Identification of CD8.SUP.+ .T cells that are CD161.SUP.hi .and/or IL18R(α).SUP.hi .and have rapid drug efflux capacity

Abstract

This invention provides, among other things, methods for the identification and isolation of viable putative long-lived antigen-specific memory CD8.sup.+ T cell subsets (CMhi and EMhi) with high surface expression of CD161 and/or IL-18R and the capacity to rapidly efflux the fluorescent dye Rh123.

Claims

1. A composition comprising an isolated population of long-lived human memory CD8.sup.+ CD161.sup.hi IL-18R.sup.hi T cells, wherein: the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cells make up at least 30% of the total CD8+ T cells in the composition; and the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cells comprise CD95.sup.hi memory cells, wherein the CD95.sup.hi memory cells are capable of proliferating in response to IL-7 or IL-15 and comprise a population of cells comprising an engineered immunoreceptor.

2. The composition of claim 1, wherein the population of long-lived memory CD8+ CD161.sup.hiIL-18R.sup.hi T cells comprises high CD28 surface expression and high MDR-1 mRNA levels as compared to a CD8+ T cell population with low surface expression of IL-18R.

3. The composition of claim 1, wherein the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cells are CD127.sup.+, CD25.sup., bcl2.sup.hi, perforin.sup.neg/low, granzyme A.sup.int, granzyme B.sup.int/neg and NKG2D.sup.int.

4. The composition of claim 1, wherein the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cell population has increased expression of CD43, CD44, CD46, CD148, and CD162 as compared to a CD8+ T cell population with low surface expression of IL-18R.

5. The composition of claim 1, wherein the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cell population lacks expression of CD57, CD103, and CD69 as compared to a CD8+ T cell population with low surface expression of IL-18R.

6. The composition of claim 1, wherein the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cell population increased expression of CD122 as compared to a CD8+ T cell population with low surface expression of IL-18R.

7. The composition of claim 1, wherein the CD95.sup.hi memory cells comprise CD62L.sup.+, CD45RA.sup.int/neg, CD45RO.sup.int/hi central memory cells.

8. The composition of claim 7, wherein the CD95.sup.hi memory cells further comprise CD62L.sup., CD45RA.sup.int/neg, CD45RO.sup.int/hi central memory cells.

9. The composition of claim 1, wherein the CD95.sup.hi memory cells comprise CD62L.sup., CD45RA.sup.int/neg, CD45RO.sup.int/hi central memory cells.

10. The composition of claim 1, wherein the engineered immunoreceptor is specific for a tumor-associated antigen.

11. The composition of claim 1, wherein the engineered immunoreceptor is an antigen-specific T cell receptor.

12. The composition of claim 1, wherein the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cells are at least 40% of the total CD8+ T cells in the composition.

13. The composition of claim 1, wherein the long-lived memory CD8+ CD161.sup.hi IL-18R.sup.hi T cells are at least 50% of the total CD8+ T cells in the composition.

14. The composition of claim 1, wherein the long-lived memory CD.sup.8+ CD161.sup.hi IL-18R.sup.hi T cells are at least 80% of the total CD8+ T cells in the composition.

15. The composition of claim 1, wherein the long-lived memory CD8+ CD161.sup.hiIL-18R.sup.hi T cells have enhanced proliferation in response to a cytokine selected from the group consisting of IL-12, Il-18, IL-23, or combinations thereof, as compared to a CD8+T cell population with low surface expression of IL-18R.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: CM and EM populations contain subsets that rapidly efflux Rh123 and are CD161.sup.hi and IL-18R.sup.hi as described in Example 1.

(2) FIGS. 2A-2C: CMhi and EMhi subsets are TCR.sup.+ and TCR.sup.. There is no restriction to V24. They are predominantly CD45RA.sup.int/neg, CD45RO.sup.int/hi, CD95.sup.+, CD8.sup.+, CD8.sup.+/neg, CD27.sup.+, CD28.sup.+, CD122.sup.+, perforin.sup.int/lo, granzyme A.sup.int/lo, granzyme B.sup.neg, Ki67.sup.neg, bcl-xL.sup.int/hi and bcl-2.sup.int/hi, as described in Example 2.

(3) FIG. 3: CMhi and EMhi have a surface phenotype that suggests possible derivation from the distal pole of an asymmetrically dividing nave CD8.sup.+ T cell, as described in Example 3.

(4) FIGS. 4A-4B: CMhi and EMhi express higher levels of MDR-1 mRNA than their non-effluxing counterparts and actively efflux the fluorescent chemotherapy drug, daunorubicin, as described in Example 4.

(5) FIG. 5: CMhi and EMhi are resistant to daunorubicin-induced apoptosis in vitro, as described in Example 5.

(6) FIGS. 6A-6D: CMhi and EMhi divide in response to the homeostatic cytokines, IL-7 and IL-15, and have high viability after culture in the absence of supplementary cytokines, as described in Example 6.

(7) FIG. 7: CMhi and EMhi show reduced .sup.3H-thymidine uptake in response to polyclonal TCR stimulation with OKT3, compared to their non-effluxing counterparts, as described in Example 7. .sup.3H-thymidine uptake is increased after costimulation as indicated in the Figure.

(8) FIG. 8: CMhi and EMhi have a different cytokine secretion profile compared to their non-effluxing counterparts, as described in Example 8.

(9) FIG. 9: CMhi and EMhi have decreased calcium flux in response to ionomycin, compared to their non-effluxing counterparts, as described in Example 9.

(10) FIG. 10: CMhi and EMhi subsets comprise polyclonal TCR repertoires by molecular spectratyping, as described in Example 10.

(11) FIGS. 11A-11B: Viral antigen tetramer-positive cells can be identified within CMhi and EMhi subsets and CMV-, EBV- and influenza-specific CTL responses can be generated from sorted CMhi and EMhi subsets, as described in Example 11.

(12) FIG. 12: CMhi and EMhi subsets have unique and distinct gene expression profiles as shown by the Principal Components Plot and as described in Example 12.

(13) FIGS. 13A-13B: CMhi and EMhi are rare in cord blood, peak in early adult life and are found at decreasing frequency with advancing age, as described in Example 13.

(14) The present invention is explained in greater detail below. The disclosures of all United States patent references cited herein are incorporated by reference herein in their entirety.

DETAILED DESCRIPTION

(15) I. Definitions

(16) T cells or T lymphocytes as used herein are from humans. In some embodiments the T cells are autologous (the donor and the recipient are the same individual); in some embodiments the T cells are allogeneic (the donor and recipient/s are genetically different individuals); in some embodiments the T cells are syngeneic (the donor and recipient/s are different individuals who are genetically identical).

(17) Cytotoxic T lymphocyte (CTL) as used herein refers to a T lymphocyte that expresses CD8 on the surface thereof (i.e., a CD8.sup.+ T cell).

(18) Central memory T cell (or CM) as used herein refers to a CTL that has previously been exposed to antigen (a memory CTL) and is CD62L.sup.+, CD45RA.sup.int/neg/CD45RO.sup.int/hi and CD95.sup.int/hi.

(19) Effector memory T cell (or EM) as used herein refers to a CTL that has previously been exposed to antigen (a memory CTL) and is CD62L.sup., CD45RA.sup.int/neg/CD45RO.sup.int/hi and CD95.sup.int/hi.

(20) Rapidly effluxing T lymphocytes are found within the CD8.sup.+ T cell CM and EM populations (CMhi and EMhi, respectively) with some or all of the following identifying characteristics: (a) actively and rapidly efflux the dye Rh123 in culture over a time of 30 minutes at a temperature of 37 C.; (b) high surface expression of CD161 (CD161.sup.hi) and/or IL-18R (IL-18R.sup.hi). Such cells typically include, but are not limited to, the following features: (a) high surface expression of CD127, CD28; (b) typically, but not consistently, expression of markers suggesting derivation from the distal pole complex or uropod of mitotic CD8.sup.+ T cells (higher CD43, CD44, CD46, CD148 and CD162 surface expression than non-effluxing subsets; lower CD8, CD11 a and CD50 surface expression than non-effluxing subsets); (c) CD3.sup.+/TCR.sup.+/TCR.sup.; (d) low or no surface expression of CD25, CD57, PD-1, CD103 and CD69; (e) lower surface expression of NKG2D than non-effluxing subsets; (f) intermediate/high expression of CD45RO, intermediate/negative surface expression of CD45RA and expression of CD95; (g) typically, but not consistently higher expression of bcl-2 and bcl-xL than their non-effluxing counterparts; (h) negative expression of granzyme B, intermediate expression of granzyme A and low/intermediate expression of perforin; (i) normal or low expression of CD8 and normal, low or absent expression of CD8; (j) higher expression of MDR-1 mRNA than their non-effluxing counterparts; (k) lower percentage expression of Ki67 than their non-effluxing counterparts in a healthy individual. Optionally, in some embodiments, rapidly effluxing T lymphocytes are characterized by positive surface expression of CD122. Note also that CD62L expression is variable and defines the presence of these cells in the CM or EM subsets. Such cells also typically include, but are not limited to, the following functional features: (a) low proliferation compared to non-effluxing subsets in response to stimulation with plate-bound OKT3, which can be increased after costimulation with anti-CD28 antibody or cytokines; (b) contain virus-specific clones, evidenced by tetramer binding and the capacity to identify antigen-specific CD8.sup.+ T cells after in vitro expansion of sorted subsets; (c) resistance to apoptosis during in vitro culture in the presence or absence of chemotherapeutic agents; (d) reduced secretion of IFN-, IL-2, IL-4, IL-5, IL-8, IL-10 and MIP-1 compared to their non-effluxing counterparts in response to stimulation with PMA/ionomycin or OKT3 in combination with anti-CD28 antibody or cytokines; (e) capacity to actively efflux the fluorescent chemotherapy drug, daunorubicin; (f) low, but heterogeneous, calcium flux after stimulation with ionomycin; (g) CMhi cells typically have an increased uptake of tritiated thymidine (.sup.3H-thymidine) and increased dilution of CFSE compared to their non-effluxing counterparts in response to IL-7.

(21) Efflux blockers as used herein includes, but is not limited to, PK11195, cyclosporine A, PSC833, verapamil.

(22) Effluxed drug as used herein includes, but is not limited to, doxorubicin, daunorubicin, epirubicin, methotrexate, mitoxantrone, vinblastine, vincristine, dexamethasone and derivatives, etoposide and taxanes.

(23) Antigen as used herein refers to a protein or peptide that can be recognized by the immune system.

(24) Antigen-specific as used herein refers to the nature of the highly specific recognition by the adaptive immune system of peptide fragments presented in the context of an MHC molecule.

(25) Antibody as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE and all isotypes thereof. Of the immunoglobulins, IgM and IgG are particularly preferred. The antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et al., Molec. Immunol. 26, 403-11 (1989). The antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in Reading U.S. Pat. No. 4,474,893, or Cabilly et al., U.S. Pat. No. 4,816,567. The antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in SegAl et al., U.S. Pat. No. 4,676,980.

(26) Autoimmune disease as used herein may be any autoimmune disease, including but not limited to: systemic lupus erythematosus, Hashimoto's disease, rheumatoid arthritis, graft-versus-host disease, Sjogren's syndrome, pernicious anemia, Addison's disease, scleroderma, Goodpasture's syndrome, Crohn's disease, autoimmune hemolytic anemia, sterility, myasthenia gravis, multiple sclerosis, Basedow's disease, thrombotic thrombocytopenic purpura, immune thrombocytopenic purpura, insulin-dependent diabetes mellitis, allergy, asthma, atopic disease, arteriosclerosis, myocarditis, cardiomyopathy, nephritis, and hypoplastic anemia. See, e.g., U.S. Pat. No. 7,279,160.

(27) Cancer as used herein may include, but is not limited to any pathologic variation of: cancer of the prostate, breast, bladder, stomach, oropharynx, nasopharynx, esophagus, stomach, pancreas, liver, kidneys, colon, rectum, anus, lung, thyroid, brain, hematopoietic system (including, but not limited to Hodgkin's and Non-Hodgkin's lymphoma, acute and chronic lymphoid and myeloid leukemias) and skin (including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma).

(28) Donor as used herein refers to the individual from whom rapidly effluxing T lymphocytes or other cellular product was obtained.

(29) Recipient as used herein refers to the individual who will receive rapidly effluxing T lymphocytes or other treatment.

(30) Enriched as used herein to describe amounts of cell types in a mixture refers to the subjecting of the mixture of the cells to a physical process or step, which results in an increase in the number of the enriched type, as compared to the same mixture before that physical process or step. For cell types that are few in number, those cells may be enriched five, ten, twenty, thirty, forty, or fifty fold (times) or more, yet still be a relatively small number of total cells (e.g., total number T cells) in the enriched population (e.g., the enriched cells being at least 1, 5, 10, 20, or 30 percent of the total cells in the preparation, or more, up to 40 or 50 percent of the total cells in the preparation or more). In other embodiments, the enriched cells may be enriched to a point that they become at least 40, 50, 60, 70 or 80 percent of the total cells in the preparation, or more, up to 90, 95, or 99 percent of the total cells in the preparation, or more).

(31) Toxic agent as used herein includes, but is not limited to, radioisotopes, therapeutic drugs, and toxins or cytotoxins. See, e.g., U.S. Pat. No. 6,274,118.

(32) Radioisotope as used herein includes but is not limited to. .sup.227Ac, .sup.211At, .sup.131Ba, .sup.77Br, .sup.109Cd, .sup.51Cr, .sup.67Cu, .sup.165Dy, .sup.155Eu, .sup.153Gd, .sup.198Au, .sup.166Ho, .sup.113mIn, .sup.115mIn, .sup.123I, .sup.125I, .sup.131I, .sup.189Ir, .sup.191Ir, .sup.192Ir, .sup.194Ir, .sup.52Fe, .sup.55Fe, .sup.59Fe, .sup.177Lu, .sup.109Pd, .sup.32P, .sup.226Ra, .sup.186Re, .sup.188Re, .sup.153Sm, .sup.46Sc, .sup.47Sc, .sup.72Se, .sup.75Se, .sup.105Ag, .sup.89Sr, .sup.35S, .sup.177Ta, .sup.117mSn, .sup.121Sn, .sup.166Yb, .sup.169Yb, .sup.90Y, .sup.212Bi, .sup.119Sb, .sup.197Hg, .sup.97Ru, .sup.100Pd, .sup.101mRh, and .sup.212Pb.

(33) Therapeutic drug as used herein includes but is not limited to Adriamycin, Chlororambucil, Daunorubicin, Leucovorin, Folinic acid, Methotrexate, Mitomycin C, Neocarzinostatin, Melphalan Vinblastine, Mitocyn, Mechlorethamine, Fluorouracil, Floxuridine, Idarubicin, Doxorubicin, Epirubicin, Cisplatin, Cannustine, Cyclophosphamide, Bleomycin, Vincristine and Cytarabine.

(34) Toxin or cytotoxin as used herein includes but is not limited to diptheria toxin, ricin toxin, monensin, verrucarin A, abrin, saporin, vinca alkaloids, tricothecenes, and pseudomonas exotoxin A, and pokeweed viral protein. See, e.g., U.S. Pat. No. 6,630,576.

(35) Pharmaceutically acceptable as used herein means that the compound or composition is suitable for administration to a subject to achieve the treatments described herein, without unduly deleterious side effects in light of the severity of the disease and necessity of the treatment.

(36) II. Identification

(37) Identification of Rh123-Effluxing CM and EM Populations. PBMC or T lymphocytes from any tissue can be collected in accordance with known techniques and loaded with Rh123 (or alternate similarly-effluxed fluorescent dye) at 5-10 g/ml in RPMI 1640/10% BSA (efflux buffer) on ice for 30 minutes, washed three times and cultured in efflux buffer for 30 minutes at 37 C. A control sample cultured in the presence of vinblastine (a competitive antagonist of Rh123 efflux) can be used to establish and gate active efflux of Rh123. The PBMC can then be surface labeled with fluorochrome-conjugated antibodies (e.g., to CD4, CD16, TCR, V24, CD8, CD95 and CD62L), allowing identification of Rh123.sup.lo effluxing CM or EM populations by fluorescence-activated cell sorting (FACS) analysis.

(38) Identification of CD161.sup.hi and/or IL-18R.sup.hi CM and EM Populations. High expression of CD161 and/or IL-18R on CM and EM populations can be used as a surrogate marker for the ability to rapidly efflux Rh123 in in vitro culture. T lymphocytes can be collected in accordance with known techniques from any tissue and labeled with fluorochrome-conjugated antibodies to CD161 and/or IL-18R, and other markers (e.g., to CD4, CD16, TCR, V24, CD8, CD95 and CD62L), allowing identification of CD161.sup.hi and/or IL-18R.sup.hi effluxing CM (CMhi) or EM (EMhi) populations by fluorescence-activated cell sorting (FACS) analysis.

(39) III. Isolation

(40) Isolation of Rh123-Effluxing CM and EM Populations. PBMC or T lymphocytes from any tissue can be collected in accordance with known techniques and enriched or depleted by known techniques such as affinity binding to antibodies such as in flow cytometry, immunomagnetic separation and/or affinity binding. For example, CD8.sup.+ CTL may be isolated by positive immunomagnetic separation. The positively selected CD8.sup.+ T cell fraction can be loaded with Rh123 (or alternate similarly-effluxed fluorescent dye) at 5-10 g/ml in efflux buffer on ice for 30 minutes, washed three times and cultured in efflux buffer for 30 minutes at 37 C. A control sample cultured in the presence of vinblastine (a competitive antagonist of Rh123 efflux) can be used to establish and gate active efflux of Rh123. The PBMC can then be labeled with antibodies (e.g., to CD4, CD16, TCR, V24, CD8, CD95 and CD62L), allowing identification and isolation of CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+/CD62L.sup.+/Rh123.sup.lo effluxing CMhi or CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+/CD62L.sup./Rh123.sup.lo effluxing EMhi populations by fluorescence-activated cell sorting (FACS) analysis.

(41) Isolation of CD161.sup.hi and/or IL-18R.sup.hi CM and EM Populations. High expression of CD161 and/or IL-18R on CM and EM populations can be used as a surrogate marker for the ability to rapidly efflux Rh123 in in vitro culture. T lymphocytes from any tissue can be collected in accordance with known techniques and enriched or depleted by known techniques such as affinity binding to antibodies such as in flow cytometry, immunomagnetic separation and/or affinity binding. For example, CD8.sup.+ CTL may be isolated by positive immunomagnetic separation. The positively selected CD8.sup.+ T cell fraction can be labeled with antibodies (e.g., to CD4, CD16, TCR, V24, CD8, CD95, CD62L and CD161 and/or IL-18R), allowing identification and isolation of CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+/CD62L.sup.+/CD161.sup.hi or CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+/CD62L.sup.+/IL-18R.sup.hi effluxing CMhi or CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+/CD62L.sup./CD161.sup.hi or CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+/CD62L.sup./IL- 18R.sup.hi effluxing EMhi populations by fluorescence-activated cell sorting (FACS) analysis.

(42) IV. Kits. Kits useful for carrying out all or parts of the methods of the invention (particularly the steps of identifying or isolating cell populations) may take any of a variety of forms. Typically the kits would include the necessary antibodies or antibody conjugates for the procedure. Isolation of these cells is a multistep process. Many combinations of kits are possible, depending on the desired product (pure CD8.sup.+ CMhi cells, pure CD8.sup.+ EMhi cells or enriched CD8.sup.+ CMhi and EMhi cells) and method of identification of CMhi and EMhi (Rh123 effluxing and/or CD161.sup.hi and/or IL-18R.sup.hi). The kit may be used for identification and analysis, non-clinical grade or clinical grade isolation of Rh123 effluxing or non-effluxing populations. The kit can be packaged in any suitable container and optionally include instructions for carrying out all or parts of the methods described herein. Hence, a kit could include, but is not limited to, any components necessary for any combination of the following steps:

(43) (i) Initial identification, enrichment and/or isolation of memory CD8.sup.+ T cells (positive or negative immunomagnetic selection or cell sorting)

(44) (ii) Removal of contaminating cells after immunomagnetic separation (e.g., antibodies to non-CD8.sup.+ T cells, streptavidin-fluorochrome conjugates, goat anti-mouse-fluorochrome conjugates).

(45) (iii) Identification, enrichment and/or isolation of CM and EM subsets (e.g., fluorochrome-conjugated CD95 and CD62L)

(46) (iv) Identification, enrichment and/or isolation of effluxing (Rh123/efflux buffer/efflux blocking agents) or CD161.sup.hi and/or IL-18R.sup.hi populations

(47) V. Compositions and Methods for Therapy

(48) CMhi and EMhi cells may be used in an autologous, allogeneic or syngeneic setting. CMhi and EMhi populations may be used or targeted for, but are not limited to, the following indications;

(49) (a) for transfer of antigen-specific immunity in cancer, infectious diseases or immunodeficiency

(50) (b) for targeted ablation of immunity in autoimmune diseases, graft versus host disease or graft rejection

(51) (c) for therapeutic gene delivery

(52) VI. Transfer of Antigen-Specific Immunity

(53) In some embodiments, the lymphocytes of the invention may be used to confer immunity to individuals. By immunity is meant an increase of one or more factors associated with a response to infection by a pathogen, or to a tumor, to which the lymphocyte response is directed.

(54) Subjects that can be treated by the present invention are human. The subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.

(55) Subjects that can be treated include subjects afflicted with cancer, including but not limited to hematopoietic cancers, cancers of the colon, lung, liver, breast, prostate, ovary, skin (including melanoma), bone, and brain etc. In some embodiments the tumor associated antigens are known, including, but not limited to melanoma, breast cancer, squamous cell carcinoma, colon cancer, leukemia, myeloma and prostate cancer (in these embodiments memory T cells can be isolated or engineered by introducing the T cell receptor genes). In other embodiments the tumor associated antigens can be targeted with genetically modified T cells expressing an engineered immunoreceptor. Examples include but are not limited to B cell lymphoma, breast cancer, prostate cancer, and leukemia.

(56) Subjects that can be treated also include subjects afflicted with, or at risk of developing, an infectious disease, including but not limited to viral, bacterial, and protozoal infections.

(57) Subjects that can be treated include immunodeficient patients, including but not limited to transplant patients, afflicted with a viral infection. Such viral infections may include, but are not limited to, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, influenza or parainfluenza, varicella, human herpes virus type 6 (HHV6) or HHV7, respiratory syncytial virus (RSV) or BK polyomavirus infections.

(58) Cells prepared as described above can be utilized in methods and compositions for adoptive immunotherapy in accordance with known techniques, or variations thereof that will be apparent to those skilled in the art based on the instant disclosure (see, e.g., US Patent Application Publication No. 2003/0170238 to Gruenberg et al; see also U.S. Pat. No. 4,690,915 to Rosenberg; see also U.S. Pat. No. 6,040,177 to Riddell).

(59) CMhi and EMhi may be used for therapy of autologous, allogeneic or syngeneic recipients and may be harvested in the presence or absence of in vivo stimulation, and administered with or without in vitro manipulation or in vivo stimulation after administration.

(60) In vivo stimulation before harvesting CMhi and/or EMhi may involve, but is not limited to, the use of drugs, vaccines or cytokines. In vitro manipulation may involve, but is not limited to, by culture or other methods, any combination of subset enrichment, antigen-specific enrichment, expansion, feeder cell (e.g., irradiated LCL or PBMC) treatment, cytokine treatment, antibody treatment, small molecule treatment or transduction with antigen-specific TCR genes or other therapeutic, suicide and/or knockdown genes. In vivo stimulation may involve, but is not limited to, vaccination with commercial vaccines or research cellular or non-cellular reagents, cytokine administration or drug administration.

(61) CMhi or EMhi cells may be infused with or without isolation and enrichment. The use and method of enrichment or isolation will reflect the requirements of the product. In some embodiments, the cells are formulated by first harvesting them from the donor or their culture medium, and then washing and concentrating the cells in a medium and container system suitable for administration (a pharmaceutically acceptable carrier) in a treatment-effective amount. Suitable infusion medium can be any isotonic medium formulation, such as normal saline, Normosol R (Abbott), Plasma-Lyte A (Baxter), 5% dextrose in water or Ringer's lactate. The infusion medium can be supplemented with human serum albumin, human serum or other nutrients.

(62) The amount of cells to be infused is variable and may range from <10 antigen-specific cells in the absence of enrichment or in vitro expansion to greater than 10.sup.12 cells after enrichment and in vitro expansion (see, e.g., U.S. Pat. No. 6,040,177 to Riddell et al. at column 17). The cells may be administered by a single infusion or by multiple infusions over a range of time. However, since different individuals are expected to vary in responsiveness, the type and amount of cells infused, the route of administration, the number of infusions and the time range over which multiple infusions are given are determined by the attending physician as dictated by circumstance, physical and laboratory examination.

(63) A typical example of the use of the invention in adoptive immunotherapy would be treatment in the setting of allogeneic or autologous hematopoietic stem cell transplantation (HSCT). In the setting of severe immunosuppression, allogeneic and autologous HSCT patients are at great risk of contracting opportunistic infections. The invention may be used to transfer immune memory to the recipient to protect them against potentially life-threatening infection. CMhi and EMhi cells may be isolated from the transplant donor (in the case of allogeneic HSCT) or the patient (in the case of autologous HSCT), selected or engineered for specificity to infection-associated antigens, expanded or not, and returned to the patient in the post transplant setting. The subsets would proliferate with or without differentiation in the post-transplant lymphopenic environment and reconstitute immune memory. The selection of CMhi and EMhi specific for infections would allow reconstitution of immunity without causing graft versus host disease.

(64) VII. Targeted Ablation

(65) A further aspect of the invention is a method of ablating long-lived memory CD8.sup.+ T cells in a human subject by selectively depleting actively effluxing CMhi and/or EMhi cells in the subject as treatment for autoimmune disease or graft versus host disease. The selective depletion may be carried out by any suitable technique, such as by administering to the subject an antibody that selectively binds to CD161 and/or IL-18R in an amount effective to treat the disease or by administering inhibitors of drug efflux in combination with cytotoxic drugs.

(66) The antibody, either monoclonal or polyclonal of any isotype, or pharmaceutical composition containing the same, can be formulated in multiple different carriers, or conjugated to many possible toxins, including but not limited to radioisotopes, ricin or diphtheria toxin, as is known in the art. The antibody could be administered by various routes including but not limited to, intravenously, intraperitoneally or intracisternally. A therapeutic antibody of the invention is administered in an effective amount to treat the disease, at a suitable schedule, though these will vary somewhat with the particular disease, formulation, route of administration, and condition of the subject, as is known in the art.

(67) CMhi and EMhi may be ablated by administration of cytotoxic drugs in combination with inhibitors of cytotoxic drug efflux. In vitro studies show that CMhi and EMhi are protected from chemotherapy-induced apoptosis and this protection is lost in the presence of ABC-cotransporter (drug efflux pump) inhibition. Prevention of cytotoxic drug efflux from CMhi and EMhi may be used to reduce or ablate these cell populations in vivo. Cytotoxic drugs and drug efflux inhibitors may be administered in recognized or novel protocols as is known in the art.

(68) VIII. Therapeutic Gene Delivery

(69) In some embodiments, the lymphocytes of the invention may be used to deliver therapeutic genes to individuals. Therapeutic genes could include, but are not limited to, genes encoding costimulatory (e.g., CD80) or inhibitory molecules (PD-L1), cytokines (e.g., IL-7), apoptosis-inducing signals or congenitally deficient or abnormal genes (e.g., Factor VIII gene in severe hemophilia). The delivery of T cells transfected with genes encoding antigen-specific TCR is discussed above in Transfer of antigen-specific immunity.

(70) Isolation, in vitro manipulation, formulation and administration for therapeutic gene delivery will encompass similar considerations, as discussed above in Transfer of antigen-specific immunity. Gene delivery to isolated T cells would likely be performed in vitro, but may include methods to target long-lived memory cells in vivo. Utilizing recombinant infectious virus particles for gene delivery is a preferred approach to the transduction of T lymphocytes of the present invention. The viral vectors which have been used in this way include virus vectors derived from simian virus 40, adenoviruses, adeno-associated virus (AAV), retroviruses and lentiviruses and modifications thereof. Thus, gene transfer and expression methods are numerous but essentially function to introduce and express genetic material in mammalian cells. Several of the above techniques, amongst others, have been used to transduce hematopoietic or lymphoid cells, including calcium phosphate transfection, protoplast fusion, electroporation, and infection with recombinant adenovirus, adeno-associated virus and retrovirus vectors. Primary T lymphocytes have been successfully transduced by electroporation, retroviral infection and lentiviral infection.

EXPERIMENTAL

Example 1

CM and EM Populations Contain Subsets That Rapidly Efflux Rh123 and are CD161.SUP.hi .and IL-18R.SUP.hi

(71) PBMC were separated from fresh peripheral blood by density gradient centrifugation, washed in RPMI 1640/10% bovine serum albumin (herein known as efflux buffer) and resuspended at 110.sup.6/ml in ice cold efflux buffer with 10 g/ml Rh123. PBMC were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer, with or without vinblastine, for 30 minutes at 37 C. At 30 minutes, PBMC were washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with antibodies to CD4, CD16, TCR, V24, CD3, CD8, CD95, CD62L, CD161 and IL-18R for 20 minutes on ice. After washing in ice cold FACS buffer, samples were analyzed on a BD FacsARIA flow cytometer.

(72) CM and EM subsets were identified as CD62L.sup.+ or CD62L.sup. events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD3.sup.+/CD8.sup.+/CD95.sup.+ population. Rh123 fluorescence was identified with appropriate compensation and a 530/30 emission filter after laser excitation at 488 nm. Rh123-effluxing events were defined as those with fluorescence lower than the mean fluorescence intensity identified after culture in the presence of vinblastine efflux blockade.

(73) The results in FIG. 1 demonstrate that many lymphocytes have the capacity to efflux Rh123; however only small subsets of CM and EM CD8.sup.+ T lymphocytes have the capacity to rapidly efflux Rh123 over a 30 minute period. Efflux is blocked by vinblastine, a non-fluorescent substrate for MDR-1 and MRP-1, demonstrating specificity of Rh123 efflux. CM and EM cells rapidly effluxing Rh123 express high levels of IL-18R and CD161.

Example 2

CMhi and EMhi Subsets are TCR.SUP.+ and TCR^{.. There is No Restriction to V24. They are Predominantly CD45RA.SUP.int/neg., CD45RO.SUP.int/hi., CD95.SUP.+., CD8.SUP.+., CD8.SUP.+/neg., CD25.SUP.neg., CD27.SUP.+., CD56.SUP.pos/neg ., CD57.SUP.neg., CD28.SUP.hi., CD122.SUP.+., CD127.SUP.hi., PD-1.SUP.neg., CD103.SUP.neg., NKG2D.SUP.int/lo., perforin.SUP.lo/int., granzyme A.SUP.int., granzyme B.SUP.neg., Ki67.SUP.neg., bcl-xL.SUP.hi .and bcl-2.SUP.hi..}

(74) PBMC were separated from fresh peripheral blood by density gradient centrifugation and resuspended in PBS. Surface labeling was performed with antibodies to CD4, CD16, TCR, V24, CD3, CD8, CD95, CD62L, CD161 and other antibodies as indicated for 20 minutes on ice. After washing in cold PBS, surface-labeled samples were analyzed on a BD LSR-2 flow cytometer. Samples for intracellular staining were fixed in BD Cytofix before washing, permeabilization and labeling in BD Perm/wash buffer, then analyzed as above.

(75) CMhi and EMhi subsets were identified as CD62L.sup.+/CD161.sup.hi or CD62L.sup./ 161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD3.sup.+/CD8.sup.+ and CD95.sup.+ populations.

(76) The results in FIGS. 2a-c) demonstrate the unique phenotype of CMhi and EMhi subsets of CD8.sup.+ TCR.sup.+ T cells. The phenotype is consistent with a memory population and is similar to murine memory stem cells as described by Zhang et al (Nat Med, 2005). The phenotype is different from that expected for other characterized lymphoid populations, such as NK cells and invariant NKT cells. CMhi and EMhi have a similar phenotype, but can be immunophenotypically differentiated by the expression of CD62L. Only the phenotype of EMhi is shown for clarity in FIGS. 2a) and b). FIG. 2c) is shown to illustrate the higher expression of bcl-2 and bcl-xl and lower expression of Ki67 in CMhi and EMhi, compared to CMlo and EMlo, from all donors tested.

Example 3

CMhi and EMhi Have a Surface Phenotype That Suggests Derivation from the Distal Pole of an Asymmetrically Dividing CD8.SUP.+ T Cell or Uropod of a Polarized CD8.SUP.+ T Cell

(77) PBMC were separated from fresh peripheral blood by density gradient centrifugation, washed in efflux buffer and resuspended at 110.sup.6/ml in ice cold efflux buffer with 5 g/ml rhodamine 123. PBMC were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer for 30 minutes at 37 C. At 30 minutes, PBMC were washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with antibodies to CD3, CD4, CD8, CD45RA, CD45RO, CD62L, CD16 and either CD11a, CD43, CD44, CD46, CD148 or CD162 for 20 minutes on ice. After washing in ice cold FACS buffer, samples were analyzed on a BD FacsARIA flow cytometry. CM and EM subsets were identified as CD62L.sup.+ or CD62L.sup. events, respectively, in the CD4.sup./CD16.sup./CD3.sup.+/CD8.sup.+/CD45RA.sup.int/neg/CD45RO.sup.+ population. Establishment of rapid efflux was performed as described in Example 1.

(78) FIG. 3 indicates that CMhi have a phenotype consistent with derivation from the memory distal pole of a dividing CD8.sup.+ T cell or uropod of a polarized CD8+ T cell. The phenotype of CMhi is similar to the phenotype of EMhionly FACS profiles gated on CM CD8.sup.+ T cells are shown for clarity. The MFIs of CMhi and CMlo populations are shown in red. After TCR signaling in the presence of appropriate costimulation and/or adhesion molecules, an immune synapse forms between the APC and T cell. CD8 and CD11 a (amongst other cell surface proteins) actively localize to the immune synapse (Proximal markers) and CD43, CD44, CD46, CD148 and CD162 are excluded from the synapse and form the distal pole complex (Distal markers). A similar structure to the distal pole complex, the uropod, is also formed on stimulation of some T cells with chemokines and has a similar pattern of expression of cell surface markers. It is unknown whether these surface molecules remain localized within or outside the immune synapse or uropod until or beyond the first cell division; however the phenotype of CMhi and EMhi, while variable, could be consistent with cell populations that are derived from the distal pole of a dividing memory cell or the uropod.

Example 4

CMhi and EMhi Express Higher Levels of MDR-1 mRNA Than Their Non-effluxing Counterparts and Actively Efflux the Fluorescent Chemotherapy Drug, Daunorubicin

(79) In FIG. 4(a), CMhi and EMhi and their non-effluxing IL-18R.sup.lo/neg counterparts (CMlo and EMlo) were isolated using negative immunomagnetic selection of CD8.sup.+ T cells with biotinylated antibodies to non-CD8.sup.+ T cells, followed by surface labeling with fluorochrome-conjugated streptavidin to identify non-CD8.sup.+ T cells, CD95, CD62L and IL-18R and sorting on a BD FacsARIA flow sorter. CM and EM CD8.sup.+ T cells were defined as streptavidin.sup./CD95.sup.+/CD62L.sup.+ or streptavidin.sup./CD95.sup.+/CD62L.sup., respectively. CMhi and EMhi were defined by high expression of IL-18R. Expression of mdr 1 in isolated CMhi, CMlo, EMhi and EMlo subsets was determined by quantitative polymerase chain reaction, using the following primers and probes: MDR1 forwardGGA AGC CAA TGC CTA TGA CTT TA; MDR1 reverseGAA CCA CTG CTT CGC TTT CTG; MDR1 probe6FAM-TGA AAC TGC CTC ATA AAT TTG ACA CCC TGG-TAMRA. Results shown are normalized to GAPDH expression. The mean/SE from 3 normal donors are shown.

(80) In FIG. 4(b), PBMC were resuspended at 110.sup.6/ml in efflux buffer and loaded with the fluorescent chemotherapy drug, daunorubicin, at 2.5 M for 20 minutes at 37 C., before washing three times and effluxing with or without vinblastine 25 M for 1 hour at 37 C. PBMC were then washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with antibodies to CD16, CD3, CD8, CD95, CD62L and CD161 or IL-18R for 20 minutes on ice. After washing in ice cold FACS buffer, samples were analyzed on a BD LSR-2 flow cytometer.

(81) The data indicate that CMhi and EMhi express high levels of mRNA for MDR-1, the ATP-binding cassette (ABC) co-transporter responsible for specific and active efflux of Rh123 and daunorubicin. The capacity of CMhi and EMhi (defined by either high IL-18R or CD161 expression) to actively and specifically efflux a chemotherapy drug is shown by the inhibition of efflux in the presence of a competitive agonist for MDR-1 protein, vinblastine. Inhibition was also achieved with two other MDR-1 channel blockers, PK11195 and cyclosporine A (data not shown).

Example 5

CMhi and EMhi are Resistant to Daunorubicin-Induced Apoptosis In Vitro

(82) CMhi and EMhi and their non-effluxing IL-18R.sup.lo/neg counterparts (CMlo and EMlo) were isolated using negative immunomagnetic selection of CD8.sup.+ T cells with biotinylated antibodies to non-CD8.sup.+ T cells, followed by surface labeling with fluorochrome-conjugated streptavidin to identify non-CD8.sup.+ T cells, CD95, CD62L and IL-18R and sorting on a BD FacsARIA flow sorter. CM and EM CD8.sup.+ T cells were defined as streptavidin.sup./CD95.sup.+/CD62L.sup.+ or streptavidin.sup./CD95.sup.+/CD62L.sup., respectively. CMhi and EMhi were defined by high expression of IL-18R. Effluxing and non-effluxing CM and EM subsets were cultured for 44 hours in the presence or absence of daunorubicin (an anthracycline chemotherapeutic agent, effluxed by the ABC-B1 cotransporter, MDR-1), with or without MDR-1 blockade with the peripheral benzodiazepine receptor antagonist, PK11195. Cultures were harvested, washed twice with cold PBS and stained with Annexin V and DAPI before analysis.

(83) The results in FIG. 5 indicate that CMhi and EMhi subsets are resistant to apoptosis induced by culture with daunorubicin at pharmacological concentrations (0.1 uM). Chemoresistance is mediated by efflux pumps as inhibition of MDR-1 with PK11195 results in increased cell death. Culture with PK11195 alone does not impair viability.

Example 6

CMhi and EMhi Divide in Response to the Homeostatic Cytokines, IL-7 and IL-15, and Have High Viability After Culture in the Absence of Supplementary Cytokines

(84) In FIGS. 6(a-c), PBMC were separated from fresh peripheral blood by density gradient centrifugation. CD8.sup.+ T cells were positively selected using CD8 Microbeads (Miltenyi) and resuspended at 110.sup.6/ml in ice cold efflux buffer with 10 g/ml Rh123. CD8.sup.+ T cells were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer for 30 minutes at 37 C. Vinblastine was added to control samples to establish the presence of efflux). CD8.sup.+ T cells were then washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCR, V24, CD8, CD95, CD62L and CD161. CMhi and EMhi subsets were identified as CD62L.sup.+/Rh123.sup.lo/CD161.sup.hi or CD62L.sup./Rh123.sup.lo/CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+ population. CMlo and EMlo subsets were identified as CD62L.sup.+/Rh123.sup.hi/CD161.sup.int/neg or CD62L.sup./Rh123.sup.hi/CD161.sup.int/neg events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ population. The gating strategy is shown in FIG. 6a). Subsets were isolated using a BD FacsARIA flow sorter and proliferation in response to IL-7 was determined by .sup.3H-thymidine uptake or CF SE dilution assays. Proliferation in response to IL-15 was determined by CFSE dilution assay. The .sup.3H-thymidine proliferation assay was performed by culturing for 5 days in CTL medium supplemented with IL-7 then pulsing overnight with .sup.3H-thymidine before harvesting and counting. The CFSE-dilution assay was performed by loading the cells with CFSE and culturing for 10 days, before viability labeling with DAPI and analysis on a BD LSR2 flow cytometer.

(85) In FIG. 6(d), PBMC from non-lymphopenic healthy donors (n=8) or lymphopenic acute myeloid leukemia patients (n=6) at the nadir (day 11 to day 22) of induction therapy were analyzed for Ki67 expression on memory subsets as described in Example 2. The fold change in the percent Ki67 expression of CD8.sup.+ T cell subsets between non-lymphopenic healthy donors and lymphopenic patients is depicted.

(86) The results indicate that the CMhi and EMhi subsets have the capacity to enter the cell cycle and undergo division in response to IL-7 (FIG. 6b) and IL-15 (FIG. 6c) stimulation. CMhi and EMhi are also recruited more effectively into the cell cycle than CMlo and EMlo in lymphopenic chemotherapy patients (FIG. 6d), suggesting high sensitivity to lymphopenia-induced IL-7 and IL-15-mediated proliferation. In addition, CMhi and EMhi maintain higher viability in culture in the absence of supplementary cytokines than their non-effluxing counterparts (FIG. 6b). IL-7 and IL-15 are critical for the maintenance of long term memory and survival of memory T cells and the CMhi and EMhi subsets are sensitive to signaling by IL-7 and IL-15.

Example 7

CMhi and EMhi Show Reduced .SUP.3.H-thymidine Uptake, Compared to Their Non-effluxing Counterparts, in Response to Polyclonal TCR Stimulation with OKT3, and Can be Rescued After Costimulation as Indicated in FIG. 7

(87) CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMC were separated from fresh peripheral blood by density gradient centrifugation. CD8.sup.+ T cells were positively selected using CD8 paramagnetic beads and resuspended at 110.sup.6/ml in ice cold efflux buffer with 10 g/ml Rh123. CD8.sup.+ T cells were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer, with or without vinblastine, for 30 minutes at 37 C. At 30 minutes, CD8.sup.+ T cells were washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCR, V24, CD8, CD95, CD62L and CD161. CMhi and EMhi subsets were identified as CD62L.sup.+/Rh123.sup.lo/CD161.sup.hi or CD62L.sup./Rh123.sup.lo/CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+ population. CMlo and EMlo subsets were identified as CD62L.sup.+/Rh123.sup.hi/CD161.sup.int/neg or CD62L.sup./Rh123.sup.hi/CD161.sup.int/neg events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ population. Subsets were isolated using a BD FacsARIA flow sorter and cultured in 96 well plates at 10,000-30,000 per well in 200 l CTL in the indicated conditions. OKT3 was plate-bound by incubating at 1000 ng/ml in 100 l PBS per well for 6 hours at 4 C., then washing twice with 200 ul cold PBS before plating the sorted subsets. Anti-CD28 was plate-bound (at 5 g/ml) with OKT3, as above. Cytokine concentrations were as follows: IL-7, 2 ng/ml; IL12, 10 ng/ml; IL-15, 1 ng/ml, IL-18, 80 ng/ml; IL-23, 10 ng/ml. Culture with cytokines in the absence of cytokine costimulation resulted in minimal proliferation. Data for the proliferation of the CMhi subset with IL-12 alone or OKT3/IL-12 is not available.

Example 8

CMhi and EMhi Have a Different Cytokine Secretion Profile Compared to Their Non-effluxing Counterparts

(88) CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMC were separated from fresh peripheral blood by density gradient centrifugation. CD8.sup.+ T cells were positively selected using CD8 paramagnetic beads and resuspended at 110.sup.6/ml in ice cold efflux buffer with 10 g/ml Rh123. CD8.sup.+ T cells were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer, with or without vinblastine, for 30 minutes at 37 C. At 30 minutes, CD8.sup.+ T cells were washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCR, V24, CD8, CD95, CD62L and CD161. CMhi and EMhi subsets were identified as CD62L.sup.+/Rh123.sup.lo/CD161.sup.hi or CD62L.sup./Rh123.sup.lo/CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+ population. CMlo and EMlo subsets were identified as CD62L.sup.+/Rh123.sup.hi/CD161.sup.int/neg or CD62L.sup./Rh123.sup.hi/CD161.sup.int/neg events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ population. Subsets were isolated using a BD FacsARIA flow sorter and plated in 200 l CTL medium at 60,000 cells per well. Polyclonal stimulation was performed by culturing isolated subsets with either PMA (5 ng/ml)/ionomycin (1 g/ml) or plate-bound OKT3/anti-CD28 (prepared as described in Example 7) for 20 hours. Cytokine secretion was detected in culture supernatant using a Luminex Cytokine Array assay.

(89) These experiments show that CMhi and EMhi secrete less IL-2, IL-4, IL-6, IL-8, IL-10, IFN- and MIP-1 and more IL-17 than their non-effluxing counterparts in response to polyclonal stimulation.

Example 9

CMhi and EMhi Have Decreased Calcium Flux in Response to Ionomycin, Compared to Their Non-effluxing Counterparts

(90) PBMC were separated from fresh peripheral blood by density gradient centrifugation at room temperature and incubated at 110.sup.7/ml in CTL medium supplemented with Indo-1AM (Sigma) 10 M and probenicid 4 mM for 30 minutes at 37 C. The PBMC were washed once in CTL medium at 25 C. Surface labeling was performed in CTL medium with antibodies to CD4, CD16, TCR, V24, CD8, CD62L and CD161 for 10 minutes at room temperature. After washing in room temperature CTL medium, surface-labeled samples were warmed to 37 C. for 4 minutes before high speed acquisition on a BD LSR-2 flow cytometer equipped with UV, violet, blue, green and red lasers. After 30 seconds acquisition, the sample was removed from the aspiration port, ionomycin was added to a final concentration of 5 g/ml, the sample was returned and acquisition was continued at 20,000 events/second. CMhi and EMhi subsets were identified as CD62L.sup.+/CD161.sup.hi or CD62L.sup./CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ populations. CMlo and EMlo subsets were identified as CD62L.sup.+/CD161.sup.int/neg or CD62L.sup./CD161.sup.int/neg events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ populations. Relative intracellular calcium concentration was measured as the ratio of Indo-1AM fluorescence in the UV violet (405 nm):UV blue (505 nm) detectors and is plotted as the mean ratio versus time (seconds) for appropriately gated subsets.

(91) This experiment demonstrates that CMhi and EMhi subsets have a different capacity to flux calcium in response to the calcium ionophore, ionomycin, compared to their non-effluxing counterparts. Calcium flux is a proximal signaling event downstream from antigen-specific TCR ligation.

Example 10

CMhi and EMhi Subsets Comprise Polyclonal TCR Repertoires by Molecular Spectratyping, as Described in Example 10

(92) CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMC were separated from fresh peripheral blood by density gradient centrifugation. CD8.sup.+ T cells were positively selected using CD8 paramagnetic beads and resuspended at 110.sup.6/ml in ice cold efflux buffer with 10 g/ml Rh123. CD8.sup.+ T cells were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer, with or without vinblastine, for 30 minutes at 37 C. At 30 minutes, CD8.sup.+ T cells were washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCR, V24, CD8, CD95, CD62L and CD161. CMhi and EMhi subsets were identified as CD62L.sup.+/Rh123.sup.lo/CD161.sup.hi or CD62L.sup./Rh123.sup.lo/CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+ population. CMlo and EMlo subsets were identified as CD62L.sup.+/Rh123.sup.hi/CD161.sup.int/neg or CD62L.sup./Rh123.sup.hi/CD161.sup.int/neg events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ population. Nave CD8.sup.+ T cells were identified as CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup./CD62L.sup.+ events. Subsets were isolated using a BD FacsARIA flow sorter.

(93) Molecular V spectratyping was performed on isolated subsets and nave CD8.sup.+ T cells by multiplex RT-PCR and Genescan analysis of TCR V fragments.

(94) These experiments show polyclonal TCR V usage in the effluxing CMhi and EMhi subsets, demonstrating that the CD8.sup.+ T cells within the CMhi and EMhi subsets express diverse TCR that are potentially specific for a broad range of antigens.

Example 11

Viral Antigen Tetramer-Positive Cells Can be Identified within CMhi and EMhi Subsets and CMV-, EBV- and Influenza-Specific CTL Responses Can be Generated from Sorted CMhi and EMhi Subsets, as Described in Example 11

(95) PBMC were separated from fresh peripheral blood by density gradient centrifugation and resuspended in PBS. Surface labeling was performed with antibodies to CD4, CD16, TCR, CD8, CD95, CD62L, CD161 and an APC-labeled HLA-A*0201; NLV peptide tetramer to allow identification of CD8.sup.+ T cells specific for the NLV peptide from the pp65 antigen of CMV. After washing in cold PBS, surface-labeled samples were analyzed on a BD LSR-2 flow cytometer. CMhi and EMhi subsets were identified as CD62L.sup.+/CD161.sup.hi or CD62L.sup./CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./CD8.sup.+/CD95.sup.+ populations. Data is shown in FIG. 11a).

(96) To demonstrate that rare tetramer-positive events seen in the CMhi and EMhi subsets ex vivo were viral antigen-specific CTL, we expanded antigen-specific CTL in vitro from isolated CMhi and EMhi subsets by culture with autologous activated peptide-pulsed monocyte-derived DC (MoDC). CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. MoDC were generated by culture of CD14.sup.+ monocytes, isolated using CD14-specific paramagnetic beads, with GM-CSF (800 U/ml) and IL-4 (1000 U/ml) for 5 days. Mature MoDC were generated by culture to day 7 with additional GM-CSF (800 U/ml)/IL-4 (1000 U/ml) and IL-1(2 ng/ml), IL-6 (1000 U/ml), PGE.sub.2 (1000 ng/ml) and TNF (10 ng/ml). Activated MoDC were pulsed in RPMI1640 (Gibco) for 2 hours at room temperature with 1 g/ml HLA-A*0201-restricted NLVPMVATV, GLCTLVAML or GILGFVFTL peptides, derived from CMV, EBV or influenza, respectively. MoDC were washed 3 times in RPMI1640 and irradiated (3500 cGy) before use.

(97) CMhi, CMlo, EMhi and EMlo subsets were plated in 96 well plates with irradiated, activated, peptide-pulsed MoDC at a T:DC ratio of 4:1 in 200 ml CTL medium supplemented with IL-2 (10 U/ml), IL7 (1 ng/ml) and IL-15 (100 pg/ml). Cytokine and half medium exchanges were performed on days 4 and 7 and analysis by CD8, DAPI and tetramer staining was performed on day 10. Data is shown in FIG. 11b).

(98) This experiment demonstrates that rare virus-specific tetramer-positive CD8.sup.+ T cells can be identified within rapidly effluxing CMhi and EMhi populations ex vivo and that rare viral antigen-specific CD8.sup.+ T cells can be identified after in vitro stimulation of isolated effluxing CMhi and EMhi subsets. Despite the fact that effluxing CMhi and EMhi are refractory to stimulation with OKT3 (Example 7), proliferation can be rescued by culturing with cytokines. The use of activated MoDC and cytokine supplementation in Example 11 allows expansion of antigen-specific CTL from effluxing subsets in vitro.

Example 12

CMhi and EMhi Subsets Have Unique and Distinct Gene Expression Profiles

(99) CMhi, CMlo, EMhi and EMlo were isolated as described in Example 6. PBMC were separated from fresh peripheral blood by density gradient centrifugation. CD8.sup.+ T cells were positively selected using CD8-specific paramagnetic beads and resuspended at 110.sup.6/ml in ice cold efflux buffer with 10 g/ml Rh123. CD8.sup.+ T cells were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer, with or without vinblastine, for 30 minutes at 37 C. At 30 minutes, CD8.sup.+ T cells were washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCR, V24, CD8, CD95, CD62L and CD161. CMhi and EMhi subsets were identified as CD62L.sup.+/Rh123.sup.lo/CD161.sup.hi or CD62L.sup./Rh123.sup.lo/CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+ population. CMlo and EMlo subsets were identified as CD62L.sup.+/Rh123.sup.hi/CD161.sup.int/neg or CD62L.sup./Rh123.sup.hi/CD161.sup.int/neg events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ population. Subsets were isolated using a BD FacsARIA flow sorter.

(100) cRNA was generated from isolated subsets and gene expression array studies were performed, using the Illumina HumanWG-6 expression beadchip array. The data are displayed on a Principal Components Plot to illustrate the gene expression relationships of CMhi and EMhi in relation to non-effluxing CD8.sup.+ T cell subsets. The data show that rapidly effluxing (CMhi and EMhi) subsets have gene expression profiles that are distinct from those of nave or non-effluxing memory (CMlo and EMlo) CD8.sup.+ T cells. In addition, the separation of CMhi and EMhi clusters suggests that, despite their similar phenotype, CD62L.sup.+ (CMhi) and CD62L.sup. (CMlo) effluxing CD8.sup.+ T cells have different gene expression profiles.

Example 13

CMhi and EMhi are Rare in Cord Blood, Peak in Early Adult Life and are Found at Decreasing Frequency with Advancing Age

(101) PBMC were separated from fresh peripheral blood or cord blood by density gradient centrifugation. CD8.sup.+ T cells were positively selected using CD8 Microbeads (Miltenyi) and resuspended at 110.sup.6/ml in ice cold efflux buffer with 10 g/ml Rh123. CD8.sup.+ T cells were incubated for 30 minutes on ice before washing three times in ice cold efflux buffer and resuspending in pre-warmed efflux buffer for 30 minutes at 37 C. Vinblastine was added to control samples to establish the presence of efflux). CD8.sup.+ T cells were then washed once in ice cold PBS/0.2% BSA (FACS buffer) and labeled with fluorochrome-conjugated antibodies to CD4, CD16, TCR, V24, CD8, CD95, CD62L and CD161. CMhi and EMhi subsets were identified as CD62L.sup.+/Rh123.sup.lo/CD161.sup.hi or CD62L.sup./Rh123.sup.lo/CD161.sup.hi events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+ population. CMlo and EMlo subsets were identified as CD62L/Rh123hi/CD161.sup.int/neg or CD62L.sup./Rh123.sup.hi/CD161.sup.int/neg events, respectively, in the CD4.sup./CD16.sup./TCR.sup./V24.sup./CD8.sup.+/CD95.sup.+ population. The gating strategy is shown in FIG. 6a). Samples were assayed using a BD FacsARIA flow cytometer. The frequency of CMhi and EMhi phenotype cells as a percentage of CD8.sup.+ T cells is shown in cord blood compared to adult peripheral blood in FIG. 13a). The percentage of effluxing (top) and non-effluxing (bottom) cells in the parental CM and EM compartments are shown in FIG. 13b). Each point represents a single healthy donor.

(102) The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.