Amplification of non-hematopoietic tissue-resident γδ T cells and use of said cells

Abstract

To provide: methods for expansion of non-hematopoietic tissue-resident γδ T cells; and populations of non-hematopoietic tissue-resident γδ T cells and uses thereof.SOLUTION: This invention provides methods for expansion of non-hematopoietic tissue-resident γδ T cells in vitro by culturing lymphocytes obtained from non-hematopoietic tissue of humans or non-human animals in the presence of interleukin-2 (IL-2) and / or interleukin-15 (IL-15) and the absence of TCR activation or co-stimulation signals, without any direct contact with stromal or epithelial cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for amplifying non-hematopoietic tissue-resident γδ T cells ex vivo. "Non-hematopoietic tissue-resident γδ T cells" means a subset of T lymphocytes that reside in non-hematopoietic tissues, rather than in lymphoid organs and blood. Such cells include non-Vδ2 cells, such as Vδ1, Vδ3, and Vδ5 cells. The present invention also relates to the use of these cells in adoptive T cell therapy and chimeric antigen receptor therapy, as well as their use in screening methods for checkpoint regulators. The present invention also relates to cells produced as a result of ex vivo amplification of non-hematopoietic tissue-resident γδ T cells. [Background technology]

[0002] Growing interest in T-cell immunotherapy for cancer has focused particularly on the apparent ability of subsets of CD8+(1-4) and CD4+αβ(5,6) T cells to recognize cancer cells and mediate host-protective functional potential, especially when desuppressed by clinically mediated antagonism of inhibitory pathways exerted by PD1(7,8), CTLA4(9,10) and other receptors(11). Nevertheless, many questions remain. For example, there appear to be numerous major clinical situations in which such therapies seem to be ineffective (11); serious adverse events (AEs) occur in many cases (12); the ability to predict efficacy or AEs is extremely limited (13); and there is little explanation for the interaction that enables the host to sense tumor cells (so-called "immunogenicity"), which must precede the activation of conventional antigen-specific CD8+ and CD4+αβ T cell responses.

[0003] In relation to these, numerous scientists and clinicians are similarly beginning to re-evaluate the potential of γδ T cells, a third lineage of somatically-produced receptor-bearing lymphocytes that, like αβ T cells and B cells, is evolutionarily highly conserved. Essentially, there are two subgroups of human γδ T cells; one is dominant in human peripheral blood, with the majority expressing the Vδ2 T cell receptor (TCR); the other is dominant in non-hematopoietic tissues, with the majority expressing the Vδ1 TCR, and a relatively small population expressing TCRs containing Vδ3 or Vδ5 chains or some other non-Vδ2 chain (14).

[0004] In most adults, Vδ2 cells constitute only a small, highly diverse component (0.01–5%) of blood T cells during the quiescent phase. However, after sensitization with a wide range of substances, including numerous bacteria and parasites, these cells rapidly amplify, temporarily reaching up to approximately 25% of CD3+ cells (14). The primary basis of this response is Vδ2 TCR-mediated recognition of low molecular weight "phospho moieties," including hydroxyl-methylbuta-2-enylpyrophosphate (HMBPP) (15), which are intermediates in key microbial synthesis pathways of cholesterol and other lipids used to modify proteins (e.g., by geranylation or farnesylation). In primates, this synthesis occurs via the mevalonate pathway, one of its intermediates, isopentenylpyrophosphate (IPP), is expressed at very high levels in virus-infected and transformed cells, and is also a target of Vδ2 TCR-mediated recognition (16).

[0005] In addition, most Vδ2 T cells express high levels of the NKG2D receptor, which, when encapsulated with NKG2D ligands (e.g., MICA, MICB, and ULBP), can activate or co-stimulate (in conjunction with the TCR) the cytolytic capacity of cells. These ligands are host proteins that are upregulated when cells are exposed to factors such as oxidative stress, osmotic stress, or ultraviolet light. These factors promote hyper-active signaling in the epidermal growth factor receptor (EGFR) pathway, which is also commonly dysregulated in human solid tumors (17).

[0006] The ability of Vδ2 T cells to detect transformed cells using TCR and / or NKG2D (18-20), along with their potent cytolytic ability and evident ability to present antigens to CD8+ T cells (21), has collectively led to the view that Vδ2 T cells can be clinically utilized to deliver cancer immunotherapy. This can be achieved by adoptive cell transfer, in which case the MHC-free status of γδ T cells significantly and beneficially limits graft-versus-host disease (GvHD) (22). To achieve this, vascular commensal Vγ9Vδ2 γδ T cells can be amplified ex vivo by adding cytokines (such as interleukin (IL)-2), along with exogenous TCR activators (such as phospho moieties (e.g., BrHPP)), or with clinically approved bisphosphonates (e.g., zoledronic acid) (which inhibits farnesyl pyrophosphate synthase in the mevalonate pathway, thereby inducing the accumulation of IPP, the TCR activating moiety). However, chronic activation of Vγ9Vδ2 cells mediated by factors such as BrHPP can gradually lead to cellular exhaustion and a decrease in cytotoxicity.

[0007] Alternatively, the patient's own γδ T cells can be activated in situ using pharmacologically modified forms of HMBPP or clinically approved aminobisphosphonates. These approaches have treated over 250 cancer patients, and while they appear safe, complete remission has been rare. One major concern regarding the limited clinical efficacy of the cells is their tendency to become irreversibly exhausted due to chronic antigen exposure. A second major concern is their apparent inefficiency in homing to solid tumors and the tissues that hold them (23).

[0008] Chimeric antigen receptor T cell (CAR-T) therapy has shown clinical promise for B-cell malignancies. However, with regard to treating solid tumors, the performance of CAR-T cells has so far been lower than predicted, with low efficiency in producing a complete tumor response and a high incidence of cytotoxicity outside the tumor (24). Regarding peripheral blood γδ T cells, the main obstacle to the success of CAR-T approaches to solid tumors is that systemic CAR-T cells are probably inefficient in migrating to the site of the malignant tumor and commensating there in a functionally effective state (25). In addition, based on conventional αβ T cells, CAR-T cells must overcome immunosuppressive signaling in the tumor microenvironment (e.g., those transmitted via the PD1 receptor).

[0009] γδ T cells can be transduced using tumor-reactive chimeric antigen-specific TCRs while retaining their innate ability to recognize transformed cells using receptors such as NKG2D, potentially offering advantages in CAR-T approaches. In other words, γδ T cells can simultaneously possess both tumor compatibility (TCR)-mediated and innate (NKG2D)-mediated activity. However, the efficiency with which human blood γδ T cells can home to tumors in solid tissues and maintain their active state within them remains insufficient. This consideration has prompted a more detailed examination of γδ T cells, which are normally commensal in non-hematopoietic tissues.

[0010] Such T cells migrate to non-hematopoietic tissues as part of their development, thereby differentiating them from T cells that infiltrate tissues after systemic priming (e.g., tissue-resident TCRαβ+ memory T cells (so-called TRM cells)). Tissue-resident γδ T cells have been best studied in mice, where they have been shown to be common in the skin, intestines, and germline tissues, among other sites. A large number of such cells have been shown to retain innate-like functional ability, thereby enabling them to respond to sensitization through activation of the NKG2D receptor. We have recently obtained data demonstrating that human skin and intestines also harbor a large proportion of non-hematopoietic tissue-resident γδ T cells with innate-like activity. It is also striking that studies of malignancies, inflammation, atopy, allergies, and other pathological conditions formed in non-hematopoietic tissues have largely overlooked the potential influence of these innate-like human T cells resident in the tissues where the lesions occur.

[0011] Human γδ T cells commensal to non-hematopoietic tissues have been largely unstudied due to the difficulty in cell harvesting caused by their localization and the lack of established methods for culturing them. From the relatively limited information available, this subtype includes a diverse range of cells with non-MHC-restricted cytolyticity, which do not express Vγ2-containing TCRs and therefore do not react at all to low molecular weight phospho moieties. While little is known about the precise TCR specificity of such cells, available data suggest that the cells are reactive to autoantigens such as endothelial protein C receptor (EPCR), which is overexpressed by cytomegalovirus (CMV)-infected cells and numerous solid tumors (32). Non-hematopoietic tissue-associated γδ T cells also commonly express NKG2D (14). Given these characteristics, as well as the physiological commensalization of cells in non-hematopoietic tissues such as skin and intestines, adoptive transfer of such cells to cancer patients may be considerably more effective in targeting solid tumors and potentially other immunopathologies.

[0012] To utilize non-Vδ2 cells in immunotherapy, either a means of amplifying these cells in situ or a means of recovering them and amplifying them ex vivo before reinjection is required. Since there are no known TCR activators proven capable of amplifying large numbers of non-Vδ2 cells in situ, the latter approach is employed. To overcome the difficulty of limited availability of non-hematopoietic tissue, some researchers have attempted to amplify very small numbers of non-Vδ2 cells from blood, assuming these cells are equivalent to tissue-resident non-Vδ2 cells, and that Vδ2-expressing cells are the dominant subset. The small number of non-Vδ2 γδ T cells found in the blood appear to amplify considerably during active CMV infection, show superior responsiveness to CMV compared to Vδ2 T cells, and may protect the human fetus in cases of intrauterine CMV infection. In addition, CMV-reactive non-Vδ2 γδ T cells appear to protect transplant patients from CMV reactivation and reduce the risk of secondary malignancies during immunosuppression, through cross-reactivity with transformed cells (26). Similarly, there is data suggesting that γδ T cells play a beneficial role in controlling HIV infection, in this case non-Vδ2 γδ T cells are amplified in the blood compared to Vδ2 T cells (24).

[0013] Blood resident non-Vδ2 cells can be amplified ex vivo (41-44) by adding exogenous factors that directly activate TCR signaling (for example, by using substances such as anti-CD3 antibodies, pan-γδ-TCR specific antibodies or phytohemagglutinin (PHA)), or by co-culturing stimulated non-Vδ2 T cells with artificial antigen-presenting cells (aAPCs) (in this case, direct contact between γδ T cells and aAPCs is required for ex vivo non-Vδ2 T cell amplification). Alternatively, cells have been amplified (28) by promoting NKG2D receptor signaling using immobilized recombinant MICA (NKG2D ligand), similar to that used to maintain the proliferation of ex vivo γδ T cell cultures derived from epithelial cancer infiltrating lymphocytes (TILs). Overall, current methods for ex vivo amplification of Vδ2-expressing blood γδ T cells or non-Vδ2 blood γδ T cells always require the addition of substances that promote activation of the TCR and / or NKG2D receptor, together with auxiliary cytokines such as interleukin-2 (IL-2) (41-44). This combination of receptor activation signals and cytokines reflects the standard approach widely adopted by society for culturing and amplifying T cells. To date, methods for significantly amplifying γδ T cells resident in non-hematopoietic tissues have not been described. Such methods are described herein. Summary of the Invention

[0014] As part of the phenotypic and functional characterization of human skin T cells, the inventors isolated distinct large populations of γδ T cells that normally reside in non-hematopoietic tissues and have unique properties compared to αβ T cells and blood-resident γδ T cells. The inventors found that the cells show strong TCR-independent innate-like responses against NKG2D ligands and cytokines. While efforts to expand primary αβ T cells have generally used co-culture with other supportive cells as a source of beneficial growth factors (29), the inventors unexpectedly showed that γδ T cells resident in the skin and other non-hematopoietic tissues are greatly and specifically suppressed by co-culturing these cells in contact with autologous skin fibroblasts and potentially other stromal components such as keratinocytes and endothelial cells. Removal of such interactions allows for the amplification of such cells in large numbers and rapidly for potential clinical applications.

[0015] Furthermore, compared to current efforts to expand blood-derived and tumor-derived γδ T cells, the inventors showed that such non-hematopoietic tissue-resident γδ T cells can be expanded without the intentional addition of any exogenous substances that activate their TCR or NKG2D signaling pathways.

[0016] This specification discloses novel means for efficiently and reproducibly isolating and expanding γδ T cells from non-hematopoietic tissues of human or non-human animals such as the skin and intestine. Expansion is promoted by disrupting the contact of non-hematopoietic tissue-derived non-Vδ2 T cells with autologous fibroblasts and potentially other stromal components, and maintained by culturing in interleukin-2 (IL-2) and / or interleukin-15 (IL-15).

[0017] The amplification of αβ T cells or Vδ2-expressing T cells or NK cells is highly selective, as it is not induced by disrupting their contact with autologous fibroblasts (Figures 3A, 3C, and 3D). This amplification of non-hematopoietic tissue-resident γδ T cells by liberation from fibroblast-mediated checkpoint regulation also triggers the "spontaneous" activation of the cells' effector potential (Figures 5A and 5B), which is highly desirable in the context of antitumor activity. These developments allow non-hematopoietic tissue-resident γδ T cells to be amplified in culture and activated for conceivable use as an "off-the-shelf" cell injector into patients. Simultaneously, the development of antibodies or other forms of inhibitors of checkpoint regulation of tissue-resident γδ T cells by fibroblasts (or other stromal or epithelial cells) should allow non-hematopoietic tissue-resident γδ T cells to be activated in situ (e.g., in the bodies of cancer patients) via checkpoint blockade.

[0018] The ability to obtain and cultivate γδ T cells from non-hematopoietic tissues (e.g., skin) is clearly different from that of blood-derived Vδ1 T cells. For example, skin-derived Vδ1 T cells exhibit preceding T cell activation markers such as CD69 expression, ICOS, and TIM3 positivity, and show little to no expression of the classical costimulatory molecule CD28 (Figure 10A). Furthermore, they show high expression of NKG2D. In contrast, human blood-derived Vδ1 T cells do not express CD69 or TIM3, express only slight levels of ICOS, and are somewhat positive for CD28. Moreover, NKG2D expression by blood-derived Vδ1 T cells is much lower than that by skin-derived Vδ1 T cells, and skin-derived Vδ1 T cells show innate responsiveness to NKG2D ligands such as recombinant MICA in the absence of T cell receptor stimulation, while blood-derived Vδ1 T cells do not (Figure 10B). As described herein, non-hematopoietic tissue-derived γδ T cells may also show elevated expression of CCR3, CD39, CD11b, IL-13, and / or CD9 compared to blood-derived Vδ1 T cells or other lymphocyte populations.

[0019] In a first aspect, the present invention provides a method for amplifying non-hematopoietic tissue-resident γδ T cells in vitro, comprising the step of culturing lymphocytes obtained from non-hematopoietic tissue of a human or non-human animal in the presence of IL-2 and / or interleukin-15 (IL-15), wherein the lymphocytes do not come into direct contact with stromal or epithelial cells during culture.

[0020] Preferably, the lymphocytes do not come into direct contact with fibroblasts during culture.

[0021] γδ T cells are normally commensal in non-hematopoietic tissues in vivo.

[0022] Preferably, the method includes the step of culturing lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of IL-2 and IL-15.

[0023] In some embodiments, lymphocytes obtained from human or non-human animal non-hematopoietic tissues can be cultured in the absence of TCR activators or costimulatory factors that induce T cell activation. For example, lymphocytes can be cultured in the absence of TCR pathway agonists (e.g., CD3 activators such as anti-CD3 antibodies) and in the presence or absence of CD28 activators.

[0024] The addition of such exogenous activators of TCR signaling is not necessary for amplification of non-hematopoietic tissue-resident γδ T cells using the method of the present invention. Accordingly, a suitable γδ amplification medium for use in the method of the present invention may be one that lacks T cell activation activity (e.g., αβ T cell or blood γδ T cell activation activity) and does not activate or co-stimulate the TCR.

[0025] For example, the γδ amplification medium may be exogenously supplemented with TCR pathway agonists, or may be substantially free of substances or factors that activate T cell signaling, such as TCR activators or costimulators. The γδ amplification medium may contain IL-2 and / or IL-15. In some embodiments, the γδ amplification medium may contain one or more additional growth factors, such as cytokines, in addition to IL-2 and / or IL-15. Suitable growth factors do not exhibit T cell activation activity. In other embodiments, the γδ amplification medium may lack growth factors other than IL-2 and / or IL-15; for example, the γδ amplification medium may consist of a basal medium supplemented with IL-2 and / or IL-15.

[0026] In one embodiment, lymphocytes obtained from human or non-human animal non-hematopoietic tissue can be cultured in the absence of stromal cells or epithelial cells. For example, stromal cells or epithelial cells can be removed prior to culture. Preferably, lymphocytes obtained from human or non-human animal non-hematopoietic tissue can be cultured in the absence of fibroblasts. For example, fibroblasts can be removed prior to culture.

[0027] Lymphocytes can be obtained from any suitable human or non-human animal non-hematopoietic tissue, such as skin, gastrointestinal tract (e.g., colon or ileum), mammary gland tissue, lungs, liver, pancreas, adipose tissue, or prostate.

[0028] Non-hematopoietic tissue-resident γδ T cells are preferably non-Vδ2 cells, most commonly expressing a TCR containing the Vδ1 chain, i.e., Vδ1 cells. Non-hematopoietic tissue-resident γδ T cells can also include so-called double-negative (DN) γδ T cells, which are defined as expressing a γδ TCR that does not contain either the Vδ1 or Vδ2 chain.

[0029] The method optionally includes the step of obtaining lymphocytes from human or non-human animal non-hematopoietic tissue. For example, lymphocytes can be obtained from a sample of human or non-human animal non-hematopoietic tissue. The method may include the steps of providing a sample of human or non-human animal non-hematopoietic tissue and separating lymphocytes from the non-hematopoietic cells of the sample to produce a lymphocyte population substantially free of stromal cells.

[0030] In a second aspect, the present invention provides a method for amplifying γδ T cells, comprising the steps of: (i) providing a population of γδ T cells obtained from non-hematopoietic tissue; and (ii) culturing the γδ T cells substantially without interstitial cell contact to produce an amplified population of γδ T cells.

[0031] A population of γδ T cells obtained from non-hematopoietic tissue may be a substantially pure population of γδ T cells.

[0032] Populations of γδ T cells obtained from non-hematopoietic tissue are preferably non-Vδ2 cells, most commonly expressing a TCR containing a Vδ1 chain, i.e., Vδ1 cells. Populations of γδ T cells may also include DN γδ T cells.

[0033] Populations of γδ T cells obtained from non-hematopoietic tissue may express one or more additional tissue-resident γδ T cell markers, such as CLA, IL13, CCL1, CD103, and CCR8. In some embodiments, populations of γδ T cells obtained from non-hematopoietic tissue may express Vδ1 + CCR8 + It may contain γδ T cells.

[0034] γδ T cells can be cultured in the absence of contact with stromal cells to create an amplified population of γδ T cells (i.e., there is no contact between γδ T cells and stromal cells in the cell culture).

[0035] γδ T cells can be cultured in the absence of TCR activation or co-stimulatory signals. In some embodiments, the culture step can be carried out in stromal cell-conditioned medium or in the presence of IL-2, IL-15, or a combination thereof. For example, γδ T cells can be cultured in γδ amplification medium containing IL-2 and / or IL-15. A suitable γδ amplification medium may not activate or co-stimulate the TCR. For example, a γδ amplification medium may not contain or substantially contain substances or factors that activate T cell signaling, such as TCR pathway agonists, TCR activators, or co-stimulators. A γδ amplification medium may contain IL-2 and / or IL-15. In some embodiments, in addition to IL-2 and / or IL-15, a γδ amplification medium may contain one or more additional growth factors, such as cytokines. A suitable growth factor does not exhibit T cell activation activity. In other embodiments, the γδ amplification medium may lack growth factors other than IL-2 and / or IL-15; for example, the γδ amplification medium may consist of a basal medium supplemented with IL-2 and / or IL-15.

[0036] In a third aspect, the present invention provides a method for amplifying γδ T cells, comprising the steps of: (i) providing non-hematopoietic tissue containing non-hematopoietic cells and γδ T cells; (ii) separating γδ T cells from the non-hematopoietic cells to produce a population containing γδ T cells that substantially does not contain stromal cells; and (iii) culturing the population from step (ii) in the absence of a TCR activation signal or a co-stimulatory signal to produce an amplified population of γδ T cells.

[0037] γδ T cells can be isolated, for example, from αβ T cells.

[0038] The population in step (ii) may be a substantially pure population of γδ T cells.

[0039] The γδ T cells in the population in step (ii) may include non-Vδ2 γδ T cells, most commonly those expressing a TCR containing the Vδ1 chain, i.e., Vδ1 γδ T cells. The γδ T cells in the population in step (ii) may also include DN γδ T cells.

[0040] The γδ T cells in the population in step (ii) may also include γδ T cells expressing one or more additional tissue-resident γδ T cell markers, such as CLA, CD103, and CCR8. In some embodiments, the γδ T cells in the population in step (ii) may also include Vδ1 + CCR8 + It may contain γδ T cells.

[0041] The culture step in step (iii) may be substantially free from stromal cell contact with the population in step (ii) and / or may be in the absence of TCR activation signals or co-stimulatory signals. For example, the culture step may be carried out without contact between γδ T cells and stromal cells. In some embodiments, the culture step in step (iii) may be performed in stromal cell-conditioned medium or in the presence of IL-2, IL-15, or a combination thereof.

[0042] In some embodiments, γδ T cells can be cultured in a γδ amplification medium containing IL-2 and / or IL-15. A suitable γδ amplification medium may not activate or co-stimulate the TCR. For example, the γδ amplification medium may not contain or substantially contain substances or factors that activate T cell signaling (e.g., TCR activators or co-stimulators, such as TCR pathway agonists). In some embodiments, the γδ amplification medium may contain one or more additional growth factors, such as cytokines, in addition to IL-2 and / or IL-15. A suitable growth factor does not exhibit T cell activating activity. In other embodiments, the γδ amplification medium may consist of a basal medium supplemented with IL-2 and / or IL-15.

[0043] An amplified population of γδ T cells according to the second or third embodiment may contain at least 5 times, at least 10 times, at least 15 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times, or at least 10000 times more γδ T cells than γδ T cells obtained from non-hematopoietic tissue or isolated from non-hematopoietic cells. The amplified population can be generated within 3, 5, 7, 10, 14, 21, or 28 days of culture.

[0044] γδ T cells in the amplified population are preferably Vδ2 - These are T cells. The amplified population of γδ T cells contains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% Vδ1 +Cells may be included. An amplified population of γδ T cells may have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of it be positive for one, two, or all three of CCR4, CCR8, and CD103. For example, an amplified population may be at least 10% positive for CD103, at least 30% positive for CCR4, and at least 60% positive for CCR8.

[0045] In a fourth aspect, the present invention provides non-hematopoietic tissue-resident γδ T cells or populations thereof obtained by the method of the first, second, or third aspect of the present invention.

[0046] In a fifth aspect, the present invention progresses through the following steps: (i) the step of culturing non-hematopoietic tissue resident γδ T cells in vitro by directly contacting stromal cells or epithelial cells (e.g., fibroblasts) in the presence and absence of the test compound, or the step of culturing non-hematopoietic tissue resident γδ T cells in vitro by directly contacting stromal cells or epithelial cells (e.g., fibroblasts), wherein the expression of the test gene in the γδ T cells and / or stromal cells or epithelial cells (e.g., fibroblasts) is altered; and (ii) A step of determining the proliferation rate or activation rate of non-hematopoietic tissue-resident γδ T cells in the presence or absence of the test compound, or in the presence or absence of changes in the expression of the test gene in stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells; or a step of determining the mortality rate of stromal cells or epithelial cells (e.g., fibroblasts) in the presence or absence of the test compound, or in the presence or absence of changes in the expression of the test gene in stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells. A method for screening checkpoint regulators of non-hematopoietic tissue-resident γδ T cells, wherein, If the T cell proliferation rate or activation rate is higher in the presence of the test compound than in the absence of the test compound, or if the test gene expression change in stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells is higher than in the absence of the test gene change in stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells, and / or if the stromal cell or epithelial cell (e.g., fibroblast) mortality rate is higher in the presence of the test compound than in the absence of the test compound, or if the test gene expression change in stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells is higher than in the absence of the test gene change in fibroblasts and / or γδ T cells, then the test compound may be a checkpoint regulator, or the test gene may be a candidate checkpoint gene or its regulator. The above method is provided.

[0047] The expression of test genes in γδ T cells and / or stromal or epithelial cells (e.g., fibroblasts) can be altered, for example, by RNA targeting substances such as small interfering RNA (siRNA) or small hairpin RNA (shRNA), or by gene editing (e.g., using the CRISPR / Cas system).

[0048] In a sixth aspect, the present invention provides a method for treating a subject with adoptive T cell therapy, comprising the step of administering non-hematopoietic tissue-resident γδ T cells obtained by a method according to a first, second, or third aspect of the present invention to a subject in need thereof. The subject is preferably human.

[0049] The subjects are preferably human cancer patients or patients with viral infections (e.g., CMV-infected patients or HIV-infected patients).

[0050] In a seventh aspect, the present invention provides non-hematopoietic tissue-resident γδ T cells obtained by the method of the first, second or third aspect of the present invention for use in a method of treating a human or non-human animal by adoptive T cell therapy. The non-hematopoietic tissue-resident γδ T cells can have one or more of the following characteristics: (i) exhibit the phenotype CD69 high , ICOS high , TIM3 high and CD28 low / absent ; or (ii) upregulate one or more of CCR3, CD11b, CD9 and CD39; (iii) produce IFN-γ in response to NKG2D ligands in the absence of a TCR agonist; (iv) produce IL-13 in the absence of a TCR agonist; (v) produce one or more of IFN-γ, TNF-α and GM-CSF in response to TCR activation; (vi) produce no or substantially no IL-17 in response to TCR activation; (vii) grow in a culture medium containing IL-2 without additional growth factors; (viii) exhibit a cytotoxic T cell response in the absence of a TCR agonist and / or (ix) exhibit selective cytotoxicity against tumor cells compared to normal cells.

[0051] In a preferred embodiment, the human is a human cancer patient or a virus-infected patient (e.g., a CMV-infected patient or an HIV-infected patient), wherein the CMV or HIV infection is a MICA-related infection.

[0052] In an eighth aspect, the present invention provides a method of treating a subject by chimeric antigen receptor therapy, comprising the step of administering to a subject in need thereof non-hematopoietic tissue-resident γδ T cells obtained by the method of the first, second or third aspect of the present invention. The subject is preferably human.

[0053] In a preferred embodiment, the subject is a human cancer patient.

[0054] In a ninth aspect, the present invention provides non-hematopoietic tissue resident γδ T cells obtained by a method according to a first, second, or third aspect of the present invention for use in a method of treating a human or non-human animal by chimeric antigen receptor therapy.

[0055] In a preferred embodiment, the human is a human cancer patient.

[0056] Each of the above embodiments can be combined with one or more of the other embodiments.

[0057] These or other embodiments of the present invention will be described in further detail below. [Brief explanation of the drawing]

[0058] [Figure 1A] This figure shows that human skin contains a clear population of resident γδ T cells. Figure 1A: Resident lymphocytes were isolated using the organ-type cell culture "Clark protocol" published by Clark et al. (29). Among CD45+ cells, anti-CD3 was used to stain T cells, and anti-CD56 antibody was used to identify NK cells (CD3-CD56+). Among CD3+ cells, antibody against the pan-γδ T cell receptor was used to identify resident γδ T cells, and anti-CD8alpha was used to identify the proportion of conventional CD4 and CD8-positive αβ T cells within the CD3+ pan-γδ TCR gate. [Figure 1B]This figure shows that human skin contains a clear population of resident γδ T cells. Figure 1B outlines these experiments with 7-10 donors using the Clark protocol. This protocol allowed lymphocytes in human skin to remain in contact with cutaneous fibroblasts, with either no cytokines added or interleukin-2 (IL-2), interleukin-15 (IL-15), or IL-2 and IL-15 added. This demonstrated that the use of cytokines did not alter the composition of resident cutaneous lymphocytes, except for a slightly larger γδ T cell population when IL-15 or IL-2 and IL-15 were added to the culture, thus demonstrating the effectiveness of the Clark protocol. Lymphocyte composition after 3 weeks of organ-type skin culture is shown as an outline for 4 donors, using the described cytokines. [Figure 1C] This figure shows that human skin contains a distinct population of resident γδ T cells. Figure 1C: Resident γδ cells in the skin mainly consist of Vδ1-expressing γδ T cells (76.24% ± 17.3%), a small population of Vδ2-expressing T cells (3.06% ± 6.1%), and a population of pan-γδ TCR-positive cells (also referred to herein as double-negative (DN) γδ T cells) that are negative for staining for Vδ1 and Vδ2 (20.7% ± 13.97%). Control staining of healthy volunteer blood showed that the dominant population of γδ T cells in the blood expressed the Vδ2 TCR chain, demonstrating the strong localization of human γδ T cells. [Figure 1D] This figure shows that human skin contains a clear population of resident γδ T cells. Figure 1D: Skin resident γδ T cells show markers previously associated with chronically activated T cells, although these markers do not necessarily reflect chronic activation and are characteristic indicators of tissue residentity. Histograms show staining of the described markers against γδ T cells (filled histogram) and appropriate isotype controls for each antibody (white histogram). [Figure 2A-1]This figure shows that cutaneous resident γδ T cells directly induced from human skin via the Clark protocol exhibit a so-called TH1-biased response to activation by conventional means for T cell activation, and similarly to a TH1-biased response to activation by NKG2D ligand alone. Figure 2A: Cutaneous resident γδ T cells show activation and strong expression of the NK cell-related receptor NKG2D (filled histogram; compared to isotypes represented by white histogram). Upon activation with plate-bound recombinant MICA (one of the known ligands for the NKG2D receptor), cutaneous γδ T cells respond independently of TCR ligation and without any other stimuli, as the response is resolved in the presence of a blocking NKG2D antibody. Cells were stimulated for 6 hours in the presence of brefeldin A and 100 units of IL-2 / mL, and then analyzed for degranulation by staining for CD107a. TNFα and INF-γ production was analyzed by permeabilization after surface staining and subsequent staining of intracellular cytokines. Phorbol 12-myristate 13-acetate (P) was used in combination with ionomycin (I) as a positive control for T cell activation. [Figure 2A-2] Continuation of Figure 2A. [Figure 2B]Figure 2B: Resident γδ T cells in human skin, directly induced via the Clark protocol, exhibit a so-called TH1-biased response to activation by conventional means for T cell activation, and similarly to a TH1-biased response to activation by NKG2D ligand alone. γδ T cells were harvested using the Clark protocol, stimulated with PMA and ionomycin for 6 hours in the presence of brefeldin A, and stained for intracellular cytokines. Freshly isolated γδ T cells from human skin produce TNFα and IFN-γ upon stimulation, but only small or undetectable amounts of cytokines associated with TH2 or TH-17 cells (e.g., IL-4, IL-17A, IL-13, IL-22), whereas conventional CD4+ αβ T cells exhibit a much more diverse cytokine production. [Figure 2C] This figure shows that cutaneous commensal γδ T cells directly induced from human skin via the Clark protocol exhibit a so-called TH1-biased response to activation by conventional means for T cell activation, and similarly to a TH1-biased response to activation by NKG2D ligand alone. Figure 2C: Among lymphocytes directly induced from human skin, various levels of NKG2D receptors are expressed by γδ T cells, conventional CD8a+ αβ T cells, and NK cells. Of these cells, NK cells respond to exposure to NKG2D ligand alone, but among T cells, only the γδ T cell population shows a cytokine response to stimulation with NKG2D ligand in the absence of any TCR stimulation (see the upper panel of the flow cytometry dot plot). The response can be blocked using a soluble blocking anti-NKG2D antibody, indicating that the response is exclusively mediated by the NKG2D receptor. [Figure 2D]This figure shows that cutaneous commensal γδ T cells directly induced from human skin via the Clark protocol exhibit a so-called TH1-biased response to activation by conventional means for T cell activation, and similarly to a TH1-biased response to activation by NKG2D ligand alone. Figure 2D: Of the cutaneous commensal γδ T cells, only Vδ1 γδ T cells and DN γδ T cells exhibit an innate ability to be activated by recombinant MICA alone (indicated by *). Vδ2-expressing T cells, which are found in small numbers in the skin, do not show such a response. [Figure 3A-1] This figure shows that only cutaneous resident γδ T cells respond to separation from the cutaneous stroma with strong activation and proliferation. Figure 3A: Cutaneous resident lymphocytes were isolated using the Clark protocol. After 3 weeks of organ-type culture, cutaneous lymphocytes were harvested and separated from any residual cutaneous cells, including fibroblasts, and transferred to tissue culture wells at a density of 1 million lymphocytes / mL, with 100 U / mL of IL-2 added. After another 3 weeks, resident γδ T cells were significantly amplified and enriched in the cutaneous lymphocyte culture. This marked proliferation was limited to cutaneous resident γδ T cells, as evidenced by the fact that the majority of Vδ1+ T cells proliferated an average of 127.18 times in 3 weeks, while conventional αβ T cells proliferated only an average of 5.21 times (which is lower than 20 times). [Figure 3A-2] Continuation of Figure 3A. [Figure 3B-1]This figure shows that only cutaneous commensal γδ T cells respond to separation from the skin stroma with strong activation and proliferation. Figure 3B: Cutaneous commensal Vδ1+ T cells respond to tissue removal by strongly upregulating the marker Ki-67 (indicating the cell cycle) over 14 days (isotype controls are shown by dashed white histograms; Ki-67 expression on day 0 is shown by a white histogram; Ki-67 expression on day 7 is shown by a light gray histogram; Ki-67 expression on day 14 is shown by a dark gray histogram). Furthermore, cutaneous commensal Vδ1 T cells, which are mostly negative for IL-2 receptor alpha (CD25) when in contact with the skin stroma, upregulate CD25 after separation from the tissue (isotype controls: dashed histogram, day 0 staining: light gray histogram, day 7 staining: dark gray histogram). [Figure 3B-2] Continuation of Figure 3B. [Figure 3C] This figure shows that only cutaneous commensal γδ T cells respond to separation from the cutaneous stroma with strong activation and proliferation. Figure 3C: The rapid cell cycle, indicated by the median fluorescence intensity (MFI) of Ki-67, is observed only in cutaneous commensal γδ T cells, represented by Vδ1+ T cells, and not in conventional αβ T cells or NK cells, in which the MFI actually decreases over 14 days. [Figure 3D-1] This figure shows that only cutaneous resident γδ T cells respond to isolation from the cutaneous stroma with strong activation and proliferation. Figure 3D: Cutaneous lymphocytes isolated from stromal cells show a significantly enriched population of resident γδ T cells after 3 weeks of culture. This γδ T cell population contains a majority of Vδ1-positive cells (77.49% ± 17.04) and pan-γδ TCR-positive DN T cells (21.46% ± 16.92). The initial small population of Vδ2 T cells seen in freshly recovered cutaneous lymphocytes using the Clark protocol decreases and is almost lost after 3 weeks of amplification of tissue γδ T cells (0.6% ± 1.204). [Figure 3D-2] Continuation of Figure 3D. [Figure 4A-1] This figure shows that resident cutaneous γδ T cells respond to tissue removal and are suppressed via a contact-dependent mechanism by cutaneous stromal cells, particularly fibroblasts. Figure 4A: Mixed cutaneous lymphocytes were cultured in organ-type cultures according to the Clark protocol and harvested after 3 weeks. The mixed lymphocytes were then seeded on a confluent layer of autologous cutaneous fibroblasts and in transwells to control for the presence of soluble inhibitors produced by fibroblasts. After 14 days, amplification factors, calculated via the absolute number of cells present, were measured for γδ T cells and conventional αβ T cells. Resident cutaneous γδ T cells showed significant proliferation when isolated from tissue and in the presence of fibroblasts, but only when there was no direct cell contact with autologous fibroblasts. Conventional αβ T cells did not show such a response under any of the conditions tested. [Figure 4A-2] Continuation of Figure 4A. [Figure 4B] This figure shows that resident cutaneous γδ T cells respond to tissue removal and are suppressed via a contact-dependent mechanism by cutaneous stromal cells, particularly fibroblasts. Figure 4B: Mixed lymphocytes obtained from organ-type cultures were seeded on a monolayer of autologous fibroblasts (light gray histogram) or in empty wells (dark gray histogram), and cultured for 7 days with the addition of IL-2. Resident cutaneous Vδ1+ T cells (left panel) and panγδ TCR+ DN T cells (right panel) remained quiescent in the direct presence of fibroblasts, but showed strong activation when isolated from cutaneous organ-type cultures and in the absence of fibroblasts, as indicated by the upregulated expression (MFI) of CD25, the TH-related transcription factor T-bet, and the cell cycle marker Ki-67 (dashed white histograms represent the corresponding isotype controls). [Figure 5A]This figure shows that amplified cutaneous γδ T cells exhibit signs of disinhibition and acquisition of potent cytotoxicity. Figure 5A: Resident cutaneous γδ T cells were amplified for 14 days after isolation from organ-type cell culture. The γδ T cells were then negatively sorted by flow cytometry by excluding all conventional T cells stained with a pan-αβ TCR monoclonal antibody. 150,000 sorted γδ T cells were subsequently seeded in double repeats into 96-well flat-bottom culture plates and cultured for 24 hours without the addition of cytokines or any activating ligands. The supernatant was collected and the produced cytokines were analyzed using a cytokine array based on Affymetrix LUMINEX®. [Figure 5B] This figure shows that amplified cutaneous γδ T cells exhibit signs of disinhibition and acquisition of potent cytotoxicity. Figure 5B: Negatively sorted γδ T cells were also seeded on cancer cell lines seeded 1 day prior at a concentration of 10,000 cells / well. Negatively sorted conventional cutaneous αβ T cells were used as a control. T cells were seeded in the presence of 100 U / mL of IL-2, and in and without blocking NKG2D antibody, at the effector:target ratios described. Resident cutaneous γδ T cells showed superior killing power against malignant cell lines compared to conventional αβ T cells, as indicated by caspase-cleaved epithelium-specific cytokeratin 18 (CK18) release (measured via ELISA). Cytotoxicity was at least partially mediated through the NKG2D receptor, as indicated by its reduction in cultures containing antibodies blocking the NKG2D receptor. [Figure 6A] This figure shows the analysis of tissue-resident γδ T cells in the human gut. Figure 6A: By applying the Clark protocol, isolation of gut-resident lymphocytes became possible. Mixed-type gut lymphocytes typically contain mainly Vδ1 cells, but also include a large population of tissue-resident γδ T cells that also contain Vδ2 cells and double-negative γδ T cells. [Figure 6B]This figure shows the analysis of tissue-resident γδ T cells in the human gut. Figure 6B: γδ T cells isolated from intestinal organ-type cultures show a similar response to skin-derived γδ T cells, as they upregulate Ki-67 over time once separated from the intestinal stroma. [Figure 6C] This figure shows the analysis of tissue-resident γδ T cells in the human gut. Figure 6C: Gut-derived γδ T cells respond to innate-like stimuli such as recombinant MICA by producing IFN-γ and by degranulation, as measured by CD107a upregulation. [Figure 6D] This figure shows the analysis of tissue-resident γδ T cells in the human gut. Figure 6D: γδ T cells isolated from intestinal organ-type cultures show a similar response to skin-derived γδ T cells and amplify over time in the cell culture, as can be seen from the overall enrichment in lymphocyte cultures without contact with the intestinal stroma. [Figure 7A] This figure shows the tissue phenotype of amplified skin-derived γδ T cells. Figure 7A: Skin-derived γδ T cells are positively stained for cutaneous lymphocyte antigen (CLA), skin homing chemokine receptors CCR4 and CCR8. [Figure 7B] This figure shows the tissue phenotype of amplified skin-derived γδ T cells. Figure 7B: Expression levels differ for amplified γδ T cells induced from skin or blood, respectively. [Figure 8]The figure shows that deinhibition of skin-derived γδ T cells without any TCR stimulation results in spontaneous TH-1 cytokine production, and, interestingly, production of the atopic cytokine IL-13, in contrast to fresh TCR-activated γδ T cells. Consistent with freshly induced γδ T cells, deinhibited and amplified γδ T cells produce negligible amounts of TH-2-related cytokines (e.g., IL-4 and IL-5). Skin-derived γδ T cells were amplified for 14 days and negatively sorted by eliminating conventional αβ T cells. 150,000 mixed-type γδ T cells were cultured in double repeats in 96-well flat-bottom plates at a density of 1 million cells / mL from four donors, without any stimulation or cytokine addition. The supernatant was collected after 24 hours and analyzed using a cytokine array based on Affymetrix LUMINEX®. [Figure 9A] This figure shows that amplified and negatively sorted skin-derived γδ T cells exhibit strong cytotoxicity against various human tumor cell lines (Figure 9A: HCT1954, Figure 9B: HCT116, Figure 9C: MD231) co-cultured with them, as measured by the release of caspase-cleaved cytokeratin 18 from target cells using ELISA. [Figure 9B] This figure shows that amplified and negatively sorted skin-derived γδ T cells exhibit strong cytotoxicity against various human tumor cell lines (Figure 9A: HCT1954, Figure 9B: HCT116, Figure 9C: MD231) co-cultured with them, as measured by the release of caspase-cleaved cytokeratin 18 from target cells using ELISA. [Figure 9C] This figure shows that amplified and negatively sorted skin-derived γδ T cells exhibit strong cytotoxicity against various human tumor cell lines (Figure 9A: HCT1954, Figure 9B: HCT116, Figure 9C: MD231) co-cultured with them, as measured by the release of caspase-cleaved cytokeratin 18 from target cells using ELISA. [Figure 10A]Figure 10A shows that fresh, unamplified skin-derived Vδ1 T cells exhibit markers of previous T cell activation. Skin-derived Vδ1 T cells highly express CD69, ICOS, and TIM3, and low express CD28. Furthermore, they show high expression of the activation marker NKG2D. This phenotype is maintained by skin-derived Vδ1 T cells during amplification in vitro. In contrast, Vδ1 T cells induced from human blood do not exhibit these signs of activation, do not express CD69 or TIM3, and express only low levels of ICOS. Compared to skin-derived Vδ1 T cells, NKG2D expression is much lower in blood-derived Vδ1 T cells, while blood-derived Vδ1 T cells express the co-stimulatory molecule CD28. [Figure 10B] Figure 10B shows that fresh, unamplified skin-derived Vδ1 T cells exhibit markers of prior T cell activation. Only skin-derived Vδ1 T cells are responsive to NKG2D ligands such as recombinant MICA in the absence of any other stimuli, such as ligands for the T cell receptor. Blood-derived Vδ1 or Vδ2 T cells do not exhibit such responsiveness to innate stimuli. Cells were seeded in 96-well plates with recombinant MICA or anti-CD3 antibody or both, as described. Cells were cultured for 6 hours, with the last 4 hours in IL-2 100 U / mL and BFA, followed by surface antigen staining, permeabilization, and intracellular staining for IFN-γ. [Figure 11] This figure shows that skin-derived Vδ1 T cells express only small levels of CD16 but also significant expression of the high-affinity IgG receptor CD64. Therefore, in addition to direct cytotoxic activity, tissue-derived Vδ1 T cells may also be used to enhance the efficacy of monoclonal antibody therapies such as CD20 therapy or Her2 therapy, as they will recognize tumor cells that have been antibody-guided and opsonized to the malignant and metastatic side and induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC). The results shown are from one representative donor (out of four). [Figure 12]This figure shows the amplification of Vδ1 T cells in IL-2 (left panel), IL-15 (center panel), and IL-2 + IL-5 (right panel). Freshly isolated skin-derived lymphocytes were cultured in RPMI medium containing 10% FCS and 1% Pen / Strep in 96-well flat-bottom plates and supplemented with either IL-2, IL-15, or IL-2 + IL-15 for 7 days each. Both IL-2 and IL-15, as well as combinations of the two cytokines, induced Vδ1 T cell proliferation, as indicated by the shift in Ki-67 staining compared to isotype (true negative) staining in the absence of stromal cells. Ki-67 stains only cells remaining in the G0 phase of the cell cycle and is generally associated with proliferation. [Figure 13] Flow cytometry results showing the expression of CD9, CCR3, and CD39 on the surface of amplified Vδ1 γδ T cells on day 21. The amplified skin-derived Vδ1 T cells maintained high levels of the cell surface markers CCR3, CD39, and CD9, as shown by (dark histogram) compared to the corresponding isotype staining (true negative, white histogram). [Figure 14] This figure shows the mRNA expression of CCR3 and CD9 in skin-derived Vδ1 T cells (dark bars) and blood-derived Vδ1 T cells (light bars). Skin-derived Vδ1 T cells were amplified as disclosed herein, and blood-derived Vδ1 T cells were amplified using an antibody (20 μg / mL) against the plate-bound Vδ T cell receptor. After amplification, Vδ1 T cells were isolated using fluorescence-activated cell sorting (FACS), and RNA was isolated from three donors for both groups (blood = gray, skin = black). All mRNA was sequenced, and the expression levels of the described mRNA were normalized and log 2 converted. All expression levels are shown as direct comparisons and as ratios to GAPDH (a common housekeeping gene that is highly expressed in most human cells). [Figure 15]This figure shows IL-13 mRNA expression in skin-derived Vδ1 T cells (dark bars) and blood-derived Vδ1 T cells (light bars). Skin-derived Vδ1 T cells were amplified as disclosed herein, and blood-derived Vδ1 T cells were amplified using a plate-bound high dose antibody against the Vδ T cell receptor (20 μg / mL). After amplification, Vδ1 T cells were isolated using FACS, and RNA was isolated from three donors for both groups (blood = gray, skin = black). Total mRNA was sequenced, and mRNA expression levels for IL-13 were normalized and log 2 converted. Expression levels are shown as direct comparisons and as a ratio to GAPDH. [Figure 16A] This figure shows cytokine production in skin-derived Vδ1 T cells after TCR stimulation with PMA / ionomycin (Figure 16A) or anti-CD3 (Figure 16B). Following isolation and amplification, skin-derived Vδ1 T cells were purified using fluorescence-activated cell sorting (FACS). 150,000 Vδ1 T cells were seeded in double replicates in 96-well flat-bottom plates from three donors and stimulated for 24 hours with either plate-bound CD3 (5 μg / mL) or PMA / ionomycin. The supernatant was analyzed for the absolute amounts of the described cytokines using the LUMINEX® platform. [Figure 16B] This figure shows cytokine production in skin-derived Vδ1 T cells after TCR stimulation with PMA / ionomycin (Figure 16A) or anti-CD3 (Figure 16B). Following isolation and amplification, skin-derived Vδ1 T cells were purified using fluorescence-activated cell sorting (FACS). 150,000 Vδ1 T cells were seeded in double replicates in 96-well flat-bottom plates from three donors and stimulated for 24 hours with either plate-bound CD3 (5 μg / mL) or PMA / ionomycin. The supernatant was analyzed for the absolute amounts of the described cytokines using the LUMINEX® platform. [Modes for carrying out the invention]

[0059] Gamma-delta T cells (γδ T cells) represent a small subset of T cells that express a specific, characteristic T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain.

[0060] There are two main subtypes of human γδ T cells: one is dominant in peripheral blood, and the other is dominant in non-hematopoietic tissues.

[0061] The non-hematopoietic localization of the second human γδ T cell subtype has made harvesting difficult, and there have been no established means for culturing these cells. To address this need, the present invention relates to a method for amplifying non-hematopoietic tissue-resident γδ T cells (or, as referred herein, non-hematopoietic tissue-specific γδ T cells). These γδ T cells are normally commensal in non-hematopoietic tissues. Non-hematopoietic tissue-resident γδ T cells for use as described herein can be derived from or obtained from non-hematopoietic tissues. Non-hematopoietic tissues may contain non-hematopoietic cells and γδ T cells.

[0062] The methods described herein provide means for amplifying γδ T cells derived from any human or non-human animal non-hematopoietic tissue that can be taken from a patient, including skin, gastrointestinal tract (e.g., colon), mammary gland tissue, lungs, prostate, liver, spleen, and pancreas. γδ T cells may also be commensal in human cancer tissue, such as tumors of the breast and prostate. In some embodiments, the γδ T cells may be derived from human cancer tissue. In other embodiments, the γδ T cells may be derived from non-hematopoietic tissue other than human cancer tissue.

[0063] The dominant γδ T cells in the blood are mainly Vδ2 T cells, while the dominant γδ T cells in non-hematopoietic tissues are mainly Vδ1 T cells, thus Vδ1 T cells constitute approximately 70-80% of the non-hematopoietic tissue resident γδ T cell population. However, some Vδ2 T cells are also found in non-hematopoietic tissues (e.g., the intestines), and they may constitute approximately 10-20% of γδ T cells (Figure 6). Some γδ T cells resident in non-hematopoietic tissues do not express either Vδ1 TCR or Vδ2 TCR, and we have named them double-negative (DN) γδ T cells. These DN γδ T cells appear to be mostly Vδ3-expressing T cells, with a small number expressing Vδ5 T cells.

[0064] Therefore, the γδ T cells that are normally commensal in non-hematopoietic tissues and amplified by the method of the present invention are preferably non-Vδ2 T cells, such as Vδ1 T cells, including a relatively small amount of DN γδ T cells.

[0065] As used herein, “double-negative” γδ T cells (DN γδ T cells) mean γδ T cells that express the γδ receptor (i.e., stain positively for the pan-TCR) but are negative for the Vδ1 and Vδ2 receptors. DN γδ T cells include those expressing Vδ receptors other than Vδ1 and Vδ2 (e.g., Vδ3, Vδ4, Vδ5, or Vδ8). Cells are positive for a marker (e.g., Vδ1) if they express a higher marker than negative control cells as determined by standard FACS gating. + ) can be characterized as follows.

[0066] Methods described herein may include the step of culturing lymphocytes obtained from human or non-human animal non-hematopoietic tissue in vitro.

[0067] Lymphocytes can be obtained from any suitable human or non-human animal non-hematopoietic tissue. Non-hematopoietic tissue is tissue other than blood, bone marrow, or thymic tissue. In some embodiments, γδ T cells are not obtained from samples of certain types of bodily fluids such as blood or synovial fluid. Examples of such suitable human or non-human animal non-hematopoietic tissues include skin or parts thereof (e.g., dermis, epidermis), the gastrointestinal tract (e.g., gastrointestinal epithelium, colon, small intestine, stomach, appendix, cecum, or rectum), mammary gland tissue, lungs (preferably, in this case, the tissue is not obtained by bronchoalveolar lavage), prostate, liver, spleen, and pancreas. γδ T cells may also be commensal in human cancer tissue (e.g., breast cancer and prostate cancer). In some embodiments, γδ T cells are not obtained from human cancer tissue. Non-hematopoietic tissue samples can be obtained by standard techniques, for example, by explantation (e.g., biopsy).

[0068] Lymphocytes can be obtained by any preferred method that allows for the isolation of lymphocytes from human or non-human animal non-hematopoietic tissues. One such method is described in the literature by Clark et al. (29), which describes a three-dimensional skin explant protocol for the isolation of lymphocytes from human skin. The explant can be attached to a synthetic scaffold to facilitate the release of lymphocytes from the explant onto the scaffold. A synthetic scaffold means a non-natural three-dimensional structure suitable for supporting cell growth. Synthetic scaffolds can be constructed from materials such as polymers (e.g., natural or synthetic polymers, e.g., polyvinylpyrrolidone, polymethyl methacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (tantalum, titanium, platinum and metals in the same element group as platinum, niobium, hafnium, tungsten, and combinations of their alloys). To enhance cell adhesion, migration, survival, or proliferation, biological factors (e.g., collagen (e.g., type I or type II collagen), fibronectin, laminin, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, antibodies and their fragments, cytokines (e.g., interleukin-2 (IL-2) or interleukin-15 (IL-15)), and combinations thereof) can be coated onto the scaffold surface or encapsulated within the scaffold material according to methods known in the art. This method and others can be used to isolate lymphocytes from several other non-hematopoietic tissue types (e.g., intestine, prostate, and breast). Other examples of preferred methods include enzymatic digestion of tissue and the “crawl-out” method described by Carrasco et al. (30) (finely chopping the tissue and adding IL-2 so that lymphocytes “crawl out”).

[0069] As mentioned above, any suitable non-hematopoietic tissue can be used, such as skin, gastrointestinal tract (e.g., colon), mammary gland tissue, lungs, prostate, liver, spleen, and pancreas.

[0070] Non-hematopoietic tissue-resident γδ T cells are preferably obtained from human tissue. However, they may also be obtained from non-hematopoietic tissues of any preferred non-human animal, such as mice, rats, dogs, horses, and pigs.

[0071] The crucial steps are the deliberate isolation of non-hematopoietic tissue-resident T cells (e.g., within a mixed lymphocyte population, which may include αβ T cells, γδ2 T cells, and non-γδ2 T cells, for example) from non-hematopoietic cells (e.g., stromal cells, particularly fibroblasts) in the tissue from which the T cells were obtained (e.g., after several days or weeks of culture), and the subsequent culture step of the cells as lymphocytes in cytokines as described below. This allows for the selective and significant amplification of tissue-derived γδ1 T cells and DN γδ T cells over the following days and weeks.

[0072] As used herein, “separation,” “separated,” or “to separate” means an action that disrupts or interferes with physical contact between different cell populations. Separation can be performed, for example, by strongly pipetting a mixed population of cells to disrupt membrane-to-membrane binding, or, as described by Carrasco et al. (30), by inducing “crawl-out” of a population of cells from a tissue matrix, for example, by culturing with chemokines or cytokines. Separation can be maintained during culture using a transwell culture system or by similar culture methods that disrupt physical contact between different cell populations.

[0073] As used herein, “substantially pure” means a purity of more than 90% by number, mass, or volume. “Substantially free” means having less than 5% of a given component by number, mass, or volume. Lymphocytes obtained from human or non-human animal non-hematopoietic tissue can be cultured for at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 2 weeks, at least 3 weeks, or at least 4 weeks.

[0074] The method comprises the step of culturing lymphocytes obtained from human or non-human animal non-hematopoietic tissue in the presence of IL-2. The concentration of IL-2 is preferably at least 10 international units / mL (IU / mL, or U / mL), at least 20 U / mL, at least 30 U / mL, at least 40 U / mL, at least 50 U / mL, at least 60 U / mL, at least 70 U / mL, at least 80 U / mL, at least 90 U / mL, or at least 100 U / mL.

[0075] The use of IL-2 to promote the amplification of skin-derived γδ T cells is not obvious because these cells express the high-affinity IL-2 receptor known as CD25 at very low levels (Figure 1D). However, this receptor can be upregulated on a distinct subset of γδ T cells by dissociating it from other cell types such as stromal cells or epithelial cells (e.g., fibroblasts) (see Figures 3B and 4B), thereby making the cells highly sensitive to IL-2.

[0076] As used herein, “IL-2” means wild-type IL-2 (e.g., innate or recombinant) or a substance that acts as an agonist to one or more IL-2 receptor (IL-2R) subunits (e.g., IL-2 mutant proteins (muteins), long-acting IL-2 analogs, their subunits, or their receptor complexes). Such substances can assist in the proliferation of the IL-2-dependent cell line CTLL-2 (33; American Type Culture Collection (ATCC®) TIB 214). Mature human IL-2 exists as a 133-amino acid sequence (lacking an additional 20 N-terminal amino acid signal peptide), as described in Fujita et al. (34). IL-2 mutant proteins are polypeptides that have undergone specific substitutions to the interleukin-2 protein, such as those described in U.S. Patent Application Publication No. 2014 / 0046026, but retain their ability to bind to IL-2Rβ. IL-2 mutant proteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more other residues in the intrinsic IL-2 polypeptide chain. According to this disclosure, any such insertion, deletion, substitution, and modification results in an IL-2 mutant protein that retains IL-2Rβ binding activity. Exemplary mutant proteins may include substitutions of one, two, three, four, five, six, seven, eight, nine, ten, or more amino acids.

[0077] Nucleic acids encoding human IL-2 can be obtained by conventional procedures such as polymerase chain reaction (PCR). The amino acid sequence of human IL-2 (Gene ID 3558) can be found in Genbank under registration locator NP_000577.2 GI: 28178861. The amino acid sequence of murine (mouse (Mus musculus)) IL-2 (Gene ID 16183) can be found in Genbank under registration locator NP_032392.1 GI: 7110653.

[0078] Since the addition of IL-15 in combination with IL-2 enhances the amplification of proliferative non-hematopoietic tissue resident γδ T cells compared to IL-2 alone, lymphocytes are preferably cultured in the presence of both IL-2 and IL-15. The concentration of IL-15 is preferably at least 10 ng / mL.

[0079] IL-15, like IL-2, is a known T cell growth factor that can assist in the proliferation of CTLL-2, an IL-2-dependent cell line. IL-15 was first reported by Grabstein et al. (35) as a 114-amino acid mature protein. The term "IL-15," as used herein, refers to innate or recombinant IL-15 and its mutant proteins, analogs, subunits, or complexes thereof (e.g., receptor complexes, e.g., the sushi peptide as described in International Publication No. 2007 / 046006), each of which will stimulate the proliferation of CTLL-2 cells. In CTLL-2 proliferation assays, the supernatant of cells transfected with in-frame fusions of recombinantly expressed precursors and mature IL-15 can induce CTLL-2 cell proliferation.

[0080] When used herein, the term IL-15 also refers to IL-15 derived from various mammalian species, including, for example, humans, monkeys, cattle, pigs, horses, and mice. When referred herein, an IL-15 “mutein” or “variant” is a polypeptide substantially homologous to the sequence of the innate mammalian IL-15, but having a different amino acid sequence due to amino acid deletions, insertions, or substitutions. Variants may contain conserved substitutions, meaning that a given amino acid residue is replaced by a residue with similar physicochemical properties. Examples of conserved substitutions include substitutions of aliphatic residues to other residues (such as substituting Ile, Val, Leu, or Ala with each other), or substitutions of polar residues to other residues (such as between Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions, such as substitutions of entire regions with similar hydrophobic properties, are well known. Naturally occurring IL-15 variants are also included in the present invention. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the IL-15 protein, while retaining IL-15 binding properties. Alternative splicing of mRNA can produce cleaved but biologically active IL-15 proteins. Variations contributing to proteolysis include, for example, differences in the N-terminus or C-terminus of expression in different types of host cells, resulting from the proteolytic removal of one or more terminal amino acids (typically 1 to 10 amino acids) from the IL-15 protein.

[0081] Human IL-15 can be obtained by following the procedure described by Grabstein et al. (35) or by conventional procedures such as polymerase chain reaction (PCR). The human IL-15 cDNA was deposited with ATCC® on February 19, 1993, and was assigned registration number 69245.

[0082] The amino acid sequence of human IL-15 (Gene ID 3600) can be found in Genbank under registration locator NP000576.1 GI: 10835153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2). The amino acid sequence of murine (mouse (Mus musculus)) IL-15 (Gene ID 16168) can be found in Genbank under registration locator NP_001241676.1 GI: 363000984.

[0083] Lymphocytes can be cultured in the absence of IL-6, IL-23, and IL-1B, or in the presence of low concentrations of these cytokines (e.g., less than 20 ng / mL), because the addition of this combination of cytokines appears to reduce the proliferation of non-hematopoietic tissue-resident γδ T cells. This is surprising, as it would have been expected that these cytokines would promote proliferation.

[0084] Lymphocytes obtained from non-hematopoietic tissue can be cultured in the absence of substances that activate T cell signaling (e.g., T cell receptor (TCR) pathway agonists). For example, lymphocytes obtained from non-hematopoietic tissue can be cultured in a medium that does not support or induce the proliferation or activation of αβ T cells and blood-resident γδ T cells. A suitable medium may be free from or substantially free from TCR agonists or other substances that activate T cell signaling. In contrast, the culture of hematopoietic tissue-derived γδ T cells requires the presence of substances that activate T cell signaling, such as zoledronate (41, 42), or anti-CD3 antibodies, such as OKT3 (43).

[0085] Substances that activate T cell signaling refer to compounds that induce the proliferation or activation of T cells, such as αβ T cells and / or resident γδ T cells, through TCR signaling or co-stimulation. T cell signaling regulators function through the sequential activation of Src-related protein tyrosine kinases (PTKs), LcKs and Fyn, and 70kDA zeta chain (TCR)-related protein kinases (ZAP70). These PTKs cause phosphorylation of polypeptides, including linker activators (LATs), to T cells, which leads to downstream stimulation via extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs), and activated T cell nuclear factor (NFATs). For example, co-stimulation via CD28 and CD45 can enhance phosphorylation and strengthen the TCR signaling pathway. In other words, any substance that targets a part of the TCR or co-stimulatory pathway can activate T cell signaling. Substances that activate T cell signaling can be soluble or membrane-bound and can be presented on cells, such as artificial antigen-presenting cells (aAPCs). Suitable aAPCs for activating T cell signaling are known in the art (44).

[0086] In some embodiments, lymphocytes can be cultured in the absence of exogenously added T cell receptor pathway agonists, such as CD3 and / or CD28 activators (e.g., anti-CD3 and / or anti-CD28 monoclonal antibodies); phytohemagglutinin (PHA); concanavalin A, synthetic phosphoantigens (BrHPP (bromohydrin pyrophosphate), 2M3B1PP (2-methyl-3-butenyl-1-pyrophosphate), HMBPP ((E)-4-hydroxy-3-methyl-buta-2-enylpyrophosphate), or IPP (isopentenyl pyrophosphate)); N-bisphosphonates (e.g., zoledronate); recombinant CD70; anti-CD2 monoclonal antibodies; anti-CD27 monoclonal antibodies; anti-pan-TCRγδ antibodies; anti-CD277 monoclonal antibodies; or artificial antigen-presenting cells (aAPCs). Substances that activate T cell signaling include cell surface-bound molecules such as antigen-presenting cells (APCs) or MHC or HLA complexes bound to artificial APCs. A suitable method for activating T cells by exogenously adding TCR pathway agonists is well known in the art and is outlined in Figure 1 of Deniger et al.'s paper (44).

[0087] For example, lymphocytes can be cultured in a medium that does not contain or substantially contains exogenously added T cell receptor pathway agonists. The addition of such T cell receptor signaling activators is not required for amplification of non-hematopoietic tissue-resident γδ T cells using the method of the present invention. In contrast, amplification of hematopoietic tissue-derived γδ T cells requires the presence of both IL-2 and a T cell receptor signaling activator (such as zoledronate) (41, 42).

[0088] In some embodiments, lymphocytes can be cultured in a conditioned medium derived from stromal cell cultures to provide nutrients for γδ T cell growth.

[0089] In some embodiments, γδ T cells can be cultured in a γδ amplification medium containing IL-2 and / or IL-15. A preferred γδ amplification medium lacks T cell activation activity (e.g., αβ T cell or blood γδ T cell activation activity) and may not contain or substantially contain, for example, TCR agonists or costimulators. In some embodiments, the γδ amplification medium may contain one or more additional growth factors, such as cytokines, in addition to IL-2 and / or IL-15. Preferred growth factors do not exhibit T cell activation activity. In other embodiments, the γδ amplification medium may lack growth factors other than IL-2 and / or IL-15; for example, the γδ amplification medium may consist of a basal medium supplemented with IL-2 and / or IL-15.

[0090] Numerous basal culture media suitable for use in the amplification of γδ T cells are available, particularly complete media such as AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies). Other media elements such as serum, serum proteins, and selective agents such as antibiotics can be added to the media. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES (pH 7.2), 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1 × MEM non-essential amino acids; Life Technologies), and 10 μL / L β-mercaptoethanol. IL-2 and / or IL-15 can be added to the basal medium at standard concentrations readily determined by those skilled in the art by conventional experimental techniques.

[0091] For simplicity, cells are cultured at 37°C in a suitable culture medium in a humidified atmosphere containing 5% CO2.

[0092] γδ T cells can be cultured in any suitable system, including a fermenter, airlift incubator, roller bottle, culture bag or culture dish, and other bioreactors, particularly hollow fiber bioreactors, as described herein. The use of such systems is well known in the art.

[0093] Methods and techniques for culturing lymphocytes are well known in the art (36-39).

[0094] During culture, lymphocytes do not come into direct contact with stromal or epithelial cells. This is because direct contact between lymphocytes and stromal or epithelial cells appears to inhibit the amplification of tissue-resident γδ T cells.

[0095] Stromal cells are non-hematopoietic connective tissue cells of any organ that assist the function of parenchymal cells in that organ. Examples of stromal cells include fibroblasts, pericytes, mesenchymal cells, keratinizing cells, endothelial cells, and non-hematopoietic tumor cells. Preferably, lymphocytes do not come into direct contact with fibroblasts during culture.

[0096] Epithelial cells are non-hematopoietic cells that line the lumens and surfaces of blood vessels and organs throughout the body. They are normally flattened, columnar, or cubic in shape and can be arranged as a single layer of cells or as layers of two or more cells.

[0097] Fibroblasts and / or other stromal or epithelial cells are preferably present in lymphocyte culture because factors secreted by these cells can promote the amplification of non-hematopoietic tissue resident γδ T cells, but they are not in direct contact with lymphocytes because direct contact inhibits the amplification of non-hematopoietic tissue resident γδ T cells. For example, lymphocytes can be cultured in a transwell, which allows for the physical separation of lymphocytes and fibroblasts. Examples of fibroblast cell lines that can be used include human foreskin fibroblasts (e.g., BJ(ATCC(registered trademark) CRL-2522)). TM)), normal skin fibroblasts (e.g., CCD-1059Sk (ATCC® CRL-2072 TM )) and lung fibroblasts (e.g., HEL 299 (ATCC(registered trademark) CRL-137) TM )) are cited as examples.

[0098] The Clark protocol allows for the recovery and separation of non-hematopoietic tissue-resident lymphocytes from stromal cells such as cutaneous fibroblasts, for example, by thorough pipetting. The recovered lymphocytes can be further washed through a 40 μm nylon mesh to retain fibroblast aggregates that may have become loosely bound during the process. Lymphocytes can also be isolated using fluorescence or magnetic cell sorting, for example, with a CD45 antibody. To minimize T cell activation, lymphocytes can also be sorted based on their forward and lateral scattering properties. Lymphocytes can then be grown in isolation from or in the presence of stromal cells (e.g., fibroblasts) without direct contact. For example, lymphocytes can be grown in a Transwell basket containing a confluent monolayer of fibroblasts in a lower cell culture well to allow for the exchange of soluble growth factors produced by fibroblasts without allowing direct contact. Alternatively, fibroblasts can be cultured in a Transwell basket while lymphocytes are grown in the lower cell culture well. To provide nutrients for lymphocyte amplification, a culture medium conditioned with non-hematopoietic cells (e.g., fibroblasts) can also be used.

[0099] The conditioned medium contains soluble factors secreted by non-hematopoietic cells (e.g., stromal cells such as fibroblasts). The conditioned medium may or may not contain the cells that secreted these factors. For example, γδ T cells can be cultured in the presence of cells that secrete conditioned factors during γδ T cell culture. Alternatively, non-hematopoietic cells can be removed from the medium prior to γδ T cell culture, leaving the factors they secreted in the medium. Examples of conditioned mediums include media to which previously prepared non-hematopoietic cell factors have been added (e.g., as a concentrate or lyophilized powder).

[0100] Stromal cells or epithelial cells are preferably present in the lymphocyte culture (but not in direct contact with the lymphocytes), but they can be removed so that the lymphocytes are cultured in the absence of stromal cells or epithelial cells (for example, in the absence of fibroblasts).

[0101] In some embodiments, following amplification of non-hematopoietic tissue resident γδ T cells in the absence of T cell signaling-activating substances such as exogenously added T cell receptor pathway agonists, as described above, the amplified γδ T cells may be further cultured in the presence of one or more T cell signaling-activating substances (such as exogenously added T cell receptor pathway agonists) and / or one or more growth factors (such as cytokines). If necessary, after amplification and optional further culture as described herein, non-hematopoietic tissue resident γδ T cells may be isolated or further purified, stored, mixed with other reagents (such as pharmaceutically acceptable excipients), and / or used.

[0102] Non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention can be distinguished from other blood-derived γδ T cells in that they respond to NKG2D ligand (MICA), which is strongly associated with malignancies, in the absence of any T cell receptor stimulating ligands, for example, by increased production of TNFα, IFNγ, and CD107a (Figures 2A-2D, 10A, and 10B). These cells also perform a cytotoxic T cell response without activation of T cell receptors by exogenous agents or ligands, and are therefore cytotoxic in the absence of stimulation (Figures 3 and 5). This means that non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention are unique in their ability to respond and proliferate in the absence of exogenous substances that activate T cell receptor signaling, compared to other γδ T cells, αβ T cells, or NK cells (Figure 3). Non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention were also positively stained for CD69 and PD-1, lacked CD28 expression, and showed only low levels of CD25 (see Figure 1D). This combination of markers is not expressed by blood-derived γδ T cells. Furthermore, these cells showed relatively high expression of tissue-homing receptors such as CCR4 and CCR8 compared to amplified blood-derived Vd2 γδ T cells (Figure 7B). Non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention may be cultured in the presence of IL-2 and / or IL-15 without the use of TCR agonists or other growth factors. For example, non-hematopoietic tissue-resident γδ T cells can be grown in a medium consisting of RPMI 1640 medium supplemented with IL-2.

[0103] In other words, non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention may have one or more of the following characteristics: (i) Phenotype CD69 high ICOS high , TIM3 high and CD28 low / absent To show, (ii) Upregulate one or more of the following: CCR3, CD39, CD11b, and CD9. (iii) In the absence of a TCR agonist, it produces IFN-γ in response to the NKG2D ligand, (iv) To produce IL-13 in the absence of a TCR agonist, (v) In response to TCR activation, produce one or more of IFN-γ, TNF-α, and GM-CSF, (vi) Not producing any IL-17 at all or substantially not producing it in response to TCR activation, (vii) Growing in a culture medium containing IL-2 without additional growth factors, (viii) Exhibiting a cytotoxic T cell response in the absence of a TCR agonist, and / or (ix) It exhibits selective cytotoxicity against tumor cells compared to normal cells.

[0104] Preferably, non-hematopoietic tissue-resident γδ T cells produced by the method of the present invention produce IL-13 in the absence of a TCR agonist and / or produce IFN-γ in response to an NKG2D ligand in the absence of a TCR agonist.

[0105] The γδ T cells obtained by the method of the present invention can be used in a screening method for checkpoint regulators of non-hematopoietic tissue-resident γδ T cells. Identifying such checkpoint regulators may be useful for developing cancer immunotherapies, since checkpoint regulators are potential targets for cancer therapy.

[0106] To determine whether a test compound is a checkpoint regulator, non-hematopoietic tissue-resident γδ T cells can be cultured in vitro in direct contact with stromal or epithelial cells (e.g., fibroblasts) in the presence or absence of the test compound. The proliferation rate or activation level of the non-hematopoietic tissue-resident γδ T cells is determined in the presence and absence of the test compound. If the proliferation rate or activation level is higher in the presence of the test compound than in the absence of the test compound, the test compound may be a candidate checkpoint regulator. This is because the test compound can overcome contact inhibition by stromal or epithelial cells (e.g., fibroblasts).

[0107] The test compounds are selected based on their ability to modulate checkpoints with respect to tissue-resident T cells.

[0108] A clear increase in the proliferation of non-hematopoietic tissue-resident γδ T cells may also be due to the inhibition of cell death, which can be measured by an increase in T cell counts or their markers, and by a decrease in the expression of programmed cell death markers. Increased activation can be assessed by measuring the release of cytokines (such as IFN-γ) secreted by activated tissue-resident γδ T cells.

[0109] Alternatively, non-hematopoietic tissue-resident γδ T cells can be cultured in vitro in direct contact with stromal cells or epithelial cells (e.g., fibroblasts), in which case the expression of the test gene is altered in the γδ T cells and / or stromal cells, epithelial cells (e.g., fibroblasts), or both cell types. The expression of the test gene in γδ T cells and / or stromal cells or epithelial cells (e.g., fibroblasts) can be altered, for example, by RNA targeting substances such as small interfering RNA (siRNA) or small hairpin RNA (shRNA), or by gene editing (e.g., using the CRISPR / Cas system). The proliferation rate or activation level of non-hematopoietic tissue-resident γδ T cells is determined in the presence and absence of altered expression of the test gene in stromal cells, epithelial cells (e.g., fibroblasts) and / or γδ T cells. If the proliferation rate or activation level is higher in the presence of changes in the expression of the test gene in stromal or epithelial cells (e.g., fibroblasts) and / or γδ T cells than in the absence of changes in the test gene in stromal or epithelial cells (e.g., fibroblasts) and / or γδ T cells, the test gene may be a candidate checkpoint gene.

[0110] Alternatively, if the rate of cell death in stromal or epithelial cells (e.g., fibroblasts) is higher in the presence of the test compound than in the absence of the test compound, or higher in the presence of changes in the expression of the test gene in stromal or epithelial cells (e.g., fibroblasts) and / or γδ T cells than in the absence of changes in the test gene in stromal or epithelial cells (e.g., fibroblasts) and / or γδ T cells, then the test compound may be a checkpoint regulator, or the test gene may be a candidate checkpoint gene. The rate of cell death can be measured, for example, by quantifying the molecules released by the dying cells. A higher rate of cell death in the presence of the test compound or in the presence of changes in the expression of the test gene in stromal or epithelial cells (e.g., fibroblasts) and / or γδ T cells indicates that a checkpoint that was suppressing cell killing has been released, and therefore it can be concluded that the test compound may be a checkpoint regulator, or the test gene may be a candidate checkpoint gene.

[0111] Examples of fibroblast cell lines that can be used in each embodiment include human foreskin fibroblasts (e.g., BJ(ATCC(registered trademark) CRL-2522)). TM )), normal skin fibroblasts (e.g., CCD-1059Sk (ATCC® CRL-2072 TM )) and lung fibroblasts (e.g., HEL 299 (ATCC(registered trademark) CRL-137) TM )) are cited as examples.

[0112] To identify a test compound as a candidate checkpoint regulator, or a test gene as a candidate checkpoint gene, the proliferation and / or activation rate of γδ T cells in the presence of the test compound or the alteration of the test gene can be at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, or at least 5 times higher than in the absence of the test compound or the alteration of the test gene. The cell cycle is determined by the absolute number of cells on day 0 and day 7, the levels of Ki-67 and CD25 expression (these are cell cycle markers), and the cell culture dye (CFSE or CELLTRACE). TM It can be measured by a number of means, such as using violet. Cell activation can be measured by the production of effector proteins such as IFN-γ. A clear increase in the proliferation of non-hematopoietic tissue resident γδ T cells may also be due to the inhibition of cell death and can be measured by an increase in T cell count or its markers, and by a decrease in the expression of programmed cell death markers.

[0113] The γδ T cells obtained by the method of the present invention can be used as a pharmaceutical, for example, for adoptive T cell therapy. This includes transferring the γδ T cells obtained by the method of the present invention into a patient. The therapy may be autologous, i.e., the γδ T cells can be transferred back into the same patient from which they were obtained, or the therapy may be homogeneous, i.e., γδ T cells derived from one person can be transferred into a different patient. The treatment method may include the following steps: A step of providing a sample of non-hematopoietic tissue obtained from a donor individual. To produce an amplified population, the steps of culturing γδ T cells derived from the sample as described above; and A step of administering the amplified population of γδ T cells to a recipient individual.

[0114] The donor and recipient individuals may be the same or different.

[0115] γδ T cells can be administered to patients or subjects in need by any preferred method. For example, γδ T cells can be administered intravenously or via an intratumoral route to patients or subjects in need of treatment.

[0116] The patients or subjects targeted for treatment are preferably patients with human cancer or viral infections (e.g., patients infected with CMV or HIV).

[0117] Because γδ T cells are MHC-independent, they do not recognize the host into which they are transplanted as exogenous, meaning they are less likely to cause graft-versus-host disease. This means they can be used "off the shelf" or, for example, transplanted into any recipient for allogeneic adoptive T cell therapy.

[0118] Since they normally reside in non-hematopoietic tissues, tissue-resident Vδ1 T cells and DN γδ T cells are also more likely to home to and remain within tumor masses than their systemic blood-resident counterparts, and adoptive transfer of these cells may be more effective in targeting solid tumors and potentially other non-hematopoietic tissue-associated immune lesions.

[0119] In some embodiments, a method for treating an individual having a tumor in non-hematopoietic tissue may include the following steps: A step of providing a sample of non-hematopoietic tissue obtained from a donor individual, As described above, the steps include culturing γδ T cells derived from the sample to produce an amplified population; and A step of administering the amplified population of γδ T cells to an individual having a tumor.

[0120] Non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention express NKG2D and respond to NKG2D ligands (e.g., MICA) strongly associated with malignant tumors. They also exhibit a cytotoxic profile in the absence of activation and may therefore be effective in killing tumor cells. For example, non-hematopoietic tissue-resident γδ T cells obtained as described herein can express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzyme A and B, and perforin in the absence of activation. IL-17A may not be expressed.

[0121] Accordingly, the findings reported herein provide compelling evidence regarding the practicality and suitability for the clinical application of non-hematopoietic tissue-resident γδ T cells obtained by the method of the invention as an "off-the-shelf" immunotherapy reagent. These cells retain innate-like killing ability, are MHC-free, and exhibit improved tumor homing and / or retention within tumors compared to other T cells.

[0122] Non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention can also be used for CAR-T therapy. This involves generating genetically modified T cell receptors (TCRs) to reprogram T cells with novel specificity (e.g., specificity of monoclonal antibodies). Genetically modified TCRs can make T cells specific to malignant tumor cells and thus useful for cancer immunotherapy. For example, T cells can recognize cancer cells that express tumor antigens, such as tumor-associated antigens (TAAs), which are not expressed by normal somatic cells derived from the subject's tissue. In other words, CAR-modified T cells can be used, for example, for adoptive T cell therapy in cancer patients.

[0123] The use of blood-resident γδ T cells for CAR has been described. However, non-hematopoietic tissue-resident γδ T cells obtained by the method of the present invention may be a particularly good vehicle for CAR-T approaches because they can be transduced using chimeric antigen-specific TCRs while retaining their innate ability to recognize transformed cells, and may have better tumor penetration and persistence than either blood-resident γδ T cells or conventional systemic αβ T cells. Furthermore, the absence of MHC-dependent antigen presentation in these cells reduces the likelihood of graft-versus-host disease and allows them to target tumors expressing low levels of MHC. Similarly, the independence of these cells from conventional costimulation (e.g., via entanglement with CD28) facilitates the targeting of tumors expressing low levels of ligands for costimulatory receptors.

[0124] Cancer can be characterized by the abnormal proliferation of malignant cancer cells and includes leukemia (such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL)), lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma), and solid tumors (such as sarcoma, skin cancer, melanoma, bladder cancer, brain cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, adrenal cancer, stomach cancer, testicular cancer, gallbladder and bile duct cancer, thyroid cancer, thymic cancer, bone cancer, and cerebral cancer).

[0125] Cancer cells in a cancer patient's body may be immunologically different from the normal somatic cells of that individual (i.e., cancerous tumors may be immunogenic). For example, cancer cells may elicit a systemic immune response in a cancer patient's body to one or more antigens expressed by the cancer cells. The antigens that elicit an immune response may be tumor antigens or may be shared by normal cells. A patient with cancer may present at least one identifiable sign, symptom, or laboratory result sufficient to make a diagnosis of cancer according to clinical criteria known in the art. Examples of such clinical criteria can be found in medical textbooks such as Harrison's Principles of Internal Medicine (40). In some cases, the diagnosis of cancer in an individual may involve the identification of a specific cell type (e.g., cancer cells) in a sample of bodily fluids or tissue taken from that individual.

[0126] Suitable patients, subjects, or individuals for the treatment described above may be rodents (e.g., guinea pigs, hamsters, rats, mice), murids (e.g., mice), canids (e.g., dogs), felines (e.g., cats), equids (e.g., horses), primates, simians (e.g., monkeys or apes), monkeys (e.g., marmosets or baboons), apes (e.g., gorillas, chimpanzees, orangutans or gibbons), or mammals such as humans.

[0127] In some preferred embodiments, the patient, subject, or individual is human. In other preferred embodiments, non-human mammals, in particular mammals conventionally used as models to demonstrate therapeutic efficacy in humans (e.g., rodents, primates, pigs, canids, or rabbits), may be used.

[0128] In some embodiments, patients, subjects, or individuals may have minimal residual disease (MRD) after initial cancer treatment.

[0129] Treatment may be any treatment or therapy, whether in humans or animals (e.g., in veterinary applications), that achieves some desired therapeutic effect (e.g., inhibition or delay of disease progression) and includes, but is greater than what would be expected in the absence of treatment, a reduction in the rate of disease progression, a cessation of the rate of disease progression, improvement of the disease, cure or remission of the disease (whether partial or complete remission), prevention, delay, reduction or cessation of one or more symptoms and / or signs of the disease, or extension of the survival of the subject or patient.

[0130] Treatment as a preventative measure (i.e., prevention) is also included. For example, a patient, subject, or individual who is susceptible to cancer or at risk of developing or recurring cancer may be treated as described herein. Such treatment may prevent or delay the development or recurrence of cancer in the patient, subject, or individual.

[0131] In particular, treatment may include inhibiting cancer growth, including complete remission of cancer, and / or inhibiting cancer metastasis. Cancer growth generally refers to any one of many indicators of change within the cancer, such as a shift to a more developed form. Indicators for measuring the inhibition of cancer growth include a decrease in cancer cell viability, a decrease in tumor volume or morphology (e.g., as determined by computed tomography (CT), ultrasound, or other imaging methods), delayed tumor growth, destruction of the tumor vascular system, improved performance on delayed-type hypersensitivity skin tests, increased activity of cytolytic T lymphocytes, and decreased levels of tumor-specific antigens. Reduced immunosuppression in cancerous tumors in an individual may improve the individual's ability to resist cancer growth, especially the growth of cancer already present in the subject, and / or reduce the tendency for cancer growth within the individual.

[0132] Other aspects and embodiments of the present invention provide the above aspects and embodiments in which the term "including" is replaced with the term "consisting of," and the above aspects and embodiments in which the term "including" is replaced with the term "substantially consisting of."

[0133] This application should be understood to disclose all combinations of any of the above aspects and embodiments, unless the context requires otherwise. Similarly, this application discloses all combinations of preferred and / or optional features, either individually or together with any of the other aspects, unless the context requires otherwise.

[0134] Modifications to the above embodiments, further embodiments and their modifications will be apparent to those skilled in the art by reading this disclosure, and indeed, they fall within the scope of the present invention.

[0135] All references and sequence database entries mentioned herein are incorporated herein by reference in their entirety for all purposes.

[0136] As used herein, “and / or” should be understood as each of two specified features or components, which may or may not be combined with the other. For example, “A and / or B” should be understood as each of (i) A, (ii) B, and (iii) A and B, as if each were described separately herein.

[0137] The present invention encompasses the following embodiments: 1. A method for amplifying non-hematopoietic tissue-resident γδ T cells in vitro, comprising the step of culturing lymphocytes obtained from non-hematopoietic tissue of a human or non-human animal in the presence of interleukin-2 (IL-2) and / or interleukin-15 (IL-15), wherein the lymphocytes do not come into direct contact with stromal cells or epithelial cells during culture. 2. The method according to Embodiment 1, wherein the lymphocytes do not come into direct contact with fibroblasts during culture. 3. The method according to Embodiment 1 or 2, comprising the step of culturing lymphocytes obtained from non-hematopoietic tissue of a human or non-human animal in the presence of IL-2. 4. The method according to Embodiment 1 or 2, comprising the step of culturing lymphocytes obtained from non-hematopoietic tissue of a human or non-human animal in the presence of interleukin-15 (IL-15). 5. The method according to Embodiment 1 or 2, comprising the step of culturing lymphocytes obtained from non-hematopoietic tissue of a human or non-human animal in the presence of IL-2 and IL-15. 6. The method according to any one of Embodiments 1 to 5, comprising the step of culturing the lymphocytes in the absence of a TCR activation signal or a co-stimulatory signal. 7. The method according to any one of Embodiments 1 to 6, comprising the step of culturing the lymphocytes in the absence of a T cell receptor pathway agonist. 8. The method according to any one of Embodiments 1 to 7, wherein the lymphocytes are cultured in the absence of stromal cells or epithelial cells. 9. The method according to Embodiment 8, wherein the stromal cells or epithelial cells are removed prior to culture. 10. The method according to Embodiment 8, wherein the lymphocytes are cultured in the absence of fibroblasts. 11. The method according to Embodiment 10, wherein the fibroblasts are removed prior to culture. 12. The method according to any one of Embodiments 1 to 11, wherein the lymphocytes are cultured in a γδ amplification medium containing IL-2 and / or IL-15. 13. The method according to Embodiment 12, wherein the γδ amplification medium does not activate or co-stimulate T cell receptors. 14. The method according to Embodiment 12 or 13, wherein the γδ amplification medium lacks growth factors other than IL-2 and / or IL-15. 15. The method according to any one of Embodiments 12 to 14, wherein the γδ amplification medium consists of a basal medium supplemented with IL-2 and / or IL-15. 16. The method according to any one of Embodiments 1 to 15, wherein the lymphocytes are obtained from the skin, the digestive tract (e.g., the colon), mammary gland tissue, the lungs, the liver, the pancreas, or the prostate. 17. The method according to any one of Embodiments 1 to 16, wherein the γδ T cells are non-Vδ2 cells. 18. The method according to Embodiment 17, wherein the γδ T cell is a Vδ1 cell. 19. The method according to Embodiment 17, wherein the γδ T cells are double-negative (DN) γδ T cells. 20. The method according to any one of Embodiments 1 to 19, comprising the step of obtaining lymphocytes from human or non-human animal tissue. 21. The method according to Embodiment 20, wherein the lymphocytes are obtained from a sample of non-hematopoietic tissue of a human or non-human animal. 22. The method according to Embodiment 20 or 21, wherein the tissue comprises non-hematopoietic cells and lymphocytes. 23. The method according to any one of Embodiments 20 to 22, comprising the steps of providing a sample of non-hematopoietic tissue from a human or non-human animal, and separating lymphocytes from the non-hematopoietic cells of the sample to produce a population of lymphocytes substantially free of stromal cells. 24. The following steps: (i) the step of providing a population of γδ T cells obtained from non-hematopoietic tissue; and (ii) A step of culturing the γδ T cells under conditions that substantially do not involve contact with stromal cells to produce an amplified population of γδ T cells. A method for amplifying γδ T cells, including the method described above. 25. The method according to Embodiment 24, wherein the population of γδ T cells obtained from non-hematopoietic tissue is a substantially pure population of γδ T cells. 26. The method according to Embodiment 24 or 25, wherein the population of γδ T cells obtained from non-hematopoietic tissue is non-Vδ2 cells. 27. The method according to Embodiment 26, wherein the population of non-Vδ2 cells includes Vδ1 cells. 28. The method according to Embodiment 26 or 27, wherein the population of non-Vδ2 cells includes DN γδ T cells. 29. The aforementioned population of γδ T cells obtained from non-hematopoietic tissue is Vδ1 + CLA + CCR8 + CD103 + The method according to any one of embodiments 24 to 27, comprising γδ T cells. 30. The method according to any one of embodiments 24 to 29, wherein the culture step of step (ii) is carried out without contact between γδ T cells and stromal cells. 31. The method according to any one of Embodiments 24 to 30, wherein the γδ T cells of step (ii) are cultured under conditions that substantially do not contain a TCR activation signal or a costimulatory signal. 32. The method according to any one of embodiments 24 to 31, wherein the culture step of step (ii) is carried out in the absence of a TCR activation signal or a co-stimulatory signal. 33. The method according to any one of embodiments 24 to 32, wherein the culture step of step (ii) is carried out in interstitial cell-conditioned medium. 34. The method according to any one of embodiments 24 to 33, wherein the γδ T cell culture step of step (ii) is carried out in the presence of IL-2, IL-15, or a combination thereof. 35. The method according to any one of embodiments 24 to 34, wherein the population of γδ T cells is obtained from a sample of non-hematopoietic tissue of a human or non-human animal. 36. The method according to any one of embodiments 24 to 35, wherein the non-hematopoietic tissue comprises non-hematopoietic cells and γδ T cells. 37. The method according to any one of embodiments 24 to 36, comprising the step of separating the γδ T cells from the non-hematopoietic cells to produce an isolated lymphocyte population containing γδ T cells that substantially does not contain stromal cells. 38. The method according to any one of embodiments 24 to 37, wherein the γδ T cells are cultured in a γδ amplification medium containing IL-2 and / or IL-15. 39. The method according to any one of embodiments 24 to 38, wherein the γδ amplification medium does not activate or co-stimulate T cell receptors. 40. The method according to any one of embodiments 24 to 39, wherein the γδ amplification medium lacks growth factors other than IL-2 and / or IL-15. 41. The method according to any one of Embodiments 24 to 40, wherein the γδ amplification medium consists of a basal medium supplemented with IL-2 and / or IL-15. 42. The following steps: (i) A step of providing non-hematopoietic tissue containing non-hematopoietic cells and γδ T cells; (ii) the step of isolating the γδ T cells from the non-hematopoietic cells to produce an isolated population containing γδ T cells that is substantially free of stromal cells; and (iii) A step of culturing the isolated population from step (ii) in the absence of a TCR activation signal or a co-stimulatory signal to produce an amplified population of γδ cells. A method for amplifying γδ T cells, including the method described above. 43. The method according to Embodiment 42, wherein the separation step of step (ii) includes separating the γδ T cells from the αβ T cells. 44. The method according to Embodiment 42 or 43, wherein the isolated population in step (ii) is a substantially pure population of γδ T cells. 45. The method according to any one of embodiments 42 to 44, wherein the isolated population in step (ii) is non-Vδ2 cells. 46. ​​The method according to Embodiment 45, wherein the population of non-Vδ2 cells includes Vδ1 cells. 47. The method according to embodiment 45 or 46, wherein the population of non-Vδ2 cells includes DN γδ T cells. 48. The separated population in step (ii) is Vδ1 + CLA + CCR8 + CD103 + The method according to any one of embodiments 42 to 47, comprising a population of γδ T cells. 49. The method according to any one of embodiments 42 to 48, wherein the culture step of step (iii) is carried out substantially without contact with stromal cells. 50. The method according to any one of embodiments 42 to 48, wherein the culture step of step (iii) is carried out without contact between the γδ T cells and the stromal cells. 51. The method according to any one of Embodiments 42 to 50, wherein the culture step of step (iii) substantially does not include a TCR activation signal or a co-stimulatory signal. 52. The method according to any one of Embodiments 42 to 51, wherein the culture step of step (iii) is carried out in the absence of a TCR activation signal and a co-stimulatory signal. 53. The method according to any one of Embodiments 42 to 52, wherein the culture step of step (iii) is carried out in interstitial cell-conditioned medium. 54. The method according to any one of Embodiments 42 to 53, wherein the culture step of step (iii) is carried out in the presence of IL-2, IL-15, or a combination thereof. 55. The method according to Embodiment 54, wherein the isolated population is cultured in a γδ amplification medium containing IL-2 and / or IL-15. 56. The method according to Embodiment 55, wherein the γδ amplification medium does not activate or co-stimulate T cell receptors. 57. The method according to Embodiment 55 or 56, wherein the γδ amplification medium lacks growth factors other than IL-2 and / or IL-15. 58. The method according to any one of embodiments 55 to 57, wherein the γδ amplification medium consists of a basal medium supplemented with IL-2 and / or IL-15. 59. The method according to any one of embodiments 24 to 58, wherein the amplified population of γδ T cells contains at least 20 times the number of γδ T cells obtained from non-hematopoietic tissue within 14 days of culture. 60. The method according to Embodiment 59, wherein the amplified population of γδ T cells contains at least twice the number of γδ T cells obtained from non-hematopoietic tissue within 7 days of culture. 61. Of the amplified population of γδ T cells, at least 50% are Vδ1 + The method according to any of embodiments 24 to 60, wherein the cell is a cell. 62. Of the aforementioned amplified population of γδ T cells, at least 70% are Vδ1 + The method according to embodiment 61, wherein the cell is a cell. 63. Of the aforementioned amplified population of γδ T cells, at least 90% are Vδ1 + The method according to embodiment 62, wherein the cell is a cell. 64. The method according to any one of embodiments 24 to 63, wherein at least 10% of the amplified population of γδ T cells are positive for CCR4, CCR8, and CD103. 65. The method according to Embodiment 63, wherein at least 30% of the amplified population of γδ T cells are positive for CCR4 and CCR8. 66. The method according to Embodiment 65, wherein at least 60% of the amplified population of γδ T cells are positive for CCR8. 67. The γδ T cells are Vδ2 - The method according to any one of embodiments 24 to 66, wherein the cell is a cell. 68. Non-hematopoietic tissue-resident γδ T cells obtained by the method described in any of Embodiments 1 to 67. 69. The following characteristics: (i) Phenotype CD69 high ICOS high , TIM3 high and CD28 low / absent To show, (ii) Upregulate one or more of the following: CCR3, CD39, CD11b, and CD9. (iii) In the absence of a TCR agonist, it produces IFN-γ in response to the NKG2D ligand, (iv) To produce IL-13 in the absence of a TCR agonist, (v) In response to TCR activation, produce one or more of IFN-γ, TNF-α, and GM-CSF, (vi) Not producing any IL-17 at all or substantially not producing it in response to TCR activation, (vii) Growing in a culture medium containing IL-2 without additional growth factors, (viii) Exhibiting a cytotoxic T cell response in the absence of a TCR agonist, and / or (ix) Shows selective cytotoxicity against tumor cells compared to normal cells. Non-hematopoietic tissue-resident γδ T cells possessing one or more of the following types. 70. The following steps: (i) the step of culturing non-hematopoietic tissue resident γδ T cells in vitro by directly contacting stromal cells or epithelial cells (e.g., fibroblasts) in the presence and absence of the test compound, or the step of culturing non-hematopoietic tissue resident γδ T cells in vitro by directly contacting stromal cells or epithelial cells (e.g., fibroblasts), wherein the expression of the test gene in the γδ T cells and / or stromal cells or epithelial cells (e.g., fibroblasts) is altered; and (ii) A step of determining the proliferation rate or activation rate of non-hematopoietic tissue resident γδ T cells in the presence or absence of the test compound, or in the presence or absence of changes in the expression of the test gene in fibroblasts and / or γδ T cells, or a step of determining the mortality rate of stromal cells or epithelial cells (e.g., fibroblasts) in the presence or absence of the test compound, or in the presence or absence of changes in the expression of the test gene in stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells, wherein the proliferation rate or activation rate of T cells is higher in the presence or absence of the test compound than in the absence of changes in the test gene in stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells If the test compound is higher in the presence of altered expression of the test gene in T cells, and / or if the mortality rate of stromal cells or epithelial cells (e.g., fibroblasts) is higher in the presence of the test compound than in the absence of the test compound, or if the mortality rate of stromal cells or epithelial cells (e.g., fibroblasts) and / or γδ T cells is higher in the presence of altered expression of the test gene in fibroblasts and / or γδ T cells than in the absence of altered expression of the test gene in fibroblasts and / or γδ T cells, then the test compound may be a checkpoint regulator, or the test gene may be a candidate checkpoint gene, as described above. A screening method for checkpoint inhibitors, including [specific example]. 71. Non-hematopoietic tissue-resident γδ T cells obtained by any of the methods described in Embodiments 1 to 67 for use in therapeutic methods for humans or non-human animals by adoptive T cell therapy. 72. Non-hematopoietic tissue resident γδ T cells for use according to Embodiment 71, wherein the human is a patient with human cancer or a patient with a viral infection such as CMV infection or HIV infection. 73. A method for treating a subject with adoptive T cell therapy, comprising the step of administering non-hematopoietic tissue resident γδ T cells obtained by any of the methods described in Embodiments 1 to 67 to a subject in need thereof. 74. The method according to Embodiment 73, wherein the subject is a human cancer patient or a patient with a viral infection such as CMV35 infection or HIV infection. 75. Non-hematopoietic tissue resident γδ T cells obtained by any of the methods described in Embodiments 1 to 67, for use in therapeutic methods for humans or non-human animals by chimeric antigen receptor therapy. 76. Non-hematopoietic tissue resident γδ T cells for use according to Embodiment 75, wherein the human is a human cancer patient. 77. A method for treating a subject by chimeric antigen receptor therapy, comprising the step of administering non-hematopoietic tissue commensal γδ T cells obtained by any method described in any of Embodiments 1 to 67 to a subject in need thereof. 78. The method according to embodiment 77, wherein the subject is a human cancer patient. 79. A method for treating a subject with adoptive T cell therapy, comprising the step of administering non-hematopoietic tissue-resident γδ T cells as described in Embodiment 69. 80. The method according to Embodiment 79, wherein the subject is a human cancer patient or a patient with a viral infection such as CMV35 infection or HIV infection. 81. A method for treating a subject by chimeric antigen receptor therapy, comprising the step of administering non-hematopoietic tissue resident γδ T cells as described in Embodiment 69, wherein the cells express chimeric antigen receptors. 82. The method according to Embodiment 81, wherein the subject is a human cancer patient. Some aspects and embodiments of the present invention are illustrated here by reference to the drawings described above. [Examples]

[0138] method Isolation of human skin-derived lymphocytes by three-dimensional explant culture. The three-dimensional skin explant protocol was established as described in other literature (29). A 9mm × 9mm × 1.5mm Cellfoam matrix (Cytomatrix Pty Ltd, Victoria, Australia) was autoclaved and subsequently incubated in a 100mg / mL rat tail type I collagen (BD Biosciences) solution (in PBS) at room temperature for 30 minutes, followed by a single rinse in PBS. Adult human skin samples were obtained within 3–6 hours of skin surgery. Subcutaneous fat was removed, and the remaining skin tissue was cut into fragments approximately 1mm × 1mm in size. Approximately five skin fragments / explants were positioned and pressed onto the surface of each matrix. Each matrix was placed in a separate well of a 24-well plate (Corning) containing 2 mL of "Skin-T" medium (10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimal essential medium (MEM) non-essential amino acids (1×; Life Technologies), and Iskov modified Dulbecco's medium (IMDM; Life Technologies) containing 3.5 μL / L 2-mercaptoethanol (Life Technologies)). Amphotericin (2.5 μg / mL; Life Technologies) was added to the medium for the first 7 days of culture. The culture medium was changed three times a week. To provide nutrients, 1 mL of the medium from the top of each well was aspirated and replaced with fresh medium. IL-2 and IL-15 were added at each medium change from the start of the culture until lymphocyte isolation on day 21. Human recombinant IL-2 (Proleukin; Novartis Pharmaceutical UK Ltd) was added at 100 IU / mL.Human recombinant IL-15 (Biolegen) was added at a concentration of 20 ng / mL. Up to 96 wells (four 24-well plates) were set up for each donor during culture.

[0139] To isolate lymphocytes, the matrix was transferred to 50 mL centrifuge tubes (Corning) containing 10 mL of Hanks equilibrium salt solution (HBSS; Life Technologies) with 0.01 mM HEPES (maximum 12 matrix tubes / centrifuge tube). The matrix was rinsed with cell suspension using a 10 mL pipette, and the cell suspension was placed in a new 50 mL centrifuge tube (Corning) through a 70 μm filter (BD Biosciences). This matrix "washing" was repeated two more times. The culture medium was also aspirated from the culture wells and placed in a new 50 mL centrifuge tube (Corning) through a 70 μm filter (BD Biosciences). The wells were washed two more times with 1 mL of 0.01 mM HEPES / HBSS and placed through a 70 μm filter (BD Biosciences). Subsequently, the cells were isolated by centrifugation (1600 rpm, 15 minutes). The pellet was resuspended in "Skin-T" medium. The final cell pellet was resuspended in Skin-T medium for subsequent flow cytometry analysis or functional studies. If cell counts were required, leukocytes were counted at this stage by either (1) trypan blue staining (0.4%) (Life Technologies) and hemocytometer, or (2) CASY® Model TT cell counter and analyzer (Roche).

[0140] To isolate lymphocytes, the matrix was transferred to 50 mL centrifuge tubes (Corning) containing 10 mL of Hanks equilibrium salt solution (HBSS; Life Technologies) with 0.01 mM HEPES (maximum 12 matrix tubes / centrifuge tube). The matrix was rinsed with cell suspension using a 10 mL pipette, and the cell suspension was placed in a new 50 mL centrifuge tube (Corning) through a 70 μm filter (BD Biosciences). This matrix "washing" was repeated two more times. The culture medium was also aspirated from the culture wells and placed in a new 50 mL centrifuge tube (Corning) through a 70 μm filter (BD Biosciences). The wells were washed two more times with 1 mL of 0.01 mM HEPES / HBSS and placed through a 70 μm filter (BD Biosciences). Subsequently, the cells were isolated by centrifugation (1600 rpm, 15 minutes). The pellet was resuspended in "Skin-T" medium. The final cell pellet was resuspended in Skin-T medium for subsequent flow cytometry analysis or functional studies. If cell counts were required, leukocytes were counted at this stage by either (1) trypan blue staining (0.4%) (Life Technologies) and hemocytometer, or (2) CASY® Model TT cell counter and analyzer (Roche).

[0141] Because primary intestinal samples are easily contaminated, the acquired biopsy samples were first washed in IMDM containing 10% FCS, penicillin 500 U / mL, streptomycin 500 μg / mL, gentamicin 100 μg / mL, amphotericin B 12.5 μg / mL, and metronidazole 5 μg / mL, and then washed twice before being chopped and placed on a grid. Intestinal grid cultures were grown in Gut-T medium (IMDM, 10% FCS, penicillin 100 U / mL, streptomycin 100 μg / mL, gentamicin 20 μg / mL, metronidazole 1 μg / mL). For the first week of growth, the inventors also used amphotericin B 2.5 μg / mL, as with skin samples. The medium contained IL-2 (100 IU / mL) and IL-15 (20 ng / mL) and was changed three times a week. Because the structure of the intestines was less flexible than that of the skin, lymphocytes could be collected after one week.

[0142] Amplification of tissue γδ T cells For amplification of human skin-derived γδ T cells, mixed lymphocytes harvested 3-4 weeks after grid culture were washed in PBS, centrifuged, and then mixed into Roswell Park Memorial Institute 1640 medium (RPMI-1640; Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), minimal essential medium (MEM) non-essential amino acids (1 ×; Life Technologies), and 10 μL / L 2-mercaptoethanol (Life Technologies), with 1 × 10⁶ cells added. 6 The cells were resuspended at a density of / mL and either IL-2 (100 IU / mL) alone or IL-2 + IL-15 (20 ng / mL) was added.5 96-well flat-bottom plate (Corning) or 2 x 10 6 Cells were seeded in 12-well plates (Corning) at a rate of / well (for amplification). Cells were monitored daily by microscopy, nutrient-supplied with fresh medium, and cytokines were added three times weekly. When full confluence was reached or cell aggregates began to appear, cells were divided 1:1 into additional wells and plates. Cells were harvested and analyzed using flow cytometry, or used for functional assays after 7, 14, or 21 days, depending on the assay. If pure γδ T cells were required, cells were resuspended in 1 mL of FACS buffer (PBS, containing 2% heat-inactivated fetal bovine serum and 0.01 M EDTA), stained for αβ T cell receptors (Biolegend, clone IP26, 1:50) on ice in the dark for 30 minutes, and all negative cells were sorted using an Aria sorter (BD Biosciences) running DIVA.

[0143] Co-culture with fibroblasts For each grid culture setup, the inventors prepared two Petri dishes (100 x 25 mm, Corning) with several marks made using a surgical scalpel. Sliced ​​skin pieces were placed in the marks. After drying in the air for 5-10 minutes, the skin pieces were allowed to adhere properly to the dish, and 10 mL of Skin-T medium was added. The medium was changed weekly, and after 3 weeks of growth, primary fibroblasts were harvested following treatment with ACCUTASE® (Life Technologies). The fibroblasts were divided into 1 x 10⁶ cells. 4 For a 48-well plate, or 2 × 10⁻⁶ in the case of a Transwell experiment. 4 The cells were seeded in the bottom chamber of a 24-well plate. After 2-3 days, the fibroblasts reached confluence and were treated with RPMI and the described cytokines, resulting in 2 × 10⁶ cells in a 48-well plate. 5 For mixed cutaneous lymphocytes, or in the case of a 24-well plate, bottom well and transwell, 3 × 10⁶ 5We added these lymphocytes and started the co-culture experiment.

[0144] Flow cytometry Flow cytometry was performed using the following antibodies in combination with the listed fluorescent dyes: Ki-67-BV421, CD3-BV510, Vδ1-PeVio770, TIM-3-PE, CD9-PE, CCR3-BV421, and CD39-BV421. All samples were always stained for viability using eFluor770 NIR. Commercial antibodies were purchased from Biolegend or Miltenyi. Viability dyes (near-infrared) were obtained from eBioscience. Ki-67 staining was performed on cells fixed and permeabilized using the Foxp3 staining buffer set (eBioscience). After each experiment was completed, the cell population was washed in PBS and divided in half. Cells were stained for viability using eFluor770 NIR, washed, and then TrueStain (Biolegend) was used to avoid nonspecific binding of the staining antibody. Half of the samples were stained for the described surface markers, while the other half were stained for cell lineage markers only (CD3, Vδ1) and with isotype controls corresponding to the surface markers used. This means that matching mouse isotype antibodies conjugated to the same fluorescent dye were used at the same concentrations. The isotype controls do not bind to known human antigens and therefore show nonspecific binding or false positives, also known as true negatives. Each histogram (dark) is shown in comparison to its corresponding isotype control (white, dashed line). The data summary shows the percentage (%) of cells that were positive for the compared described markers, i.e., stained at a higher level than the isotype. Flow cytometry data analysis was performed using FlowJo (version 10.1).

[0145] RNA sequencing Human skin-derived Vδ1 T cells and human blood Vδ1 T cells (after T cell receptor-induced amplification) were sorted (FACS), centrifuged, and the cell pellet was resuspended in RLT buffer. RNA was prepared using the RNA-Micro-plus kit (QIAGEN). RNA libraries were prepared using the KAPA Stranded RNA-seq Kit with RiboErase (HMR) (KAPA BIOSYSTEMS). Paired-end sequencing was performed using HiSeq 2500 (Illumina) with rapid run chemistry (read length: 100 bp). 101 base pair paired-end reads were aligned and quantified using RSEM (v1.2.11) with Bowtie2. Reads were aligned to the human transcriptome, and count values ​​were log2 transformed and quantile normalized.

[0146] Cytokine quantification Human skin-derived Vδ1 T cells were stimulated for 24 hours with PMA and ionomycin or plate-bound anti-CD3 mAb (OKT3, 5 μg / mL). The supernatant was then collected and analyzed using ProcartaPlex Human Cytokine & Chemokine Panel 1A (34 plex) (eBioscience). The assay was analyzed using Luminex FlexMap3D (Luminex). Data were analyzed in Microsoft Excel, and the mean values ​​from three donors (double-repeat experiment) are shown. Error bars indicate the standard deviation.

[0147] Amplification of blood-derived γδ T cells Blood-derived γδ T cells within PBMCs can only be amplified when stimulated with a TCR ligand (for Vδ2, e.g., IPP, HMBPP, bisphosphonate) (41, 42) or with the addition of an antibody to crosslink the TCR receptor (mAb) or TCR-associated kinase CD3 (43). The same effect of TCR crosslinking can also be achieved using lectins such as PHA. In the absence of such TCR stimulants, γδ T cells in PBMCs survive for several days but cannot be amplified and remain in their initial composition as a T cell subset with little diversity.

[0148] To isolate PBMCs, whole blood from healthy volunteers was used. The blood was layered on Ficoll and then centrifuged at 400g for 20 minutes to separate red blood cells, plasma, and white lymohocytes / monocytes. White blood cells were carefully collected through a stripett and washed four times in cold PBS. Cells were placed in RPMI-1640 medium (Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies), L-glutamine (292 μg / mL; Life Technologies), penicillin (100 units / mL; Life Technologies), streptomycin (100 μg / mL; Life Technologies), N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES; 0.01 M; Life Technologies), sodium pyruvate (1 mM; Life Technologies), and minimal essential medium (MEM) non-essential amino acids (1 ×; Life Technologies), with 1 × 10⁶ cells. 6The cells were resuspended at a density of / mL and IL-2 (100 IU / mL) was added. 90 minutes prior to cell migration, the cells were transferred to 24-well plates coated with a pan-γδ TCR monoclonal antibody (20 μg / mL, clone B1, Biolegend). The cells were grown for 14 days, with the medium changed every 2-3 days and fresh cytokines added. Upon reaching confluence, the cells were divided 1:1. Under these conditions, after 14 days, the original small population of γδ T cells was significantly enriched, consisting mainly of Vδ2 T cells but also including Vδ1 T cells (up to 30% of all γδ T cells), and normally highly activated through their TCRs (as indicated by the upregulation of CD69 and CD25). Vδ1 T cells could then be isolated using FACS for functional, phenotypic, or genetic analysis.

[0149] result Human γδ T cells are abundant in the skin, express non-Vδ2 TCRs, and are involved in the human lymphoid stress surveillance response. Using the Clark protocol (29), the inventors obtained a lymphocyte population of an average of 240,000 cells per grid using human surplus skin samples supplemented with IL-2 and IL-15 to enable proliferation of tissue-resident lymphocytes over a period of 3 weeks. Consistent with previous reports (29), the inventors were able to identify a distinctly different subset of skin-resident lymphocytes, along with the majority of cells expressing conventional αβ TCRs, mostly of the tissue-resident "TRM" type. Overall, 59.9% (±8.6) of the CD45+ cells were CD4+, 18.3% (±2.8) were CD8+ αβ T cells, and the fraction included 8.7% (±3.6) of NK cells. In addition, the inventors identified a substantial population of γδ T cells (average 8.513%, ±6.564% of CD45+ cells) in their donors (Figures 1A and 3D). This subset representation of lymphocytes after organ-type culture was highly reproducible in approximately 100 donors and comparable to fresh digested skin samples, differing only in a slight increase in the γδ population, but was practically useful and provided a much larger and purer lymphocyte population compared to standard tissue digestion protocols. According to the literature on tissue localization of human γδ T cells based on their TCR delta chains, the majority of human cutaneous γδ T cells expressed Vδ1 TCR chains paired with various γ chains, as identified by flow cytometry. This was in contrast to the majority of peripheral blood γδ T cells, which showed a single-specific TCR heterodimer of Vδ2 chains linked to Vγ9 and were hardly present in human skin samples. However, it is important to note that a significant subset did not express either Vδ1 TCR or Vδ2 TCR, which led to the term "double-negative" γδ T cells (Figure 1C).

[0150] Resident γδ T cells grown in this manner exhibited a non-terminally differentiated memory phenotype, lacked CD45RA expression, and expressed various levels of the costimulatory molecule CCR7. Compared to conventional systemic T cells, the strong expression of the surface protein CD69 and its associated programmed expression of cell death receptor 1 (PD-1); low to absent levels of IL-2 receptor α (CD25); and the absence of the costimulatory molecule CD28 describe a situation of pre-activated or chronically activated T cells (Figure 1D). Consistent with their tissue localization, Vδ1 and DN cells showed expression of skin and tissue homing markers such as CLA, CCR4, CCR8, and integrin αE (CD103) (see Figure 7). This combination of tissue homing markers is considered to demonstrate its usefulness in immunotherapy settings. In addition, cutaneous resident γδ T cells exhibit high levels of expression for the activating receptor NKG2D (Figure 2A), suggesting a possible role for these cells in the lymphoid stress monitoring response. NKG2D ligands such as MICA, MICB, and ULBP are upregulated by cells in response to DNA damage, EGF receptor activation, and oxidative stress, respectively, and therefore, NKG2D-expressing T cells may be able to identify and eliminate stressed or transformed cells, thereby maintaining tissue homeostasis. In line with this principle, we found that cutaneous resident γδ T cells amplified by the present invention are activated upon exposure to recombinant ligands for the NKG2D receptor (MICA, ULBP2), demonstrating degranulation as measured by the upregulation of the lysosome-associated membrane protein CD107a (Figure 2A). This innate characteristic was limited to Vδ1+ T cells and DN γδ T cells, as other tissue-resident T cells (Figure 2C) and systemic γδ T cells lacked this response (Figure 10B).

[0151] Overall, activated cutaneous commensal Vδ1+ T cells and DN γδ T cells, when activated by PMA / ionomycin or NKG2D ligands (e.g., recombinant MICA protein), executed a pro-inflammatory TH1-biased cytokine program (positively staining for IFN-γ, TNF-α, and GM-CSF) (Figures 2A and 2B), thereby revealing the cellular innate response. Indeed, the response to MICA was almost completely neutralized by blocking the NKG2D receptor with antibodies (Figures 2B and 2C).

[0152] γδ T cells are known to secrete IL-17 in certain disease conditions, such as psoriasis, and in some types of tumors. γδ T cells amplified by the present invention produced low levels of IL-17 or no IL-17 at all, even upon thorough activation (Figures 2B and 8). Conversely, tissue-resident CD4-expressing αβ T cells produced IL-17 upon TCR activation (Figure 2B). Overall, αβ T cells exhibited a far more diverse cytokine repertoire in response to PMA / ionomycin compared to Vδ1+ T cells and DN γδ T cells (which were limited to TH1-biased programs associated with host protection).

[0153] Isolation from tissue triggers activation and significant amplification of human tissue γδ T cells. To further study human tissues, γδ T cells, and their biology, the inventors transferred mixed cutaneous lymphocytes to cell culture wells and added IL-2 to maintain long-term viability. Interestingly, the inventors quickly found that Vδ1 T cells, isolated from stromal and epithelial cells present in organ-type cultures, uniquely showed signs of activation and proliferation without any other stimulation. Within less than 7 days, Vδ1+ T cells and DN γδ T cells uniquely and significantly upregulated nuclear factor Ki-67 and increased surface expression of IL-2 receptor α(CD25) (Figures 3B and 4B). Notably, over a three-week period and in the presence of IL-2 alone, tissue-derived Vδ1+ T cells and DN γδ T cells proliferated more than all other T cell subsets, reaching up to 65% of total cutaneous lymphocytes and increasing in number by an average of 127.18 times, while αβ T cells increased only 5.21 times (p=0.0124) when measured by absolute cell number (Figure 3A). Cell cycle-associated nuclear factor Ki-67 MFI increased in Vδ1+ T cells and DN γδ T cells from 2664.5 (±1876.1) to 8457.7 (±4574.2) over 14 days, while in αβ T cells, MFI decreased from 592.8 (±390.5) to 284.7 (±140.1) over the same period (Figure 3C). This phenomenon of selective proliferation of cutaneous commensal γδ T cells can be further aided with additional recombinant IL-15, which increased lymphocyte viability and total number.

[0154] Skin γδ T cells are significantly suppressed by fibroblasts in a contact-dependent manner. The remarkable amplification of Vδ1+ T cells and DN γδ T cells described in the previous paragraph did not occur at all in organ-type culture systems where a large amount of fibroblast proliferation was present. Therefore, the inventors grew autologous fibroblasts to directly investigate whether their co-culture with Vδ1+ T cells and DN γδ T cells inhibits T cell amplification. After 3 weeks of grid culture, the inventors seeded mixed cutaneous lymphocytes into wells that were either empty or contained a confluent monolayer of pre-achieved fibroblasts, and in each case, IL-2 was exogenously added to the culture medium to maintain T cell growth. In addition, the inventors used transwells, which prevent T lymphocytes from directly contacting fibroblasts in the same well, but allow T lymphocytes to be affected by any soluble factors produced by fibroblasts. During 14 days of co-culture, Vδ1+ T cells and DN γδ T cells began to proliferate in wells without fibroblasts and in wells where direct contact between T cells and fibroblasts was prevented. As previously observed, αβ T cell proliferation was low under all conditions. Notably, when T cells were brought into direct contact with fibroblasts, the growth rate of Vδ1+ T cells and DN γδ T cells over two weeks decreased significantly from 22.6 times (SEM 8.07) times in wells without fibroblast contact to 3.3 times (SEM 0.17) times (Figure 4A). This contact-mediated inhibition was further confirmed by the absence of upregulation of CD25, Ki-67, and the transcription factor T-bet in Vδ1 over a 7-day period compared to lymphocytes grown alone (Figure 4B). Some forms of tissue-mediated regulation of the immune system are clearly fundamental to maintaining tissue homeostasis, because without this regulation, persistent inflammation is likely to occur. The suppressive regulation of Vδ1+ T cells and DN γδ T cells by stromal fibroblasts appears to be one example of such regulation.

[0155] In summary, the phenotypes of resident skin Vδ1+ T cells and DN γδ T cells, as well as their outstanding functional potential, reflect pre-activation T cells normally suppressed by adjacent skin fibroblasts, via a contact-dependent mechanism characterized by the following. By inactivating this mechanism by freeing T cells from contact with fibroblasts, a remarkable amplification of Vδ1+ T cells and DN γδ T cells becomes selectively possible, while other T cells in the skin remain unaffected.

[0156] Removal of contact-mediated inhibition promotes cytotoxic TH1-biased cytokine responses by cutaneous Vδ1 T cells. Mixed skin-derived lymphocytes were amplified for 14 days, and fluorescence-associated cell sorting was used to remove αβ T cells from γδ T cells, thereby obtaining γδ T cells with up to 90% purity. These highly enriched cells were placed in cell culture wells at a concentration of 150,000 cells / well in RPMI medium containing 10% FCS, and the supernatant was collected after 24 hours and evaluated for a wide range of effector cytokines using a LUMINEX®-based array. Completely unexpectedly, the amplified γδ T cells (induced solely by their isolation from fibroblasts) spontaneously produced large amounts of TH1-related cytokines such as IFN-γ (12,383.46±16,618.90 pg / mL) and GM-CSF (4,316.73±4,534.96 pg / mL), as well as the inflammatory chemokines CCL4 (14,877.34±10,935.64 pg / mL) and CCL3 (1,303.07±757.23 pg / mL) (Figure 5A).

[0157] Furthermore, the cells spontaneously produced large amounts of IL-13, which is associated with atopic responses, during amplification, in contrast to freshly isolated skin-derived TCR-activated γδ T cells; other cytokines (e.g., IL-17A) were produced at much lower levels or not at all (Figure 8). The high effector capacity of these cells could be further increased after stimulation with recombinant MICA (NKG2D ligand), anti-CD3 crosslinking, or PMA / ionomycin. To evaluate the cytotoxicity of amplified γδ T cells against malignant target cells, we used established transformed cell lines in a 24-hour co-culture experiment. Vδ1+ T cells and DN γδ T cells showed very high cytotoxic activity against HeLa cells (cervical cancer) and Caco2 cells (colon cancer) in a dose-dependent manner, far exceeding that of conventional tissue αβ T cells (Figure 5B). Furthermore, γδ cell-mediated cytotoxicity can be strongly inhibited by blocking the NKG2D receptor using a soluble monoclonal antibody, indicating that this receptor is at least partially involved in tumor surveillance by disinhibiting human skin-derived γδ T cells. The inventors further confirmed the cytotoxicity of other targets: HCT1954 cells, MDAMB231 cells (both from breast cancer), and HCT116 cells (from colon cancer) (Figure 9).

[0158] Human gut tissue-resident γδ T cells The inventors have identified a non-hematopoietic tissue-resident population of γδ T cells expressing the Vδ1 T cell receptor in human colon-derived grid cultures (Figure 6). Using three donors, the inventors were able to amplify these cells over a period of 4–5 weeks using the same method as used for skin cells (15). During amplification, colon-derived Vδ1+ T cells and DN γδ T cells showed similar Ki-67 upregulation patterns after their isolation from fibroblast-rich organelle cell cultures. Similarly, colon-resident Vδ1+ T cells and DN γδ T cells were strongly stimulated by ligand supply to the NKG2D receptor. Hematopoietic γδ T cells have been previously reported to be well-equipped to perform antibody-dependent cell-mediated cytotoxicity via CD16 expression, demonstrating targeted and enhanced cytotoxicity against CD20-positive B-cell lymphomas when combined with rituximab. Similarly, chronic lymphocytic leukemia (CLL) and HER2-positive breast cancer cells were more effectively killed when targeted with monoclonal antibodies (31). To evaluate the ability of skin-derived Vδ1 T cells to target antibody-opsonized target cells, we checked the expression levels of three IgG1-related Fc receptors: CD16, CD32, and CD64. Skin-derived Vδ1 T cells express only low levels of the Fc receptor CD16 but show good expression levels for the high-affinity IgG receptor CD64 (Figure 11). Therefore, tissue-derived Vδ1 T cells may be well-suited to be used as adjuvants to monoclonal antibody therapies such as CD20 therapy or Her2 therapy, as they would recognize opsonized tumor cells induced by antibodies on the malignant and metastatic side and kill targets via ADCC.

[0159] References JPEG0007860161000001.jpg190145JPEG0007860161000002.jpg71149

Claims

1. A composition comprising a population of skin-derived γδ T cells for treating a subject in need thereof, wherein the population of skin-derived γδ T cells is obtained by a method comprising the step of culturing lymphocytes obtained from human or non-human animal skin tissue in the presence of interleukin-15 (IL-15), wherein the lymphocytes do not come into direct contact with stromal or epithelial cells during culture, the skin-derived γδ T cells of the population (i) express CD69 and PD-1, (ii) lack CD28 expression, and the subject is a human cancer patient or a viral infection patient.

2. The composition according to claim 1, wherein the stromal cells or epithelial cells include fibroblasts.

3. The composition according to claim 1 or 2, wherein the method comprises the step of culturing lymphocytes obtained from skin tissue in the presence of IL-2.

4. The composition according to any one of claims 1 to 3, wherein the method comprises the step of culturing the lymphocytes in the absence of a TCR activation signal or a co-stimulatory signal.

5. The composition according to any one of claims 1 to 4, wherein the stromal cells or epithelial cells are removed prior to culture.

6. The composition according to any one of claims 1 to 5, wherein the lymphocytes are cultured in the absence of fibroblasts.

7. The composition according to claim 6, wherein the fibroblasts are removed prior to culture.

8. The composition according to any one of claims 1 to 7, wherein the lymphocytes are cultured in a γδ amplification medium containing IL-15.

9. The composition according to claim 8, wherein the γδ amplification medium further contains IL-2.

10. The composition according to claim 8 or 9, wherein the γδ amplification medium does not activate or co-stimulate T cell receptors.

11. The composition according to any one of claims 1 to 10, wherein the γδ T cells are non-Vδ2 cells, and the non-Vδ2 cells include Vδ1 cells or double-negative (DN) γδ T cells.

12. The composition according to any one of claims 1 to 11, wherein the cancer includes colon cancer, cervical cancer, or breast cancer.

13. The composition according to any one of claims 1 to 11, wherein the virus comprises CMV35 or HIV.

14. The composition according to any one of claims 1 to 13, wherein the cells express a chimeric antigen receptor.

15. The composition according to any one of claims 1 to 14, wherein the γδ T cells express CLA, CCR4 and CCR8.

16. The composition according to any one of claims 1 to 15, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the γδ T cells in the population of skin-derived γδ T cells are Vδ1+ cells.

17. The γδ T cells are (i) The phenotype shows CD69 high, ICOS high, TIM3 high, and CD28 low / absent, (ii) Upregulate one or more of the following: CCR3, CD39, CD11b, and CD9. (iii) In the absence of a TCR agonist, it produces IFN-γ in response to the NKG2D ligand, (iv) To produce IL-13 in the absence of a TCR agonist, (v) In response to TCR activation, produce one or more of IFN-γ, TNF-α, and GM-CSF, (vi) Not producing any IL-17 at all or substantially not producing it in response to TCR activation, (vii) Growing in a culture medium containing IL-2 without additional growth factors, (viii) Whether they exhibit a cytotoxic T cell response in the absence of a TCR agonist, (ix) It exhibits selective cytotoxicity against tumor cells compared to normal cells, or (x) represents one of the combinations (i) to (ix). The composition according to any one of claims 1 to 16.

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