Massive monoclonal expansion of functionally stable human regulatory T cells (THER5P.826)

Abstract

Abstract Adoptive Treg transfer is a promising therapeutic strategy for autoimmune diseases. Most current approaches rely on the enrichment and in vitro expansion of non-clonal, presumably thymus-derived Treg cells. Limitations include low expansion rates, contaminating effector cells, and unstable suppressive activity. Here, we hypothesized that cloning of human CD4+CD127lowCD25highFoxP3+HELIOS+ cells yields a highly pure cellular product with stable suppressor function throughout repeated re-expansion cycles. Cloning was performed through selection for CD4+CD127lowCD25+ cells by magnetic bead isolation and expansion in limiting dilution conditions. Suppressor function was tested in suppression assays of CD3/CD28-stimulated allogeneic Teff cells. Clonality was assessed by Vbeta staining. To demonstrate validity of our approach for a human autoimmune disease we also cloned Treg from a patient with Behcet's disease, a multisystem vasculitis. Cloning efficiency was 2-5/100 seeded cells. Treg clones were homogenously CD4+CD127lowCD25highFoxP3+HELIOS+, and clonality was confirmed by staining for a unique TCR Vbeta chain. Clones maintained at least 80% suppression at a 1:4 Treg:Teff ratio over 8 weeks in in vitro culture. Expansion rate was up to 9.8 billion-fold. Our results indicate that massive expansion of human antigen-specific Treg clones is a feasible approach for the generation of large numbers of functionally stable regulatory T cells for preclinical and clinical testing.