IMMUNOTHERAPEUTIC USES OF EX VIVO GENERATED FOXP3+ REGULATORY T CELLS

Abstract

The present invention relates to therapeutic uses of ex vivo generated Foxp3+ regulatory T cells. The inventors showed the presence of Foxp3+ expressing T cells in tumor infiltrating lymphocytes (TILs) isolated from luminal-B breast cancer. The inventors performed an ex vivo generation and expansion of specific CD3+ TCRy8+ expressing Foxp3: CD3+ TCRy8+ T cells maintain their Foxp3 level and their suppressive activity, after a further 21-day-culture. They also showed that tumor Ag-specific CD3+ TCR Va24+ T cells maintain their ability to perform suppressive function in pro-inflammatory conditions. In particular, the present invention relates to immunotherapeutic uses of at least one of ex vivo generated Foxp3+regulatory T cells population selected among a MHCII restricted CD4+Foxp3+regulatory T cells population, a y8 Foxp3+regulatory T cells population and an invariant Foxp3+regulatory T cells population.

Claims

1. An immunogenic product, a pharmaceutical composition or a vaccine composition comprising at least one inactivated ex vivo generated Foxp3.sup.+ regulatory T cell population selected from the group consisting of a MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cell population, a Foxp3.sup.+ regulatory T cell population and an invariant Foxp3.sup.+ regulatory T cell population.

2. The pharmaceutical composition comprising of claim 1, further comprising at least one pharmaceutically acceptable excipient.

3. The vaccine composition of claim 1, further comprising at least one adjuvant.

4. The pharmaceutical composition of claim 2, wherein said at least one ex vivo generated regulatory T cells population remains stable when placed in inflammatory condition.

5. A method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an immunogenic product, pharmaceutical composition or vaccine composition according to claim 1.

6. A method for preparing the immunogenic product, pharmaceutical composition or vaccine composition according to claim 1, comprising: identifying from a tumor sample obtained from a subject at least one overrepresented regulatory T cell population selected from the group consisting of a MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cell population, a Foxp3.sup.+ regulatory T Bells cell population and an invariant Foxp3.sup.+ regulatory T cell population, ex vivo generating the at least one overrepresented regulatory T cell population, and inactivating the at least one ex vivo generated regulatory T cell population.

7. A method of performing cell therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to claim 4.

8. A method of treating inflammatory or autoimmune diseases or for preventing transplant rejection or graft versus host disease (GVHD) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition according to claim 4.

9. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 1, wherein the at least one inactivated ex vivo generated regulatory T cell population is obtained or expanded by a method comprising: for the MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cell population: culturing CD3.sup.+ CD4.sup.+ CD25.sup.+ T cells in the presence of a TCR cell activator and the following agents: i) a 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, for at least 5 days; for the Foxp3.sup.+ regulatory T cell population: culturing CD3.sup.+ TCR.sup.+ T cells in the presence of a T cell activator and the following agents: i) a 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 from the group consisting of IL-2, IL-7, IL-15 and TSLP, for at least 5 days; for the invariant Foxp3.sup.+ regulatory T cell population: culturing CD3.sup.+ V24.sup.+ T cells in the presence of an invariant T cell activator and the following agents: i) a 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 from the group consisting of IL-2, IL-7, IL-15 and TSLP, for at least 5 days.

10. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 9, wherein the TCR cell activator is a polyclonal TCR cell activator; the T cell activator is a polyclonal T cell activator; and the invariant T cell activator is a polyclonal invariant T cell activator.

11. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 9, wherein the TCR cell activator is an antigen-specific TCR cell activator; the T cell activator is an antigen-specific T cell activator, and the invariant T cell activator is an antigen-specific invariant T cell activator.

12. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 9, wherein the cAMP activator is prostaglandin E2 (PGE2), an EP2 or EP4 agonist, a membrane adenine cyclase activator or a metabotropic glutamate receptor agonist.

13. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 9, wherein the TGF pathway activator is TGF, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-mullerian hormone (AMH), activin or nodal.

14. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 9, wherein the mTOR inhibitor is rapamycin, a rapamycin analog, 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 or WJDO08.

15. (canceled)

16. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 10, wherein the polyclonal TCRP cell activator is an anti-CD3 antibody or an anti-TCR antibody.

17. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 10, wherein the polyclonal T cell activator is an anti-TCR antibody or a non peptide phosphoantigen.

18. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 10, wherein the polyclonal invariant T cell activator is a V24 activator.

19. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 11, wherein the antigen-specific TCR cell activator is tolerogenic dendritic cells (DCs) pulsed with at least one self-peptide antigen.

20. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 11, wherein the antigen-specific T cell activator is tolerogenic dendritic cells (DCs) pulsed with at least one bisphosphonate.

21. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 20, wherein the at least one bisphosphonate is at least one aminobisphosphonate.

22. The immunogenic product, pharmaceutical composition or vaccine composition according to claim 11, wherein the antigen-specific invariant T cell activator is tolerogenic dendritic cells (DCs) expressing CD1 and pulsed with at least one non peptide lipid antigen.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0445] 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.

[0446] FIG. 2: 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. Representation of the Foxp3 expression level by the MFI in the CD3.sup.+ CD4.sup.+ TCR.sup.+ restricted T cells and in the CD3.sup.+ CD4.sup.+ TCR.sup.+ unrestricted T cells.

[0447] FIG. 3: Positive correlation of Foxp3.sup.+ expression in lymphocytes present in the TILs and a poor clinical outcome in breast cancer. 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.+ restricted T cells and in the CD3.sup.+ CD4.sup.+ TCR.sup.+ unrestricted T cells.

[0448] FIG. 4: 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.

[0449] FIG. 5: 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.

[0450] FIG. 6: In vitro induction of tumor-Ag specific CD3.sup.+ TCR V24.sup.+ CD1-restricted T cells (invTreg) from stimulated naive CD3.sup.+ TCR V24.sup.+ T cells with different nTreg polarizing medium. Naive CD3.sup.+ TCR V24.sup.+ T cells were stimulated for 21 days with tumorapoptotic breast tumor cell line pulsed autologous tDC as described in FIG. 3 in presence of IL-2 (100 IU/ml) and IL-15 (10 ng/ml). Where indicated, TGF, RAPA and PGE2 were added. (A) Overlay histogram displaying Foxp3 expression profiles of each of the generated invTreg. (B) Frequency and (C) expression level (evaluated by MFI) of Foxp3 in CD3.sup.+ T cell culture. Dashed black line represents in (B) and (C) the frequency and the expression level of FOXP3 in naive Treg phenotypically defined by the expression a high level of CD45RA and CD25 and a low level of CD127.

[0451] FIG. 7: Combination of TGF, RAPA and PGE2 induce the establishment and the expansion of tumor Antigen specific FOXP3.sup.+ CD3.sup.+ TCR V24.sup.+ CD1-restricted T cell cells committed to exclusively exert regulatory activity, with an autologous MLR assay. CD3.sup.+ TCR V24.sup.+ CD45RA.sup.+ T cells were stimulated with autologous tolerogenic DC pulsed with apoptotic breast tumor cell lines in presence of IL-2, IL-15 and nTreg polarizing medium. After 21 days of in vitro expansion in nTreg polarizing medium, suppressive capacity of ex vivo generated Tumor Ag-specific invariant Foxp3.sup.+ Treg was evaluated in the presence of (A) a low or (B) high inflammatory medium. Fresh nave Treg were used as control.

[0452] FIG. 8: Analysis of Foxp3.sup.+ expression in human MHCII restricted CD4.sup.+ Foxp3.sup.+ CD4.sup.+ regulatory T cells (Treg) generated ex vivo from polyclonally stimulated naive CD4.sup.+ T cells with different nTreg polarizing medium. Naive CD4.sup.+ T cells were stimulated for 12 days with plate-bound anti-CD3 (4 g/ml) in presence of IL-2 (100 IU/ml). Where indicated, TGF (5 ng/ml), RAPA (10 nM) and PGE2 (1 M) were added. (A) Overlay histogram displaying Foxp3 expression profiles of each of the generated pTreg. (B) Frequency and (C) expression level (evaluated by MFI) of Foxp3 in CD4.sup.+ T cell culture.

[0453] FIG. 9: Comparative analysis of in vitro suppressive capacity of human Treg generated with different nTreg polarizing medium. Suppressive capacity of ex vivo generated Treg was evaluated (A) in quiescent and (B) in inflammatory context with the standard polyclonal nTreg assay. CFSE-labeled conventional T cells (Tconv) were cocultured with ex vivo generated Treg at different ratio. Percent inhibition of TconvCFSE proliferation by Treg was depicted. Fresh Treg and Tconv were used as control.

[0454] FIG. 10: Combination of TGF, RAPA and PGE2 induce the establishment and the expansion of cultured Treg committed to exclusively exert regulatory activity. After 21 days of ex vivo generation in nTreg or TH-17 polarizing medium, suppressive capacity of ex vivo generated OVA-specific Treg was evaluated in the presence of a high inflammatory context inducing medium. Fresh Treg were used as control.

[0455] FIG. 11: IL-17 production by stimulated OVA-ex vivo generated Treg. Specific-Treg (A) induced after the first 21 days of culture in nTreg polarizing medium or (B) expanded for 3 weeks in nTreg or TH-17 polarizing medium were tested for their IL-17-producing capacity upon stimulation with aCD3 Ab and aCD28 Ab for 2 days in IMDM medium containing IL-2, IL-1, IL-6, IL-21, and IL-23 cytokines. IL-17 was detected in supernatant culture by ELISA.

EXAMPLES

[0456] The present invention is further illustrated by the following examples.

[0457] Materials and Methods

[0458] Human Blood Sample.

[0459] Blood samples from healthy individuals originated from Etablissement Francais du Sang (EFS, Paris). Blood cells are collected using standard procedures.

[0460] Human Tumor Sample.

[0461] Tumor tissue sample originated from patient with luminal A and Luminal B Breast cancer (Institut Jean Godinot, Reims).

[0462] Cell Purification and Culture.

[0463] 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).

[0464] 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%. Subsequently, 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).

[0465] CD14.sup.+ monocytes are isolated from PBMCs by positive selection using a MACS system.

[0466] 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).

[0467] CD3.sup.+ CD45RA.sup.+ invTCR V24.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 V24.sup.+ T cells are stained with anti-CD3 (UCHT-1) V450 anti-invariant TCR V24-JQ (6B11)-PE (inv TCR V24-JQ (Becton Dickinson) and anti-CD45RA (T6D11)-FITC (Miltenyi Biotec).

[0468] 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).

[0469] 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%.

[0470] 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.

[0471] TIL Isolation.

[0472] 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.

[0473] Ex Vivo Generation of Polyclonal Functionally Committed FOXP3 Expressing Regulatory T Cells.

[0474] Ex Vivo Generation of Polyclonal Functionally Committed FOXP3 Expressing CD3.sup.+ TCR.sup.+ MHCII Restricted T Cells:

[0475] On day 0, T cells are seeded at 2.510.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, TGF5 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, TGF5 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.+.

[0476] Ex Vivo Generation of Polyclonal Functionally Committed FOXP3 Expressing Invariant T Cells:

[0477] On day 0, T cells are seeded at 110.sup.3/well in 96-well plates and stimulated with plate-bound anti-inv TCR V24-JQ (6B11) mAb (2 g/ml) in the presence of CD3-feeder (2.510.sup.5). Cells are cultured in IMDM-5 media with PGE2 1 M, TGF5 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 V24-JQ (6B11) mAb (2 g/ml) in the presence of CD3-feeder, PGE2 1 M, TGF5 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 V24.sup.+ RA.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.

[0478] Ex Vivo Generation of Polyclonal Functionally Committed FOXP3 Expressing TCR.sup.+ T Cells:

[0479] On day 0, T cells are seeded at 110.sup.3/well in 96-well plates and stimulated with plate-bound anti-TCR mAb (2 g/ml) in the presence of CD3-feeder (2.510.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, TGF5 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, TGF5 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.+.

[0480] Ex Vivo Generation of Antigen Specific Functionally Committed FOXP3 Expressing T Cells:

[0481] Ex Vivo Generation of Antigen (Ovalbumun) Specific Functionally Committed Foxp3 Expressing CD3.sup.+ TCR.sup.+ MHCII Restricted T Cells: [0482] 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 310.sup.5/ml in IMDM-5 and 3) pulsed in IMDM-5 with specific Ag (OVA). [0483] 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 310.sup.5/ml in IMDM-5 in the presence of 210.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, TGF5 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.

[0484] Ex Vivo Generation of Tumor-Antigen Specific Functionally Committed FOXP3 Expressing CD3.sup.+ invTCR V24.sup.+ CD1d-Restricted T Cells: [0485] 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 210.sup.6/per vial vial-in 90% FBS 10% DMSO. [0486] b) Ex vivo generation and expansion of tumor-antigen specific functionally committed Foxp3 expressing CD3.sup.+ invTCR V24.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 310.sup.5/ml in IMDM-5 in the presence of 210.sup.5 irradiated autologous feeders, PGE2 1 M, and Rapa 10 nM. Purified CD3.sup.+ CD45RA.sup.+ invTCR V24.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, TGF5 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 V24.sup.+ cells that became CD45RO.sup.+ express Foxp3.sup.+.

[0487] Ex Vivo Generation of Phospho-Antigen Specific Functionally Committed FOXP3 Expressing CD3.sup.+ TCR.sup.+ Unrestricted T Cells: [0488] 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 granulocyte macrophage 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. [0489] 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 310.sup.5/ml in IMDM-5 in the presence of 210.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, TGF5 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.+.

[0490] In Vitro Generation of Stimulator Cells for MLR Assay:

[0491] 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 210.sup.6/per vialin 90% FBS10% 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.

[0492] IL-17 Detection by ELISA.

[0493] The presence of IL-17 in the culture supernatant is measured by ELISA. The recognition of IL-17 by an anti-IL-17 antibody may be carried out by conventional methods known in the art such as a sandwich ELISA anti-IL-17. The ELISA is developed by any colorimetric means known in the art such as for example using a detection antibody labelled with biotin, a poly-streptavidin HRP amplification system and an o-phenylenediamine dihydrochloride substrate solution.

[0494] One example of said method is the following: [0495] coating a plate with the capture antibody, such as for example an anti-IL17 antibody, [0496] blocking the plate with a blocking buffer (such as, for example, casein 2% in PBS) during 90 min at 37 C., [0497] incubating the plate during 90 min at 37 C. with a dilution series of IL-17 standard, samples or negative controls, [0498] incubating the plate 90 min at 37 C. with the detection antibody such as for example a biotinylated anti-IL-17 antibody, [0499] incubating the plate with streptavidin-HRP during 30 min at 37 C. and developing the complex with an o-phenylenediamine dihydrochloride (OPD) substrate solution during 30 min. After stopping the enzymatic reaction, the intensity of the resulting color is determined by spectrophotometric methods at 490 nm.

[0500] The person skilled in the art considers that an IL-17 level inferior to 200 ng/ml, 100 ng/ml, 50 ng/ml corresponds to no secretion or low secretion of IL-17 after calculation with the standard curve.

[0501] Flow Cytometry Analysis.

[0502] Mab Labeling.

[0503] The following conjugated mAbs are used. 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 V24-JQ (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 (0323) (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% NaN.sub.3) (FACS buffer). Foxp3 and CTLA-4 intracellular staining 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).

[0504] CFSE Staining.

[0505] 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 110.sup.7 cells/mL. The labeling are 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.

[0506] 7-AAD (7-Amino-Actinomycin D) Staining.

[0507] 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.sup.+7-AAD.sup. FSC.sup.+ cells.

[0508] Functional Assays.

[0509] T-Cell Proliferation.

[0510] 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, contained 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.

[0511] Standard Polyclonal Cell-Cell Contact Treg Suppression Assay:

[0512] CFSE-labeled Tconv (410.sup.4 per well), used as responder cells, are cultured with CD3-feeder (410.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.

[0513] Autologous MLR Suppression Assay:

[0514] CFSE-labeled Tconv CD4.sup.+ CD25.sup. T cells (510.sup.4) are stimulated either with 110.sup.4 pulsed iDC in RPMI-5 media or with 510.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.

[0515] Measurement of DNA Methylation:

[0516] 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.

[0517] Measurement of Histone Acetylation:

[0518] 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 TrisHCl, 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 TrisHCl, 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 with 1 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 antibody-bead 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).

[0519] Results

[0520] a) Presence of FOXP3.sup.+ Expressing T Cells in Tumor Infiltrating Lymphocytes (TILs) Isolated from Luminal-B Breast Cancer.

[0521] 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. 2). Moreover, a positive correlation is observed between a high percentage of Foxp3 expression in CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells and in CD3.sup.+ CD4.sup.+ TCR.sup.+ unrestricted T cells, and a poor clinical outcome in breast cancer (FIG. 3).

[0522] 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(3.sup.+ T cells (FIG. 4).

[0523] b) Ex Vivo Generation and Expansion of Specific CD3.sup.+ TCR.sup.+ Expressing Foxp3 Committed to Exclusively Exert Regulatory Activity.

[0524] 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.

[0525] FIG. 5 shows that naive CD3.sup.+ TCR.sup.+ 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.

[0526] c) Optimal Conditions for Inducing Foxp3 Expression in Invariant Tcells

[0527] Starting from naive CD3.sup.+ invTCR V24.sup.+ T cells isolated from human PBMCs, different nTreg polarizing medium were assessed for their capacity to induce the expression the differentiation of Foxp3.sup.+ cells with suppressive function.

[0528] FIG. 6 shows that cultured naive CD3.sup.+ invTCR V24.sup.+ T cells exhibit a variable level of Foxp3 dependent on their culture condition of stimulation. Polarizing medium comprising the combination of IL-2, TGF, PGE2 and rapamycin results in a higher Foxp3 expression over combinations of IL-2, TGF and rapamycin, IL-2 and PGE2, or IL-2 alone. Moreover, the combination of IL-2, TGF, PGE2 and rapamycin results in an optimal intensity of Foxp3 expression in the invTCR V24.sup.+ T cells, as compared to the other combinations.

[0529] Furthermore, it is interesting to note that only naive CD3.sup.+ invTCR V24.sup.+ T cells stimulated with the polarizing medium comprising the combination of IL-2, TGF, PGE2 and rapamycin express level and intensity of Foxp3 similar or higher to those of blood nave regulatory T cells (CD3.sup.+ TCR CD4.sup.+ CD127.sup./low CD45RA.sup.+ CD25.sup.+), corresponding to our positive control.

[0530] d) Tumor Ag-Specific CD3.sup.+ TCR V24.sup.+ T Cells Maintain their Ability to Perform Suppressive Function in Pro-Inflammatory Conditions

[0531] FIG. 7A shows that tumor Ag-specific memory invTCR V24.sup.+ T cells ex vivo generated and expanded in the presence of the nTreg polarizing medium above described are endowed of a higher suppressive activity than fresh Foxp3 expressing CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells when using an autologous MLR coculture assay.

[0532] Furthermore, FIG. 7B shows that these tumor Ag-specific invTCR V24.sup.+ T cells still maintain their suppressive activity, when the autologous MLR coculture assay are performed in presence of a high inflammatory medium containing IL-2 IL-1 IL-6, IL-21 IL-23 cytokines, while fresh Foxp3 expressing CD3.sup.+ CD4.sup.+ TCR(3.sup.+ MHCII restricted T cells lose their suppressive activity.

[0533] e) Optimal Conditions for Inducing Foxp3 Expression in Naive CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII Restricted T Following Polyclonal and Antigen-Specific Activation.

[0534] Starting from naive conventional CD4.sup.+ T cells (CD3.sup.+ CD4.sup.+ CD127.sup.+ CD45RA.sup.+ CD25.sup. TCR(3.sup.+ MHCII restricted) isolated from human PBMCs, different nTreg polarizing medium were assessed for their capacity to induce the differentiation of Foxp3.sup.+ cells with suppressive function.

[0535] FIG. 8 shows that, when ex vivo activated polyclonally with anti-CD3 mAbs, naive conventional CD4.sup.+ T cells exhibit a variable level of Foxp3 dependent on their culture condition of stimulation. Polarizing medium comprising the combination of IL-2, TGF and rapamycin or IL-2, TGF, rapamycin and PGE2 results in a higher Foxp3 expression over combinations of IL-2 and PGE2, or IL-2 alone (B). Moreover, the combination of IL-2, TGF, rapamycin and PGE2 results in an optimal intensity of Foxp3 expression in the CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells, as compared to the other combinations (C).

[0536] It is interesting to note that only naive conventional CD4.sup.+ T cells, stimulated with the polarizing medium comprising the combination of IL-2, TGF, PGE2 and rapamycin, express level and intensity of Foxp3 similar or higher to those of blood nave regulatory T cells (CD3.sup.+ TCR(r CD4.sup.+ CD127.sup./low CD45RA.sup.+ CD25.sup.+), corresponding to our positive control.

[0537] We next evaluated the functional suppressive capacity of the Foxp3 expressing CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells polyclonally stimulated. FIG. 9A shows that CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells, ex vivo generated and expanded for 21 days, using polyclonal stimulation, in the presence of the nTreg polarizing medium comprising the combination of IL-2, TGF, PGE2 and rapamycin, display a higher suppressive activity compared with both those generated in the presence of the nTreg polarizing medium comprising the combination of IL-2, TGF, rapamycin without PGE2 and fresh FOXP3 expressing CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells, when using the standard polyclonal cell-cell contact Treg suppression assay. Furthermore, FIG. 9B shows that these 21-day-expanded-FOXP3 expressing CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells still maintain their suppressive activity, when the functional suppressive assay is performed in presence of a highly-inflammatory medium containing IL-2 IL-1 IL-6, IL-21 IL-23 cytokines, while fresh FOXP3 expressing CD3.sup.+ CD4.sup.+ TCR.sup.+ MHCII restricted T cells lose their suppressive capacity under these culture condition of stimulation.

[0538] To confirm that the Ova-specific CD3.sup.+ TCR(3.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 or TH-17 polarizing medium (IMDM medium containing IL-2 IL-1 IL-6, IL-21 IL-23 cytokines) and were tested for 1) their functional suppressive capacity in the presence of a high inflammatory context (FIG. 10) and 2) for their IL-17-producing capacity when stimulated through CD3 and CD28 as described above (FIG. 11). After a further 21-day-culture either in nTreg or TH-17 polarizing medium, Ova-specific CD3.sup.+ TCR.sup.+ MHCII restricted T cells not only still retain, in a high inflammatory context, functional suppressive activity (FIG. 10), but also produce low level of IL-17 (FIG. 11B). By contrast fresh Foxp3 expressing CD3.sup.+ TCR.sup.+ MHCII restricted T cells lose their suppressive function while producing IL-17 in this inflammatory context.