Methods of expansion of skin vdelta1+ gammadelta t cells and use of same

Abstract

The present disclosure relates to the expansion of gamma delta (γδ) T cells, e.g., Vδ1+ γδ T cells. In particular, the present disclosure provides improved methods and processes for expanding highly active γδ T cells (e.g., Vδ1+ γδ T cells) from skin with the addition of one or more cytokines, such as IL-2, IL-15, and TGF-β.

Description

ATTORNEY DOCKET NO.: MIL-054WO1METHODS OF EXPANSION OF SKIN VDELTA1+ GAMMADELTA T CELLS AND USE OF SAMECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to, and the benefit of U. S. Provisional Application No: 63 / 722,540, filed on November 19, 2024, and U. S. Provisional Application No: 63 / 827,068, filed on June 20, 2025, both of which are incorporated herein by reference in their entireties.BACKGROUND

[0002] Immune cells (e.g, T cells) are a critical component of the body’s defense against foreign pathogens (e.g., bacteria and viruses) and malignant cancer cells. T cells can be divided into two subclasses: “conventional” alpha beta (aP) T cells and “unconventional” gamma delta (y5) T cells. y5 T cells comprise approximately 1-5% of the total population of PMBCs in the blood and can be found in higher proportions in secondary lymphoid organs (e.g., skin). These immune cells have also been found in various cancerous tissue types, including lung and melanoma to a similar or greater extent that CD8+ tumor infiltrating lymphocytes have been found. Particularly, y5 T cells have a demonstrated capacity for cytotoxicity by secreting large amounts of pro-inflammatory cytokines (e.g., TNFs, ILs, and TGFs). Recent studies also demonstrate that the majority of y5 T cells infiltrating cancer tissues is V51+ T cells. These observations suggest enormous potential of y5 T cells, e.g, V51+ T cells as effectors for next-generation immunotherapies.

[0003] To obtain y5 T cells for use in therapeutic applications, processes and methods to expand y5 T cells are useful for expanding y5 T cells mediated cell therapy.SUMMARY OF INVENTION

[0004] The present disclosure provides, in part, improved methods of producing skin-derived V51+y5 T cells that have enhanced cytotoxic activity, compositions comprising skin-derived V51+y5 T cells and uses for the same in adoptive cell therapy and for example, in treating cancer. The improved methods of the present disclosure, in part, to relate to expansion of skin-derived y5 T cells (e.g., V51+ y5 T cells) with co-culture cytokines, such as IL-2 and IL-15, together with other cytokines that are specifically related to the skin microenvironment (e.g., TGF-P).ATTORNEY DOCKET NO.: MIL-054WO1

[0005] The present disclosure provides, in part, a method of producing skin-derived V51+y5 T cells comprising expanding y5 T cells isolated from skin in the presence of one or more cytokines and TGF-P, wherein the expanded skin-derived V51+y5 T cells are selectively enriched for Tissue Resident memory cells (TRM cells). The present disclosure provides, in part, a method of expanding skin-derived V51+y5 T cells comprising culturing skin y5 T cells in a culture medium supplemented with TGF-P, wherein the expanded skin-V51+y5 T cells are characterized with expression of one or more-skin specific-Tissue Resident memory cells (TRM) markers (e.g., CD103+, CD101+ AHR+, CXCR4+ and / or Vimentin+) and one or more skin-homing receptors e.g., CCR4, CCR6, CCR8, CCCR10 and CLA). The present disclosure provides in part, a method of expanding skin-derived V51+y5 T cells comprising culturing y5 T cells isolated from skin in the presence of one or more cytokines and TGF-P, wherein the expanded skin-derived V51+y5 T cells produce high levels of XCL1 and one or more chemokines of CCL3, CCL4 and CCL5, as compared to skin-derived V51+y5 T cells expanded without TGF-p.

[0006] Among other things, the expanded skin-derived V51+y5 T cells of the present disclosure have an uncharacteristic transcriptomic profile as compared to other ex vivo V51+ y5 T cells, and, in particular, show increased expression of genes that play a role in the TGF-P pathway including a variety of Tissue Resident Memory (TRM) receptors, skin-tropism markers, natural killer receptors (NKRs), and chemoattractant molecules. For example, skin-derived V51+ y5 T cells of the present disclosure, express more XCL1 and Tel, one or more NK cell receptors (e.g., NKG2D), but express less IFNy and TNFa. The unique transcriptomic profile is advantageous in functional aspects such as targeting and cytolytic activity. For example, the expanded skin-derived V51+ y5 T cells have higher cytolytic potentials, as compared to skin-derived V51+ y5 T cells expanded without TGF-p. The skin expanded V51+ cells gain the potential to produce chemoattractants, which are not inhibited by the addition of TGF-P, and facilitate recruitment to the tumor microenvironment, such that cells cultured in the presence of exemplary cytokines, IL-2 and IL-15, together with TGF-P are able to kill just as well or better than those cultured only in the presence of cytokines, IL-2 and IL- 15, but in the absence of TGF-p.

[0007] In some aspects, provided herein is a method of producing skin-derived V51+y5 T cells comprising expanding y5 T cells isolated from skin in the presence of one or more cytokines and TGF-P, wherein the expanded skin-derived V51+y5 T cells are selectively enriched for Tissue Resident memory cells (TRM cells).ATTORNEY DOCKET NO.: MIL-054WO1

[0008] In some aspects, provided herein is a method of expanding skin-derived V51+y5 T cells comprising culturing skin y5 T cells in a culture medium supplemented with TGF-P, wherein the expanded skin-V51+y5 T cells are characterized with expression of one or more skin specific-Tissue Resident memory cells (TRM) markers and skin-homing receptors.

[0009] In some embodiments, the expanded skin-derived V51+y5 T cells are CD103+, CD101+ AHR+, CXCR4+ and / or Vimentin+. In some embodiments, the expanded skin-derived V51+y5 T cells are CD103+. In some embodiments, the expanded skin-derived V51+y5 T cells are CD101+ AHR+. In some embodiments, the expanded skin-derived V51+y5 T cells are CXCR4+. In some embodiments, the expanded skin-derived V51+y5 T cells are Vimentin+.

[0010] In some embodiments, the expanded skin-derived V51+y5 T cells express one or more skin-homing receptors of CCR4, CCR6, CCR8, CCCR10 and CLA. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR4. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR6. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR8. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR10. In some embodiments, the expanded skin-derived V51+y5 T cells express CLA.

[0011] In some aspects, provided herein is a method of expanding skin-derived V51+y5 T cells comprising culturing y5 T cells isolated from skin in the presence of one or more cytokines (e.g., IL-2, IL-15) and TGF-P, wherein the expanded skin-derived V51+y5 T cells produce a high level of XCL1 and one or more chemokines of CCL3, CCL4 and CCL5, as compared to skin-derived V51+y5 T cells expanded without TGF-p.

[0012] In some embodiments, the culture medium comprises IL-2 and IL-15.

[0013] In some embodiments, the skin cells are expanded for at least 21 days.

[0014] In some embodiments, the skin cells are first expanded in the presence of IL-2 and IL-15 for 18 days and then expanded in the presence of IL-2, IL-15 and TGF-P for at least three days.

[0015] In some embodiments, the skin cells are cultured with IL-2, IL- 15 and TGF-p.

[0016] In some embodiments, the skin cells are cultured in a medium comprising a high concentration of glucose. In some embodiments, the expanded skin-derived V51+y5 T cells produce XCL1.

[0017] In some embodiments, the expanded skin-derived V51+y5 T cells produce Tel.ATTORNEY DOCKET NO.: MIL-054WO1

[0018] In some embodiments, the expanded skin-derived V51+y5 T cells produce XCL2.

[0019] In some embodiments, the expanded skin-derived V51+y5 T cells produce less IFNy and TNFa, as compared to expanded skin-derived V51+y5 T cells without TGF-p.

[0020] In some embodiments, the expanded skin-derived V51+y5 T cells produce one or more chemokines CCL3, CCL4 and CCL5.

[0021] In some embodiments, the expanded skin-derived V51+y5 T cells express one or more NK cell receptors.

[0022] In some embodiments, the expanded skin-derived V51+y5 T cells express NKp44, NKp46, NKG2D, NKG2A, NKG2C and / or CD94.

[0023] In some embodiments, the expanded skin-derived V51+y5 T cells express NKG2D.

[0024] In some embodiments, the expanded skin-derived V51+y5 T cells are cytolytic.

[0025] In some embodiments, the expanded skin-derived V51+y5 T cells have higher cytolytic potentials, as compared to skin-derived V51+y5 T cells expanded without TGF-p.

[0026] In some embodiments, the V51+y5 T cells are from human skin.

[0027] In some embodiments, the expanded skin-derived V51+y5 T cells are for allogeneic cell therapy.

[0028] In some embodiments, the method further comprises cryopreservation of the expanded V51+y5 T cells.

[0029] In some embodiments, provided herein is a population of skin-derived V51+y5 T cells produced by the method of the present disclosure.

[0030] In some embodiments, provided herein is a composition comprising the skin-derived V51+y5 T cells of the present disclosure.

[0031] In some aspects, provided herein is a method of treating a subject with a cancer comprising administering to the subject the expanded skin-derived V51+y5 T cells or the composition of the present disclosure.

[0032] In some embodiments, the treatment is allogeneic cell therapy.

[0033] In some embodiments, the subject has a solid tumor.

[0034] In some aspects, provided herein is use of the expanded skin-derived V51+y5 T cells of the present disclosure for the treatment of a cancer.ATTORNEY DOCKET NO.: MIL-054WO1

[0035] In some aspects, provided herein is a skin-derived V51+y5 T cell that produces XCL-1, wherein the skin derived V51+y5 T cell produces XCL-1 upon stimulation, in less time as compared to a skin derived CD8+ T cell.

[0036] In some embodiments, the skin-derived V51+y5 T cell produces one or more chemokines CCL3, CCL4 and CCL5.

[0037] In some embodiments, provided herein is a method of preparing CD 103+ positive human V51+TRM cell for use in immunotherapy, said method comprising isolating V51+y5 T cell from human skin and culturing the isolated skin-derived cells in the presence of (a) IL-2 and IL-15, and (b) TGF-p.

[0038] In some embodiments, the CD103+ positive human V51+TRM cells have enhanced cytotoxicity as compared to the V51+TRM cells prepared without TGF-p.BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1A - FIG. 1I shows that exemplary y5 T cells that are expanded from skin explants have a unique transcriptomic profile relative to their ex vivo counterparts. FIG. 1A shows the exemplary expansion process at a glance for y5 and CD8 T cells from blood and skin FIG. 1B is a principal component analysis, showing that the environment from which the exemplary cells are expanded from (e.g., ex vivo, blood, or skin) greatly influences the transcriptome of the cells, seemingly to a greater degree than the cell lineage. FIG. 1C -FIG. IE are MA plots of exemplary cells expanded from skin expanded V51 versus ex vivo skin V51 (FIG. 1C), skin expanded V51 versus blood V51 (FIG. 1D), or ex vivo skin V51 versus blood V51 (FIG. 1E) showing the differences in gene expression. FIG. IF is a heat map of the Z score of all differentially expressed genes in V51 and CD8 cells from various exemplary tissues in different donors. FIG. 1G - FIG. II demonstrates a comparison of pathways activated between cells expanded from skin expanded V51 versus ex vivo skin V51 (FIG. 1G), skin expanded V51 versus blood V51 (FIG. 1H), or ex vivo skin V51 versus blood V51 (FIG. 1I).

[0040] FIG. 2A - FIG. 2H demonstrate that exemplary skin expanded y5 and CD8 T cells retain a resident memory T cell (TRM) phenotype. FIG. 2A - FIG. 2C shows that exemplary ex vivo skin expanded y5 and CD8 T cells exhibit a composite skin specific TRM transcriptional profile. FIG. 2D is a heat map depicting the expression of genes associated with aP TRM cells in exemplary V51 and CD8 cells from different donors. FIG. 2E demonstrates that the addition of TGF-P to cultures with cytokines (IL-2 / IL-15) after 18 daysATTORNEY DOCKET NO.: MIL-054WO1for a 3-day incubation period or a 21-day incubation period in comparison to cultures with cytokines alone enhances the expression of exemplary TRM markers (mean MFI in FIG. 2F and representative flow cytometry staining in FIG. 2G). FIG. 2G demonstrates that the addition of TGF-P to cultures with cytokines (IL-2 / IL-15) enhances the expression of some skin-tropism receptors on expanded V51 cells.

[0041] FIG. 3A - FIG. 3K shows that exemplary V51 cells directly expanded from skin retain Tel and XCL1 potentials seen in ex vivo skin expanded and CD8 counterparts. FIG.3A depicts the level of various ligand transcripts in V51 and CD8 T cells expanded directly from skin, ex vivo from skin, or blood. FIG. 3B depicts that exemplary skin expanded V51 cells produce XCL1 upon TCR-stimulation. FIG. 3C is the exemplary flow cytometry staining of gated V51 cells showing the production of XCL1. FIG. 3D - FIG. 3F shows that exemplary skin expanded V51 cell produce XCL1 upon TCR stimulation and produce it earlier in comparison to colocalized CD8 T cells. FIG. 3D - FIG. 3F shows that the addition of TGF-P to cultures with cytokines (IL-2 / IL-15) augments the production of XCL1 (FIG.3D), ZFN-y (FIG. 3E), and TNFa (FIG. 3F) from exemplary skin-expanded cells. FIG. 3G-FIG. 3H show the quantification of XCL1 production upon stimulation when cultured with IL-2 / IL-15 or IL-2 / IL-15 / TGF-P. FIG. 3I - FIG. 3J depicts that prolonged exposure of exemplary skin expanded V51 cells to TGF-P significantly reduces their capacity to produce exemplary cytokines, IFNy and TNFa (representative flow cytometry staining in FIG. 3K).

[0042] FIG. 4A - FIG. 41 show that skin expanded V51 cells gain increased potential to produce exemplary chemoattractants, CCL3, CCL4 and CCL5 in comparison to their ex vivo counterparts. FIG. 4A depicts the levels of transcripts of CCL3, CCL4, and CCL5 of V51 cells expanded from skin, ex vivo skin explants, and blood in comparison to their CD8 counterparts. FIG. 4B - FIG. 4E depict the percentage of chemoattractant form from exemplary skin-expanded T cells. FIG. 4F - FIG. 4H show that exemplary culture conditions (18 days with IL-2 / IL-15, 3 days with IL-2 / IL-15 / TGF-P, 21 days with IL-2 / IL-15, and 21 days with IL-2 / IL-15 / TGF-P) are sufficient to induce the expression of CCL4 and CLL5, and that addition of TGF-P does not impair the expression of these chemokines. FIG. 41 is exemplary representative flow cytometry staining of cells expressing CCL3, CCL4, or CCL5 in each culture condition.

[0043] FIG. 5A - FIG. 5K demonstrate that exemplary skin expanded V51 cells cultured kill just as well, or better, than their counterparts that are cultured with IL-2 and IL-15. FIG.5A depicts the number of transcripts of cytolytic genes in V51 and CD8 T cells expanded from skin, ex vivo expanded skin, and blood. FIG. 5B quantifies the percentage of perforinATTORNEY DOCKET NO.: MIL-054WO1and granulysin positive V51 and CD8 T cells expanded from skin (representative flow cytometry staining in FIG. 5C). FIG. 5D and FIG. 5E shows the exemplary V51 cells cultured with cytokines (IL-2 / IL-15; FIG. 5D) and TGF-P (FIG. 5E) that were negatively sorted for killing assays after depletion of aP T cells. FIG. 5F - FIG. 5I shows the time course and quantification of the killing of HT-29 colorectal adenocarcinoma cells and A375 malignant melanoma cells by exemplary skin expanded V51 cells. FIG. 5J - FIG. 5K shows staining (FIG. 5J) and quantification of cytokines (FIG. 5K) expressed by cells cultured with and without TGF-P, further demonstrating the ability of skin-expanded V51 cells to kill target cancer cells with addition of TGF-p.

[0044] FIG. 6A - FIG. 6F is the quantification of exemplary y5 T cells expanded from skin donors (patients undergoing primary resection following a diagnosis of breast cancer or ductal carcinoma-in-situ, followed by breast reconstruction; four had neoadjuvant chemotherapy prior to donation) by flow cytometry in comparison to healthy donors. FIG.6A - FIG. 6F depict graphs of % CD3+ cells (FIG. 6A), % Pan y5+ cells (FIG. 6B), % V51+ cells (FIG. 6C), % CD107a+ cells (FIG. 6D), % IFNy+ cells (FIG. 6E) and % TNFa+ cells (FIG. 6F) in patients that underwent cosmetic procedure and a bulk RNAseq donor. All panels show that cancer patient and healthy donor skin expanded y5 T cells behaved comparably.

[0045] FIG. 7A - FIG. 7F shows that exemplary expanded skin-derived V51+cell transcript levels were comparable across healthy donors and breast cancer patients for six genes: CCL5 (FIG. 7A), XCL1 / 2 (FIG. 7B), IFNG (FIG. 7C), TOX (FIG. 7D), PRF1 (FIG.7E), and KLRK1 (FIG. 7F).

[0046] FIG. 8A - FIG. 8F show exemplary transcriptomic maps of various pathways comparing PBMCs, ex vivo skin expanded, and skin expanded V51+y5 T cells from different donors, including the mTORcl pathway (FIG. 8A), TNFa signaling pathway via NFKB (FIG. 8B), EMT pathway (FIG. 8C), E2F pathway (FIG. 8D), IFNy signaling pathway (FIG. 8E), and the TGF-P pathway (FIG. 8F).

[0047] FIG. 9A - FIG. 9B show the exemplary skin-specific TRM signatures associated with ex vivo skin-expanded V51+y5 T cells (FIG. 9A) and barrier TRM genes (FIG. 9B) versus PBMC derived V51+y5 T cells. These results indicate the upregulation of 25 mRNAs, including CXCR4 and CCR4, representative of skin-specific TRM.

[0048] FIG. 10A - FIG. 10D show that skin expanded V51+y5 T cells and CD8+T cells transcribe high levels of PEC AMI (FIG. 10A), which encodes CD31, and has a higherATTORNEY DOCKET NO.: MIL-054WO1surface expression of CD31 as percent positive cells (FIG. 10B) and MFI (FIG. 10C). FIG.10D shows flow cytometry sorting of V51+y5 T cells and CD8+T cells.

[0049] FIG. 11A - FIG. 11D demonstrates that the viability (FIG. 11 A) or recovery of CD3+ T cells (FIG. 11B), pan y5 T cells (FIG. 11C) or specifically V51+ T cells (FIG. 11D) expanded from skin were not impacted by exposure to TGF-p.

[0050] FIG. 12A - FIG. 12C demonstrates that skin expanded V51+y5 T cells exposed to TGF-P for three days following 18 day culturing with IL-2 / IL-15 (FIG. 12A), or TGF-P for three weeks (FIG. 12C) increased the percentage of V51+y5 T cells expressing tissue-associated homing receptors relative to IL-2 / IL-15 without TGF-P (FIG. 12B).

[0051] FIG. 13 is a transcriptomic map of exemplary cells expanded from PBMCs, ex vivo skin and skin expressed transcripts of TRM markers. This map further shows that TRM transcripts were upregulated and skin V51+cells expressed less exhaustion markers than their CD8+counterparts.

[0052] FIG. 14A - FIG. 14C show transcriptomic heat maps of exemplary V51+y5 T cells expanded from ex vivo skin explants and skin donors, demonstrating the V51+subtype skewed towards more the cytolytic transcriptomic signature. FIG. 14A shows the heat map for cytotoxicity transcripts. FIG. 14B shows the heat map for intermediate (bipotent progenitor of wound healing and CRC surveillance) phenotype transcripts. FIG. 14C shows the heat map for wound healing transcripts.

[0053] FIG. 15A - FIG. 15H shows the phenotypic skewing of exemplary V51+y5 T cells expanded from PBMCs, ex vivo skin explants, and skin donors. FIG. 15A is a table that shows most transcription factors that might drive skewed phenotypes were either expressed at low levels or not enriched in any cell population. FIG. 15B - FIG. 15D are transcriptomic maps similar to those of FIG. 14A - FIG. 14C, showing a dynamic trichotomy between ex vivo skin and skin-expanded CD8+cells. FIG. 15E is an MA plot visualization showing the similarity of the transcriptomes between ex vivo expanded and skin expanded V51+cells. FIG. 15F and FIG. 15G show the levels of TRAC, CD8A, CD8B, CD2, CD28, TRDV1 and TRGC1 in V51+and CD8+cells respectively, wherein the V51+cells showed consistently higher expression of these transcripts. FIG. 15H shows the consistently higher expression of different transcripts in ex vivo V51+and CD8+cells, including significantly higher XCL1 expression.

[0054] FIG. 16A - FIG. 16L shows the exemplary tumor cell targeting potential of skin expanded V51+y5 T cells. FIG. 16A shows intracellular staining detected for Granzyme A (GZMA), Granzyme B (GZMB), and NKG7 in all V51+and CD8 expansion cells. FIG. 16BATTORNEY DOCKET NO.: MIL-054WO1and FIG. 16C show that expanded V51+cells express mRNAs for several natural killer receptors (NKR). FIG. 16D and FIG. 16E (FACS data) show expression of various NKRs were upregulated in skin expanded V51 cells, however TGF-P exposure significantly diminished NKp30 expression. FIG. 16F shows expression of skin expanded V51 cells expressed “innateness” transcripts at a higher basal level than their CD8 counterparts. FIG.16G and FIG. 16H show percent expression of 4- IBB on cell surface. FIG. 161 shows expression of TMSB4X in V51 skin expanded cells. FIG. 16J shows percent TRGV postsort. FIG. 16K and FIG. 16L show FACS data of cells expanded from donors without (FIG.16K) or with exposure to TGF-P (FIG. 16L), respectively, wherein the skin expanded cells show high TCR diversity.DEFINITIONS

[0055] In order that the present disclosure can be more readily understood, certain terms are first defined. Unless otherwise defined herein, scientific and technical terms used in connection with the present application shall have the meanings that are commonly understood by those of ordinary skill in the art.

[0056] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The terms “comprise,” “comprises” and “comprising” and variations thereof (e.g., “comprises / comprising,” “includes / including”) should be understood to imply the inclusion of a stated component, feature, element or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element or step or group of components, features, elements or steps. Any one of the terms "comprising," consisting essentially of, "and" consisting of may be substituted with either of the other two terms, while retaining their ordinary meaning.

[0057] Administer'. As used herein, the term “administer” and its various grammatical forms refers to giving, applying or bringing a composition into contact with a subject.Administration can be accomplished by any of a number of routes, such as, for example, topical, oral, subcutaneous, intramuscular, intraperitoneal, intravenous, intrathecal, and intradermal.

[0058] Allogeneic cell therapy: As used herein, the term “allogeneic cell therapy” refers to a type of cellular therapy that uses cells from a donor (e.g., a healthy person) to treat a patient. Cells from a single donor can be used to treat one or more patients. Donor cells (e.g., V51+ y5 T cells isolated from a donor’s skin) can undergo extensive expansion and screeningATTORNEY DOCKET NO.: MIL-054WO1before establishing a master cell bank, which is the basis for generating allogeneic cell therapies. Cells from the donor can be modified (e.g., expressing a chimeric antigen receptor (CAR)) or unmodified. The cells can be derived from multiple potential cell sources, including but not limited to donated tissues (e.g., skin). An advantage of allogeneic strategies, among other things, is “off-the-shelf’ supply. For example, in urgent medical situations, allogeneic therapies utilize donor’s cells, allowing storage and immediate availability to treat multiple patients. Exemplary allogeneic cell therapies include CAR-T cell therapy.Allogeneic CAR-T cell therapy involves engineering T cells from a donor to express chimeric receptors that target cancer cells which are then infused into patients. In the context of the present invention, the y5 T cells may be produced for the manufacture of a medicament for treating various cancers.

[0059] Biopsy. As used herein, the term “biopsy” refers to a sample of tissue taken directly from a subject / donor. In some embodiments, the biopsy can be a skin biopsy from a tissue donor for expansion of cells.

[0060] Cancer-. As used herein, the term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers are described herein and include but are not limited to, leukemia, lymphoma, multiple myeloma, ovarian cancer, breast cancer, endometrial cancer, colon cancer (colorectal cancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer, bile duct cancer, esophageal cancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma of the head and neck (head and neck cancer), testicular cancer, cancer of the endocrine gland, cancer of the adrenal gland, cancer of the pituitary gland, cancer of the skin, cancer of soft tissues, cancer of blood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancer of hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma, neuroblastoma, melanoma, myelodysplastic syndrome, and a sarcoma and the like.

[0061] Cell population. As used herein, the term "cell population" refers to a number of cells obtained by isolation directly from a suitable source, usually from a mammal. The isolated cell population may be subsequently cultured in vitro. Those of ordinary skill in the art will appreciate that various methods for isolating and culturing cell populations for useATTORNEY DOCKET NO.: MIL-054WO1with the present invention and various numbers of ceils in a cell population that are suitable for use in the present invention.

[0062] Chemokine As used herein, the term “chemokine” refers to a large group of proteins that stimulate the movement of certain types of white blood cells (e.g, yS T cells) and attracts them to areas of inflammation to help fight infections, inflammatory conditions, and other diseases. It should be noted that a ‘■‘chemokine” is a type of cytokine.

[0063] Culture'. The term “culture” or “cell culture” or “culturing” refers to the maintenance, growth and / or differentiation of cells in an in vitro environment. In various methods described herein, cells are cultured in a particular cell culture medium (or “media” in case of a plural) which facilitates or promotes the growth or differentiation of one type of cell into a different type of cell. A cell culture medium acts as a source of nutrients, hormones and / or other factors helpful to propagate and / or sustain the cells.

[0064] Cytokine-. As used herein, the term “cytokine” refers to a large class of secreted proteins, peptides, and / or glycoproteins that are essential in cell-to-cell communication. In the broadest sense of the term, cytokines are proteins that are secreted cells of the immune system to control growth and function of other immune cells, as well as the inflammatory response. The term also encompasses several classes of proteins, including but not limited to, tumor necrosis factors (TNFs), interferons (IFNs), transforming growth factors (TGFs) and interleukins (ILs). In various embodiments, the cytokines used for expansion of immune cells include, but are not limited to IL-2, IL- 15, and TGF-p.

[0065] Cytolytic-. As used herein, the term “cytolytic” refers to the ability of a cell (e.g., y5 cells) to kill a target pathogen or aberrant cell.

[0066] Derived. As used herein, the term “derived” refers to obtaining something from a specified source. In the present disclosure, the term “derived” refers to obtaining y5 cells from non-hematopoietic tissue (e.g., skin).

[0067] Enhance'. As used herein, the term "enhance" refers to an increase or improvement in the function or activity of a cell after administration or contacting witha combination described herein compared to the cell prior to such administration or contact.

[0068] Expansion: As used herein, the term “expansion” or “expanding” refers to a method of growing a cell population in tissue culture that increases the number of cells in that population. Cells that have undergone ex vivo expansion are referred to as “expanded.” A synonym that may be used for “expansion” in this context is “proliferation.”ATTORNEY DOCKET NO.: MIL-054WO1

[0069] Expanded population of Gamma Delta T cells'. As used herein, “expanded” or “expanded population of gamma delta T cells” includes populations of cells which are larger or contain a larger number of cells than a non-expanded population. Such populations may be large in number, small in number or a mixed population with the expansion of a proportion or particular cell type within the population. It will be appreciated that the term “expansion step” refers to processes which result in expansion or an expanded population. Thus, expansion or an expanded population may be larger in number or contain a larger number of cells compared to a population which has not had an expansion step performed or prior to any expansion step. It will be further appreciated that any numbers indicated herein to indicate expansion (e.g., fold-increase or fold-expansion) are illustrative of an increase in the number or size of a population of cells or the number of cells and are indicative of the amount of expansion.

[0070] Explant: As used herein, the term “explant” refers to a cell, organ, or piece of tissue which has been transferred from animals or plants to a nutrient medium.

[0071] Ex vivo: As used herein, the term “ex vivo" refers to events that occur outside of a living organism, e.g., in tissue taken from an organism in an external environment.

[0072] Gamma delta (yd) T cells'. Gamma delta (y5) T cells refer to a subset of T cells that express their surface a distinct and defining T cell receptor (TCR). This TCR is composed of one gamma chain (y) and one delta (5) chain. The heterodimer y5 TCR can recognize antigens. y5 T cells can display a pre-activated and memory phenotype and the high frequency of these cells enables rapid responses without the presence of cognate TCR agonists and / or cellular expansion. In humans, y5 T cells are classified according to their V5 gene segment used. Three V5 genes V51-3; and seven functional Vy gene segments: Vy2-5, Vy8, Vy9, and Vyl 1, exist in human y5 T cells. The y5 TCR repertoire is also associated with the tissue distribution. For example, V51+ T cells are abundant in the epithelium in human.

[0073] Immune cells'. As used herein, the term “immune cell” or “immune cells” refers to cells of the immune system, including, but not limited to, aP T cells, y5 T cells, NK cells, T / NK cells, dendritic cells, macrophages, B cells, neutrophils, erythrocytes, monocytes, basophils, neutrophils, mast cells, eosinophils, and any combination thereof.

[0074] In vitro'. As used herein, the term “in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.ATTORNEY DOCKET NO.: MIL-054WO1

[0075] In vivo'. As used herein, the term “in vivo” refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cellbased systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).

[0076] MA Plot'. An MA plot is an application of a Bland-Altman plot for visual representation of genomic data. The plot visualizes the differences between measurements taken in two samples, by transforming the data onto M (log ratio) and A (mean average) scales, then plotting these values.

[0077] Principal Component Analysis: As used herein, the term “Principal Component Analysis” refers to a dimensionality reduction method that is often used to reduce the dimensionality of large data sets, by transforming a large set of variables into a smaller one that still contains most of the information in the large set.

[0078] Therapeutically effective amount'. As used herein, the term “therapeutically effective amount” means an amount effective, at dosages, frequency of administration and for duration of time necessary to achieve the desired results such that one or more symptoms or biomarkers is improved after treatment.

[0079] Tissue-resident memory T cells (TRM cells): As used herein, the term “Tissueresident memory T cells (TRM cells)” refers to a type of memory T cells that remains in tissues instead of recirculating through the body. TRM cells are found in skin, lungs, gut, reproductive lining and other barrier tissues to block entry of pathogens. They play a role in immune surveillance, tumor immunity and fast response to re-exposure by pathogens, detecting and responding quickly, is in part due to expression of CD69.DETAILED DESCRIPTION

[0080] The present disclosure provides, among other things, improved methods for expanding V51+ y5 T cells which are derived from skin. In particular, the y5 T cells are V51 + y5 T cells that upregulate the expression of a variety of Tissue Resident Memory (TRM) receptors, skin-tropism markers, natural killer receptors (NKRs), and chemoattractant molecules. The present disclosure also provides compositions comprising y5 T cells and methods of use thereof. The improved processes as described herein, relate to expansion of skin-derived y5 T cells (e.g., V51+ y5 T cells) with co-culture cytokines, such as IL-2 and IL-ATTORNEY DOCKET NO.: MIL-054WO115, together with other cytokines that are specifically related to the skin microenvironment (e.g., TGF-P).

[0081] Particularly, V51+ y5 T cells expanded from whole skin tissue have a unique transcriptomic profile, and genes that play a role in the TGF-P pathway are highly upregulated. While V51+ y5 T cells from ex vivo whole skin retain a generic tissue resident memory T cell (TRM) profile, addition of TGF-P to cultures restores / enhances the expression of TRM markers and skin-tropism receptors on expanded V51+ cells. Further, V51+ y5 T cells expanded from whole skin tissue closely resemble co-localised tissue resident CD8 T cells but have higher levels of effector potentials and are more innate-like and produce XCL1 more rapidly. Furthermore, skin expanded V51+ cells gain the potential to produce chemoattractants, which are not inhibited by the addition of TGF-P, increasing their immunoregulatory potentials and facilitating their recruitment to a tumor microenvironment and effective cytotoxic activity. Cells cultured in IL-2 / IL-15 / TGF-P also produce high levels of XCL-1 and are able to kill just as well or better than cells not cultured in the presence of TGF-P but rather cultured in IL-2 / IL-15 alone.

[0082] In some aspects, provided herein is a method of producing skin-derived V51+y5 T cells comprising expanding y5 T cells isolated from skin in the presence of one or more cytokines and TGF-P, wherein the expanded skin-derived V51+y5 T cells are selectively enriched for Tissue Resident memory cells (TRM cells).

[0083] In some aspects, provided herein is a method of expanding skin-derived V51+y5 T cells comprising culturing skin y5 T cells in a culture medium supplemented with TGF-P, wherein the expanded skin-V51+y5 T cells are characterized with expression of one or more skin specific-Tissue Resident memory cells (TRM) markers and skin-homing receptors.

[0084] In some aspects, provided herein is a method of expanding skin-derived V51+y5 T cells comprising culturing y5 T cells isolated from skin in the presence of one or more cytokines (e.g., IL-2, IL-15) and TGF-P, wherein the expanded skin-derived V51+y5 T cells produce a high level of XCL1 and one or more chemokines of CCL3, CCL4 and CCL5, as compared to skin-derived V51+y5 T cells expanded without TGF-p.

[0085] In some aspects, provided herein is a method of treating a subject with a cancer comprising administering to the subject the expanded skin-derived V51+y5 T cells prepared by the method of the present disclosure.

[0086] In some aspects, provided herein is use of the expanded skin-derived V51+y5 T cells of the present disclosure for the treatment of a cancer.ATTORNEY DOCKET NO.: MIL-054WO1

[0087] In some aspects, provided herein is a skin-derived V51+y5 T cell that produces XCL-1, wherein the skin derived V51+y5 T cell produces XCL-1 upon stimulation, in less time as compared to a skin derived CD8+ T cell.Methods of Expanding Skin-Derived V81+y T Cells

[0088] Human y§ T-cells use three main vS (v31, v32, v33) and at most six Vy region genes to make their TCRs. Among the four subtypes of human y5 T cells defined by the TCR 5 chain, V51+ y5 T cells is one of the two subtypes that are the most predominant. V51+ y5 T cells, for example, recognize target cells and mediate anti-tumor activity through the direct lysis of transformed cells. The present disclosure provides improved methods for expanding skin-derived V51+ y5 T cells.

[0089] In one aspect, the present disclosure provides methods for expanding skin-derived V51+ y 5 T cells from a cell population. The cell population may be a mixed cell population comprising y5 T cells isolated from skin tissue. In some embodiments, the present disclosure provides methods for expanding V51+ y5 T cells from a cell population derived from whole skin biopsies from donors. Methods and processes of the present disclosure are for expanding y5 T cells (e.g., V51+ y5 T cells), for example, for use in adoptive cell therapy.

[0090] In some embodiments, the methods described herein involve culturing a cell population comprising y5 T cells with cytokines that induce expansion (e.g. IL-2 and IL-15) and are important in the skin's microenvironment (<?.g, TGF-p).

[0091] In some aspects, provided herein is a method of producing skin-derived V51+y5 T cells comprising expanding y5 T cells isolated from skin in the presence of one or more cytokines and TGF-P, wherein the expanded skin-derived V51+y5 T cells are selectively enriched for Tissue Resident memory cells (TRM cells).

[0092] Transforming growth factor beta (TGF-P) is a multifunctional cytokine belonging to the transforming growth factor superfamily that includes three different mammalian isoforms (TGF-P 1 to 3, HGNC symbols TGFB1, TGFB2, TGFB3) and many other signaling proteins. In the present disclosure, addition of TGF-P expands skin-derived V51+y5 T cells with a unique transcriptomic profile and functional improvements in targeting and killing tumors.

[0093] In various methods and processes disclosed herein, the skin-derived cells being expanded are cultured in any suitable cell culture medium supplemented with for example, cytokines for cell culture. In some aspects, provided herein is a method of expanding skin-derived V51+y5 T cells comprising culturing skin y5 T cells in a culture mediumATTORNEY DOCKET NO.: MIL-054WO1supplemented with TGF-P, wherein the expanded skin-V51+y5 T cells are characterized with expression of one or more-skin specific-Tissue Resident memory cells (TRM) markers and skin-homing receptors.

[0094] In some embodiments, the cells are from human skin.

[0095] In some aspects, provided herein is a method of expanding skin-derived V51+y5 T cells comprising culturing y5 T cells isolated from skin in the presence of one or more cytokines (e.g., IL-2, IL-15) and TGF-P, wherein the expanded skin-derived V51+y5 T cells produce a high level of XCL1 and one or more chemokines of CCL3, CCL4 and CCL5, as compared to skin-derived V51+y5 T cells expanded without TGF-p.Expansion Protocol

[0096] In accordance with methods and processes for preparing y5 T cells, a cell population that has been treated exemplary cytokines, IL-2 and IL-15, and with the addition of TGF-P is used to expand skin-derived y5 T cells. In one aspect, a cell population is treated with IL-2, IL-15, and TGF-p. The expansion stimulates proliferation of y5 T cells and increases the cell number. In some embodiments, the expansion further selectively enriches particular cell types.

[0097] In some embodiments, the culture medium for expanding cells comprises interleukins, such as IL-2 and IL- 15. In some embodiments, the culture medium further comprises TGF-p. In some embodiments, the culture medium comprises IL-2, IL-15 and TGF-p.

[0098] In some embodiments, the skin cells are expanded for at least 21 days.

[0099] In some embodiments, the skin cells are first expanded in the presence of IL-2 and IL-15 for 18 days and then expanded in the presence of IL-2, IL-15 and TGF-P for at least three days.

[0100] In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 14 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 15 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 16 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 17 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 18 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 19 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 20ATTORNEY DOCKET NO.: MIL-054WO1days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 21 days.

[0101] In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 1 to 21 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 1 day. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 2 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 3 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 4 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 5 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 6 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 7 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 8 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 9 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 10 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 11 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 12 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 13 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 14 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 15 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 16 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 17 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 18 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 19 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL- 15 for 20 days. In some embodiments, the y5 T cells expanded from skin are cultured with IL-2 and IL-15 for 21 days.

[0102] In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for at least three days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL- 15 and TGF-P for three days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 3-12 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 3 days. In some embodiments, the skin cells areATTORNEY DOCKET NO.: MIL-054WO1subsequently expanded in the presence of IL-2, IL- 15 and TGF-P for 4 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 5 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 6 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL- 15 and TGF-P for 7 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 8 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 9 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL- 15 and TGF-P for 10 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 11 days. In some embodiments, the skin cells are subsequently expanded in the presence of IL-2, IL-15 and TGF-P for 12 days.

[0103] In some embodiments, the y5 T cells are expanded from skin are cultured with IL-2 and IL-15 for 18 days, then IL-2, IL-15, and TGF-P for 3 days. In some embodiments the y5 T cells are expanded from skin are cultured with IL-2, IL- 15, and TGF-P for 21 days.Tissue resident memory T cells

[0104] In some embodiments, the expanded V51+ y5 T cells are selectively enriched with Tissue resident memory cells (TRM).

[0105] Tissue resident memory (TRM) cells are resident at epithelial sites where most solid tumors develop and are thought to be involved in cancer immunity from the development stage. They can persist as durable memory cells and maintain potent recall functions, while also recognizing a wide variety of antigens. Further, receptor phenotypes that are reflective of higher cytotoxic potential and ability to produce cytokines and chemokines make immune cells more tractable for targeting aberrant cells, such as cancer. These phenotypes make these immune cells highly desirable for adoptive cell therapies.

[0106] In some embodiments, V51+ T cells expanded from skin express tissue resident memory (TRM) receptors. The TRM homing receptors include, but are not limited to CD103, CD101, AHR, CXCR4, and Vimentin. In some embodiments, the addition of TGF-P to cultures restores and / or enhances the expression of TRM receptors. In some embodiments, the expanded V51+ y5 T cells express at least one or more receptors from CD103, CD101, AHR, CXCR4, and Vimentin. In some embodiments, the expanded skin-derived V51+ y5 T cells express two, three, four, or five receptors of CD 103, CD101, AHR, CXCR4, and Vimentin.ATTORNEY DOCKET NO.: MIL-054WO1

[0107] In some embodiments, the expanded V51+ y5 T cells are CD103 positive. In some embodiments, the expanded V51+ y5 T cells are CD101 positive. In some embodiments, the expanded V51+ y5 T cells are AHR positive. In some embodiments, the expanded V51+ y5 T cells are CXCR4 positive. In some embodiments, the expanded V51+ y5 T cells are vimentin positive.Skin homing receptors

[0108] As used herein, “skin homing receptors” is used interchangeably with “skin tropism receptors.” In some embodiments, the V51+ y5 T cells expanded from skin express skin-tropism or skin-homing receptors. In some embodiments, the addition of TGF-P to cultures restores and / or enhances the expression of some skin-tropism receptors. In some embodiments, the expanded skin-derived V51+y5 T cells express one or more skin-homing receptors. In some embodiments, the one or more skin-homing receptors are CCR4, CCR6, CCR8, CCR10 and CLA. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR4. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR6. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR8. In some embodiments, the expanded skin-derived V51+y5 T cells express CCR10. In some embodiments, the expanded skin-derived V51+y5 T cells express CLA.

[0109] In some embodiments, the expanded skin-derived V51+y5 T cells express at least two, three, four or five skin-homing receptors. In some embodiments, the expanded skin-derived V51+y5 T cells express at least two skin-homing receptors. In some embodiments, the expanded skin-derived V51+y5 T cells express at least three skin-homing receptors. In some embodiments, the expanded skin-derived V51+y5 T cells express at least four skinhoming receptors. In some embodiments, the expanded skin-derived V51+y5 T cells express at least five skin-homing receptors.

[0110] In some embodiments, the one or more skin-homing receptors are CCR4 and CCR6. In some embodiments, the one or more skin-homing receptors are CCR4 and CCR8. In some embodiments, the one or more skin-homing receptors are CCR4 and CCR10. In some embodiments, the one or more skin-homing receptors are CCR4 and CLA.

[0111] In some embodiments, the one or more skin-homing receptors are CCR6 and CCR8. In some embodiments, the one or more skin-homing receptors are CCR6 and CCR10. In some embodiments, the one or more skin-homing receptors are CCR6 and CLA.

[0112] In some embodiments, the one or more skin-homing receptors are CCR8 and CCR10. one or more skin-homing receptors are CCR8 and CLA.ATTORNEY DOCKET NO.: MIL-054WO1

[0113] In some embodiments, the one or more skin-homing receptors are CCR4, CCR6 and CCR8. In some embodiments, the one or more skin-homing receptors are CCR4, CCR6 and CCR10. In some embodiments, the one or more skin-homing receptors are CCR4, CCR6 and CLA.Cytolytic function of Skin-Derived Vdl+ T cells

[0114] In some embodiments, the V51+ T cells expanded from skin are cytolytic. In some embodiments, the V51+ T cells expanded from skin have a greater cytolytic potential than cells expanded from ex vivo skin explants.

[0115] In some embodiments, V51+ T cells expanded from skin express cytokines and other effector molecules to a greater degree than V51+ T cells obtained from ex vivo skin explants. In some embodiments, V51+ T cells expanded from skin express cytokines and other effector molecules to a greater degree than CD8 T cells expanded from skin. In some embodiments, the cytokines produced are XCL1, XCL2, and IFNy. In some embodiments, V51+ T cells expanded from skin produce XCL1 upon TCR stimulation. In some embodiments, V51+ T cells expanded from skin produce XCL2 upon TCR stimulation. In some embodiments, V51+ T cells expanded from skin produce IFNy upon TCR stimulation. In some embodiments, V51+ T cells expanded from skin produce XCL1 upon TCR stimulation earlier than colocalized CD8 T cells. In some embodiments, addition of TGF-P to V51+ T cells expanded from skin augments the production of XCL1. In some embodiments, addition of TGF-P to V51+ T cells expanded from skin reduces their ability to produce IFNy and TNFa.

[0116] In some embodiments, V51+ T cells expanded from skin express various chemokines. In some embodiments, V51+ T cells expanded from skin express chemokines to a greater degree than CD8 T cells. In some embodiments, V51+ T cells expanded from skin express chemokines to a greater degree than V51+ T cells expanded from ex vivo skin explants.

[0117] In some embodiments, the expanded skin-derived V51+y5 T cells express XCL1. In some embodiments, the expanded skin-derived V51+y5 T cells express Tel.

[0118] In some embodiments, the expanded skin-derived V51+y5 T cells express less IFNy and TNFa, as compared to expanded skin-derived V51+y5 T cells without TGF-p. In some embodiments, the expanded skin-derived V51+y5 T cells express less IFNy, as compared to expanded skin-derived V51+y5 T cells without TGF-p. In some embodiments,ATTORNEY DOCKET NO.: MIL-054WO1the expanded skin-derived V51+y5 T cells express less TNFa, as compared to expanded skin-derived V51+y5 T cells without TGF-p.

[0119] In some embodiments, the expanded skin-derived V51+y5 T cells express one or more chemokines CCL3, CCL4 and CCL5. In some embodiments, the expanded skin-derived V51+y5 T cells express CCL3. In some embodiments, the expanded skin-derived V51+y5 T cells express CCL4. In some embodiments, the expanded skin-derived V51+y5 T cells express CCL5.

[0120] In some embodiments, the expanded skin-derived V51+y5 T cells express CCL3 and CCL4. In some embodiments, the expanded skin-derived V51+y5 T cells express CCL3 and CCL5. In some embodiments, the expanded skin-derived V51+y5 T cells express CCL4 and CCL5. In some embodiments, the expanded skin-derived V51+y5 T cells express CCL3, CCL4 and CCL5.

[0121] In some embodiments, the expanded skin-derived V51+y5 T cells express chemokines that are chemoattractants for CCR5+ and CCR1+, XCR1+ dendritic cells (DCs), Macrophages and T cells. In some embodiments, the expanded skin-derived V51+y5 T cells show increased recruitment to a tumor microenvironment.

[0122] In some embodiments, the expanded skin-derived V51+y5 T cells express one or more NK cell receptors. In some embodiments, V51+ T cells expanded from skin express NKp30, NKp44, NKp46, NKG2A, NKG2C, NKG2D, and / or CD94. In some embodiments, the expanded skin-derived V51+y5 T cells express NKG2D. In some embodiments, V51+ T cells expanded from skin express NKp30. In some embodiments, V51+ T cells expanded from skin express NKp44. In some embodiments, V51+ T cells expanded from skin express NKp30, NKp46. In some embodiments, V51+ T cells expanded from skin express NKG2A. In some embodiments, V51+ T cells expanded from skin express NKG2C. In some embodiments, V51+ T cells expanded from skin express CD94.

[0123] In some embodiments, the expanded skin-derived V51+y5 T cells are cytolytic.

[0124] In some embodiments, the expanded skin-derived V51+y5 T cells have higher cytolytic potentials, as compared to skin-derived V51+y5 T cells expanded without TGF-p.

[0125] In some embodiments, the V51+y5 T cells are from human skin.

[0126] In some embodiments, the expanded skin-derived V51+y5 T cells are for allogeneic cell therapy.

[0127] In some embodiments, the method further comprises cryopreservation of the expanded V51+y5 T cells.ATTORNEY DOCKET NO.: MIL-054WO1

[0128] In some embodiments, V51+ T cells expanded from skin kill target cells just as well or better when cultured with IL-2, IL-15, and TGF-P than IL-2 and IL-15 alone.Skin-Derived Vdl+ y T cell Compositions

[0129] In some aspects, provided herein is a population of skin-derived V51+y5 T cells produced by the method of the present disclosure.

[0130] In some aspects, provided herein is a composition comprising the skin-derived V51+y5 T cells of the present disclosure.

[0131] In some aspects, the present disclosure provides a pharmaceutical composition comprising y5 T cells manufactured by methods and processes described herein. In some embodiments, the pharmaceutical composition comprises skin-derived V51+ y5 T cells manufactured by methods and processes described herein. The term “pharmaceutical formulation” refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. A “pharmaceutically acceptable carrier’’ refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is no toxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. In some aspects, the choice of carrier is determined in part by the particular cell and / or by the method of admini tration. Accordingly, there are a variety of sui table form ulati ons.

[0132] In some embodiments, the pharmaceutical composition comprises skin-derived V51+ y5 T cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some embodiments is monitored by periodic assessment of treated subjects.

[0133] In some embodiments, the composition comprises a desired cell dosage. The desired cell dosage can be delivered by a single bolus administration of the composition, by multiple bolus administrations of the composition, or by continuous infusion administration of the composition. The cells may be administered using standard administration techniques, formulations, and / or devices. Provided are formulations and devices, such as syringes and vials, for storage and administration of the compositions. Cells can be administered via localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or parenteral administration. When administering a therapeutic composition (e.g., a pharmaceutical composition containing a genetically modifiedATTORNEY DOCKET NO.: MIL-054WO1immunoresponsive cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).

[0134] In some embodiments, compositions comprising cells are provided as sterile liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which may in some aspects be buffered to a selected pH. Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

[0135] In some embodiments, the pharmaceutical composition comprises predominantly skin-derived V51+ y5 T cells.

[0136] In some embodiments, the pharmaceutical composition comprises a buffer, a salt, and an excipient. In some embodiments, the pharmaceutical composition comprises a buffer. In some embodiments, the pharmaceutical composition comprises a salt. In some embodiments, the pharmaceutical composition comprises an excipient.

[0137] In some embodiments, the skin-derived V51+ y5 T cells are reconstituted in a pharmaceutically acceptable carrier.Methods of Use of d T Cells

[0138] In another aspect, the present disclosure provides methods of use of y5 T cells (e.g., V51+ y5 T cells) prepared by a method described herein.

[0139] In some aspects, provided herein is a method of treating a subject with a cancer comprising administering to the subject the expanded skin-derived V51+y5 T cells prepared by the method of the present disclosure.

[0140] In some embodiments, the treatment is allogeneic cell therapy.

[0141] In some aspects, provided herein is use of the expanded skin-derived V51+y5 T cells of the present disclosure for the treatment of a cancer.

[0142] In some aspects, provided herein is a skin-derived V51+y5 T cell that produces XCL-1, wherein the skin derived V51+y5 T cell produces XCL-1 upon stimulation, in less time as compared to a skin derived CD8+ T cell.

[0143] In some embodiments, the skin-derived V51+y5 T cell produces one or more chemokines CCL3, CCL4 and CCL5.ATTORNEY DOCKET NO.: MIL-054WO1

[0144] In some embodiments, provided herein is a method of preparing CD 103+ positive human V51+TRM cell for use in immunotherapy, said method comprising isolating V51+y5 T cell from human skin and culturing the isolated skin-derived cells in the presence of (a) IL-2 and IL-15, and (b) TGF-p.Adoptive Cell Therapy

[0145] In accordance with the present disclosure, V51+ y5 T cells prepared by a method described herein may be used for applications in adoptive cell therapy.

[0146] T cells for adoptive cell therapy can be isolated from the skin of a subject, grown in vitro! ex vivo at large numbers and optionally modified to enhance targeting of cancers, then reintroduced at therapeutically effective doses into the same or different patient. T cells engineered to express a CAR (CAR-T) or a TCR, and tumor infiltrating lymphocyte (TIL) therapies fall under the umbrella of allogeneic T cell therapy. Reintroduction of these T cells into a subject in need thereof is critical for augmenting the immune response against diseases.

[0147] In some embodiments, adoptive cell therapy is allogeneic cell therapy. Allogeneic cell therapy is a type of immunotherapy in which T cells obtained from one subject (donor) are given to a different subject to help the immune system fight diseases such as cancers.

[0148] In some embodiments, V51+ y5 T cells are derived from a donor and prepared for allogeneic cell therapy for treating one or more patients.

[0149] In some embodiments, V51+ y5 T cells are used for treatment of a disease, wherein the disease is an autoimmune / rheumatic disease, cancer and / or other malignancy.

[0150] In some embodiments, the autoimmune / rheumatic disease includes, but is not limited to rheumatic arthritis, systemic lupus erythematosus, systemic sclerosis, ankylosing spondylitis, and juvenile idiopathic arthritis.

[0151] In some embodiments, the cancers include, but are not limited to, carcinoma, lymphoma, glioblastoma, melanoma, sarcoma, and leukemia, myeloma, or lymphoid malignancies. More particular examples of such cancers are noted below and include: squamous cell cancer (e.g., epithelial squamous cell cancer), Ewing sarcoma, Wilms tumor, astrocytomas, glioblastomas, lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma multiforme, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, hepatocellular carcinoma, neuroendocrine tumors, medullary thyroid cancer,ATTORNEY DOCKET NO.: MIL-054WO1differentiated thyroid carcinoma, breast cancer, ovarian cancer, colon cancer, rectal cancer, endometrial cancer or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, anal carcinoma, penile carcinoma, as well as head-and-neck cancer. The term “cancer” includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).

[0152] Other examples of cancers or malignancies include, but are not limited to: Acute Childhood Lymphoblastic Leukemia, Acute Lymphoblastic Leukemia, Acute Lymphocytic Leukemia, Acute Myeloid Leukemia, Adrenocortical Carcinoma, Adult (Primary) Hepatocellular Cancer, Adult (Primary) Liver Cancer, Adult Acute Lymphocytic Leukemia, Adult Acute Myeloid Leukemia, Adult Hodgkin's Lymphoma, Adult Lymphocytic Leukemia, Adult Non-Hodgkin's Lymphoma, Adult Primary Liver Cancer, Adult Soft Tissue Sarcoma, AIDS-Related Lymphoma, AIDS-Related Malignancies, Anal Cancer, Astrocytoma, Bile Duct Cancer, Bladder Cancer, Bone Cancer, Brain Stem Glioma, Brain Tumors, Breast Cancer, Cancer of the Renal Pelvis and Ureter, Central Nervous System (Primary) Lymphoma, Central Nervous System Lymphoma, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Childhood (Primary) Hepatocellular Cancer, Childhood (Primary) Liver Cancer, Childhood Acute Lymphoblastic Leukemia, Childhood Acute Myeloid Leukemia, Childhood Brain Stem Glioma, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Childhood Extracranial Germ Cell Tumors, Childhood Hodgkin's Disease, Childhood Hodgkin's Lymphoma, Childhood Hypothalamic and Visual Pathway Glioma, Childhood Lymphoblastic Leukemia, Childhood Medulloblastoma, Childhood Non-Hodgkin's Lymphoma, Childhood Pineal and Supratentorial Primitive Neuroectodermal Tumors, Childhood Primary Liver Cancer, Childhood Rhabdomyosarcoma, Childhood Soft Tissue Sarcoma, Childhood Visual Pathway and Hypothalamic Glioma, Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Colon Cancer, Cutaneous T-Cell Lymphoma, Endocrine Pancreas Islet Cell Carcinoma, Endometrial Cancer, Ependymoma, Epithelial Cancer, Esophageal Cancer, Ewing's Sarcoma and Related Tumors, Exocrine Pancreatic Cancer, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Eye Cancer, Female Breast Cancer, Gaucher's Disease, Gallbladder Cancer, Gastric Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Tumors, Germ Cell Tumors, Gestational TrophoblasticATTORNEY DOCKET NO.: MIL-054WO1Tumor, Hairy Cell Leukemia, Head and Neck Cancer, Hepatocellular Cancer, Hodgkin's Lymphoma, Hypergammaglobulinemia, Hypopharyngeal Cancer, Intestinal Cancers, Intraocular Melanoma, Islet Cell Carcinoma, Islet Cell Pancreatic Cancer, Kaposi's Sarcoma, Kidney Cancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer, Lymphoproliferative Disorders, Macroglobulinemia, Male Breast Cancer, Malignant Mesothelioma, Malignant Thymoma, Medulloblastoma, Melanoma, Mesothelioma, Metastatic Occult Primary Squamous Neck Cancer, Metastatic Primary Squamous Neck Cancer, Metastatic Squamous Neck Cancer, Multiple Myeloma, Multiple Myeloma / Plasma Cell Neoplasm, Myelodysplastic Syndrome, Myelogenous Leukemia, Myeloid Leukemia, Myeloproliferative Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin's Lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Occult Primary Metastatic Squamous Neck Cancer, Oropharyngeal Cancer, Osteo- / Malignant Fibrous Sarcoma, Osteosarcoma / Malignant Fibrous Histiocytoma, Osteosarcoma / Malignant Fibrous Histiocytoma of Bone, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic Cancer, Paraproteinemias, Polycythemia vera, Parathyroid Cancer, Penile Cancer, Pheochromocytoma, Pituitary Tumor, Primary Central Nervous System Lymphoma, Primary Liver Cancer, Prostate Cancer, Rectal Cancer, Renal Cell Cancer, Renal Pelvis and Ureter Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary Gland Cancer, Sarcoidosis Sarcomas, Sezary Syndrome, Skin Cancer, Small Cell Lung Cancer, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Neck Cancer, Stomach Cancer, Supratentorial Primitive Neuroectodermal and Pineal Tumors, T-Cell Lymphoma, Testicular Cancer, Thymoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Transitional Renal Pelvis and Ureter Cancer, Trophoblastic Tumors, Ureter and Renal Pelvis Cell Cancer, Urethral Cancer, Uterine Cancer, Uterine Sarcoma, Vaginal Cancer, Visual Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, and any other hyperproliferative disease, besides neoplasia, located in an organ system listed above.

[0153] In some embodiments, V51+ y5 T cells are administered intravenously, intramuscularly, intraci sternal magna, intrathecally, intraperitoneally, intradermally or subcutaneously. In some embodiments, V51+ y5 T cells are administered intravenously. In some embodiments, V51+ y5 T cells are administered intramuscularly. In some embodiments, V51+ y5 T cells are administered intracistemal magna. In some embodiments, V51+ y5 T cells are administered intrathecally. In some embodiments, V51+ y5 T cells are administeredATTORNEY DOCKET NO.: MIL-054WO1intraperitoneally. In some embodiments, V51+ y5 T cells are administered intradermally. In some embodiments, V51+ y5 T cells are administered subcutaneously.

[0154] The composition comprising y5 T cells can be administered to a patient in need thereof one or more times per day, week, month (e.g., one or more times every 2 weeks), or year depending on the severity of the disease and change in disease state of the patient during the treatment. Generally, it is expected that a typical dosage would include 5 x 105to 5 x 1012(e.g., 5 x 105, 5 x 106, 5 x 107, 5 x 108, 5 x 109, 5 x 1010, 5 x 1011, or 5 x 1012) T cells. Severity of symptoms, change in symptoms, patient response to treatment, any adverse effects of treatment, and / or effect of any additional treatment(s), can be used to determine the frequency of treatment and the dosage, i.e., the number of T cells to be administered to a patient.

[0155] The composition comprising V51+ y5 T cells can be administered in any medically useful form.EXAMPLESExample 1. Skin Expanded V81 Cells Exhibit Unique Transcriptomic Profile from ex vivo Counterparts

[0156] This example shows that expanded skin-derived V51 cells have a unique transcriptome in comparison to ex vivo counterparts, demonstrating a powerful impact of the anatomical environment on the transcriptomes of V51+ T cells, much more than cell lineage.

[0157] V51 and CD8 T cells were obtained from either blood, ex vivo skin explants, or whole skin biopsies (FIG. 1A). A principal component analysis of these cells showed that the environment from which the cells are derived influences the transcriptome, and that skin-derived cells were very different from blood-derived V51+ cells that were not expanded (FIG. IB). Exemplary MA plots comparing the culture conditions further reflect this fact (FIG. 1C - FIG. IE). Further, the V51 and CD8 T cells obtained from whole skin biopsies (FIG. IF), differentially expressed genes that are upregulated in the TGF-P pathway, suggesting that, in comparison to blood, this pathway plays an important role in the skin’s microenvironment (FIG. 1G - FIG. 1H)

[0158] Furthermore, by flow cytometry, skin T cells expanded from breast cancer donors behaved comparably to those derived from healthy patients undergoing cosmetic procedures (FIG. 6A - FIG. 6F)ATTORNEY DOCKET NO.: MIL-054WO1

[0159] FIG. 8A - FIG. 8F show exemplary transcriptomic maps of various pathways comparing PBMCs, ex vivo skin expanded, and skin expanded V51+y5 T cells from different donors, comparing expression of genes in the mTORcl pathway (FIG. 8A), TNFa signaling pathway via NFKB (FIG. 8B), EMT pathway (FIG. 8C), E2F pathway (FIG. 8D), IFNy signaling pathway (FIG. 8E), and the TGF-P pathway (FIG. 8F).

[0160] Overall, results showed that expanded skin-derived V51 cells have a unique transcriptome in comparison to ex vivo counterparts.Example 2. Skin Expanded Cells Retain a TRM Phenotype and TGF-P augments the expression of TRM Markers and Skin-Tropism Receptors

[0161] This example demonstrates that exemplary immune cells that are expanded from whole skin biopsies retain some aspects of a resident memory T cell (TRM) phenotype and that addition of TGF-P restores or enhances the expression of those and additional TRM markers and skin-tropism receptors.

[0162] FIG. 2A - FIG. 2C and FIG. 2D are representative heat maps of various genes that are expressed in cells that express a TRM phenotype and skin-tropism related receptors. Skin expanded cells retain a TRM phenotype, despite the loss of some related genes that are enriched in their ex vivo counterparts. Cells from whole skin were expanded in culture over the course of three weeks, wherein the culture medium contained a mixture of IL-2 and IL- 15 alone, IL-2 and IL-15 with TGF-P added during the entirety of the three weeks, or IL-2 and IL-15 alone for 18 days with IL-2, IL-15, and TGF-P added at the 18-day mark for 3 days. In cultures where TGF-P was added, the percentage of TRM receptors and some skin-tropism receptors were enhanced on skin expanded V51 cells as determined by representative flow cytometry staining (see FIG. 2E - FIG. 2H, FIG. 7A - FIG. 7F, and FIG. 12A - FIG. 12C)

[0163] Further, FIG. 9A - FIG. 9B show the exemplary skin-specific TRM signatures associated with ex vivo skin-expanded V51+y5 T cells (FIG. 9A) and barrier TRM genes (FIG. 9B) versus PBMC derived V51+y5 T cells. About 25 out of 28 top skin-specific TRM signatures were upregulated, including CXCR4 and CCR4; however, 17 out of 26 representative generic signatures were also upregulated. These results indicated that ex vivo skin-expanded V51+y5 T cells showed expression of TRM markers.

[0164] FIG. 10A - FIG. 10D demonstrate that expansion cells transcribe high levels of PEC AMI, which encodes CD31, an inhibitory receptor that facilitates TGFP mediated suppression of T cell responses to TCR stimulation in vitro. PEC AMI transcription was significantly higher in V51+ cells versus CD8+ cells expanded from the same skin samples;ATTORNEY DOCKET NO.: MIL-054WO1surface CD31 expression was on a greater frequency of expanded V51+versus CD8+cells (mean expression = 79.68 % and 67.48 % in V51+and CD8+cells, respectively, n=5); and the expression levels were higher (mean gMFI = 15245 and 10870 in V51+and CD8+cells, respectively, n=5), albeit not reaching statistical significance

[0165] It was also observed that exposure to TGF-P had little to no impact on the viability of the cells (FIG. 11A) or recovery of CD3+ T cells (FIG. 11B), pan y5+ T cells (FIG. 11C) or V5+ T cells (FIG. 11D).

[0166] The results in FIG. 12A - FIG. 12C demonstrated that skin expanded V51+y5 T cells exposed to TGF-P for three days following 18 day culturing with IL-2 / IL-15 (FIG. 12A), or TGF-P for three weeks (FIG. 12C) increased the percentage of V51+y5 T cells expressing tissue-associated homing receptors relative to IL-2 / IL-15 without TGF-P (FIG.12B)

[0167] FIG. 13 further depicts a transcriptomic map, showing that skin expanded V51 cells express several key TRM markers, including PDCD1 (which encodes PD1, an important target for blockade therapies), NRA1, NRA2, and NRA3. This map also shows that these cells expressed less exhaustion markers, notably, TOX than their CD8 T cell ex vivo counterparts. The skin expanded cells did not show an exhausted phenotype.Example 3. Skin Expanded V81 Cells Retain Tel andXCLl Potentials of ex vivo skin Vdl cells

[0168] This example shows that exemplary skin expanded V51 cells retain Tel and XCL1 cytolytic potentials of their ex vivo V51 counterparts and the impact of the addition of exogenous TGF-P has on cytokine production.

[0169] FIG. 3A shows the levels of various transcripts made in V51 and CD8 T cells expanded from skin tissue. Particularly, XCL1 is upregulated in V51 cells expanded from skin tissue in comparison to colocalized CD8 T cells and is comparable to V51 cells that are examined directly from skin. Further, in skin expanded V51 cells cultured in IL-2 and IL-15 show an increase in XCL1 produced and XCL1+ cells upon T cell receptor (TCR) stimulation by assays known in the art and representative flow cytometry staining (see FIG.3B and FIG. 3C) In comparison to their colocalized CD8 counterparts, V51 cells expanded from skin produce XCL1 upon TCR stimulation and produce it earlier, while other exemplary cytokine production (e.g., IFN-y and TNF-a) were comparable in time and production levels (FIG. 3D - FIG. 3G)

[0170] As alluded to above, addition of exogenous TGF-P to cultures containing cytokines (IL-2 and IL- 15) has a direct effect on the ability of the V51 cells to produceATTORNEY DOCKET NO.: MIL-054WO1XCL1. In fact, addition of TGF-P to the cultures over the course of 21 days augments the XCL1 production of skin expanded V51 cells (see FIG. 3H - FIG. 31). It is also noted that exposure of skin expanded V51 cells to TGF-P reduces their ability to express IFNy and TNFa (FIG. 3J - FIG. 3K)

[0171] Furthermore, a basic difference in the biochemical status of the cell populations was reflected in the enrichment and upregulation of core mTORcl pathway, TNFa signaling pathway, epithelial-to-mesenchymal transition (EMT) pathway, e2F pathway, IFNy response pathway, and TGFP signaling pathway genes see FIG. 8 A - FIG. 8F and FIG. 9 A - FIG.9B)Example 4. Skin Expanded V81 Cells Gain the Potential to Produce Chemoattractants CCL3, CCL4, and CCL5

[0172] This example shows that exemplary skin expanded V51 cells gain the potential to produce various chemoattractants in comparison to their ex vivo counterparts.

[0173] Skin expanded V51 cells produced higher amounts of transcripts of CCL3, CCL4, and CCL5 in comparison to their CD8 counterparts and cells from ex vivo explants and blood (FIG. 4A - FIG. 4E). Additionally, present culture conditions (IL-2 and IL- 15 over 21 days) are sufficient enough to produce CCL4 and CCL5, while the addition of exogenous TGF-P did not impact the expression of CCL3 / CCL4 / CCL5 (FIG. 4F - FIG. 41).Example 5. Skin Expanded V81 Cells Cultured in TGF-P Kill just as Well / Better than their Counterparts Cultured in IL-2 / IL-15

[0174] This example shows that exemplary skin expanded V51 cells can have increased cytolytic potential when cultured with cytokines (IL-2 and IL-15) and TGF-p.

[0175] FIG. 5A - FIG. 5C show that skin expanded V51 cells express various cytokines, wherein the level of PRF1 and GNLY transcripts are and expression of perforin and granulysin significantly higher than skin colocalized CD8 T cells, respectively. These data show that the skin expanded V51 cells have the capacity to be more cytolytic than their CD8 T cell counterpart.

[0176] To demonstrate this capacity for killing, an assay was designed wherein the cultures (Condition 1: IL-2 / IL-15 for 3-week expansion; Condition 2: IL-2 / IL-15 / TGF-P for 3-week expansion) were depleted of aP T cells and negatively sorted for y5 T cells (FIG. 5D - FIG. 5E), and co-cultured with either HT-29 colorectal adenocarcinoma (FIG. 5F - FIG.5G) or A375 malignant melanoma (FIG. 5H - FIG. 51) cells in an Incucyte. The results of this assay show that the V51 cells cultured with TGF-P were able to efficiently kill both cell lines at least just as well and seemingly better than their counterparts cultured without TGF-p.ATTORNEY DOCKET NO.: MIL-054WO1Further, FIG. 5J - FIG. 5K shows staining of skin biopsy (SB) donors of A375 cells cultured in Condition 1 or Condition 2, and the presence of cytokine positive cells after anti-CD3 stimulation was quantified. The images on the left show an enhanced killing capacity when cultured with TGF-P, while the quantifications of cytokine positive V51 cells after stimulation show a marked decrease in various cytokines, but not perforin and granzyme B. This is consistent with addition of TGF-P reducing production of different cytokines.Example 6. Skin Expanded V81 Cells Have a Skewed Cytolytic Phenotype

[0177] This example shows that exemplary skin expanded V51 cells have a skewed cytolytic phenotype in comparison to ex vivo expanded counterparts and CD8 counterparts.

[0178] FIG. 14A - FIG. 14C show transcriptomic heat maps of transcripts that are associated with a cytolytic phenotype (FIG. 14A), an intermediate phenotype that may be a bipotent progenitor associated with host-beneficial CRC surveillance and wound healing factors (FIG. 14B), and wound healing phenotype (FIG. 14C). Nonetheless, skin V51+ ex vivo cells showed relatively consistent expression across each donor of many of the core signature genes that define each functional subtype. Strikingly, V51 cell expansion overtly skewed the transcriptomic program toward the cytolytic signature.

[0179] It is reflected in FIG. 15A that the transcription factors that might drive skewed phenotypes of ex vivo expanded and / or skin expanded V51+cells were expressed at low levels, and importantly not enriched in any cell population (z.e., PBMCs, ex vivo, or skin expanded). In CD8+cells expanded in the same fashion; a dynamic trichotomy applies (see FIG. 15B - FIG. 15D). Exemplary MA analysis (FIG. 15E) also demonstrates that the transcriptomes of CD8+aP T cells and ex vivo expanded or skin expanded V51+y5 T cells are similar, further bolstering the premise that the V51 cells have an increased cytolytic potential.

[0180] Of the other differences many related to their distinct lineages, e.g., higher transcript levels for TRAC, CD8A, CD8B, CD2, CD28 in CD8 cells, while V51+cells were significantly enriched for TRDV1 and TRGC1. Nonetheless, there were other mRNAs were also consistently expressed to higher levels by V51 cells (FIG. 15F - FIG. 15G), e.g., HCSTXCL1, HCST, VAV3, HIF1 A, or vice versa, expressed at lower levels in V51 cells (FIG. 15H)Example 7. Tumor Cell Targeting Potential by Skin Expanded, Innate-Like Skin V81 T Cells

[0181] This example shows that exemplary skin expanded V51 cells have an innate profile that increases their potential to target tumor cells.ATTORNEY DOCKET NO.: MIL-054WO1

[0182] Intracellular staining detected for GZMA, GZMB, and NKG7 showed in essentially all V51+ and CD8 ex vivo expanded or skin expanded cells being positive (FIG.16A) whereas for perforin, a rate-determining regulator of killing, between 80% and 100% of V51+expansion cells were positive at baseline, significantly more than paired CD8+expansion T cells, with a similar trend apparent for granulysin, albeit with greater variation in the percentage of positive cells (see FIG. 5 A and FIG. 5C).

[0183] Skin expanded V51 cells expressed mRNAs for several NKRs see FIG. 16B and FIG. 16C) and were validated at the protein level for NKp30, NKp44, NKG2D, NKG2A, NKG2C and CD94 (FIG. 16D and FIG. 16E). Cells expanded the presence of TGFp showed a substantial decrease in expression of NKp30 (FIG. 16D and FIG. 16E).

[0184] The cells at baseline also expressed many of the other remaining “innateness” genes, including TYROBP, FCERIG, HOPX, CRT AM, and TNFRSF9 (encodes 4- IBB) all of which were upregulated by the expansion protocol relative to cells ex vivo and were expressed at significantly higher levels than expansion CD8 T cells (FIG. 16F), as validated at the protein level for 4- IBB (FIG. 16G and FIG. 16H). The innateness was reflected in the fact that the skin expanded V51+cells highly expressed TMSB4X transcripts (FIG. 161). The combination of innateness and cytolytic potentials suggested that expansion V51+cells might be able to target tumor cells in a non-MHC restricted fashion, and rapidly, without any requirement clonal expansion. To test this, we purified V51+cells from expansion cultures of five independent donors, by depleting aP T cells and then checking for purity post-sort (FIG.16J). TCR diversity was also reflected (as determined by TGRV expression levels in PBMC, ex vivo skin expanded V51+, and skin expanded V51+cells), with TCR Vy usage being comparable to that of V51+PBMCs (FIG. 16K and FIG. 16L).ATTORNEY DOCKET NO.: MIL-054WO1EQUIVALENTS AND SCOPE

[0185] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of examples only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed.

Claims

ATTORNEY DOCKET NO.: MIL-054WO1CLAIMS1. A method of producing skin-derived V51+y5 T cells comprising expanding y5 T cells isolated from skin in the presence of one or more cytokines and TGF-P, wherein the expanded skin- derived V51+y5 T cells are selectively enriched for Tissue Resident memory cells (TRM cells).

2. A method of expanding skin-derived V51+y5 T cells comprising culturing skin y5 T cells in a culture medium supplemented with TGF-P, wherein the expanded skin-V51+y5 T cells are characterized with expression of one or more-skin specific-Tissue Resident memory cells (TRM) markers and skin-homing receptors.

3. The method of claim 1 or 2, wherein the expanded skin-derived V51+y5 T cells are CD103+, CD101+ AHR+, CXCR4+ and / or Vimentin+.

4. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells express one or more skin-homing receptors of CCR4, CCR6, CCR8, CCCR10 and CLA.

5. A method of expanding skin-derived V51+y5 T cells comprising culturing y5 T cells isolated from skin in the presence of one or more cytokines and TGF-P, wherein the expanded skin-derived V51+y5 T cells produce high-level of XCL1 and one or more chemokines of CCL3, CCL4 and CCL5, as compared to skin-derived V51+y5 T cells expanded without TGF-p.

6. The method of any one of the preceding claims, wherein the culture medium comprises IL-2 and IL-15.

7. The method of any one of the preceding claims, wherein the skin cells are expanded for at least 21 days.

8. The method of any one of the preceding claims, wherein the skin cells are first expanded in the presence of IL-2 and IL-15 for 18 days and then expanded in the presence of IL-2, IL-15 and TGF-P for at least three days.

9. The method of any one of claims 1-7, wherein the skin cells are cultured with IL-2, IL- 15 and TGF-p.ATTORNEY DOCKET NO.: MIL-054WO110. The method of any one of claims 1-9, wherein the skin cells are cultured in a medium comprising a high concentration of glucose.

11. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells produce XCL1.

12. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells produce Tel.

13. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells produce XCL2.

14. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells produce less IFNy and TNFa, as compared to expanded skin-derived V51+y5 T cells without TGF-p.

15. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells produce one or more chemokines CCL3, CCL4 and CCL5.

16. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells express one or more NK cell receptors.

17. The method of claim 16, wherein the expanded skin-derived V51+y5 T cells express NKp44, NKp46, NKG2D, NKG2A, NKG2C and / or CD94.

18. The method of claim 17, wherein the expanded skin-derived V51+y5 T cells express NKG2D.

19. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells are cytolytic.

20. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells have higher cytolytic potentials, as compared to skin-derived V51+y5 T cells expanded without TGF-p.

21. The method of any one of the preceding claims, wherein the V51+y5 T cells are from human skin.ATTORNEY DOCKET NO.: MIL-054WO122. The method of any one of the preceding claims, wherein the expanded skin-derived V51+y5 T cells are for allogeneic cell therapy.

23. The method of any one of the preceding claims, further comprising cry opreservation of the expanded V51+y5 T cells.

24. A population of skin-derived V51+y5 T cells produced by the method of any one of the preceding claims.

25. A composition comprising the skin-derived V51+y5 T cells of claim 24.

26. A method of treating a subject with a cancer comprising administering to the subject the skin-derived V51+y5 T cells of claim 24 or the composition of claim 25.

27. A method of inhibiting tumor growth and viability in a subject comprising administering to the subject the skin-derived V51+y5 T cells of claim 24 or the composition of claim 25.

28. The method of claim 27, wherein the treatment is allogeneic cell therapy.

29. The method of any one of claims 26-28, wherein the subject has a solid tumor.

30. Use of the expanded skin-derived V51+y5 T cells of any one of the preceding claims for the treatment of a cancer.

31. A skin-derived V51+y5 T cell that produces XCL-1, wherein the skin derived V51+y5 T cell produces XCL1 upon stimulation, in less time as compared to a skin derived CD8+ T cell.

32. The skin-derived V51+y5 T cell of claim 31, wherein the skin-derived V51+y5 T cell produces one or more chemokines CCL3, CCL4 and CCL5.

33. A method of preparing CD103+ positive human V51+TRM cells for use in immunotherapy, the method comprising isolating V51+y5 T cell from human skin and culturing the isolated skin-derived cells in the presence of (a) IL-2 and IL-15, and (b) TGF-p.

34. The method of claim 33, wherein the CD103+ positive human V51+TRM cells have enhanced cytotoxicity as compared to the V51+TRM cells prepared without TGF-p.

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