title: Generation of Human Alloantigen-Specific Regulatory T Cells Under Good Manufacturing Practice-Compliant Conditions for Cell Therapy.
abstract: Natural regulatory T cells (Tregs) may have a great therapeutic potential to induce tolerance in allogeneic cells and organ transplantations. In mice, we showed that alloantigen-specific Tregs (spe-Tregs) were more efficient than polyclonal Tregs (poly-Tregs) in controlling graft-versus-host disease (GVHD). Here we describe a clinical-grade compliant method for generating human spe-Tregs. Tregs were enriched from leukapheresis products with anti-CD25 immunomagnetic beads, primed twice by allogeneic mature monocyte-derived dendritic cells (mDCs), and cultured during 3 weeks in medium containing interleukin 2 (IL-2), IL-15, and rapamycin. After 3 weeks of culture, final cell products were expanded 8.3-fold from the initial CD25(+) purifications. Immunophenotypic analyses of final cells indicate that they were composed of 88 ± 2.6% of CD4(+) T cells, all expressing Treg-specific markers (FOXP3, Helios, GARP, LAP, and CD152). Spe-Tregs were highly suppressive in vitro and also in vivo using a xeno-GVHD model established in immunodeficient mice. The specificity of their suppressive activity was demonstrated on their ability to significantly suppress the proliferation of autologous effector T cells stimulated by the same mDCs compared to third-party mDCs. Our data provide evidence that functional alloantigen Tregs can be generated under clinical-grade compliant conditions. Taking into account that 130 × 10(6) CD25(+) cells can be obtained at large scale from standard leukapheresis, our cell process may give rise to a theoretical final number of 1 × 10(9) spe-Tregs. Thus, using our strategy, we can propose to prepare spe-Tregs for clinical trials designed to control HLA-mismatched GVHD or organ transplantation rejection.
authors.affiliation: AP-HP, University Hospital La Pitié-Salpêtrière, Department of Biotherapies, Paris, France.
authors.forename: Mustapha
authors.initials: M
authors.lastname: Cheraï
authors.forename: Yamina
authors.initials: Y
authors.lastname: Hamel
authors.forename: Claude
authors.initials: C
authors.lastname: Baillou
authors.forename: Soumia
authors.initials: S
authors.lastname: Touil
authors.forename: Maude
authors.initials: M
authors.lastname: Guillot-Delost
authors.forename: Frédéric
authors.initials: F
authors.lastname: Charlotte
authors.forename: Laila
authors.initials: L
authors.lastname: Kossir
authors.forename: Ghislaine
authors.initials: G
authors.lastname: Simonin
authors.forename: Sébastien
authors.initials: S
authors.lastname: Maury
authors.forename: José L
authors.initials: JL
authors.lastname: Cohen
authors.forename: François M
authors.initials: FM
authors.lastname: Lemoine
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D064987:Cell- and Tissue-Based Therapy / Q000379:methods; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000276:immunology; D005260:Female; D006086:Graft vs Host Disease / Q000276:immunology* / Q000517:prevention & control* / Q000628:therapy; D006801:Humans; D007108:Immune Tolerance / Q000276:immunology; D018189:Immunomagnetic Separation; D019409:Interleukin-15 / Q000494:pharmacology; D007376:Interleukin-2 / Q000494:pharmacology; D053645:Interleukin-2 Receptor alpha Subunit / Q000378:metabolism; D007519:Isoantigens / Q000276:immunology*; D007937:Leukapheresis; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008875:Middle Aged; D016377:Organ Transplantation / Q000379:methods; D020123:Sirolimus / Q000494:pharmacology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000637:transplantation*; D055815:Young Adult
chemical_list: C569274:IL15 protein, human; C508594:IL2 protein, human; C508017:IL2RA protein, human; D019409:Interleukin-15; D007376:Interleukin-2; D053645:Interleukin-2 Receptor alpha Subunit; D007519:Isoantigens; D020123:Sirolimus