EX VIVO GENERATION OF GAMMA DELTA FOXP3+ REGULATORY T CELLS AND THERAPEUTIC USES THEREOF

Abstract

The present invention relates to a method for ex vivo generating and expanding ?? Foxp3.sup.+ regulatory T cells, and therapeutic uses thereof. The inventors performed the induction of Foxp3? expression in ex vivo human induced tumor-antigen specific CD4+ TCR?? unrestricted T cells and the induction of autologous CD8-mediated T-cell responses against tumor-antigen specific FOXP3 expressing CD4+ TCR?? unrestricted T cells. The inventors developed a method to ex vivo generated and expanded antigen specific Foxp3 expressing CD3+ TCR?? unrestricted T cells, committed to exclusively exert regulatory activity, whichever culture condition of stimulation is. In particular, the present invention relates to a method for generating ex vivo ?? Foxp3+ regulatory T cells having the following phenotype: CD3+ TCR?? Foxp3+.

Claims

1. A method for generating ex vivo ?? Foxp3.sup.+ regulatory T cells having the following phenotype: CD3.sup.+ TCR??.sup.+ Foxp3.sup.+, comprising culturing CD3.sup.+ TCR??.sup.+ T cells in the presence of a ?? T cell activator and the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator, ii) a TGF? (Transforming growth factor beta) pathway activator, and iii) a mTOR inhibitor, and optionally iv) at least one cytokine selected from the group consisting of IL-2, IL-7, IL-15 and TSLP, and/or v) at least one TET enzyme activator and/or vi) at least one DNMT inhibitor, for at least 5 days.

2. The method according to claim 1, wherein the ?? T cell activator is a polyclonal ?? T cell activator.

3. The method according to claim 1, wherein the ?? T cell activator is an antigen-specific ?? T cell activator.

4. The method according to claim 1, wherein the cAMP activator is selected from the group consisting of prostaglandin E2 (PGE2), an EP2 or EP4 agonist, a membrane adenine cyclase activator and a metabotropic glutamate receptors agonist.

5. The method according to claim 1, wherein the TGF? pathway activator is selected from the group consisting of TGF?, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-mullerian hormone (AMH), activin and nodal.

6. The method according to claim 1, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, rapamycin analogs, wortmannin; theophylline; caffeine; epigallocatechin gallate (EGCG), curcumin, resveratrol; genistein, 3,3-diindolylmethane (DIM), LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), PP242, PP30, Torin1, Ku-0063794, WAY-600, WYE-687, WYE-354, GNE477, NVP-BEZ235, PI-103, XL765 and WJD008.

7. The method according to claim 1, further comprising an expansion step, wherein the ?? Foxp3.sup.+ regulatory T cells are cultured in the presence of a ?? T cell activator and the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator, ii) a TGF? (Transforming growth factor beta) pathway activator, iii) a mTOR inhibitor, and optionally iv) at least one cytokine selected in from the group consisting of IL-2, IL-7, IL-15 and TSLP, and optionally v) at least one TET enzymes activator and/or at least one DNMT inhibitor, for at least 5 days.

8. An ex vivo generated ?? Foxp3.sup.+ regulatory T cell population obtained by the method according to claim 1.

9. An ex vivo generated and expanded ?? Foxp3.sup.+ regulatory T cell population obtainable by the method according to claim 7, wherein said ?? Foxp3.sup.+ regulatory T cells have the phenotype CD3.sup.+ TCR ??.sup.+ Foxp3.sup.+ or the phenotype CD3.sup.+ TCR ??.sup.+ Foxp3.sup.+ IL-1R1.sup.?.

10. The ex vivo generated ?? Foxp3.sup.+ regulatory T cell population of claim 9, wherein said ex vivo generated ?? Foxp3.sup.+ regulatory T cells remain functionally stable during inflammation.

11-12. (canceled)

13. A pharmaceutical composition comprising inactivated ?? Foxp3.sup.+ regulatory T cells having the following phenotype: CD3.sup.+ TCR??.sup.+ Foxp3.sub.+, blebs of ?? Foxp3.sup.+ regulatory T cells having the following phenotype: CD3.sup.+ TCR??.sup.+ Foxp3.sup.+, or immunogenic dendritic cells loaded with blebs of ?? Foxp3.sup.+ regulatory T cells having the following phenotype: CD3.sup.+ TCR??.sup.+ Foxp3.sup.+; and at least one pharmaceutically acceptable excipient.

14. The pharmaceutical composition of claim 13, wherein the pharmaceutical composition is a vaccine and comprises at least one adjuvant.

15. A method of treating cancer or an inflammatory or autoimmune disease, performing cell therapy, or treating transplant rejection or graft versus host disease (GVDH) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 13.

16-18. (canceled)

19. The method according to claim 2, wherein the polyclonal ?? T cell activator is an anti-TCR ?? antibody or a non-peptide phosphoantigen.

20. The method according to claim 3, wherein the antigen-specific ?? T cell activator is tolerogenic dendritic cells (DCs) pulsed with at least one bisphosphonate.

21. The method of claim 20, wherein the at least one bisphosphonate comprises at least one aminobisphosphonate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0337] FIG. 1: Different frequencies and phenotypic characteristics between FOXP3.sup.+ and FOXP3.sup.? CD3.sup.+ T cell populations, as defined by their variable TCR recognition in human peripheral blood (PBMCs) and in TIL isolated from breast tumor.

[0338] FIG. 2: Analysis of Foxp3.sup.+ expression in ex vivo human induced tumor-antigen specific FOXP3 expressing CD4.sup.+ TCR??.sup.+ unrestricted T cells. Apoptotic tumor antigen (Ag)-pulsed tolerogenic DCs (tDCs) were used to generate and expand specific pTreg from naive CD4.sup.+ T cells in the presence of IL-2 (100 IU/ml) and the nTreg polarizing medium composed of TGF? (5 ng/ml), PGE2 (1 ?M) and Rapa (10 nM). Unloaded tDC were used as control. (A) Frequency and (B) expression level (evaluated by MFI) of Foxp3 in CD4.sup.? T cell culture.

[0339] FIG. 3: Phenotypic differences between the Foxp3 expressing CD3.sup.+ CD4.sup.+ ?? T cells unrestricted T cells isolated from BC biopsies and the currently described ?? T cells' subtypes. Foxp3 expressing CD3.sup.+ CD4.sup.+ ?? T cells' phenotypic identification was performed by flow cytometry using antibody against CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), pan ?? (clone IMMU510) and Foxp3+(clone 259D).

[0340] FIG. 4: TCR V? usage among CD3.sup.+ CD4.sup.+ ?? T cells expressing FOXP3.sup.+ isolated from BC biopsies. Identification of TCR V?? chains was performed by flow cytometry using antibody against CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), pan ?? (clone IMMU510), TCR V?1 (REA173), TCR V?2 (REA771) and Foxp3.sup.? (clone 259D).

[0341] FIG. 5: Suppressive capacity of the Foxp3 expressing CD3.sup.+ CD4.sup.+ ?? T cells unrestricted T cells isolated from BC biopsies. CFSE-labeled Tconv (TconvCFSE) were cocultured with sorted CD3.sup.+ CD4.sup.+ ?? T cells at different ratios. Percent inhibition of TconvCFSE proliferation by CD4.sup.+ ?? T cells was depicted. Circulating fresh Treg from health donor were used as control.

[0342] FIG. 6: Generation of autologous CD8.sup.+ T cell lines functionally committed to lyse specific pathogenic CD4.sup.+ T cells, i.e. tumor-antigen specific FOXP3 expressing CD4.sup.+ TCR??.sup.+ unrestricted T cells. The capacity of a CD8.sup.+ T cell clone to lyse its inducing pathogenic CD4.sup.+ T cell clone is evaluated with the classical 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay as previously described. In brief, 4 days after stimulation, pathogenic CD4.sup.| target cells or an autologous lymphoblastoid line were labeled with

[0343] CFSE and placed at 3?10.sup.4 per well in 96-well U-bottomed plates in triplicate. CD8.sup.+ Effector T cells (5:1 E:T ratio) were added, and incubation was carried out at 37? C. for 6 hours. At the end of the experiment, dead cells were labeled with 7-AAD to detect lysed cells. Cytolytic activity against target cells was analyzed based on regions showing double-positive staining CFSE and 7-AAD, using a FACSCalibur instrument. CD8.sup.+ T cell clone cytolytic activity (%) was calculated as cells positive for both CFSE and 7-AAD/total CFSE positive cells, after subtracting the spontaneous lysis (%) in negative control. The percentage of cytolytic activity was then calculated using the following equation: Cytolytic activity (%) [dead target cells (%)?spontaneous death (%)]?100/[100?spontaneous death (%)].

[0344] FIG. 7: Analysis of Foxp3.sup.+ expression in lymphocytes present in the TILs extracted from luminal A and B breast subtypes. Tumor tissue from patient with luminal-A and luminal B was minced with scalpels and enzymatically digested by overnight incubation in collagenase Type IV. Expression of FOXP3 marker in lymphocytes present in the isolated TIL was determined by flow cytometric analysis. FIG. 8: Analysis of Foxp3.sup.+ expression in lymphocytes present in the TILs extracted from 3 different breast cancers' subgroups: tumor tissue from patient with luminal A (n=3), luminal B (n=3) and patients with triple-negative breast cancer (TNBC) (n=2) was minced with scalpels and enzymatically digested by overnight incubation in collagenase Type IV. Expression of FOXP3 marker in lymphocytes present in the isolated TIL was determined by flow cytometric analysis. Representation of the percentage of FOXP3 expression in the CD3.sup.+CD4.sup.+TCR??.sup.+ unrestricted T cells.

[0345] FIG. 9: Multiparametric flow cytometry analysis of lymphocytes present in the TILs from luminal A and B breast subtypes. Lymphocytes present in the TIL were stained at the cell surface using Abs directed against CD3, CD4, CD25, CD56, CD161. After fixation and permeabilization Foxp3 and CTLA4 were stained intracellularly.

[0346] FIG. 10: Phenotype and functional suppressive capacity of ex vivo generated Ag specific CD3.sup.+ TCR??.sup.+ T cells from stimulated naive CD3.sup.+ TCR??.sup.+ T cells. Naive CD3.sup.+ TCR??.sup.+ T cells were stimulated with zoledronic acid-treated-autologous tDCs, in presence of the nTreg polarizing medium and IL-2 (100 IU/ml) and IL-15 (10 ng/ml). (A) Overlay histogram displaying Foxp3 expression profiles and (B) suppressive capacity of Ag specific CD3.sup.+ TCR??.sup.+ T cells expanded for 21 or 42 days.

EXAMPLES

[0347] The present invention is further illustrated by the following examples.

Materials and Methods

[0348] Human Blood Sample. Blood samples from healthy individuals originated from Etablissement Fran?ais du Sang (EFS, Paris). Blood cells are collected using standard procedures.

[0349] Human tumor sample. Tumor tissue sample originated from patient with Luminal A and Luminal B Breast cancer (Institut Jean Godinot, Reims).

Cell Purification and Culture.

[0350] Peripheral blood mononuclear cells (PBMCs) are isolated by density gradient centrifugation on Ficoll-Hypaque (Pharmacia). PBMCs are used either as fresh cells or stored frozen in liquid nitrogen. T-cell subsets and T cell-depleted accessory cells (?CD3 cells) are isolated from either fresh or frozen PBMCs. T cell-depleted accessory cells (?CD3 cells) are isolated by negative selection from PBMCs by incubation with anti-CD3-coated Dynabeads (Dynal Biotech) and are irradiated at 3000 rad (referred to as ?CD3-feeder).

[0351] CD4.sup.+ T cells are negatively selected with a CD4.sup.+ T-cell isolation kit (Miltenyi Biotec, yielding CD4.sup.+ T-cell populations at a purity of 96-99%. Sub-sequently, selected CD4.sup.+ T cells are labeled with anti-CD4 (13B8.2)-FITC (Beckman Coulter), anti-CD25(4E3)-APC (Miltenyi Biotec), and anti-CD127(R34.34)-PE (Beckman Coulter) before being sorted into CD4.sup.+CD127.sup.?/loCD25.sup.high(pTregs) and CD4.sup.+CD127.sup.+CD25.sup.neg/dim [conventional helper CD4 T cells (Tconv)] subpopulations using a FACSAria III Cell Sorter (Becton Dickinson).

[0352] CD14.sup.+ monocytes are isolated from PBMCs by positive selection using a MACS system.

[0353] CD3.sup.+ CD4.sup.+ CD127.sup.? CD45RA.sup.+ CD25.sup.? TCR??.sup.+ MHCII restricted (naive conventional CD4.sup.+ T cells) are isolated from PBMCs after magnetic enrichment (MACS system: CD4 microbeads) and FACs sorting. Before the sorting step, enriched CD3.sup.+ CD4.sup.+ T cells are stained with anti-CD4 (13B8.2)-FITC (Beckman Coulter), anti-CD25(4E3)-APC (Miltenyi Biotec), and anti-CD127(R34.34)-PE (Beckman Coulter), anti-TCR ??-BV421 (IP26) (Biolegend).

[0354] CD3.sup.+ CD45RA.sup.+ invTCR V?24.sup.? CD1-restricted T cells are isolated from PBMCs after magnetic enrichment (MACS system: anti-iNKT microbeads and FACS sorting. Before the sorting step, enriched CD3.sup.+ invTCR V?24.sup.+T cells are stained with anti-CD3 (UCHT-1) V450 anti-invariant TCR V?24-J?Q (6B11)-PE (inv TCR V?24-J?Q (Becton Dickinson) and anti-CD45RA (T6D11)-FITC (Miltenyi Biotec).

[0355] CD3.sup.+ CD45RA.sup.+ CD27.sup.+ TCR??.sup.+ unrestricted T cells are isolated from PBMCs after magnetic enrichment (MACS system: TCR??.sup.+ T cell isolation kit) and FACS sorting. Before the sorting step, enriched CD3.sup.+ TCR??.sup.+ T cells are stained with anti-CD3 (UCHT-1) V450, anti-TCR pan??.sup.+ PE (IMMU510) (Beckman Coulter), anti-CD27-APC efluor 780 (O323) (ebioscience) and anti-CD45RA (T6D11)-FITC (Miltenyi Biotec).

[0356] T cell subsets are cultured either in IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (IMDM-5 media) in hypoxia 2%.

[0357] Breast cancer cell line and culture. The human breast cancer cell line MCF-7 was obtained from the American Type Culture Collection (USA). Cells are maintained in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS). MCF-7 cells are treated with 5 ?g/ml Doxorubicin for 24 h or by ? irradiation (20 Gy). Extent of apoptosis is monitored by flow cytometric analysis (FACS). Cells are extensively washed prior to feeding DCs.

[0358] TIL isolation. Tumor tissue was minced with scalpels and enzymatically digested by overnight incubation in collagenase Type IV (2 mg/mL, Roche Diagnostic GmbH) in DMEM High Glucose medium supplemented with 2 mM glutamine (Gibco), 50 mg/mL gentamycin and 0.25% Human Serum Albumin, at 37? C. on a rotary shaker.

Ex Vivo Generation of Polyclonal Functionally Committed FOXP3 Expressing Regulatory T Cells.

[0359] Ex vivo generation of polyclonal functionally committed FOXP3 expressing CD3.sup.+ TCR??.sup.+ MHCII restricted T cells: On day 0, T cells are seeded at 2.5?10.sup.5/well in 48-well plates and stimulated with plate-bound anti-CD3 mAb (4 ?g/ml) in the presence of ?CD3-feeder (1 M). Cells are cultured in IMDM-5 media (IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES) with PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM. On day 2, IL-2 (100 IU/ml) are added to the culture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml). On day 11, these CD4.sup.+ T-cell lines were further expanded by restimulation with plate-bound anti-CD3 Abs (4 ?g/ml). The restimulations were performed in the presence of ?CD3-feeder, PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM and IL-2 (100 UI/ml). Then every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml). On day 20, the phenotype of the expanded CD4.sup.+ T cells was assessed by flow cytometry. 75% of the stimulated naive conventional T cells that became CD45RO.sup.+ express FOXP3.sup.+.

[0360] Ex vivo generation of polyclonal functionally committed FOXP3 expressing invariant T cells: On day 0, T cells are seeded at 1?10.sup.3/well in 96-well plates and stimulated with plate-bound anti-inv TCR V?24-J?Q (6B11) mAb (2 ?g/ml) in the presence of ?CD3-feeder (2.5?10.sup.5). Cells are cultured in IMDM-5 media with PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM, IL-2 (100 UI/ml) and IL-15 (10 ng/ml). Every three days, IL-2 (100 UI/ml) and IL-15 (10 ng/ml) are added to the culture. On day 12, T cells are further expanded by restimulation with plate-bound anti-anti-inv TCR V?24-J?Q (6B11) mAb (2 ?g/ml) in the presence of ?CD3-feeder, PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM IL-2 (100 UI/ml) and IL-15 (10 ng/ml). Then every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml) and IL-15 (10 ng/ml). On day 21, cells are analyzed by flow cytometry. 70% of the stimulated CD3+ invTCR V?24.sup.+ RA.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.

[0361] Ex vivo generation of polyclonal functionally committed FOXP3 expressing TCR??.sup.+ T cells: On day 0, T cells are seeded at 1?10.sup.3/well in 96-well plates and stimulated with plate-bound anti-TCR?? mAb (2 ?g/ml) in the presence of ?CD3-feeder (2.5?10.sup.5). Cells are cultured in IMDM-5 media (IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES) with PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM, IL-2 (100 UI/ml) and IL-15 (10 ng/ml). Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml) and IL-15 (10 ng/ml). On day 11, T cells were further expanded by restimulation with plate-bound anti-pan TCR ?? Abs (2 ?g/ml). The restimulations were performed in the presence of ?CD3-feeder, PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM and IL-2 (100 UI/ml) and IL-15 (10 ng/ml). Then every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml) and IL-15 (10 ng/ml). On day 21, cells are analyzed by flow cytometry. 65% of the stimulated CD3.sup.+ CD45RA.sup.+ CD27.sup.+ TCR??.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.|.

Ex Vivo Generation of Antigen Specific Functionally Committed FOXP3 Expressing T Cells.

[0362] Ex Vivo Generation of Antigen (Ovalbumin) Specific Functionally Committed FOXP3 Expressing CD3.sup.+ TCR??.sup.+ MHCII Restricted T Cells: [0363] a) In vitro generation of ovalbumin-loaded tolerogenic DC from CD14.sup.+ monocytes (termed tolerogenic monocyte-derived DC (Tol-Mo-DC): monocytes are cultured in 48-well flat-bottom plates containing 0.5 ml of AIMV per well supplemented with 100 ng/ml recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4 for the generation of immature DC. At day 3, 500 ?l of the medium containing cytokines was added. On day 6, Tol-Mo-DC are 1) removed from the wells, washed twice with IMDM-5 (IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES, 2) added to wells of a 48-well plate at a concentration of 3?10.sup.5/ml in IMDM-5 and 3) pulsed in IMDM-5 with specific Ag (OVA). [0364] b) Ex vivo generation and expansion of specific functionally committed FOXP3 expressing CD3.sup.+ TCR??.sup.+ MHCII restricted T cells: On day 0, ovalbumin pulsed tDC are 1) washed twice with IMDM-5 and 2) added to wells of a 48-well plate at a concentration of 3?10.sup.5/ml in IMDM-5 in the presence of 2?10.sup.5 irradiated autologous feeders, PGE2 1 ?M, and Rapa 10 nM. Purified naive conventional CD4.sup.+ T cells (isolated from the previously frozen PBMC by FACS) are added to the pulsed tDC. On day 1, IL-2 (100 IU/ml) and TGF? (5 ng/ml) are added to the coculture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml (T cell cloning medium). On day 12, these T-cells are further expanded by restimulation with ova-pulsed tDC in the presence of ?CD3-feeder, PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM, IL-2 (100 UI/ml). Once T cells begin to expand, they can be split every 2 to 3 days with T cell cloning medium and irradiated feeder. On day 21, cells are analyzed by flow cytometry. 85% of the stimulated naive conventional CD4.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.?. To confirm that the Ova-specific memory CD3.sup.+ TCR??.sup.? MHCII restricted T cells are committed to exclusively exert regulatory activity, whatever culture condition of stimulation, after 21 days of expansion in nTreg polarizing medium, the ova-specific-pTreg are further cultured for 3 weeks either in nTreg polarizing medium (comprising the combination of IL-2, TGF?, PGE2 and rapamycin) or TH-17 polarizing medium (IMDM medium containing IL-2 IL-1 IL-6, IL-21 IL-23 cytokines). The 21-day-expanded-Foxp3 expressing CD3.sup.+ CD4.sup.+ TCR??.sup.+ MHCII restricted T cells are stimulated with plate-bound anti-CD3 mAb (4 ?g/ml) in the presence of ?CD3-feeder (1 M) in 48-well plates and every three days, half of the supernatant volume is discarded and replaced with fresh T cell cloning medium or TH-17 polarizing medium for 21 days.

[0365] Ex Vivo Generation of Specific Tumor Phospho-Antigen Functionally Committed FOXP3 Expressing CD3.sup.+ TCR??.sup.+ Unrestricted T Cells:

[0366] In vitro generation of tumor-loaded tolerogenic DC from CD14.sup.? monocytes (termed tolerogenic monocyte-derived DC (tDC)): monocytes are cultured in 48-well flat-bottom plates containing 0.5 ml of AIMV per well supplemented with 100 ng/ml recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4. At day 3, 500 ?l of the medium containing cytokines are added. At day 5, a portion of tDCs are co-cultured with apoptotic MCF-7 cells at a DC/tumor cell ratio of 1:2 for 24 h in AIMV with GM-CSF (100 ng/mL), IL-4 (10 ng/mL). Another portion of tDC are freezed at 2?10.sup.6/per vialin 90% FBS ?10% DMSO.

[0367] Ex Vivo Generation and Expansion of Tumor-Phospho-Antigen Specific Functionally Committed Foxp3 Expressing CD3.sup.+ TCR??.sup.+ Unrestricted T Cells:

[0368] On day 0, tumor-antigen pulsed tDC are 1) washed twice with IMDM-5 and 2) added to wells of a 48-well plate at a concentration of 3?10.sup.5/ml in IMDM-5 in the presence of 2?10.sup.5 irradiated autologous feeders, PGE2 1 ?M, and Rapa 10 nM. Purified CD3.sup.+ CD45RA.sup.+ TCR??.sup.+ unrestricted T cells (isolated from the previously frozen PBMC by FACS) are added to the pulsed tDC. On day 1, IL-2 (100 IU/ml) and TGF? (5 ng/ml) are added to the coculture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml) (T cell cloning medium). On day 12, these T-cells are further expanded by restimulation with tumor Ag-pulsed tDC in the presence of ?CD3-feeder, PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM and IL-2 (100 UI/ml). Once T cells begin to expand, they can be split every 2 to 3 days with T cell cloning medium and irradiated feeder. On day 21, cells are analyzed by flow cytometry. 75% of the stimulated naive CD3.sup.+ CD45RA.sup.+ TCR??.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.

[0369] Ex Vivo Generation of Tumor-Antigen Specific Functionally Committed FOXP3 Expressing CD3.sup.+ invTCR V?24.sup.+ CD1d-Restricted T Cells: [0370] a) In vitro generation of tumor-loaded Tolerogenic DC from CD14.sup.+ monocytes (termed tolerogenic monocyte-derived DC (tDC): monocytes are cultured in 48-well flat-bottom plates containing 0.5 ml of AIMV per well supplemented with 100 ng/ml recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4 and AM580 (100 nM) for the generation of immature DC expressing CD1d. At day 3, 500 ?l of the medium containing cytokines are added. At day 5, a portion of tDCs are co-cultured with apoptotic MCF-7 cells at a DC/tumor cell ratio of 1:2 for 24 h in AIMV with GM-CSF (100 ng/mL), IL-4 (10 ng/mL). Another portion of tDC are freezed at 2?10.sup.6/per vial vialin 90% FBS ?10% DMSO. [0371] b) Ex vivo generation and expansion of tumor-antigen specific functionally committed Foxp3 expressing CD3.sup.+ invTCR V?24.sup.+ CD1d-restricted T cells: On day 0, tumor-antigen pulsed tDC are 1) washed twice with IMDM-5 and 2) added to wells of a 48-well plate at a concentration of 3?10.sup.5/ml in IMDM-5 in the presence of 2?10.sup.5 irradiated autologous feeders, PGE2 1 ?M, and Rapa 10 nM. Purified CD3.sup.+ CD45RA.sup.+ invTCR V?24.sup.+ CD1-restricted T cells (isolated from the previously frozen PBMC by FACS) are added to the pulsed tDC. On day 1, IL-2 (100 IU/ml), IL-15 (10 ng/ml) and TGF? (5 ng/ml) are added to the coculture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml) and IL-15 (10 ng/ml) (T cell cloning medium). On day 12, these T-cells are further expanded by restimulation with tumor Ag-pulsed tDC in the presence of ?CD3-feeder, PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM, IL-2 (100 UI/ml) and IL-15 (10 ng/ml). Once T cells begin to expand, they can be split every 2 to 3 days with T cell cloning medium and irradiated feeder. On day 21, cells are analyzed by flow cytometry. 75% of the stimulated CD3.sup.? CD45RA.sup.+ invTCR V?24.sup.+ cells that became CD45RO.sup.+ express Foxp3.sup.+.

[0372] Ex Vivo Generation of Phospho-Antigen Specific Functionally Committed FOXP3 Expressing CD3.sup.+ TCR??.sup.+ Unrestricted T Cells: [0373] a) In vitro generation of Tolerogenic DC from CD14.sup.+ monocytes (termed tolerogenic monocyte-derived DC (Tol-Mo-DC): monocytes are cultured in 48-well flat-bottom plates containing 0.5 ml of AIMV per well supplemented with 100 ng/ml recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4 for the generation of immature DC. At day 3, 500 ?l of the medium containing cytokines was added. On day 6, generated Tol-Mo-DC are removed from the wells, washed twice with IMDM-5 (IMDM supplemented with 5% SVF, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES, freezed or used for the generation and expansion of phospho-antigen specific functionally committed FOXP3 expressing CD3.sup.? TCR??.sup.+ unrestricted T cells. [0374] b) Ex vivo generation and expansion of phospho-antigen specific functionally committed FOXP3 expressing CD3.sup.? TCR??.sup.+ unrestricted T cells: On day 0, tDC are added to wells of a 48-well plate at a concentration of 3?10.sup.5/ml in IMDM-5 in the presence of 2?10.sup.5 irradiated autologous feeders, PGE2 1 ?M, and Rapa 10 nM and zoledronic acid (100 nM). Purified CD3.sup.+ CD45RA.sup.? TCR??.sup.+ unrestricted T cells (isolated from the previously frozen PBMC by FACS) are added to the pulsed tDC. On day 1, IL-2 (100 IU/ml), IL-15 (10 ng/ml) and TGF? (5 ng/ml) are added to the coculture. Every three days, half of the supernatant volume is discarded and replaced with fresh IMDM-5 with IL-2 (100 UI/ml) and IL-15 (10 ng/ml) (T cell cloning medium). On day 12, these T-cells are further expanded by restimulation with tDC in the presence of ?CD3-feeder, PGE2 1 ?M, TGF? 5 ng/ml, Rapa 10 nM, IL-2 (100 UI/ml), IL-15 (10 ng/ml) and zoledronic acid (100 nM). Once T cells begin to expand, they can be split every 2 to 3 days with T cell cloning medium and irradiated feeder. On day 21, cells are analyzed by flow cytometry. 75% of the stimulated CD3.sup.+ CD45RA.sup.+ TCR??.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.

[0375] In vitro generation of stimulator cells for MLR assay: monocytes are cultured in 48-well flat-bottom plates containing 0.5 ml of RPMI-5 per well supplemented with 20 ng/ml recombinant human granulocytemacrophage colony-stimulating factor (GM-CSF) and 20 ng/ml human recombinant IL-4 for the generation of immature DC (iDC). At day 3, 500 ?l of the medium containing cytokines are added. At day 5, a portion of iDC are co-cultured with apoptotic MCF-7 cells at a DC/tumor cell ratio of 1:2 for 24 h in RPMI 1640 supplemented with GM-CSF (20 ng/mL), IL-4 (20 ng/mL) and 5% FBS. Another portion of iDC are freezed at 2?10.sup.6/per vialin 90% FBS ?10% DMSO. When indicated, pulsed DCs are matured with tumor necrosis factor ? (TNF-?; 20 ng/mL final) and PGE2 (1 ?M) for 2 days (mDC). In some experiments, TNF and PGE2 (at the same concentrations), or lipopolysaccharide (LPS; 10-1000 ng/mL; Sigma) are added directly to MLRs. Antigen-loaded DC stimulators are irradiated at 30 Gy.

[0376] In vitro generation of TAP-inhibited stimulator cells for MLR assay: matured DC, obtained as described above, are electroporated with 20 ?g of RNA synthesized from the pGem4Z vector containing the UL49.5 gene from BHV-1. (ref: Lampen M H, Verweij M C, Querido B, van der Burg S H, Wiertz E J, van Hall T. CD8.sup.| T cell responses against TAP-inhibited cells are readily detected in the human population. (J Immunol. 2010 Dec. 1; 185(11):6508-17.)

[0377] Apoptotic T cells-DC cocultures: immature DCs were cultured alone or with apoptotic cells (3 apoptotic cells: 1 iDC) for 16 h. DCs were then purified by immunomagnetic depletion of apoptotic T cells using anti-CD3-coated microbeads (Miltenyi Biotec), electroporated or not with 20 ?g of synthesized RNA and incubated in RPMI-5 supplemented with 20 ng/ml GM-CSF, 20 ng/ml human recombinant IL-4 and the maturation cocktail (TNF-? 20 ng/ml and PGE2 1 u?M) for 24 hours.

Flow Cytometry Analysis

[0378] mAb labeling. The following conjugated mAbs are used.

[0379] a) for CD3.sup.+ T cells: anti-CD4(SK3)-PerCP-eFluor 710, anti-TCR ?? (IP26)-APC (ebioscience), anti-CD25 (B1.49.9)-PeCy55, anti-CD127 (R34.34)-APC-AF700 (Beckman Coulter), anti-CD3 (UCHT1)-BB515 anti-invariant TCR V?24-J?Q (6B11)-PE, anti-Foxp3 (259D/C7)-PE-CF594 and anti-CD152 (BNI3)-BV421, anti-CD161 (DX12) BV605 and anti-CD56 (NCAM 16.2) BU395 (Becton Dickinson), anti-TCR ??-BV421 (IP26) (Biolegend), anti-TCR pan ??.sup.+ PE (IMMU510) (Beckman Coulter) and anti-CD27-APC efluor 780 (O323) (ebioscience). Cells are stained for surface markers (at 4? C. in the dark for 30 min) using mixtures of Ab diluted in PBS containing BSA/NaN.sub.3 (0.5% BSA, 0.01% NaN3) (FACS buffer). Foxp3 and CTLA-4 intracellular stainings are performed with FOXP3 staining kit obtained from ebioscience according to the manufacturer's instructions. Appropriate isotype control Abs are used for each staining combination. Samples are acquired on a BD LSR FORTESSA flow cytometer using BD FACSDIVA 8.0.1 software (Becton Dickinson). Results are expressed in percentage (%) or in mean fluorescence intensity (MFI).

[0380] b) for the induced specific Treg: presence of IL-1R1 on induced Treg was evaluated with the monoclonal anti-Foxp3 (259D/C7)-PE-CF594 Ab and the polyclonal anti-IL1R1-PE (R&D system, FAB269P).

[0381] CFSE staining. Tconv are stained with 1 ?M carboxy-fluorescein succinimidyl ester (CFSE) (CellTrace cell proliferation kit; Molecular Probes/Invitrogen) in PBS for 8 min at 37? C. at a concentration of 1?10.sup.7 cells/mL. The labeling is stopped by washing the cell twice with RPMI 1640 culture medium containing 10% FBS. Cells are then resuspended at the desired concentration and subsequently used for proliferation assays.

[0382] 7-AAD (7-amino-actinomycin D) staining. Apoptosis of stimulated CFSE-labeled or unlabeled nTregs and Tconv was determined using the 7-AAD assay. Briefly, cultured cells are stained with 20 ?g/mL nuclear dye 7-AAD (Sigma-Aldrich) for 30 min at 4? C. FSC/7-AAD dot plots distinguish living (FSC.sup.high/7-AAD.sup.?) from apoptotic (FSC.sup.high/7-AAD.sup.+) cells and apoptotic bodies (FSC.sup.low/7-AAD.sup.+) and debris ((FSC.sup.low/7-AAD.sup.?). Living cells are identified as CD3+ 7-AAD.sup.? FSC.sup.+ cells.

[0383] Phenotypic characteristics of the Foxp3 expressing CD3+ CD4+ ?? T cells unrestricted T cells isolated from BC biopsies: TCR ?? T cells' subset identification was performed by flow cytometry. The panel included antibody against CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), pan ?? (clone IMMU510), TCR V?1 (REA173), TCR V?2 (REA771) and Foxp3+ (clone 259D).

Functional Assays.

[0384] T-cell proliferation. T-cell proliferation is assessed CFSE dilution assay in RPMI supplemented with 5% FBS, 100 IU/ml penicillin/streptomycin, 1 mM sodium pyruvate, 1 mM nonessential amino acids, glutamax and 10 mM HEPES (RPMI-5 media) in normoxia. At coculture completion, stimulated CFSE-labeled Tconv are harvested, costained with anti-CD3 mAb and 7-AAD, and the percentage of living proliferating cells (defined as CFSE low fraction) in gated CD3.sup.+ 7-AAD.sup.? cells is determined by flow cytometry.

[0385] T cell apoptosis induction: tumor-antigen specific functionally committed FOXP3 expressing CD3.sup.+ TCR??.sup.+ unrestricted T cells are generated ex vivo as described above. Then tumor-antigen specific stimulated-T cells were irradiated (240 mJ/cm.sup.2) at 254 nm (UV-C) and cultured for 6 hours before coculture with immature DCs. Apoptosis was confirmed by 7-AAD staining. On average, 75% of cells are 7-AAD.sup.+.

[0386] Standard polyclonal cell-cell contact Treg suppression assay: CFSE-labeled Tconv (4?10.sup.4 per well), used as responder cells, are cultured with ?CD3-feeder (4?10.sup.4 per well) in the presence or absence of defined amounts of Foxp3 T cells (blood Treg or ex vivo generated T cells) for 4 to 5 d. Cultures are performed in round-bottom plates coated with 0.2 ?g/mL anti-CD3 mAb in 200 ?L of complete RPMI medium. Results are expressed as the percentage of proliferating CFSE low T cells or as a percentage of suppression calculated as follows: (100?[(percentage of Tconv CFSE low cells?percentage of Tconv CFSE low in coculture with nTregs)/percentage of Tconv CSFE low cells].

[0387] Autologous MLR suppression assay: CFSE-labeled Tconv CD4.sup.+CD25.sup.? T cells (5?10.sup.4) are stimulated either with 1?10.sup.4 pulsed iDC in RPMI-5 media or with 5?10.sup.3 pulsed mDC in IMDM-5 media supplemented with IL-2 (20 IU/ml) IL-1b (10 ng/ml), IL-6 (30 ng/ml), IL-21 (50 ng/ml) and IL-23 (30 ng/ml) in the presence or absence of defined amounts of Foxp3 T cells (blood Treg or ex vivo generated T cells) for 5 to 6 d. When indicated, culture is performed in IMDM-5 media supplemented with IL-2 (20 IU/ml) IL-1? (10 ng/ml), IL-6 (30 ng/ml), IL-21 (50 ng/ml) and IL-23 (30 ng/ml). Results are expressed as the percentage of proliferating CFSE low T cells or as a percentage of suppression calculated as follows: (100?[(percentage of Tconv CFSE low cells?percentage of Tconv CFSE low in coculture with nTregs)/percentage of Tconv CSFE low cells. Classical 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay: target cells were labeled with CFSE as described above and placed at 3?10.sup.4 per well in 96-well U-bottomed plates in triplicate. CD8.sup.+ effector T cells (5:1 E:T ratio) were added, and incubation was carried out at 37? C. for 6 hours. At the end of the experiment, dead cells were labeled with 7-AAD to detect lysed cells. Cytolytic activity against target cells was analyzed based on regions showing double-positive staining CFSE and 7-AAD, using a FACSCalibur instrument. CD8.sup.+ T cell clone cytolytic activity (%) was calculated as cells positive for both CFSE and 7-AAD/total CFSE positive cells, after subtracting the spontaneous lysis (%) in negative control. The percentage of cytolytic activity was then calculated using the following equation: Cytolytic activity (%) [dead target cells (%)-spontaneous death (%)]?100/[100-spontaneous death (%)].

[0388] Measurement of DNA methylation: Classically, a stable Treg genetic signature consisted of highly demethylated CpG islands within the conserved non-coding sequence 2 (CNS2) of the Treg specific demethylation region (TSDR). DNA methylation analysis of the TSDR region of the gene FOXP3 was evaluated by quantitative PCR after bisulfite treatment of genomic DNA as previously described by Christopher Fuhrman (Fuhrman et al, Divergent Phenotypes of Human Regulatory T Cells Expressing the Receptors TIGIT and CD226, 2015, Journal of immunology). Briefly Nucleotides were isolated with AllPrep DNA/RNA Mini Kit (Qiagen) or DNeasy tissue kit (Qiagen), as appropriate. Bisulfite treatment of genomic DNA was performed on 500 ng DNA with the EZ DNA Methylation Kit (Zymo Research). DNA standards originated from unmethylated bisulfite-converted human EpiTect control DNA (Qiagen) or universally methylated bisulfite-converted human control DNA (Zymo Research). To obtain a large quantity of standard, the TSDR was PCR-amplified using the following reaction: 50 ?l reaction volume containing 25 ?l of ZymoTaq PreMix buffer (Zymo Research) and 0.5 ?M each of the primers FOXP3_TSDRfwd (5-ATATTTTTAGATAGGGATATGGAGATGATTTGTTTGG-3 SEQ ID NO: 1) and FOXP3_TSDRrev (5-AATAAACATCACCTACCACATCCACCAACAC-3-SEQ ID NO: 2). After incubation at 95? C. for 10 min, amplification was performed as follows: 50 cycles at 95? C. for 30 s, 55? C. for 30 s, and 72? C. for 1 min. Amplified PCR products were purified with the QIAquick Gel Extraction Kit (Qiagen). The concentration of purified control TSDR DNA was determined with a GE NanoVue spectrophotometer (GE Healthcare Life Sciences). TSDR real-time PCR was performed with probes that targeted methylated or demethylated target sequences. The reaction was performed in 96-well white trays with a Roche LightCycler 480 system (Roche Diagnostics). Each reaction contained 10 ?l LightCycler 480 Probes Master Mix (Roche), 10 ng of bisulfite converted DNA sample or standards, 1 ?M of each primer, and 150 nM of each probe with a final reaction value of 20 ?l. The probes used for amplification were TSDR-Forward 5-GGTTTGTATTTGGGTTTTGTTGTTATAGT-3 (SEQ ID NO: 3) and TSDR-Reverse 5-CTATAAAATAAAATATCTACCCTCTTCTCTTCCT-3 (SEQ ID NO: 4). The probes for target sequence detection were FAM-labeled methylated probe, FAM-CGGTCGGATGCGTC-MGB-NFQ (SEQ ID NO: 5), or VIC-labeled unmethylated probe, VIC-TGGTGGTTGGATGTGTTG-MGB-NFQ (SEQ ID NO: 6). All samples were tested in triplicate. The protocol for real-time amplification is as follows: after initial denaturation at 95? C. for 10 min, the samples were subjected to 50 cycles at 95? C. for 15 s and at 61? C. for 1 min. Fourteen different ratios of fully methylated and demethylated template were used as real-time standards. A six-order polynomial equation was used to extrapolate the percentage of cells demethylated at the TSDR for each sample.

[0389] Measurement of histone acetylation: Histone acetylation analysis of the four different sites of FOXP3 gene was evaluated by ChIP assay, as previously described by Ling Lu (Ling Lu et al, PNAS 2014). Briefly, 50,000 cells of each treated nTreg cell sample were harvested and cross-linked with 1% formaldehyde, and then lysed with 120 ?L of lysis buffer [50 mM Tris.HCl, pH 8.0, 10 mM EDTA, 1% (wt/vol) SDS, protease inhibitor mix (1:100 dilution; Sigma), 1 mM PMSF, 20 mM Na-butyrate]. The chromatin in the lysate was sonicated to 500-800-bp fragments and then diluted with 800 ?L of RIPA ChIP buffer [10 mM Tris.HCl, pH 7.5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% (vol/vol) Triton X-100, 0.1% (wt/vol) SDS, 0.1% (wt/vol) Na-deoxycholate, protease inhibitor mix (1:100 dilution; Sigma), 1 mM PMSF, and 20 mM Na-butyrate]. Dynabeads protein G (10 ?L; Invitrogen) was incubated with1 ?g of H3K4me3 (Abcam) or H3K9ac (Cell Signaling) or normal rabbit IgG negative control ChIP-grade antibodies for 2 h separately. Then, 100 ?L of the sheared chromatin was immunoprecipitated with pretreated antibodybead complexes and another 100 ?L of the sheared chromatin for total input DNA extraction separately. Immunoprecipitated DNA was quantified by real-time PCR with following primers: promoter, 5-ACC GTA CAG CGT GGT TTT TC-3 (SEQ ID NO: 7) and 5-CTA CCT CCC TGC CAT CTC CT-3 (SEQ ID NO: 8); CNS1, 5-CCC AAG CCC TAT GTG TGATT-3 (SEQ ID NO: 9) and 5-GTG TGT CAG GCC TTG TGC TA-3 (SEQ ID NO: 10) ; CNS2, 5-GTC CTC TCC ACAACC CAA GA-3 (SEQ ID NO: 11) and 5-GAC ACC ACG GAG GAA GAG AA-3 (SEQ ID NO: 12); and CNS3, 5-AGG TGC CGA CCT TTA CTG TG-3 (SEQ ID NO: 13) and 5-ACA ATA CGG CCT CCT CCT CT-3 (SEQ ID NO: 14).

[0390] Results

[0391] a) Induction of Foxp3.sup.+ Expression in Ex Vivo Human Induced Tumor-Antigen Specific CD4.sup.+ TCR?? Unrestricted T Cells.

[0392] Optimal conditions are set up for inducing tumor-antigen specific FOXP3.sup.+ expressing CD4.sup.+ TCR?? unrestricted T cells, as described before. FIG. 2 shows that apoptotic tumor antigen -pulsed tolerogenic DCs (tumor Ag loaded tDC), in presence of IL-2 and the nTreg polarizing medium composed of TGF?, PGE2 and rapamycin are able to induce high levels of Foxp3.sup.+ expression (in frequency in FIG. 2A and in MFI in FIG. 2B) in antigen specific stimulated naive conventional CD4.sup.+ T cells (Na?ve Treg), while non-pulsed tDCs (unloaded tDC), in presence of the same polarizing medium, are unable to induce Foxp3+expression in naive conventional CD4.sup.+ T cells.

[0393] b) Specific Recruitment of Pathogenic CD4.sup.+ ?? T Cells Expressing Foxp3 in Human Breast Cancer.

[0394] A novel ?? T cells' subset exhibiting CD4 and Foxp3 expression has been identified in the TIL isolated from breast cancer (BC) biopsies. Indeed, while ?? T cells expression Foxp3 are rare in PBMCs from normal individuals (<1%), they are strongly enriched in the TIL purified from BC biopsies (around 10-fold) as shown in FIG. 3.

[0395] As generally human ??T cells are divided into two major structural subsets according to their TCR ? chain usage: V?1 and V?2 T cells, V?1 being the predominant tissue resident cells whereas V?2 the major subset in peripheral blood, we have investigated the TCR V? chain in this new CD4.sup.+ Foxp3.sup.+ ??T subset by flow cytometry. FIG. 4 shows that most of the pathogenic CD4.sup.+ ?? T cells expressing Foxp3 (>85%) are V?1negV?2neg.

[0396] We have next evaluated the functional suppressive capacity of the Foxp3 expressing CD3.sup.+ CD4.sup.+ ?? T cells unrestricted T cells isolated from BC biopsies. FIG. 5 shows that, similar to fresh Treg, these CD4.sup.+ ?? T cells expressing FOXP3.sup.+ display suppressive activity when using the standard polyclonal cell-cell contact Treg suppression assay.

[0397] c) Induction of Autologous CD8-Mediated T-Cell Responses Against Tumor-Antigen Specific FOXP3 Expressing CD4.sup.+ TCR?? Unrestricted T Cells.

[0398] A culture system is established in which inflammatory DC (inf DC) loaded with apoptotic pathogenic CD4.sup.+ T cells cocultured with autologous CD3.sup.+ na?ve T cells are able to induce the generation of CD8.sup.+ T-cell lines against pathogenic CD4.sup.+ T cells used to load the dendritic cells. FIG. 6 shows that the two CD8.sup.+ clones induced with apoptotic pathogenic CD4.sup.+ T cells loadedinf DC (mDC) or -TAP-inhibited DC respectively are able to lyse their specific targets, their inducing pathogenic CD4.sup.+ T cell clone. However, when both CD8.sup.+ clones are tested against an autologous EBV cell line, they are unable to lyse this target (FIG. 6).

[0399] d) Presence of FOXP3.sup.+ Expressing T Cells in Tumor Infiltrating Lymphocytes (TILs) Isolated from Luminal B Breast Cancer.

[0400] Luminal A and B subtypes are both estrogen-receptor-positive (ER+) and low-grade, with luminal A tumors growing very slowly and luminal B tumors growing more quickly. Luminal A tumors have the best prognosis. Luminal B tumors are associated with a poor clinical outcome. We examined by flow cytometry the phenotype of lymphocytes in the TIL isolated from both luminal subtypes breast cancer and found the presence of Foxp3 expression in CD3.sup.+ CD4.sup.+ TCR??.sup.+ MHCII restricted and CD3.sup.+ CD4.sup.+ TCR??.sup.+ unrestricted T cells. No Foxp3 was detected in TILs extracted from luminal A breast tumor (FIG. 7). Moreover, a positive correlation is observed between a high percentage of Foxp3 expression in CD3.sup.? CD4.sup.+ TCR??.sup.+ unrestricted T cells and a poor clinical outcome in breast cancer (FIG. 8).

[0401] Foxp3 expressing CD3.sup.+ CD4.sup.+ TCR??.sup.+ MHCII restricted T cells and Foxp3 expressing CD3.sup.+ TCR??.sup.+ unrestricted T cells represent approximately 20% of the CD3.sup.+ TCR?? T cells and 23% of the CD3.sup.+ TCR??.sup.+ respectively in the studied sample. Foxp3 expressing CD3.sup.+ TCR??.sup.+ T cells present a same phenotypic profile as Foxp3.sup.+ CD3.sup.+ TCR??.sup.+ T cells. These Foxp3.sup.+ TCR??.sup.? T cell population express levels of Foxp3, CD25 and CTLA4 similar to those of Foxp3.sup.| CD3.sup.| TCR??.sup.? T cells (FIG. 9).

[0402] d) Ex Vivo Generation and Expansion of Specific CD3.sup.+ TCR??.sup.+ Expressing Foxp3 Committed to Exclusively Exert Regulatory Activity.

[0403] As studies suggested that the suppressive potential of antigen-specific Treg was much greater than that of polyclonal Treg, we set up a method to ex vivo generated and expanded antigen specific Foxp3 expressing CD3.sup.| TCR??.sup.| unrestricted T cells, committed to exclusively exert regulatory activity, whichever culture condition of stimulation is.

[0404] FIG. 10 shows that naive CD3+ TCR??+ T cells (CD3.sup.+CD45RA.sup.+ CD27.sup.+ TCR??.sup.+ T cells) stimulated with zoledronic acid-treated-autologous tDCs, in presence of the nTreg polarizing medium comprising the combination of IL-15, IL-2, TGF?, PGE2 and rapamycin, express Foxp3 after 21 days expansion and exhibit significant functional suppressive activity, as assessed by the standard polyclonal cell-cell contact Treg suppression assay. Interestingly the 21-day-expanded FOXP3 expressing CD3.sup.? TCR ??.sup.+ T cells maintain their Foxp3 level and their suppressive activity, after a further 21-day-culture in nTreg polarizing medium.