amount effective to generate an expanded population of γδ T cells.

[0110] In some embodiments, an amount of IL-2 effective to generate an expanded population of γδ T cells is between 1 IU / mL and 2,000 IU / mL (e.g., between 5 IU / mL and 1,000 IU / mL, between 10 IU / mL and 500 IU / mL, between 20 IU / mL and 400 IU / mL, between 50 IU / mL and 250 IU / mL, or about 100 IU / mL, e.g., between 5 IU / mL and 10 IU / mL, 10IU / mL~20IU / mL, 20IU / mL~30IU / mL, 30IU / mL~40IU / mL, 40IU / mL~50IU / mL, 50IU / mL~60IU / mL, 60IU / m L~70IU / mL, 70IU / mL~80IU / mL, 80IU / mL~90IU / mL, 90IU / mL~100IU / mL, 100IU / mL~120IU / mL, 120IU / mL~140IU / m IL-2 may be administered in the presence of an IL-2 antagonist, such as IL-2 antagonists, IL-2 antagonists, or IL-2 antagonists. In some embodiments, the effective amount of IL-2 for generating an expanded population of γδ T cells is about 100 IU / mL.

[0111] In some embodiments, an expanded population of γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DNA) is / are selected from the group consisting of: An effective amount of IL-4 to generate T cells is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 250 ng / mL, 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, 500 ng / mL to 1,000 ng / mL). In some embodiments, the amount of IL-4 effective for generating expanded γδ T cells is about 5 ng / mL.

[0112] In some embodiments, an amount of IL-15 effective to generate an expanded population of γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 250 ng / mL, For example, 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, or 500 ng / mL to 1,000 ng / mL. In some embodiments, the amount of IL-15 effective for generating expanded γδ T cells is about 10 ng / mL.

[0113] In some embodiments, an amount of IL-21 effective to generate an expanded population of γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, 100 ng / mL to 250 ng / mL, For example, 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, or 500 ng / mL to 1,000 ng / mL. In some embodiments, the amount of IL-21 effective for generating expanded γδ T cells is about 10 ng / mL.

[0114] Substitution or addition of other factors in the expansion culture of non-hematopoietic tissue-resident γδ T cells is also provided herein. For example, in some embodiments, one or more factors selected from the group consisting of IL-6, IL-7, IL-8, IL-9, IL-12, IL-18, IL-33, IGF-1, IL-1β, human platelet lysate (HPL), and stromal cell-derived factor-1 (SDF-1) are included in addition to or in place of any of IL-2, IL-4, IL-15, and IL-21. Appropriate concentrations of each factor are provided in Table 2 of Example 3.

[0115] It will be understood that the amount of each of the above cytokines required to generate an expanded population of γδ T cells will depend on the concentration of one or more of the other cytokines. For example, if the concentration of IL-2 is increased or decreased, the concentration of IL-15 will be correspondingly decreased or increased. As noted above, an amount effective to generate an expanded population herein refers to the combined effect of all factors on the expansion of the cells.

[0116] In some embodiments, γδ T cells are exposed to each factor simultaneously (e.g., γδ T cells are exposed to IL-2, IL-4, IL-15, and IL-21 simultaneously, e.g., for 5 days). In other instances, γδ T cells are exposed to a particular factor before culturing with another factor. For example, an expansion culture may receive additional factors gradually over the course of the expansion, or γδ T cells may be transferred from a culture containing one factor or group of factors to another.

[0117] In some embodiments, γδ T cells are expanded in culture for a period of several hours (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days). In certain embodiments, a population of γδ T cells is expanded for 14-21 days. Thus, in some embodiments, between 28 and 56 days, or about 41 days, may be required for the isolation and expansion steps, including the isolation culture period (e.g., 1-40 days, e.g., 14-21 days).

[0118] The expansion method provides an expanded population of γδ T cells that is more numerous than the reference population. In some embodiments, the expanded population of γδ T cells is more numerous than the population of γδ T cells isolated before the expansion step (e.g., at least 2 times the number, at least 3 times the number, at least 4 times the number, at least 5 times the number, at least 6 times the number, at least 7 times the number, at least 8 times the number, at least 9 times the number, at least 10 times the number, at least 15 times the number, at least 20 times the number, at least 25 times the number, at least 30 times the number, at least 35 times the number, at least 40 times the number, at least 50 times the number, at least 60 times the number, at least 70 times the number, at least 80 times the number, at least 90 times the number, at least 100 times the number, at least 200 times the number, at least 300 times the number, at least 400 times the number, at least 500 times the number, at least 600 times the number, at least 700 times the number, at least 800 times the number, at least 900 times the number, at least 1,000 times the number, at least 5,000 times the number, at least 10,000 times the number, or more, compared to the population of isolated γδ T cells before the expansion step).

[0119] Thus, the present invention provides a means to generate large populations of non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) with high efficiency (e.g., by excluding stromal cell contact and / or TCR stimulation, or by culturing in the presence of an effective amount of a factor). In some embodiments, the expansion step described herein expands γδ T cells with a short cell number doubling time, where the doubling time is given by the formula: [Number 1] Doubling time = duration × log(2) / (log(final concentration) - log(initial concentration)) Armed with the information herein, e.g., in Example 3 below, those skilled in the art will recognize that the present invention provides methods for expanding γδ T cells derived from non-hematopoietic tissues (e.g., γδ T cells derived from skin and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) having a doubling time in cell number of less than 5 days (e.g., less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less than 3.7 days, less than 3.6 days, less than 3.5 days, less than 3.4 days, less than 3.3 days, less than 3.2 days, less than 3.1 days, less than 3.0 days, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32 hours).

[0120] In some embodiments, within 7 days of culture, the expanded population of γδ T cells (e.g., an expanded population of V51 T cells and / or DN T cells) comprises at least 10-fold more γδ T cells compared to the population of isolated γδ T cells prior to expansion (e.g., at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, or at least 8,000-fold more). In some embodiments, within 14 days of culture, the expanded population of γδ T cells (e.g., an expanded population of Vδ1 T cells and / or DN T cells) comprises at least 20-fold more γδ T cells compared to the population of isolated γδ T cells prior to expansion (e.g., an expanded population of Vδ1 T cells and / or DN T cells). at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold greater in number compared to the population of T cells).In some embodiments, within 21 days of culture, the expanded population of γδ T cells (e.g., an expanded population of Vδ1 T cells and / or DN T cells) comprises at least 50-fold more γδ T cells compared to the population of isolated γδ T cells prior to expansion (e.g., at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold more). In some embodiments, within 28 days of culture, the expanded population of γδ T cells (e.g., an expanded population of V51 T cells and / or DN T cells) comprises at least 100-fold more γδ T cells compared to the population of isolated γδ T cells prior to expansion (e.g., at least 110-fold, at least 120-fold, at least 130-fold, at least 140-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, at least 10,000-fold, at least 12,000 times, or at least 15,000 times as many).

[0121] Non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) expanded by the methods described herein may have a phenotype suitable for anti-tumor potential. In some embodiments, the expanded population of γδ T cells (e.g., skin-derived V51 T cells) exhibits higher mean CD27 expression than a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells has an average CD27 expression that is at least 2-fold higher compared to the population of isolated γδ T cells (e.g., at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 5,000-fold, at least 10,000-fold, at least 20,000-fold, or more, compared to the population of isolated γδ T cells).

[0122] A distinct portion of the expanded γδ T cell population (skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) has upregulated CD27 abundance, whereas other portions have low or no CD27 abundance. - In this case, the CD27 + For example, the expanded γδ T cell population may have a higher frequency of CD27 T cells compared to the isolated γδ T cell population before expansion. +The γδ T cells may have at least a 5% higher frequency of CD27 T cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100%, compared to the isolated population of γδ T cells prior to expansion). + In some embodiments, the frequency of CD27 in the expanded population compared to the isolated population of γδ T cells is higher. + The number of cells may be increased, for example, the expanded population of γδ T cells may have at least twice the number of CD27 T cells compared to the isolated population of γδ T cells prior to expansion. + cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater than or equal to CD27 compared to the isolated γδ T cell population prior to expansion). + high frequency of cells).

[0123] The expansion methods provided herein, in some embodiments, result in a population of non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) that have reduced TIGIT expression compared to a reference population (e.g., the isolated population of γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells exhibits a lower mean TIGIT expression level than the reference population (e.g., the isolated population of γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells exhibits at least 10% reduced mean TIGIT expression compared to the isolated population of γδ T cells (e.g., at least 20% reduced, at least 30% reduced, at least 40% reduced, at least 50% reduced, at least 60% reduced, at least 70% reduced, at least 80% reduced, at least 90% reduced, or up to 100% reduced compared to the isolated population of γδ T cells).

[0124] A distinct portion of the expanded γδ T cell (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) population expresses TIGIT, e.g., high levels of TIGIT, while other portions express low levels of TIGIT or no TIGIT. - In this case, the TIGIT expression level in the expanded population compared to the isolated γδ T cell population may be higher. + For example, the expanded population of γδ T cells may have a lower frequency of TIGIT T cells than the isolated population of γδ T cells before expansion. + The frequency of TIGIT cells may be at least 5% lower (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% lower compared to the isolated population of γδ T cells prior to expansion). +In some embodiments, the TIGIT frequency of the expanded population is lower than that of the isolated γδ T cell population before expansion. + The number of cells may be low, e.g., TIGIT in isolated γδ T cell populations before expansion. + TIGIT of the expanded γδ T cell population compared to the number of cells + The number of cells should be at least 10% lower (e.g., TIGIT in the isolated γδ T cell population before expansion). + at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% TIGIT compared to the number of cells + small number of cells).

[0125] In some embodiments, the expanded population of γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) contains a high frequency of CD27 + cells and low frequency of TIGIT + In some embodiments, the expanded population of γδ T cells has a high frequency of CD27 cells compared to a reference population (e.g., compared to the isolated population of γδ T cells prior to expansion). + TIGIT - For example, the expanded population of γδ T cells has at least a 5% higher frequency of CD27 T cells compared to the isolated population of γδ T cells prior to expansion. + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher frequency of CD27 T cells compared to the isolated γδ T cell population prior to expansion). + TIGIT -In some embodiments, the CD27 + TIGIT - The number of cells may be expanded, e.g., the expanded population of γδ T cells may contain at least twice as many CD27 T cells as the isolated population of γδ T cells prior to expansion. + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher frequency of CD27 T cells compared to the isolated γδ T cell population prior to expansion). + TIGIT - cell).

[0126] In some embodiments, CD27 in a population of expanded γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DNT cells) + The mean expression of TIGIT in a population of γδ T cells is lower compared to a reference population. + The γδ T cell population can be determined by a reference population (e.g., isolated CD27 T cells prior to expansion). + In some embodiments, the mean expression of TIGIT is lower compared to the population of γδ T cells. + The γδ T cell population was isolated using CD27 + At least 10% lower TIGIT expression compared to the γδ T cell population (e.g., isolated CD27 + at least 20% lower, at least 30% lower, at least 40% lower, at least 50% lower, at least 60% lower, at least 70% lower, at least 80% lower, at least 90% lower, or up to 100% lower compared to the population of γδ T cells).

[0127] Additionally or alternatively, TIGIT expression in a population of expanded γδ T cells (e.g., skin-derived γδ T cells or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DNT cells) can be detected. - The median CD27 expression level in γδ T cell populations is higher compared to reference populations. For example, amplified TIGIT - The γδ T cell population was isolated from TIGIT cells before expansion. - Compared to the γδ T cell population, CD27 + The frequency of cells is at least 5% higher (e.g., isolated TIGIT - at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher CD27 compared to the γδ T cell population + In some embodiments, the isolated TIGIT - CD27 in the expanded population compared with the γδ T cell population + The number of cells may be expanded, e.g., isolated TIGIT cells before expansion. - Compared with the γδ T cell population, amplified TIGIT - The γδ T cell population expresses at least twice as many CD27 + cells (e.g., isolated TIGIT cells prior to amplification). - at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% higher CD27 compared to the γδ T cell population + cell frequency).

[0128] Increased or decreased expression of other markers, including CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64, may additionally or alternatively be used to characterize a population of expanded γδ T cells from one or more non-hematopoietic tissues (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells). In some cases, the expanded population of γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DNT cells) has a higher mean expression of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2, for example, compared to the isolated population of γδ T cells prior to expansion. Additionally or alternatively, the expanded population of γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, such as V51 T cells and / or DNT cells) has a higher frequency of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30 and CD2 compared to the isolated population of γδ T cells. In some embodiments, the expanded population of γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, such as V51 T cells and / or DNT cells) has a lower mean expression of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64 compared to an isolated population of γδ T cells.Similarly, compared to the isolated population of γδ T cells, the expanded population has a reduced number of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64.

[0129] Thus, non-hematopoietic tissue-resident γδ T cells generated by the methods of the present invention may have one or more of the following properties: (i) display a phenotype of high CD69, high TIM3, and low / absent CD28; (ii) upregulate one or more of CCR3, CD39, CD11b, and CD9; (iii) produce IFN-γ in response to NKG2D ligands in the absence of TCR agonist; (iv) produce IL-13 in the absence of TCR agonist; (v) produce one or more of IFN-γ, TNF-α, and GM-CSF in response to TCR activation; (vi) do not, or substantially do not, produce IL-17 in response to TCR activation; (vii) grow in medium containing IL-2 in the absence of additional growth factors; (viii) display a cytotoxic TCR cell response in the absence of TCR agonist; and / or (ix) display selective cytotoxicity against tumor cells over normal cells.

[0130] In some instances, non-hematopoietic tissue-resident γδ T cells generated by the methods of the present invention produce IL-13 in the absence of a TCR agonist and / or produce IFN-γ in response to NKG2D in the absence of a TCR agonist.

[0131] Numerous basal media suitable for use in expanding γδ T cells are available, including complete media such as AIM-V, Iscoves' medium, and RPMI-1640 (Life Technologies). The media may be supplemented with other media components, such as serum, serum proteins, and selection agents such as antibiotics. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser, 1×MEM Non-Essential Amino Acids, Life Technologies), and 10 μL / L β-mercaptoethanol. Conveniently, cells are cultured in the appropriate medium at 37°C in a humidified atmosphere containing 5% CO2.

[0132] γδ T cells can be cultured as described herein in any suitable system, including stirred tank fermentors, airlift fermentors, roller bottles, culture bags or dishes, and other bioreactors, particularly hollow fiber bioreactors. The use of such systems is well known in the art. General methods and techniques for culturing lymphocytes are well known in the art.

[0133] The methods described herein can include multiple selection steps, e.g., multiple depletion steps. Enrichment of a T cell population by negative selection can be achieved, for example, by combining antibodies against surface markers unique to the cells being negatively selected. In some methods, cells are sorted and / or selected by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the cells being negatively selected.

[0134] IV. Pharmaceutical Compositions and Methods of Treatment The γδ T cells obtained by the methods of the present invention can be used as a medicine, for example, for adoptive T cell therapy. This involves transplanting the γδ T cells obtained by the methods of the present invention into a patient. The treatment can be an autologous transplant, i.e., transplanting the γδ T cells back into the same patient from which they were removed, or an allogeneic transplant, i.e., transplanting γδ T cells from one person into a different patient. In examples involving allogeneic transplantation, the γδ T cells can be substantially free of αβ T cells. For example, αβ T cells can be depleted from a population of γδ T cells, e.g., after expansion, using any suitable means known in the art (e.g., by negative selection, e.g., using magnetic beads). A method of treatment can include providing a sample of non-hematopoietic cells obtained from a donor, culturing γδ T cells from the sample to generate an expanded population, and administering the expanded population of γδ T cells to an individual recipient.

[0135] The patient or subject to be treated is preferably a human cancer patient (e.g., a human cancer patient undergoing treatment for a solid tumor) or a viral infection patient (e.g., a CMV-infected or HIV-infected patient). In some cases, the patient has been and / or is undergoing treatment for a solid tumor.

[0136] Because tissue-resident Vδ1 T and DN γδ T cells normally reside in non-hematopoietic tissues, they are more likely to home to and be retained in tumor masses compared with systemic blood-resident cells, making adoptive transfer of these cells more effective in targeting solid tumors and other potential non-hematopoietic tissue-associated immune diseases.

[0137] Because γδ T cells are not MHC restricted, they do not recognize the recipient as foreign, meaning they are less likely to cause graft-versus-host disease, meaning they can be used "off the shelf" and transplanted into any recipient, for example for allogeneic adoptive T cell therapy.

[0138] Non-hematopoietic tissue-resident γδ T cells obtained by the methods of the present invention express NKG2D and respond to NKG2D ligands (e.g., MICA) that are strongly associated with malignant tumors. They also exhibit cytotoxicity even in the absence of activation, making them effective at killing tumor cells. For example, the non-hematopoietic tissue-resident γδ T cells obtained herein express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzymes A and B, and perforin in the absence of activation. IL-17A may not be expressed.

[0139] Thus, the findings reported herein provide a compelling rationale for the practicality and suitability of non-hematopoietic tissue-resident γδ T cells obtained by the methods of the present invention as "off-the-shelf" immunotherapeutic agents for clinical application: these cells have innate killing capacity, are not MHC-restricted, and exhibit better homing and / or retention within tumors than other T cells.

[0140] In some embodiments, a method of treating an individual having a tumor in a non-hematopoietic tissue may comprise the steps of providing a sample of said non-hematopoietic tissue from a donor individual, culturing γδ T cells from said sample to generate an expanded population, and administering the expanded population of γδ T cells to said individual having a tumor.

[0141] Pharmaceutical compositions may comprise the non-hematopoietic tissue-resident expanded γδ T cells described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline and phosphate buffered saline; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides; or amino acids such as glycine; antioxidants; chelating agents such as EDTA and glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryopreservation solutions that can be used in the pharmaceutical compositions of the present invention may include, for example, DMSO. The compositions can be formulated, for example, for intravenous administration.

[0142] In certain embodiments, the pharmaceutical composition is substantially free of contaminants, e.g., no detectable levels of contaminants such as endotoxins or mycoplasma.

[0143] In some cases, a therapeutically effective amount of expanded T cells obtained by any of the above methods can be administered to a subject (e.g., to treat cancer, e.g., to treat a solid tumor). In some cases, a therapeutically effective amount of expanded γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) can be 10×10 per administration. 12 cells (e.g., 9 x 10 per dose) 12 Less than 8 × 10 cells per dose 12 Less than 7 × 10 cells per dose 12 Less than 6 × 10 cells per dose 12 Less than 5 × 10 cells per dose 12 Less than 4 × 10 cells per dose 12 Less than 3 x 10 cells per dose 12 Less than 2 × 10 cells per dose 12 Less than 1 × 10 cells per dose 12 Less than 9 × 10 cells per dose 11 Less than 8 × 10 cells per dose 11 Less than 7 × 10 cells per dose 11Less than 6 × 10 cells per dose 11 Less than 5 × 10 cells per dose 11 Less than 4 × 10 cells per dose 11 Less than 3 x 10 cells per dose 11 Less than 2 × 10 cells per dose 11 Less than 1 × 10 cells per dose 11 Less than 9 × 10 cells per dose 10 Less than 7.5 × 10 cells per dose 10 Less than 5 × 10 cells per dose 10 Less than 2.5 × 10 cells per dose 10 Less than 1 × 10 cells per dose 10 Less than 7.5 × 10 cells per dose 9 Less than 5 × 10 cells per dose 9 Less than 2.5 × 10 cells per dose 9 Less than 1 × 10 cells per dose 9 Less than 7.5 × 10 cells per dose 8 Less than 5 × 10 cells per dose 8 Less than 2.5 × 10 cells per dose 8 Less than 1 × 10 cells per dose 8 Less than 7.5 × 10 cells per dose 7 Less than 5 × 10 cells per dose 7 Less than 2.5 x 10 cells per dose 7 Less than 1 × 10 cells per dose 7 Less than 7.5 × 10 cells per dose 6 Less than 5 × 10 cells per dose 6 Less than 2.5 × 10 cells per dose 6 Less than 1 × 10 cells per dose 6 Less than 7.5 × 10 cells per dose 5 Less than 5 × 10 cells per dose 5 Less than 2.5 × 10 cells per dose 5 Less than 1 x 10 cells per dose 5 less than a cell).

[0144] In some embodiments, a therapeutically effective amount of expanded γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is greater than or equal to 10×10 over the course of treatment. 12 cells (e.g., 9 x 10 over the course of treatment). 12 Less than 8 × 10 cells 12 Less than 7 × 10 cells per dose 12 Less than 6 × 10 cells 12 Less than 5 × 10 cells per dose 12 Less than 4 × 10 cells 12 Less than 3 × 10 cells 12 Less than 2 × 10 cells 12 Less than 1 x 10 cells 12 Less than 9 × 10 cells 11 Less than 8 × 10 cells 11 Less than 7 × 10 cells 11 Less than 6 × 10 cells 11 Less than 5 × 10 cells 11 Less than 4 × 10 cells 11 Less than 3 × 10 cells 11 Less than 2 × 10 cells 11 Less than 1 x 10 cells 11 Less than 9 × 10 cells 10 Less than 7.5 × 10 cells 10 Less than 5 × 10 cells 10 Less than 2.5 × 10 cells 10 Less than 1 x 10 cells 10 Less than 7.5 × 10 cells 9 Less than 5 × 10 cells 9 Less than 2.5 × 10 cells 9 Less than 1 x 10 cells 9 Less than 7.5 × 10 cells 8 Less than 5 × 10 cells 8 Less than 2.5 × 10 cells 8 Less than 1 x 10 cells 8 Less than 7.5 × 10 cells 7 Less than 5 × 10 cells 7 Less than 2.5 x 10 cells 7 Less than 1 x 10 cells 7 Less than 7.5 × 10 cells 6 Less than 5 × 10 cells 6 Less than 2.5 × 10 cells 6Less than 1 x 10 cells 6 Less than 7.5 × 10 cells 5 Less than 5 × 10 cells 5 Less than 2.5 × 10 cells 5 Less than or equal to 1 x 10 cells 5 less than a cell).

[0145] In some embodiments, one dose of the expanded non-hematopoietic tissue-resident γδ T cells described herein is about 1×10 6 , 1.1×10 6 , 2 × 10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 or 5×10 8 In some embodiments, a dose of expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises at least about 1×10 cells / kg. 6 , 1.1×10 6 , 2 × 10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 or 5×10 8 In some embodiments, a single dose of expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises up to about 1×10 cells / kg. 6 , 1.1×10 6 , 2 × 10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7, 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 or 5×10 8 In some embodiments, one dose of expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises about 1.1 x 10 cells / kg. 6 ~1.8×10 7 In some embodiments, one dose of expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises about 1×10 cells / kg. 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 × 10 9 In some embodiments, a dose of expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises at least about 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 10 9 , or 5 × 10 9 In some embodiments, a single dose of expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises up to about 1×10 7 , 2 × 10 7 , 5×10 7 , 1×10 8 , 2 × 10 8 , 5×10 8 , 1×10 9 , 2 × 109 , or 5 × 10 9 Contains cells.

[0146] In one embodiment, the subject receives 10 mg / kg of the subject's body weight. 4 ~10 6 In one embodiment, the subject receives an initial administration of a population of non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) of the expanded non-hematopoietic tissue-resident γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells). In one embodiment, the subject receives an initial administration of a population of non-hematopoietic tissue-resident γδ T cells (e.g., 10 / kg of the subject's body weight). 4 ~10 6 an initial administration of γδ T cells, e.g., 10 per kg of subject body weight 4 ~10 5 γδ T cells) followed by one or more (e.g., 2, 3, 4, or 5) subsequent administrations of expanded non-hematopoietic tissue-resident γδ T cells (e.g., 10 / kg body weight of the subject followed by one or more subsequent administrations of 4 ~10 6 administration of γδ T cells, e.g., 10 per kg of subject body weight 4 ~10 5 In one embodiment, the subsequent administration or administrations are administered less than 15 days after the previous administration, e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, e.g., less than 4, 3, or 2 days after the previous administration. In one embodiment, the subject receives a total of 10 γδ T cell populations over the course of at least three administrations of γδ T cell populations. 6 For example, a subject may receive 1×10 γδ T cells. 5 Initial dose of γδ T cells, 3 × 10 5 a second dose of γδ T cells, and 6 × 10 5 A third dose of γδ T cells is received, and for example, each dose is administered less than 4, 3, or 2 days after the previous dose.

[0147] The non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention can also be used in CAR-T therapy. This involves generating modified T cell receptors (TCRs) to reprogram T cells with new specificities, such as those of monoclonal antibodies. Modified TCRs can make T cells specific to malignant cells and are therefore useful in cancer immunotherapy. For example, T cells can recognize cancer cells that express tumor antigens, such as tumor-associated antigens that are not expressed on normal somatic cells from the target tissue. Thus, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients.

[0148] The use of blood-resident γδ T cells for CAR-T has been previously reported. However, non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention are likely to be particularly good vehicles for CAR-T approaches because they can be transduced with chimeric antigen-specific TCRs while retaining their inherent ability to recognize transformed cells. They are also likely to have superior tumor penetration and retention capabilities compared to blood-resident γδ T cells or conventional systemic αβ T cells. Furthermore, because they do not require MHC-dependent antigen presentation, they reduce the potential for host-versus-graft remission and can target tumors expressing low levels of MHC. Similarly, because they do not rely on conventional costimulation (e.g., costimulation via CD28), they can also target tumors expressing low levels of costimulatory receptor ligands.

[0149] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an angiogenesis inhibitor, or a combination of two or more thereof. The additional therapeutic agent can be administered simultaneously with, before, or after administration of the expanded γδ T cells. The additional therapeutic agent can be an immunotherapeutic agent that acts on targets within the subject (e.g., the subject's own immune system) and / or on the transplanted γδ T cells.

[0150] Administration of the compositions can be carried out in any convenient manner. The compositions described herein can be administered to a patient intraarterially, subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously, or intraperitoneally, e.g., by intradermal or subcutaneous injection. Compositions of non-hematopoietic resident γδ T cells may also be injected directly into a tumor, lymph node, or site of infection. [Example]

[0151] In most adults, Vδ2 cells constitute a small and highly diverse component of circulating T cells (0.01–5%) at rest, but after challenge with a wide range of agents, including many bacteria and parasites, they rapidly expand and transiently represent up to CD3 + V52 TCR-mediated recognition of low molecular weight "phosphomoieties," including hydroxyl-methylbut-2-enyl pyrophosphate (HMBPP), an intermediate in the key microbial synthetic pathway of cholesterol and other lipids used to modify proteins (e.g., by geranylation or farnesylation). In primates, this synthesis occurs via the mevalonate pathway, one intermediate of which, isopentenyl pyrophosphate (IPP), is expressed at very high levels in virally infected and transformed cells and is also a target of V52 TCR-mediated recognition.

[0152] In addition, most V52 T cells express high levels of the NKG2D receptor, which can activate or costimulate (together with the T cell receptor (TCR)) the cell's cytolytic potential when engaged with NKG2D ligands (e.g., MICA, MICB, and ULBP). These ligands are host proteins that are upregulated when cells are exposed to factors such as oxidative or osmotic stress or ultraviolet light. These factors promote hyper-active signaling of the epidermal growth factor receptor (EGFR) pathway, which is also commonly dysregulated in human solid tumors.

[0153] The ability of Vδ2 T cells to detect transformed cells using the TCR and / or NKG2D is mediated by their potent cytolytic potential, and CD8 + Together with their apparent ability to present antigen to T cells, these findings have led to the idea that Vδ2 T cells could be clinically exploited to deliver cancer immunotherapy. This can be achieved by adoptive cell transfer, in which the lack of MHC restriction of γδ T cells notably and beneficially limits graft-versus-host disease (GvHD). To achieve this, blood-resident Vγ9Vδ2 γδ T cells can be expanded ex vivo by adding cytokines (e.g., interleukin-2 (IL-2)), together with exogenous TCR activators (e.g., phosphomoieties (e.g., BrHPP)), or together with clinically approved bisphosphonates (e.g., zoledronic acid), which inhibit farnesyl pyrophosphate synthase in the mevalonate pathway, thereby inducing the accumulation of the TCR-activating moiety, IPP. However, chronic activation of Vγ9Vδ2 γδ T cells via factors such as BrHPP can eventually lead to cellular exhaustion and a loss of cytotoxic potential.

[0154] Alternatively, the patient's own γδ T cells can be activated in situ using a pharmacologically modified form of HMBPP or a clinically approved aminobisphosphonate. More than 250 patients have been treated with these approaches, and although they appear to be safe, complete remissions have been rare. One major concern regarding the cells' limited clinical effectiveness is their tendency to become irreparably exhausted by chronic antigen exposure. A second major concern is their seemingly inefficient homing to solid tumors and the tissues that support them.

[0155] Chimeric antigen receptor T cell (CAR-T) therapy has shown clinical promise for B-cell malignancies. However, for the treatment of solid tumors, CAR-T cell performance has currently been lower than expected, demonstrating inefficient tumor response and a high incidence of extratumoral cytotoxicity. Regarding peripheral blood γδ T cells, a major obstacle to the success of CAR-T approaches for solid tumors is the likely inefficiency of systemic CAR-T cells to migrate to and reside at the site of malignancy in a functionally effective manner. Additionally, based on conventional αβ T cells, CAR-T cells must overcome immunosuppressive signals in the tumor microenvironment (e.g., those mediated through the PD1 receptor).

[0156] There are potential advantages to using γδ T cells in CAR-T approaches because they can be transduced with tumor-reactive chimeric antigen-specific TCRs while retaining their innate ability to recognize transformed cells using receptors such as NKG2D. That is, γδ T cells can be made to simultaneously possess tumor-compatible (TCR)-mediated and innate (NKG2D)-mediated functions. However, the problem remains that human blood γδ T cells may be inefficient in homing to and maintaining active tumors within solid tissues. This consideration invites a more detailed examination of γδ T cells, which are normally resident in nonhematopoietic tissues.

[0157] Such T cells migrate to nonhematopoietic tissues as part of their development, thereby differing from T cells that infiltrate tissues after systemic priming (e.g., tissue-resident TCRαβ memory T cells (so-called TRM cells)). Tissue-resident γδ T cells have been best studied in mice, where they have been shown to be common in the skin, gastrointestinal tract, and reproductive system tissues, among other sites. Large numbers of such cells have been shown to retain an innate-like functional capacity, thereby enabling them to respond to sensitization through activation of the NKG2D receptor. The inventors have recently obtained data demonstrating that human skin and intestine similarly harbor large proportions of nonhematopoietic tissue-resident γδ T cells with innate-like activity. Studies of malignancies, inflammation, atopy, allergies, and other pathological conditions formed in nonhematopoietic tissues have not fully addressed the potential impact of these innate-like human T cells resident in the tissues where the pathology occurs.

[0158] Human γδ T cells resident in nonhematopoietic tissues have been largely unstudied because their localization makes cell collection difficult and there are no established means of culturing them. This subtype comprises a variety of cells with non-MHC-restricted cytolytic activity that do not express Vδ2-containing TCRs and therefore do not respond at all to low-molecular-weight phosphomoieties. While the precise TCR specificity of such cells is largely unknown, available data suggest that they are reactive to autoantigens such as endothelial protein C receptor (EPCR), which is overexpressed by cytomegalovirus (CMV)-infected cells and many solid tumors. Nonhematopoietic tissue-associated γδ T cells also commonly express NKG2D. Given these properties and the physiological presence of these cells in nonhematopoietic tissues such as the skin and gastrointestinal tract, adoptive transfer of such cells into cancer patients may be significantly more effective in targeting solid tumors and potentially other immunopathologies.

[0159] To exploit non-V52 cells for immunotherapy, a means is needed to either expand them in situ or harvest and expand them ex vivo before reinfusion. The latter approach has been adopted because no known TCR activators have demonstrated the ability to expand large numbers of non-V52 cells in situ. To overcome the challenge of limited availability of non-hematopoietic tissues, some researchers have attempted to expand very small numbers of non-V52 T cells from the blood, where V52-expressing cells are the predominant subset, assuming these cells are equivalent to tissue-resident non-V52 T cells. The small number of non-V52 γδ T cells found in the blood expands significantly during active CMV infection, exhibits superior reactivity to CMV compared with V52 T cells, and appears to protect the human fetus in cases of intrauterine CMV infection. Additionally, CMV-reactive non-Vδ2 γδ T cells appear to protect transplant recipients from CMV reactivation during immunosuppression through cross-reactivity with transformed cells, reducing the risk of secondary malignancies. Similarly, data suggest that γδ T cells may play a beneficial role in controlling HIV infection, where non-Vδ2 γδ T cells are expanded in the blood relative to Vδ2 T cells.

[0160] Blood-resident non-V52 cells have been expanded ex vivo by adding exogenous factors that directly activate TCR signaling (e.g., using substances such as anti-CD3 antibodies, pan-γδ TCR-specific antibodies, or phytohemagglutinin (PHA)) or by coculturing stimulated non-V52 T cells with artificial antigen-presenting cells (aAPCs), in which direct contact between γδ T cells and aAPCs is required for ex vivo expansion. Alternatively, cells have been expanded by promoting NKG2D receptor signaling using immobilized recombinant MICA (an NKG2D ligand), similar to that used to maintain the growth of ex vivo γδ T cell cultures derived from epithelial carcinoma-infiltrating lymphocytes (TILs). Taken together, current methods for ex vivo expansion of V52-expressing or non-V52 blood γδ T cells always require the addition of substances that promote TCR and / or NKG2D receptor activation, along with supplemental cytokines such as IL-2. This combination of receptor-activating signals and cytokines reflects the standard approach for culturing and expanding T cells that has been widely adopted by society. To date, no method has been described for appreciably expanding γδ T cells resident in non-hematopoietic tissues. Such a method is described herein.

[0161] As part of the phenotypic and functional characterization of human nonhematopoietic tissue-resident γδ T cells (e.g., skin γδ T cells), we isolated a distinct, large population of γδ T cells that normally reside in nonhematopoietic tissues and have unique properties compared with αβ T cells and blood-resident γδ T cells. We found that the cells exhibited potent, TCR-independent, innate-like responses to NKG2D ligands and cytokines. While efforts to expand primary αβ T cells have typically used coculture with other supportive cells as a source of beneficial growth factors, we unexpectedly showed that skin and other nonhematopoietic tissue-resident γδ T cells were profoundly and specifically suppressed by coculture of these cells in contact with autologous skin fibroblasts and potentially other stromal components (such as keratinocytes and endothelial cells). Elimination of such interactions allows for the rapid expansion of the cells in large quantities for potential clinical applications.

[0162] Furthermore, in comparison to previous efforts to expand blood- and tumor-derived γδ T cells, we have shown that such non-hematopoietic tissue-resident γδ T cells can be expanded without the intentional addition of any exogenous substances that activate their TCR or NKG2D signaling pathways.

[0163] Disclosed herein is a novel means for the efficient and reproducible isolation and expansion of γδ T cells from non-hematopoietic tissues of humans or non-human animals, such as the skin and gut. Expansion is facilitated by disrupting contact of non-hematopoietic tissue-derived non-V52 T cells with autologous fibroblasts and potentially other stromal components, and is maintained by culture in IL-2, IL-15, IL-4, and / or IL-21.

[0164] The following examples are put forward to provide those of ordinary skill in the art with a complete disclosure and description of how the methods and compounds claimed herein can be performed, made, and evaluated, and are intended to be merely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention.

[0165] Example 1 Analysis Method Unless otherwise specified, the following methods were utilized to obtain the results in the following examples.

[0166] Flow cytometry Flow cytometry was performed using the following antibody-fluorochrome conjugates: Ki-67-BV421, CD3-BV510, Vδ1-PeVio770, TIM-3-PE, CD9-PE, CCR3-BV421, and CD39-BV421. Samples were also stained for viability using eFluor 770 NIR. Commercially available antibodies were purchased from Biolegend or Miltenyi. Viability dyes (near-infrared) were obtained from eBioscience. Ki-67 staining was performed on fixed and permeabilized cells using Foxp3 Staining Buffer Set (eBioscience). At the completion of each experiment, cell populations were washed in PBS and split in half. Cells were stained for viability using eFluor 770 NIR, washed, and subsequently stained with TrueStain (Biolegend) to avoid nonspecific binding of the staining antibody. Half of the samples were stained for the indicated surface markers, while the other half were stained for lineage markers only (CD3, Vδ1) and with isotype controls corresponding to the surface markers used. Matching mouse isotype antibodies conjugated to the same fluorochromes were used at the same concentrations. The isotype controls do not bind to known human antigens and therefore represent nonspecific binding or false positives. Histograms are shown relative to their corresponding isotype controls or in FMO (Figures 1D, 2A, 3B, 4B, 6B, 7A, and 11-13). Data summaries show the percentage of cells positive for the compared indicated markers, i.e., staining at a level higher than the isotype. Flow cytometry data analysis was performed using FlowJo (version 10.1).

[0167] RNA sequencing Human skin-derived Vδ1 T cells and human blood Vδ1 T cells (after T cell receptor-induced amplification) were sorted (FACS), centrifuged, and the cell pellet was resuspended in RLT buffer. RNA was prepared using the RNA-Micro-plus kit (QIAGEN). RNA libraries were generated using the KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KAPA BIOSYSTEMS). Paired-end sequencing on a HiSeq 2500 (Illumina) was performed using rapid run chemistry (read length: 100 bp). 101-base-pair paired-end reads were aligned and quantified using RSEM (v1.2.11) with Bowtie2. Reads were aligned to the human transcriptome, and counts were log2-transformed and quantile-normalized.

[0168] Cytokine quantification Human skin-derived V51 T cells were stimulated with PMA and ionomycin or plate-bound anti-CD3 mAb (OKT3, 5 μg / mL) for 24 hours. Afterward, supernatants were collected and analyzed using ProcartaPlex Human Cytokine & Chemokine Panel 1A (34 plex) (eBioscience). Assays were analyzed using Luminex FlexMap3D (Luminex). Data were analyzed in Microsoft Excel and represent the average of three donors (duplicates). Error bars indicate standard deviation.

[0169] Co-culture with fibroblasts For each grid culture setup, we prepared two Petri dishes (100 × 25 mm, Corning) by scraping them in several places with a scalpel. The chopped skin pieces were placed on the scrapes. After drying in air for 5-10 minutes, the skin pieces were attached normally to the dishes and 10 mL of Skin-T medium was added. The medium was changed weekly, and after 3 weeks of growth, primary fibroblasts were harvested following treatment with ACCUTASE® (Life Technologies). Fibroblasts were cultured at 1 × 10 4 in a 48-well plate, or 2 x 10 in the case of transwell experiments 4 After 2–3 days, fibroblasts reached confluence and were cultured at 2 × 10 cells per well in RPMI and the indicated cytokines in a 48-well plate at a density of 2 × 10 cells per well. 5 10 mixed cutaneous lymphocytes, or 3 x 10 in the case of a 24-well plate, bottom wells, and transwells 5 Lymphocytes were added to initiate the co-culture experiment.

[0170] Expansion of blood-derived γδ T cells Blood-derived γδ T cells within PBMCs can be expanded only when stimulated with TCR ligands (e.g., IPP, HMBPP, bisphosphonates in the case of Vδ2) or the addition of antibodies to crosslink the TCR receptor (mAb) or the TCR-associated kinase CD3. The same effect of TCR crosslinking can also be achieved with lectins such as PHA. In the absence of such TCR stimuli, γδ T cells in PBMCs survive for several days but fail to expand and remain in their original composition of T cell subsets with limited diversity.

[0171] To isolate PBMCs, blood from healthy volunteers was used; whole blood was layered onto Ficoll and then centrifuged at 400 g for 20 minutes to separate red blood cells, plasma, and white blood lymphocytes / monocytes. White blood cells were carefully collected through a stripette and washed four times in cold PBS. Cells were cultured at 1 × 10 in RPMI-1640 medium (Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), and minimal essential medium (MEM) non-essential amino acids (1×; Life Technologies). 6 The cells were resuspended at a density of 1 / mL and supplemented with IL-2 (100 IU / mL). Ninety minutes before cell transfer, the cells were transferred to 24-well plates coated with a pan-γδ TCR monoclonal antibody (20 μg / mL, clone B1, Biolegend). Cells were grown for 14 days, with medium changes and fresh cytokines added every 2–3 days. Upon reaching confluence, the cells were split 1:1. Under these conditions, after 14 days, the original minor population of γδ T cells, normally highly activated through their TCR (as indicated by upregulation of CD69 and CD25) and consisting primarily of Vδ2 T cells, is significantly enriched for Vδ1 T cells (up to 30% of all γδ T cells), although they also comprise Vδ1 T cells, which can be subsequently isolated using FACS for functional or phenotypic analyses (e.g., genetic analysis).

[0172] Example 2 Isolation of non-hematopoietic tissue-resident γδ T cells from the skin and gastrointestinal tract A three-dimensional skin explant protocol was constructed using the Clark protocol. Cellfoam matrices (Cytomatrix Pty Ltd, Victoria, Australia) or equivalent with dimensions of 9 mm x 9 mm x 1.5 mm were autoclaved and then incubated in 100 mg / mL rat tail type I collagen (BD Biosciences) solution in PBS for 30 minutes at room temperature, followed by a single rinse in PBS. Adult human skin samples were obtained within 3–6 hours of skin surgery. Subcutaneous fat was removed, and the remaining skin tissue was minced into pieces approximately 1 mm x 1 mm in size. Approximately five skin fragments / explants were placed and pressed onto the surface of each matrix. Each matrix was placed in a separate well of a 24-well plate (Corning) containing 2 mL of "Skin-T" medium (Iscove's Modified Dulbecco's Medium (IMDM; Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimal essential medium (MEM) non-essential amino acids (1×; Life Technologies), and 3.5 μL / L 2-mercaptoethanol (Life Technologies). For the first 7 days of culture, amphotericin (2.5 μg / mL; Life Technologies) was added. Technologies) was added to the medium. The medium was changed three times a week by aspirating the top 1 mL of medium from each well and replacing it with fresh medium.Human recombinant IL-2 (PROLEUKIN®; Novartis Pharmaceutical UK Ltd) at 100 IU / mL and human recombinant IL-15 (Biolegend) at 20 ng / mL were added to the medium at the beginning of the culture and continued until lymphocyte isolation after 21–35 days, as shown in Table 1. A maximum of 96 wells (four 24-well plates) were set up in culture for each donor.

[0173] To isolate lymphocytes, the matrices were transferred to 50 mL centrifuge tubes (Corning) containing 10 mL of Hank's Balanced Salt Solution (HBSS; Life Technologies) containing 0.01 mM HEPES (maximum of 12 matrices / tube). The matrices were washed with the cell suspension using a 10 mL pipette, and the cell suspension was passed through a 70 μm filter (BD Biosciences) into a new 50 mL centrifuge tube (Corning). This washing of the matrices was repeated two more times. The medium from the culture wells was also aspirated and passed through a 70 μm filter (BD Biosciences) into a new 50 mL centrifuge tube (Corning). The wells were washed two more times with 1 mL of 0.01 mM HEPES / HBSS and passed through a 70 μm filter (BD Biosciences). Cells were then isolated by centrifugation (1600 rpm, 15 min). The pellet was resuspended in "Skin-T" medium. The final cell pellet was resuspended in "Skin-T" medium for subsequent flow cytometry analysis or functional studies. If cell counts were required, leukocytes were counted at this stage by either: (1) trypan blue staining (0.4%) (Life Technologies) and a hemocytometer, or (2) a CASY® Model TT cell counter and analyzer (Roche). Exemplary study results are shown in Table 1 below.

[0174] [Table 1]

[0175] Because raw gut samples are prone to contamination, the biopsy samples were first washed twice with IMDM containing 10% FCS, penicillin (500 units / mL), streptomycin (500 μg / mL), gentamicin (100 μg / mL), amphotericin B (12.5 μg / mL), and metronidazole (5 μg / mL) before being minced and placed on the scaffolds. Gut scaffold cultures were grown in "Gut-T" medium (IMDM, 10% FCS, penicillin 100 units / mL, streptomycin 100 μg / mL, gentamicin 20 μg / mL, and metronidazole 1 μg / mL). As with skin samples, amphotericin B 2.5 μg / mL was also used for the first week of culture. The medium was supplemented with IL-2 (100 IU / mL) and IL-15 (20 ng / mL) and was changed three times a week. Because the structure of the digestive tract is looser than that of the skin, lymphocytes were extracted after one week.

[0176] Example 3 Characterization of non-hematopoietic tissue-resident γδ T cells Human γδ T cells are abundant in the skin and are predominantly Vδ2 - and is involved in the human lymphatic stress surveillance response Figures 1A-1D show that human skin contains a prominent population of resident γδ T cells. Using the Clark protocol, we expanded tissue-resident lymphocytes over a 3-week period using human surplus skin samples supplemented with IL-2 and IL-15. The average yield was 240,000 lymphocytes per scaffold. Consistent with previous reports, we were able to identify distinct skin-resident lymphocyte subsets, with the majority of cells expressing the conventional αβ TCR, mostly of the tissue-resident "TRM" type. Overall, CD45 + 59.9% (±8.6%) of the cells were CD4 + and 18.3% (±2.8%) were CD8 + The majority of the NK cell fraction was αβ T cells, and comprised 8.7% (±3.6%) of the NK cell fraction. In addition, we observed a significant population of γδ T cells (CD45 +An average of 8.513% (±6.564%) of the cells was found in our donors (Figure 1A and Figure 3D). This lymphocyte profile was highly reproducible across approximately 100 donors and comparable to freshly digested skin samples after organotypic culture, differing only in a slight increase in the γδ population, but providing a practically useful, much larger and purer lymphocyte population compared to standard tissue digestion protocols. Consistent with literature on the tissue localization of human γδ T cells based on their TCR delta chain, the majority of human skin γδ T cells expressed the Vδ1 TCR chain paired with various γ chains, as identified by flow cytometry. This contrasts with the majority of peripheral blood γδ T cells, which displayed a monospecific TCR heterodimer of the Vδ2 chain linked to Vγ9 and were largely absent in human skin samples. However, it is important to note a subset that expressed neither the V51 nor the V52 TCR, designated here as "double-negative" γδ T cells (DN γδ T cells) (Figure 1C).

[0177] Skin-resident γδ T cells grown in this manner displayed a non-terminally differentiated memory phenotype, did not express CD45RA, and expressed varying levels of the costimulatory molecule CCR7. Compared to conventional systemic T cells, skin-resident γδ T cells exhibited high expression of the surface marker CD69, along with expression of programmed death receptor 1 (PD-1); low to absent levels of IL-2 receptor α (CD25); and a lack of the costimulatory molecule CD28, suggesting prior or chronic activation (Figure 1D). Consistent with their tissue localization, Vδ1 and DN cells exhibited expression of skin and tissue-homing markers such as CCR4, CCR8, and integrin αE (CD103) (Figure 7). This combination of tissue-homing markers may prove beneficial in immunotherapy settings. In addition, skin-resident γδ T cells exhibit high levels of expression of the activating receptor NKG2D (Figure 2A), suggesting a possible role for these cells in lymphoid stress surveillance responses. NKG2D ligands, such as MICA, MICB, and ULBP, are upregulated by cells in response to DNA damage, EGF receptor activation, and oxidative stress, respectively, and may therefore enable NKG2D-expressing T cells to identify and eradicate stressed or transformed cells, thereby maintaining tissue homeostasis. In line with this principle, we found that skin-resident γδ T cells expanded by the method of the present invention were activated upon exposure to recombinant ligands for the NKG2D receptor (MICA, ULBP2), which demonstrated degranulation as measured by upregulation of the lysosome-associated membrane protein CD107a (Figure 2A). This innate-like feature was consistent with Vδ 1 T cells, as other tissue-resident T cells (Fig. 2C) and systemic γδ T cells were defective in this response. + T cells and DN γδ T cells (Fig. 10B).

[0178] Overall, activated skin-resident Vδ1 +T cells and DN γδ T cells, when activated with PMA / ionomycin or NKG2D ligands (e.g., recombinant MICA protein), executed a proinflammatory, Th1-biased cytokine program (staining positive for IFN-γ, TNF-α, and GM-CSF) (Figures 2A and 2B), demonstrating an innate-like response of the cells. Indeed, the response to MICA was almost completely abolished by blocking the NKG2D receptor with an antibody (Figures 2B and 2C).

[0179] γδ T cells are known to secrete IL-17 in certain disease conditions, such as psoriasis, and in some types of tumors. γδ T cells expanded by the methods of the present invention produced low levels or no IL-17, even upon extensive activation (FIGS. 2B and 8). Conversely, tissue-resident CD4-expressing αβ T cells produced IL-17 upon TCR activation (FIG. 2B). Overall, αβ T cells secreted Vδ1 + Compared with T cells and DN γδ T cells, which were restricted to a Th1-biased program associated with host protection, they displayed a much more diverse cytokine repertoire in response to PMA / ionomycin.

[0180] Isolation from tissue leads to activation and significant proliferation of human tissue γδ T cells To further study human tissue γδ T cells, mixed cutaneous lymphocytes were transferred to cell culture wells and supplemented with IL-2 to maintain long-term viability. Interestingly, when separated from the stromal and epithelial cells present in organotypic culture, Vδ1 T cells uniquely showed signs of activation and proliferation without any added stimulation. Over a 7-day period, Vδ1 T cells + T cells and DN γδ T cells uniquely and significantly upregulated nuclear factor Ki-67 and increased surface expression of IL-2 receptor α (CD25) (Figures 3B and 4B). Strikingly, over a 3-week period and in the presence of IL-2 alone, tissue-derived Vδ 1 T cells were significantly upregulated. +T cells and DN γδ T cells expanded more than all other T cell subsets, representing up to 65% of all cutaneous lymphocytes and increasing in number by an average of 127.18-fold, whereas αβ T cells, as measured by absolute cell number, only increased 5.21-fold (p = 0.0124) (Fig. 3A). The MFI of the cell cycle-associated nuclear factor Ki-67 was significantly higher than that of Vδ 1 cells. + For T cells and DN γδ T cells, the MFI increased from 2,664.5 (±1,876.1) to 8,457.7 (±4,574.2) over 14 days, whereas for αβ T cells, the MFI decreased from 592.8 (±390.5) to 284.7 (±140.1) over the same period (Figure 3C). This phenomenon of selective skin-resident γδ T cell proliferation could be further enhanced with additional recombinant IL-15, which increased lymphocyte survival and total numbers.

[0181] Cutaneous γδ T cells are significantly suppressed by fibroblasts in a contact-dependent manner Vδ 1 above + No significant expansion of Vδ1 T cells or DN γδ T cells occurred in the organotypic culture system in the presence of abundant fibroblast proliferation. + To directly examine whether coculture with T cells and DN γδ T cells inhibits T cell proliferation, autologous fibroblasts were grown. After 3 weeks of scaffold culture, mixed dermal lymphocytes were seeded into wells that were empty or contained a previously established confluent monolayer of fibroblasts. In each case, exogenous IL-2 was added to the medium to maintain T cell proliferation. In addition, a transwell was used, which prevents T lymphocytes from directly contacting fibroblasts in the same well but allows T cells to be influenced by soluble factors secreted by fibroblasts. During 14 days of coculture, Vδ1 expression was significantly increased in wells without fibroblasts and in wells where T cells were prevented from directly contacting fibroblasts. +T cells and DN γδ T cells began to proliferate. As before, αβ T cell proliferation was low under all conditions. When T cells were placed in direct contact with fibroblasts, Vδ 1 + The viability of T cells and DN γδ T cells over a 2-week period was significantly reduced from 22.6 (±8.07)-fold in wells without fibroblast contact to 3.3 (±0.17)-fold (Figure 4A). This contact-mediated inhibition was further confirmed by the absence of upregulation of CD25, Ki-67, and the transcription factor T-bet in Vδ1 T cells over a 7-day period compared to lymphocytes grown alone (Figure 4B). Some form of tissue-mediated control of the immune system appears to be fundamental for maintaining tissue homeostasis, since without this control, the potential for persistent inflammation would arise. Vδ1 T cell proliferation by stromal fibroblasts + The downregulation of T cells and DN γδ T cells appears to be an example of such control.

[0182] Taken together, skin-resident Vδ 1 + The phenotype of T cells and DN γδ T cells, as well as their distinct functional potential, reflects pre-activated T cells that are normally suppressed by adjacent dermal fibroblasts through a contact-dependent mechanism. By inactivating this mechanism by releasing T cells from contact with fibroblasts, Vδ 1 + It selectively allows the expansion of T cells and DN γδ T cells, while leaving other T cells in the skin unaffected.

[0183] Release of contact-mediated inhibition promotes cytotoxic TH1-biased cytokine responses by cutaneous Vδ1 T cells Mixed-type skin-derived lymphocytes were expanded for 14 days, and fluorescence-associated cell sorting was used to deplete αβ T cells from γδ T cells, allowing for up to 90% purity. These highly enriched cells were plated into cell culture wells at a concentration of 150,000 cells / well in RPMI medium containing 10% FCS. Supernatants were collected after 24 hours and evaluated for a broad range of effector cytokines using a LUMINEX®-based array. Completely unexpectedly, expanding γδ T cells (induced solely by their isolation from fibroblasts) spontaneously produced high amounts of TH1-associated cytokines, such as IFN-γ (12,383.46 ± 16,618.90 pg / mL), GM-CSF (4,316.73 ± 4,534.96 pg / mL), and the pro-inflammatory chemokines CCL4 (14,877.34 ± 10,935.64 pg / mL) and CCL3 (1,303.07 ± 757.23 pg / mL) (Figure 5A).

[0184] Furthermore, during expansion, and in contrast to freshly isolated skin-derived TCR-activated γδ T cells, the cells spontaneously produced high amounts of IL-13, which is associated with atopic responses. Other cytokines, such as IL-17, were produced at much lower levels or not at all (Figure 8). The high effector potential of the cells could be further enhanced after stimulation with recombinant MICA (NKG2D ligand), anti-CD3, or PMA / ionomycin. To evaluate the cytotoxic potential of expanded γδ T cells against malignant target cells, we used established transformed cell lines in 24-hour coculture experiments. Vδ 1 +T cells and DNγδ T cells exhibited extremely high cytotoxic activity against HeLa cells (cervical cancer) and Caco2 cells (colon cancer) in a dose-dependent manner, far exceeding that of conventional tissue-derived αβ T cells (Figure 5B). Furthermore, γδ cell-mediated cytotoxicity could be strongly inhibited by blocking the NKG2D receptor with a soluble monoclonal antibody, indicating that this receptor is at least partially involved in tumor surveillance by disinhibiting human skin-derived γδ T cells. Furthermore, we confirmed the cytotoxic activity of these cells using other targets: HCT1954 cells (breast cancer), MDAMB231 cells (breast cancer), and HCT116 cells (colon cancer) (Figure 9).

[0185] Non-hematopoietic tissue-resident γδ T cells generated by the methods of the present invention can be further distinguished from other blood-derived γδ T cells in that they respond to NKG2D ligand (MICA). Non-hematopoietic tissue-resident γδ T cells are strongly associated with malignant tumors in the absence of T cell receptor ligand stimulation, e.g., by increased production of TNFα, IFNγ, and CD107a (Figures 2 and 10). They also execute T cell cytotoxic responses in the absence of exogenously administered or ligand-mediated T cell receptor activation, i.e., they are cytotoxic in the absence of stimulation (Figures 3 and 5). Compared with other γδ T cells, αβ T cells, or NK cells, non-hematopoietic tissue-resident γδ T cells generated by the methods of the present invention are unique in their response and proliferative capacity in the absence of the addition of exogenous agents that activate T cell receptor signaling (Figure 3). Furthermore, non-hematopoietic tissue-resident γδ T cells generated by the method of the present invention stained positively for CD69 and PD-1, lacked CD28 expression, and expressed only low levels of CD25 (see Figure 1D). This combination of markers is not expressed on blood-derived γδ T cells. Furthermore, they showed higher expression of tissue-homing receptors, such as CCR4 and CCR8, compared with blood-derived expanded Vd2 γδ T cells (Figure 7B).

[0186] Tissue-resident γδ T cells in the human gastrointestinal tract We also identified a population of non-hematopoietic tissue-resident γδ T cells from human colon that expressed the Vδ1 T cell receptor (Figure 6). In three donors, we were able to expand these cells over a period of 4–5 weeks using the same method as used for skin cells. During expansion, colon-derived Vδ1 + T cells and DN γδ T cells showed similar Ki-67 upregulation patterns after their isolation from fibroblast-enriched organotypic cell cultures. + T cells and DN γδ T cells were potently stimulated by the provision of ligands for the NKG2D receptor. Blood-derived γδ T cells are fully capable of antibody-dependent cell-mediated cytotoxicity via CD16 expression, demonstrating enhanced targeted cytotoxicity against CD20-positive B-cell lineage lymphomas when combined with rituximab. Similarly, chronic lymphocytic leukemia (CLL) and HER2-positive breast cancer cells are more effectively killed when targeted with monoclonal antibodies. To assess the ability of skin-derived Vδ1 T cells to target antibody-opsonized target cells, we quantified the expression levels of three IgG1-related Fc receptors: CD16, CD32, and CD64. Skin-derived Vδ1 T cells express only low levels of the Fc receptor CD16 but show good expression of the high-affinity IgG receptor CD64. Thus, tissue-derived Vδ1 T cells may well be capable of being used as adjuvants to monoclonal antibody therapies such as CD20 or Her2 therapy, as they will be directed to the side of malignancies and metastases by antibodies, recognize opsonized tumor cells, and kill targets via ADCC.

[0187] Example 4 Optimization of expansion conditions for non-hematopoietic tissue-resident γδ T cells Expansion of skin-derived γδ T cells After 3–4 weeks of scaffold culture, mixed lymphocytes were removed, washed with PBS, centrifuged, and cultured at a cell concentration of 1 × 10 in Roswell Park Memorial Institute 1640 medium (RPMI-1640; Life Technologies) supplemented with 10% filtered, heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine N-2-ethanesulfonate (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimal essential medium (MEM) non-essential amino acid solution (1×; Life Technologies), and 50 μM 2-mercaptoethanol (Life Technologies). 6 The initial population of Vδ1 cells was resuspended at 100 cells / mL. + The cells were 1.12% of the lymphocytes. 2 × 10 cells were cultured in a 96-well flat-bottom plate (Corning). 5 cells / well, or 2 x 10 in a 24-well plate 6 Cells / well were seeded and expanded by adding factors at the concentrations shown in Table 2.

[0188] [Table 2]

[0189] Cells were monitored microscopically daily and fed with fresh medium and cytokines three times per week. Upon complete confluence and cell aggregation, cells were split 1:1 into additional wells and plates as needed. After 21 days, cells were harvested using ACCUTASE® (eBioscience) and counted and analyzed using flow cytometry. Figures 17A and 17B show representative flow cytometry plots before and after expansion. Table 3 shows the relative CD27 and TIGIT expression levels in expanded cells from each treatment group, as well as the corresponding fold expansion in Figure 17C. The final V51 expansion fold was calculated from the total V51 counts before and after expansion (%CD3 + pan-γδ + Vδ1 + cells ÷ 100) × (total number of cells).

[0190] [Table 3]

[0191] As shown in Figure 17C, the addition of other factors increased V51 T cell expansion compared with IL-2 and IL-15 alone. Because V51 T cell yields were high in response to IL-2, IL-15, IL-4, and IL-21, we further investigated combinations of these factors, as shown in Figures 17D–17H.

[0192] Early and terminal Vδ1 + The phenotype of each T cell population, including expression of CD27 and TIGIT, was measured using mean fluorescence intensity (MFI), and samples from each group were averaged by taking the median MFI. + The expression levels of CD27 and TIGIT by T cells are shown in Table 4.

[0193] [Table 4]

[0194] As shown in Figure 18A, the addition of other factors increased CD27 expression, as measured by mean fluorescence intensity, compared with IL-2 and IL-15 alone. Notably, the addition of IL-4 and IL-21 increased CD27 MFI approximately 8-fold compared with IL-2 and IL-15 alone. Furthermore, the quadruple combination of IL-2, IL-15, IL-4, and IL-21 induced the highest CD27 expression compared with other combinations (Figures 18B and 18D). Notably, low CD27 expression on T cells is often associated with an exhausted, terminally differentiated phenotype with little potential for further long-term proliferation.

[0195] Amplified Vδ1 + A distinct trend was observed for TIGIT expression by T cells (Figure 19). Notably, TIGIT expression was decreased in response to IL-4 and IL-21 in combination with IL-2 and IL-15. To further explore this trend, TIGIT expression was plotted against CD27 expression (Figure 20). A negative correlation was observed between TIGIT and CD27. High TIGIT expression makes T cells more susceptible to inhibition by the tumor microenvironment. The tumor microenvironment expresses the T cell ligand poliovirus receptor (PVR; CD155) at elevated levels.

[0196] Example 5: Incubation of four cytokines as an alternative to serum in cell culture Traditionally, when producing tissue- or tumor-derived T cells, it is common to supplement the culture medium with multiple blood-derived serum and plasma components. However, the use of these serum or plasma components can be undesirable due to batch-to-batch variations in these components, their high cost, limited supply given the high demand across the advanced therapy medicinal products (ATMP) industry, and the increased risk of cross-contamination with adventitious substances from these components. Therefore, after successfully identifying cytokines that support the expansion and enrichment of desired γδ T cells as described herein, we tested whether the use of such cytokines could eliminate the need for multiple serum / plasma components typically used to expand tissue-derived γδ T cells. To further test this, we released immune cells from skin samples and tested whether plasma / serum was still required to support the expansion and enrichment of γδ T cells+ / -serum. To do this, we harvested the released cells and seeded them into two media on "day 0." The first medium contained animal-derived component-free (ADCF) medium (TexMACS, Miltenyi) supplemented with 10% human serum and cytokines. The second medium contained the same ADCF medium / cytokine mixture, standard defined supplement (including CTS™, purified human serum albumin, recombinant insulin, and transferrin), but without serum. The results of this study are shown in Figure 21. Surprisingly, comparable enrichment and fold expansion were observed with or without serum. This demonstrates that tissue-derived γδ T cells can be expanded and enriched without animal-derived factors or human serum.

[0197] This approach was then evaluated for the preferential expansion of γδ T cells from mixed lymphocyte populations derived from standard organotypic culture. If achievable, such preferential expansion would be highly desirable, particularly if this protocol resulted in the final expanded lymphocyte population containing greater than 50% γδ T cells. Indeed, previous conventional enrichment protocols required the use of depletion or enrichment techniques, such as magnetic immunodepletion (e.g., from Miltenyi or Dynal) or flow cytometry sorting (e.g., from BD Biosciences), to physically separate the minority γδ cells or physically deplete the majority of αβ cells. Instead, this study evaluated whether the method of the present invention could enrich for γδ T cells present in mixed lymphocyte populations without the need for such physical separation or depletion protocols. Surprisingly, such enrichment was achieved using these expansion methods due to the selectivity of the protocol for expanding γδ cells over other cell types present in the initial population. This resulted in more enriched and purer γδ T cells, representing 50% of all cells present in the culture. Furthermore, as shown in Figure 21 and Figures 22A-22D, this enrichment was achieved with or without serum, and γδ T cells were expanded over 100-fold, increasing the purity of γδ T cells from less than 50% to greater than 50% in all sites of this tissue sample.

[0198] Isolation and Amplification Methods After 3 weeks of scaffold culture, mixed lymphocytes were obtained from the tissue using the Clark protocol above and an equivalent procedure described in Example 2. The characteristics of the obtained cells were equivalent to those described in Example 3. The harvested cells were washed with HBSS + HEPES, centrifuged, and resuspended in TexMACS medium (Miltenyi) containing either (i) IL-2 (100 IU / L), IL-4 (Biolegend, 5 ng / mL), IL-15 (Biolegend, 20 ng / mL), and IL-21 (Biolegend, 5 ng / mL) plus 10% human AB serum (Life Science Productions), or (ii) IL-2 (100 IU / L), IL-4 (Biolegend, 5 ng / mL), IL-15 (Biolegend, 20 ng / mL), and IL-21 (Biolegend, 5 ng / mL) plus 5% CTS™ (Thermo Fisher Scientific). Both serum-containing and serum-free media were supplemented with penicillin / streptomycin antibiotics (100 units / mL and 100 μg / mL, respectively, Life Technologies). Cells were then seeded at 2 million cells / well into 24-well plates (Corning). Once confluent, cells were expanded / passaged by splitting between 1 / 2 and 1 / 4 into new wells containing the same medium. On day 21, cells were harvested using an ACCUTASE® cell separator (Thermo-Fisher) and analyzed by flow cytometry to determine final cell characteristics, as shown in Figure 21 and Figures 22A-22D.

[0199] Example 6 Functional relevance of TIGIT expression As shown in Figure 23, TIGIT is constitutively expressed on gut-resident Vδ1 cells. Data were generated using Vδ1 cells isolated from the gut by conventional cell digestion. Constitutive TIGIT expression on tissue-resident γδ T cells is not exclusively associated with skin-derived Vδ1 cells, and TIGIT expression is not an artifact of the Clark protocol (grid-based isolation procedure).

[0200] Furthermore, the poliovirus receptor (PVR) specifically inhibited TCR signaling, as measured by IFNγ (Fig. 24A) and TNFα (Fig. 24B) expression in cells cultured with IL-2 and IL-15 alone. The PVR inhibitory effect was also observed in TIGIT-negative Vδ1 cells, as measured by IFNγ (Fig. 25A) or TNFα (Fig. 25B) expression in cells cultured in the presence of IL-2, IL-15, IL-4, and IL-21. + / Vδ3 + This is because TIGIT negativity due to the four mixed cytokines prevents TIGIT-mediated Vδ1 + / Vδ3 + This shows that inhibition of T cell activation is preferentially prevented.

[0201] Example 7 Replacement of IL-2 with IL-9 in expansion cultures Skin tissue from three donors (TS052, TS056, and SK073) was arranged on 9 mm grids and cultured for 3 weeks in medium supplemented with IL-2 and IL-15. Isolated lymphocytes were cultured in medium supplemented with IL-2, IL-4, IL-15, and IL-21 (Figures 26A and 26B, left bars) or IL-4, IL-9, IL-15, and IL-21 (Figures 26A and 26B, right bars). The final yield of γδ T cells / grid (Figure 26A) and Vδ1 cells / grid (Figure 26B) after 3 weeks of expansion was calculated.

[0202] As shown in Figures 27A to 27C, IL-9 significantly increased the fold increase in cutaneous γδ T cells (Figure 27A), γδ TCR + Percentage of T cells (Figure 27B) and Vδ1 + This was sufficient to replace the function of IL-2, as measured by the percentage of T cells (Figure 27C). Skin tissue was derived from six donors (SK073, SK075, SK077, TS052, TS053, and TS056). Two cytokine cocktails contained IL-2 and IL-15, and four cytokine cocktails contained IL-2, IL-15, IL-21, and IL-4.

[0203] As shown in Figures 28A and 28B, IL-9 plays a key role in the expansion of cutaneous γδ T cells through the role of Vδ1 + Mean fluorescence intensity (MFI) of CD27 expression on T cells (Figure 28A) and Vδ1 + This was sufficient to replace the function of IL-2, as measured by the MFI-corrected CD27 expression on T cells (Figure 28B). No differences in CD27 expression were observed compared to standard culture conditions. Skin tissue was derived from four donors (SK073, TS052, TS053, and TS056). Two cytokine cocktails contained IL-2 and IL-15, and four cytokine cocktails contained IL-2, IL-15, IL-21, and IL-4.

[0204] Expansion of skin-derived γδ T cells After 3 weeks of scaffold culture, mixed lymphocytes were removed, washed with phosphate-buffered saline (PBS), centrifuged, and cultured at a cell concentration of 1 × 10 in RPMI-1640 supplemented with 10% filtered, heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine N-2-ethanesulfonate (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimal essential medium (MEM) non-essential amino acid solution (1×; Life Technologies), and 50 mM 2-mercaptoethanol (Life Technologies). 6 The cells were resuspended at 2 × 10 cells / mL in a 24-well plate (Corning). 6Cells / well were seeded and expanded by adding cytokines to the following final concentrations: IL-2: 100 U / mL, IL-4: 5 ng / mL, IL-9: 10 ng / mL, IL-15: 20 ng / mL and IL-21: 10 ng / mL.

[0205] Cells were monitored microscopically daily and given fresh medium and cytokines three times a week by replacing 1 mL of medium with 1 mL of fresh medium containing cytokines at double the strength (i.e., IL-2: 200 U / mL, IL-4: 10 ng / mL, IL-9: 20 ng / mL, IL-15: 40 ng / mL, and IL-21: 20 ng / mL).

[0206] Upon reaching full confluence and cell aggregation, cells were split 1:1 into additional wells and plates as needed. After 21 days, cells were harvested using ACCUTASE® (eBioscience) and counted and analyzed by flow cytometry. Final numbers of γδ T cells and Vδ1 cells were calculated using pre- and post-expansion counts, and the final cell yield per grid after expansion was shown.

[0207] Other embodiments All publications, patents, and patent applications mentioned in this specification are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0208] While the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications, and this application generally covers any variations, uses, or adaptations of the invention in accordance with the principles of the invention, including departures from the present disclosure that come within known or customary practice in the art to which this invention pertains and that may apply to the essential features recited in the claims.

[0209] Other embodiments are within the scope of the following claims. The present invention includes the following embodiments. (1) Follow these steps: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; (ii) administering the γδ T cells in an effective amount (a) IL-2 or IL-9; (b) IL-15; and (c) IL-21 for at least 5 days to generate an expanded population of γδ T cells; A method for expanding γδ T cells, comprising: (2) The method according to (1), wherein step (ii) further comprises culturing γδ T cells in the presence of IL-4. (3) Follow these steps: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; (ii) culturing the γδ T cells in the presence of IL-2, IL-15, and at least one factor selected from the group consisting of IL-21, stromal cell-derived factor (SDF), IL-1β, IL-12, IL-18, and IL-33 for at least 5 days to generate a population of expanded γδ T cells. (4) The method according to any one of (1) to (3), wherein step (ii) comprises culturing γδ T cells in the absence of an exogenous TCR pathway agonist. (5) The method according to any one of (1) to (4), wherein step (ii) comprises culturing γδ T cells in a serum-free medium. (6) after step (i), further comprising separating the γδ T cells from non-hematopoietic cells to generate a population of isolated γδ T cells; and Step (ii) comprises: (a) Culture of γδ T cells with virtually no contact with stromal cells; (b) culture of γδ T cells in substantial absence of contact with tumor cells; and / or (c) culture of γδ T cells with virtually no contact with supportive cells; The method according to any one of (1) to (5). (7) Follow these steps: (i) providing non-hematopoietic tissue containing non-hematopoietic cells and γδ T cells; (ii) separating γδ T cells from non-hematopoietic cells to obtain an isolated population of γδ T cells; (iii) culturing the γδ T cells in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-21, SDF, IL-1β, IL-12, IL-18, and IL-33 for at least 5 days to generate an expanded population of γδ T cells; A method for expanding γδ T cells, comprising: (8) The method according to (7), wherein the non-hematopoietic tissue is obtained from a human or non-human animal subject. (9) The method according to (7) or (8), wherein step (ii) comprises culturing γδ T cells in the presence of IL-2, IL-15 and IL-21. (10) The method according to any one of (7) to (9), wherein step (ii) comprises culturing γδ T cells in a serum-free medium. (11) The method according to any one of (7) to (10), wherein step (iii) comprises culturing γδ T cells under conditions in which there is substantially no contact between stromal cells and the γδ T cells. (12) The method according to any one of (7) to (11), wherein step (iii) comprises culturing γδ T cells in the absence of an exogenous TCR pathway agonist. (13) The method according to any one of (6) to (12), wherein the step of separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells on a synthetic scaffold constructed to release cells from non-hematopoietic tissue. (14) The method according to any one of (6) to (13), wherein the step of separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells and non-hematopoietic cells in the presence of IL-2 and / or IL-15. (15) The method according to any one of (6) to (14), wherein the isolated lymphocyte population comprises an isolated γδ T cell population, and the isolated γδ T cell population comprises an isolated Vδ1 T cell population. (16) The method according to (15), wherein 1 to 10% of the isolated lymphocyte population are γδ T cells before expansion. (17) The method according to (15) or (16), wherein 1 to 10% of the isolated lymphocyte population are Vδ1 T cells before expansion. (18) The method according to any one of (6) to (17), wherein at least 80% of the isolated γδ T cell population are Vδ1 T cells before expansion. (19) The method according to any one of (6) to (18), wherein less than 10% of the isolated γδ T cell population is Vδ2 T cells before expansion. (20) The method according to any one of (6) to (19), wherein αβ T cells and / or NK cells are removed from the separated population of γδ T cells. (21) Before expansion, the isolated γδ T cell population has at least 10% CCR3 + cells, at least 10% CCR4 + cells, at least 10% CCR7 + cells, at least 10% CCR8 + cells, or at least 10% CD103 + The method according to any one of (6) to (20), comprising cells. (22) Prior to expansion, the isolated γδ T cell population exhibits a high frequency of CCR3 T cells compared to a reference blood-resident Vδ2 T cell population. + cells, CCR4 + cells, CCR7 + cells and / or CCR8 + The method according to any one of (6) to (21), comprising cells. (23) The isolated Vδ1 T cell population has a high frequency of NKG2D compared to a reference blood-resident Vδ1 T cell population. + cells, CD56 + cells, CD69 +Cells and / or TIM3 + The method according to any one of (14) to (22), comprising cells. (24) The method according to any one of (1) to (23), wherein the expanded population of γδ T cells contains at least 20 times the number of γδ T cells in the population of isolated γδ T cells before expansion, after culturing for 14 days or less. (25) The method according to any one of (1) to (24), wherein the expanded population of γδ T cells contains at least 50 times the number of γδ T cells in the population of isolated γδ T cells before expansion, after culturing for 21 days or less. (26) The method according to any one of (1) to (25), wherein the expanded population of γδ T cells comprises an expanded population of Vδ1 T cells. (27) The method according to (26), wherein, within 14 days of culture, the expanded population of Vδ1 T cells contains at least 20 times the number of Vδ1 T cells compared to the population of isolated Vδ1 T cells before expansion. (28) The method according to (26) or (27), wherein, within 21 days of culture, the expanded population of Vδ1 T cells contains at least 50 times the number of Vδ1 T cells compared to the population of isolated Vδ1 T cells before expansion. (29) The method according to any one of (1) to (28), wherein the expanded population of γδ T cells expresses CD27. (30) The method according to (29), wherein the expanded population of γδ T cells exhibits a higher median CD27 expression level than the isolated population of γδ T cells. (31) The method according to (30), wherein the expanded population of γδ T cells exhibits at least twice the median CD27 expression level as compared to the isolated population of γδ T cells. (32) The expanded γδ T cell population expressed a higher frequency of CD27 compared with the isolated γδ T cell population. + The method according to (29), comprising cells. (33) The expanded γδ T cell population expresses at least 5% higher frequency of CD27 compared with the isolated γδ T cell population. + The method according to (32), comprising cells. (34) The method according to any one of (26) to (28), wherein the expanded population of Vδ1 T cells expresses CD27. (35) The method according to (34), wherein the expanded Vδ1 T cell population exhibits a higher median CD27 expression level than the isolated Vδ1 T cell population. (36) The method according to (35), wherein the expanded population of Vδ1 T cells exhibits at least twice the median CD27 expression level compared to the isolated population of Vδ1 T cells. (37) The expanded Vδ1 T cell population expressed a higher frequency of CD27 compared with the isolated Vδ1 T cell population. + The method according to any one of (34) to (36), comprising cells. (38) The expanded Vδ1 T cell population expresses at least 5% higher frequency of CD27 compared with the isolated Vδ1 T cell population. + The method according to (37), comprising cells. (39) The method according to any one of (1) to (38), wherein the expanded population of γδ T cells exhibits a lower average TIGIT expression level than the isolated population of γδ T cells. (40) The method according to (39), wherein the expanded population of γδ T cells exhibits a mean TIGIT expression level that is at least 50% lower than that of the isolated population of γδ T cells. (41) The expanded γδ T cell population showed a lower frequency of TIGIT than the isolated γδ T cell population. + The method according to (39) or (40), comprising cells. (42) The method according to (41), wherein the expanded population of γδ T cells has a frequency of TIGIT+ cells that is at least 20% lower than the isolated population of γδ T cells. (43) The method according to any one of (26) to (28) or (34) to (38), wherein the expanded population of Vδ1 T cells exhibits a lower average TIGIT expression level than the isolated population of Vδ1 T cells. (44) The method according to (43), wherein the expanded population of Vδ1 T cells exhibits a mean TIGIT expression level that is at least 50% lower than the isolated population of Vδ1 T cells. (45) The expanded Vδ1 T cell population exhibited a lower frequency of TIGIT than the isolated Vδ1 T cell population. + The method according to any one of (26) to (28) or (34) to (38), comprising a cell. (46) The expanded Vδ1 T cell population had at least 20% lower frequency of TIGIT than the isolated Vδ1 T cell population. + The method according to (45), comprising cells. (47) The method according to any one of (1) to (46), wherein the average expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 is higher in the expanded population of γδ T cells than in an isolated population of γδ T cells. (48) The method according to any one of (1) to (47), wherein the expanded population of γδ T cells contains cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 at a higher frequency than the isolated population of γδ T cells. (49) The method according to any one of (1) to (48), wherein the average expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 in the expanded population of γδ T cells is lower than that in an isolated population of γδ T cells. (50) The method according to any one of (1) to (49), wherein the frequency of cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 in the expanded population of γδ T cells is lower than that in the isolated population of γδ T cells. (51) The method according to any one of (26) to (28), (34) to (38), (43), or (45), wherein the average expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 is higher in the expanded population of Vδ1 T cells compared to the isolated population of Vδ1 T cells. (52) The method according to any one of (26) to (28), (34) to (38), (43), (45), or (51), wherein the expanded population of γδ T cells has a higher frequency of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 than the isolated population of γδ T cells. (53) The method according to any one of (26) to (28), (34) to (38), (43), (45), (51), or (52), wherein the average expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 is lower in the expanded population of γδ T cells compared to an isolated population of γδ T cells. (54) The method according to any one of (26) to (28), (34) to (38), (43), (45), or (51) to (53), wherein cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 are at a lower frequency in the expanded population of γδ T cells compared to the isolated population of γδ T cells. (55) The method according to any one of (8) to (54), wherein step (iii) comprises culturing γδ T cells substantially without contact with stromal cells. (56) The method according to any one of (8) to (55), wherein step (iii) comprises culturing γδ T cells substantially in the absence of contact with support cells. (57) The method according to any one of (8) to (55), wherein step (iii) comprises culturing γδ T cells substantially without contact with tumor cells. (58) The method according to any one of (1) to (57), wherein the non-hematopoietic tissue is not a tumor tissue. (59) The method according to any one of (1) to (58), wherein the non-hematopoietic tissue is skin. (60) Expanded γδ T cells obtained by any one of the methods (1) to (59). (61) A population of isolated γδ T cells in which at least 50% of the γδ T cells in the isolated population express CD27 and substantially no TIGIT. (62) The isolated population of γδ T cells of claim 61, wherein at least 50% of the γδ T cells of the isolated population express Vδ1. (63) A pharmaceutical composition comprising the expanded γδ T cells according to (60) or the isolated population of γδ T cells according to (61) or (62). (64) The pharmaceutical composition according to (63) for use in a method for treating cancer or an infectious disease in a subject. (65) Use of the pharmaceutical composition according to (63) in the manufacture of a medicament for treating cancer or an infectious disease in a subject. (66) A method for treating a subject by adoptive T cell therapy, comprising a step of administering to a subject in need of treatment an effective amount of the expanded γδ T cells obtained by the method according to any one of (1) to (59), the expanded γδ T cells according to claim 60, the isolated population according to (61) or (62), or the pharmaceutical composition according to (63). (67) The therapeutically effective dose of expanded γδ T cells is 10 × 10 per dose. 12 The method according to (66), wherein the cell is subcellular. (68) The method according to (66) or (67), which comprises administering one or more additional therapeutic agents to a subject in need of said treatment. (69) The method of (68), wherein the one or more additional therapeutic agents are selected from the group consisting of immunotherapeutic agents, cytotoxic agents, growth inhibitory agents, radiotherapeutic agents, anti-angiogenic agents, and combinations thereof. (70) The method according to (68) or (69), wherein the one or more additional therapeutic agents are administered simultaneously with the expanded γδ T cells. (71) The method according to (68) or (69), wherein the one or more additional therapeutic agents are administered after administration of the expanded γδ T cells. (72) The method according to any one of (68) to (71), wherein the additional therapeutic agent is an immunotherapeutic agent. (73) A method for treating a subject by adoptive T cell therapy, comprising the step of administering a therapeutically effective amount of the pharmaceutical composition according to (63) to a subject in need of treatment. (74) The method according to any one of (66) to (73), wherein the subject is a human. (75) The method according to (74), wherein the human is a human cancer patient. (76) The method according to (75), wherein the human cancer patient is undergoing treatment for a solid cancer. (77) The method according to (76), wherein the human is undergoing treatment for a viral infection.

Claims

1. Steps below: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; (ii) administering the γδ T cells in an effective amount (a) IL-2 or IL-9; (b) IL-15; and (c) IL-21 for at least 5 days to generate an expanded population of γδ T cells; A method for expanding γδ T cells, comprising:

2. 2. The method of claim 1, wherein step (ii) further comprises culturing the γδ T cells in the presence of IL-4.

3. Steps below: (i) providing a population of γδ T cells obtained from a non-hematopoietic tissue; (ii) culturing the γδ T cells in the presence of IL-2, IL-15, and at least one factor selected from the group consisting of IL-21, stromal cell-derived factor (SDF), IL-1β, IL-12, IL-18, and IL-33 for at least 5 days to generate a population of expanded γδ T cells.

4. The method of any one of claims 1 to 3, wherein step (ii) comprises culturing γδ T cells in the absence of an exogenous TCR pathway agonist.

5. The method of any one of claims 1 to 4, wherein step (ii) comprises culturing γδ T cells in serum-free medium.

6. after step (i), further comprising separating the γδ T cells from non-hematopoietic cells to produce an isolated population of γδ T cells; and Step (ii) comprises: (a) Culture of γδ T cells with virtually no contact with stromal cells; (b) a culture of γδ T cells that is substantially free from contact with tumor cells; and / or (c) culture of γδ T cells in the substantial absence of contact with supportive cells; The method according to any one of claims 1 to 5.

7. Steps below: (i) providing non-hematopoietic tissue containing non-hematopoietic cells and γδ T cells; (ii) separating γδ T cells from non-hematopoietic cells to obtain an isolated population of γδ T cells; (iii) culturing the γδ T cells in the presence of IL-2, IL-15, and a factor selected from the group consisting of IL-21, SDF, IL-1β, IL-12, IL-18, and IL-33 for at least 5 days to generate an expanded population of γδ T cells; A method for expanding γδ T cells, comprising:

8. 8. The method of claim 7, wherein the non-hematopoietic tissue is obtained from a human or non-human animal subject.

9. 9. The method of claim 7 or 8, wherein step (ii) comprises culturing γδ T cells in the presence of IL-2, IL-15 and IL-21.

10. The method of any one of claims 7 to 9, wherein step (ii) comprises culturing γδ T cells in serum-free medium.

11. The method of any one of claims 7 to 10, wherein step (iii) comprises culturing γδ T cells under conditions in which there is substantially no contact between stromal cells and said γδ T cells.

12. The method of any one of claims 7 to 11, wherein step (iii) comprises culturing γδ T cells in the absence of exogenous TCR pathway agonist.

13. 13. The method of any one of claims 6 to 12, wherein the step of separating γδ T cells from non-hematopoietic cells comprises culturing γδ T cells on a synthetic scaffold constructed to release cells from non-hematopoietic tissue.

14. The method of any one of claims 6 to 13, wherein the step of separating γδ T cells from non-hematopoietic cells comprises culturing the γδ T cells and the non-hematopoietic cells in the presence of IL-2 and / or IL-15.

15. 15. The method of any one of claims 6 to 14, wherein the separated population of lymphocytes comprises a separated population of γδ T cells, and said separated population of γδ T cells comprises a separated population of Vδ1 T cells.

16. 16. The method of claim 15, wherein 1-10% of the separated lymphocyte population are γδ T cells before expansion.

17. 17. The method of claim 15 or 16, wherein 1-10% of the separated lymphocyte population are Vδ1 T cells before expansion.

18. The method of any one of claims 6 to 17, wherein at least 80% of the isolated population of γδ T cells are Vδ1 T cells before expansion.

19. The method of any one of claims 6 to 18, wherein less than 10% of the isolated population of γδ T cells are Vδ2 T cells prior to expansion.

20. The method of any one of claims 6 to 19, wherein αβ T cells and / or NK cells are depleted from the separated population of γδ T cells.

21. Prior to expansion, the isolated γδ T cell population contains at least 10% CCR3 + cells, at least 10% CCR4 + cells, at least 10% CCR7 + cells, at least 10% CCR8 + cells, or at least 10% CD103 + The method of any one of claims 6 to 20, comprising cells.

22. Prior to expansion, the isolated γδ T cell population exhibited a high frequency of CCR3 T cells compared to a reference blood-resident Vδ2 T cell population. + cells, CCR4 + cells, CCR7 + cells and / or CCR8 + The method of any one of claims 6 to 21, comprising cells.

23. the isolated population of Vδ1 T cells expresses a high frequency of NKG2D compared to a reference population of blood-resident Vδ1 T cells; + cells, CD56 + cells, CD69 + Cells and / or TIM3 + The method of any one of claims 14 to 22, comprising cells.

24. The method according to any one of claims 1 to 23, wherein the expanded population of γδ T cells contains at least 20 times the number of γδ T cells as compared to the population of isolated γδ T cells before expansion, after culturing for 14 days or less.

25. The method according to any one of claims 1 to 24, wherein the expanded population of γδ T cells contains at least 50 times the number of γδ T cells as compared to the population of isolated γδ T cells before expansion, after culturing for 21 days or less.

26. The method of any one of claims 1 to 25, wherein the expanded population of γδ T cells comprises an expanded population of Vδ1 T cells.

27. 27. The method of claim 26, wherein, within 14 days of culture, the expanded population of V51 T cells comprises at least 20 times the number of V51 T cells compared to the population of isolated V51 T cells prior to expansion.

28. 28. The method of claim 26 or 27, wherein, within 21 days of culture, the expanded population of V51 T cells comprises at least 50-fold more V51 T cells than the population of isolated V51 T cells prior to expansion.

29. The method of any one of claims 1 to 28, wherein the expanded population of γδ T cells expresses CD27.

30. 30. The method of claim 29, wherein the expanded population of γδ T cells exhibits a higher median CD27 expression level than the isolated population of γδ T cells.

31. 31. The method of claim 30, wherein the expanded population of γδ T cells exhibits at least 2-fold higher median CD27 expression compared to the isolated population of γδ T cells.

32. The expanded γδ T cell population exhibited a higher frequency of CD27 compared with the isolated γδ T cell population. + 30. The method of claim 29, comprising cells.

33. The expanded γδ T cell population has at least a 5% higher frequency of CD27 compared to the isolated γδ T cell population. + 33. The method of claim 32, comprising cells.

34. The method of any one of claims 26 to 28, wherein the expanded population of Vδ1 T cells expresses CD27.

35. 35. The method of claim 34, wherein the expanded population of V51 T cells exhibits a higher median CD27 expression than the isolated population of V51 T cells.

36. 36. The method of claim 35, wherein the expanded population of V51 T cells exhibits at least a 2-fold higher median CD27 expression compared to the isolated population of V51 T cells.

37. The expanded Vδ1 T cell population exhibited a higher frequency of CD27 compared with the isolated Vδ1 T cell population. + The method of any one of claims 34 to 36, comprising cells.

38. The expanded Vδ1 T cell population has at least a 5% higher frequency of CD27 compared with the isolated Vδ1 T cell population. + 38. The method of claim 37, comprising cells.

39. The method of any one of claims 1 to 38, wherein the expanded population of γδ T cells exhibits a lower mean expression level of TIGIT than the isolated population of γδ T cells.

40. 40. The method of claim 39, wherein the expanded population of γδ T cells exhibits a mean TIGIT expression level that is at least 50% lower than the isolated population of γδ T cells.

41. The expanded γδ T cell population had a lower frequency of TIGIT than the isolated γδ T cell population. + 41. The method of claim 39 or 40, comprising cells.

42. 42. The method of claim 41, wherein the expanded population of γδ T cells has a frequency of TIGIT+ cells that is at least 20% lower than the isolated population of γδ T cells.

43. 39. The method of any one of claims 26 to 28 or 34 to 38, wherein the expanded population of Vδ1 T cells exhibits a lower mean expression of TIGIT than the isolated population of Vδ1 T cells.

44. 44. The method of claim 43, wherein the expanded population of V51 T cells exhibits a mean TIGIT expression that is at least 50% lower than the isolated population of V51 T cells.

45. The expanded Vδ1 T cell population exhibited a lower frequency of TIGIT than the isolated Vδ1 T cell population. + 39. The method of any one of claims 26 to 28, or 34 to 38, comprising a cell.

46. The expanded Vδ1 T cell population has at least 20% lower frequency of TIGIT than the isolated Vδ1 T cell population. + 46. ​​The method of claim 45, comprising cells.

47. 47. The method of any one of claims 1 to 46, wherein the expanded population of γδ T cells has a higher mean expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to an isolated population of γδ T cells.

48. 48. The method of any one of claims 1 to 47, wherein the expanded population of γδ T cells has a higher frequency of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to the isolated population of γδ T cells.

49. 49. The method of any one of claims 1 to 48, wherein the expanded population of γδ T cells has a lower mean expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to the isolated population of γδ T cells.

50. 50. The method of any one of claims 1 to 49, wherein cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 are at a lower frequency in the expanded population of γδ T cells compared to the isolated population of γδ T cells.

51. 46. ​​The method of any one of claims 26 to 28, 34 to 38, 43 or 45, wherein the expanded population of V51 T cells has a higher mean expression level of one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to the isolated population of V51 T cells.

52. 52. The method of any one of claims 26 to 28, 34 to 38, 43, 45, or 51, wherein the expanded population of γδ T cells has a higher frequency of cells expressing one or more markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to the isolated population of γδ T cells.

53. 53. The method of any one of claims 26 to 28, 34 to 38, 43, 45, 51 or 52, wherein the expanded population of γδ T cells has a reduced mean expression level of one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1 and CD64 compared to an isolated population of γδ T cells.

54. 54. The method of any one of claims 26 to 28, 34 to 38, 43, 45, or 51 to 53, wherein cells expressing one or more markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 are at a lower frequency in the expanded population of γδ T cells compared to the isolated population of γδ T cells.

55. 55. The method of any one of claims 8 to 54, wherein step (iii) comprises culturing γδ T cells substantially in the absence of contact with stromal cells.

56. 56. The method of any one of claims 8 to 55, wherein step (iii) comprises culturing the γδ T cells substantially in the absence of contact with feeder cells.

57. 56. The method of any one of claims 8 to 55, wherein step (iii) comprises culturing γδ T cells substantially in the absence of contact with tumor cells.

58. The method of any one of claims 1 to 57, wherein the non-hematopoietic tissue is not tumor tissue.

59. 59. The method of any one of claims 1 to 58, wherein the non-hematopoietic tissue is skin.

60. 60. Expanded γδ T cells obtained by the method of any one of claims 1 to 59.

61. An isolated population of gamma delta T cells, wherein at least 50% of the gamma delta T cells of the isolated population express CD27 and do not substantially express TIGIT.

62. 62. The isolated population of γδ T cells of claim 61, wherein at least 50% of the γδ T cells of the isolated population express Vδ1.

63. 63. A pharmaceutical composition comprising the expanded γδ T cells of claim 60 or the isolated population of γδ T cells of claim 61 or 62.

64. 64. The pharmaceutical composition of claim 63 for use in a method for treating cancer or an infectious disease in a subject.

65. 64. Use of the pharmaceutical composition of claim 63 in the manufacture of a medicament for the treatment of cancer or an infectious disease in a subject.

66. 64. A method of treating a subject by adoptive T cell therapy, comprising administering to a subject in need thereof a therapeutically effective amount of expanded γδ T cells obtained by the method of any one of claims 1 to 59, the expanded γδ T cells of claim 60, the isolated population of claim 61 or 62, or the pharmaceutical composition of claim 63.

67. The therapeutically effective dose of expanded γδ T cells is 10 × 10 per dose. 12 67. The method of claim 66, wherein the cell is subcellular.

68. 68. The method of claim 66 or 67, comprising administering to a subject in need of said treatment one or more additional therapeutic agents.

69. 69. The method of claim 68, wherein the one or more additional therapeutic agents are selected from the group consisting of immunotherapeutic agents, cytotoxic agents, growth inhibitory agents, radiotherapeutic agents, anti-angiogenic agents, and combinations thereof.

70. 70. The method of claim 68 or 69, wherein the one or more additional therapeutic agents are administered simultaneously with the expanded γδ T cells.

71. 70. The method of claim 68 or 69, wherein the one or more additional therapeutic agents are administered after administration of the expanded γδ T cells.

72. 72. The method of any one of claims 68 to 71, wherein the additional therapeutic agent is an immunotherapeutic agent.

73. 64. A method of treating a subject with adoptive T cell therapy, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition of claim 63.

74. 74. The method of any one of claims 66 to 73, wherein the subject is a human.

75. 75. The method of claim 74, wherein the human is a human cancer patient.

76. 76. The method of claim 75, wherein the human cancer patient is undergoing treatment for a solid tumor.

77. 77. The method of claim 76, wherein the human is undergoing treatment for a viral infection.

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