Human LAP(+)GARP(+)FOXP3(+) regulatory T cells attenuate xenogeneic graft versus host disease

Wang, Huihui; Song, Hyo; Pham, Anha V.; Cooper, Laurence J.; Schulze, Janika J.; Olek, Sven; Tran, Dat Q.

The level of methylation at the promoter region upstream of FOXP3 termed Treg-specific demethylated region (TSDR) has provided specific differentiation between activated T effector cells and bona fide Tregs [19, 20] . Several studies have demonstrated that FOXP3 − cells are fully methylated at this site, while iTregs are intermediate and tTregs are fully demethylated [21, 22] . The level of demethylation at the TSDR region is critical for establishment of a stable Treg lineage and correlation with suppressive function. In this study, we demonstrate that after 30 days expansion the purified LAP + Tregs are highly demethylated with an average of 95% demethylation in the TSDR region, while the CD25 + cells with mean of 45% demethylation ( Figure 5A ). The LAP -nonTregs are highly methylated, with less than 20% demethylation even when the population contains close to 60% FOXP3 + and HELIOS + cells ( Figure 5B and C). It should be noted that the LAPpopulations still contain LAP + cells ranging from 10-50% which represent most of the demethylated TSDR and HELIOS signals (Figure 1 and 2).