In vitro assessment of cord blood–derived proinsulin-specific regulatory T cells for cellular therapy in type 1 diabetes

Abstract Background Antigen-specific regulatory T cells (Tregs) have proven to be effective in reversing established autoimmunity in type 1 diabetes (T1D). Cord blood (CB) can serve as an efficient and safe source for Tregs for antigen-specific immunomodulation in T1D, a strategy that is yet to be explored. Therefore, we assessed the potential of CB in generation of proinsulin (PI)-specific Tregs by using HLA class II tetramers. Methods We analyzed the frequency of PI-specific natural Tregs (nTregs) and induced Tregs (iTregs) derived from the CB as well as peripheral blood (PB) of patients with T1D and healthy control subjects. For this, CD4+CD25+CD127 low and CD4+CD25-T cells were cultured in the presence of PI-derived peptides, transforming growth factor (TGF)-β and rapamycin. PI-specific Tregs were then selected using allele-specific HLA II tetramers loaded with PI-derived peptides, followed by suppression assays. Results Following stimulation, we observed that CB harbors a significantly higher frequency of PI-specific Tregs than PB of subjects with T1D ( P  = 0.0003). Further, the proportion of PI-specific Tregs was significantly higher in both the nTreg ( P  = 0.01) and iTreg ( P  = 0.0003) compartments of CB as compared with PB of subjects with T1D. In co-culture experiments, the PI-specific Tregs suppressed the proliferation of effector T cells significantly ( P  = 0.0006). The expanded nTregs were able to retain hypomethylation status at their Tregs-specific demethylated region (TSDR), whereas iTregs were unable to acquire the characteristic demethylation pattern. Conclusion Our study demonstrates that CB can serve as an excellent source for generation of functional antigen-specific Tregs for immunotherapeutic approaches in subjects with T1D.