iTregBet displayed an activated and suppressive phenotype after allergen-specific and cross-reactive stimulation
In order to identify the phenotype of allergen-specific iTreg in more detail, we performed a flow cytometric analysis of iTregBet, thereby gating on proliferating (activated) and non-proliferating iTregBet in the setting of suppressor assays after Bet- and Cor-specific stimulation (by allergen-loaded mDC) (Figure 4, gating strategy see Supplementary Figure 6). Allergen (Bet or Cor)-specifically restimulated proliferating iTregBet populations exhibited significantly higher percentages of CD45RO+ (enhanced differentiation into a memory phenotype) and activated CD25+ and HLA-DR+ cells compared to Bet- or Cor-specifically stimulated non-proliferating iTregBet or Tresp, respectively (Figure 4A). Compared to non-proliferating iTreg and to Tresp, the proliferating iTregBet population was characterized by a significantly enhanced expression of CTLA-4, TNFR2, PD-1, IL-10 and ICOS, molecules known to be involved in the immunosuppressive capacity of regulatory T cells, confirming the regulatory phenotype of allergen-stimulated iTreg (Figure 4B). These results emphasized the activation state induced by allergen-specific (mDCBet) or cross-reactive (mDCCor) stimulation as prerequisite for iTreg-mediated suppressive activity. For further characterization, the expression of CD49b and LAG3 as parameters for Tr1 differentiation and Treg function were investigated (Figure 4C).45–48 Compared to Tresp, iTregBetshowed an increase in CD49b+LAG3+cells after mDCBet stimulation, although this was not significant under mDCCor stimulation.