Ex vivo generation of MHCII restricted CD4.SUP.+ .FOXP3.SUP.+ .regulatory T cells and therapeutic uses thereof

Abstract

The present invention relates to a method for ex vivo generating and expanding MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cells, and therapeutic uses thereof. The inventors here demonstrated the optimal conditions for inducing Foxp3 expression in naive CD3+ CD4+ TCRαβ+ MHCII restricted T following polyclonal or following antigen-specific activation. They also developed an experimental procedure to generate autologous CD8+ T cell lines functionally committed to lyse tumor-antigen specific FOXP3 expressing TCRαβ+ MHCII restricted T cells, pathogenic CD4+ T cells that favour tumor cell immune evasion. In particular, the present invention relates to a method for generating ex vivo MHCII restricted CD4+ Foxp3+ regulatory T cells having the following phenotype: CD3+ CD4+ Foxp3+.

Claims

1. A method for generating ex vivo MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cells having the following phenotype: CD3.sup.+ CD4.sup.+ Foxp3.sup.+ comprising culturing in a first culture medium CD3.sup.+ CD4.sup.+ CD25.sup.− T cells in the presence of a TCRαβ cell activator and adding regulatory T cell differentiation agents to the first culture medium, wherein said regulatory T cell differentiation agents consist of: i) PGE2, ii) TGFβ (Transforming growth factor beta), iii) rapamycin and iv) IL-2, for at least 5 days, and expanding in a second culture medium the MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cells in the presence of the TCRαβ cell activator and adding regulatory T cell differentiation agents to the second culture medium, wherein said regulatory T cell differentiation agents consist of: i) PGE2, ii) TGFβ (Transforming growth factor beta), iii), rapamycin and iv) IL-2, for at least 5 days; wherein the TCRαβ cell activator is a polyclonal TCR αβ cell activator or an antigen-specific TCRαβ cell activator, with the caveat that the TCRαβ cell activator is not tolerogenic dendritic cells (DCs) pulsed with at least one self-peptide antigen; and wherein said expanding step produces MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cells that remain stable when placed in inflammatory conditions.

2. The method according to claim 1, wherein the polyclonal TCRαβ cell activator is an anti-CD3 antibody or an anti-TCRαβ antibody.

3. The method of claim 1, wherein the MHCII restricted CD4.sup.+ Foxp3.sup.+ regulatory T cells produced in said expanding step lack IL-1R1 expression.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) 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.

(2) FIG. 2: 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 μg/ml) in presence of IL-2 (100 IU/ml). Where indicated, TGFβ (5 ng/ml), RAPA (10 nM) and PGE2 (1 μM) 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.

(3) FIG. 3: 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.

(4) FIG. 4: Phenotype of ex vivo generated Ag-specific Treg after 21 days of culture. Naive CD4.sup.+ T cell were stimulated with (A) non-pulsed autologous tDCs or (B) with OVA-pulsed autologous tDCs, in presence of IL-2 and defined nTreg polarizing medium. Stimulated CD4.sup.+ T cells were stained at the cell surface using Abs directed against CD45RA, CD25, CD26, CD39. After fixation and permeabilization Foxp3 and CTLA4 were stained intracellularly.

(5) FIG. 5: Contamination of ex vivo generated OVA-specific Treg with unstimulated naive CD4+ T cells. Cells were stained with CD45RA, Foxp3, CTL14, CD26 and CD25.

(6) FIG. 6: CD154 expression analysis on nave CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ T cells primed with Ova pulsed tDC and Foxp3 expression in expanded Ova specific generated Treg. A-Frequency of CD154.sup.+ expression among primed nave CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ T cells, 16 h after their stimulation with either unpulsed tDC or Ova pulsed as described in Material and Methods. B-Foxp3 expression in ex vivo generated Ag-specific Treg after 21 days of culture in nTreg polarizing medium.

(7) FIG. 7: Suppressive capacity of ex vivo generated OVA-specific Treg after 21 days of culture evaluated with the standard polyclonal nTreg assay. After magnetic depletion of resting CD4.sup.+ naive T cells, suppressive capacity of expanded pTreg, was evaluated (A) in quiescent and (B) in inflammatory context. CFSE-labeled Tconv (TconvCFSE) were cocultured with ex vivo generated Tregs at different ratios under the indicated polyclonal stimulations. Proliferation of TconvCFSE was evaluated by the CFSE dilution assay. Fresh Treg were used as control.

(8) FIG. 8: Suppressive capacity of ex vivo generated OVA-specific Treg after 21 days of culture evaluated with an autologous MLR assay. After magnetic depletion of CD4.sup.+ naive T cells, suppressive capacity of ex vivo generated Treg, was evaluated (A) in low and (B) high inflammatory context. CFSE-labeled Tconv (TconvCFSE) were cocultured with ex vivo generated Tregs at different ratios under the indicated stimulations. Proliferation of TconvCFSE was evaluated by the CFSE dilution assay and express as proliferation index (IP). Fresh Treg were used as control.

(9) FIG. 9: Combination of TGFβ, 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 as described in FIG. 7. Fresh Treg were used as control.

(10) FIG. 10: 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.

(11) FIG. 11: Analysis of IL-1R1 expression in human MHCII restricted CD4.sup.+ Foxp3.sup.+ CD4.sup.+ regulatory T cells (Treg) ex vivo expanded or in vitro induced with different nTreg polarizing medium from conventional or naive CD4.sup.÷ T cells either after polyclonal or antigen-specific stimulation. Frequency of IL-1R1 expression was evaluated by flow cytometry on the following regulatory T cells population: a) ex vivo resting Tregs isolated from PBMCs, b) ex vivo expanded Tregs from Treg isolated from PBMCs with polyclonal stimulation, c) polyclonal in vitro induced Treg in the presence of Rapa and TGFβ from conventional T cells isolated from PBMCs and d) in vitro induced Ova-specific CD3+ FOXP3.sup.+ T cells in presence of Rapa, TGFβ and PGE2 isolated from nave CD4.sup.+ T cells. We found that IL-1R1 is preferentially expressed on resting, polyclonal expanded/induced Tregs when compared to the induced Ova-specific CD3.sup.+ FOXP3.sup.+ T cells. We also observe that the stability of the suppressive function is inversely correlated with the IL-1R1 expression.

(12) FIG. 12: Analysis of Foxp3.sup.+ expression in ex vivo human induced tumor-antigen specific FOXP3 expressing TCRαβ.sup.+ MHCII restricted T cells. Apoptotic tumor 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 TGFβ (5 ng/ml), PGE2 (1 μM) 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.

(13) FIG. 13: 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 TCRαβ.sup.+ MHCII restricted T cells. The capacity of a CD8.sup.+ T cell clone to lyse its inducing pathogenic CD4+ 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 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 (%)].

(14) FIG. 14: 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.+ TCRαβ.sup.+ restricted T cells.

EXAMPLES

(15) The present invention is further illustrated by the following examples.

(16) Materials and Methods

(17) Human Blood Sample. Blood samples from healthy individuals originated from Etablissement Francais du Sang (EFS, Paris). Blood cells are collected using standard procedures.

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

(19) Cell Purification and Culture.

(20) 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 (ΔCD3 cells) are isolated from either fresh or frozen PBMCs. T cell-depleted accessory cells (ΔCD3 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 ΔCD3-feeder).

(21) CD3.sup.+ T cells are positively selected with a CD3 beads isolation kit (Miltenyi Biotec). Subsequently, selected CD3.sup.+ T cells are labeled with anti-CD3 (SK7)-FITC (Becton Dickinson), anti-CD45RA.sup.+ (REA562)-FITC (Miltenyi Biotec), and anti-CD27(0323)-APC efluor780 (ebioscience) before being sorted into CD3.sup.+ RA.sup.+ CD27.sup.+ T cells.

(22) 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).

(23) CD14.sup.+ monocytes are isolated from PBMCs by positive selection using a MACS system.

(24) CD3.sup.+ CD4.sup.+ CD127.sup.+ CD45RA.sup.+ CD25.sup.− TCRαβ.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 αβ-BV421 (IP26) (Biolegend).

(25) CD3.sup.+ CD45RA.sup.+ invTCR Vα24.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 Vα24.sup.+ T cells are stained with anti-CD3 (UCHT-1) V450 anti-invariant TCR Vα24-JαQ (6B11)-PE (inv TCR Vα24-JαQ (Becton Dickinson) and anti-CD45RA (T6D11)-FITC (Miltenyi Biotec).

(26) CD3.sup.+ CD45RA.sup.+ CD27.sup.+ TCRγδ6.sup.+ unrestricted T cells are isolated from PBMCs after magnetic enrichment (MACS system: TCRγδ.sup.+ T cell isolation kit) and FACS sorting. Before the sorting step, enriched CD3.sup.+ TCRγδ.sup.+ T cells are stained with anti-CD3 (UCHT-1) V450, anti-TCR panγδ.sup.+ PE (IMMU510) (Beckman Coulter), anti-CD27-APC efluor 780 (0323) (ebioscience) and anti-CD45RA (T6D11)-FITC (Miltenyi Biotec).

(27) 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%.

(28) 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 μg/ml Doxorubicin for 24 h or by γ irradiation (20 Gy). Extent of apoptosis is monitored by flow cytometric analysis (FACS). Cells are extensively washed prior to feeding DCs.

(29) 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.

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

(31) Ex vivo generation of polyclonal functionally committed FOXP3 expressing CD3.sup.+ TCRαβ.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 μg/ml) in the presence of ΔCD3-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 μM, TGFβ 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 μg/ml). The restimulations were performed in the presence of ΔCD3-feeder, PGE2 1 μM, TGFβ 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.+.

(32) 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 Vα24-JαQ (6B11) mAb (2 μg/ml) in the presence of ΔCD3-feeder (2.5×10.sup.5). Cells are cultured in IMDM-5 media with PGE2 1 μM, TGFβ5 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 Vα24-JαQ (6B11) mAb (2 μg/ml) in the presence of ΔCD3-feeder, PGE2 1 μM, TGFβ 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 Vα24.sup.+ RA.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.

(33) Ex vivo generation of polyclonal functionally committed FOXP3 expressing TCRγδ.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-TCRγδ mAb (2 μg/ml) in the presence of ΔCD3-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 μM, TGFβ 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 γδ Abs (2 μg/ml). The restimulations were performed in the presence of ΔCD3-feeder, PGE2 1 μM, TGFβ 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.+ TCRγδ.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.

(34) Ex Vivo Generation of Antigen Specific Functionally Committed FOXP3 Expressing T Cells:

(35) Ex vivo generation of antigen (Ovalbumin) specific functionally committed Foxp3 expressing CD3.sup.+ TCRαβ.sup.+ MHCII restricted T cells: 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 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 μl 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). b) Ex vivo generation and expansion of specific functionally committed FOXP3 expressing CD3.sup.+ TCRαβ.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 μM, 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 TGFβ (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 ΔCD3-feeder, PGE2 1 μM, TGFβ 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.+ TCRαβ.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, TGFβ, 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.+ TCRαβ.sup.+ MHCII restricted T cells are stimulated with plate-bound anti-CD3 mAb (4 μg/ml) in the presence of ΔCD3-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.
Ex Vivo Generation of Tumor-Antigen Specific Functionally Committed FOXP3 Expressing CD3.sup.+ TCRαβ.sup.+ MHCII Restricted T Cells: 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 granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4. At day 3,500 μl 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 frozen at 2×10.sup.6/per vial in 90% FBS-10% DMSO. P b) Ex vivo generation and expansion of tumor-antigen specific functionally committed Foxp3 expressing CD3.sup.+ TCRαβ.sup.+ MHCII 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 μM, and Rapa 10 nM. Purified CD3.sup.+ CD45RA.sup.+ TCRαβ.sup.+ MHCII 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) and TGFβ (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 ΔCD3-feeder, PGE2 1 μM, TGFβ 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. 88% of the stimulated naive conventional CD4.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.
Ex Vivo Generation of Tumor-Antigen Specific Functionally Committed FOXP3 Expressing CD3.sup.+ invTCR Vα24.sup.+ CD1d-Restricted T Cells: 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 granulocyte-macrophage 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 μl 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 frozen at 2×10.sup.6/per vial in 90% FBS-10% DMSO. b) Ex vivo generation and expansion of tumor-antigen specific functionally committed Foxp3 expressing CD3.sup.+ invTCR Vα24.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 μM, and Rapa 10 nM. Purified CD3.sup.+ CD45RA.sup.+ invTCR Vα24.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 TGFβ (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 ΔCD3-feeder, PGE2 1 μM, TGFβ 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 Vα24.sup.+ cells that became CD45RO.sup.+ express Foxp3.sup.+.
Ex Vivo Generation of Specific Phospho-Antigen Functionally Committed FOXP3 Expressing CD3.sup.+ TCRγδ.sup.+ Unrestricted T Cells: 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 μl 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, frozen or used for the generation and expansion of phospho-antigen specific functionally committed FOXP3 expressing CD3.sup.+ TCRγδ.sup.+ unrestricted T cells. b) Ex vivo generation and expansion of phospho-antigen specific functionally committed FOXP3 expressing CD3.sup.+ TCRγδ.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 μM, and Rapa 10 nM and zoledronic acid (100 nM). Purified CD3.sup.+ CD45RA.sup.+ TCRγδ.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 TGFβ (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 ΔCD3-feeder, PGE2 1 μM, TGFβ 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.+ TCRγδ.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.
Ex Vivo Generation of Specific Tumor Phospho-Antigen Functionally Committed FOXP3 Expressing CD3.sup.+ TCRγδ.sup.+ Unrestricted T Cells: 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 granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml human recombinant IL-4. At day 3, 500 μl of the medium containing cytokines is 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 frozen at 2×10.sup.6/per vial-in 90% FBS-10% DMSO. b) Ex vivo generation and expansion of tumor-phospho-antigen specific functionally committed Foxp3 expressing CD3.sup.+ TCRγδ.sup.+ unrestricted 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 μM, and Rapa 10 nM. Purified CD3.sup.+ CD45RA.sup.+ TCRγδ.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 TGFβ (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 ΔCD3-feeder, PGE2 1 μM, TGFβ 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.+ TCRγδ.sup.+ T cells that became CD45RO.sup.+ express Foxp3.sup.+.
Ex Vivo Expansion of Treg from Treg Isolated from PBMCs According to Classical Protocol Described in the Literature:

(36) CD4.sup.+ CD127.sup.−/loCD25.sup.high (Tregs) are stimulated with plate-bound anti-CD3 mAb (4 μg/ml), soluble anti-CD28 Ab (4 μg/ml) in the presence of ΔCD3-feeder (1 M) and IL-2 (100 UI/ml) and Rapamycin (100 nM). Cells are cultured in IMDM-5 media.

(37) Ex Vivo Induction and Generation of Treg from Conventional Helper CD4+ T Cells Isolated from PBMCs According to Classical Protocol Described in the Literature:

(38) CD4.sup.+ CD127.sup.+ CD25.sup.neg/dim [conventional helper CD4 T cells (Tconv)] are stimulated with plate-bound anti-CD3 mAb (4 μg/ml), soluble anti-CD28 Ab (4 μg/ml) in the presence of ΔCD3-feeder (1 M) TGFβ (5 ng/ml) and Rapamycin (100 nM). Cells are cultured in IMDM-5 media. 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 μg/ml) and anti-CD28 Abs. The restimulations were performed in the presence of ΔCD3-feeder, TGFβ 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).

(39) Improved Ova-Specific Activation and Expansion of CD3+ CD4+ TCR□β+ MHCII Restricted T Cells Expressing Foxp3:

(40) 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 μM, 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 in the presence of soluble anti-CD28 Abs (1 μg/ml—clone CD28.2) and CD40-Abs (1 μg/ml—clone G28.5). After 16 h of stimulation, cells are washed with PBS (0.5% BSA) and stained for 10 min with anti-CD154 (clone 5C8)-PE and anti-CD4(SK3)-PerCP-eFluor 710. The stained cells are incubated with PE-conjugated microbeads (Miltenyi Biotec) and enriched by using MACS columns (Miltenyi Biotec). Isolated CD154+ T cells are then restimulated and expanded under the same optimal conditions as those described above.

(41) 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 granulocyte-macrophage colony-stimulating factor (GM-CSF) and 20 ng/ml human recombinant IL-4 for the generation of immature DC (iDC). At day 3, 500 μl 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 vial—in 90% FBS-10% DMSO. When indicated, pulsed DCs are matured with tumor necrosis factor α (TNF-α; 20 ng/mL final) and PGE2 (1 μM) 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.

(42) In vitro generation of TAP-inhibited stimulator cells for MLR assay: matured DC, obtained as described above, are electroporated with 20 μg 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+T cell responses against TAP-inhibited cells are readily detected in the human population. J Immunol. 2010 Dec. 1; 185(11):6508-17.)

(43) Apoptotic T Cells-DC Cocultures.

(44) 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 μg 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-α 20 ng/ml and PGE2 1 μM) for 24 hours.

(45) IL-17 Detection by ELISA.

(46) 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 detection antibody labelled with biotin, a poly-streptavidin HRP amplification system and an o-phenylenediamine dihydrochloride substrate solution.

(47) One example of said method is the following: coating a plate with the capture antibody, such as for example an anti-IL17 antibody, blocking the plate with a blocking buffer (such as casein 2% in PBS for example) during 90 min at 37° C., incubating the plate during 90 min at 37° C. with a dilution series of IL-17 standard, samples or negative controls, incubating the plate 90 min at 37° C. with the detection antibody such as for example a biotinylated anti-IL-17 antibody, 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.

(48) 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.

(49) Flow Cytometry Analysis.

(50) mAb labeling. The following conjugated mAbs are used.

(51) a) for CD3.sup.+ T cells: anti-CD4(SK3)-PerCP-eFluor 710, anti-TCRαβ (IP26)-APC (ebioscience), anti-CD25 (B1.49.9)-PeCy55, anti-CD127(R34.34)-APC-AF700 (Beckman Coulter), anti-CD3(UCHT1)-BB515 anti-invariant TCR Vα24-JαQ (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 αβ-BV421 (IP26) (Biolegend), anti-TCR pan γδ.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 ebio science 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).

(52) 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-IL-1R1-PE (R&D system, FAB269P).

(53) CFSE staining. Tconv are stained with 1 μM 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 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.

(54) 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 μg/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.

(55) Functional Assays.

(56) 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.

(57) T cell apoptosis induction: tumor-antigen specific functionally committed FOXP3 expressing TCRαβ.sup.+ MHCII restricted T cells are generated ex vivo as described above. Then tumor-antigen specific stimulated-T cells were irradiated (240 mJ/cm2) 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+.

(58) Standard polyclonal cell-cell contact Treg suppression assay: CFSE-labeled Tconv (4×10.sup.4 per well), used as responder cells, are cultured with ΔCD3− 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 μg/mL anti-CD3 mAb in 200 μL 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.

(59) 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 are performed in IMDM-5 media supplemented with IL-2 (20 IU/ml) IL-1β (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.

(60) 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 μl reaction volume containing 25 μl of ZymoTaq PreMix buffer (Zymo Research) and 0.5 μM 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 μl LightCycler 480 Probes Master Mix (Roche), 10 ng of bisulfite converted DNA sample or standards, 1 μM of each primer, and 150 nM of each probe with a final reaction value of 20 μl. 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.

(61) 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 μL, 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 8004, 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 μL; Invitrogen) was incubated with 1 μg of H3K4me3 (Abcam) or H3K9ac (Cell Signaling) or normal rabbit IgG negative control ChIP-grade antibodies for 2 h separately. Then, 100 μL of the sheared chromatin was immunoprecipitated with pretreated antibody-bead complexes and another 100 μL 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).

(62) Classical 7-AAD/CFSE Cell-Mediated Cytotoxicity Assay: target cells were labeled with CFSE as described above and placed at 3×104 per well in 96-well U-bottomed plates in triplicate. CD8+ Effector T cells (5:1 E:T ratio) were added, and incubation was carried out at 37° C. for 6 hr. 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+ 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 (%)]
Results
a) Optimal Conditions for Inducing Foxp3 Expression in Naive CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII Restricted T Following Polyclonal.

(63) Starting from naive conventional CD4.sup.+ T cells (CD3.sup.+ CD4.sup.+ CD127.sup.+ CD45RA.sup.+ CD25.sup.− TCRαβ.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.

(64) FIG. 2 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, TGFβ and rapamycin or IL-2, TGFβ, 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, TGFβ, rapamycin and PGE2 results in an optimal intensity of Foxp3 expression in the CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII restricted T cells, as compared to the other combinations (C).

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

(66) We next evaluated the functional suppressive capacity of the Foxp3 expressing CD3.sup.+ CD4.sup.+ TCRαβ+ MHCII restricted T cells polyclonally stimulated. FIG. 3A shows that CD3.sup.+ CD4.sup.+ TCRαβ.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, TGFβ, 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, TGFβ, rapamycin without PGE2 and fresh FOXP3 expressing CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII restricted T cells, when using the standard polyclonal cell-cell contact Treg suppression assay. Furthermore, FIG. 3B shows that these 21-day-expanded-FOXP3 expressing CD3.sup.+ CD4.sup.+ TCRαβ.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.+ TCRαβ.sup.+ MHCII restricted T cells lose their suppressive capacity under these culture condition of stimulation.

(67) b) Optimal Conditions for Inducing Foxp3 Expression in Naive CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII Restricted T Cells Following Antigen-Specific Activation.

(68) 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.+ CD4.sup.+ TCRαβ.sup.+ MHCII restricted T cells, committed to exclusively exert regulatory activity, whichever culture condition of stimulation.

(69) FIG. 4 shows that OVA-pulsed autologous tDCs, in presence of the nTreg polarizing medium comprising the combination of IL-2, TGFβ, PGE2 and rapamycin are able to stimulate naive conventional CD4+ T cells, (increase expression of CD25 and loss of CD45RA marker), while non-pulsed autologous tDCs, in presence of the same polarizing medium, were unable to stimulate them (absence of CD25 expression and persistence of CD45RA marker). Furthermore, naive conventional CD4.sup.+ T cells, when specifically activated and expanded for 21 days with OVA-pulsed autologous tDCs, in presence of the nTreg polarizing medium described above, are able to express similar level and intensity of Foxp3 to those displayed by blood nave regulatory T cells (CD3.sup.+ TCRαβ.sup.+ CD4.sup.+ CD127.sup.−/low CD45RA.sup.+ CD25.sup.+), corresponding to our positive control (FIG. 5).

(70) To improve the antigen specific activation and expansion of nave CD3.sup.+ CD4.sup.+ TCRαβ+ MHCII restricted T cells expressing Foxp3, 16 h after their priming with OVA-pulsed autologous tDCs in the presence of soluble anti-CD28 Abs (1 m/ml) and CD40-Abs (1 μg/ml), CD154 expressing nave CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ T cells are sorted (FIG. 6). Isolated CD154.sup.+ T cells are then restimulated and expanded under the same optimal conditions as those described above. Using this strategy, we are able to ex vivo induce and generate highly specific functionally committed FOXP3 expressing CD3.sup.+ TCRαβ+ MHCII restricted T cells lines.

(71) In addition, these 21-day-expanded-ova-specific CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII restricted T cells display a similar suppressive activity compared with fresh Foxp3 expressing CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII restricted T cells, when using both the standard polyclonal cell-cell contact Treg suppression assay (FIG. 7A) and the autologous MLR suppression assay (FIG. 8A).

(72) Furthermore Ova-specific CD3.sup.+ TCRαβ.sup.+ MHCII restricted T cells maintain their ability to perform suppressive function in pro-inflammatory conditions. When the both functional suppressive assay (FIG. 7B, FIG. 8B) are 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.+ TCRαβ.sup.+ MHCII restricted T cells lose their suppressive capacity under these culture condition of stimulation, the 21-day-expanded-Foxp3 expressing CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII restricted T cells still maintain their suppressive activity. The maintenance of their suppressive capacity in a high inflammatory context could be ascribed to the fact that they produce low level of IL-17 after 21 days of expansion in the nTreg polarizing medium, when stimulated through CD3 and CD28 in the presence of IMDM medium containing IL-2 IL-1 IL-6, IL-21 IL-23 cytokines (FIG. 10A)

(73) To confirm that the Ova-specific CD3.sup.+ TCRαβ3.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. 9) and 2) for their IL-17-producing capacity when stimulated through CD3 and CD28 as described above (FIG. 10). After a further 21-day-culture either in nTreg or TH-17 polarizing medium, Ova-specific CD3.sup.+ TCRαβ.sup.+ MHCII restricted T cells not only still retain, in a high inflammatory context, functional suppressive activity (FIG. 9), but also produce low level of IL-17 (FIG. 10B). By contrast fresh Foxp3 expressing CD3.sup.+ TCRαβ.sup.+ MHCII restricted T cells lose their suppressive function while producing IL-17 in this inflammatory context.

(74) The absence of IL-1R1 expression on the Ova-specific CD3.sup.+ TCRαβ.sup.+ MHCII restricted T cells could be explained why these specific induced Treg are stable and function effectively in an inflammatory environment. Indeed, as depicted in FIG. 11, when we assessed the expression of IL-1R1 on different population of Treg: a) ex vivo resting Tregs isolated from PBMCs, b) ex vivo expanded Tregs from Treg isolated from PBMCs with polyclonal stimulation, c) polyclonal in vitro induced Treg in the presence of Rapa and TGFβ from conventional T cells isolated from PBMCs and d) in vitro induced Ova-specific CD3.sup.+ FOXP3+ T cells in presence of RAPA, TGFβ and PGE2 isolated from naïve CD4+ T cells, We found that IL-1R1 is preferentially expressed on resting, polyclonal expanded/induced Tregs when compared to the induced Ova-specific CD3.sup.+ FOXP3.sup.+ T cells. We also observe that the stability of the suppressive function is inversely correlated with the IL-1R1 expression.

(75) c—Induction of Autologous CD8-Mediated T-Cell Responses Against Pathogenic CD4.sup.+ T Cells Using Apoptotic CD4.sup.+ T Cell-Loaded Dendritic Cells.

(76) We have developed an experimental procedure to generate autologous CD8.sup.+ T cell lines functionally committed to lyse tumor-antigen specific FOXP3 expressing TCRαβ.sup.+ MHCII restricted T cells, pathogenic CD4.sup.+ T cells that favour tumor cell immune evasion.

(77) We have first set up optimal conditions for inducing tumor-antigen specific FOXP3.sup.+ expressing TCRαβ.sup.+ MHCII restricted T cells, as described before. FIG. 12 shows that apoptotic tumor cell lines-pulsed autologous tDCs (“tumor Ag loaded tDC”), in presence of the nTreg polarizing medium comprising the combination of IL-2, TGFβ, PGE2 and rapamycin are able to induce high levels of Foxp3.sup.+ expression (in frequency in FIG. 12A and in MFI in FIG. 12B) in antigen specific stimulated naive conventional CD4.sup.+ T cells (“Nave Treg”) while non-pulsed autologous tDCs (“unloaded tDC”), in presence of the same polarizing medium, were unable to induce Foxp3.sup.+ expression in naive conventional CD4.sup.+ T cells.

(78) Then, we have established a culture system in which inflammatory DC (inf DC) loaded with apoptotic pathogenic CD4.sup.+ T cells cocultured with autologous CD3.sup.+ naïve 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. 13 shows that the two CD8.sup.+ clones induced with apoptotic pathogenic CD4.sup.+ T cells loaded—inf 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.

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

(80) 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.+ TCRαβ.sup.+ MHCII restricted T cells. No Foxp3 was detected in TILs extracted from luminal A breast tumor. Moreover, a positive correlation is observed between a high percentage of expression of Foxp3 in CD3.sup.+ CD4.sup.+ TCRαβ.sup.+ MHCII restricted T cells and a poor clinical outcome of breast cancer (FIG. 14).