Ex vivo generation of γδ Foxp3
The present invention relates to a method for ex vivo generating and expanding γδ Foxp3 + regulatory T cells, and therapeutic uses thereof. The inventors performed the induction of Foxp3+ expression in ex vivo human induced tumor-antigen specific CD4+ TCRγδ unrestricted T cells and the induction of autologous CD8-mediated T-cell responses against tumor-antigen specific FOXP3 expressing CD4+ TCRγδ unrestricted T cells. The inventors developed a method to ex vivo generated and expanded antigen specific Foxp3 expressing CD3+ TCRγδ+ unrestricted T cells, committed to exclusively exert regulatory activity, whichever culture condition of stimulation is. In particular, the present invention relates to a method for generating ex vivo γδ Foxp3+ regulatory T cells having the following phenotype: CD3+ TCRγδ+ Foxp3+.
1. A method for generating ex vivo γδ Foxp3 + regulatory T cells having the following phenotype: CD3 + TCRγδ + Foxp3 + , comprising
culturing CD3 + TCRγδ + T cells in the presence of a γδ T cell activator and the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator, wherein the cAMP activator is selected from the group consisting of prostaglandin E2 (PGE2), an EP2 or EP4 agonist, a membrane adenine cyclase activator and a metabotropic glutamate receptors agonist, ii) a TGFβ (Transforming growth factor beta) pathway activator, wherein the TGFβ pathway activator is selected from the group consisting of TGFβ, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs), anti-müllerian hormone (AMH), activin and nodal, and iii) a mTOR inhibitor, wherein the mTOR inhibitor is selected from the group consisting of rapamycin, rapamycin analogs, wortmannin; theophylline; caffeine; epigallocatechin gallate (EGCG), curcumin, resveratrol; genistein, 3,3-diindolylmethane (DIM), LY294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one), PP242, PP30, Torin1, Ku-0063794, WAY-600, WYE-687, WYE-354, GNE477, NVP-BEZ235, PI-103, XL765 and WJD008, and optionally
iv) at least one cytokine selected from the group consisting of IL-2, IL-7, IL-15 and TSLP, and/or
v) at least one TET enzyme activator selected from the group consisting of vitamin C and a NaHS hydrogen sulfide releasing agent and/or
vi) at least one DNMT inhibitor selected from the group consisting of 2-(1,3-dioxo-1,2-dihydro-2H-isoindol-2-yl)-3-(1H-indol-3-yl) propanoic acid (RG108), 5-aza-22-deoxycytidine (DAC) and 5-azacytidine (SAC),
for at least 5 days,
wherein the γδ T cell activator is an antigen-specific γδ T cell activator comprising tolerogenic dendritic cells (DCs) pulsed with at least one bisphosphonate and wherein the tolerogenic DCs are pulsed with the at least one bisphosphonate prior to being added to the CD3 + TCRγδ + T cells.
2. The method according to claim 1 , further comprising an expansion step, wherein the γδ Foxp3 + regulatory T cells are cultured in the presence of a γδ T cell activator and the following agents: i) an cAMP (Cyclic adenosine monophosphate) activator, ii) a TGFβ (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, and optionally v) at least one TET enzymes activator and/or at least one DNMT inhibitor, for at least 5 days, wherein the γδ T cell activator, the cAMP activator, the TGFβ pathway activator, the mTOR inhibitor, the TET enzymes activator, and the DNMT inhibitor are as defined in claim 1 .
3. The method of claim 1 , wherein the at least one bisphosphonate comprises at least one aminobisphosphonate.
4. The method of claim 1 , wherein the tolerogenic DCs are derived from monocytes.