Methods and materials for expanding tumor-infiltrating gamma-delta T cells

Abstract

The present specification relates to methods and materials for culturing and expanding tumor-infiltrating γδ T cells (e.g., tumor-infiltrating γδ T cells). For example, large numbers of tumor-infiltrating γδ T cells (e.g., Vδ1 T cells) from tissue (e.g., tumor samples) obtained from a mammal with cancer, an autoimmune condition, or an infectious disease can be cultured and expanded. + Methods and materials are provided for expanding populations of such tumor-infiltrating γδ T cells (tumor-infiltrating γδ T cells in which the tumor is predominantly resident in the γδ T cell population). Also provided are methods and materials for treating cancer in a mammal (e.g., a human) using such tumor-infiltrating γδ T cells and / or such populations.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 257,805, filed October 20, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference into) the disclosure of this application.

[0002] 1.Technical Field The present specification relates to methods and materials for culturing and expanding tumor-infiltrating gamma-delta (γδ) T cells (e.g., tumor-infiltrating γδ T cells). For example, the present specification describes methods and materials for culturing and expanding tumor-infiltrating γδ T cells (e.g., Vδ1 T cells) from tissue (e.g., tumor samples) obtained from a mammal with cancer. + Provided herein are methods and materials for expanding populations of such tumor-infiltrating γδ T cells (tumor-infiltrating γδ T cells in which the T cell population is predominantly γδ T-cells). Also provided herein are methods and materials for using such tumor-infiltrating γδ T cells and / or such populations to treat cancer in a mammal (e.g., a human). [Background technology]

[0003] 2. Background information Cancer immunotherapy, including adoptive cell therapy (ACT) using tumor-infiltrating lymphocytes (TILs), relies on T cell effector function. These αβ receptor (TCR)-expressing cells target cancer cells by recognition of peptide or lipid antigens presented by major histocompatibility complex (MHC) classes I and II and MHC-like CD1 molecules. TIL therapy, which involves lymphodepletion, adoptive transfer of ex vivo expanded autologous TILs, and post-infusion administration of high-dose interleukin-2 (IL-2), has produced durable complete responses in patients with treatment-resistant metastatic melanoma, cervical cancer, and other epithelial cancers. Current TIL therapy protocols have produced objective clinical responses, particularly complete responses, in many treated patients, and improved understanding of the mechanisms of response to treatment may broaden the application of these treatments (Dafni et al., Ann. Oncol., 30:1902-1913 (2019)).

[0004] Clinical manifestations of cancer often occur several years after cancer immunoediting, with the emergence of poorly immunogenic tumor cell variants, many of which have lost class I MHC molecules (Schreiber et al., Science, 331:1565-1570 (2011)). Despite efforts to reinvigorate immune responses by ACT, genomic instability in cancer cells promotes a Darwinian selection process associated with mutational downregulation or complete loss of immune-reactive tumor-associated peptide antigens that provide a means of immune escape (Dudley et al., J. Clin. Oncol., 23:2346-2357 (2005); Khong et al., Nat. Immunol., 3:999-1005 (2002); Zitvogel et al., Nat. Rev. Immunol., 6:715-727 (2006); and Orlando et al., Nat. Med., 24:1504-1506 (2018)).

[0005] As previously mentioned, immune evasion is also prevalent in several solid tumors and is mediated by reduced or absent expression of MHC class 1 antigen presentation, limiting the efficacy of αβ T cell immunotherapy (Dhatchinamoorthy et al., Front. Immunol., 12:636568 (2021); Tran et al., N. Engl. J. Med., 375:2255-2262 (2016); and Chowell et al., Science, 359:582-587 (2018)). More recently, T cell intrinsic factors, including functional exhaustion due to lack of effective co-stimulation, expression of inhibitory receptors and abrogation of stem cell-like memory differentiation, dictate persistence and response to immunotherapy (Ahmadzadeh et al., Blood, 114:1537-1544 (2009); Baitsch et al., J. Clin. Invest., 121:2350-2360 (2011); Miller et al., Nat. Immunol., 20:326-336 (2019); Sade-Feldman et al., Cell, 175:998-1013 e1020 (2018); Jansen et al., Nature, 576:465-470 (2019); and Krishna et al., Science, 370:1328-1334 (2020)). Therapeutic interventions that can overcome the challenges inherent in tumor cell immune escape and suppression paradigms could further improve immunotherapy treatment outcomes.

[0006] γδ TCR expressing cells are an evolutionarily conserved lymphocyte subset whose MHC-unrestricted recognition of pathogen-derived or host cell non-peptide metabolites and stress antigens offers a compelling opportunity for therapeutic utility in immune surveillance and cancer immunotherapy (Vantourout et al., Nat. Rev. Immunol., 13:88-100 (2013); Silva-Santos et al., Nat. Rev. Immunol., 15:683-691 (2015); Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019); Sebestyen et al., Nat. Rev. Drug Discov., 19:169-184 (2020); and Ribot et al., Nat. Rev. Immunol., 21:221-232 (2021)). γδ T cells, especially Vδ1 +The cells are primarily tissue-resident immune effectors that display diverse roles in mediating TCR- and natural cytotoxicity receptor (NCR)-dependent tumor surveillance. As such, they regulate and mediate both innate and adaptive immune responses (Vantourout et al., Nat. Rev. Immunol., 13:88-100 (2013); Silva-Santos et al., Nat. Rev. Immunol., 15:683-691 (2015); Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019); Sebestyen et al., Nat. Rev. Drug Discov., 19:169-184 (2020); Ribot et al., Nat. Rev. Immunol., 21:221-232 (2021); and Davey et al., Trends Immunol., 39:446-459 (2018)). The presence of these cells is associated with better outcomes in patients with many types of cancer. For example, leukemia patients with increased numbers of γδ T cells after bone marrow transplantation experienced longer survival (Godder et al., Bone Marrow Transplant., 39:751-757 (2007)). Furthermore, a meta-analysis of infiltrating immune cell gene expression signatures of 25 solid tumor types from The Cancer Genome Atlas (TCGA) identified γδ T cells as the most important cell type associated with favorable prognosis (Gentles et al., Nat. Med., 21:938-945 (2015)). Early and ongoing efforts to target phosphoantigen-reactive blood-resident Vγ9Vδ2 cells have established the clinical feasibility and safety of γδ cancer cell therapy (Sebestyen et al., Nat. Rev. Drug Discov., 19:169-184 (2020)). Summary of the Invention

[0007] Provided herein are methods and materials for culturing and expanding tumor-infiltrating γδ T cells (e.g., tumor-infiltrating γδ T cells). For example, provided herein are methods for expanding tumor-infiltrating γδ T cells obtained from tissue (e.g., tumor samples) to large numbers (e.g., 1×10 7 or 1×10 8 or 5×10 8 or exceeds 1×10 9 (>100) tumor-infiltrating γδ T cells (e.g., Vδ1 + The present invention provides methods and materials for obtaining tumor-infiltrating γδ T cells (in which γδ T cells are predominant) within, for example, 25 to 30 days.

[0008] As described herein, γδ T cells obtained from tumor tissue (and / or healthy tissue within 30 mm of the tumor) can be expanded in vitro using a combination of cytokines (e.g., IL-2, IL-4, and IL-15 (IL-2 / IL-4 / IL-15)) to generate a population of tumor-infiltrating γδ T cells with a desired percentage of cells with a desired phenotype. For example, the present disclosure provides methods for culturing a first population containing tumor-infiltrating γδ T cells for 5-15 days (e.g., 6-15 days, 7-15 days, 8-15 days, 9-15 days, 9-13 days, 10-12 days, or 7-10 days) in the presence of IL-2 to produce a second population of cells, and then culturing the second population of cells for 8-21 days (e.g., 10-21 days, 12-21 days, 14-21 days, 8-18 days, 8-16 days, 8-14 days, 10-20 days, 10-18 days, 12-18 days, 10-16 days, 12-16 days, or 13-15 days) in the presence of IL-2, IL-4, and IL-15 (and optionally PBMCs, such as irradiated allogeneic PBMCs, and optionally an anti-CD3 agonist antibody) to produce tumor-infiltrating γδ T cells. The present invention provides methods and materials for expanding tumor-infiltrating γδ T cells by generating an expanded population of T cells. In some cases, the expanded population of tumor-infiltrating γδ T cells is produced by (a) obtaining a tissue sample containing a tumor and / or healthy tissue that was within 30 mm of a tumor, (b) obtaining a first cell population containing tumor-infiltrating γδ T cells from the tissue, and (c) optionally, determining whether the resulting enriched population is a total CD3 +(d) enriching the first cell population (or optionally the enriched population) in the presence of IL-2, IL-4, IL-15, PBMCs (e.g., irradiated PBMCs) and anti-CD3 antibody for 8-21 days (e.g., 10-21 days, 12-21 days, 14-21 days, 8-18 days, 8-16 days, 8-14 days, 10-20 days, 10-18 days, 12-18 days, 10-16 days, 12-16 days, or 13-15 days) to obtain an expanded population of tumor-infiltrating γδ T cells.

[0009] In some cases, the CD3 + More than 85 percent of cells are γδ TCR + The population may be CD3 + Fewer than 10 percent of cells have αβ TCR + The population may be CD45 + Fewer than 10 percent of the cells may be NK cells, and the γδ TCR + More than 30 percent of cells are Vδ1 + cells, and the γδ TCR + Fewer than 60 percent of cells are Vδ1 - Vδ2 - cells, and the γδ TCR + Fewer than 25 percent of cells are Vδ2 + cells, and the γδ TCR + More than 70 percent of the cells are T EM cells, and the γδ TCR + Fewer than 25 percent of cells are T EMRA cells, and the γδ TCR + About 10 percent of cells express CD69 + CD103 + Organization-resident memory (T RM ) cells, and the γδ TCR + Approximately 50 percent of cells are CD56 + cells, and the γδ TCR +Between 1 and 40 percent of cells express CD137 + cells, and the γδ TCR + Fewer than 25 percent of cells express PD-1 + cells, and the γδ TCR + Between 5 and 40 percent of cells are BTLA + cells, and the γδ TCR + More than 60 percent of cells are NKG2D + cells, and the γδ TCR + More than 20 percent of cells are NKp46 + It may be a cell.

[0010] As also described herein, a population of tumor-infiltrating γδ T cells provided herein can be administered to a mammal (e.g., a human) having cancer to treat the cancer in the mammal. For example, a population of tumor-infiltrating γδ T cells provided herein can be administered (e.g., administered intravenously) to a mammal (e.g., a human) having cancer as adoptive cell therapy to treat the cancer, alone or in combination with (a) tumor-infiltrating αβ T cells and / or (b) one or more therapeutic agents, such as one or more checkpoint inhibitors (e.g., anti-PD-1 antibodies and / or anti-PD-L1 antibodies), IL-2, one or more lymphocyte-depleting chemotherapeutic agents (e.g., cyclophosphamide and / or fludarabine), one or more tumor-infiltrating lymphocyte enhancing agents (e.g., CpG and / or oncolytic viruses, such as vaccinia virus), brachytherapy, or a combination thereof. In such cases, the administered tumor-infiltrating γδ T cells can provide an effective immune response against cancer cells in the mammal, thereby reducing the number of cancer cells in the mammal.

[0011] In general, one aspect herein features a method for generating a cell population comprising γδ T cells. The method includes (or consists essentially of or consists of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8-21 days to obtain a second cell population, where the second cell population comprises at least 10-fold more γδ T cells than the first cell population. The γδ T cells can be human cells. The γδ T cells can be tumor-infiltrating γδ T cells. The first cell population can be (i) a population of tumor-infiltrating γδ T cells obtained from (a) a tissue comprising a tumor or (b) a healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from a healthy tissue, (iii) a population of γδ T cells obtained from an infected tissue, or (iv) a population of γδ T cells obtained from a tissue harboring autoimmune T cells. The method can include obtaining the first cell population from a tissue comprising a tumor. The method may include obtaining a first cell population from healthy tissue that was within 30 mm of a tumor. The first cell population may be a cell population that has been cultured in the presence of 50 International Units / mL to 6000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population that has been cultured in the presence of 100 International Units / mL to 4000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells by (a) depleting at least some αβ T cells or (b) isolating at least some γδ T cells. The method may include depleting at least some of the αβ T cells from the cell population to obtain a first cell population. Depletion may include positively selecting the αβ T cells and depleting the positively selected αβ T cells. The method may include isolating at least some of the γδ T cells from the cell population to obtain a first cell population. Isolation may include positively selecting the γδ T cells and isolating the positively selected γδ T cells.Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8-21 days can include culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and anti-CD3 antibody for 8-21 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 12-16 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 13-15 days. The second cell population may comprise at least 50-fold more γδ T cells than the first cell population, at least 100-fold more γδ T cells than the first cell population, at least 200-fold more γδ T cells than the first cell population, at least 300-fold more γδ T cells than the first cell population, or at least 400-fold more γδ T cells than the first cell population. The second cell population may comprise at least 1 x 10 8 The second cell population may comprise more than one γδ T cell. The IL-2 may be human IL-2. The IL-4 may be human IL-4. The IL-15 may be human IL-15. The second cell population may comprise more than one γδ T cell. + More than 85 percent of the cells express γδ TCR + The second cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The second cell population may be CD45 cells. + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The second cell population may be a γδ TCR cell. + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells are Vδ2 + The second cell population may be a γδ TCR cell. + More than 70 percent of the cells are T EM The second cell population may be a γδ TCR cell. +Fewer than 25 percent of cells were T EMRA The second cell population may be a γδ TCR cell. + Fewer than 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Fewer than 50 percent of the cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 50 percent of cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 40 percent of cells express CD137 + The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells express PD-1 + The second cell population may be a γδ TCR cell. + Between 5 and 40 percent of the cells were BTLA + The second cell population may be a γδ TCR cell. + More than 60 percent of cells express NKG2D + The second cell population may be a γδ TCR cell. + More than 20 percent of the cells express NKp46 + It may be a cell.

[0012] In another aspect, the present disclosure provides an isolated cell population comprising (or consisting essentially of or consisting of) polyclonal γδ T cells, the population being greater than or equal to 1×10 8 The present invention is characterized in that the isolated cell population comprises more than 10 γδ T cells. + More than 85 percent of the cells express γδ TCR + The cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The cell population may be CD45 +Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The γδ TCR cell population may be + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The γδ TCR cell population may be + Fewer than 25 percent of cells are Vδ2 + The γδ TCR cell population may be + More than 70 percent of the cells are T EM The γδ TCR cell population may be + Fewer than 25 percent of cells were T EMRA The γδ TCR cell population may be + Fewer than 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be + Fewer than 50 percent of the cells are CD56 + The γδ TCR cell population may be + Between 1 and 50 percent of cells are CD56 + The γδ TCR cell population may be + Between 1 and 40 percent of cells express CD137 + The γδ TCR cell population may be + Fewer than 25 percent of cells express PD-1 + The γδ TCR cell population may be + Between 5 and 40 percent of the cells were BTLA + The γδ TCR cell population may be + More than 60 percent of cells express NKG2D + The γδ TCR cell population may be + More than 20 percent of the cells express NKp46 +The cell population may be a human cell. The cells of the cell population may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The cell population may be a cell population generated using a method for generating a cell population comprising γδ T cells as described in any description or combination of descriptions from the following paragraphs.

[0013] The method may comprise (or may consist essentially of or consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-5 for 8-21 days to obtain a second cell population, wherein the second cell population comprises at least 10-fold more γδ T cells than the first cell population. The γδ T cells may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The first cell population may be (i) a population of tumor-infiltrating γδ T cells obtained from (a) a tissue comprising a tumor or (b) a healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from a healthy tissue, (iii) a population of γδ T cells obtained from an infected tissue, or (iv) a population of γδ T cells obtained from a tissue harboring autoimmune T cells. The method may comprise obtaining the first cell population from a tissue comprising a tumor. The method may comprise obtaining the first cell population from a healthy tissue that was within 30 mm of a tumor. The first cell population may be a cell population cultured in the presence of 50 International Units / mL to 6000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population cultured in the presence of 100 International Units / mL to 4000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells by (a) depleting at least some αβ T cells or (b) isolating at least some γδ T cells. The method may include depleting at least some αβ T cells from the cell population to obtain the first cell population. The depletion may include positively selecting the αβ T cells and depleting the positively selected αβ T cells. The method may include isolating at least some of the γδ T cells from the population of cells to obtain the first population of cells. The isolation may include positively selecting the γδ T cells and isolating the positively selected γδ T cells.Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8-21 days can include culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and anti-CD3 antibody for 8-21 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 12-16 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 13-15 days. The second cell population may comprise at least 50-fold more γδ T cells than the first cell population, at least 100-fold more γδ T cells than the first cell population, at least 200-fold more γδ T cells than the first cell population, at least 300-fold more γδ T cells than the first cell population, or at least 400-fold more γδ T cells than the first cell population. The second cell population may comprise at least 1 x 10 8 The second cell population may comprise more than one γδ T cell. The IL-2 may be human IL-2. The IL-4 may be human IL-4. The IL-15 may be human IL-15. The second cell population may comprise more than one γδ T cell. + More than 85 percent of the cells express γδ TCR + The second cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The second cell population may be CD45 cells. + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The second cell population may be a γδ TCR cell. + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells are Vδ2 + The second cell population may be a γδ TCR cell. + More than 70 percent of the cells are T EM The second cell population may be a γδ TCR cell. + Fewer than 25% of cells were TEMRA The second cell population may be a γδ TCR cell. + Fewer than 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Fewer than 50 percent of the cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 50 percent of cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 40 percent of cells express CD137 + The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells express PD-1 + The second cell population may be a γδ TCR cell. + Between 5 and 40 percent of the cells were BTLA + The second cell population may be a γδ TCR cell. + More than 60 percent of cells express NKG2D + The second cell population may be a γδ TCR cell. + More than 20 percent of the cells express NKp46 + It may be a cell.

[0014] In another aspect, this document features a method for providing γδ T cells to a mammal. The method includes administering to the mammal (or consists essentially of or consists of) a cell population generated as described in any statement or combination of statements in the preceding paragraphs. The mammal can be a human. The mammal can be a mammal with cancer. The cells of the first cell population can be allogeneic or autologous to the mammal administered the cell population. The method can include administering to the mammal αβ T cells.

[0015] In another aspect, this document features a method for providing γδ T cells to a mammal. The method includes administering (or consisting essentially of, or consisting of) a cell population (e.g., an isolated cell population) to the mammal. The mammal can be a human. The mammal can be a mammal with cancer, an autoimmune condition, or an infectious disease. The cells of the cell population can be allogeneic or autologous to the mammal. The method can include administering αβ T cells to the mammal. The cell population (e.g., an isolated cell population) can include (or can consist essentially of, or can consist of) polyclonal γδ T cells, the population being greater than or equal to 1×10 8 The CD3 + More than 85 percent of the cells express γδ TCR + The cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The cell population may be CD45 + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The γδ TCR cell population may be + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The γδ TCR cell population may be + Fewer than 25 percent of cells are Vδ2 + The γδ TCR cell population may be + More than 70 percent of the cells are T EM The γδ TCR cell population may be + Fewer than 25 percent of cells were T EMRA The γδ TCR cell population may be + Fewer than 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be + Between 1 and 10 percent of cells express CD69 + CD103 + T RMThe γδ TCR cell population may be + Fewer than 50 percent of the cells are CD56 + The γδ TCR cell population may be + Between 1 and 50 percent of cells are CD56 + The γδ TCR cell population may be + Between 1 and 40 percent of cells express CD137 + The γδ TCR cell population may be + Fewer than 25 percent of cells express PD-1 + The γδ TCR cell population may be + Between 5 and 40 percent of the cells were BTLA + The γδ TCR cell population may be + More than 60 percent of cells express NKG2D + The γδ TCR cell population may be + More than 20 percent of the cells express NKp46 + The cell population may be a human cell. The cells of the cell population may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The cell population may be a cell population generated using a method for generating a cell population comprising γδ T cells as described in any description or combination of descriptions from the following paragraphs.

[0016] The method may comprise (or may consist essentially of or consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-5 for 8-21 days to obtain a second cell population, wherein the second cell population comprises at least 10-fold more γδ T cells than the first cell population. The γδ T cells may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The first cell population may be (i) a population of tumor-infiltrating γδ T cells obtained from (a) a tissue comprising a tumor or (b) a healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from a healthy tissue, (iii) a population of γδ T cells obtained from an infected tissue, or (iv) a population of γδ T cells obtained from a tissue harboring autoimmune T cells. The method may comprise obtaining the first cell population from a tissue comprising a tumor. The method may comprise obtaining the first cell population from a healthy tissue that was within 30 mm of a tumor. The first cell population may be a cell population cultured in the presence of 50 International Units / mL to 6000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population cultured in the presence of 100 International Units / mL to 4000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells by (a) depleting at least some αβ T cells or (b) isolating at least some γδ T cells. The method may include depleting at least some αβ T cells from the cell population to obtain the first cell population. The depletion may include positively selecting the αβ T cells and depleting the positively selected αβ T cells. The method may include isolating at least some of the γδ T cells from the population of cells to obtain the first population of cells. The isolation may include positively selecting the γδ T cells and isolating the positively selected γδ T cells.Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8-21 days can include culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and anti-CD3 antibody for 8-21 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 12-16 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 13-15 days. The second cell population may comprise at least 50-fold more γδ T cells than the first cell population, at least 100-fold more γδ T cells than the first cell population, at least 200-fold more γδ T cells than the first cell population, at least 300-fold more γδ T cells than the first cell population, or at least 400-fold more γδ T cells than the first cell population. The second cell population may comprise at least 1 x 10 8 The second cell population may comprise more than one γδ T cell. The IL-2 may be human IL-2. The IL-4 may be human IL-4. The IL-15 may be human IL-15. The second cell population may comprise more than one γδ T cell. + More than 85 percent of the cells express γδ TCR + The second cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The second cell population may be CD45 cells. + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The second cell population may be a γδ TCR cell. + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells are Vδ2 + The second cell population may be a γδ TCR cell. + More than 70 percent of the cells are T EM The second cell population may be a γδ TCR cell. +Fewer than 25 percent of cells were T EMRA The second cell population may be a γδ TCR cell. + Fewer than 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Fewer than 50 percent of the cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 50 percent of cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 40 percent of cells express CD137 + The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells express PD-1 + The second cell population may be a γδ TCR cell. + Between 5 and 40 percent of the cells were BTLA + The second cell population may be a γδ TCR cell. + More than 60 percent of cells express NKG2D + The second cell population may be a γδ TCR cell. + More than 20 percent of the cells express NKp46 + It may be a cell.

[0017] In another aspect, the document features a method for treating cancer. The method includes (or consists essentially of or consists of) a cell population generated as described in any of the preceding paragraphs or combinations of descriptions, to a mammal having cancer. The mammal can be a human. The cells of the first cell population can be allogeneic or autologous to the mammal having cancer. The method can include administering αβ T cells to the mammal.

[0018] In another aspect, this document features a method for treating cancer. The method includes administering (or consists essentially of, or consists of) a cell population (e.g., an isolated cell population) to a mammal having cancer. The mammal can be a human. The cells of the cell population can be allogeneic or autologous to the mammal having cancer. The method can include administering αβ T cells to the mammal. The cell population (e.g., an isolated cell population) includes (or consists essentially of, or consists of) polyclonal γδ T cells, and the population can be greater than or equal to 1×10 8 The CD3 + More than 85 percent of the cells express γδ TCR + The cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The cell population may be CD45 + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The γδ TCR cell population may be + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The γδ TCR cell population may be + Fewer than 25 percent of cells are Vδ2 + The γδ TCR cell population may be + More than 70 percent of the cells are T EM The γδ TCR cell population may be + Fewer than 25 percent of cells were T EMRA The γδ TCR cell population may be + Fewer than 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be +Fewer than 50 percent of the cells are CD56 + The γδ TCR cell population may be + Between 1 and 50 percent of cells are CD56 + The γδ TCR cell population may be + Between 1 and 40 percent of cells express CD137 + The γδ TCR cell population may be + Fewer than 25 percent of cells express PD-1 + The γδ TCR cell population may be + Between 5 and 40 percent of the cells were BTLA + The γδ TCR cell population may be + More than 60 percent of cells express NKG2D + The γδ TCR cell population may be + More than 20 percent of the cells express NKp46 + The cell population may be a human cell. The cells of the cell population may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The cell population may be a cell population generated using a method for generating a cell population comprising γδ T cells as described in any description or combination of descriptions from the following paragraphs.

[0019] The method may comprise (or may consist essentially of or consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-5 for 8-21 days to obtain a second cell population, wherein the second cell population comprises at least 10-fold more γδ T cells than the first cell population. The γδ T cells may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The first cell population may be (i) a population of tumor-infiltrating γδ T cells obtained from (a) a tissue comprising a tumor or (b) a healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from a healthy tissue, (iii) a population of γδ T cells obtained from an infected tissue, or (iv) a population of γδ T cells obtained from a tissue harboring autoimmune T cells. The method may comprise obtaining the first cell population from a tissue comprising a tumor. The method may comprise obtaining the first cell population from a healthy tissue that was within 30 mm of a tumor. The first cell population may be a cell population cultured in the presence of 50 International Units / mL to 6000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population cultured in the presence of 100 International Units / mL to 4000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells by (a) depletion of at least some αβ T cells or (b) isolation of at least some γδ T cells. The method may include depleting at least some αβ T cells from the cell population to obtain the first cell population. The depletion may include positively selecting the αβ T cells and depleting the positively selected αβ T cells. The method may include isolating at least some of the γδ T cells from the population of cells to obtain the first population of cells. The isolation may include positively selecting the γδ T cells and isolating the positively selected γδ T cells.Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8-21 days can include culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and anti-CD3 antibody for 8-21 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 12-16 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 13-15 days. The second cell population may comprise at least 50-fold more γδ T cells than the first cell population, at least 100-fold more γδ T cells than the first cell population, at least 200-fold more γδ T cells than the first cell population, at least 300-fold more γδ T cells than the first cell population, or at least 400-fold more γδ T cells than the first cell population. The second cell population may comprise at least 1 x 10 8 The second cell population may comprise more than one γδ T cell. The IL-2 may be human IL-2. The IL-4 may be human IL-4. The IL-15 may be human IL-15. The second cell population may comprise more than one γδ T cell. + More than 85 percent of the cells express γδ TCR + The second cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The second cell population may be CD45 cells. + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The second cell population may be a γδ TCR cell. + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells are Vδ2 + The second cell population may be a γδ TCR cell. + More than 70 percent of the cells are T EM The second cell population may be a γδ TCR cell. +Fewer than 25 percent of cells were T EMRA The second cell population may be a γδ TCR cell. + Fewer than 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Fewer than 50 percent of the cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 50 percent of cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 40 percent of cells express CD137 + The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells express PD-1 + The second cell population may be a γδ TCR cell. + Between 5 and 40 percent of the cells were BTLA + The second cell population may be a γδ TCR cell. + More than 60 percent of cells express NKG2D + The second cell population may be a γδ TCR cell. + More than 20 percent of the cells express NKp46 + It may be a cell.

[0020] In another aspect, the document features a method for treating an autoimmune condition. The method includes administering (or consists essentially of or consists of) a cell population (e.g., an isolated cell population) to a mammal having an autoimmune condition. The mammal can be a human. The cells of the cell population can be allogeneic or autologous to the mammal having the autoimmune condition. The method can include administering αβ T cells to the mammal. The cell population (e.g., an isolated cell population) can include (or consist essentially of or consist of) polyclonal γδ T cells, the population being greater than or equal to 1×10 8 The CD3 + More than 85 percent of the cells express γδ TCR + The cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The cell population may be CD45 + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The γδ TCR cell population may be + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The γδ TCR cell population may be + Fewer than 25 percent of cells are Vδ2 + The γδ TCR cell population may be + More than 70 percent of the cells are T EM The γδ TCR cell population may be + Fewer than 25 percent of cells were T EMRA The γδ TCR cell population may be + Fewer than 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be +Fewer than 50 percent of the cells are CD56 + The γδ TCR cell population may be + Between 1 and 50 percent of cells are CD56 + The γδ TCR cell population may be + Between 1 and 40 percent of cells express CD137 + The γδ TCR cell population may be + Fewer than 25 percent of cells express PD-1 + The γδ TCR cell population may be + Between 5 and 40 percent of the cells were BTLA + The γδ TCR cell population may be + More than 60 percent of cells express NKG2D + The γδ TCR cell population may be + More than 20 percent of the cells express NKp46 + The cell population may be a human cell. The cells of the cell population may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The cell population may be a cell population generated using a method for generating a cell population comprising γδ T cells as described in any description or combination of descriptions from the following paragraphs.

[0021] The method may comprise (or may consist essentially of or consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-5 for 8-21 days to obtain a second cell population, wherein the second cell population comprises at least 10-fold more γδ T cells than the first cell population. The γδ T cells may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The first cell population may be (i) a population of tumor-infiltrating γδ T cells obtained from (a) a tissue comprising a tumor or (b) a healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from a healthy tissue, (iii) a population of γδ T cells obtained from an infected tissue, or (iv) a population of γδ T cells obtained from a tissue harboring autoimmune T cells. The method may comprise obtaining the first cell population from a tissue comprising a tumor. The method may comprise obtaining the first cell population from a healthy tissue that was within 30 mm of a tumor. The first cell population may be a cell population cultured in the presence of 50 International Units / mL to 6000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population cultured in the presence of 100 International Units / mL to 4000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells by (a) depletion of at least some αβ T cells or (b) isolation of at least some γδ T cells. The method may include depleting at least some αβ T cells from the cell population to obtain the first cell population. The depletion may include positively selecting the αβ T cells and depleting the positively selected αβ T cells. The method may include isolating at least some of the γδ T cells from the population of cells to obtain the first population of cells. The isolation may include positively selecting the γδ T cells and isolating the positively selected γδ T cells.Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8-21 days can include culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and anti-CD3 antibody for 8-21 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 12-16 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 13-15 days. The second cell population may comprise at least 50-fold more γδ T cells than the first cell population, at least 100-fold more γδ T cells than the first cell population, at least 200-fold more γδ T cells than the first cell population, at least 300-fold more γδ T cells than the first cell population, or at least 400-fold more γδ T cells than the first cell population. The second cell population may comprise at least 1 x 10 8 The second cell population may comprise more than one γδ T cell. The IL-2 may be human IL-2. The IL-4 may be human IL-4. The IL-15 may be human IL-15. The second cell population may comprise more than one γδ T cell. + More than 85 percent of the cells express γδ TCR + The second cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The second cell population may be CD45 cells. + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The second cell population may be a γδ TCR cell. + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells are Vδ2 + The second cell population may be a γδ TCR cell. + More than 70 percent of the cells are T EM The second cell population may be a γδ TCR cell. +Fewer than 25 percent of cells were T EMRA The second cell population may be a γδ TCR cell. + Fewer than 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Fewer than 50 percent of the cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 50 percent of cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 40 percent of cells express CD137 + The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells express PD-1 + The second cell population may be a γδ TCR cell. + Between 5 and 40 percent of the cells were BTLA + The second cell population may be a γδ TCR cell. + More than 60 percent of cells express NKG2D + The second cell population may be a γδ TCR cell. + More than 20 percent of the cells express NKp46 + It may be a cell.

[0022] In another aspect, this document features a method for treating an infectious disease. The method includes administering (or consisting essentially of, or consisting of) a cell population (e.g., an isolated cell population) to a mammal having an infectious disease. The mammal can be a human. The cells of the cell population can be allogeneic or autologous to the mammal having the infectious disease. The method can include administering αβ T cells to the mammal. The cell population (e.g., an isolated cell population) can include (or can consist essentially of, or can consist of) polyclonal γδ T cells, the population being greater than or equal to 1×108 The CD3 + More than 85 percent of the cells express γδ TCR + The cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The cell population may be CD45 + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The γδ TCR cell population may be + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The γδ TCR cell population may be + Fewer than 25 percent of cells are Vδ2 + The γδ TCR cell population may be + More than 70 percent of the cells are T EM The γδ TCR cell population may be + Fewer than 25 percent of cells were T EMRA The γδ TCR cell population may be + Fewer than 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The γδ TCR cell population may be + Fewer than 50 percent of the cells are CD56 + The γδ TCR cell population may be + Between 1 and 50 percent of cells are CD56 + The γδ TCR cell population may be + Between 1 and 40 percent of cells express CD137 + The γδ TCR cell population may be + Fewer than 25 percent of cells express PD-1 + The γδ TCR cell population may be + Between 5 and 40 percent of the cells were BTLA+ The γδ TCR cell population may be + More than 60 percent of cells express NKG2D + The γδ TCR cell population may be + More than 20 percent of the cells express NKp46 + The cell population may be a human cell. The cells of the cell population may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The cell population may be a cell population generated using a method for generating a cell population comprising γδ T cells as described in any description or combination of descriptions from the following paragraphs.

[0023] The method may comprise (or may consist essentially of or consist of) culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-5 for 8-21 days to obtain a second cell population, wherein the second cell population comprises at least 10-fold more γδ T cells than the first cell population. The γδ T cells may be human cells. The γδ T cells may be tumor-infiltrating γδ T cells. The first cell population may be (i) a population of tumor-infiltrating γδ T cells obtained from (a) a tissue comprising a tumor or (b) a healthy tissue that was within 30 mm of a tumor, (ii) a population of γδ T cells obtained from a healthy tissue, (iii) a population of γδ T cells obtained from an infected tissue, or (iv) a population of γδ T cells obtained from a tissue harboring autoimmune T cells. The method may comprise obtaining the first cell population from a tissue comprising a tumor. The method may comprise obtaining the first cell population from a healthy tissue that was within 30 mm of a tumor. The first cell population may be a cell population cultured in the presence of 50 International Units / mL to 6000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 3 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population cultured in the presence of 100 International Units / mL to 4000 International Units / mL of IL-2 and in the absence of IL-4 and IL-15 for 8 to 15 days prior to culture in the presence of IL-2, IL-4, and IL-15. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells. The first cell population may be a cell population enriched for tumor-infiltrating γδ T cells by (a) depleting at least some αβ T cells or (b) isolating at least some γδ T cells. The method may include depleting at least some αβ T cells from the cell population to obtain the first cell population. The depletion may include positively selecting the αβ T cells and depleting the positively selected αβ T cells. The method may include isolating at least some of the γδ T cells from the population of cells to obtain the first population of cells. The isolation may include positively selecting the γδ T cells and isolating the positively selected γδ T cells.Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 for 8-21 days can include culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and anti-CD3 antibody for 8-21 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 12-16 days. Culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 can be culturing for 13-15 days. The second cell population may comprise at least 50-fold more γδ T cells than the first cell population, at least 100-fold more γδ T cells than the first cell population, at least 200-fold more γδ T cells than the first cell population, at least 300-fold more γδ T cells than the first cell population, or at least 400-fold more γδ T cells than the first cell population. The second cell population may comprise at least 1 x 10 8 The second cell population may comprise more than one γδ T cell. The IL-2 may be human IL-2. The IL-4 may be human IL-4. The IL-15 may be human IL-15. The second cell population may comprise more than one γδ T cell. + More than 85 percent of the cells express γδ TCR + The second cell population may be CD3 + Fewer than 10 percent of cells express αβ TCR + The second cell population may be CD45 cells. + Less than 10 percent of the cells may be NK cells. + More than 30 percent of cells are Vδ1 + The second cell population may be a γδ TCR cell. + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells are Vδ2 + The second cell population may be a γδ TCR cell. + More than 70 percent of the cells are T EM The second cell population may be a γδ TCR cell. +Fewer than 25 percent of cells were T EMRA The second cell population may be a γδ TCR cell. + Fewer than 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Between 1 and 10 percent of cells express CD69 + CD103 + T RM The second cell population may be a γδ TCR cell. + Fewer than 50 percent of the cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 50 percent of cells are CD56 + The second cell population may be a γδ TCR cell. + Between 1 and 40 percent of cells express CD137 + The second cell population may be a γδ TCR cell. + Fewer than 25 percent of cells express PD-1 + The second cell population may be a γδ TCR cell. + Between 5 and 40 percent of the cells were BTLA + The second cell population may be a γδ TCR cell. + More than 60 percent of cells express NKG2D + The second cell population may be a γδ TCR cell. + More than 20 percent of the cells express NKp46 + It may be a cell.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although the present invention can be practiced using methods and materials similar or equivalent to those described herein, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0025] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief description of the drawings]

[0026] [Figure 1A-B] Photograph of representative pseudomyxoma peritonei (PMP) TIL tissue. PMP tissue from a representative patient tumor was shown using hematoxylin and eosin staining to show focal lymphocytic infiltration confined to the tumor-associated stroma. No lymphocytes are seen in the mucin pool (white). [Diagram 2] Figure 1 shows clinical variables describing PMP patients whose tumors were used for lymphocyte repertoire sequencing. A representative retrospective tumor was used for histological analysis and repertoire sequencing of low-grade PMP treated with cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). MSS = microsatellite stable; PD-L1 = programmed cell death ligand 1 positive (0.9% in selected positive patients). [Figure 3A-B]Low-grade PMP showed an increase in the BCR IgE fraction associated with TCR Vδ. After dimer-avoidance multiplex polymerase chain reaction (DAM-PCR) and next-generation sequencing of RNA isolated from resected formalin-fixed paraffin-embedded (FFPE) low-grade PMP (n=10) tumor tissues, T-cell receptor CDR3 sequences and B-cell receptor CDR3 sequences were constructed using the migec v1.2.9 MiXCR pipeline. Figure 3A shows a representative treemap of a patient's PMP tumor repertoire, where each rounded rectangle represents a unique CDR3 and the size of the rectangle corresponds to the relative frequency of the CDR3 clone across the repertoire. The mean CDR3 expression (Figure 3B), mean CDR3 amino acid length (Figure 3C), true entropy repertoire diversity (Figure 3D), and BCR immunoglobin fraction (Figure 3E) of the entire cohort. Figure 3F shows a comparison of IgE fractions between the PMP cohort, healthy donor PBMCs (HD PBMCs; n=238), and high-grade pancreatic cancer tumors (pancreatic ductal adenocarcinoma; PDAC; n=68). Figure 3G shows the correlation between TCR Vδ% and BCR IgE fractions in the PMP cohort. [Figure 3C-D]Low-grade PMP showed an increase in the BCR IgE fraction associated with TCR Vδ. After dimer-avoidance multiplex polymerase chain reaction (DAM-PCR) and next-generation sequencing of RNA isolated from resected formalin-fixed paraffin-embedded (FFPE) low-grade PMP (n=10) tumor tissue, T-cell receptor CDR3 sequences and B-cell receptor CDR3 sequences were constructed using the migec v1.2.9 MiXCR pipeline. Figure 3A shows a representative treemap of a patient's PMP tumor repertoire, where each rounded rectangle represents a unique CDR3 and the size of the rectangle corresponds to the relative frequency of the CDR3 clone across the repertoire. The mean CDR3 expression (Figure 3B), mean CDR3 amino acid length (Figure 3C), true entropy repertoire diversity (Figure 3D), and BCR immunoglobin fraction (Figure 3E) of the entire cohort. Figure 3F shows a comparison of IgE fractions between the PMP cohort, healthy donor PBMCs (HD PBMCs; n=238), and high-grade pancreatic cancer tumors (pancreatic ductal adenocarcinoma; PDAC; n=68). Figure 3G shows the correlation between TCR Vδ% and BCR IgE fractions in the PMP cohort. [Figure 3E-F]Low-grade PMP showed an increase in the BCR IgE fraction associated with TCR Vδ. After dimer-avoidance multiplex polymerase chain reaction (DAM-PCR) and next-generation sequencing of RNA isolated from resected formalin-fixed paraffin-embedded (FFPE) low-grade PMP (n=10) tumor tissue, T-cell receptor CDR3 sequences and B-cell receptor CDR3 sequences were constructed using the migec v1.2.9 MiXCR pipeline. Figure 3A shows a representative treemap of a patient's PMP tumor repertoire, where each rounded rectangle represents a unique CDR3 and the size of the rectangle corresponds to the relative frequency of the CDR3 clone across the repertoire. The mean CDR3 expression (Figure 3B), mean CDR3 amino acid length (Figure 3C), true entropy repertoire diversity (Figure 3D), and BCR immunoglobin fraction (Figure 3E) of the entire cohort. Figure 3F shows a comparison of IgE fractions between the PMP cohort, healthy donor PBMCs (HD PBMCs; n=238), and high-grade pancreatic cancer tumors (pancreatic ductal adenocarcinoma; PDAC; n=68). Figure 3G shows the correlation between TCR Vδ% and BCR IgE fractions in the PMP cohort. [Figure 3G]Low-grade PMP showed an increase in the BCR IgE fraction associated with TCR Vδ. After dimer-avoidance multiplex polymerase chain reaction (DAM-PCR) and next-generation sequencing of RNA isolated from resected formalin-fixed paraffin-embedded (FFPE) low-grade PMP (n=10) tumor tissue, T-cell receptor CDR3 sequences and B-cell receptor CDR3 sequences were constructed using the migec v1.2.9 MiXCR pipeline. Figure 3A shows a representative treemap of a patient's PMP tumor repertoire, where each rounded rectangle represents a unique CDR3 and the size of the rectangle corresponds to the relative frequency of the CDR3 clone across the repertoire. The mean CDR3 expression (Figure 3B), mean CDR3 amino acid length (Figure 3C), true entropy repertoire diversity (Figure 3D), and BCR immunoglobin fraction (Figure 3E) of the entire cohort. Figure 3F shows a comparison of IgE fractions between the PMP cohort, healthy donor PBMCs (HD PBMCs; n=238), and high-grade pancreatic cancer tumors (pancreatic ductal adenocarcinoma; PDAC; n=68). Figure 3G shows the correlation between TCR Vδ% and BCR IgE fractions in the PMP cohort. [Figure 4A-B]Data on sharing of PMP repertoires are shown. PMP T and B cell repertoires were compared to healthy donor PBMC repertoires (n=238) and shared CDR3s (public) localized to Vα, Igκ, and Igλ chains were identified (Figure 4A). Figure 4B shows the breakdown of the percentage of n=238 healthy donors with shared uCDR3s in the PMP cohort. Figure 4C shows the identification of shared CDR3 sequences in the PMP cohort that are separate from the public CDR3s, localized to the BCR chains. Figure 4D shows the generation probability of the 10 identified IgH sequences (SEQ ID NOs: 1-10 from the left) shared in the PMP cohort, with a high probability associated with random recombination, whereas a low probability associated with antigen-directed convergent evolution. Figure 4E shows the BCR immunoglobin fraction, consisting mainly of IgG, IgE, and IgA, among the 11 shared IgH PMP CDR3s identified. BCR sequences, particularly those of the heavy chain, have five distinct subtypes: IgA, D, E, G, and M, each with different antigen specificities, structural homologies, and functions. The figure shows that there are public BCRs in the PMP repertoire that are shared with the general population (2-3% of IgK and IgL). There are also CDR3 sequences shared within the BCR repertoire restricted to patients with PMP. Quantifying the probability of generation of these amino acid sequences suggests that certain shared sequences are due to random recombination, while others are due to antigen-driven recombination, thus suggesting convergent evolution of BCR clones against a common antigen to PMP tumors. Figure 4D shows this calculation for 11 IgH sequences shared within patients in the PMP cohort. Figure 4E details the immunoglobulin fraction (BCR identity) of the shared PMP IgH sequences, showing that these are primary IgG and IgE. [Figure 4C-D]Data on sharing of PMP repertoires are shown. PMP T and B cell repertoires were compared to healthy donor PBMC repertoires (n=238) and shared CDR3s (public) localized to Vα, Igκ, and Igλ chains were identified (Figure 4A). Figure 4B shows the breakdown of the percentage of n=238 healthy donors with shared uCDR3s in the PMP cohort. Figure 4C shows the identification of shared CDR3 sequences in the PMP cohort that are separate from the public CDR3s, localized to the BCR chains. Figure 4D shows the generation probability of the 10 identified IgH sequences (SEQ ID NOs: 1-10 from the left) shared in the PMP cohort, with a high probability associated with random recombination, whereas a low probability associated with antigen-directed convergent evolution. Figure 4E shows the BCR immunoglobin fraction, consisting mainly of IgG, IgE, and IgA, among the 11 shared IgH PMP CDR3s identified. BCR sequences, particularly those of the heavy chain, have five distinct subtypes: IgA, D, E, G, and M, each with different antigen specificities, structural homologies, and functions. The figure shows that there are public BCRs in the PMP repertoire that are shared with the general population (2-3% of IgK and IgL). There are also CDR3 sequences shared within the BCR repertoire restricted to patients with PMP. Quantifying the probability of generation of these amino acid sequences suggests that certain shared sequences are due to random recombination, while others are due to antigen-driven recombination, thus suggesting convergent evolution of BCR clones against a common antigen to PMP tumors. Figure 4D shows this calculation for 11 IgH sequences shared within patients in the PMP cohort. Figure 4E details the immunoglobulin fraction (BCR identity) of the shared PMP IgH sequences, showing that these are primary IgG and IgE. [Figure 4E]Data on sharing of PMP repertoires are shown. PMP T and B cell repertoires were compared to healthy donor PBMC repertoires (n=238) and shared CDR3s (public) localized to Vα, Igκ, and Igλ chains were identified (Figure 4A). Figure 4B shows the breakdown of the percentage of n=238 healthy donors with shared uCDR3s in the PMP cohort. Figure 4C shows the identification of shared CDR3 sequences in the PMP cohort that are separate from the public CDR3s, localized to the BCR chains. Figure 4D shows the generation probability of the 10 identified IgH sequences (SEQ ID NOs: 1-10 from the left) shared in the PMP cohort, with a high probability associated with random recombination, whereas a low probability associated with antigen-directed convergent evolution. Figure 4E shows the BCR immunoglobin fraction, consisting mainly of IgG, IgE, and IgA, among the 11 shared IgH PMP CDR3s identified. BCR sequences, particularly those of the heavy chain, have five distinct subtypes: IgA, D, E, G, and M, each with different antigen specificities, structural homologies, and functions. The figure shows that there are public BCRs in the PMP repertoire that are shared with the general population (2-3% of IgK and IgL). There are also CDR3 sequences shared within the BCR repertoire restricted to patients with PMP. Quantifying the probability of generation of these amino acid sequences suggests that certain shared sequences are due to random recombination, while others are due to antigen-driven recombination, thus suggesting convergent evolution of BCR clones against a common antigen to PMP tumors. Figure 4D shows this calculation for 11 IgH sequences shared within patients in the PMP cohort. Figure 4E details the immunoglobulin fraction (BCR identity) of the shared PMP IgH sequences, showing that these are primary IgG and IgE. [Figure 5A]γδ TILs sparsely infiltrate peritoneal surface malignancies. Figure 5A shows an overview of the study outline. Tumor specimens were prospectively collected from patients (n=26) with peritoneal surface malignancies undergoing cytoreductive surgery-hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). Tumor-infiltrating lymphocytes were released from spatially distinct tumor fragments (n=40 per patient) that were first cultured with high concentrations of IL-2 (3,000 IU / mL). RPMI (10% human AB serum) medium and IL-2 were replenished every 3 days in gas-permeable culture flasks (G-REX®). On day 11, following phenotyping by spectral cytometry, γδ TILs were negatively selected by magnetic bead isolation and rapidly expanded in parallel native αβ TIL cultures (1×106 cells) in combination with anti-CD3 antibodies (OKT-3 30 ng / mL), IL-2 (3,000 IU / mL), radiation (30 Gy), allogeneic healthy donor PBMCs (1:100; 1×108 cells), and other γ chain cytokines. Spectral cytometric phenotyping of expanded γδ and αβ TILs was completed on day 25. At the time of tumor fragmentation, the remaining spatially representative tumor fragments were utilized for tumor digestion (Miltenyi Biotech GentleMACS system) and cryopreserved until autologous tumor reactivity assessment by co-culture of single-cell suspensions of expanded TILs and tumor digests. Figure 5B shows a comparison of total TILs harvested on day 11 of pre-expansion (pre-REP) culture by peritoneal tumor histology (high-grade colon cancer vs. low-grade (grade 1) appendix cancer). Figure 5C provides spectral cytometry phenotyping data on day 11 for viable TIL populations (CD56+ CD3- NK cells; CD3+ γδ TCR+ cells; CD3+ αβ TCR+ CD4+ T cells; and CD3+ αβ TCR+ CD8+ T cells). Figure 5D shows the percentages of γδ Vδ chain subsets determined by spectral cytometry (CD3+, γδ TCR+, Vδ1+, Vδ2+, or Vδ1-, Vδ2- cells) on day 11 of pre-expansion (pre-REP) culture. [Figure 5B-C]γδ TILs sparsely infiltrate peritoneal surface malignancies. Figure 5A shows an overview of the study outline. Tumor specimens were prospectively collected from patients (n=26) with peritoneal surface malignancies undergoing cytoreductive surgery-hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). Tumor-infiltrating lymphocytes were released from spatially distinct tumor fragments (n=40 per patient) that were first cultured with high concentrations of IL-2 (3,000 IU / mL). RPMI (10% human AB serum) medium and IL-2 were replenished every 3 days in gas-permeable culture flasks (G-REX®). On day 11, following phenotyping by spectral cytometry, γδ TILs were negatively selected by magnetic bead isolation and rapidly expanded in parallel native αβ TIL cultures (1×106 cells) in combination with anti-CD3 antibodies (OKT-3 30 ng / mL), IL-2 (3,000 IU / mL), radiation (30 Gy), allogeneic healthy donor PBMCs (1:100; 1×108 cells), and other γ chain cytokines. Spectral cytometric phenotyping of expanded γδ and αβ TILs was completed on day 25. At the time of tumor fragmentation, the remaining spatially representative tumor fragments were utilized for tumor digestion (Miltenyi Biotech GentleMACS system) and cryopreserved until autologous tumor reactivity assessment by co-culture of single-cell suspensions of expanded TILs and tumor digests. Figure 5B shows a comparison of total TILs harvested on day 11 of pre-expansion (pre-REP) culture by peritoneal tumor histology (high-grade colon cancer vs. low-grade (grade 1) appendix cancer). Figure 5C provides spectral cytometry phenotyping data on day 11 for viable TIL populations (CD56+ CD3- NK cells; CD3+ γδ TCR+ cells; CD3+ αβ TCR+ CD4+ T cells; and CD3+ αβ TCR+ CD8+ T cells). Figure 5D shows the percentages of γδ Vδ chain subsets determined by spectral cytometry (CD3+, γδ TCR+, Vδ1+, Vδ2+, or Vδ1-, Vδ2- cells) on day 11 of pre-expansion (pre-REP) culture. [Figure 5D]γδ TILs sparsely infiltrate peritoneal surface malignancies. Figure 5A shows an overview of the study outline. Tumor specimens were prospectively collected from patients (n=26) with peritoneal surface malignancies undergoing cytoreductive surgery-hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). Tumor-infiltrating lymphocytes were released from spatially distinct tumor fragments (n=40 per patient) that were first cultured with high concentrations of IL-2 (3,000 IU / mL). RPMI (10% human AB serum) medium and IL-2 were replenished every 3 days in gas-permeable culture flasks (G-REX®). On day 11, following phenotyping by spectral cytometry, γδ TILs were negatively selected by magnetic bead isolation and rapidly expanded in parallel native αβ TIL cultures (1×106 cells) in combination with anti-CD3 antibodies (OKT-3 30 ng / mL), IL-2 (3,000 IU / mL), radiation (30 Gy), allogeneic healthy donor PBMCs (1:100; 1×108 cells), and other γ chain cytokines. Spectral cytometric phenotyping of expanded γδ and αβ TILs was completed on day 25. At the time of tumor fragmentation, the remaining spatially representative tumor fragments were utilized for tumor digestion (Miltenyi Biotech GentleMACS system) and cryopreserved until autologous tumor reactivity assessment by co-culture of single-cell suspensions of expanded TILs and tumor digests. Figure 5B shows a comparison of total TILs harvested on day 11 of pre-expansion (pre-REP) culture by peritoneal tumor histology (high-grade colon cancer vs. low-grade (grade 1) appendix cancer). Figure 5C provides spectral cytometry phenotyping data on day 11 for viable TIL populations (CD56+ CD3- NK cells; CD3+ γδ TCR+ cells; CD3+ αβ TCR+ CD4+ T cells; and CD3+ αβ TCR+ CD8+ T cells). Figure 5D shows the percentages of γδ Vδ chain subsets determined by spectral cytometry (CD3+, γδ TCR+, Vδ1+, Vδ2+, or Vδ1-, Vδ2- cells) on day 11 of pre-expansion (pre-REP) culture. [Figure 6]Table showing characteristics of prospective CRS-HIPEC patients. Clinical characteristics of patients with peritoneal surface malignancies undergoing CRS-HIPEC whose tumors were prospectively sampled for TIL culture are shown. [Figure 7A-B] Phenotypic assessment of peritoneal tumors prior to fragmentation and rapid expansion. Figure 7A shows four consecutive photographs of the dissection and fragmentation of mucinous peritoneal tumors into spatially distinct 2-3 mm3 tumor pieces. On day 11, γδ TCR+ % and total viable cell counts were compared by histological examination (Figure 7B) or prior chemotherapy (Figures 7D and 7E). Figure 7C shows the gating strategy for spectral cytometry. CD45+ immune cells were selected from live single cells. NK cells (CD3- CD56+) and T cells (CD3+) were selected. T cells were segmented by TCR αβ or γδ positivity. CD4 / CD8 or Vδ1 / Vδ2 populations were identified from the selected T cell subsets. [Figure 7C] Phenotypic assessment of peritoneal tumors prior to fragmentation and rapid expansion. Figure 7A shows four consecutive photographs of the dissection and fragmentation of mucinous peritoneal tumors into spatially distinct 2-3 mm3 tumor pieces. On day 11, γδ TCR+ % and total viable cell counts were compared by histological examination (Figure 7B) or prior chemotherapy (Figures 7D and 7E). Figure 7C shows the gating strategy for spectral cytometry. CD45+ immune cells were selected from live single cells. NK cells (CD3- CD56+) and T cells (CD3+) were selected. T cells were segmented by TCR αβ or γδ positivity. CD4 / CD8 or Vδ1 / Vδ2 populations were identified from the selected T cell subsets. [Figure 7D-E]Phenotypic assessment of peritoneal tumors prior to fragmentation and rapid expansion. Figure 7A shows four consecutive photographs of the dissection and fragmentation of mucinous peritoneal tumors into spatially distinct 2-3 mm3 tumor pieces. On day 11, γδ TCR+ % and total viable cell counts were compared by histological examination (Figure 7B) or prior chemotherapy (Figures 7D and 7E). Figure 7C shows the gating strategy for spectral cytometry. CD45+ immune cells were selected from live single cells. NK cells (CD3- CD56+) and T cells (CD3+) were selected. T cells were segmented by TCR αβ or γδ positivity. CD4 / CD8 or Vδ1 / Vδ2 populations were identified from the selected T cell subsets. [Figure 8A-B] γδ TILs display a tissue-resident effector memory phenotype with reduced PD-1 but increased expression of NKG2D and CD137 compared to αβ TILs. Peritoneal tumor-infiltrating lymphocyte spectrum phenotyping on day 11 comparing CD8 expression of αβ and γδ TILs (CD8α+, CD8β+, or double positive; FIG. 8A), memory phenotype (naive with CD62L+ CD45RO-; central memory with CD62L+, CD45RO+; effector memory with CD62L- CD45RO+; effector memory RA+ with CD62L- CD45RO-; FIGs. 8B and 8C), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 8D), activation status (CD2, CD25, CD27, CD56, CD137, or 4-1BB; FIG. 8E), exhaustion status (PD-1, LAG-3, TIGIT, BTLA, or PD-L1; FIG. 8F), and natural cytotoxicity receptor (NCR) expression (NKG2D or NKp46; FIG. 8G). FIG. 8H is a summary of the mean expression heatmaps of CD8, memory, activation, exhaustion, and natural cytotoxicity receptor (NCR) phenotypes. [Figure 8C-D]γδ TILs display a tissue-resident effector memory phenotype with reduced PD-1 but increased expression of NKG2D and CD137 compared to αβ TILs. Peritoneal tumor-infiltrating lymphocyte spectrum phenotyping on day 11 comparing CD8 expression of αβ and γδ TILs (CD8α+, CD8β+, or double positive; FIG. 8A), memory phenotype (naive with CD62L+ CD45RO-; central memory with CD62L+, CD45RO+; effector memory with CD62L- CD45RO+; effector memory RA+ with CD62L- CD45RO-; FIGs. 8B and 8C), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 8D), activation status (CD2, CD25, CD27, CD56, CD137, or 4-1BB; FIG. 8E), exhaustion status (PD-1, LAG-3, TIGIT, BTLA, or PD-L1; FIG. 8F), and natural cytotoxicity receptor (NCR) expression (NKG2D or NKp46; FIG. 8G). FIG. 8H is a summary of the mean expression heatmaps of CD8, memory, activation, exhaustion, and natural cytotoxicity receptor (NCR) phenotypes. [Fig. 8E-F] γδ TILs display a tissue-resident effector memory phenotype with reduced PD-1 but increased expression of NKG2D and CD137 compared to αβ TILs. Peritoneal tumor-infiltrating lymphocyte spectrum phenotyping on day 11 comparing CD8 expression of αβ and γδ TILs (CD8α+, CD8β+, or double positive; FIG. 8A), memory phenotype (naive with CD62L+ CD45RO-; central memory with CD62L+, CD45RO+; effector memory with CD62L- CD45RO+; effector memory RA+ with CD62L- CD45RO-; FIGs. 8B and 8C), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 8D), activation status (CD2, CD25, CD27, CD56, CD137, or 4-1BB; FIG. 8E), exhaustion status (PD-1, LAG-3, TIGIT, BTLA, or PD-L1; FIG. 8F), and natural cytotoxicity receptor (NCR) expression (NKG2D or NKp46; FIG. 8G). FIG. 8H is a summary of the mean expression heatmaps of CD8, memory, activation, exhaustion, and natural cytotoxicity receptor (NCR) phenotypes. [Figure 8G-H] γδ TILs display a tissue-resident effector memory phenotype with reduced PD-1 but increased expression of NKG2D and CD137 compared to αβ TILs. Peritoneal tumor-infiltrating lymphocyte spectrum phenotyping on day 11 comparing CD8 expression of αβ and γδ TILs (CD8α+, CD8β+, or double positive; FIG. 8A), memory phenotype (naive with CD62L+ CD45RO-; central memory with CD62L+, CD45RO+; effector memory with CD62L- CD45RO+; effector memory RA+ with CD62L- CD45RO-; FIGs. 8B and 8C), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 8D), activation status (CD2, CD25, CD27, CD56, CD137, or 4-1BB; FIG. 8E), exhaustion status (PD-1, LAG-3, TIGIT, BTLA, or PD-L1; FIG. 8F), and natural cytotoxicity receptor (NCR) expression (NKG2D or NKp46; FIG. 8G). FIG. 8H is a summary of the mean expression heatmaps of CD8, memory, activation, exhaustion, and natural cytotoxicity receptor (NCR) phenotypes. [Figure 9A] Representative flow diagram showing αβ and γδ TIL percent positive cells gated by fluorescence minus one (FMO) controls and unstimulated PBMCs (negative control) with respect to CD8 (FIG. 9A), activation status (CD56 and CD137 or 4-1BB; FIG. 9B), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 9C), exhaustion status (PD-1 and BTLA; FIG. 9D), and natural cytotoxicity receptor expression (NKG2D; FIG. 9E). [Figure 9B] Representative flow diagram showing αβ and γδ TIL percent positive cells gated by fluorescence minus one (FMO) controls and unstimulated PBMCs (negative control) with respect to CD8 (FIG. 9A), activation status (CD56 and CD137 or 4-1BB; FIG. 9B), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 9C), exhaustion status (PD-1 and BTLA; FIG. 9D), and natural cytotoxicity receptor expression (NKG2D; FIG. 9E). [Figure 9C]Representative flow diagram showing αβ and γδ TIL percent positive cells gated by fluorescence minus one (FMO) controls and unstimulated PBMCs (negative control) with respect to CD8 (FIG. 9A), activation status (CD56 and CD137 or 4-1BB; FIG. 9B), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 9C), exhaustion status (PD-1 and BTLA; FIG. 9D), and natural cytotoxicity receptor expression (NKG2D; FIG. 9E). [Figure 9D] Representative flow diagram showing αβ and γδ TIL percent positive cells gated by fluorescence minus one (FMO) controls and unstimulated PBMCs (negative control) with respect to CD8 (FIG. 9A), activation status (CD56 and CD137 or 4-1BB; FIG. 9B), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 9C), exhaustion status (PD-1 and BTLA; FIG. 9D), and natural cytotoxicity receptor expression (NKG2D; FIG. 9E). [Figure 9E] Representative flow diagram showing αβ and γδ TIL percent positive cells gated by fluorescence minus one (FMO) controls and unstimulated PBMCs (negative control) with respect to CD8 (FIG. 9A), activation status (CD56 and CD137 or 4-1BB; FIG. 9B), tissue-resident memory phenotype (CD69+, CD103+, or double positive; FIG. 9C), exhaustion status (PD-1 and BTLA; FIG. 9D), and natural cytotoxicity receptor expression (NKG2D; FIG. 9E). [Figure 10A-B]Use of IL-4 and IL-15 for rapid expansion of γδ TILs. 1×10 6 negatively selected γδ TILs or native αβ TILs were cultured in gas-permeable flasks (G-REX®) with anti-CD3 antibody (OKT-3, 30 ng / mL), irradiated (30 Gy) allogeneic PBMC feeder cells (1:100), IL-2 (3,000 IU / mL), RPM1640 (5% human serum), and other indicated γ-chain cytokines (IL-4 100 ng / mL; IL-7 20 ng / mL; IL-15 70 ng / mL; or combinations thereof) and expanded in culture for 14 days, with cytokines and medium replaced every 3 days. Total viable cell counts under individual and various cytokine combinations were measured at days 7, 10, and 14 of the rapid expansion protocol ( FIG. 10A ). Negatively selected γδ TIL populations expanded for 14 days with IL-2 / IL-4 / IL-15 contained minimal NK (CD3- CD56+) and αβ T (CD3+ αβ TCR+) cells, as well as γδ T cells (CD3+ γδ TCR+) that were predominantly Vδ1+ or Vδ1-δ2- cells, as assessed by spectral cytometry (Figure 10B). The mean percent change in absolute percent positive cells between phenotyping on day 11 (i.e., before expansion) and day 14 after expansion (i.e., day 25 total) for αβ TILs (expanded using anti-CD3 antibody (OKT-3, 30 ng / mL), irradiated (30 Gy) allogeneic PBMC feeder cells (1:100), IL-2 (3,000 IU / mL), and RPM1640 (5% human serum)) and γδ TILs (expanded using the IL-2 / IL-4 / IL-15 combination) is shown in FIG. 10C. Statistics show significant changes from day 11 to day 25 of culture as a result of IL-2 / IL-4 / IL-15 expansion. [Figure 10C]Use of IL-4 and IL-15 for rapid expansion of γδ TILs. 1×10 6 negatively selected γδ TILs or native αβ TILs were cultured in gas-permeable flasks (G-REX®) with anti-CD3 antibody (OKT-3, 30 ng / mL), irradiated (30 Gy) allogeneic PBMC feeder cells (1:100), IL-2 (3,000 IU / mL), RPM1640 (5% human serum), and other indicated γ-chain cytokines (IL-4 100 ng / mL; IL-7 20 ng / mL; IL-15 70 ng / mL; or combinations thereof) and expanded in culture for 14 days, with cytokines and medium replaced every 3 days. Total viable cell counts under individual and various cytokine combinations were measured at days 7, 10, and 14 of the rapid expansion protocol ( FIG. 10A ). Negatively selected γδ TIL populations expanded for 14 days with IL-2 / IL-4 / IL-15 contained minimal NK (CD3- CD56+) and αβ T (CD3+ αβ TCR+) cells, as well as γδ T cells (CD3+ γδ TCR+) that were predominantly Vδ1+ or Vδ1-δ2- cells, as assessed by spectral cytometry (Figure 10B). The mean percent change in absolute percent positive cells between phenotyping on day 11 (i.e., before expansion) and day 14 after expansion (i.e., day 25 total) for αβ TILs (expanded using anti-CD3 antibody (OKT-3, 30 ng / mL), irradiated (30 Gy) allogeneic PBMC feeder cells (1:100), IL-2 (3,000 IU / mL), and RPM1640 (5% human serum)) and γδ TILs (expanded using the IL-2 / IL-4 / IL-15 combination) is shown in FIG. 10C. Statistics show significant changes from day 11 to day 25 of culture as a result of IL-2 / IL-4 / IL-15 expansion. [Figure 11A]Fold expansion and phenotyping of rapidly expanded γδ and αβ TILs. FIG. 11A shows the fold expansion of negatively selected γδ TILs (e.g., negatively selected by αβ TCR ablation) and native αβ TILs after 14 days of culture with the indicated combinations of different cytokines. FIG. 11B shows representative flow plots of expanded negatively selected γδ TILs expanded in IL-2, IL-4, and IL-15 for 14 days of culture. FIG. 11C shows the phenotype of cells present after IL-2-only expansion of native αβ TIL populations. FIG. 11D shows an overview of the average expression heatmaps of memory markers, activation markers, exhaustion markers, and natural cytotoxicity receptors (NCRs) in IL-2-expanded positively selected αβ TILs and IL-2 / IL-4 / IL-15-expanded negatively selected γδ TILs after 14 days of expansion. Statistics show that there is a significant difference between γδ and αβ TILs at day 25 of culture as a result of IL-2 / IL-4 / IL-15 expansion. [Figure 11B] Fold expansion and phenotyping of rapidly expanded γδ and αβ TILs. FIG. 11A shows the fold expansion of negatively selected γδ TILs (e.g., negatively selected by αβ TCR ablation) and native αβ TILs after 14 days of culture with the indicated combinations of different cytokines. FIG. 11B shows representative flow plots of expanded negatively selected γδ TILs expanded in IL-2, IL-4, and IL-15 for 14 days of culture. FIG. 11C shows the phenotype of cells present after IL-2-only expansion of native αβ TIL populations. FIG. 11D shows an overview of the average expression heatmaps of memory markers, activation markers, exhaustion markers, and natural cytotoxicity receptors (NCRs) in IL-2-expanded positively selected αβ TILs and IL-2 / IL-4 / IL-15-expanded negatively selected γδ TILs after 14 days of expansion. Statistics show that there is a significant difference between γδ and αβ TILs at day 25 of culture as a result of IL-2 / IL-4 / IL-15 expansion. [Figure 11C-D]Fold expansion and phenotyping of rapidly expanded γδ and αβ TILs. FIG. 11A shows the fold expansion of negatively selected γδ TILs (e.g., negatively selected by αβ TCR ablation) and native αβ TILs after 14 days of culture with the indicated combinations of different cytokines. FIG. 11B shows representative flow plots of expanded negatively selected γδ TILs expanded in IL-2, IL-4, and IL-15 for 14 days of culture. FIG. 11C shows the phenotype of cells present after IL-2-only expansion of native αβ TIL populations. FIG. 11D shows an overview of the average expression heatmaps of memory markers, activation markers, exhaustion markers, and natural cytotoxicity receptors (NCRs) in IL-2-expanded positively selected αβ TILs and IL-2 / IL-4 / IL-15-expanded negatively selected γδ TILs after 14 days of expansion. Statistics show that there is a significant difference between γδ and αβ TILs at day 25 of culture as a result of IL-2 / IL-4 / IL-15 expansion. [Figure 12A-B]MHC-independent γδ TCR-mediated autologous tumor recognition. TILs were thawed and rested overnight in IL-2 (3,000 IU / mL) medium, then washed twice with PBS and co-cultured. Autologous tumor reactivity was assessed by co-culturing 1×105 14-day rapidly expanded αβ TILs (natural αβ TILs expanded with IL-2) or γδ TILs (negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15) with 1×105 tumor digest cells in cytokine-free medium for 24 h in 96-well plates. IFNγ production was assessed in culture supernatants by ELISA. Figure 12A shows IFNγ production of expanded αβ or γδ TILs after non-specific CD3 / CD28 stimulation (Dynabeads, positive control), after co-culture with autologous PBMCs (1×105 cells, negative control), or after co-culture with tumor digest. MHC-unrestricted TIL reactivity of expanded γδ and αβ TILs was assessed with K562 leukemia cancer cell lines and a panel of colon cancer cell lines (HCT116, RKO, SW480, and SW80) passaged twice before coculture (Figure 12B). In a subset of patients, γδ TILs were cultured with autologous tumor digests in the presence of blocking antibodies (isotype control mouse IgG 10 μg / mL, anti-MHC-1 (W6 / 32 10 μg / mL), anti-γδ TCR (7A5 3 μg / mL), or anti-NKG2D (1D11, 10 μg / mL)) (Figure 12C). Figure 12D shows the correlation between IFNγ production and the percentage of Vδ1 γδ TILs after expansion. [Fig. 12C-D]MHC-independent γδ TCR-mediated autologous tumor recognition. TILs were thawed and rested overnight in IL-2 (3,000 IU / mL) medium, then washed twice with PBS and co-cultured. Autologous tumor reactivity was assessed by co-culturing 1×105 14-day rapidly expanded αβ TILs (natural αβ TILs expanded with IL-2) or γδ TILs (negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15) with 1×105 tumor digest cells in cytokine-free medium for 24 h in 96-well plates. IFNγ production was assessed in culture supernatants by ELISA. Figure 12A shows IFNγ production of expanded αβ or γδ TILs after non-specific CD3 / CD28 stimulation (Dynabeads, positive control), after co-culture with autologous PBMCs (1×105 cells, negative control), or after co-culture with tumor digest. MHC-unrestricted TIL reactivity of expanded γδ and αβ TILs was assessed with K562 leukemia cancer cell lines and a panel of colon cancer cell lines (HCT116, RKO, SW480, and SW80) passaged twice before coculture (Figure 12B). In a subset of patients, γδ TILs were cultured with autologous tumor digests in the presence of blocking antibodies (isotype control mouse IgG 10 μg / mL, anti-MHC-1 (W6 / 32 10 μg / mL), anti-γδ TCR (7A5 3 μg / mL), or anti-NKG2D (1D11, 10 μg / mL)) (Figure 12C). Figure 12D shows the correlation between IFNγ production and the percentage of Vδ1 γδ TILs after expansion. [Figure 13] Table showing the expression of NKG2D ligands in cancer cell lines. Z-scores of natural killer receptor ligand mRNAs in tested cancer cell lines were queried from the Cancer Line Encyclopedia. Cell lines with stable or upregulated MICA and MICB allow recognition of γδ TILs. [Figure 14] 1 is a table describing the clinical characteristics of patients by resected melanoma, whose tumor specimens were utilized for tumor digestion and TIL expansion. [Figure 15A-B]High dose IL-2 expands γδ TCR+ TILs the most during pre-rapid expansion protocol (pre-REP). Cryopreserved melanoma tumor digests (n=15) were thawed and plated (5×106 cells / well) in G-REX® culture wells with complete medium and high dose IL-2 (3,000 IU / mL, n=15, black), a combination of γ-chain cytokines (IL-2 3,0000 IU / mL, IL-4 100 ng / mL, IL-15 70 ng / mL, n=15, red), or a combination of γ-chain cytokines and anti-CD137 mAb (urelumab, 10 μg / mL, n=10, black and white). Cultures were supplemented with cytokines or antibodies on days 4 and 8, and 50% of the medium was replaced on day 8. On day 11, expanded TILs were harvested through a 70 μm filter, counted (A, B), and analyzed by spectral cytometry for TIL populations (B–E). [Figure 15C-D] High dose IL-2 expands γδ TCR+ TILs the most during pre-rapid expansion protocol (pre-REP). Cryopreserved melanoma tumor digests (n=15) were thawed and plated (5×106 cells / well) in G-REX® culture wells with complete medium and high dose IL-2 (3,000 IU / mL, n=15, black), a combination of γ-chain cytokines (IL-2 3,0000 IU / mL, IL-4 100 ng / mL, IL-15 70 ng / mL, n=15, red), or a combination of γ-chain cytokines and anti-CD137 mAb (urelumab, 10 μg / mL, n=10, black and white). Cultures were supplemented with cytokines or antibodies on days 4 and 8, and 50% of the medium was replaced on day 8. On day 11, expanded TILs were harvested through a 70 μm filter, counted (A, B), and analyzed by spectral cytometry for TIL populations (B–E). [Figure 15E]High dose IL-2 expands γδ TCR+ TILs the most during pre-rapid expansion protocol (pre-REP). Cryopreserved melanoma tumor digests (n=15) were thawed and plated (5×106 cells / well) in G-REX® culture wells with complete medium and high dose IL-2 (3,000 IU / mL, n=15, black), a combination of γ-chain cytokines (IL-2 3,0000 IU / mL, IL-4 100 ng / mL, IL-15 70 ng / mL, n=15, red), or a combination of γ-chain cytokines and anti-CD137 mAb (urelumab, 10 μg / mL, n=10, black and white). Cultures were supplemented with cytokines or antibodies on days 4 and 8, and 50% of the medium was replaced on day 8. On day 11, expanded TILs were harvested through a 70 μm filter, counted (A, B), and analyzed by spectral cytometry for TIL populations (B–E). [Figure 16A] Vδ1 TILs are associated with improved pan-cancer survival. Figure 16A shows the mean expression (logarithm of transcripts per million (TPM)) of γδ TIL subsets (TRDV1, TRDV2, and TRDV3 genes) and αβ TILs (TRBC2 beta chain 2 constant region) in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database for bulk RNA sequencing of primary tumors. Figure 16B-G shows Kaplan-Meier survival analysis with normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median in selected tumor types in which autologous TIL therapy is tested (SKCM = cutaneous melanoma; HNSC = head and neck squamous cell carcinoma, LUAD+LUSC = lung adenocarcinoma and lung squamous cell carcinoma, BRCA = breast cancer, CESC = cervical squamous cell carcinoma and cervical adenocarcinoma). Log rank P values ​​are displayed with 95 CI of survival estimates. [Figure 16B-C]Vδ1 TILs are associated with improved pan-cancer survival. Figure 16A shows the mean expression (logarithm of transcripts per million (TPM)) of γδ TIL subsets (TRDV1, TRDV2, and TRDV3 genes) and αβ TILs (TRBC2 beta chain 2 constant region) in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database for bulk RNA sequencing of primary tumors. Figure 16B-G shows Kaplan-Meier survival analysis with normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median in selected tumor types in which autologous TIL therapy is tested (SKCM = cutaneous melanoma; HNSC = head and neck squamous cell carcinoma, LUAD+LUSC = lung adenocarcinoma and lung squamous cell carcinoma, BRCA = breast cancer, CESC = cervical squamous cell carcinoma and cervical adenocarcinoma). Log rank P values ​​are displayed with 95 CI of survival estimates. [Fig. 16D-E] Vδ1 TILs are associated with improved pan-cancer survival. Figure 16A shows the mean expression (logarithm of transcripts per million (TPM)) of γδ TIL subsets (TRDV1, TRDV2, and TRDV3 genes) and αβ TILs (TRBC2 beta chain 2 constant region) in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database for bulk RNA sequencing of primary tumors. Figure 16B-G shows Kaplan-Meier survival analysis with normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median in selected tumor types in which autologous TIL therapy is tested (SKCM = cutaneous melanoma; HNSC = head and neck squamous cell carcinoma, LUAD+LUSC = lung adenocarcinoma and lung squamous cell carcinoma, BRCA = breast cancer, CESC = cervical squamous cell carcinoma and cervical adenocarcinoma). Log rank P values ​​are displayed with 95 CI of survival estimates. [Fig. 16F-G]Vδ1 TILs are associated with improved pan-cancer survival. Figure 16A shows the mean expression (logarithm of transcripts per million (TPM)) of γδ TIL subsets (TRDV1, TRDV2, and TRDV3 genes) and αβ TILs (TRBC2 beta chain 2 constant region) in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database for bulk RNA sequencing of primary tumors. Figure 16B-G shows Kaplan-Meier survival analysis with normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median in selected tumor types in which autologous TIL therapy is tested (SKCM = cutaneous melanoma; HNSC = head and neck squamous cell carcinoma, LUAD+LUSC = lung adenocarcinoma and lung squamous cell carcinoma, BRCA = breast cancer, CESC = cervical squamous cell carcinoma and cervical adenocarcinoma). Log rank P values ​​are displayed with 95 CI of survival estimates. [Figure 17A] Survival benefit of Vδ1 in additional primary cancers. TRDV1 tumor (T) and normal (N) tissue expression for the entire cohort was plotted against the tumor types with highest Vδ1 expression: lung adenocarcinoma (LUAD), kidney renal cell carcinoma (KIRC), breast cancer (BRCA), and cervical squamous cell carcinoma and adenocarcinoma (CESC) (Figure 17A). Figures 17B-G show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for additional selected tumor types. Log rank P values ​​are displayed along with the 95 CI of survival estimates. TCGA=Full Cancer Genome Atlas GBM=glioblastoma;HNSC=head and neck squamous cell carcinoma;SKCM=cutaneous melanoma;ESCA=esophageal cancer;LUAD=lung adenocarcinoma;LUSC=lung squamous cell carcinoma;BRCA=breast cancer;MESO=mesothelioma;LIHC=liver hepatocellular carcinoma;STAD=gastric adenocarcinoma;PAAD=pancreatic ductal adenocarcinoma ;KIRC=kidney renal cell carcinoma;BLCA=bladder urothelial carcinoma;COAD=colorectal adenocarcinoma;READ=rectal adenocarcinoma;OV=ovarian serous cystadenocarcinoma;UCEC=uterine corpus endometrial carcinoma;CESC=cervical squamous cell carcinoma and cervical adenocarcinoma;PRAD=prostatic adenocarcinoma;and SARC=sarcoma. [Fig. 17B-C]Survival benefit of Vδ1 in additional primary cancers. TRDV1 tumor (T) and normal (N) tissue expression for the entire cohort was plotted against the tumor types with highest Vδ1 expression: lung adenocarcinoma (LUAD), kidney renal cell carcinoma (KIRC), breast cancer (BRCA), and cervical squamous cell carcinoma and adenocarcinoma (CESC) (Figure 17A). Figures 17B-G show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for additional selected tumor types. Log rank P values ​​are displayed along with the 95 CI of survival estimates. TCGA=Full Cancer Genome Atlas GBM=glioblastoma;HNSC=head and neck squamous cell carcinoma;SKCM=cutaneous melanoma;ESCA=esophageal cancer;LUAD=lung adenocarcinoma;LUSC=lung squamous cell carcinoma;BRCA=breast cancer;MESO=mesothelioma;LIHC=liver hepatocellular carcinoma;STAD=gastric adenocarcinoma;PAAD=pancreatic ductal adenocarcinoma ;KIRC=kidney renal cell carcinoma;BLCA=bladder urothelial carcinoma;COAD=colorectal adenocarcinoma;READ=rectal adenocarcinoma;OV=ovarian serous cystadenocarcinoma;UCEC=uterine corpus endometrial carcinoma;CESC=cervical squamous cell carcinoma and cervical adenocarcinoma;PRAD=prostatic adenocarcinoma;and SARC=sarcoma. [Fig. 17D-E]Survival benefit of Vδ1 in additional primary cancers. TRDV1 tumor (T) and normal (N) tissue expression for the entire cohort was plotted against the tumor types with highest Vδ1 expression: lung adenocarcinoma (LUAD), kidney renal cell carcinoma (KIRC), breast cancer (BRCA), and cervical squamous cell carcinoma and adenocarcinoma (CESC) (Figure 17A). Figures 17B-G show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for additional selected tumor types. Log rank P values ​​are displayed along with the 95 CI of survival estimates. TCGA=Full Cancer Genome Atlas GBM=glioblastoma;HNSC=head and neck squamous cell carcinoma;SKCM=cutaneous melanoma;ESCA=esophageal cancer;LUAD=lung adenocarcinoma;LUSC=lung squamous cell carcinoma;BRCA=breast cancer;MESO=mesothelioma;LIHC=liver hepatocellular carcinoma;STAD=gastric adenocarcinoma;PAAD=pancreatic ductal adenocarcinoma ;KIRC=kidney renal cell carcinoma;BLCA=bladder urothelial carcinoma;COAD=colorectal adenocarcinoma;READ=rectal adenocarcinoma;OV=ovarian serous cystadenocarcinoma;UCEC=uterine corpus endometrial carcinoma;CESC=cervical squamous cell carcinoma and cervical adenocarcinoma;PRAD=prostatic adenocarcinoma;and SARC=sarcoma. [Fig. 17F-G]Survival benefit of Vδ1 in additional primary cancers. TRDV1 tumor (T) and normal (N) tissue expression for the entire cohort was plotted against the tumor types with highest Vδ1 expression: lung adenocarcinoma (LUAD), kidney renal cell carcinoma (KIRC), breast cancer (BRCA), and cervical squamous cell carcinoma and adenocarcinoma (CESC) (Figure 17A). Figures 17B-G show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for additional selected tumor types. Log rank P values ​​are displayed along with the 95 CI of survival estimates. TCGA=Full Cancer Genome Atlas GBM=glioblastoma;HNSC=head and neck squamous cell carcinoma;SKCM=cutaneous melanoma;ESCA=esophageal cancer;LUAD=lung adenocarcinoma;LUSC=lung squamous cell carcinoma;BRCA=breast cancer;MESO=mesothelioma;LIHC=liver hepatocellular carcinoma;STAD=gastric adenocarcinoma;PAAD=pancreatic ductal adenocarcinoma ;KIRC=kidney renal cell carcinoma;BLCA=bladder urothelial carcinoma;COAD=colorectal adenocarcinoma;READ=rectal adenocarcinoma;OV=ovarian serous cystadenocarcinoma;UCEC=uterine corpus endometrial carcinoma;CESC=cervical squamous cell carcinoma and cervical adenocarcinoma;PRAD=prostatic adenocarcinoma;and SARC=sarcoma. [Figure 18A-B] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18C-D] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18E-F]Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18G-H] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18I-J] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18K-L] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18M-N] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18O-P]Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Fig. 18Q-R] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Figure 18S] Correlation of TRDV1 and TRBC2 genes in the 20 most common primary solid tumor types analyzed by the GEPIA2 tool of The Cancer Genome Atlas (TCGA) database on bulk RNA sequencing of primary tumors. Pearson correlation and p-values ​​were reported. The TCGA abbreviations in Figure 17 were used. All correlations were significant. [Figure 19A-B] No survival benefit of Vδ1 in certain primary cancers. Figures 19A-H show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for selected tumor types. Log rank P values ​​are displayed with the 95 CI of survival estimates. [Fig. 19C-D] No survival benefit of Vδ1 in certain primary cancers. Figures 19A-H show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for selected tumor types. Log rank P values ​​are displayed with the 95 CI of survival estimates. [Fig. 19E-F] No survival benefit of Vδ1 in certain primary cancers. Figures 19A-H show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for selected tumor types. Log rank P values ​​are displayed with the 95 CI of survival estimates. [Fig. 19G-H] No survival benefit of Vδ1 in certain primary cancers. Figures 19A-H show Kaplan-Meier survival analysis by normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for selected tumor types. Log rank P values ​​are displayed with the 95 CI of survival estimates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The present specification relates to methods and materials for culturing and expanding tumor-infiltrating γδ T cells (e.g., tumor-infiltrating γδ T cells). For example, the present specification describes methods for expanding tumor-infiltrating γδ T cells obtained from a tissue (e.g., a tumor sample) to produce larger numbers of cells than would be acceptable for therapeutic use (e.g., 1×10 7 or 1×10 8 or 5×10 8 or exceeds 1×10 9 (>100) tumor-infiltrating γδ T cells (e.g., Vδ1 + The present invention provides methods and materials for obtaining tumor-infiltrating γδ T cells (TCR-predominant γδ T cells).

[0028] As described herein, tissue containing a tumor (or healthy tissue within 30 mm of the tumor, or healthy tissue within 20 mm of the tumor, or healthy tissue within 10 mm of the tumor) can contain tumor-infiltrating γδ T cells and can be obtained from a mammal (e.g., a human cancer patient). In some cases, one or more lymph nodes adjacent to the tumor and / or one or more tumor-draining lymph nodes can contain tumor-infiltrating γδ T cells and can be obtained from a mammal (e.g., a human cancer patient). For example, lung tissue containing a lung tumor (or healthy lung tissue within 30 mm (e.g., within 20 mm or within 10 mm) of the lung tumor or the tumor-draining lymph nodes of the lung tumor) can be obtained from a mammal (e.g., a human lung cancer patient) and used as a source of tumor-infiltrating γδ T cells. In another example, skin tissue containing a skin tumor (or healthy skin tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a skin tumor or a tumor-draining lymph node of a skin tumor) can be obtained from a mammal (e.g., a human skin cancer patient) and used as a source of tumor-infiltrating γδ T cells.Other examples of tissues that may be obtained and used as described herein include, but are not limited to, tissue containing glioblastoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of glioblastoma), tissue containing head and neck squamous cell carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of head and neck squamous cell carcinoma), tissue containing cutaneous melanoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of cutaneous melanoma), tissue containing lung adenocarcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of lung adenocarcinoma), and / or tissue containing glioblastoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of glioblastoma). 0mm)), tissue containing lung squamous cell carcinoma (or healthy tissue within 30mm (e.g., within 20mm or within 10mm) of lung squamous cell carcinoma), tissue containing breast cancer (or healthy tissue within 30mm (e.g., within 20mm or within 10mm) of breast cancer), tissue containing mesothelioma (or healthy tissue within 30mm (e.g., within 20mm or within 10mm) of mesothelioma), tissue containing liver hepatocellular carcinoma (or healthy tissue within 30mm (e.g., within 20mm or within 10mm) of liver hepatocellular carcinoma), tissue containing pancreatic ductal adenocarcinoma (or healthy tissue within 30mm (e.g., within 20mm or within 10mm) of pancreatic ductal adenocarcinoma), tissue containing renal cell carcinoma of the kidney (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of renal cell carcinoma of the kidney); tissue containing bladder urothelial carcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of bladder urothelial carcinoma of the bladder); tissue containing cervical squamous cell carcinoma and cervical adenocarcinoma (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of cervical squamous cell carcinoma and cervical adenocarcinoma); tissue containing lymph node metastasis; tissue containing peritoneal tumor (or peritoneal tumor tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a bone tumor), tissue containing a bone tumor (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a bone tumor), tissue containing an endocrine tumor (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of an endocrine tumor), tissue containing a reproductive tumor (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a reproductive tumor), or tissue containing a brain (or healthy tissue within 30 mm (e.g., within 20 mm or within 10 mm) of a brain tumor).

[0029] Once the tissue is obtained, it can be cultured in a manner that facilitates the isolation of tumor-infiltrating γδ T cells from the tissue. For example, one or more small pieces of tissue (e.g., 2-3 mm 3 1 mm) in the presence of IL-2 for 5-15 days (e.g., 6-15 days, 7-15 days, 8-15 days, 9-15 days, 9-13 days, 10-12 days, 7-10 days, or 8-14 days). In some cases, one or more pieces of tissue (e.g., 2-3 mm 3 The pieces of IL-2 can be cultured in a gas-permeable rapid expansion flask. Any suitable concentration of IL-2 can be used to facilitate isolation of tumor-infiltrating γδ T cells from tissue. For example, about 50 international units (IU) to about 6000 IU (e.g., about 100 IU to about 6000 IU, about 500 IU to about 6000 IU, about 1000 IU to about 6000 IU, about 1500 IU to about 6000 IU, about 2000 IU to about 6000 IU, about 2500 IU to about 6000 IU, about 3000 IU to about 6000 IU, about 3500 IU to about 6000 IU, about 2500 IU to about 4000 IU, or about 2500 IU to about 3500 IU) of IL-2 can be used per mL of culture medium.

[0030] In some cases, tissue (e.g., tumor tissue) is mechanically and / or enzymatically digested and a single cell tumor digest suspension is cultured for a period of time, or γδ T cells can be directly isolated at this point.

[0031] After culturing the tissue containing tumor-infiltrating γδ T cells with IL-2 for 5-15 days (e.g., 6-15 days, 7-15 days, 8-15 days, 9-15 days, 9-13 days, 10-12 days, 7-10 days, or 8-14 days), the cell population from the tissue can be harvested. In some cases, the harvested cell population can include tumor-infiltrating αβ T cells and tumor-infiltrating γδ T cells. In some cases, the harvested cell population can include a number of tumor-infiltrating αβ T cells that is greater than the number of tumor-infiltrating γδ T cells. In some cases, an anti-αβ TCR antibody, an anti-CD28 antibody, an anti-4-1BBL antibody, an anti-GITR antibody, an anti-CD27 antibody, or a combination thereof can be used to promote a cell population enriched for γδ T cells from the harvested cell population.

[0032] Once a harvested cell population is obtained, optional enrichment for γδ T cells can be performed. For example, magnetic beads containing anti-αβ TCR antibodies can be used in a negative selection process to remove αβ T cells from the harvested cell population and obtain a cell population enriched for γδ T cells. In some cases, anti-TCR γδ antibodies, anti-Vδ1 antibodies, anti-NKG2D antibodies, or a combination thereof can be used in a positive selection process to isolate γδ T cells from the harvested cell population and obtain a cell population enriched for γδ T cells.

[0033] Briefly, when an antibody is used to remove non-γδ T cells (e.g., αβ T cells) or isolate γδ T cells from a harvested cell population to obtain a cell population enriched for γδ T cells, the antibody may be biotinylated and attached to a magnetic substrate (e.g., magnetic beads) with streptavidin. In some cases, flow activated cell sorting (FACS) may be used to remove non-γδ T cells (e.g., αβ T cells) or isolate γδ T cells from a harvested cell population to obtain a cell population enriched for γδ T cells.

[0034] In some cases, the harvested cell population (or a portion thereof) can be used to expand the numbers of γδ T cells, optionally without an enrichment step.

[0035] Once a harvested cell population (optionally enriched or not enriched for γδ T cells) is obtained, the harvested cell population (or a portion thereof) can be used to carry out an expansion step to increase the number of γδ T cells present. In some cases, this expansion step can increase the starting number of γδ T cells present in the starting cell population until the number of γδ T cells present in the resulting cell population is 10-1000 fold (e.g., 10-600 fold, 20-600 fold, 30-600 fold, 40-600 fold, 50-600 fold, 75-600 fold, 100-600 fold, 200-1000 fold, 250-1000 fold, 300-1000 fold, 350-1000 fold, 400-1000 fold, 450-1000 fold, 500-1000 fold, 200-1000 fold, 250-1000 fold, 300-51000 fold, 350-1000 fold, 400-1000 fold, or 450-1000 fold) greater than the starting number. In some cases, this expansion step can increase the starting number of γδ T cells present in the starting cell population until the number of γδ T cells present in the resulting cell population is more than 200-fold higher (e.g., more than 250-fold, more than 300-fold, more than 350-fold, more than 400-fold, or more than 450-fold higher) than the starting number. In some cases, this expansion step can expand the starting number of γδ T cells present in the starting cell population until the number of γδ T cells present in the resulting cell population is 200-600 fold (e.g., 200-600 fold, 250-600 fold, 300-600 fold, 350-600 fold, 400-600 fold, 450-600 fold, 500-600 fold, 200-550 fold, 250-550 fold, 300-550 fold, 350-550 fold, 400-550 fold, 450-550 fold, 500-550 fold, 200-500 fold, 250-500 fold, 300-500 fold, 350-500 fold, 400-500 fold, or 450-500 fold) greater than the starting number. In some cases, this expansion step can increase the starting number of γδ T cells present in the starting cell population until the number of γδ T cells present in the resulting cell population is enriched for γδ T cells by more than 25 percent (e.g., more than 50 percent, more than 75 percent, or more than 95 percent).

[0036] Any suitable method can be used to promote the expansion of γδ T cells of the harvested cell population (or a portion thereof) or of a harvested cell population enriched for γδ T cells (or a portion thereof). For example, the harvested cell population (optionally enriched or not enriched for γδ T cells) or a portion thereof can be cultured in the presence of IL-2, IL-4 and IL-15 to promote the expansion of γδ T cells. The amount of IL-2 can be about 50 IU to about 6000 IU (e.g., about 100 IU to about 6000 IU, about 500 IU to about 6000 IU, about 1000 IU to about 6000 IU, about 1500 IU to about 6000 IU, about 2000 IU to about 6000 IU, about 2500 IU to about 6000 IU, about 3000 IU to about 6000 IU, about 3500 IU to about 6000 IU, about 2500 IU to about 4000 IU, or about 2500 IU to about 3500 IU) of IL-2 per mL of culture medium. The amount of IL-4 may be about 10 ng to about 200 ng (e.g., about 20 ng to about 200 ng, about 50 ng to about 200 ng, about 75 ng to about 200 ng, about 10 ng to about 150 ng, about 10 ng to about 100 ng, about 50 ng to about 150 ng, or about 90 ng to about 110 ng) of IL-4 per mL of culture medium. The amount of IL-15 may be about 10 ng to about 200 ng (e.g., about 20 ng to about 200 ng, about 50 ng to about 200 ng, about 75 ng to about 200 ng, about 10 ng to about 150 ng, about 10 ng to about 100 ng, about 50 ng to about 150 ng, about 50 ng to about 90 ng, or about 60 ng to about 90 ng) of IL-15 per mL of culture medium.

[0037] In some cases, the harvested cell population (optionally enriched or not enriched for γδ T cells) or a portion thereof can be cultured in the presence of IL-2, IL-4, and IL-15, optionally including IL-7 and / or IL-21. When optionally including IL-7, the amount of IL-7 can be about 10 ng to about 200 ng (e.g., about 20 ng to about 200 ng, about 50 ng to about 200 ng, about 75 ng to about 200 ng, about 10 ng to about 150 ng, about 10 ng to about 100 ng, about 50 ng to about 150 ng, or about 90 ng to about 110 ng) of IL-7 per mL of culture medium. When IL-21 is optionally included, the amount of IL-21 can be about 10 ng to about 200 ng (e.g., about 20 ng to about 200 ng, about 50 ng to about 200 ng, about 75 ng to about 200 ng, about 10 ng to about 150 ng, about 10 ng to about 100 ng, about 50 ng to about 150 ng, or about 90 ng to about 110 ng) of IL-21 per mL of culture medium.

[0038] Any suitable IL-2, IL-4 and IL-15 (and optionally including IL-7 and / or IL-21) can be used to expand γδ T cells as described herein. For example, when expanding human γδ T cells, human IL-2, human IL-4 and human IL-15 can be used to expand human γδ T cells. In another example, when expanding equine γδ T cells, equine IL-2, equine IL-4 and equine IL-15 can be used to expand equine γδ T cells. In another example, when expanding monkey γδ T cells, monkey IL-2, monkey IL-4 and monkey IL-15 can be used to expand monkey γδ T cells. In another example, when expanding canine γδ T cells, canine IL-2, canine IL-4 and canine IL-15 can be used to expand canine γδ T cells.

[0039] The harvested cell population (optionally enriched or not enriched for γδ T cells) or a portion thereof can be cultured in the presence of IL-2, IL-4, and IL-15 for any suitable length of time to promote the expansion of γδ T cells. For example, the harvested cell population (optionally enriched or not enriched for γδ T cells) or a portion thereof can be cultured in the presence of IL-2, IL-4, and IL-15 for 8-21 days (e.g., 10-21 days, 12-21 days, 14-21 days, 8-18 days, 8-16 days, 8-14 days, 10-20 days, 10-18 days, 12-18 days, 10-16 days, 12-16 days, or 13-15 days). In some cases, IL-2, IL-4, and IL-15 during culture can be replenished every 3 days, every 4-6 days, or every 2-3 days.

[0040] In some cases, the cultures used to expand the numbers of γδ T cells contain IL-2, IL-4, and IL-15, and may contain one or more additional agents. For example, in addition to IL-2, IL-4, and IL-15, the cultures may be treated with an anti-CD3 antibody (e.g., soluble and / or fixed anti-CD3 antibody), an anti-CD28 antibody (e.g., soluble and / or fixed anti-CD28 antibody), irradiated PBMCs (e.g., irradiated PBMCs that are autologous to the mammal being treated with the expanded γδ T cells), an agonistic anti-γδ TCR antibody (e.g., soluble and / or fixed anti-γδ TCR antibody, such as Vδ1 antibody; about 1 μg / mL; see, e.g., Zhou et al., Cell Mol. Immunol., 9(1):34-44 (2012)), an anti-4-1BB antibody (e.g., soluble and / or fixed anti-4-1BB antibody, such as urelumab; about 10 μg / mL; see, e.g., Sakellariou-Thompson et al., Clin. Cancer Res., 2012). 23(23):7263-7275 (2017)), anti-TIGIT antibodies (e.g., soluble and / or immobilized anti-TIGIT antibodies; 1 μg / mL; see, e.g., Chauvin et al., J. Clin. Invest., 125(5):2046-58 (2015)), high glucose (e.g., 5 mM-25 mM, 8 mM-20 mM, 8 mM-12 mM, or 9 mM-11 mM glucose; see, e.g., Lopes et al., Nat. Immunol., 22:179-192 (2021)), irradiated artificial antigen presenting cells (e.g., cloned K562 cells transfected with 4-1BBL, CD86, IL-15 / membrane-bound IL-15; 1:100 ratio; see, e.g., Deniger et al., Clin. Cancer, 2016, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 130, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, Res., 20(22):5708-5719 (2014)), PHA (approximately 1 μg / mL), irradiated EBV-transfected B cell lines (1:100 ratio; see, e.g., Ma et al., J. Exp. Med., 208(3):491-503 (2011)), anti-OX40 antibodies (e.g., soluble and / or immobilized anti-OX40 antibodies), phosphoinositide 3-kinase (PI 3-kinase) inhibitors (e.g., idelalisib, copanlisib, duvelisib, alpelisib, or umbralisib), CDK4 / 6 inhibitors (e.g., palbociclib, ribociclib, or abemaciclib; see, e.g., Lelliott et al., Cancer Discov., 11(10):2582-2601 (2021)), CBL-B inhibitors (e.g., NX-0255 or NX-1607; see, e.g., Rountree et al., Cancer Res., July 1, 2021 (81)(13 Supplement):1595), STS1 inhibitor (see, e.g., Hwang et al., Exp. Mole. Med., 52:750-761 (2020)), CISH (see, e.g., Palmer et al., J. Exp. Med., 212(12):2095-2113 (2015)), TET2 (see, e.g., Fraietta et al., Nature, 558(7709):307-312 (2018)), or a combination thereof. For example, in addition to IL-2, IL-4, and IL-15, the culture may contain an anti-CD3 antibody (e.g., a soluble anti-CD3 antibody) and irradiated PBMCs. The amount of anti-CD3 antibody may be about 0.1 μg to about 1 μg of anti-CD3 antibody per mL of culture medium. The amount of anti-CD28 antibody can be about 500 ng to about 5 μg of anti-CD28 antibody per mL of culture medium. The amount of irradiated PBMCs can be based on the number of input γδ T cells, such that the ratio of γδ T cells:PBMCs is about 1:25 to about 1:200 (e.g., 1:100).

[0041] After expanding the number of γδ T cells in the presence of IL-2, IL-4, and IL-15, the cells can be washed to remove any particular components of the culture medium. For example, after the expansion step is completed, the resulting cell population can be washed to remove remaining IL-2, IL-4, IL-15, anti-CD3 antibodies, and / or anti-CD28 antibodies, and / or enrich for the expanded γδ T cells. In some cases, after the expansion step, the expanded γδ T cells can be cultured in the absence of IL-2, IL-4, and / or IL-15 for any suitable time. For example, after the rapid expansion step, the expanded population of γδ T cells can be cultured in the absence of IL-2, IL-4, and / or IL-15 for 10 to 75 days (e.g., 10 to 60 days, 10 to 50 days, or 10 to 25 days). In some cases, expanded γδ T cells can be obtained from multiple donors (e.g., multiple humans) and pooled to provide a population of γδ T cells for treatment of one or more patients (e.g., one or more humans).

[0042] As described herein, cell populations containing expanded γδ T cells resulting from expansion of γδ T cells in the presence of IL-2, IL-4, and IL-15 can have a particularly desirable cellular composition. For example, in some cases, the CD3 + More than 85 percent of the cells (e.g., more than 90 percent, more than 91 percent, more than 92 percent, more than 93 percent, more than 94 percent, more than 95 percent, more than 96 percent, more than 97 percent, more than 98 percent, or more than 99 percent) are γδ TCR + In some cases, the populations provided herein may be CD3 + Fewer than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are αβ TCR + In some cases, the populations provided herein may be CD45 cells. +Less than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) can be NK cells.

[0043] In some cases, the cell population containing expanded γδ T cells resulting from the expansion of γδ T cells in the presence of IL-2, IL-4, and IL-15 as described herein may vary and may contain various proportions of not only αβ T cells, but also phenotypes NKT, NK, and B cells.

[0044] In some cases, the CD3 + More than 85 percent of the cells (e.g., more than 90 percent, more than 91 percent, more than 92 percent, more than 93 percent, more than 94 percent, more than 95 percent, more than 96 percent, more than 97 percent, more than 98 percent, or more than 99 percent) are γδ TCR + The population may be CD3 + Fewer than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are αβ TCR + It may be a cell.

[0045] In some cases, the CD3 + More than 85 percent of the cells (e.g., more than 90 percent, more than 91 percent, more than 92 percent, more than 93 percent, more than 94 percent, more than 95 percent, more than 96 percent, more than 97 percent, more than 98 percent, or more than 99 percent) are γδ TCR + The population may be CD45 + Less than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) can be NK cells.

[0046] In some cases, the CD3 + More than 85 percent of the cells (e.g., more than 90 percent, more than 91 percent, more than 92 percent, more than 93 percent, more than 94 percent, more than 95 percent, more than 96 percent, more than 97 percent, more than 98 percent, or more than 99 percent) are γδ TCR + The population may be CD3 + Fewer than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are αβ TCR + The population may be CD45 + Less than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) can be NK cells.

[0047] In some cases, the γδ TCRs of the population provided herein + More than 30 percent of the cells (e.g., more than 35 percent, more than 40 percent, more than 45 percent, more than 50 percent, more than 55 percent, more than 60 percent, more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) are Vδ1 + It may be a cell.

[0048] In some cases, the γδ TCRs of the population provided herein + Less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the cells are VDδ1 - Vδ2 - It may be a cell.

[0049] In some cases, the γδ TCRs of the population provided herein + Fewer than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are Vδ2 + It may be a cell.

[0050] In some cases, the γδ TCRs of the population provided herein + More than 30 percent of the cells (e.g., more than 35 percent, more than 40 percent, more than 45 percent, more than 50 percent, more than 55 percent, more than 60 percent, more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) are Vδ1 + cells, and the γδ TCR + Less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the cells are VDδ1 - Vδ2 - It may be a cell.

[0051] In some cases, the γδ TCRs of the population provided herein + More than 30 percent of the cells (e.g., more than 35 percent, more than 40 percent, more than 45 percent, more than 50 percent, more than 55 percent, more than 60 percent, more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) are Vδ1 + cells, and the γδ TCR + Fewer than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are Vδ2+ It may be a cell.

[0052] In some cases, the γδ TCRs of the population provided herein + More than 30 percent of the cells (e.g., more than 35 percent, more than 40 percent, more than 45 percent, more than 50 percent, more than 55 percent, more than 60 percent, more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) are Vδ1 + cells, and the γδ TCR + Less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the cells are VDδ1 - Vδ2 - cells, and the γδ TCR + Fewer than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are Vδ2 + It may be a cell.

[0053] In some cases, the γδ TCRs of the population provided herein + More than 70 percent (e.g., more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) of the cells are T EM It may be a cell.

[0054] In some cases, the γδ TCRs of the population provided herein + Less than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are T EMRA It may be a cell.

[0055] In some cases, the γδ TCRs of the population provided herein + More than 70 percent (e.g., more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) of the cells are T EM cells, and the γδ TCR + Less than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are T EMRA It may be a cell.

[0056] In some cases, the populations provided herein, after cell expansion in the presence of IL-2, IL-4, and IL-15, have a higher percentage (e.g., 2-40 percentage points higher, 5-40 percentage points higher, 10-40 percentage points higher, 15-40 percentage points higher, 20-40 percentage points higher, 5-35 percentage points higher, 5-30 percentage points higher, 5-25 percentage points higher, 5-20 percentage points higher, 5-15 percentage points higher, or 5-10 percentage points higher) of γδ TCRs than the starting population before cell expansion in the presence of IL-2, IL-4, and IL-15. + T EM The cell may have

[0057] In some cases, the populations provided herein, after cell expansion in the presence of IL-2, IL-4, and IL-15, have a lower percentage (e.g., 2-30 percentage points lower, 5-30 percentage points lower, 10-30 percentage points lower, 15-30 percentage points lower, 20-30 percentage points lower, 5-25 percentage points lower, 5-20 percentage points lower, 5-15 percentage points lower, or 5-10 percentage points lower) of γδ TCRs than the starting population before cell expansion in the presence of IL-2, IL-4, and IL-15. + T EMRA The cell may have

[0058] In some cases, the populations provided herein, after cell expansion in the presence of IL-2, IL-4, and IL-15, have a higher percentage (e.g., 2-40 percentage points higher, 5-40 percentage points higher, 10-40 percentage points higher, 15-40 percentage points higher, 20-40 percentage points higher, 5-35 percentage points higher, 5-30 percentage points higher, 5-25 percentage points higher, 5-20 percentage points higher, 5-15 percentage points higher, or 5-10 percentage points higher) of γδ TCRs than the starting population before cell expansion in the presence of IL-2, IL-4, and IL-15. + T EM cells, and a lower percentage (e.g., 2-30 percentage points lower, 5-30 percentage points lower, 10-30 percentage points lower, 15-30 percentage points lower, 20-30 percentage points lower, 5-25 percentage points lower, 5-20 percentage points lower, 5-15 percentage points lower, or 5-10 percentage points lower) of γδ TCR + T EMRA The cell may have

[0059] In some cases, the γδ TCRs of the population provided herein + Fewer than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) express CD69 + CD103 + T RM In some cases, the γδ TCRs of the populations provided herein may be + Between 1 and 10 percent of the cells (e.g., between 1 and 9 percent, between 1 and 8 percent, between 1 and 7 percent, between 1 and 6 percent, between 1 and 5 percent, between 1 and 4 percent, between 2 and 10 percent, between 3 and 10 percent, between 4 and 10 percent, between 5 and 10 percent, between 6 and 10 percent, between 2 and 8 percent, between 2 and 6 percent, between 4 and 8 percent, or between 4 and 6 percent) are CD69 + CD103 + T RM It may be a cell.

[0060] In some cases, the γδ TCRs of the population provided herein + Less than 50 percent of the cells (e.g., less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) are CD56 + In some cases, the γδ TCR cells of the populations provided herein + Between 1 and 50 percent of the cells (e.g., between 1 and 45 percent, between 1 and 40 percent, between 1 and 35 percent, between 1 and 30 percent, between 1 and 25 percent, between 1 and 20 percent, between 5 and 50 percent, between 10 and 50 percent, between 15 and 50 percent, between 20 and 50 percent, between 10 and 40 percent, between 15 and 35 percent, or between 20 and 30 percent) are CD56 + It may be a cell.

[0061] In some cases, the γδ TCRs of the population provided herein + Between 1 and 40 percent of the cells (e.g., between 1 and 35 percent, between 1 and 30 percent, between 1 and 25 percent, between 1 and 20 percent, between 1 and 15 percent, between 1 and 10 percent, between 5 and 40 percent, between 10 and 40 percent, between 15 and 40 percent, between 20 and 40 percent, between 5 and 35 percent, between 10 and 30 percent, or between 15 and 25 percent) are CD137 + It may be a cell.

[0062] In some cases, the populations provided herein have a higher percentage (e.g., 2-50 percentage points higher, 5-50 percentage points higher, 2-40 percentage points higher, 5-40 percentage points higher, 10-40 percentage points higher, 15-40 percentage points higher, 20-40 percentage points higher, 5-35 percentage points higher, 5-30 percentage points higher, 5-25 percentage points higher, 5-20 percentage points higher, 5-15 percentage points higher, or 5-10 percentage points higher) of CD137 + Gamma delta TCR + The cell may have

[0063] In some cases, the γδ TCRs of the population provided herein + Fewer than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) express PD-1 + It may be a cell.

[0064] In some cases, the populations provided herein, after cell expansion in the presence of IL-2, IL-4, and IL-15, have a lower percentage (e.g., 5 to 90 percentage points lower, 5 to 80 percentage points lower, 5 to 75 percentage points lower, 5 to 70 percentage points lower, 5 to 75 percentage points lower, 5 to 70 percentage points lower, 5 to 65 percentage points lower, 5 to 60 percentage points lower, or 5 to 70 percentage points lower) of the starting population before cell expansion in the presence of IL-2, IL-4, and IL-15. percentage points lower, 5 to 55 percentage points lower, 5 to 50 percentage points lower, 5 to 45 percentage points lower, 5 to 40 percentage points lower, 5 to 35 percentage points lower, 5 to 30 percentage points lower, 5 to 25 percentage points lower, 5 to 20 percentage points lower, 5 to 15 percentage points lower, 5 to 10 percentage points lower, 10 to 90 percentage points lower, 10 to 80 percentage points lower, 10 to 75 a percentage point lower, 10 to 70 percentage points lower, 10 to 75 percentage points lower, 10 to 70 percentage points lower, 10 to 65 percentage points lower, 10 to 60 percentage points lower, 10 to 55 percentage points lower, 10 to 50 percentage points lower, 10 to 45 percentage points lower, 10 to 40 percentage points lower, 10 to 35 percentage points lower, 10 to 30 percentage points lower, 10 to 25 percentage points lower points lower, 10 to 20 percentage points lower, 10 to 15 percentage points lower, 25 to 90 percentage points lower, 25 to 80 percentage points lower, 25 to 75 percentage points lower, 25 to 70 percentage points lower, 25 to 75 percentage points lower, 25 to 70 percentage points lower, 25 to 65 percentage points lower, 25 to 60 percentage points lower, 25 to 55 percentage points lower, 25 to 50 percentage points lower,25 to 45 percentage points lower, 25 to 40 percentage points lower, 25 to 35 percentage points lower, or 25 to 30 percentage points lower) PD-1; + Gamma delta TCR + The cell may have

[0065] In some cases, the γδ TCRs of the population provided herein + Between 5 and 40 percent of the cells (e.g., between 5 and 35 percent, between 5 and 30 percent, between 5 and 25 percent, between 5 and 20 percent, between 5 and 15 percent, between 10 and 40 percent, between 10 and 35 percent, between 10 and 30 percent, between 10 and 25 percent, between 10 and 20 percent, or between 15 and 25 percent) are BTLA + It may be a cell.

[0066] In some cases, the αβ TCR of the population provided herein + Between 5 and 40 percent of the cells (e.g., between 5 and 35 percent, between 5 and 30 percent, between 5 and 25 percent, between 5 and 20 percent, between 5 and 15 percent, between 10 and 40 percent, between 10 and 35 percent, between 10 and 30 percent, between 10 and 25 percent, between 10 and 20 percent, or between 15 and 25 percent) are BTLA + It may be a cell.

[0067] In some cases, the γδ TCRs of the population provided herein + More than 60 percent (e.g., more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) of the cells are NKG2D + It may be a cell.

[0068] In some cases, the γδ TCRs of the population provided herein +More than 20 percent of the cells (e.g., more than 25 percent, more than 30 percent, more than 35 percent, more than 40 percent, more than 45 percent, more than 50 percent, more than 55 percent, more than 60 percent, more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) express NKp46 + It may be a cell.

[0069] In some cases, the populations provided herein have a higher percentage (e.g., 5 to 90 percentage points higher, 5 to 85 percentage points higher, 5 to 80 percentage points higher, 5 to 75 percentage points higher, 5 to 70 percentage points higher, 5 to 65 percentage points higher, 5 to 60 percentage points higher, 5 to 70 percentage points higher, 5 to 75 ... ~55 percentage points higher, 5 to 50 percentage points higher, 5 to 45 percentage points higher, 5 to 40 percentage points higher, 5 to 35 percentage points higher, 5 to 30 percentage points higher, 5 to 25 percentage points higher, 10 to 90 percentage points higher, 10 to 85 percentage points higher, 10 to 80 percentage points higher, 10 to 75 percentage points higher, 10 to 70 percentage points higher, 10 to 65 percentage points higher, percentage points higher, 10 to 60 percentage points higher, 10 to 55 percentage points higher, 10 to 50 percentage points higher, 10 to 45 percentage points higher, 10 to 40 percentage points higher, 10 to 35 percentage points higher, 10 to 30 percentage points higher, 10 to 25 percentage points higher, 15 to 90 percentage points higher, 15 to 85 percentage points higher, 15 to 80 percentage points higher, 15 to 75 percentage points higher, 15 to 70 percentage points higher, 15 to 65 percentage points higher, 15 to 60 percentage points higher, 15 to 55 percentage points higher, 15 to 50 percentage points higher, 15 to 45 percentage points higher, 15 to 40 percentage points higher, 15 to 45 percentage points higher, 15 to 30 percentage points higher, 15 to 25 percentage points higher, or 20 to 40 percentage points higher) + The cell may have

[0070] In some cases, (a) a CD3 + More than 85 percent of the cells (e.g., more than 90 percent, more than 91 percent, more than 92 percent, more than 93 percent, more than 94 percent, more than 95 percent, more than 96 percent, more than 97 percent, more than 98 percent, or more than 99 percent) are γδ TCR + (b) the population of CD3 + Fewer than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are αβ TCR + (c) the population of CD45 cells; + (d) less than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) can be NK cells; + More than 30 percent of the cells (e.g., more than 35 percent, more than 40 percent, more than 45 percent, more than 50 percent, more than 55 percent, more than 60 percent, more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) are Vδ1 + (e) the population of γδ TCR cells; + Less than 60 percent (e.g., less than 55 percent, less than 50 percent, less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) of the cells are VDδ1 - Vδ2 - (f) the population of γδ TCR cells; +Fewer than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are Vδ2 + (g) a γδ TCR cell of the population provided herein; + More than 70 percent (e.g., more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) of the cells are T EM (h) the population of γδ TCR cells; + Less than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) are T EMRA (i) a γδ TCR + Fewer than 10 percent of the cells (e.g., less than 9 percent, less than 8 percent, less than 7 percent, less than 6 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) express CD69 + CD103 + T RM or a population of γδ TCR + Between 1 and 10 percent of the cells (e.g., between 1 and 9 percent, between 1 and 8 percent, between 1 and 7 percent, between 1 and 6 percent, between 1 and 5 percent, between 1 and 4 percent, between 2 and 10 percent, between 3 and 10 percent, between 4 and 10 percent, between 5 and 10 percent, between 6 and 10 percent, between 2 and 8 percent, between 2 and 6 percent, between 4 and 8 percent, or between 4 and 6 percent) are CD69 + CD103 + T RM (j) the population of γδ TCR cells; + Less than 50 percent of the cells (e.g., less than 45 percent, less than 40 percent, less than 35 percent, less than 30 percent, less than 25 percent, less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, or less than 2 percent) are CD56 + or a population of γδ TCR+ Between 1 and 50 percent of the cells (e.g., between 1 and 45 percent, between 1 and 40 percent, between 1 and 35 percent, between 1 and 30 percent, between 1 and 25 percent, between 1 and 20 percent, between 5 and 50 percent, between 10 and 50 percent, between 15 and 50 percent, between 20 and 50 percent, between 10 and 40 percent, between 15 and 35 percent, or between 20 and 30 percent) are CD56 + (k) the population of γδ TCR cells; + Between 1 and 40 percent of the cells (e.g., between 1 and 35 percent, between 1 and 30 percent, between 1 and 25 percent, between 1 and 20 percent, between 1 and 15 percent, between 1 and 10 percent, between 5 and 40 percent, between 10 and 40 percent, between 15 and 40 percent, between 20 and 40 percent, between 5 and 35 percent, between 10 and 30 percent, or between 15 and 25 percent) are CD137 + (l) a γδ TCR + Fewer than 25 percent of the cells (e.g., less than 20 percent, less than 15 percent, less than 10 percent, less than 5 percent, less than 4 percent, less than 3 percent, less than 2 percent, or less than 1 percent) express PD-1 + (m) a γδ TCR + Between 5 and 40 percent of the cells (e.g., between 5 and 35 percent, between 5 and 30 percent, between 5 and 25 percent, between 5 and 20 percent, between 5 and 15 percent, between 10 and 40 percent, between 10 and 35 percent, between 10 and 30 percent, between 10 and 25 percent, between 10 and 20 percent, or between 15 and 25 percent) are BTLA + (n) a γδ TCR cell of the population; + More than 60 percent of the cells (e.g., more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) are NKG2D + (o) a γδ TCR +More than 20 percent of the cells (e.g., more than 25 percent, more than 30 percent, more than 35 percent, more than 40 percent, more than 45 percent, more than 50 percent, more than 55 percent, more than 60 percent, more than 65 percent, more than 70 percent, more than 75 percent, more than 80 percent, more than 85 percent, more than 90 percent, or more than 95 percent) express NKp46 + It may be a cell.

[0071] In addition to providing the cell populations described herein and methods for producing those cell populations described herein, the present specification provides methods for treating any suitable disease, disorder, or condition using the cell populations described herein. For example, the cell populations described herein can be used to treat autoimmune conditions such as rheumatoid arthritis, systemic lupus erythematosus, and scleroderma, infectious diseases such as HIV infection, malaria, tuberculosis, hepatitis B, and SARS-CoV-2 infection, and / or cancer. For example, the cell populations described herein can be administered to a mammal for use in adoptive cell therapy, for example, to treat cancer. Any suitable mammal can be treated with the cell populations described herein. For example, humans, horses, cows, pigs, dogs, cats, mice, and rats can be treated with the populations of expanded tumor-infiltrating γδ T cells described herein. Any suitable number of cells can be present in the cell populations described herein that are administered to a mammal (e.g., a human) to treat cancer. For example, the cell populations described herein can be administered to a mammal (e.g., a human) in a concentration of about 1×10 7 ~Approx. 1×10 12 cells (e.g., 5 × 10 7 ~1×10 11 cells, 1 x 10 8 ~1×10 11 cells, 5 x 10 8 ~1×10 11 cells, 1 x 10 9 ~1×10 11 cells, or 1 x 10 10 ~1×10 1210 cells) and can be administered to a mammal (e.g., a human) to treat cancer. In some cases, the cell populations described herein can have a cell population of about 1 x 10 7 ~Approx. 1×10 12 pieces (e.g., 5×10 7 ~1×10 11 pieces, 1×10 8 ~1×10 11 pieces, 5×10 8 ~1×10 11 pieces, 1×10 9 ~1×10 11 Pieces or 1 x 10 10 ~1×10 12 ) γδ T cells are delivered to the mammal.

[0072] Any suitable cancer can be treated using the cell population described herein.For example, glioblastoma, head and neck squamous cell carcinoma, skin melanoma, lung adenocarcinoma, lung squamous cell carcinoma, breast cancer, mesothelioma, liver hepatocellular carcinoma, pancreatic ductal adenocarcinoma, kidney renal cell carcinoma, bladder urothelial carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, esophageal carcinoma, gastric adenocarcinoma, colorectal adenocarcinoma, rectal adenocarcinoma, ovarian serous cystadenocarcinoma, uterine endometrial carcinoma, prostate adenocarcinoma, and sarcoma can be treated using the cell population described herein.

[0073] Any suitable route of administration can be used to administer the cell populations described herein to a mammal, for example, the cell populations described herein can be administered intravenously, intraperitoneally, or intratumorally.

[0074] When treating a mammal having a condition other than cancer (e.g., an autoimmune condition), disease, or infection (e.g., HIV infection, malaria, tuberculosis, Hepatitis B, and SARS-CoV-2 infection), any suitable tissue source can be used to obtain γδ T cells. For example, when expanding γδ T cells to treat an autoimmune condition, tissue involved in the autoimmune condition that contains γδ T cells, or uninvolved tissue that contains γδ T cells (e.g., involved or uninvolved skin, liver, kidney, esophagus, small intestine, and / or colon tissue) can be used as a tissue source to obtain the γδ T cells described herein. When expanding γδ T cells to treat an infection, tissue involved in the infection that contains γδ T cells, or uninvolved tissue that contains γδ T cells (e.g., involved or uninvolved skin, liver, kidney, esophagus, small intestine, and / or colon tissue) can be used as a tissue source to obtain the γδ T cells described herein.

[0075] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0076] Example 1: Expanded tumor-infiltrating γδ T cells demonstrate potential utility for cancer adoptive cell therapy Clinical cohort and sample collection A retrospective series of n=10 low-grade (AJCC 8th edition Grade 1) PMP tumor specimens were identified from the University of Pittsburgh Medical Center Digestive Diseases Tissue Repository and pathologically examined for evidence of lymphocytic infiltration followed by immune repertoire sequencing (Figures 1A, 1B, and 2). All selected FFPE tumor specimens were obtained from patients with pathologically confirmed Grade 1 PMP who underwent CRS-HIPEC without prior treatment. Five FFPE tissue scrolls cut at a depth of 6 μm were placed into RNase / DNase-free Eppendorf tubes and stored at 4 °C until further processing.

[0077] Consenting n=26 patients with peritoneal surface malignancies (PMP or colon cancer) undergoing standard of care CRS-HIPEC at the University of Pittsburgh Medical Center completed prospective TIL expansion as part of a non-interventional tumor registry and tissue procurement clinical protocol (Figure 6). Peritoneal tumor tissue was stored at 4°C until further processing. Cryopreserved patient buffy coats were retrieved from the UPMC Gastroenterology Tissue Archive at the time of TIL autologous tumor reactivity testing.

[0078] As part of a non-interventional tumor bank clinical protocol, pre-rapid expansion protocol (pre-REP) modulation of γδ TILs was evaluated in n=15 tumor digests from patients undergoing resection of melanoma (FIG. 14).

[0079] Sequencing and analysis of the PMP immune repertoire Total RNA was extracted from FFPE tissue scrolls using a Covaris M220 focused ultrasonicator and truXTRAC FFPE Total NA Magnetic Beads Ultra Kit according to the manufacturer's protocol. Next-generation sequencing libraries covering human TCR-Vα, -Vβ, -Vγ, -Vδ, and BCR IgH, Igκ, and Igλ chains were generated using the iR-RepSeq-plus 7-Chain DAM-PCR Amplification Sequencing Kit (iRepertoire Inc). 1000 ng of extracted RNA was amplified by a Biomek-i5 workstation (Beckman Coulter) in a single assay that incorporates unique molecular identifiers (UMIs) during the reverse transcription (RT) step. Amplified libraries were multiplexed and pooled for sequencing on the Illumina NovaSeq platform using a 500-cycle kit. Each sample was assigned a total of 5 million sequencing reads. The raw data were demultiplexed and subjected to UMI-guided assembly using migec v1.2.9, and the resulting consensus fastqs were aligned and assembled into clonotypes using mixcr v3.0.14. The output T cell receptor sequences encompass FR2 through FR4 and the start of the constant region.

[0080] Raw data were analyzed using the iRmap program (iRepertoire Inc.). Total reads were normalized to generate UMIs, and unique CDR3 (uCDR3), average CDR3 length, and sample Shannon true entropy scores were compared across all seven chains. Immunoglobulin fractions of IgH chains were assembled with the TRUST algorithm and correlated with TCR and BCR repertoire metrics. Concomitant immune repertoire analysis was completed on n=68 pancreatic tumor specimens from patients undergoing neoadjuvant chemotherapy and curative resection, and n=238 healthy donor PBMCs (iRepertoire). TCR and BCR publicity of PMPs was determined by percent sharing with n=238 healthy donor PBMCs. Probability of generation of shared PMP-specific BCR IgH clonotypes was calculated using the OLGA algorithm.

[0081] TIL expansion Mucinous peritoneal tumors were dissected to remove necrotic or adipose tissue, and n = 40 spatially distinct 2-3 mm 3 Tumor fragments (Figure 7A) were placed in gas-permeable G-REX™ 100 flasks with complete medium (RPMI 1640 (Cytiva HyClone™) supplemented with 10% human AB serum (Gemini Bio), 1% GlutaMAX (Gibco), 5% Penstrep, 1.25 μg / mL amphotericin B (Gibco), 0.05 μmolar mercaptoethanol (βME)), and 3,000 IU / mL IL-2 (aldesleukin, Clinigen Therapeutics) as described elsewhere (Jin et al., J. Immunother., 35:283-292 (2012)). For experiments to expand TILs from tumor digests, cryopreserved single cell suspensions were thawed, washed twice with PBS, filtered through a 70 μM cell strainer, and cultured using a Cellometer K2 Fluorescent Cell Viability The cells were counted using a counter (Nexcelom Bioscience). 6Cells were plated in G-REX™ 6-well flasks with complete medium and the respective gamma chain cytokines (IL-4, 100 ng / mL; IL-15, 70 ng / mL; Miltenyi Biotec) or CD137 antibody (Urelumab, 10 μg / mL, Creative Biolabs). G-REX flasks were incubated at 37°C in 5% CO2 in a humidified incubator, and half of the medium was removed and replaced with fresh medium and IL-2 5 days after the start of culture. From day 5 onwards, half of the medium and IL-2 were replaced every 2 days. On day 11 of culture, TILs were filtered through a 70 μM cell strainer and counted. 2 × 10 6 10 harvested TILs were saved for phenotyping by spectral cytometry and for αβ rapid expansion protocol (REP), and the remaining TILs (up to 200 × 10 6 αβ TCR + A minimum number of 0.3 × 10 6 αβ TCR + Depleted TILs were assessed for γδ TIL purity by spectral cytometry. 6γδ and αβ TILs were expanded by the REP protocol using complete medium supplemented with 5% human AB serum and 50% CTS AIMV (Gibco) medium, mitogen OKT-3 (30 ng / mL, Miltenyi Biotec), 1:100 allogeneic irradiated feeder cells (two pooled CMV-negative healthy donors, San Diego Blood Bank), IL-2 (3,000 IU / mL) and γ-chain cytokines (IL-4, 100 ng / mL; IL-7, 20 ng / mL; IL-15, 70 ng / mL; all Miltenyi Biotec). Seven days after the start of the REP, the cultures were counted, resuspended and split 50% into new G-REX flasks and supplemented with fresh CTS AIMV medium and cytokines. On day 10 of the REP, the cells were counted again and half of the medium was removed and replaced with fresh CTS AIMV and cytokines. TILs were pooled and counted on day 14 of REP (day 25 of culture). 6 γδ and αβ TILs were stored for phenotyping by spectral cytometry, and the remaining expanded TILs were cryopreserved in 10% DMSO in fetal bovine serum using a CoolCell® (Corning) freezing system at −80°C and transferred to a liquid nitrogen freezer within 24 hours for long-term storage.

[0082] Tumor digestion n = 40 spatially distinct 2-3 mm 3 After randomization and selection of tumor fragments, the remaining tumor fragments (if available) were enzymatically and mechanically digested into single cell suspensions using a Human Tumor Dissection Kit (Miltenyi Biotec) and OctoMACS with Heaters Disassociater (Miltenyi Biotec) according to the manufacturer's protocol. The digested single cell suspensions were filtered through a 70 μm strainer, treated with 10 mL ACK lysis buffer (Gibco) for 5 min, washed twice with PBS, counted, and cryopreserved as described above.

[0083] Whole blood PBMC isolation Whole blood was collected in BD Vacutainer® EDTA tubes, diluted 1:1 with PBS, and centrifuged for 20 minutes at 1200G over 15 mL of Lymphoprep™ density gradient medium (Stemcell Technologies) in a SepMate™ 50 Tube. Plasma and mononuclear cells were removed, washed with PBS, treated with 10 mL of ACK lysis buffer (Gibco) for 5 minutes, washed twice with PBS, counted, and stored frozen as above.

[0084] Spectral cytometry Day 11 bulk TIL cultures, αβ TCR + Depleted cells and TIL cultures 25 days after REP were utilized for spectral cytometry to assess phenotypic expression of T cell memory, activation, exhaustion, and NCR. Cells were strained through a 30 μm filter, washed with cytometry buffer (2% FBS in PBS at 4° C.), incubated with Human TruStain FcX™ block (Biolegend) for 5 min, washed, and stained with a master mix of fluorescent conjugated antibodies and Brilliant Stain Buffer (BD) for 25 min at 4° C. in the dark. Samples were washed, resuspended in 200 μL of cytometry buffer, and analyzed on a 5-laser Cytek® Aurora Spectral Cytometer. Single-color spectral signatures were measured using UltraComp eBeads™ (Invitrogen) and spectrally resolved together with TIL autofluorescence spectral signatures using SpectroFlo® software. Single cells, live cells, CD45 + immune cells, CD56 + NK cells, CD3 + cells, αβ TCR + CD4 + and CD8 + T cells and γδ TCR + Vδ1 + , Vδ2 + , and Vδ1 - Vδ2 -After gating on T cells (Figure 7C), expression of phenotypic markers was assessed based on fluorescence minus one (FMO) controls in FlowJo v10.7 software. αβ and γδ TCR + Expression of phenotypic markers in TILs was assessed on days 11 and 25 of culture, and changes in expression of markers between the two time points were consistent with αβ and γδ TCR + was evaluated within the TIL population.

[0085] TIL-tumor reactivity To assess autologous tumor reactivity of expanded αβ (IL-2 only) and γδ (IL-2, IL-4, and IL-15) TILs, cryopreserved TILs were thawed, rested overnight in IL-2 (3,000 IU / mL) medium, washed twice with PBS, counted, and plated in 96-well round-bottom plates in IL-2-free complete medium alone, CD3-CD28 stimulated (Dynabeads, 2.5 μL / well, Invitrogen), 1 × 10 5 1 x 10 autologous PBMCs, or 1 x 10 5 Culture volumes were normalized to 200 μL with autologous tumor digest single cell suspensions and plated as described elsewhere (1 × 10 5cells) (Dudley et al., J. Immunother., 26:332-342 (2003)). 50 μL of supernatant was taken from duplicate co-cultures, diluted 1:2, and assessed for IFNγ production using a human IFNγ ELISA kit (Invitrogen) according to the manufacturer's instructions. TIL-autologous tumor digestion reactivity was compared between co-cultures with autologous PBMCs and paired γδ and αβ TILs. In certain cases, γδ TILs or autologous tumor digests were also incubated with blocking antibodies (TILs: isotype control mouse IgG (Invitrogen, 10 μg / mL), anti-γδ TCR (Novus Biologicals, clone 7A5, 3 μg / mL), or anti-NKG2D (BD, clone 1D11, 10 μg / mL); or tumor digests: isotype control mouse IgG (10 μg / mL) or anti-MHC-1 (Invitrogen, W6 / 32 10 μg / mL)) for 2 h prior to co-culture.

[0086] To assess MHC-unrestricted recognition of TILs, γδ and αβ TILs were cultured at least twice in complete medium at 37°C in a humidified incubator in 5% CO2 at 1 × 10 5 Cancer cell lines (K562, HCT116, RKO, SW480, or SW48; all from ATCC, authenticated and mycoplasma negative (eMyco™ plus PCR kit)) were co-cultured similarly.

[0087] Cancer Cell Encyclopedia (CCLE) analysis To assess the expression of NKG2D ligands in the cancer cell lines tested, the mRNA Z-scores of MICA, MICA, ULBP1, ULBP2, and ULBP3 were queried for K562, HCT116, RKO, SW480, and SW48 cells from the Cancer Cell Line Encyclopedia using the cBioPortal for Cancer Genomics Analysis.

[0088] TCGA analysis Tumor-specific Vδ1 infiltration and prognostic value were assessed in the 20 most common primary solid tumors (NCI) from bulk RNA sequencing data from The Cancer Genome Atlas (TCGA) using Gene Expression Profiling Interactive Analysis Server 2 (GEPIA2). Mean expression (log of transcripts per million) of γδ TIL subsets (TRDV1, TRDV2, and TRDV3) and αβ TILs (TRBC2 beta chain 2 constant region) was calculated. Kaplan-Meier survival analysis with normalized (ACTB beta actin) TRDV1 expression above (high) or below (low) the median for selected tumor types was completed along with calculation of log-rank P values ​​and 95% confidence intervals of survival estimates. TRDV1 expression was directly correlated with TRBC2 expression across selected tumors, and the corresponding Pearson correlation coefficients and P values ​​were calculated.

[0089] statistical analysis Data were expressed as mean ± standard deviation. Graphical visualization and statistical analysis were performed using Microsoft Excel and GraphPad Prism 9. Descriptive statistics, two-tailed nonparametric tests, Mann-Whitney U test (one-tailed) and Wilcoxon signed-rank test (two-tailed, all comparisons of αβ and γδ TILs) were used. Correlations were calculated using Pearson correlation coefficients and plotted using nonlinear regression and 95% confidence bands. A P value <0.05 was considered statistically significant, with a significance level of ≥0.05. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P was set at <0.0001.

[0090] result Low-grade pseudomyxoma peritonei (PMP) shows elevated TCR V.DELTA.-associated B-cell receptor (BCR) IgE fraction We identified a representative cohort (n=10) of treatment-naïve patients with low-grade (G1) PMP treated with standard-of-care cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC, Figure 2) after pathologic analysis of previously resected peritoneal tumor specimens (Figures 1A and 1B). All patients demonstrated microsatellite-stable tumors with limited programmed cell death ligand-1 (PD-L1) positivity (n=1), and seven patients required at least one follow-up CRS-HIPEC for tumor recurrence. H&E staining identified an infiltrating lymphocyte population restricted to the tumor-associated stroma and notably absent from the mucin pool (Figures 1A and 1B).

[0091] Given the limited prior understanding of the adaptive immune response to such an understudied tumor type, complete TCR and BCR sequencing (Figures 3A-3G) was completed on FFPE tumor specimens resected at initial resection surgery. Following dimer avoidance multiplex PCR (DAM-PCR) of bulk tumor RNA, cDNA library preparation, and NGS, complete unique CDR3 (uCDR3) sequences were constructed using the migec v1.2.9 MixCR pipeline for adaptive immune repertoire analysis (Han et al., Cancer Treat. Res., 180:111-147 (2020); Han et al., Methods Mol. Biol., 2055:369-397 (2020); and Bolotin et al., Nat. Methods, 12:380-381 (2015)). Representative treemaps of the TCR Vα, Vβ, Vδ, and BCR IgH, Igκ, and Igλ repertoires from one patient are shown in Figure 3A. Notably, the seven chain repertoire was composed predominantly of BCR transcripts, with only 2.06% of the reads on average representing TCR clones derived primarily from αβ T cells (Figure 3B). Vδ chains represented only 0.09% of the reads on average, and Vγ reads were not verifiably detected, as expected. As previously reported, the CDR3 length of Vδ chains (18.8 ± 2.0 amino acids) was larger and more variable than Vα chains (13.5 ± 0.2, p < 0.001) and Vβ chains (14.1 ± 0.3, p = 0.005) (Figure 3C) (Rock et al., J. Exp. Med., 179:323-328 (1994)). Vδ chains were of similar length to IgH (17.6 ± 0.5) and longer than Igκ (11.1 ± 0.03, p < 0.0001) and Igλ (12.5 ± 0.09, p < 0.0001). Calculation of the true Shannon entropy of the repertoire showed similar diversity among the chains, but Vδ was significantly reduced compared to the Vαβ and BCR repertoires (Figure 3D) (Bortone et al., Cancer Immunol. Res., 9:103-112 (2021)).

[0092] Compared to a cohort of healthy donor PBMC repertoires (n=238), this low-grade PMP cohort was found to display a highly private repertoire with only Vα (0.06% of chains), Igκ (3.7%), and Igλ (1.9%) displaying shared public CDR3s (Figure 4A-B). Analysis of shared CDR3s within the patient population identified shared putative convergent disease-specific BCR CDR3s, but not TCR CDR3s (Figure 4C). Considering that shared disease-associated CDR3s can arise from both random recombination and convergent evolution of antigen-driven recombination, we calculated the generation probability of these shared BCR CDR3s, revealing a spectrum of antigen-driven IgH CDR3s, mainly IgG or IgE (Figure 4D-E) (Murugan et al., Proc. Natl. Acad. Sci. USA, 109:16161-16166 (2012); and Sethna et al., Bioinformatics, 35:2974-2981 (2019)).

[0093] Where BCR transcripts were unexpectedly abundant, further analysis of immunoglobin fractions across the low-grade PMP IgH repertoire revealed the expected distribution dominated by IgG (53.4 ± 12.0%) and IgA (21.7 ± 9.9%) (Figure 3E). However, we observed that the IgE fraction (12.2 ± 2.1%) was abnormally elevated and substantially more abundant than that observed in the repertoires of healthy donor PBMCs (n = 238, 0.9 ± 0.6%, p < 0.0001) and high-grade pancreatic cancer tumors (n = 68, 5.6 ± 3.2%, p < 0.0001) (Figure 3F). When correlating the BCR IgE fraction with other repertoire features, we observed a strong positive correlation with Vδ expression (r = +0.81, p = 0.013), but not with Vα or Vβ chains (Figure 3G). The association with IgE expression levels and γδ TILs was intriguing because intraepithelial γδ T cells have previously been shown to be required for tumor-protective IgE class switching in response to epithelial DNA damage (Crawford et al., Nat. Immunol., 19:859-870 (2018)).

[0094] γδ TILs sparsely infiltrate peritoneal surface malignancies With the understanding that peritoneal γδ TILs exhibit diverse polyclonal and private repertoires, we performed the following to prospectively evaluate γδ TILs. Tumor specimens were collected with consent from patients (n=26) with peritoneal surface malignancies undergoing CRS-HIPEC (Figure 5A). 30.7% of patients were female, with a mean age of 59.3±12.2 years and BMI of 27.5±8.4 (Figure 6). Patients in this prospective cohort had peritoneal tumors of low-grade appendiceal cancer (n=14, 54%) and high-grade colorectal cancer (n=12, 46%). Three patients (11.5%) had previously undergone CRS-HIPEC and 14 patients (54%) had previously received systemic chemotherapy.

[0095] Mucinous peritoneal tumors were classified into spatially distinct 2-3 mm 3 Peritoneal TILs were dissected into small fragments (Figure 7A) and cultured in gas-permeable rapid expansion flasks (G-REX™). Peritoneal TILs were released with high-dose IL-2 (3,000 IU / mL) and harvested after 11 days in culture. There was no difference in the total number of surviving TILs between untreated and neoadjuvant chemotherapy-treated patients (Figure 7B), but the number of viable TILs was significantly higher in patients with low-grade appendiceal cancer (4.9 × 10 7 pieces ±6.1×10 7 cells) in high-grade colon cancer (3.9 × 10 7 pieces ±10.3×10 7 The total number of viable TILs was higher in mice treated with 100-mL immunized mice ( 100-mL cells, p = 0.028, Figure 5B ).

[0096] Multispectral flow cytometry was used to define the composition and phenotype of the bulk-expanding peritoneal TIL population. + CD3 - Natural killer (NK) cells and CD3 + T cells express CD45 + NK cells were the major component of the TIL population (Fig. 5C). +On average, they accounted for 20.2% of the cells, although certain individual patients had less than 4% and others had more than 40% NK cells (Figure 5C). + Cells (3.4±4.4%) were total CD3 + They make up a small proportion of T cells and are primarily CD4 + (57.3±21.7) or CD8 + (36.1±19.3) + The cells were CD3 + γδ TCR + The cells are predominantly Vδ1 + cells (49.0 ± 31.5%) or Vδ1 - Vδ2 - cells (27.7 ± 25.1%) and Vδ2 + cells (21.0 ± 29.5%) were γδ TCR in five patients. + Despite accounting for more than 60% of the cells, on average they were not very abundant (Figure 5D). + No substantial differences in cell phenotype were observed between patients with appendix or colon tumors, or with or without pretreatment (FIGS. 7D-E).

[0097] γδ TILs display a tissue-resident effector memory phenotype with reduced PD-1 but increased expression of NKG2D and CD137 compared with αβ TILs To better understand the phenotype of the γδ and αβ TIL populations, we assessed the expression of markers of T cell memory, differentiation, and activation, inhibitory receptors associated with T cell exhaustion, and natural cytotoxicity receptors (NCRs) (Figures 8A–H and 9A–E). - Intraepithelial CD8αβ compared with γδ T cells + γδ T cells typically display an enriched T helper type 1 (Th1) phenotype associated with intestinal homeostasis and mucosal healing (Mikulak et al., JCI Insight, 4(24):e125884 (2019); and Kadivar et al., J. Immunol., 197:4584-4592 (2016)). This subset of expanded peritoneal TILs expresses CD8α + (14.6±36.1%), CD8β + (11.0±17.4%), or CD8αβ +(6.69 ± 15.3%) γδ TILs accounted for only a small proportion of cells and were much less abundant than the corresponding αβ TILs in individual cultures ( Fig. 8 A ).

[0098] The majority of γδ TILs displayed an effector memory phenotype (T EM :CD45RO + CD62L - , 75.5 ± 15.8%). In cultures 11 days after the first resection, αβ TILs were more likely to be central memory cells (T CM :CD45RO + CD62L + , 22.4±21.7% vs. 9.1±12.0%, p<0.0001). γδ TILs also expressed terminally differentiated effector memory RA cells (T EMRA :CD45RO - , CD62L - The proportion of tissue-resident memory T cells (T ) expressing the tissue retention markers CD69 and CD103 was also relatively high in the control group (14.9 ± 12.9% vs. 4.4 ± 4.6%, p < 0.0001). RM ) display long-term protective immunity and are associated with improved outcomes after immunotherapy (Okla et al., J. Exp. Med., 218(4):e20201605 (2021)). γδ TILs express higher amounts of CD69 compared to αβ TILs. + (69.9±30.5% vs. 56.6±31.8%, p=0.003, Fig. 8D), CD103 + (25.8±24.1% vs. 16.6±19.4%, p=0.016), double positive T RM cells (20.8 ± 16.2 vs. 12.3 ± 13.0, p = 0.020).

[0099] Considering that the composition of ex vivo expanded TIL populations is highly dependent on spatial heterogeneity and on culture conditions that promote the proliferation of tumor-dominant and minority populations associated with differential tumor reactivity, we compared the expression of activating and exhausting molecules (Poschke et al., Clin. Cancer Res., 26:4289-4301 (2020)). Expanded γδ TILs (92.2%) and αβ TILs (97.4%) showed high levels of CD2 (Figure 8E), a costimulatory molecule whose signaling allows immunological synapse formation, the so-called CD2 crown, and buffers PD-1-mediated exhaustion (McKinney et al., Nature, 523:612-616 (2015); and Demetriou et al., Nat. Immunol., 21:1232-1243 (2020)). The IL-2 receptor α chain (CD25) was moderately expressed on γδ TILs (28%) and αβ TILs (32.4%). CD27, a costimulatory tumor necrosis receptor family member, has been implicated as a thymic regulator of interferon-γ (IFNγ) expression on IL-17-producing γδ T cells (Ribot et al., Nat. Immunol., 10:427-436 (2009); and Ribot et al., Cell. Mol. Life Sci., 68:2345-2355 (2011)). CD27 +Increased T cell numbers have also been associated with objective clinical responses in prior trials based on αβ TIL therapy (Rosenberg et al., Clin. Cancer Res., 17:4550-4557 (2011)). γδ TILs showed a range of CD27 expression, which on average (40.2%) was similar to that of αβ TILs (39.5%). In addition to identifying NK cells, the neural cell adhesion molecule CD56, a marker of enhanced T cell Th1 cytokine production and cytolytic capacity, was expressed to a substantially higher extent in γδ TILs (19.2±14.1%) than in αβ TILs (4.5±5.4%, p<0.0001) (Kelly-Rogers et al., Hum. Immunol., 67:863-873 (2006); Cohavy et al., J. Immunol., 178:5524-5532 (2007); and Almehmadi et al., Immunology, 142:258-268 (2014)). Upregulation of CD137 (4-1BB) has been identified as a marker of tumor-reactive T cells with enhanced clonal expansion and proliferation (Cooper et al., Eur. J. Immunol., 32:521-529 (2002); and Ye et al., Clin. Cancer Res., 20:44-55 (2014)). CD137 expression under these conditions was low in all cells but was significantly higher in γδ TILs (8.0 ± 10.5%) compared to αβ TILs (1.8 ± 2.3%, p = 0.0002).

[0100] Expression of inhibitory immune receptors is a concurrent marker of tumor reactivity, immune exhaustion, and suppressive efficacy (Ahmadzadeh et al., Blood, 114:1537-1544 (2009); Baitsch et al., J. Clin. Invest., 121:2350-2360 (2011); Miller et al., Nat. Immunol., 20:326-336 (2019); and Gros et al., J. Clin. Invest., 124:2246-2259 (2014)). With the exception of PD-L1, γδ TILs showed more variable expression of PD-1, LAG-3, TIGIT, and BTLA compared to αβ TILs (Figure 8F). PD-1 was lower in gamma delta TILs (39.4 ± 27.4%) compared with alpha beta TILs (57.7 ± 16.9%, p = 0.004). Expression of LAG3 (12.2% and 14.8%) and TIGIT (25.2% and 31.5%) was generally expressed at lower levels than PD-1 in both alpha beta and gamma delta TIL subsets. BTLA, a dual regulator of T cell costimulation and inhibition of TCR signaling, is a marker that enhances T cell viability and TIL therapeutic response and showed slightly higher expression in γδ TILs (39.5 ± 25.3%) compared to αβ TILs (26.6 ± 18.0%, p = 0.032) (Radvanyi et al., Clin. Cancer Res., 18:6758-6770 (2012); Haymaker et al., Oncoimmunology, 4:e1014246 (2015); and Ritthipichai et al., Clin. Cancer Res., 23:6151-6164 (2017)). In addition to being expressed on tumor cells, suppressor myeloid populations, and regulatory T cells, PD-L1 expression on effector T cells promotes self-tolerance and tumorigenesis in mouse models (Daley et al., Cell, 166:1485-1499 e1415 (2016); and Diskin et al., Nat. Immunol., 21:442-454 (2020)). PD-L1 expression was low in both expanded γδ TILs (3.4%) and αβ TILs (1.8%).

[0101] The innate NK cell properties of γδ T cells, including expression of the NCRs NKG2D and NKp46, confer additional responsiveness to stress antigens and antitumor potential (Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019); Wu et al., Sci. Transl. Med., 11(513):aax9364 (2019); Mikulak et al., JCI Insight, 4(24):e125884 (2019); and Foord et al., Sci. Transl. Med., 13(577):abb0192 (2021)). Expression of NKG2D was uniformly high in γδ TILs (72.8 ± 7.9%) compared to αβ TILs (38.0 ± 19.8%, p = 0.007), whereas expression of NKp46 was more heterogeneous (17.4 ± 22.4%) and did not differ from αβ TILs (23.6 ± 30.1%) (Figure 8G). A summary heatmap of the mean expression of all evaluated phenotypic markers in γδ and αβ TILs is included in Figure 8H.

[0102] Expansion of γδ TILs To explore the adoptive transfer of γδ TILs that display a favorable tissue-resident effector memory phenotype with limited exhaustion and enhanced expression of CD137 and NKG2D, we designed an expansion protocol to generate clinically viable numbers of γδ TILs. + Negative selection was performed by depletion of cells. Then, 1 × 10 6 γδ TILs (or bulk αβ TILs for comparison) were expanded for 14 days using mitogenic CD3 stimulation (OKT-3, 30 ng / mL), high concentrations of IL-2 (3,000 IU / mL), and PBMCs from irradiated allogeneic healthy donors (FIG. 10A). This IL-2-dependent expansion protocol was insufficient to expand γδ TILs (5.5-fold expansion, FIG. 11A), which may explain the limited numbers of γδ TILs observed with prior TIL therapy (Donia et al., Oncoimmunology, 1:1297-1304 (2012)).

[0103] Different combinations of cytokines were evaluated (in combination with anti-CD3 and irradiated PBMCs) to determine whether a population of γδ TILs with the desired phenotype could be obtained in adequate numbers and percentages. Addition of IL-15 (25.6-fold expansion) or IL-7 (164.3-fold expansion) increased the expansion of selected γδ TILs, whereas the combination of IL-2, IL-4, and IL-15 (453.8±100.8-fold expansion) showed a significantly enhanced γδ TIL expansion (p=0.0008) nearly comparable to that observed with IL-2-only expansion of native αβ TILs (725.5±153-fold expansion) (FIG. 11A).

[0104] Spectral cytometry phenotyping of IL-2 / IL-4 / IL-15-expanded negatively selected γδ TILs (Figure 11B) demonstrated highly purified γδ TCR + cells (CD3 + 95.3 ± 3.1% of cells (Figure 10B), and NK cells (CD45 + 2.3±2.5% of cells or αβ TCR + cells (CD3 + IL-2 / IL-4 / IL-15-expanded negatively selected γδ TILs were minimally expressed in Vδ1 + cells (γδ TCR + 63.2±28.3%) or VDδ1 - Vδ2 - cells (29.8 ± 24.2%), predominantly Vδ2 + Only a small percentage of cells (8.5 ± 10.4%) expressed IL-2-expanded naive αβ TILs, which were predominantly αβ TCR + cells (CD3 + 90.8% ± 6.5% of cells; CD8 + Cells (57.3±23.1%) or CD4 + cells (39.0 ± 22.8%), NK cells (1.27 ± 2.1%) or γδ TCR + (2.5±3.5%) cells were minimal (Figure 11C).

[0105] Expansion of negatively selected γδ TILs with IL-2 / IL-4 / IL-15 significantly increased T cell proliferation compared with negatively selected γδ TIL preparations prior to IL-2 / IL-4 / IL-15 expansion. EM There was an increased proliferation of γδ TILs (87.1±7.2% vs. 75.5±15.8%, p=0.034), and T EMRA The T EM Increasing the number of populations and T EMRA A reduction in population numbers is associated with clinical response to TIL therapy (Goff et al., J. Clin. Oncol., 34:2389-2397 (2016)).

[0106] Following expansion of negatively selected γδ TILs with IL-2 / IL-4 / IL-15 (5.3±2.7% vs. 20.8±16.2%, p<0.0001) and of naive αβ TILs with IL-2 alone (1.7±1.5% vs. 12.3±13.0%, p=0.004), CD69 + CD103 + T RMThe number of cells was reduced compared to TILs before expansion, but was higher in the γδ TIL population (p=0.004, FIG. 11D). Expression of CD2, CD25, and CD27 was generally stable in both negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15 and in native αβ TILs expanded with IL-2 alone. Expression of CD56 was increased in native αβ TILs expanded with IL-2 alone (30.3±23.3% vs. 4.5±5.3%, p=0.0007), but not in negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15 (21.6±25.8% vs. 19.2±14.1%) after expansion, as both populations showed similar levels of expression. CD137 showed increased expression after expansion in negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15 (18.2 ± 13.7% vs. 8.0 ± 10.5%, p = 0.006), and levels remained higher than those observed in native αβ TILs expanded with IL-2 alone (6.18 ± 8.9%, p = 0.036). PD-1 expression was reduced in both native αβ TILs expanded with IL-2 alone (36.2±22.5% vs. 57.7±16.9%, p=0.030) and negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15 (9.7±7.3% vs. 39.4±27.4%, p=0.0006) compared to pre-expansion, but remained at lower levels in expanded γδ TILs compared to expanded αβ TILs (p=0.002). While expression of LAG3 and TIGIT remained stable in both populations after expansion, expression of BTLA was slightly increased in native αβ TILs expanded with IL-2 alone (38.7% ± 14.1% vs. 26.6 ± 18.0%, p = 0.129), slightly decreased in negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15 (20.8 ± 9.8% vs. 39.5 ± 25.3%, p = 0.154), and was higher in expanded αβ TILs compared to expanded γδ TILs (p = 0.030).While αβ TILs did not show altered NCR expression of NKG2D and NKp46 after expansion in IL-2 alone, IL-2 / IL-4 / IL-15-expanded, negatively selected γδ TILs maintained high expression of NKG2D (77.9±14.2%) and the increase in the number of NKp46+ expressing cells (56.1±32.8% vs. 17.4±22.4%, p=0.011; post-expansion compared with pre-expansion) was greater than that observed in native αβ TILs expanded in IL-2 alone (15.7±22.3%, p=0.029).

[0107] MHC-independent, self-tumor recognition by γδ TCR Completed and ongoing trials of TIL therapy in patients with metastatic epithelial cancer have identified in vitro TIL reactivity against autologous patient tumors as a key determinant of objective clinical response (Tran et al., Science, 344:641-645 (2014); Stevanovic et al., J. Clin. Oncol. 33:1543-1550 (2015); Stevanovicr et al., Clin. Cancer Res., 25:1486-1493 (2019); Chandran et al., Lancet Oncol., 18:792-802 (2017); and Zacharakis et al., Nat. Med., 24:724-730 (2018)). To measure tumor reactivity of expanded peritoneal TILs, in patients with available specimens (n=11), IFNγ production was assessed after 24 h coculture of autologous tumor digests cryopreserved at the time of resection with either negatively selected γδ TILs expanded with IL-2 / IL-4 / IL-15 or native αβ TILs expanded with IL-2 alone at a 1:1 ratio (Figure 12A). After nonspecific stimulation with anti-CD3 / anti-CD28 mAb-coated beads, both αβ TILs (1556±849pg / mL) and γδ TILs (1638±1023pg / mL) produced similar levels of IFNγ. Both αβ TILs (135.8±103.5 vs. 27.4±18.4 pg / mL, p=0.002) and γδ TILs (380.7±207.6 vs. 25.2±12.1 pg / mL, p=0.001) produced significantly more IFNγ during coculture with autologous tumor digests compared to TILs cocultured with autologous PBMCs. γδ TILs showed greater autologous tumor reactivity compared to paired αβ TILs (p=0.009). Notably, 6 of 11 (55%) αβ TIL and 10 of 11 (91%) γδ TIL populations produced >100 pg / mL IFNγ after co-culture with tumor digesta, a hypothetical threshold for screening TIL reactivity associated with clinical tumor regression (Chandran et al., Lancet Oncol., 18:792-802 (2017)).

[0108] Given that γδ TILs have an MHC-nonrestricted TCR, we also assessed their reactivity against a series of HLA-mismatched cancer cell lines (Figure 12B). Compared with αβ TILs, which cannot recognize such mismatched cell lines, γδ TILs produced significantly more IFNγ when cultured with K562 leukemia cell line and a series of colon cancer cell lines (HCT116, RKO, and SW480). The reactivity of γδ TILs against SW48 colon cancer cell line was significantly lower than that of other cancer cell lines and did not differ from that observed in αβ TIL / SW48 cocultures. This led to the hypothesis that the lack of reactivity of γδ TILs against SW48 line was due to reduced production or expression of γδ TCR or NKG2D antigen. Analysis of mRNA expression of known ligands of the NKG2D receptor in evaluated cell lines in the Cancer Cell Encyclopedia (CCLE) showed that expression of MICA and MICB was stable or increased in K562, HCT116, RKO, and SW480, whereas expression of MICA (-0.35 Z score) and MICB (-1.01 Z score) was decreased in the SW48 line (Figure 13) (Barretina et al., Nature, 483:603-607 (2012)).

[0109] Given the established role of NCR-mediated recognition of γδ T cells on target cells and the uniformly high expression of NKG2D within this cohort of expanded peritoneal γδ TILs, we sought to identify its role in mediating autologous tumor reactivity along with γδ TCR (Silva-Santos et al., Nat. Rev. Immunol., 15:683-691 (2015); and Silva-Santos et al., Nat. Rev. Cancer, 19:392-404 (2019)). After co-culture of IL-2 / IL-4 / IL-15-expanded, negatively selected γδ TILs with autologous tumor digests (n=7), we used combinations of anti-MHC-1 (W6 / 32), anti-NKG2D (1D11), anti-γδ TCR (7A5), or isotype control (mouse IgG) mAbs to block corresponding receptor binding and signaling (FIG. 12C). Addition of anti-MHC-1 mAb showed no difference in γδ TIL IFNγ production, confirming MHC-independent recognition, whereas addition of anti-γδ TCR mAb significantly reduced IFNγ production compared with blocking with an isotype control. Addition of anti-NKG2D antibody had only a minimal effect on IFNγ production, and blocking in combination with γδ TCR did not further reduce it, suggesting an involvement of γδ TCR in mediating autologous tumor reactivity.

[0110] To identify additional factors associated with autologous tumor reactivity of γδ TILs, we compared the phenotypic characteristics of IL-2 / IL-4 / IL-15-expanded and negatively selected γδ TILs with IFNγ production after coculture with autologous tumor digest. The percent Vδ1 composition positively correlated with IFNγ production (r=+0.719, p=0.012), supporting previous reports of enhanced antitumor potential of Vδ1 cells over that observed in other γδ subsets (Figure S12D) (Deniger et al., Clin. Cancer Res., 20:5708-5719 (2014); Fisher et al., Clin. Cancer Res., 20:5720-5732 (2014); and Cordova et al., PLoS One, 7:e49878 (2012)).

[0111] Rapid pre-expansion protocol modulation of gamma chain cytokines and CD137 engagement does not improve expansion of gamma delta TILs Given the enhanced autologous tumor reactivity of γδ TILs compared to αβ TILs, we sought a method to specifically expand γδ TILs during the pre-REP culture period to determine the input number of γδ TILs available for REP. Given the increased numbers of γδ TILs after isolation and culture with IL-2, IL-4, and IL-15 during REP, this γ-chain combination was evaluated in a retrospective cohort of cryopreserved tumor digests (n=15, FIG. 14) from consented patients who underwent resection following initial diagnosis or neoadjuvant therapy for melanoma. In addition to the gamma chain combinations, a humanized agonistic monoclonal antibody targeting the CD137 receptor (urelumab, 10 μg / mL) was evaluated given the high expression of CD137 on gammadelta TILs and previous reports of enhanced TIL expansion through CD137 engagement (Hall et al., J. Immunother. Cancer, 4:61 (2016); Sakellariou-Thompson et al., Clin. Cancer Res., 23:7263-7275 (2017); Poch et al., Oncoimmunology, 7:e1476816 (2018); Tavera et al., J. Immunother., 41:399-405 (2018)).

[0112] The combination of γ chains is CD3 + Alpha-beta TCR + Increases in CD4 and CD8 TILs increased the total number of expanded TILs that survived after 11 days of culture, but not γδ TCR TILs, with or without CD137 stimulation, compared with IL-2 alone. + or Vδ1 + No differences in cell numbers were observed (FIGS. 15A-E). Although no improvement in γδ TIL expansion was identified with γ-chain combination or CD137 engagement, these results support the continued use of high doses of IL-2 or combinations with other γ-chain cytokines in the pre-REP process to expand γδ TIL.

[0113] Tumor-specific Vδ1 infiltration and improved survival rate Multiple clinical studies of TIL therapy identifying increased infusion numbers of tumor-reactive T cells associated with targeted clinical responses, and the previously described sparse infiltration of γδ TILs, have been performed to identify target indications in which γδ TILs are increased and to determine the impact on long-term survival (Radvanyi et al., Clin. Cancer Res., 18:6758-6770 (2012); Goff et al., J. Clin. Oncol., 34:2389-2397 (2016) and Chandran et al., Lancet Oncol., 18:792-802 (2017)). Using bulk RNA sequencing data from The Cancer Genome Atlas (TCGA) for the 20 most prevalent solid tumors, the expression of γδ TILs and αβ TILs was identified with the Gene Expression Profiling Interactive Analysis 2 (GEPIA 2) tool (Figure 16A) (Tang et al., Nucleic Acids Res., 47:W556-W560 (2019); and Siegel et al., CA Cancer J. Clin., 71:7-33 (2021)). Primary γδ T cell subsets (Vδ1 + cell, Vδ2 + cells, and Vδ3 +Infiltrating γδ TILs were identified with the corresponding Vδ genes (TRDV1, TRDV2, and TRDV3), whereas αβ TILs were identified with the Vβ2 constant region of the TCR (TRBC2). This method allowed for the unequivocal identification of the γδ TIL population, as previously utilized RNA gene signatures of γδ T cells have been shown to erroneously include other immune effector subsets during classification of γδ T cells (Gentles et al., Nat. Med., 21:938-945 (2015); and Tosolini et al., Oncoimmunology, 6:e1284723 (2017)). Across most tumor types, expression of TRDV1 tended to be higher than TRDV2 or TRDV3. Ovarian serous cystadenocarcinoma (OV) was the only indication that showed significant expression of TRDV3. TRDV1 was differentially expressed by tumor type, being highest in lung adenocarcinoma (LUAD, median 0.5 log of transcripts per million (TPM)), kidney renal cell carcinoma (KIRC, 0.5 Log TPM), breast cancer (BRCA, 0.4 Log TPM), and cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC, 0.4 Log TPM). TRDV1 expression in the entire cohort of these selected tumors is shown in Figure 17A. Glioblastoma (GBM), liver hepatocellular carcinoma (LIHC), bladder urothelial carcinoma (BLCA), uterine endometrial carcinoma (UCEC), and prostate adenocarcinoma (PRAD) showed the lowest expression of TRDV1 (median Log TPM of TRDV1 = 0). Across all tumor types, expression of TRDV1 positively correlated with expression of TRBC2 (Figure 18A-S).

[0114] Given the predominant infiltration of the Vδ1 subset throughout the tumor compared to other γδ T cell subsets, and its association with autologous tumor reactivity, we assessed the prognostic impact of TRDV1 expression on overall survival in selected tumors. After normalizing TRDV1 expression to beta-actin (ACTB), the cohort was divided into high and low expressers based on the median TRDV1 expression levels in individual tumor types. When including all TCGA tumors analyzable on the GEPIA 2 server, high TRDV1 expression was associated with significantly improved survival (p<0.00001, Figure 16B). High expression of TRDV1 was similarly associated with significantly improved survival in 12 of the 20 solid tumors profiled, including cutaneous melanoma (SKCM, p = 0.0006), head and neck squamous cell carcinoma (HNSC, p = 0.002), lung adenocarcinoma and squamous cell carcinoma (LUSC, p = 0.0004), breast cancer BRCA (p = 0.007), esophageal cancer CESC (p = 0.014), and pancreatic ductal adenocarcinoma (PDAC, p = 0.077), all of which are current indications for TIL therapy (Figures 16C-G and 17B-G). Increased TRDV1 was also associated with improved survival in patients with GBM, mesothelioma (MESO), LIHC, KIRC, and BLCA. In the remaining eight tumors, high expression of TRDV1 was not associated with a substantial improvement in survival (Figures 19A-H).

[0115] Taken together, these results provided herein demonstrate that tumor-infiltrating γδ T cells display a diverse and patient-specific repertoire, a tissue-resident effector memory phenotype, and exquisite autologous tumor reactivity, and can exert their full TCR repertoire against cancer, either alone or in parallel with αβ TILs.

[0116] Other embodiments Although the invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for generating a cell population comprising γδ T cells, the method comprising culturing a first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-5 for 8 to 21 days to obtain a second cell population, wherein the second cell population comprises at least 10-fold more γδ T cells than the first cell population.

2. The method of claim 1, wherein the γδ T cells are human cells.

3. The method of claim 1, wherein the γδ T cells are tumor-infiltrating γδ T cells.

4. The first cell population: (i) a population of tumor-infiltrating γδ T cells obtained from (a) tissue containing a tumor or (b) healthy tissue that was within 30 mm of the tumor; (ii) a population of γδ T cells obtained from healthy tissue; (iii) a population of γδ T cells obtained from infected tissue, or (iv) a population of γδ T cells obtained from tissues harboring autoimmune T cells The method of claim 1, wherein

5. 2. The method of claim 1, wherein the first cell population is a cell population enriched for tumor-infiltrating γδ T cells by (a) depleting at least some αβ T cells or (b) isolating at least some γδ T cells.

6. 2. The method of claim 1, wherein culturing the first cell population comprising γδ T cells for 8 to 21 days in the presence of IL-2, IL-4, and IL-15 comprises culturing the first cell population comprising γδ T cells for 8 to 21 days in the presence of IL-2, IL-4, IL-15, irradiated PBMCs, and anti-CD3 antibody.

7. 2. The method of claim 1, wherein culturing the first cell population comprising γδ T cells in the presence of IL-2, IL-4, and IL-15 is for 12 to 16 days.

8. the second cell population: at least 50-fold more γδ T cells than said first cell population; at least 100-fold more γδ T cells than said first cell population; at least 200-fold more γδ T cells than said first cell population; at least 300-fold more γδ T cells than said first cell population; or at least 400 times more γδ T cells than said first cell population; 2. The method of claim 1, comprising:

9. the second cell population is 1×10 8 2. The method of claim 1, comprising more than γδ T cells.

10. 2. The method of claim 1, wherein the IL-2 is human IL-2, the IL-4 is human IL-4, and the IL-15 is human IL-15.

11. The second cell population CD3 + More than 85 percent of cells are γδ TCR + The method of claim 1, wherein the cell is a cell.

12. The second cell population CD3 + Fewer than 10 percent of cells have αβ TCR + The method of claim 1, wherein the cell is a cell.

13. CD45 of the second cell population + 10. The method of claim 1, wherein less than 10 percent of the cells are NK cells.

14. γδ TCR of said second cell population + More than 30 percent of cells are Vδ1 + The method of claim 1, wherein the cell is a cell.

15. γδ TCR of said second cell population + Fewer than 60 percent of cells are Vδ1 - Vδ2 - The method of claim 1, wherein the cell is a cell.

16. γδ TCR of said second cell population + Fewer than 25 percent of cells are Vδ2 + The method of claim 1, wherein the cell is a cell.

17. γδ TCR of said second cell population + More than 70 percent of the cells are T EM The method of claim 1, wherein the cell is a cell.

18. γδ TCR of said second cell population + Fewer than 25 percent of cells are T EMRA The method of claim 1, wherein the cell is a cell.

19. An isolated cell population comprising polyclonal γδ T cells, comprising 1×10 8 An isolated cell population comprising more than γδ T cells.

20. A composition for providing γδ T cells to a mammal, comprising a cell population produced as described in claim 1.

21. A composition for providing γδ T cells to a mammal, comprising the cell population of claim 19.

22. A composition for treating cancer in a mammal, comprising a cell population produced as described in claim 1.

23. A composition for treating cancer in a mammal, comprising the cell population of claim 19.

24. A composition for treating an autoimmune condition in a mammal, comprising the cell population of claim 19.

25. A composition for treating an infectious disease in a mammal, comprising the cell population of claim 19.

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