Circulating T helper cell subsets
Flow cytometry data revealed that the proportion of Th1 cells, defined as CD3+CD4+CD45RA-Tbet+cells, decreased between baseline and three years after treatment in the groups receiving ILIT with birch and placebo and 5-grass and placebo (p<0.05, Fig. 3a-b). An increase was observed after one year in the group receiving birch and 5-grass treatment (p<0.05, Fig. 3c). The proportion of Th2 cells, defined as CD3+CD4+CD45RA-GATA3+cells, increased three years after treatment in the group receiving birch and placebo ILIT (Fig. 3d, p<0.01) and in the group receiving both treatments (Fig. 3f, p<0.05). No other changes were observed in the group that received 5-grass and placebo ILIT (Fig. 3e). The proportion of Th17 cells, defined as CD3+CD4+ CD45RA-RORC+ cells, decreased from baseline to three years post ILIT, independent of treatment (Fig. 3g-i, p<0.05). A significant reduction in Th17 cell frequencies was also observed from one year to three years after ILIT (p<0.01). The Th17 memory population was significantly higher at baseline in the birch and placebo group than in the other treatment groups (p<0.001, Fig. S2a in the Supplementary information). One year after treatment, a higher proportion of Th17 memory cells was observed in patients receiving 5-grass and placebo ILIT than in the birch and placebo group (p<0.05, Fig. S2b in the Supplementary information). No significant differences between the treatment groups were observed at three years (Fig. S2c in the Supplementary information). The proportion of CD4dimCD25hiFoxP3+Tregs (Fig. 4a-c) and activated Tregs, defined as CD3+CD4+CD45RA-FoxP3++cells (Fig. 4d-f), significantly increased between baseline and three years, independent of treatment. In contrast, the proportion of resting Tregs, defined as CD3+CD4+CD45RA+FoxP3+cells, was not affected by ILIT (data not shown).