title: Ex vivo-expanded but not in vitro-induced human regulatory T cells are candidates for cell therapy in autoimmune diseases thanks to stable demethylation of the FOXP3 regulatory T cell-specific demethylated region.
abstract: Regulatory T cell (Treg) therapy is a promising approach for transplant rejection and severe autoimmunity. Unfortunately, clinically meaningful Treg numbers can be obtained only upon in vitro culture. Functional stability of human expanded (e)Tregs and induced (i)Tregs has not been thoroughly addressed for all proposed protocols, hindering clinical translation. We undertook a systematic comparison of eTregs and iTregs to recommend the most suitable for clinical implementation, and then tested their effectiveness and feasibility in rheumatoid arthritis (RA). Regardless of the treatment, iTregs acquired suppressive function and FOXP3 expression, but lost them upon secondary restimulation in the absence of differentiation factors, which mimics in vivo reactivation. In contrast, eTregs expanded in the presence of rapamycin (rapa) retained their regulatory properties and FOXP3 demethylation upon restimulation with no stabilizing agent. FOXP3 demethylation predicted Treg functional stability upon secondary TCR engagement. Rapa eTregs suppressed conventional T cell proliferation via both surface (CTLA-4) and secreted (IL-10, TGF-β, and IL-35) mediators, similarly to ex vivo Tregs. Importantly, Treg expansion with rapa from RA patients produced functionally stable Tregs with yields comparable to healthy donors. Moreover, rapa eTregs from RA patients were resistant to suppression reversal by the proinflammatory cytokine TNF-α, and were more efficient in suppressing synovial conventional T cell proliferation compared with their ex vivo counterparts, suggesting that rapa improves both Treg function and stability. In conclusion, our data indicate Treg expansion with rapa as the protocol of choice for clinical application in rheumatological settings, with assessment of FOXP3 demethylation as a necessary quality control step.
authors.affiliation: Translational Research Unit, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; SingHealth Translational Immunology and Inflammation Centre, SingHealth, 169856 Singapore; maurarossetti@gmail.com.
authors.forename: Maura
authors.identifier: http://orcid.org/0000-0003-3347-6200
authors.initials: M
authors.lastname: Rossetti
authors.affiliation: Translational Research Unit, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; SingHealth Translational Immunology and Inflammation Centre, SingHealth, 169856 Singapore;
authors.forename: Roberto
authors.identifier: http://orcid.org/0000-0001-8282-7658
authors.initials: R
authors.lastname: Spreafico
authors.affiliation: SingHealth Translational Immunology and Inflammation Centre, SingHealth, 169856 Singapore;
authors.forename: Suzan
authors.initials: S
authors.lastname: Saidin
authors.affiliation: SingHealth Translational Immunology and Inflammation Centre, SingHealth, 169856 Singapore;
authors.forename: Camillus
authors.initials: C
authors.lastname: Chua
authors.affiliation: Translational Research Unit, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037;
authors.forename: Maryam
authors.initials: M
authors.lastname: Moshref
authors.affiliation: SingHealth Translational Immunology and Inflammation Centre, SingHealth, 169856 Singapore;
authors.forename: Jing Yao
authors.initials: JY
authors.lastname: Leong
authors.affiliation: Department of Rheumatology and Immunology, Singapore General Hospital, 169608 Singapore; Duke-National University of Singapore Graduate Medical School, 169857 Singapore; and Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore.
authors.forename: York Kiat
authors.initials: YK
authors.lastname: Tan
authors.affiliation: Department of Rheumatology and Immunology, Singapore General Hospital, 169608 Singapore; Duke-National University of Singapore Graduate Medical School, 169857 Singapore; and Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore.
authors.forename: Julian
authors.initials: J
authors.lastname: Thumboo
authors.affiliation: Translational Research Unit, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037;
authors.forename: Jorg
authors.initials: J
authors.lastname: van Loosdregt
authors.affiliation: Translational Research Unit, Sanford-Burnham Medical Research Institute, La Jolla, CA 92037; SingHealth Translational Immunology and Inflammation Centre, SingHealth, 169856 Singapore; Duke-National University of Singapore Graduate Medical School, 169857 Singapore; and.
authors.forename: Salvatore
authors.initials: S
authors.lastname: Albani
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000628:therapy*; D060908:CTLA-4 Antigen / Q000276:immunology; D049109:Cell Proliferation; D064987:Cell- and Tissue-Based Therapy / Q000379:methods*; D002478:Cells, Cultured; D019175:DNA Methylation; D005260:Female; D051858:Forkhead Transcription Factors / Q000378:metabolism*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D016753:Interleukin-10 / Q000276:immunology; D007378:Interleukins / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D008297:Male; D051379:Mice; D008875:Middle Aged; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D020123:Sirolimus / Q000494:pharmacology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology* / Q000637:transplantation*; D016212:Transforming Growth Factor beta / Q000276:immunology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D060908:CTLA-4 Antigen; C418974:FOXP3 protein, human; D051858:Forkhead Transcription Factors; C508609:IL10 protein, human; D007166:Immunosuppressive Agents; D007378:Interleukins; D011948:Receptors, Antigen, T-Cell; D016212:Transforming Growth Factor beta; D014409:Tumor Necrosis Factor-alpha; C532417:interleukin-35, human; D016753:Interleukin-10; D020123:Sirolimus