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.