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

Abstract

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. 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. 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. 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.