Methods for isolating and expanding cells

Abstract

The present invention relates to a method for isolating lymphocytes (particularly γδ T cells) from a non-hematopoietic tissue sample, comprising culturing a non-hematopoietic tissue sample, which is an intact biopsy obtained from the non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15); and recovering the cultured population of lymphocytes from the non-hematopoietic tissue sample. Subsequent expansion methods, as well as the isolated population of cells obtained by the method and uses thereof, are also provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods for the isolation and / or expansion of non-hematopoietic tissue resident lymphocytes, in particular γδ T cells, including non-V52 cells, e.g. V51, V53, and V55 cells, and such non-hematopoietic tissues include skin and gastrointestinal. It will be appreciated that such isolated and / or expanded non-hematopoietic tissue resident lymphocytes are highly useful in adoptive T cell therapy, chimeric receptor therapy, etc. The present invention also relates to cells produced by the methods described herein. [Background technology]

[0002] BACKGROUND OF THEINVENTION Growing interest in T cell immunotherapy of cancer has focused on the apparent ability of subsets of CD8+ and CD4+ αβ T cells to recognize cancer cells and mediate host defensive functional potential, particularly when disinhibited by clinically mediated antagonism of inhibitory pathways mediated by PD-1, CTLA-4, and other receptors. However, αβ T cells are MHC restricted, which can result in graft-versus-host disease.

[0003] Gamma delta T cells (γδ T cells) represent a subset of T cells that express a distinctive and definitive γδ T cell receptor (TCR) on their surface. This TCR is composed of one gamma (γ) chain and one delta (δ) chain. Human γδ TCR chains are selected from three main δ chains, Vδ1, Vδ2, and Vδ3, and six γ chains. Human γδ T cells can be broadly classified based on their TCR chains, as specific γ and δ types are often, but not exclusively, found in cells in one or more tissue types. For example, most blood-resident γδ T cells express Vδ2 TCRs, e.g., Vγ9Vδ2, which are less common among tissue-resident γδ T cells, which often use Vδ1 in the skin and Vγ4 in the gastrointestinal tract.

[0004] Most methods for isolating lymphocytes rely on isolating these cell types from blood. Non-hematopoietic tissue-resident lymphocytes, such as αβ T cells, γδ T cells, and NK cells, may have properties that are particularly suitable for certain applications, e.g., targeting non-hematopoietic tumors and other targets. However, isolating such tissue-resident lymphocytes in clinically relevant quantities has been difficult, especially in the last decade. 8 This remains a challenge given that clinical doses ranging from 100 to 150 cells are required for many indications. Importantly, significant cell loss during production means that many more starting cells must be generated.

[0005] Because non-hematopoietic tissue resident lymphocytes, particularly αβ T cells, γδ T cells, and NK cells, are not readily obtainable in large numbers, they have not been adequately characterized or studied for therapeutic applications. Thus, there is a need in the art for methods to isolate and expand non-hematopoietic tissue resident lymphocytes, particularly γδ T cells, in sufficient quantities to study and potentially apply them as therapeutics, e.g., adoptive T cell therapy.

[0006] Clark et al. (2006) J. Invest. Dermatol. 126(5): 1059-70 describes a method for isolating skin-resident T cells from normal and diseased skin. However, the method described therein results in a relatively low yield of isolated cells, particularly due to the presence of animal products, i.e., only 1 cm of tissue is isolated. 2 10 cells per 6The method described in Clark et al. uses a comminuted sample, which leads to the intentional destruction of the structural integrity of the tissue sample. WO2017072367 and WO2018 / 202808 relate to a method for expanding non-hematopoietic tissue resident γδ T cells in vitro by culturing lymphocytes obtained from non-hematopoietic tissue in the presence of at least interleukin-2 (IL-2) and / or interleukin-15 (IL-15). WO2015189356 describes a composition for expanding lymphocytes obtained from a sample obtained by apheresis, comprising at least two cytokines selected from IL-2, IL-15, and IL-21. Therefore, there remains a need for a method for isolating tissue-resident non-hematopoietic lymphocytes, for example from skin, which yields a larger amount of cells suitable for clinical use. Summary of the Invention

[0007] (Summary of the invention) According to a first aspect of the present invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-section of at least 2 mm obtained from the non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15); and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

[0008] According to a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-section of at least 2 mm obtained from the non-hematopoietic tissue, in the presence of IL-2 and IL-15; and (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. A method is provided that includes:

[0009] According to a further aspect of the invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) at least 2 mm2 obtained from nonhematopoietic tissue 2 Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of ​​100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

[0010] According to a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) at least 2 mm2 obtained from nonhematopoietic tissue 2 Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of ​​100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. A method is provided that includes:

[0011] According to a further aspect of the invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) at least 2 mm2 obtained from nonhematopoietic tissue 3 Culturing a non-hematopoietic tissue sample, which is an intact biopsy, having a volume of about 100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

[0012] According to a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) at least 2 mm2 obtained from nonhematopoietic tissue 3 Culturing a non-hematopoietic tissue sample, which is an intact biopsy, having a volume of about 100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. A method is provided that includes:

[0013] According to a further aspect of the invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) placing a non-hematopoietic tissue sample in a container comprising a gas permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

[0014] According to a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) placing a non-hematopoietic tissue sample in a container comprising a gas permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering the population of cultured γδ T cells from the non-hematopoietic tissue sample. A method is provided that includes:

[0015] According to a further aspect of the invention there is provided a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) isolating a population of lymphocytes from said non-hematopoietic tissue sample according to the methods defined herein; and (ii) further culturing the population of lymphocytes for at least 5 days to produce an expanded population of lymphocytes. A method is provided that includes:

[0016] According to a further aspect of the invention there is provided a method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) isolating a population of γδ T cells from said non-hematopoietic tissue sample according to the methods defined herein; and (ii) further culturing the population of γδ T cells for at least 5 days to produce an expanded population of γδ T cells. A method is provided that includes: [Brief description of the drawings]

[0017] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1: Initial study comparing total cell yield from 3 mm punch biopsies and standard skin comminution methods. [Diagram 2] Figure 2: γδ cell yield from isolated punch biopsies of various sizes in AIM-V containing 5% human AB serum in 24-well plates compared to comminuted controls sampled with a scalpel. [Diagram 3] Figure 3: Total cell yield per biopsy (top graph) and total cell yield per plate (bottom graph) using various culture vessels containing AIM-V with 5% human AB serum. [Figure 4] FIG. 4: Comparison of total cell yields after 2 weeks (top graph) or 3 weeks (bottom graph) of isolation in AIM-V with the indicated serum supplements in G-REX6. [Diagram 5] Figure 5: Total cell yield and percentage of Vδ1 cells isolated using AIM-V medium containing 5% or 10% 5% serum replacement (SR) versus human AB serum (AB) in G-REX6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description of the Invention According to a first aspect of the present invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-section of at least 2 mm obtained from the non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15); and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

[0019] According to a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-section of at least 2 mm obtained from the non-hematopoietic tissue, in the presence of IL-2 and IL-15; and (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. A method is provided that includes:

[0020] References herein to "isolation" or "isolating" of cells, particularly lymphocytes and / or γδ T cells, refer to a method or process whereby cells are removed, separated, purified, enriched, or otherwise removed from a tissue or pool of cells. Such references will be understood to include the terms "isolated," "depleted," "purified," "enriched," and similar terms. Isolation of lymphocytes and / or γδ T cells includes isolation or separation of cells from an intact non-hematopoietic tissue sample or from stromal cells of a non-hematopoietic tissue (e.g., fibroblasts or epithelial cells). Such isolation may alternatively or additionally include isolation or separation of γδ T cells from other hematopoietic cells (e.g., αβ T cells or other lymphocytes). Isolation may be of a defined duration, for example beginning when the tissue explant or biopsy is placed into isolation culture and ending when the cells are harvested from the culture, for example by centrifugation or other means to transfer the isolated cell population to an expansion culture or used for other purposes, or when the original tissue explant or biopsy is removed from the culture. The isolation step may be at least about 3 days to about 45 days. In one embodiment, the isolation step is at least about 10 days to at least 28 days. In a further embodiment, the isolation step is at least 14 days to at least 21 days. Therefore, the isolation step may be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, about 35, about 40, or about 45 days. It can be understood that during this isolation step, the proliferation of the isolated cells may be insignificant, but that does not necessarily mean that the proliferation of the cells is absent. In fact, it is recognized that for some skilled artisans, the isolated cells may begin to divide and generate a plurality of such cells within the isolation vessel containing the tissue and / or scaffold.

[0021] Thus, reference herein to "isolated lymphocytes", "isolated lymphocyte population", "population of isolated lymphocytes", "isolated lymphocytes", "isolated lymphocyte population", "population of isolated lymphocytes", "isolated γδ T cells", "isolated γδ T cell population", "population of isolated γδ T cells", "isolated γδ T cells", "isolated γδ T cell population", or "population of isolated γδ T cells" will be understood to refer to hematopoietic cells or populations of hematopoietic cells comprising γδ cells that have been isolated, separated, depleted, purified or enriched from a non-hematopoietic tissue sample of origin such that the cells are substantially free of contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Similarly, reference herein to an "isolated or separated population of V51 T cells" refers to hematopoietic cells comprising V51 T cells that have been isolated, separated, depleted, purified or enriched from a non-hematopoietic tissue sample of origin such that the cells are substantially free of contact with non-hematopoietic cells or cells contained within intact non-hematopoietic tissue. Thus, isolation or separation refers to the separation, separation, removal, purification, or enrichment of hematopoietic cells (e.g., γδ T cells or other lymphocytes) from non-hematopoietic cells (e.g., stromal cells, fibroblasts, and / or epithelial cells).

[0022] The method for isolating lymphocytes and / or γδ T cells as defined herein may comprise disruption (e.g., mincing) of tissue followed by separation of lymphocytes and / or γδ T cells from other cell types. Preferably, the method for isolating lymphocytes and / or γδ T cells as defined herein may comprise "crawling" of lymphocytes and / or γδ T cells and other cell types from the tissue matrix of an intact non-hematopoietic tissue sample or explant or biopsy, where the tissue resident lymphocytes physically leave the tissue matrix without the need for disruption of the tissue matrix. By maintaining the integrity of the tissue matrix, it has surprisingly been found that the tissue resident lymphocytes preferentially escape the tissue matrix with little or no escape of inhibitory cell types such as fibroblasts, which are retained in the explant or biopsy, which can be easily removed at the end of the isolation later. Thus, in some embodiments, the use of an intact non-hematopoietic tissue sample or tissue matrix releases a small number of fibroblasts from the tissue into the culture. Such "crawling" methods utilizing intact non-hematopoietic tissue or tissue matrix have the advantage of reducing the need for excessive processing of the non-hematopoietic tissue sample or tissue matrix, and may provide the unexpected advantage of maintaining the structural integrity of the non-hematopoietic tissue or tissue matrix, and resulting in higher isolated cell yields.

[0023] Thus, the method for isolating non-hematopoietic tissue-derived lymphocytes as defined herein includes a method for isolating non-hematopoietic tissue-derived lymphocytes from an intact biopsy or explant of non-hematopoietic tissue. Such an intact biopsy or explant is one in which the structural integrity of the biopsy or explant has not been intentionally disrupted within the perimeter of the resection that removes the biopsy or explant from the tissue sample. Such an intact biopsy or explant has a three-dimensional structure that is largely maintained, except for minor disruptions caused by manipulation. Thus, the intact biopsy or explant has not been mechanically disrupted, for example by grinding or mincing, nor has it been chemically or enzymatically disrupted, for example. However, disrupted tissue may be used in the isolation method of the present invention. In one embodiment, the isolated lymphocytes are αβ T cells. In an alternative embodiment, the isolated lymphocytes are γδ T cells. In another embodiment, the isolated lymphocytes are NK cells. It can be understood that multiple types of lymphocytes may be isolated from the same isolation process.

[0024] Methods for isolating lymphocytes and / or γδ T cells utilizing the "crawling" method as defined herein may comprise culturing cells and / or non-hematopoietic tissue in the presence of cytokines and / or chemokines sufficient to induce the isolation or separation of γδ T cells and / or other lymphocytes as defined herein. Thus, in one embodiment of the invention, isolation of lymphocytes and / or γδ T cells from non-hematopoietic tissue comprises culturing the non-hematopoietic tissue in the presence of IL-2 and IL-15.

[0025] As used herein, "IL-2" refers to native or recombinant IL-2 or variants thereof that act as agonists (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof) of one or more IL-2 receptor (IL-2R) subunits. Such agents can support the growth of the IL-2-dependent cell line, CTLL-2 (33; American Type Culture Collection (ATCC®) TIB 214). Mature human IL-2 occurs as a 133 amino acid sequence (minus a signal peptide of an additional 20 N-terminal amino acids) as described by Fujita et al., Cell 1986. 46.3:401-407. IL-2 muteins are polypeptides in which certain substitutions have been made to the interleukin-2 protein while retaining the ability to bind to IL-2Rβ, such as those described in US 2014 / 0046026. IL-2 muteins can be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites in other residues of the native IL-2 polypeptide chain. According to the present disclosure, any such insertions, deletions, substitutions, and modifications result in an IL-2 mutein that retains IL-2Rβ binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0026] 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) is found in Genbank under the accession locator NP_000577.2 GI: 28178861. The murine (Mus musculus) IL-2 amino acid sequence (Gene ID 16183) is found in Genbank under the accession locator NP_032392.1 GI: 7110653.

[0027] IL-2 can also refer to IL-2 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. Variants can include conservatively substituted sequences, meaning that a given amino acid residue is replaced with a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as between Lys and Arg; between Glu and Asp; or between Gln and Asn. Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic characteristics, are well known. Natural IL-2 variants are also encompassed by the present invention. Examples of such variants are proteins that arise by alternate mRNA splicing events or by proteolytic cleavage of the IL-2 protein, where the IL-2 binding properties are retained. Alternate splicing of the mRNA can result in a truncated, but biologically active, IL-2 protein. Mutations resulting from proteolysis include, for example, differences in the N- or C-terminus upon expression in various types of host cells due to proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-2 protein. In some embodiments, the protein can be modified at its termini or internally with chemical groups, such as, for example, polyethylene glycol, to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the protein can be modified at its termini or internally with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577).

[0028] As used herein, "IL-15" refers to native or recombinant IL-15 or variants thereof that act as agonists of one or more IL-15 receptor (IL-15R) subunits (e.g., mutants, muteins, analogs, subunits, receptor complexes, fragments, isoforms, and peptidomimetics thereof). IL-15, like IL-2, is a known T cell growth factor that can support the proliferation of the IL-2-dependent cell line, CTLL-2. IL-15 was first reported by Grabstein et al. (Grabstein et al., Science 1994. 264.5161: 965-969) as a mature protein of 114 amino acids. As used herein, the term "IL-15" refers to native or recombinant IL-15 and its muteins, analogs, subunits, or complexes thereof (e.g., receptor complexes, e.g., sushi peptides described in WO 2007 / 046006), each of which is capable of stimulating the proliferation of CTLL-2 cells. In a CTLL-2 proliferation assay, supernatants from cells transfected with recombinantly expressed in-frame fusions of precursor and mature IL-15 are capable of inducing CTLL-2 cell proliferation.

[0029] Human IL-15 can be obtained by following the procedure described by Grabstein et al. (Grabstein et al., Science 1994. 264.5161: 965-969) or by conventional procedures such as polymerase chain reaction (PCR). A human IL-15 cDNA was deposited with the ATCC on February 19, 1993 and assigned the accession number 69245.

[0030] The amino acid sequence of human IL-15 (Gene ID 3600) is found in Genbank under the accession locators NP000576.1 GI: 10835153 (isoform 1) and NP_751915.1 GI: 26787986 (isoform 2). The murine (Mus musculus) IL-15 amino acid sequence (Gene ID 16168) is found in Genbank under the accession locator NP_001241676.1 GI: 363000984.

[0031] IL-15 can also refer to IL-15 from various mammalian species, including, for example, human, monkey, cow, pig, horse, and mouse. An IL-15 "mutein" or "variant" as referred to herein is a polypeptide having an amino acid sequence that is substantially homologous to the sequence of a native mammalian IL-15, but differs from the native mammalian IL-15 polypeptide due to amino acid deletion, insertion, or substitution. A variant can include a conservatively substituted sequence, meaning that a given amino acid residue is replaced with a residue having similar physicochemical characteristics. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as the substitution of Ile, Val, Leu, or Ala for each other, or the substitution of one polar residue for another, such as the substitution between Lys and Arg; the substitution between Glu and Asp; or the substitution between Gln and Asn. Other such conservative substitutions are well known, such as the substitution of entire regions with similar hydrophobic characteristics. Natural IL-15 variants are also encompassed by the present invention. Examples of such variants are proteins resulting from alternative mRNA splicing events or from proteolytic cleavage of the IL-15 protein, where IL-15 binding properties are retained. Alternative splicing of the mRNA can result in a truncated but biologically active IL-15 protein. Mutations resulting from proteolysis include differences in the N- or C-terminus upon expression in various types of host cells, for example, by proteolytic removal of one or more terminal amino acids (usually 1-10 amino acids) from the IL-15 protein. In some embodiments, the protein can be modified at its termini or interior with chemical groups, such as polyethylene glycol, to alter its physical properties (Yang et al., Cancer 1995. 76: 687-694). In some embodiments, the protein can be modified at its termini or interior with additional amino acids (Clark-Lewis et al., PNAS 1993. 90:3574-3577).

[0032] It will be appreciated that in certain embodiments, the isolation of lymphocytes and / or γδ T cells by the methods defined herein may further comprise culturing the non-hematopoietic tissue in the presence of at least one additional cytokine. In certain embodiments, the methods defined herein may further comprise culturing the non-hematopoietic tissue in the presence of at least one additional agent, such as a chemokine. It will be further appreciated that the chemokine will be selected depending on the γδ T cells or other lymphocytes being isolated. Furthermore, the chemokine will vary and be selected depending on the non-hematopoietic tissue used for the isolation of γδ T cells or lymphocytes.

[0033] In certain embodiments, the methods defined herein typically involve administering at least 10 IU / mL, such as at least 100 IU / mL (e.g., 10 IU / mL to 1,000 IU / mL, 20 IU / mL to 800 IU / mL, 25 IU / mL to 750 IU / mL, 30 IU / mL to 700 IU / mL, 40 IU / mL to 600 IU / mL, 50 IU / mL to 500 IU / mL, 75 IU / mL to 250 IU / mL, or 100 IU / mL to IL-2 at a concentration of 200 IU / mL, e.g., 10 IU / mL to 20 IU / mL, 20 IU / mL to 30 IU / mL, 30 IU / mL to 40 IU / mL, 40 IU / mL to 50 IU / mL, 50 IU / mL to 75 IU / mL, 75 IU / mL to 100 IU / mL, 100 IU / mL to 150 IU / mL, 150 IU / mL to 200 IU / mL, 200 IU / mL to 500 IU / mL, or 500 IU / mL to 1,000 IU / mL). In certain embodiments, the methods defined herein typically include IL-2 at a concentration of less than 1,000 IU / mL, e.g., less than 500 IU / mL. In some embodiments, the methods include IL-2 at a concentration of about 100 IU / mL.

[0034] In further embodiments, the methods defined herein typically involve a concentration of at least 0.1 ng / mL, such as at least 10 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, or 100 ng / mL to 250 ng / mL, such as 0.1 ng / mL to 1. In some embodiments, the method comprises a concentration of IL-15 of about 0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 100 ng / mL, 20 ng / mL to 50 ng / mL, 40 ng / mL to 70 ng / mL, 50 ng / mL to 100 ng / mL, 50 ng / mL to 60 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, or 500 ng / mL to 1,000 ng / mL). In further embodiments, the method as defined herein typically comprises a concentration of IL-15 of less than 500 ng / mL, e.g., less than 100 ng / mL. In some embodiments, the method comprises a concentration of IL-15 of about 50 ng / mL.

[0035] In some embodiments, isolation of lymphocytes and / or γδ T cells from the non-hematopoietic tissue sample comprises culturing in the presence of both IL-2 and IL-15, each at any of the concentrations described above. Optionally, the concentration of IL-2 is about 100 IU / mL and the concentration of IL-15 is 55 ng / mL.

[0036] Reference herein to "non-hematopoietic tissue" or "non-hematopoietic tissue sample" includes skin (e.g., human skin) and gastrointestinal (e.g., human gastrointestinal). Non-hematopoietic tissue is tissue other than blood, bone marrow, or thymus tissue. In one embodiment, the non-hematopoietic tissue sample is skin (e.g., human skin). In a further embodiment, the non-hematopoietic tissue sample is gastrointestinal or gastrointestinal (e.g., human gastrointestinal or human gastrointestinal). In some embodiments, lymphocytes and / or γδ T cells are not obtained from a sample of a particular type of biological fluid, e.g., blood or synovial fluid. In some embodiments, the non-hematopoietic tissue sample from which lymphocytes and / or γδ T cells are isolated according to the methods defined herein is skin (e.g., human skin), which can be obtained by methods known in the art. Alternatively, the methods for isolating lymphocytes and / or γδ T cells provided herein can be applied to the digestive tract (e.g., colon or gastrointestinal), breast, lung, prostate, liver, spleen, pancreas, uterus, vagina, and other skin membranes, mucosa, or serous membranes. The lymphocytes and / or γδ T cells can be resident in a human cancer tissue sample, e.g., a breast or prostate tumor. In some embodiments, the lymphocytes and / or γδ T cells can be from a human cancer tissue sample (e.g., solid tumor tissue). In other embodiments, the lymphocytes and / or γδ T cells can be from a non-hematopoietic tissue sample other than human cancer tissue (e.g., tissue that does not contain a significant number of tumor cells). For example, the lymphocytes and / or γδ T cells can be from an area of ​​skin (e.g., healthy skin) away from adjacent or neighboring cancer tissue. Thus, in some embodiments, the γδ T cells are not obtained from human cancer tissue. In further embodiments, the lymphocytes are not obtained from human cancer tissue.

[0037] In one embodiment, the non-hematopoietic tissue sample of the methods defined herein is obtained from a human. In an alternative embodiment, the non-hematopoietic tissue sample of the methods defined herein is obtained from a non-human animal subject.

[0038] Methods for obtaining such tissues are known in the art. Examples of such methods include scalpel explants or punch biopsies, which may vary in size. In some embodiments, non-hematopoietic tissue samples are obtained by punch biopsy.

[0039] In some embodiments of the present invention, the non-hematopoietic tissue sample is an intact biopsy. Reference herein to an "intact" biopsy or "explant" includes tissues and tissue samples that are substantially unbroken or undisrupted, whereby the structural integrity of the biopsy or explant is not intentionally disrupted within the perimeter of the incision that removes the biopsy or explant from the tissue sample. Such intact biopsies or explants have a three-dimensional structure that is largely maintained except for minor disruptions caused by manipulation. Thus, the intact biopsy or explant is not mechanically disrupted, for example, by mincing or chopping, nor is it chemically or enzymatically disrupted, for example. An intact biopsy or intact tissue sample may include the entire tissue, the complete tissue, a portion of the tissue, or all elements of the tissue. For example, in one embodiment, the intact biopsy includes all layers of the skin. In a further embodiment, the biopsy includes the epithelial and dermal layers of the skin. In such embodiments where the biopsy is intact, it will be understood that the separation and distinction of such layers is maintained. Thus, reference herein to "intact" further includes full thickness biopsies of non-hematopoietic tissue samples.

[0040] Thus, in one particular embodiment of the present invention, the non-hematopoietic tissue sample is not comminuted. In a further embodiment, the intact biopsy is a punch biopsy. In yet a further embodiment, the intact biopsy is obtained by punch biopsy. The embodiment presented herein in which the non-hematopoietic tissue sample is an intact biopsy provides the surprising advantage of obtaining a large number of isolated or separated cells from a non-comminuted and / or intact non-hematopoietic tissue sample. Moreover, as shown herein, cells obtained from a non-comminuted and / or intact non-hematopoietic tissue sample according to the methods defined herein can retain a phenotype useful for subsequent expansion and / or manipulation methods known in the art.

[0041] In further embodiments, the intact biopsy is skin (e.g., human skin) or the intact biopsy is gastrointestinal (e.g., human gastrointestinal). In one embodiment, the non-hematopoietic tissue sample has a minimum cross-section of at least 2 mm. "Minimum cross-section" will be understood to refer to the smallest or shortest length measured through the center of gravity of the tissue sample. "Maximum cross-section" will be further understood to refer to the largest or longest length measured through the center of gravity of the tissue sample. The term "center of gravity" as used herein is the average or mean location of all points of the tissue sample. According to further embodiments, the non-hematopoietic tissue sample will be understood to have a minimum cross-section of at least 2 mm, at least 3 mm, at least 4 mm, at least at least 5 mm, at least 6 mm, at least 7 mm, or at least 8 mm. In further embodiments, the non-hematopoietic tissue sample has a minimum cross-section of 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, or 3 mm or less. In one embodiment, the non-hematopoietic tissue sample has a minimum cross-section of 2 mm to 8 mm, inclusive, for example, 2 mm to 4 mm. In one particular embodiment, the non-hematopoietic tissue sample has a minimum cross-section of about 3 mm. In one particular embodiment, the non-hematopoietic tissue sample has a cross-section of about 3 mm. According to further embodiments, it will be appreciated that the non-hematopoietic tissue sample has a maximum cross-section of at least 2 mm, at least 3 mm, at least 4 mm, at least at least 5 mm, at least 6 mm, at least 7 mm, or at least 8 mm. In further embodiments, the non-hematopoietic tissue sample has a maximum cross-section of 8 mm or less, 7 mm or less, 6 mm or less, 5 mm or less, 4 mm or less, 3 mm or less, or 2 mm or less. In one embodiment, the non-hematopoietic tissue sample has a maximum cross-section of 1 mm to 8 mm, inclusive, for example, 2 mm to 4 mm. In one particular embodiment, the non-hematopoietic tissue sample has a maximum cross-section of about 3 mm.

[0042] According to a further embodiment, the non-hematopoietic tissue sample is at least 2 mm 2"Minimum cross-sectional area" will be understood to refer to the area of ​​the smallest cross-section measured around the center of gravity of the tissue sample. "Maximum cross-sectional area" will be further understood to refer to the area of ​​the largest cross-section measured around the center of gravity of the tissue sample. As used herein, the term "center of gravity" is the average or mean location of all points of the tissue sample. In a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of ​​at least 2 mm 2 , at least 3 mm 2 , at least 4 mm 2 , at least 5 mm 2 , at least 6mm 2 , at least 7mm 2 , at least 8mm 2 , at least 9mm 2 , or at least 10 mm 2 In a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of ​​50 mm 2 Below, 40mm 2 Below, 30mm 2 Below, 25mm 2 Below, 20mm 2 Below, 15mm 2 Below, 10mm 2 Less than or equal to 8mm 2 In one embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of ​​2 mm 2 ~50mm 2 , for example, 3 mm 2 ~12mm 2 In one particular embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of ​​about 7 mm 2 In a further embodiment, the non-hematopoietic tissue sample has a minimum cross-sectional area of ​​at least 2 mm 2 , at least 3 mm 2 , at least 4 mm 2 , at least 5 mm 2 , at least 6mm 2 , at least 7mm 2 , at least 8mm 2 , at least 9mm 2 , or at least 10 mm 2In a further embodiment, the non-hematopoietic tissue sample has a maximum cross-sectional area of ​​50 mm 2 Below, 40mm 2 Below, 30mm 2 Below, 25mm 2 Below, 20mm 2 Below, 15mm 2 Below, 10mm 2 Less than or equal to 8mm 2 In one embodiment, the non-hematopoietic tissue sample has a maximum cross-sectional area of ​​1 mm 2 ~50mm 2 , for example, 3 mm 2 ~12mm 2 In one particular embodiment, the non-hematopoietic tissue sample has a maximum cross-sectional area of ​​about 7 mm 2 has a maximum cross-sectional area of

[0043] According to a further embodiment, the non-hematopoietic tissue sample is at least 2 mm 3 In a further embodiment, the non-hematopoietic tissue sample has a volume of at least 4 mm 3 , at least 5 mm 3 , at least 8mm 3 , at least 10 mm 3 , at least 15 mm 3 , at least 20 mm 3 , at least 25mm 3 , at least 30 mm 3 , at least 35mm 3 , at least 40mm 3 , at least 50 mm 3 , or at least 60 mm 3 In a further embodiment, the non-hematopoietic tissue sample has a volume of 250 mm 3 Below, 200mm 3 For example, 180 mm 3 Below, 1600mm 3 Below, 140mm 3 Below, 120mm 3 Below, 100mm 3 Below, 80mm 3 Below, 60mm 3 Below, 50mm 3 Less than or equal to 40mm3 In one embodiment, the non-hematopoietic tissue sample has a volume of 5 mm 3 ~250mm 3 , for example, 15mm 3 ~65mm 3 In one particular embodiment, the non-hematopoietic tissue sample has a volume of about 35 mm 3 has a volume of

[0044] In one embodiment, the non-hematopoietic tissue sample is a punch biopsy. The punch biopsy may be of any shape, but is advantageously of circular cross section, and is preferably at least 1 mm in diameter. In yet a further embodiment, the non-hematopoietic tissue sample comprises a punch biopsy with a diameter of at least 2 mm, for example, at least 3 mm, at least 4 mm, at least 5 mm, at least 6 mm, at least 7 mm, or at least 8 mm. In a further embodiment, the non-hematopoietic tissue sample comprises a punch biopsy with a diameter of 8 mm or less, for example, at least 7 mm, at least 6 mm, at least 5 mm, or at least 3 mm. In one embodiment, the non-hematopoietic tissue sample comprises a punch biopsy with a diameter of 1 mm to 8 mm, for example, at least 2 mm to 4 mm. In a particular embodiment, the non-hematopoietic tissue sample comprises a punch biopsy with a diameter of 3 mm.

[0045] In certain embodiments, the non-hematopoietic tissue sample comprises a biopsy (e.g., a punch biopsy, particularly a punch biopsy of a circular cross section) with a size, area, volume, and / or diameter as defined above, the maximum depth being determined by the site where the biopsy is obtained (although the depth may be reduced). In one embodiment, the biopsy is a skin biopsy and comprises the epidermis and dermis layers. In a further embodiment, the biopsy is substantially free of subcutaneous fat. Thus, in one embodiment, the biopsy comprises the epidermis and dermis layers and is substantially free of a layer of subcutaneous fat. In a further embodiment, the biopsy is free of subcutaneous fat. Alternatively, the subcutaneous fat is not removed and is therefore present (or at least partially present) in the biopsy. Thus, in yet a further embodiment, the biopsy consists of the epidermis and dermis layers. In one embodiment, the biopsy comprises the full thickness of the non-hematopoietic tissue sample.

[0046] Thus, according to one aspect of the present invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) At least 2 mm 2 Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of ​​100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering said lymphocytes from said non-hematopoietic tissue. A method is provided that includes:

[0047] According to a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) At least 2 mm 2 Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of ​​100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering said γδ T cells from said non-hematopoietic tissue. A method is provided that includes:

[0048] According to one aspect of the invention, there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising the steps of: (i) At least 2 mm 3 Culturing a non-hematopoietic tissue sample, which is an intact biopsy, having a volume of about 100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering said lymphocytes from said non-hematopoietic tissue. A method is provided that includes:

[0049] According to yet a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) At least 2 mm 3 Culturing a non-hematopoietic tissue sample, which is an intact biopsy, having a volume of about 100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering said γδ T cells from said non-hematopoietic tissue sample. A method is provided that includes:

[0050] The method of the invention comprises culturing a non-hematopoietic tissue sample as defined herein. References herein to "culturing" include the addition of cells and / or non-hematopoietic tissue sample to a medium comprising growth factors and / or essential nutrients required and / or preferred by the cells and / or non-hematopoietic tissue sample, including cells isolated, separated, removed, purified or enriched from the non-hematopoietic tissue sample. It will be understood that such culture conditions may be adapted in light of the cells or cell population to be isolated from the non-hematopoietic tissue sample according to the invention, or may be adapted in light of the cells or cell population to be isolated and expanded from the non-hematopoietic tissue sample.

[0051] In certain embodiments, the culture of the non-hematopoietic tissue sample is for a period of time sufficient for isolation of γδ T cells from the non-hematopoietic tissue sample. In an alternative embodiment, the culture of the non-hematopoietic tissue sample is for a period of time sufficient for isolation of lymphocytes other than γδ T cells (e.g., αβ T cells and / or NK (natural killer) cells) from the non-hematopoietic tissue sample. In certain embodiments, the culture period according to the methods defined herein is at least 14 days. In certain embodiments, the culture period according to the methods defined herein is less than 45 days, such as less than 30 days, such as less than 25 days. In further embodiments, the culture period according to the methods defined herein is between 14 and 35 days, such as between 14 and 21 days. In yet a further embodiment, the culture period according to the methods defined herein is about 21 days.

[0052] In certain embodiments of the invention, the lymphocytes and / or γδ T cells isolated according to the methods defined herein are recovered from the culture of the non-hematopoietic tissue sample after culturing the non-hematopoietic tissue sample. Recovery of lymphocytes and / or γδ T cells as defined herein may comprise physical recovery of lymphocytes and / or γδ T cells from the culture, isolation of lymphocytes and / or γδ T cells from other lymphocytes (e.g., αβ T cells, γδ T cells, and / or NK cells), or isolation and / or separation of lymphocytes and / or γδ T cells from stromal cells (e.g., fibroblasts). In one embodiment, the lymphocytes and / or γδ T cells are recovered by mechanical means (e.g., pipetting). In a further embodiment, the lymphocytes and / or γδ T cells are recovered by magnetic separation and / or labeling. In yet a further embodiment, the lymphocytes and / or γδ T cells are recovered by flow cytometric techniques, e.g., FACS. Thus, in certain embodiments, γδ T cells are recovered by specific labeling of the γδ T cells. In further embodiments, lymphocytes are recovered by specific labeling of the lymphocytes to distinguish them from other cells in the culture. It will be appreciated that such recovery of lymphocytes and / or γδ T cells may include physical removal of the non-hematopoietic tissue sample from the culture, transfer to a separate culture vessel, or transfer to separate or different culture conditions.

[0053] It will be appreciated that such recovery of lymphocytes and / or γδ T cells is performed after a period sufficient to obtain a population of lymphocytes and / or γδ T cells isolated from the non-hematopoietic tissue sample. In certain embodiments, the lymphocytes and / or γδ T cells are recovered after at least 1 week, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days from the culture of the non-hematopoietic tissue sample. Suitably, the lymphocytes and / or γδ T cells are recovered within 40 days, e.g., within 38 days, within 36 days, within 34 days, within 32 days, within 30 days, within 28 days, within 26 days, or within 24 days. In one embodiment, the lymphocytes and / or γδ T cells are recovered after at least 14 days from the culture of the non-hematopoietic tissue sample. In a further embodiment, the lymphocytes and / or γδ T cells are recovered after 14 to 21 days from the culture of the non-hematopoietic tissue sample.

[0054] In certain embodiments of the present invention, the non-hematopoietic tissue sample is cultured in a medium that is substantially free of serum (e.g., serum-free medium or medium containing serum replacement (SR)). Thus, in one embodiment, the non-hematopoietic tissue sample is cultured in serum-free medium. Such serum-free medium may also include serum replacement medium, in which the serum replacement is based on chemically defined components to avoid the use of human or animal-derived serum. In an alternative embodiment, the non-hematopoietic tissue sample is cultured in a medium containing serum (e.g., human AB serum or fetal bovine serum (FBS)). In one embodiment, the non-hematopoietic tissue sample is cultured in a medium containing serum replacement. In one embodiment, the non-hematopoietic tissue sample is cultured in a medium that does not contain animal-derived products.

[0055] It will be appreciated that the embodiment according to the invention in which the non-hematopoietic tissue sample is cultured in serum-free medium has the advantage of avoiding problems with serum filtration, precipitation, contamination and supply. Furthermore, animal derived products are not preferred for use in the manufacture of clinical grade human therapeutics. As seen herein, the inventors have also surprisingly found that the use of serum-free medium for the isolation of cells, in particular V51 γδ cells, substantially increases the number of cells obtained from the non-hematopoietic tissue sample compared to the use of medium containing AB serum. In particular, the isolation of γδ T cells from a non-hematopoietic tissue sample cultured in serum-free medium increases the yield of V51 cells.

[0056] In one embodiment, the method defined herein is carried out in an isolation vessel. Reference to an "isolation vessel" refers to a vessel containing a non-hematopoietic tissue sample for the isolation of lymphocytes and / or γδ T cells, optionally further comprising a synthetic scaffold. It should be noted that the isolation vessel can only be used for the isolation method and not for further amplification steps.

[0057] In one embodiment, the methods defined herein are carried out in a vessel (e.g., an isolation vessel) comprising a gas permeable material. Such a material is permeable to gases such as oxygen, carbon dioxide, and / or nitrogen, allowing gas exchange between the contents of the vessel and the surrounding environment. Reference herein to a "vessel" will be understood to include culture dishes, culture plates, single-well dishes, multi-well dishes, multi-well plates, flasks, multi-layer flasks, bottles (e.g., roller bottles), bioreactors, bags, tubes, and the like. Such vessels are known in the art for use in methods involving the expansion of non-adherent cells and other lymphocytes. However, as shown herein, vessels comprising gas permeable materials surprisingly also find utility in the isolation of γδ T cells, which are typically considered to be adherent. The use of such vessels for culture has been found to greatly increase the yield of isolated γδ T cells from non-hematopoietic tissue samples. Such vessels have also been found to preferentially support γδ T cells and other lymphocytes over other stromal cells (e.g., epithelial cells), including fibroblasts and adherent cell types. Thus, in one embodiment, a container comprising a gas permeable material as defined herein preferentially supports γδ T cells as well as other lymphocytes (e.g., αβ T cells and / or NK cells). In a further embodiment, fibroblasts and / or other stromal cells (e.g., epithelial cells) are not present in cultures performed in a container comprising a gas permeable material.

[0058] Such a container that includes a gas permeable material may further include a gas permeable material that is non-porous. Thus, in one embodiment, the gas permeable material is non-porous. In some embodiments, the gas permeable material is a membrane film such as silicone, fluoroethylene polypropylene, polyolefin, or ethylene vinyl acetate copolymer. Furthermore, such a container may only include a portion of a gas permeable material, a gas permeable membrane film, or a non-porous gas permeable material. Thus, according to yet a further embodiment, a container includes a lid, a bottom, and at least one sidewall, wherein at least a portion of the bottom of the container includes a gas permeable material that is substantially horizontal when the lid is above the bottom. In one embodiment, a container includes a lid, a bottom, and at least one sidewall, wherein at least a portion of the bottom includes a gas permeable material that is substantially horizontal when the lid is above the bottom. In further embodiments, the container comprises a lid, a bottom, and at least one sidewall, wherein the at least one sidewall comprises a gas permeable material that may be in a vertical plane when the lid is above the bottom, or in a horizontal plane when the lid is not above the bottom. In such embodiments, it will be understood that only a portion of the bottom or the sidewall may comprise a gas permeable material. Alternatively, the entire bottom or the entire sidewall may comprise a gas permeable material. In still further embodiments, the lid of the container comprising a gas permeable material may be sealed, for example, by the use of an O-ring. It will be understood that such embodiments prevent leakage or reduce evaporation of the contents of the container. Thus, in some embodiments, the container comprises a liquid-tight container comprising a gas permeable material to allow gas exchange. In an alternative embodiment, the lid of the container comprising a gas permeable material is in a horizontal plane and above the bottom, and is not sealed. Thus, in some embodiments, the lid is configured to allow gas exchange from the lid of the container. In further embodiments, the bottom of the gas permeable container is configured to allow gas exchange from the bottom of the container. In still further embodiments, the container comprising the gas permeable material is a liquid-tight container and may further comprise an inlet and an outlet or exhaust tube.Thus, in some embodiments, the container comprises a gas permeable material, comprising a lid, a bottom, and optionally at least one sidewall, wherein at least a portion of the lid and the bottom comprises a gas permeable material, and at least a portion of at least one sidewall, if present, comprises a gas permeable material.Examples of containers are described in WO2005035728 and US9255243, which are incorporated herein by reference.These containers are also commercially available, for example, G-REX® cell culture devices provided by Wilson Wolf Manufacturing, such as G-REX 6-well plate, G-REX 24-well plate, and G-REX 10 container.

[0059] Thus, according to one aspect of the present invention there is provided a method for isolating lymphocytes from a non-hematopoietic tissue sample comprising the steps of: (i) placing a non-hematopoietic tissue sample in a container comprising a gas permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. A method is provided that includes:

[0060] According to a further aspect of the invention there is provided a method for isolating γδ T cells from a non-hematopoietic tissue sample comprising the steps of: (i) placing a non-hematopoietic tissue sample in a container comprising a gas permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering the population of cultured γδ T cells from the non-hematopoietic tissue sample. A method is provided that includes:

[0061] In one embodiment, the non-hematopoietic tissue sample is placed on a synthetic scaffold. As used herein, "synthetic scaffold", "scaffold" and "grid" are used interchangeably and refer to a non-natural three-dimensional structure suitable for supporting cell growth. The non-hematopoietic tissue sample can be placed on or attached to a synthetic scaffold to promote lymphocyte egress from the explant onto the scaffold. The synthetic scaffold can be constructed from natural and / or synthetic materials such as polymers (e.g., natural or synthetic polymers, e.g., polyvinylpyrrolidone, polymethylmethacrylate, methylcellulose, polystyrene, polypropylene, polyurethane), ceramics (e.g., tricalcium phosphate, calcium aluminate, calcium hydroxyapatite), or metals (e.g., tantalum, titanium, platinum, and metals in the same elemental group as platinum, niobium, hafnium, tungsten, and combinations of alloys thereof). In one embodiment of the present invention, the synthetic scaffold is tantalum coated. Biological factors (e.g., collagen (e.g., collagen I or collagen II), fibronectin, laminin, integrins, angiogenic factors, anti-inflammatory factors, glycosaminoglycans, vitrogens, antibodies and fragments thereof, cytokines (e.g., IL-2, IL-15, and combinations thereof) can be coated onto the scaffold surface, encapsulated within the scaffold material, or added to the media to enhance cell attachment, migration, survival, or proliferation according to methods known in the art. This and other methods can be used to isolate lymphocytes from several other non-hematopoietic tissue types, e.g., skin, gastrointestinal, prostate, and breast.

[0062] In one embodiment, the non-hematopoietic tissue sample is placed on a synthetic scaffold inside a vessel used to isolate lymphocytes from the non-hematopoietic tissue sample. In a further embodiment, the synthetic scaffold is configured to promote lymphocyte and / or γδ T cell egress from the non-hematopoietic tissue sample to the bottom of the vessel. Such an embodiment has the advantage of allowing isolation and / or separation of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells) from the non-hematopoietic tissue sample and / or stromal cells (e.g., fibroblasts and / or epithelial cells). Furthermore, such an embodiment allows recovery of lymphocytes (e.g., γδ T cells, αβ T cells, and / or NK cells) from the non-hematopoietic tissue sample to the bottom of the culture vessel. In a particular embodiment, the synthetic scaffold is configured to promote the egress of γδ T cells from the non-hematopoietic tissue sample. In a further embodiment, the synthetic scaffold is configured to promote the egress of lymphocytes, e.g., αβ T cells and / or NK cells, from the non-hematopoietic tissue sample.

[0063] Thus, in one embodiment of the methods defined herein, the synthetic scaffold is configured to promote lymphocyte egress from a non-hematopoietic tissue sample to the bottom of a culture vessel. In a further embodiment of the methods defined herein, the synthetic scaffold is configured to promote γδ T cell egress from a non-hematopoietic tissue sample to the bottom of a culture vessel.

[0064] The methods of the present invention provide a much greater total cell yield than previously described. In one embodiment, the total isolated cell count is at least 10% of the tissue sample. 6 cells / cm 2 , at least 2 × 10 6 cells / cm 2 , at least 5 × 10 6 cells / cm 2 , at least 10 x 10 6 cells / cm 2 , at least 20 x 10 6 cells / cm 2 , at least 30 x 10 6 cells / cm2 , at least 40 x 10 6 cells / cm 2 , at least 50×10 6 cells / cm 2 , at least 60×10 6 cells / cm 2 , at least 70×10 6 cells / cm 2 , at least 80×10 6 cells / cm 2 , at least 90×10 6 cells / cm 2 , at least 100×10 6 cells / cm 2 , at least 150×10 6 cells / cm 2 , at least 200 x 10 6 cells / cm 2 In a specific embodiment, the total isolated cell number is at least 50×10 6 cells / cm 2 In another embodiment, the total isolated cell number is at least 100 x 10 6 cells / cm 2 It is.

[0065] The predominant γδ T cells in blood are primarily Vδ2 T cells, whereas the predominant γδ T cells in non-hematopoietic tissues are primarily Vδ1 T cells, such that Vδ1 T cells comprise 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 gastrointestinal tract, where they may comprise approximately 10-20% of the γδ T cells. Some γδ T cells resident in non-hematopoietic tissues do not express either the Vδ1 or Vδ2 TCR and are referred to herein as double-negative (DN) γδ T cells. These DN γδ T cells are likely to express mostly Vδ3, with a minority of Vδ5-expressing T cells. Therefore, γδ T cells that are naturally resident in non-hematopoietic tissues and γδ T cells isolated by the methods of the invention are preferably non-Vδ2 T cells, such as Vδ1 T cells, and comprise fewer DN γδ T cells.

[0066] Thus, in one preferred embodiment, the γδ T cells isolated by the method defined herein comprise a population of V51 T cells. In one embodiment, the γδ T cells isolated by the method defined herein comprise a population of DN γδ T cells. In one embodiment, the γδ T cells isolated by the method defined herein comprise a population of V53 T cells. In one embodiment, the γδ T cells isolated by the method defined herein comprise a population of V55 T cells.

[0067] A γδ T cell can also be defined by the type of γ chain it expresses. In a further embodiment, the γδ T cells isolated by the methods defined herein comprise a population of Vγ4 T cells. In most cases, the Vγ4 T cells are obtained from a gastrointestinal tissue sample.

[0068] The isolation method provides a population of isolated γδ T cells that is more numerous (e.g., at least 2 times the number, at least 3 times the number, at least 4 times the number, at least 5 times the number, at least 6 times the number, at least 7 times the number, at least 8 times the number, at least 9 times the number, at least 10 times the number, at least 15 times the number, at least 20 times the number, at least 25 times the number, at least 30 times the number, at least 35 times the number, at least 40 times the number, at least 50 times the number, at least 60 times the number, at least 70 times the number, at least 80 times the number, at least 90 times the number, at least 100 times the number, at least 200 times the number, at least 300 times the number, at least 400 times the number, at least 500 times the number, at least 600 times the number, at least 700 times the number, at least 800 times the number, at least 900 times the number, at least 1,000 times the number, at least 5,000 times the number, at least 10,000 times the number).

[0069] In some embodiments, the population of γδ T cells isolated according to the method of the present invention has a low percentage of cells expressing TIGIT. For example, the population of isolated γδ T cells may have a frequency of TIGIT+ cells of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. Alternatively, the population of isolated γδ T cells may have a frequency of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of TIGIT+ cells. In some embodiments, the population of isolated γδ T cells has a frequency of TIGIT+ cells of less than 80%. Thus, in one embodiment, the population of isolated γδ T cells has a frequency of about 70% of TIGIT+ cells. In a further embodiment, the population of isolated γδ T cells has a frequency of TIGIT+ cells of less than 60%. In yet a further embodiment, the isolated population of γδ T cells has a frequency of TIGIT+ cells of about 30%.Thus, in one embodiment, the isolated γδ T cells do not substantially express TIGIT.

[0070] In some embodiments, the isolated population of V51 T cells has a low frequency of TIGIT+ cells. For example, the isolated population of V51 T cells may have a frequency of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% of TIGIT+ cells other than the population of V51 T cells. Alternatively, the isolated population of V51 T cells may have a frequency of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% of TIGIT+ cells. In some embodiments, the isolated population of V51 T cells has a frequency of less than 80% of TIGIT+ cells. Thus, in one embodiment, the isolated population of V51 T cells has a frequency of about 70% of TIGIT+ cells. In a further embodiment, the isolated population of V51 T cells has a frequency of TIGIT+ cells of less than 60%. In yet a further embodiment, the isolated population of V51 T cells has a frequency of TIGIT+ cells of about 30%. Thus, in one embodiment, the isolated V51 T cells do not substantially express TIGIT.

[0071] In some embodiments, the population of γδ T cells isolated according to the methods of the invention expresses CD27. For example, the population of isolated γδ T cells may have a frequency of CD27+ cells of more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%. Alternatively, the population of isolated γδ T cells may have a frequency of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the population of isolated γδ T cells has a frequency of CD27+ cells of more than 10%. Thus, in one embodiment, the population of isolated γδ T cells has a frequency of CD27+ cells of about 20%. In a further embodiment, the population of isolated γδ T cells has a frequency of CD27+ cells of more than 20%. In one embodiment, the population of isolated γδ T cells has a frequency of CD27+ cells of about 20%.

[0072] In some embodiments, the isolated population of V51 T cells expresses CD27. In a further embodiment, the isolated γδ T cells express CD27. In some embodiments, the isolated population of V51 T cells has a frequency of CD27+ cells of more than 10%, more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, or more than 90%. Alternatively, the isolated population of γδ T cells may have a frequency of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the isolated population of V51 T cells has a frequency of CD27+ cells of more than 10%. Thus, in one embodiment, the isolated population of V51 T cells has a frequency of CD27+ cells of about 20%. In a further embodiment, the population of isolated V51 T cells has a frequency of CD27+ cells of more than 20%. In one embodiment, the population of isolated V51 T cells has a frequency of CD27+ cells of about 20%.

[0073] In some embodiments of any of the foregoing aspects, the isolated population of γδ T cells has greater surface expression of one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to a reference population (e.g., compared to a population of γδ T cells isolated using an alternative method). Additionally or alternatively, the isolated population of γδ T cells may have a greater frequency of cells expressing one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to the reference population. In particular, the marker is selected from CD45RA and CD25. In some embodiments, the isolated population of γδ T cells has a lower surface expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to a reference population. Additionally or alternatively, the isolated population of γδ T cells may have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to a reference population.

[0074] In some embodiments, the isolated population of V51 T cells has greater surface expression of one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 relative to a reference population. In some embodiments, the isolated population of γδ T cells has a greater frequency of cells expressing one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CDlb, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 relative to the reference. In some embodiments, the isolated population of γδ T cells has a lower surface expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to a reference population. In other embodiments, the isolated population of γδ T cells has a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to a reference population.

[0075] When isolated from non-hematopoietic tissues (e.g., skin), γδ T cells are usually part of a larger population of lymphocytes that contains, for example, αβ T cells, B cells, and natural killer (NK) cells. In some embodiments, 1%-10% of the isolated lymphocyte population are γδ T cells (e.g., 1-10% of the isolated skin-derived lymphocyte population are γδ T cells). In most cases, the γδ T cell population (e.g., skin-derived γδ T cell population) comprises a large population of Vδ1 T cells. In some embodiments, 1-10% of the isolated lymphocyte (e.g., skin-derived lymphocyte) population are Vδ1 T cells (e.g., Vδ1 T cells can represent more than 50%, more than 60%, more than 70%, more than 80%, or more than 90% of the isolated population of γδ T cells). In some cases, less than 10% of the population of isolated γδ T cells are V52 T cells (e.g., less than 10% of the population of isolated skin-derived γδ T cells are V52 T cells).

[0076] Non-V51 T cells or non-DN T cells, such as V52 T cells, αβ T cells, B cells, or NK cells, may be removed from the isolated population of γδ T cells (e.g. before, during or after the expansion step).

[0077] Isolated γδ T cells (e.g., γδ T cells isolated from the skin, e.g., V51 T cells isolated from the skin) have a distinct phenotype from the corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived V52 T cells). For example, the isolated population of γδ T cells may express higher levels of CCR3, CCR4, CCR7, CCR8, or CD103 than a reference population, e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells or a population of corresponding hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived V52 T cells). In some embodiments, the isolated population of γδ T cells expresses at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR3, CCR4, CCR7, CCR8, or CD103. +cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR4 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR7 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CCR8 + cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of CD103 + The isolated population of γδ T cells may express one or more, two or more, three or more, four or more, five or more, or all six of CCR3, CCR4, CCR7, CCR8, or CD103.

[0078] In some embodiments, the isolated population of γδ T cells (e.g., skin-derived γδ T cells and / or skin-derived V51 T cells) expresses higher levels of NKGD2, CD56, CD69, and / or TIM3 than a reference population, e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells and / or a corresponding population of hematopoietic tissue-derived cells (e.g., blood-derived γδ T cells and / or blood-derived V52 T cells). In some embodiments, the isolated population of γδ T cells expresses at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of NKGD2, CD56, CD69, and / or TIM3. + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD56 + cells; at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more CD69 + cells; and / or at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of TIM3+ The isolated population of γδ T cells may express one or more, two or more, three or more, four or more, or all five of NKGD2, CD56, CD69, and / or TIM3.

[0079] A population of isolated non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or skin-derived V51 T cells) can also be characterized by function. Functional assays known in the art can be performed to determine functional differences between any non-hematopoietic tissue-derived cells of the invention (e.g., isolated γδ T cells, a population of skin-derived V51 T cells, or a population of expanded γδ T cells and / or skin-derived V51 T cells) and reference cells (e.g., a population of TCR-activated non-hematopoietic tissue-resident γδ T cells or a corresponding hematopoietic tissue-derived cell, e.g., a population of blood-derived γδ T cells and / or blood-derived V52 T cells). Such assays can include proliferation assays, cytotoxicity assays, binding assays, assays measuring persistence and / or location, etc.

[0080] Thus, in one aspect of the invention, the method for isolating a lymphocyte and / or γδ T cell population defined herein yields a population comprising a surface phenotype consistent with a non-exhausted lymphocyte and / or γδ T cell population.

[0081] According to one aspect of the present invention there is provided a population of isolated lymphocytes (e.g. skin-derived αβ T cells and / or NK cells) obtainable by any of the methods defined herein.

[0082] According to one aspect of the invention there is provided a population of isolated lymphocytes (e.g. skin-derived αβ T cells and / or NK cells) obtained by any of the methods defined herein.

[0083] According to a further aspect of the invention there is provided an isolated population of γδ T cells obtainable by any of the methods defined herein.

[0084] According to a further aspect of the invention there is provided an isolated population of γδ T cells obtainable by any of the methods defined herein.

[0085] In one embodiment, the isolated population comprises more than 5% γδ T cells, for example, 7%-12% γδ T cells. In one embodiment, the isolated population comprises Vδ1 cells, wherein less than 50%, for example, less than 40%, of the Vδ1 cells express TIGIT. In one embodiment, the isolated population comprises Vδ1 cells, wherein more than 50%, for example, more than 60%, of the Vδ1 cells express CD27.

[0086] The isolated non-hematopoietic tissue resident lymphocytes may be suitable for use without further expansion, or may be expanded in a further step.

[0087] In certain embodiments, the invention features methods of expanding non-hematopoietic tissue resident lymphocytes and / or γδ T cells (e.g., skin-derived αβ T cells, NK cells, γδ T cells, and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells). These methods can be performed in vitro. In some embodiments, γδ T cells are expanded from a population of γδ T cells isolated from a non-hematopoietic tissue sample according to the methods defined herein. Generally, non-hematopoietic tissue resident γδ T cells can expand spontaneously when physical contact with stromal cells (e.g., skin fibroblasts) is removed. The methods defined herein can be used to induce such separation, resulting in disinhibition of γδ T cells and inducing expansion. In certain embodiments, lymphocytes (e.g., skin-derived αβ T cells and / or NK cells, gastrointestinal-derived αβ T cells and / or NK cells) are expanded from a population of lymphocytes isolated from a non-hematopoietic tissue sample according to the methods defined herein.

[0088] As used herein, reference to "expanded" or "expanded lymphocyte and / or γδ T cell populations" includes populations of cells that are larger than a non-expanded population or contain a greater number than a non-expanded population. Such populations may be greater in number, smaller in number, or mixed populations with expansion of some or particular cell types within the population. It will be understood that the term "expansion step" refers to a process that results in an expanded or expanded population. Thus, an expanded or expanded population may be greater in number or contain more cells compared to a population in which no expansion step has been performed or prior to any expansion step. It will be further understood that any numbers provided herein to indicate expansion (e.g., fold increase or expansion) are indicative of an increase in the number or size of a population of cells or number of cells, and are indicative of the amount of expansion.

[0089] Thus, in one embodiment, lymphocytes and / or γδ T cells isolated according to the methods of the invention are expanded. Such expansion may comprise culturing the γδ T cells in the presence of IL-2 and IL-15. Alternatively, expansion may comprise culturing the γδ T cells in the presence of IL-9 and IL-15. It will be appreciated that any expansion step is carried out for a period of time effective to produce an expanded population of lymphocytes and / or γδ T cells. In one embodiment, the period of time effective to produce an expanded population of lymphocytes and / or γδ T cells is at least 5 days. Thus, in one embodiment, expansion comprises culturing the γδ T cells in the presence of IL-2 and IL-15 in an amount effective to produce an expanded population of γδ T cells for at least 5 days. In an alternative embodiment, expansion comprises culturing the γδ T cells in the presence of IL-9 and IL-15 in an amount effective to produce an expanded population of γδ T cells for at least 5 days. In further embodiments, expansion comprises culturing the lymphocytes and / or γδ T cells for a period of time (e.g., at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or longer, e.g., between 5 and 40 days, between 7 and 35 days, between 14 and 28 days, or about 21 days) in an amount effective to produce an expanded population of lymphocytes and / or γδ T cells. In some embodiments, lymphocytes and / or γδ T cells are expanded in culture for several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, or 21 hours) to about 35 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 days). In one embodiment, lymphocytes and / or γδ T cells are expanded for 14 to 21 days.Thus, the isolation and expansion process, including the isolation culture period (eg, 1-40 days, such as 14-21 days), can last, in some embodiments, 28-56 days, or about 41 days.

[0090] In further embodiments, the expansion comprises culturing the γδ T cells for at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 21 days, at least 28 days, or for a longer period, e.g., from 5 to 40 days, from 7 to 35 days, from 14 to 28 days, or about 21 days. In one embodiment, the expansion step comprises culturing the γδ T cells for at least 10 days, 15 days, or 20 days to produce an expanded population. In one embodiment, the expansion step comprises culturing the γδ T cells for 5 to 25 days, e.g., from 14 to 21 days. In a further embodiment, the expansion step comprises culturing the γδ T cells for about 20 days.

[0091] In some embodiments, a typical amount of IL-2 effective to generate an expanded population of γδ T cells is between 1 IU / mL and 2,000 IU / mL (e.g., between 5 IU / mL and 1,000 IU / mL, between 10 IU / mL and 500 IU / mL, between 20 IU / mL and 400 IU / mL, between 50 IU / mL and 250 IU / mL, or about 100 IU / mL, e.g., between 5 IU / mL and 10 IU / mL, between 10 IU / mL and 20 IU / mL, between 20 IU / mL and 30 IU / mL, between 30 IU / mL and 40 IU / mL, between 40 IU / mL and 50 IU / mL, between 50 IU / mL and 60 IU / mL, between 60 IU / mL and 70 IU / mL, between 70 IU / mL and 80 IU / mL). / mL, 80IU / mL~90IU / mL, 90IU / mL~100IU / mL, 100IU / mL~120IU / mL, 120IU / mL~140IU / mL, 140IU / mL~150IU / mL, 150IU / mL~175IU / mL, 175IU / mL~200IU / mL, 20 0IU / mL~300IU / mL, 300IU / mL~400IU / mL, 400IU / mL~500IU / mL, 500IU / mL~1,000IU / mL, 1,000IU / mL~1,500IU / mL, 1,500IU / mL~2,000IU / mL, or more). In some embodiments, the amount of IL-2 effective to generate an expanded population of γδ T cells is about 100 IU / mL.

[0092] In some embodiments, an exemplary amount of IL-15 effective to generate an expanded population of γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is at least 0.1 ng / mL (e.g., 0.1 ng / mL to 10,000 ng / mL, 1.0 ng / mL to 1,000 ng / mL, 5 ng / mL to 800 ng / mL, 10 ng / mL to 750 ng / mL, 20 ng / mL to 500 ng / mL, 50 ng / mL to 400 ng / mL, or 100 ng / mL to 250 ng / mL). 10 ng / mL, e.g., 0.1 ng / mL to 1.0 ng / mL, 1.0 ng / mL to 5.0 ng / mL, 5.0 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 50 ng / mL, 50 ng / mL to 100 ng / mL, 100 ng / mL to 200 ng / mL, 200 ng / mL to 500 ng / mL, or 500 ng / mL to 1,000 ng / mL). In some embodiments, the amount of IL-15 effective to generate an expanded population of γδ T cells is about 10 ng / mL.

[0093] Substitution or addition of other factors in the expansion of non-hematopoietic tissue resident γδ T cells can also be used. Such additional or alternative factors for the expansion of lymphocytes such as αβ T cells or NK cells are known in the art. In one embodiment, such factors are used in the expansion to selectively promote the expansion of γδ T cells. In a further embodiment, such factors are used in the expansion to selectively promote the expansion of lymphocytes such as αβ T cells and / or NK cells.

[0094] It will be appreciated that the amount of each of the above cytokines required to produce an expanded population of γδ T cells will depend on the concentration of one or more of the other cytokines. For example, if the concentration of IL-2 is increased or decreased, the concentration of IL-15 may be decreased or increased accordingly, respectively. As above, an amount effective to produce an expanded population herein refers to the combined effect of all factors on cell expansion.

[0095] The expansion method provides a population of expanded γδ T cells that is more numerous than a reference population. In some embodiments, the expanded population of γδ T cells is more numerous than the population of isolated γδ T cells prior to the expansion step (e.g., the population of isolated γδ T cells prior to the expansion step). at least 2 times as many, at least 3 times as many, at least 4 times as many, at least 5 times as many, at least 6 times as many, at least 7 times as many, at least 8 times as many, at least 9 times as many, at least 10 times as many, at least 15 times as many, at least 20 times as many, at least 25 times as many, at least 30 times as many, at least 35 times as many, at least 40 times as many, at least 50 times as many, at least 60 times as many, at least 70 times as many, at least 80 times as many, at least 90 times as many, at least 100 times as many, at least 200 times as many, at least 300 times as many, at least 400 times as many, at least 500 times as many, at least 600 times as many, at least 700 times as many, at least 800 times as many, at least 900 times as many, at least 1,000 times as many, at least 5,000 times as many, at least 10,000 times as many, or more).

[0096] In one embodiment, the expansion step comprises culturing the isolated γδ T cells in the substantial absence of stromal cell contact, hi a further embodiment, the expansion step comprises culturing the isolated γδ T cells in the substantial absence of fibroblast cell contact.

[0097] It will be understood that the expansion methods provided herein also apply to the expansion of other lymphocytes (e.g., αβ T cells and / or NK cells). In such embodiments, the expansion step comprises culturing the isolated lymphocytes in the presence of relevant growth factors and / or nutrients (e.g., cytokines and / or chemokines) to produce a population of expanded lymphocytes (e.g., αβ T cells and / or NK cells).

[0098] In one embodiment, the method of expanding a population of γδ T cells defined herein comprises culturing γδ T cells or other lymphocytes in serum-free medium. In a further embodiment, the method of expanding a population of γδ T cells defined herein comprises culturing γδ T cells in medium containing a serum replacement. It will therefore be appreciated that the expansion of such γδ T cells in serum-free medium or serum replacement-containing medium obtains similar advantages as those described above.

[0099] In some embodiments, substantial TCR pathway activation is absent during the expansion step (e.g., no exogenous TCR pathway activators are included in the culture). In one embodiment, the expansion step comprises the absence of exogenous TCR pathway agonists. Further provided herein is a method of expanding γδ T cells isolated according to the methods defined herein, wherein the expansion method does not involve contact with feeder cells, tumor cells, and / or antigen presenting cells. Thus, in a further embodiment of the methods defined herein, the expansion of γδ T cells comprises culturing the γδ T cells in the absence of substantial stromal cell contact.

[0100] Also provided are means to generate large populations of non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) at high rates (e.g., by removing stromal cell contact and / or TCR stimulation, or by culturing in the presence of an effective amount of a factor). In some embodiments, the expansion steps described herein expand γδ T cells with a low population doubling time given by the following formula:

number

[0101] In view of the information provided herein, the skilled artisan will appreciate that the present invention is a method for expanding non-hematopoietic tissue-derived γδ T cells (e.g. skin-derived γδ T cells and / or non-V52 T cells, such as V51 T cells and / or DN T cells) with a population doubling time of less than 5 days (e.g. less than 4.5 days, less than 4.0 days, less than 3.9 days, less than 3.8 days, less than 3.7 days, less than 3.6 days, less than 3.5 days, less than 3.4 days, less than 3.3 days, less than 3.2 days, less than 3.1 days, less than 3.0 days, less than 2.9 days, less than 2.8 days, less than 2.7 days, less than 2.6 days, less than 2.5 days, less than 2.4 days, less than 2.3 days, less than 2.2 days, less than 2.1 days, less than 2.0 days, less than 46 hours, less than 42 hours, less than 38 hours, less than 35 hours, less than 32 hours).

[0102] In some embodiments, within 7 days of culturing, the expanded population of γδ T cells (e.g., the expanded population of V51 T cells and / or DN T cells) comprises at least 10-fold the number of γδ T cells compared to the population of isolated γδ T cells prior to expansion (e.g., at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, or at least 8,000-fold the number of γδ T cells compared to the population of isolated γδ T cells prior to expansion). In some embodiments, within 14 days of culture, the expanded population of γδ T cells (e.g., an expanded population of V51 T cells and / or DN T cells) exhibits at least a 20-fold increase in the number of γδ T cells (e.g., an expanded population of V51 T cells and / or DN T cells) compared to the population of isolated γδ T cells prior to expansion. In some embodiments, the population of γδ T cells comprises at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, or at least 10,000 times the number of γδ T cells) compared to the population of T cells.In some embodiments, within 21 days of culture, the expanded population of γδ T cells (e.g., the expanded population of V51 T cells and / or DN T cells) comprises at least 50-fold more γδ T cells than the population of isolated γδ T cells prior to expansion (e.g., at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 600-fold, at least 700-fold, at least 800-fold, at least 900-fold, at least 1,000-fold, at least 2,000-fold, at least 3,000-fold, at least 4,000-fold, at least 5,000-fold, at least 6,000-fold, at least 7,000-fold, at least 8,000-fold, at least 9,000-fold, or at least 10,000-fold more γδ T cells than the population of isolated γδ T cells prior to expansion). In some embodiments, within 28 days of culture, the expanded population of γδ T cells (e.g., an expanded population of V51 T cells and / or DN T cells) exhibits at least 100-fold higher numbers of γδ T cells (e.g., an expanded population of V51 T cells and / or DN T cells) than the population of isolated γδ T cells prior to expansion. In some embodiments, the population of γδ T cells comprises at least 110 times, at least 120 times, at least 130 times, at least 140 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 2,000 times, at least 3,000 times, at least 4,000 times, at least 5,000 times, at least 6,000 times, at least 7,000 times, at least 8,000 times, at least 9,000 times, at least 10,000 times, at least 12,000 times, or at least 15,000 times the number of γδ T cells) compared to the population of T cells.

[0103] Non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) expanded by the methods provided herein can have a phenotype well suited for anti-tumor efficacy. In some embodiments, the expanded population of γδ T cells (e.g., skin-derived V51 T cells) has a greater average expression of CD27 than a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells has an average expression of CD27 that is at least twice as high as the population of isolated γδ T cells (e.g., at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, at least 150 times, at least 200 times, at least 300 times, at least 400 times, at least 500 times, at least 600 times, at least 700 times, at least 800 times, at least 900 times, at least 1,000 times, at least 5,000 times, at least 10,000 times, at least 20,000 times, or more, as compared to the population of isolated γδ T cells).

[0104] A distinct fraction of the expanded population of γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) can upregulate CD27, while another fraction can upregulate CD27 low or CD27 negative In this case, the CD27 expression in the expanded population compared to the isolated γδ T cell population positive The frequency of cells can be greater, for example, the expanded population of γδ T cells has at least a 5% greater frequency of CD27 positivecells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater frequency of CD27 T cells compared to the frequency of the isolated population of γδ T cells prior to expansion). positive In some embodiments, the expanded population may have CD27 T cells relative to the isolated population of γδ T cells. positive The number of cells can be increased, for example, the expanded population of γδ T cells can have at least twice as many CD27 positive The expanded population of γδ T cells may have a frequency of CD27+ cells of greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, or greater than 90%. Alternatively, the expanded population of γδ T cells may have a frequency of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In certain embodiments, the expanded population of γδ T cells has a frequency of CD27+ cells of greater than 50%.

[0105] The expansion methods provided herein, in some embodiments, produce a population of expanded non-hematopoietic tissue-derived γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) that has lower expression of TIGIT compared to a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells has lower average expression of TIGIT than a reference population (e.g., a population of isolated γδ T cells prior to the expansion step). In some embodiments, the expanded population of γδ T cells has average expression of TIGIT at least 10% less than the population of isolated γδ T cells (e.g., at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, or up to 100% less than the population of isolated γδ T cells). The expanded population of γδ T cells may have a frequency of TIGIT+ cells of less than 90%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. Alternatively, the expanded population of γδ T cells may have a frequency of about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10%. In certain embodiments, the isolated population of γδ T cells has a frequency of TIGIT+ cells of less than 80%.

[0106] In some embodiments, the expanded population of γδ T cells (e.g., skin-derived γδ T cells or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is a population of cells expressing a high number or frequency of CD27 + Cells and low frequency of TIGIT + In some embodiments, the expanded population of γδ T cells has a higher frequency of CD27 + TIGIT -For example, the expanded population of γδ T cells has a frequency of CD27 + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater frequency of CD27 T cells compared to the frequency of the isolated population of γδ T cells prior to expansion). + TIGIT - In some embodiments, the expanded population may have CD27 T cells relative to the isolated population of γδ T cells. + TIGIT - The number of cells can be increased, for example, the expanded population of γδ T cells can have at least twice as many CD27 + TIGIT - cells (e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater frequency of CD27 T cells compared to the frequency of the isolated population of γδ T cells prior to expansion). + TIGIT - The present invention may include a method for producing a cellular endothelial cell.

[0107] Optionally, CD27 expression within a population of expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) + The average expression of TIGIT in a population of γδ T cells is low compared to a reference population. + The population of γδ T cells can be determined by comparing it with a reference population (e.g., isolated CD27 T cells prior to the expansion step). + In some embodiments, the expanded CD27+ A population of γδ T cells was isolated using CD27 + At least 10% less than the population of γδ T cells (e.g., isolated CD27 + The population of γδ T cells has an average expression of TIGIT that is at least 20% less, at least 30% less, at least 40% less, at least 50% less, at least 60% less, at least 70% less, at least 80% less, at least 90% less, or up to 100% less than the population of γδ T cells.

[0108] Additionally or alternatively, TIGIT expression in a population of expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-Vδ2 T cells, e.g., Vδ1 T cells and / or DN T cells) may be determined. - The median expression of CD27 in the γδ T cell population is higher than in the reference population. - The γδ T cell population was isolated from TIGIT - A frequency of CD27 that is at least 5% greater than the frequency of the γδ T cell population + Cells (e.g., isolated TIGIT cells before expansion) - a frequency of CD27 that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater than the frequency of the population of γδ T cells; + In some embodiments, the isolated TIGIT - CD27 in the expanded population compared with the γδ T cell population + The number of cells can be increased, e.g., expanded TIGIT - The γδ T cell population was isolated from TIGIT - At least twice as many CD27 + Cells (e.g., isolated TIGIT cells before expansion) -a frequency of CD27 that is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or up to 100% greater than the frequency of the population of γδ T cells; + The present invention may include a method for producing a cellular endothelial cell.

[0109] Increased or decreased expression of other markers can additionally or alternatively be used to characterize one or more expanded populations of non-hematopoietic tissue-derived γδ T cells (e.g. skin-derived γδ T cells and / or non-V52 T cells, e.g. V51 T cells and / or DN T cells), including CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, CD2, NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64. Optionally, the expanded population of γδ T cells (e.g. skin-derived γδ T cells and / or non-V52 T cells, e.g. V51 T cells and / or DN T cells) has, for example, a greater average expression of one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to the population of isolated γδ T cells prior to expansion. Additionally or alternatively, the expanded population of γδ T cells may have a greater frequency of cells expressing one or more of the markers selected from the group consisting of CD124, CD215, CD360, CTLA4, CD1b, BTLA, CD39, CD45RA, Fas ligand, CD25, ICAM-1, CD31, KLRG1, CD30, and CD2 compared to the population of isolated γδ T cells. In some embodiments, the expanded population of γδ T cells has a lower average expression of one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells. The expanded population may also have a lower frequency of cells expressing one or more of the markers selected from the group consisting of NKp44, NKp46, ICAM-2, CD70, CD28, CD103, NKp30, LAG3, CCR4, CD69, PD-1, and CD64 compared to the population of isolated γδ T cells.

[0110] Thus, the non-hematopoietic tissue resident γδ T cells produced by the methods of the invention may have one or more of the following characteristics: (i) CD69 high , TIM3 high , and CD28 low / absent (ii) upregulate one or more of CCR3, CD39, CD11b, and CD9; (iii) produce IFN-γ in response to an NKG2D ligand 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 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 over normal cells.

[0111] Optionally, non-hematopoietic tissue resident γδ T cells generated by the methods of the 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.

[0112] Many basal culture media suitable for use in the expansion of γδ T cells are available, in particular media such as AIM-V, Iscoves medium, and RPMI-1640 (Life Technologies). The media may be supplemented with other media factors as defined herein, such as serum, serum proteins, and selection agents, antibiotics. For example, in some embodiments, RPMI-1640 medium contains 2 mM glutamine, 10% FBS, 10 mM HEPES, pH 7.2, 1% penicillin-streptomycin, sodium pyruvate (1 mM; Life Technologies), non-essential amino acids (e.g., 100 μM Gly, Ala, Asn, Asp, Glu, Pro, and Ser; 1×MEM Non-Essential Amino Acids (Life Technologies)), and 10 μl / L β-mercaptoethanol. In an alternative embodiment, AIM-V medium may be supplemented with serum replacement and amphotericin B from CTS Immune. In certain embodiments as defined herein, the medium may be further supplemented with IL-2 and IL-15. Advantageously, during isolation and / or expansion, the cells are cultured in a suitable culture medium at 37° C. in a humidified atmosphere containing 5% CO2.

[0113] According to a further aspect of the present invention, there is provided a method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample, comprising: (i) isolating a population of lymphocytes from said non-hematopoietic tissue sample according to the methods defined herein; and (ii) further culturing the population of lymphocytes (e.g., for at least 5 days) to produce an expanded population of lymphocytes. A method is provided that includes:

[0114] In one embodiment, the lymphocytes comprise αβ T cells. Thus, according to a further aspect of the present invention, there is provided a method for isolating and expanding αβ T cells from a non-hematopoietic tissue sample comprising: (i) isolating a population of αβ T cells from said non-hematopoietic tissue sample according to the methods defined herein; and (ii) further culturing the population of αβ T cells (e.g., for at least 5 days) to produce an expanded population of αβ T cells. A method is provided that includes:

[0115] The culturing in step (ii) may be by selective expansion, e.g., by selecting culture conditions under which αβ T cells are preferentially expanded over other cell types present in the isolated population of step (i). Alternatively, the expansion conditions may be non-selective and non-target cells (e.g., cells other than αβ T cells) are removed after the culturing in step (ii). Alternatively, the expansion conditions are non-selective and removal of non-target cells (e.g., cells other than αβ T cells) is performed prior to the culturing in step (ii). Note that the objective of these embodiments is to expand the total number of αβ T cells while also increasing their proportion in the population.

[0116] In one embodiment, the lymphocytes comprise NK cells. Thus, according to a further aspect of the present invention, there is provided a method for isolating and expanding NK cells from a non-hematopoietic tissue sample comprising: (i) isolating a population of NK cells from a non-hematopoietic tissue sample according to the methods defined herein; and (ii) further culturing the population of NK cells (e.g., for at least 5 days) to produce an expanded population of NK cells. A method is provided that includes:

[0117] The culturing in step (ii) may be by selective expansion, e.g., by selecting culture conditions under which NK cells are preferentially expanded over other cell types present in the isolated population of step (i). Alternatively, the expansion conditions may be non-selective and non-target cells (e.g., cells other than NK cells) are removed after the culturing in step (ii). Alternatively, the expansion conditions are non-selective and removal of non-target cells (e.g., cells other than NK cells) occurs prior to the culturing in step (ii). It should be noted that the objective of these embodiments is to expand the total number of NK cells while also increasing their proportion in the population.

[0118] In one embodiment, the lymphocytes comprise γδ T cells. Thus, according to a further aspect of the invention, there is provided a method for the isolation and expansion of γδ T cells from a non-hematopoietic tissue sample comprising: (i) isolating a population of γδ T cells from a non-hematopoietic tissue sample according to the methods defined herein; and (ii) further culturing the population of γδ T cells (e.g., for at least 5 days) to produce an expanded population of γδ T cells. A method is provided that includes:

[0119] The culturing in step (ii) may be by selective expansion, e.g., by selecting culture conditions under which γδ T cells are preferentially expanded over other cell types present in the isolated population of step (i). Alternatively, the expansion conditions may be non-selective and non-target cells (e.g., cells other than γδ T cells) are removed after the culturing in step (ii). Alternatively, the expansion conditions are non-selective and removal of non-target cells (e.g., cells other than γδ T cells) is performed prior to the culturing in step (ii). Note that the objective of these embodiments is to expand the total number of γδ T cells while also increasing their proportion in the population.

[0120] According to one aspect of the invention there is provided an expanded isolated population of lymphocytes (e.g., skin-derived αβ T cells and / or NK cells) obtained by any of the methods defined herein.

[0121] According to a further aspect of the present invention there is provided an expanded isolated population of lymphoid cells obtainable by any of the methods defined herein.

[0122] According to yet a further aspect of the invention there is provided an expanded isolated population of γδ T cells obtained by any of the methods defined herein.

[0123] According to yet a further aspect of the present invention there is provided an isolated expanded population of γδ T cells obtainable by any of the methods defined herein.

[0124] In one embodiment, the isolated population comprises more than 50% γδ T cells, such as more than 75% γδ T cells, in particular more than 85% γδ T cells. In one embodiment, the isolated population comprises Vδ1 cells, wherein less than 50%, such as less than 25%, of the Vδ1 cells express TIGIT. In one embodiment, the isolated population comprises Vδ1 cells, wherein more than 50%, such as more than 60%, of the Vδ1 cells express CD27.

[0125] The lymphocytes and / or γδ T cells obtained by the method of the invention can be used as a medicine, for example, for adoptive T cell therapy. This involves transplantation of lymphocytes and / or γδ T cells obtained by the method of the invention into a patient. The therapy can be autologous, i.e., γδ T cells can be transplanted back into the same patient from which they were obtained, or the therapy can be allogeneic, i.e., γδ T cells from one person can be transplanted into a different patient. When involving allogeneic transplantation, the γδ T cells can be substantially free of αβ T cells. For example, αβ T cells can be removed from the γδ T cell population after expansion, using any suitable means known in the art (e.g., by negative selection, e.g., using magnetic beads). The method of treatment can include; providing a sample of non-hematopoietic tissue obtained from a donor individual; culturing γδ T cells from said sample to produce an expanded population; and administering the expanded population of γδ T cells to a recipient individual.

[0126] The patient or subject to be treated is preferably a human cancer patient (e.g., a human cancer patient undergoing treatment for a solid tumor) or a viral infection patient (e.g., a CMV-infected or HIV-infected patient). Optionally, the patient has a solid tumor and / or is undergoing treatment for a solid tumor.

[0127] Because tissue-resident V51 T and DN γδ T cells normally reside in non-hematopoietic tissues, they are also more likely to home to and be retained in 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 immunopathologies.

[0128] As γδ T cells are non-MHC restricted, they do not recognise the host into which they are transplanted as foreign, meaning that they are unlikely to cause graft-versus-host disease, which means that γδ T cells can be used "off the shelf" and transplanted into any recipient, for example for allogeneic adoptive T cell therapy.

[0129] 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) that are strongly associated with malignant tumors. Non-hematopoietic tissue resident γδ T cells also express a cytotoxic profile in the absence of any activation and are therefore likely to be effective in killing tumor cells. For example, non-hematopoietic tissue resident γδ T cells obtained as described herein may express one or more, preferably all, of IFN-γ, TNF-α, GM-CSF, CCL4, IL-13, granulysin, granzymes A and B, and perforin in the absence of any activation. IL-17A may not be expressed.

[0130] The findings reported herein therefore provide strong evidence for the practicality and suitability of the clinical application of non-hematopoietic tissue-resident γδ T cells obtained by the methods of the invention as "off the shelf" immunotherapeutic reagents: these cells possess innate-like killing, are not MHC restricted, and show improved homing to and / or retention within tumors compared to other T cells.

[0131] In some embodiments, a method for treating an individual having a tumor in a non-hematopoietic tissue may comprise: providing a sample of said non-hematopoietic tissue obtained from a donor individual, culturing γδ T cells from said sample to produce an expanded population, and; administering the expanded population of γδ T cells to the individual having the tumor.

[0132] A pharmaceutical composition may comprise the expanded non-hematopoietic tissue-resident γδ T cells described herein in combination with one or more pharma- ceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include a buffer, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. Cryoprotective solutions that may be used in the pharmaceutical compositions of the invention include, for example, DMSO. The compositions may be formulated, for example, for intravenous administration.

[0133] In one embodiment, the pharmaceutical composition is substantially free, eg, absent, detectable levels of contaminants, eg, endotoxins or mycoplasma.

[0134] Optionally, a therapeutically effective amount of expanded γδ T cells obtained by any of the above methods can be administered to a subject in a therapeutically effective amount (e.g., for the treatment of cancer, e.g., for the treatment of a solid tumor). Optionally, a therapeutically effective amount of expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is greater than or equal to 10×10 per dose. 12 Fewer than 10 cells (e.g., 9 x 10 per dose) 12 Less than 8 x 10 cells per dose 12 Less than 7 x 10 cells per dose 12 Less than 6 x 10 cells per dose 12 Less than 5 x 10 cells per dose 12Less than 4 x 10 cells per dose 12 Less than 3 x 10 cells per dose 12 Less than 2 x 10 cells per dose 12 Less than 1 x 10 cells per dose 12 Less than 9 x 10 cells per dose 11 Less than 8 x 10 cells per dose 11 Less than 7 x 10 cells per dose 11 Less than 6 x 10 cells per dose 11 Less than 5 x 10 cells per dose 11 Less than 4 x 10 cells per dose 11 Less than 3 x 10 cells per dose 11 Less than 2 x 10 cells per dose 11 Less than 1 x 10 cells per dose 11 Less than 9 x 10 cells per dose 10 Less than 7.5 x 10 cells per dose 10 Less than 5 x 10 cells per dose 10 Less than 2.5 x 10 cells per dose 10 Less than 1 x 10 cells per dose 10 Less than 7.5 x 10 cells per dose 9 Less than 5 x 10 cells per dose 9 Less than 2.5 x 10 cells per dose 9 Less than 1 x 10 cells per dose 9 Less than 7.5 x 10 cells per dose 8 Less than 5 x 10 cells per dose 8 Less than 2.5 x 10 cells per dose 8 Less than 1 x 10 cells per dose 8 Less than 7.5 x 10 cells per dose 7 Less than 5 x 10 cells per dose 7 Less than 2.5 x 10 cells per dose 7 Less than 1 x 10 cells per dose 7 Less than 7.5 x 10 cells per dose 6 Less than 5 x 10 cells per dose 6 Less than 2.5 x 10 cells per dose 6 Less than 1 x 10 cells per dose6 Less than 7.5 x 10 cells per dose 5 Less than 5 x 10 cells per dose 5 Less than 2.5 x 10 cells per dose 5 Less than 1 x 10 cells or 1 x 10 per dose 5 In some embodiments, a therapeutically effective amount of expanded γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is less than 10×10 cells during the course of treatment. 12 Fewer than 9 × 10 cells (e.g., during the course of treatment) 12 Less than 8 x 10 cells 12 Less than 7 x 10 cells 12 Less than 6 x 10 cells 12 Less than 5 x 10 cells 12 Less than 4 x 10 cells 12 Less than 3 x 10 cells 12 Less than 2 x 10 cells 12 Less than 1 x 10 cells 12 Less than 9 x 10 cells 11 Less than 8 x 10 cells 11 Less than 7 x 10 cells 11 Less than 6 x 10 cells 11 Less than 5 x 10 cells 11 Less than 4 x 10 cells 11 Less than 3 x 10 cells 11 Less than 2 x 10 cells 11 Less than 1 x 10 cells 11 Less than 9 x 10 cells 10 Less than 7.5 x 10 cells 10 Less than 5 x 10 cells 10 Less than 2.5 x 10 cells 10 Less than 1 x 10 cells 10 Less than 7.5 x 10 cells 9 Less than 5 x 10 cells 9 Less than 2.5 x 10 cells 9 Less than 1 x 10 cells 9 Less than 7.5 x 10 cells 8 Less than 5 x 10 cells 8 Less than 2.5 x 10 cells 8Less than 1 x 10 cells 8 Less than 7.5 x 10 cells 7 Less than 5 x 10 cells 7 Less than 2.5 x 10 cells 7 Less than 1 x 10 cells 7 Less than 7.5 x 10 cells 6 Less than 5 x 10 cells 6 Less than 2.5 x 10 cells 6 Less than 1 x 10 cells 6 Less than 7.5 x 10 cells 5 Less than 5 x 10 cells 5 Less than 2.5 x 10 cells 5 Less than 1 x 10 cells or 1 x 10 5 (fewer than 10 cells).

[0135] In some embodiments, the dose of expanded non-hematopoietic tissue resident γδ T cells described herein is about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5 × 10 8 In some embodiments, the dose of expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises at least about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5 × 10 8In some embodiments, the dose of expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is up to about 1×10 6 , 1.1×10 6 , 2×10 6 , 3.6×10 6 , 5×10 6 , 1×10 7 , 1.8×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , or 5 × 10 8 In some embodiments, the dose of expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises about 1.1×10 6 ~1.8×10 7 In some embodiments, the dose of expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) comprises about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , or 5 × 10 9 In some embodiments, the dose of expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is at least about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , or 5 × 10 9In some embodiments, the dose of expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells) is up to about 1×10 7 , 2×10 7 , 5×10 7 , 1×10 8 , 2×10 8 , 5×10 8 , 1×10 9 , 2×10 9 , or 5 × 10 9 Contains cells.

[0136] In one embodiment, the subject is administered 10 4 ~10 6 10 expanded non-hematopoietic tissue resident γδ T cells (e.g., skin-derived γδ T cells and / or non-V52 T cells, e.g., V51 T cells and / or DN T cells). In one embodiment, the subject receives an initial administration of a population of non-hematopoietic tissue resident γδ T cells (e.g., 10 4 ~10 6 An initial administration of 10 γδ T cells per kg of subject body weight, for example 4 ~10 5 γδ T cells), and one or more (e.g., 2, 3, 4, or 5) subsequent administrations of expanded non-hematopoietic tissue resident γδ T cells (e.g., 10 4 ~10 6 10 expanded non-hematopoietic tissue resident γδ T cells per kg body weight of the subject, for example 4 ~10 5 In one embodiment, the subject receives a total of about 100 or more subsequent administrations of expanded non-hematopoietic tissue resident γδ T cells per kg body weight of the subject over the course of at least three administrations of the population of γδ T cells. In one embodiment, the one or more subsequent administrations are administered less than 15 days, e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, e.g., less than 4, 3, or 2 days, since the previous administration. In one embodiment, the subject receives a total of about 100 or more subsequent administrations of expanded non-hematopoietic tissue resident γδ T cells per kg body weight of the subject over the course of at least three administrations of the population of γδ T cells. 6 γδ T cells, for example, a subject may receive 1×10 5Initial administration of γδ T cells, 3 × 10 5 A second administration of 6 x 10 γδ T cells, and 5 and a third administration of γδ T cells, eg, each administration being administered less than 4, 3, or 2 days after the previous administration.

[0137] The non-hematopoietic tissue resident γδ T cells obtained by the method of the present invention can also be genetically modified for enhanced therapeutic properties, e.g., CAR-T therapy. This involves the generation of modified T cell receptors (TCRs) that reprogram T cells with new specificities, e.g., the specificity of monoclonal antibodies. The modified TCRs can create T cells that are specific for malignant cells and therefore useful for cancer immunotherapy. For example, the T cells can recognize cancer cells that express tumor antigens, e.g., tumor-associated antigens, that are not expressed by normal somatic cells derived from the target tissue. Thus, CAR-modified T cells can be used, for example, in adoptive T cell therapy for cancer patients.

[0138] 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 are likely to be a particularly good vehicle for the CAR-T approach, since they can retain their natural-like ability to recognize transformed cells while being transduced with a chimeric antigen-specific TCR, and are likely to have better tumor penetration and retention capabilities than either blood-resident γδ T cells or conventional systemic αβ T cells. Furthermore, its lack of MHC-dependent antigen presentation reduces the potential for graft-versus-host disease and allows it to target tumors that express low levels of MHC. Similarly, its independence from conventional co-stimulation, for example by CD28 engagement, enhances the targeting of tumors that express low levels of ligands of co-stimulatory receptors.

[0139] In some embodiments, one or more additional therapeutic agents can be administered to the subject. The additional therapeutic agent can be selected from the group consisting of an immunotherapeutic agent, a cytotoxic agent, a growth inhibitory agent, a radiotherapeutic agent, an antiangiogenic agent, or a combination of two or more of these agents. The additional therapeutic agent can be administered simultaneously with, prior to, or after administration of the expanded γδ T cells. The additional therapeutic agent can be an immunotherapeutic agent that can act on targets in the subject's body (e.g., the subject's own immune system) and / or on the transplanted γδ T cells.

[0140] Administration of the compositions can be performed in any convenient manner. The compositions described herein can be administered to a patient by intraarterial, subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous injection, or intraperitoneally, e.g., intradermal or subcutaneous injection. Compositions of non-hematopoietic tissue resident γδ T cells can be injected directly into a tumor, lymph node, or site of infection.

[0141] It will be understood that all of the embodiments described herein are applicable to all aspects of the present invention.

[0142] As used herein, the term "about" as used herein includes values ​​below and including 10% greater than the specified value and values ​​above and including 10% less than the specified value, preferably values ​​below and including 5% greater than and including 5% less than the specified value, and particularly values ​​as specified. The term "between" includes values ​​in the specified range.

[0143] Certain aspects and embodiments of the present invention will now be described, by way of example, and with reference to the above-mentioned drawings. EXAMPLES

[0144] (Example) (Example 1. Analysis method) Unless otherwise stated, the following methods were used to produce the results in the following examples.

[0145] (Flow Cytometry) Flow cytometry was performed using the following antibody-fluorophore conjugates: Ki-67-BV421, CD3-BV510, Vδ1-PeVio770, TIM-3-PE, CD9-PE, CCR3-BV421, and CD39-BV421. Samples were also stained for viability using eFluor770NIR. Commercial antibodies were purchased from Biolegend or Miltenyi. Viability dyes (near IR) were from eBioscience. Ki-67 staining was performed on fixed and permeabilized cells using Foxp3 staining buffer set (eBioscience). At the end of each experiment, cell populations were washed in PBS and split in half. Cells were stained for viability with eFluor770 NIR, washed, and then stained with TrueStain (Biolegend) to avoid non-specific binding of the staining antibodies. Half of the samples were stained for the indicated surface markers and the other half for lineage markers only (CD3, Vδ1) and with equivalent isotype controls for the surface markers. Matched mouse isotype antibodies conjugated to the same fluorophores were used at the same concentrations. Isotype controls do not bind known human antigens and therefore represent non-specific binding or false positives. Data summaries show the percentage of cells that stained positive for the indicated markers compared and therefore at a higher level than the isotype. Flow cytometry data analysis was performed in FLOWJO (version 10.1).

[0146] (Population analysis) Skin-resident lymphocytes were isolated using the methods described herein. Within CD45+ cells, anti-CD3 was used to stain T cells, and anti-CD56 antibodies were used to identify NK cells, CD3- CD56+, respectively. Within CD3+ cells, antibodies against the pan-γδ T cell receptor were used to identify skin-resident γδ T cells, and anti-CD8α was used to identify the proportion of conventional CD4 and CD8 positive αβ T cells within the CD3+, pan-γδ TCR-gate.

[0147] (Determination of total cell number) Total cell counts were performed using an NC-250 Nucleocounter (Chemometec, Copenhagen Denmark) and the manufacturer's instructions.

[0148] Example 2. Isolation of lymphocytes from human skin samples A three-dimensional skin explant protocol was established, which is described herein. Tantalum-coated reticulated vitreous carbon scaffolds (also called grids) (Ultramet, California, USA) with dimensions of 20 mm x 1.5 mm or equivalent were autoclaved, then washed and thoroughly immersed in PBS before use.

[0149] Complete isolation medium was prepared containing 1 L AIM-V medium (Gibco, Life Technologies), 50 mL serum replacement from CTS Immune (Life Technologies), human recombinant IL-2 (Miltenyi Biotech, Cat no 130-097-746) and human recombinant IL-15 (Miltenyi Biotech, Cat no 130-095-766) at the following concentrations: Complete isolation medium containing 10 mL Amphotericin B (250 μg / mL, Life Technologies) was used for the first 7 days of culture ("+AMP"). The target final concentrations of cytokines in the complete isolation medium are: Table 1: Final concentrations of cytokines in complete isolation medium [Table 1]

[0150] Adult skin samples were obtained and shipped and processed within 48 hours of collection. Excess subcutaneous fat and hair were removed from the samples with a scalpel and tweezers. The skin sample was placed with the epidermal side facing up and an appropriate size punch biopsy was used to cut the skin, holding the skin around the biopsy with sterile tweezers.

[0151] Three biopsies were placed epidermal side up, evenly spaced, and attached to the surface of one tantalum-coated carbon grid. Using sterile tweezers, the grids were transferred into tissue culture vessels with gas-permeable membranes, such as wells of a G-REX 6-well plate (Wilson Wolf Manufacturing) containing 30 mL of complete isolation medium (+AMP), or into a G-REX100 bioreactor (Wilson Wolf Manufacturing) containing 300 mL of complete isolation medium (+AMP). One grid was placed in each well of a G-REX 6-well plate, three grids were placed in a G-REX10 bioreactor, or ten grids were placed in a G-REX100 bioreactor. Cultures were incubated at 37° C. in a 5% CO2 incubator.

[0152] Unless otherwise noted, medium was changed every 7 days by gently aspirating the top medium and replacing it with 2x complete isolation medium (no AMP), avoiding disturbing the cells on the bottom of the plate or bioreactor.

[0153] To isolate lymphocytes, the grid containing the skin was removed from the G-REX 6-well plate or the G-REX 10 or G-REX 100 bioreactor and discarded for disposal. The cells present at the bottom of the plate or bioreactor were resuspended and transferred to a 500 mL centrifuge tube and then centrifuged (e.g., 300 g for 10 min).

[0154] If cell counts were required, lymphocytes were counted at this stage according to the protocol described in Example 1. The results of an exemplary test are shown in Table 2: Table 2. Yield of isolated lymphocytes per donor [Table 2]

[0155] Example 3. Optimization of punch biopsy size Initial studies have shown that 3-mm punch biopsy is superior to standard skin comminution (Figure 1).

[0156] The optimal punch biopsy size was further investigated by testing 1 mm, 2 mm, 3 mm, 4 mm, and 8 mm punch biopsy sizes and using scalpel-comminuted 2 mm explants as a control. Skin samples were prepared as described in Example 2. Each size was tested by attaching one biopsy, epidermis facing up, to the surface of a carbon grid and placing it in a well of a 24-well plate (Corning). Each well contained AIM-V 10% human AB serum + IL-2 and IL-15 at the concentrations listed above and standard concentrations of β-mercaptoethanol (2ME) and penicillin / streptomycin (P / S).

[0157] The medium was changed three times a week (half medium change) and the biopsies were incubated at 37° C. in a 5% CO 2 incubator for 21 days before cell harvest and cell yield analysis.

[0158] Total cell yield was determined as described in Example 1. The results are shown in Table 2. These results indicate that biopsies with a diameter of 2-4 mm provide the greatest cell yield. Table 3: Total cell yield obtained by biopsy type [Table 3]

[0159] The percentage of γδ T cells present in the cell yield was determined as described in Example 1. The results are presented in Figure 2. These results show that biopsies with a diameter of 3 mm provide the highest yield of γδ T cells.

[0160] Example 4. Optimization of isolation vessel Isolation in 24-well plates was compared to using containers containing gas-permeable materials such as G-REX 6-well plates (Wilson Wolf Manufacturing). Skin samples were prepared as described in Example 2. Biopsies were attached, epidermis facing up, to the surface of a carbon grid, which was then placed into the wells of a 24-well plate or a G-REX 6-well plate. 9 mm grids were used for the 24-well plate and 20 mm grids were used for the G-REX 6-well plate. All samples were placed in AIM-V 10% AB serum + P / S + 2ME + IL-2 + IL-15. For the 24-well plate, the medium was changed three times a week. For the G-REX 6-well plate, only one medium change per week was required. Biopsies were incubated at 37°C in a 5% CO2 incubator for 21 days before cell yield analysis.

[0161] Total cell yields per plate and per biopsy were determined as described in Example 1. Experiments showed that G-REX 6-well plates provided increased cell yields per biopsy and per plate when compared to 24-well plates (Figure 3 and Table 4). Although G-REX 6-well plates allowed for an increased amount of tissue to be cultured (2.5-fold more tissue compared to 24-well plates), the plates provided a staggering 25-fold increase in cell number. Table 4. Total cell yields obtained with 24-well vs. G-REX 6-well [Table 4]

[0162] Example 5. Optimization of isolation protocol The use of 3 mm punch biopsies cultured in G-REX vessels was further tested to optimize the isolation protocol. Skin samples were prepared as described in Example 2, obtained using 3 mm punch biopsies, and placed in G-REX 6-well plates or G-REX 10 bioreactors as described in Example 2. Biopsies were cultured in AIM-V (containing 5% serum replacement (SR), 5% human AB serum, or 5% SR / 5% AB "blend") + 2ME + P / S + IL2 / 15.

[0163] First, the duration of cell isolation was tested. Biopsies were incubated at 37° C. in a 5% CO2 incubator for 14 or 21 days before cell yield analysis. Total cell yield per grid was determined as described in Example 1. Results are shown in FIG. 4. For all media types, isolation after 3 weeks improved cell yield when compared to isolation after 2 weeks.

[0164] The use of serum replacement compared to human AB serum (5% or 10%) was also tested. Biopsies were incubated at 37° C. in a 5% CO2 incubator for 21 days prior to cell analysis. Total cell yield and % of V51 cells per grid were measured as described in Example 1. Results are shown in FIG. 5. Improved cell yield and higher proportion of V51 cells were obtained using medium supplemented with 5% serum replacement compared to human AB serum.

[0165] Example 6. Cell expansion Once cells are isolated using the above protocol, they can be expanded using methods known in the art, for example, selective expansion of γδ T cells can be achieved using the expansion methods described in WO2017072367. The present application provides the following aspects of the invention. (Aspect 1) 1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-section of at least 2 mm obtained from the non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15); and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. The method comprising: (Aspect 2) 1. A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-section of at least 2 mm obtained from the non-hematopoietic tissue, in the presence of IL-2 and IL-15; and (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. The method comprising: (Aspect 3) The method of any one of embodiments 1-2, wherein said non-hematopoietic tissue sample has a minimum cross-section of about 3 mm. (Aspect 4) The method of any one of embodiments 1 to 3, wherein said non-hematopoietic tissue sample has a maximum cross-section of 8 mm or less. (Aspect 5) The method of any one of embodiments 1 to 4, wherein said non-hematopoietic tissue sample has a maximum cross-section of 4 mm or less. (Aspect 6) 1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) at least 2 mm2 obtained from nonhematopoietic tissue 2 Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of ​​100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. The method comprising: (Aspect 7) 1. A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) at least 2 mm2 obtained from nonhematopoietic tissue 2 Culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross-sectional area of ​​100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. The method comprising: (Aspect 8) The non-hematopoietic tissue sample is about 7 mm 2 8. The method of embodiment 6 or embodiment 7, wherein the cross-sectional area of (Aspect 9) The non-hematopoietic tissue sample is 64 mm 2 The method of any one of embodiments 6-8, having a maximum cross-sectional area of: (Aspect 10) The non-hematopoietic tissue sample is 50 mm 2 The method of any one of embodiments 6-9, having a maximum cross-sectional area of: (Aspect 11) The non-hematopoietic tissue sample is 16 mm 2 The method of any one of embodiments 6-10, having a maximum cross-sectional area of: (Aspect 12) 1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) at least 2 mm2 obtained from nonhematopoietic tissue 3 Culturing a non-hematopoietic tissue sample, which is an intact biopsy, having a volume of about 100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. The method comprising: (Aspect 13) 1. A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) at least 2 mm2 obtained from nonhematopoietic tissue 3 Culturing a non-hematopoietic tissue sample, which is an intact biopsy, having a volume of about 100 μg / ml in the presence of IL-2 and IL-15; and (ii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. The method comprising: (Aspect 14) The non-hematopoietic tissue sample is at least 7 mm 3 14. The method of embodiment 12 or embodiment 13, wherein the (Aspect 15) The non-hematopoietic tissue sample is about 21 mm 3 The method of any one of embodiments 12 to 14, wherein the volume of the mixture is (Aspect 16) The non-hematopoietic tissue sample is 250 mm 3 The method of any one of embodiments 12 to 15, having the following volumes: (Aspect 17) The non-hematopoietic tissue sample is 65 mm 3 The method of any one of embodiments 12 to 16, having the following volumes: (Aspect 18) 18. The method of any one of embodiments 1-17, wherein said intact biopsy is obtained by punch biopsy. (Aspect 19) The method of embodiment 18, wherein said punch biopsy is at least 2 mm in diameter. (Aspect 20) 20. The method of embodiment 18, wherein said punch biopsy is about 3 mm in diameter. (Aspect 21) 20. The method of embodiment 18, wherein said punch biopsy is 8 mm or less in diameter. (Aspect 22) 20. The method of embodiment 18, wherein said punch biopsy is 4 mm or less in diameter. (Aspect 23) 1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) placing a non-hematopoietic tissue sample in a container comprising a gas permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. The method comprising: (Aspect 24) 1. A method for isolating γδ T cells from a non-hematopoietic tissue sample, comprising: (i) placing a non-hematopoietic tissue sample in a container comprising a gas permeable material; (ii) culturing the non-hematopoietic tissue sample in the presence of IL-2 and IL-15; and (iii) recovering the cultured population of γδ T cells from the non-hematopoietic tissue sample. The method comprising: (Aspect 25) 25. The method of embodiment 23 or embodiment 24, wherein the container comprises a liquid-tight container comprising a gas permeable material to allow gas exchange. (Aspect 26) 26. The method of any one of embodiments 23-25, wherein the bottom of the vessel is configured to allow gas exchange from the bottom of the vessel. (Aspect 27) 27. The method of any one of embodiments 23-26, wherein the non-hematopoietic tissue sample is placed on a synthetic scaffold inside the container. (Aspect 28) 28. The method of embodiment 27, wherein the synthetic scaffold is tantalum coated. (Aspect 29)

[0026] Embodiment 29. The method of embodiment 27 or embodiment 28, wherein the synthetic scaffold is configured to promote lymphocyte egress from the non-hematopoietic tissue sample to a bottom of the container. (Aspect 30) 30. The method of any one of embodiments 27-29, wherein said synthetic scaffold is configured to promote γδ T cell egress from said non-hematopoietic tissue sample to the bottom of said container. (Aspect 31) The method of any one of embodiments 1, 6, 12, and 23, wherein the population of lymphocytes recovered from the culture of said non-hematopoietic tissue sample is a population of αβ T cells. (Aspect 32) The method of any one of embodiments 1, 6, 12, and 23, wherein the population of lymphocytes recovered from the culture of said non-hematopoietic tissue sample is a population of NK cells. (Aspect 33) The method of any one of embodiments 1 to 32, wherein said non-hematopoietic tissue sample is not minced prior to culturing. (Aspect 34) The method according to any one of embodiments 1 to 33, wherein said lymphocytes or γδ T cells are harvested after at least 7 days of culture. (Aspect 35) The method according to any one of embodiments 1 to 34, wherein said lymphocytes or γδ T cells are harvested after at least 14 days of culture. (Aspect 36) The method according to any one of aspects 1 to 35, wherein the lymphocytes or γδ T cells are harvested up to 35 days prior to culture. (Aspect 37) The method according to any one of aspects 1 to 36, wherein the lymphocytes or γδ T cells are harvested up to 21 days prior to culture. (Aspect 38) The method of any one of embodiments 1 to 37, wherein said non-hematopoietic tissue sample is cultured in serum-free medium. (Aspect 39) The method according to any one of embodiments 1 to 37, wherein said non-hematopoietic tissue sample is cultured in a medium containing serum or a serum replacement. (Aspect 40) The method of any one of embodiments 1 to 39, wherein the non-hematopoietic tissue sample is skin. (Aspect 41) The method of embodiment 40, wherein said non-hematopoietic tissue sample comprises epithelial and dermal layers. (Aspect 42) The method of any one of embodiments 1 to 41, wherein said non-hematopoietic tissue sample is gastrointestinal or digestive tract. (Aspect 43) The method according to any one of aspects 1 to 42, wherein the non-hematopoietic tissue sample is obtained from a human. (Aspect 44) The method according to any one of aspects 1 to 43, wherein the IL-2 is human IL-2 or a functional equivalent thereof. (Aspect 45) The method according to any one of aspects 1 to 44, wherein said IL15 is human IL-15 or a functional equivalent thereof. (Aspect 46) The method of any one of embodiments 1 to 45, wherein said isolated population of cells comprises a population of Vδ1 T cells. (Aspect 47) 47. The method of embodiment 46, wherein said population of V51 T cells expresses CD27 and / or does not substantially express TIGIT. (Aspect 48) 48. The method of embodiment 46 or embodiment 47, wherein said population of V51 T cells has a frequency of TIGIT+ cells of less than 80%. (Aspect 49) The method of any one of embodiments 46 to 48, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of less than 60%. (Aspect 50) The method of any one of embodiments 46 to 49, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 40%. (Aspect 51) The method of any one of embodiments 46 to 50, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 30%. (Aspect 52) The method of any one of embodiments 46 to 51, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 20%. (Aspect 53) The method of any one of embodiments 46 to 52, wherein the population of Vδ1 T cells has a frequency of TIGIT+ cells of about 10%. (Aspect 54) The method of any one of aspects 46 to 53, wherein the population of Vδ1 T cells does not substantially express TIGIT. (Aspect 55) The method of any one of embodiments 46 to 54, wherein said population of Vδ1 T cells has a frequency of CD27+ cells of more than 10%. (Aspect 56) The method of any one of embodiments 46 to 55, wherein said population of Vδ1 T cells has a frequency of CD27+ cells of more than 20%. (Aspect 57) The method of any one of embodiments 46 to 56, wherein the population of Vδ1 T cells has a frequency of CD27+ cells of about 40%. (Aspect 58) The method of any one of embodiments 46 to 57, wherein the population of Vδ1 T cells has a frequency of CD27+ cells of about 80%. (Aspect 59) The method of any one of embodiments 46 to 58, wherein said population of Vδ1 T cells has a frequency of CD27+ cells of more than 80%. (Aspect 60) The method of any one of aspects 46 to 59, wherein the population of Vδ1 T cells expresses CD27. (Aspect 61) The method according to any one of aspects 1 to 60, further comprising expanding said isolated population of lymphocytes or γδ T cells. (Aspect 62) 1. A method for isolating and expanding lymphocytes from a non-hematopoietic tissue sample, comprising: (i) isolating a population of lymphocytes from the non-hematopoietic tissue sample according to the method of any one of embodiments 1 to 61; and (ii) further culturing the population of lymphocytes for at least 5 days to produce an expanded population of lymphocytes. The method comprising: (Aspect 63) 1. A method for isolating and expanding γδ T cells from a non-hematopoietic tissue sample, comprising: (i) isolating a population of γδ T cells from the non-hematopoietic tissue sample according to the method of any one of embodiments 1 to 62; and (ii) further culturing the population of γδ T cells for at least 5 days to produce an expanded population of γδ T cells. The method comprising: (Aspect 64) 64. The method of any one of aspects 61-63, wherein said expansion step comprises culturing said lymphocytes or γδ T cells in serum-free medium. (Aspect 65) 64. The method of any one of aspects 61 to 63, wherein said expansion step comprises culturing said lymphocytes or γδ T cells in a medium containing serum or a serum replacement. (Aspect 66) The method of any of embodiments 63-65, wherein said expanding step comprises culturing said γδ T cells in the substantial absence of stromal cell contact. (Aspect 67) 67. The method of any one of embodiments 63-66, wherein said expansion step comprises the absence of an exogenous TCR pathway agonist. (Aspect 68) An isolated lymphocyte population obtainable by the method according to any one of aspects 1 to 60. (Aspect 69) An isolated lymphocyte population, obtainable by the method according to any one of aspects 1 to 60. (Aspect 70) An isolated population of γδ T cells, obtainable by the method according to any one of embodiments 1 to 60. (Aspect 71) 66. An isolated and expanded lymphocyte population obtainable by the method according to any one of aspects 61 to 65. (Aspect 72) 66. An isolated and expanded lymphocyte population obtainable by the method according to any one of aspects 61 to 65. (Aspect 73) 66. An isolated and expanded population of γδ T cells obtainable by the method according to any one of embodiments 61 to 65. (Aspect 74) 66. An isolated and expanded γδ T cell population obtainable by the method according to any one of embodiments 61 to 65.

Claims

1. 1. A method for isolating lymphocytes from a non-hematopoietic tissue sample, comprising: (i) culturing a non-hematopoietic tissue sample, which is an intact biopsy having a minimum cross section of at least 2 mm obtained from the non-hematopoietic tissue, in the presence of interleukin-2 (IL-2) and interleukin-15 (IL-15), wherein the intact biopsy is obtained by punch biopsy; and (ii) recovering a population of cultured lymphocytes from the non-hematopoietic tissue sample. The method comprising:

2. The method of claim 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample comprises a population of γδ T cells.

3. (i) the non-hematopoietic tissue sample has a minimum cross-section of about 3 mm; (ii) the non-hematopoietic tissue sample has a maximum cross-section of 8 mm or less; or (iii) Both (i) and (ii); 3. The method of claim 1 or 2.

4. The method according to any one of claims 1 to 3, wherein the method is carried out in a vessel comprising a gas permeable material.

5. 5. The method of claim 4, wherein the non-hematopoietic tissue sample is placed on a synthetic scaffold inside the container.

6. 6. The method of claim 5, wherein the synthetic scaffold is tantalum coated.

7. The method of any one of claims 1 to 6, wherein the lymphocytes or γδ T cells are harvested after at least 7 days of culture.

8. The method of any one of claims 1 to 7, wherein the non-hematopoietic tissue sample is (i) skin, (ii) gastrointestinal, or (iii) gastrointestinal.

9. (i) the IL-2 is human IL-2; (ii) the IL-15 is human IL-15; or (iii) Both (i) and (ii); The method according to any one of claims 1 to 8.

10. the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample comprises a population of V51 T cells; and (i) the population of V51 T cells has a frequency of TIGIT+ cells of less than 80%; (ii) the population of V51 T cells has a frequency of CD27+ cells greater than 10%; or (iii) Both (i) and (ii); The method of any one of claims 2 to 9.

11. The method of claim 1, wherein the population of lymphocytes recovered from the culture of the non-hematopoietic tissue sample comprises a population of NK cells.

12. The method of any one of claims 1 to 11, further comprising expanding the isolated population of lymphocytes.

Images