Fig. 5
From: Activated protein C protects from GvHD via PAR2/PAR3 signalling in regulatory T-cells
aPC restricts allogenic T-cell reactivity via regulatory T-cells. a BALB/c mice were irradiated and transplanted with BM (5 × 106) and T-cells (0.4 × 106) obtained from C57BL/6 mice and Tregs (0.1 × 106) from C57BL/6 (transgene negative DEREG littermate mice) without (B6T + B6Treg + BM + DT) or with (B6T + B6Treg(aPC) + BM + DT) aPC-preincubation or from DEREG mice without (B6T + DTR-Treg + BM + DT) or with aPC-preincubation (B6T + DTR-Treg(aPC) + BM + DT). Diphtheria toxin (DT, 20 ng/g body weight) was injected on day 1 and 2 post transplantation in all groups. b aPC-preincubation of pre-existing human Tregs with aPC (T + Tr(aPC) + AgPC) increases Treg frequency to a larger extent than aPC-preincubation of Treg-depleted T-cells (T(aPC) + Tr + AgPC), Treg frequency was measured by flow cytometry after 48 h of MLR. c Preincubation of human Tregs with aPC (Teff + Treg(aPC) + AgPC) markedly reduces T-cell reactivity, as determined by thymidine incorporation in the MLR. In the absence of Tregs aPC-preincubation of effector T-cells (Teff(aPC) + AgPC) is less efficient, but the effect is comparable to that observed when combing effector T-cells with Tregs without aPC-preincubation (Teff + Treg + AgPC). Results are compared with T-cell reactivity of effector T-cells in the absence of Tregs (Teff + AgPC). d aPC induces proliferation of human Tregs: Tregs without (Treg) or with aPC-preincubation (Treg(aPC)) were labelled with eFluor450 (4 µM) and stimulated with plate bound αCD3 (10 µg/ml) and αCD28 (8 µg/ml) antibodies. Proliferation was measured on day 1 and 6 by FACS. e aPC induces human CD4+CD25+ cells: effector T-cells (CD4+CD25-) were left untreated or were preincubated with aPC (20 nM, 1 h, 37 °C). Following allogenic simulation with AgPC the frequency of CD4+CD25+ cells, determined at day 4, was increased following aPC-preincubation; results from three repeat experiments each with three biological disjunct donors. f Following preincubation of Teff-cells with aPC nuclear levels of FOXP3 are increased. Representative immunoblot (bottom) and bar graph (top) summarizing results of five independent repeat experiments. T Treg-depleted T-cells, T(aPC) Treg-depleted T-cells preincubated with aPC (20 nM, 1 h, 37 °C), AgPC allogenic antigen-presenting cells, T eff effector T-cells; T reg regulatory T-cells. Exemplary FACS images corresponding to Fig. 5e are shown in Supplementary Fig. 10. Mean value ± SEM (b, c, e, and f); *P < 0.05, **P < 0.01 (a log-rank test, b, c ANOVA; e, f t-test)
