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. 2017 Aug 21;8:311. doi: 10.1038/s41467-017-00169-4

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 8 — PAR2-PAR3 cofactoring on T-cells is required for inhibition of GvHD by aPC. a, b Lethally irradiated (11 Gy)-recipient BALB/c mice were transplanted with C57BL/6 wt-derived 5 × 10⁶ BM cells and 0.5 × 10⁶ T-cells without (B6T + BM) or with (B6T(aPC) + BM) aPC-preincubation (20 nM, 1 h, 37 °C). Alternatively, lethally irradiated BALB/c mice received 5 × 10⁶ C57BL/6 wt-derived BM cells and 0.5 × 10⁶ C57BL/6 PAR3^−/− T-cells (PAR3^−/−T + BM) or 0.5 × 10⁶ C57BL/6 PAR2^−/− T-cells (PAR2^−/−T + BM) or 5 × 10⁶ C57BL/6 wt-derived BM cells and 0.5 × 10⁶ C57BL/6 PAR3^−/− T-cells (PAR3^−/−T(aPC) + BM) or 0.5 × 10⁶ C57BL/6 PAR2^−/− T-cells (PAR2^−/−T(aPC) + BM; aPC preincubation in both cases: 20 nM, 1 h, 37 °C). aPC’s protective effect is lost in mice transplanted with PAR2-deficient or PAR3-deficient T-cells; survival (a Kaplan–Meier curve) and clinical score (b) from two (PAR2^−/−T + BM and PAR2^−/−T(aPC) + BM) or three (all other groups) independent experiments each with four mice per group are shown. c, d Lethally irradiated (11 Gy)-recipient BALB/c mice were transplanted with C57BL/6-derived 5 × 10⁶ BM cells and the following combination of C57BL/6-derived T-cells: (i) 0.4 × 10⁶ T-cells and 0.1 × 10⁶ T_regs (B6T + T_reg + BM), (ii) 0.4 × 10⁶ T-cells and 0.1 × 10⁶ T_regs with aPC-preincubation (B6T + T_reg(aPC) + BM), (iii) 0.4 × 10⁶ PAR3^−/−T-cells and 0.1 × 10⁶ PAR3^−/− T_regs (PAR3^−/−T + PAR3^−/−T_reg + BM), (iv) 0.4 × 10⁶ PAR3^−/−T-cells and 0.1 × 10⁶ PAR3^−/− T_regs with aPC-preincubation (PAR3^−/−T + PAR3^−/−T_reg(aPC) + BM), (v) 0.4 × 10⁶ PAR2^−/−T-cells and 0.1 × 10⁶ PAR2^−/− T_regs (PAR2^−/−T + PAR2^−/−T_reg + BM), or (vi) with 0.4 × 10⁶ PAR2^−/−T-cells and 0.1 × 10⁶ PAR2^−/− T_regs with aPC-preincubation (PAR2^−/−T + PAR2^−/−T_reg(aPC) + BM). aPC-preincubation was conducted as before (20 nM, 1 h, 37 °C). Loss of PAR2 or PAR3 on T_regs abrogates the GvHD protective effect of aPC. Survival (c Kaplan–Meier curve) clinical score (d) from two (PAR2^−/−T + BM and PAR2^−/−T(aPC) + BM) or three (all other groups) independent experiments, each with four mice per group, are shown. *P < 0.05, **P < 0.01, (a, c log-rank test; b, d ANOVA)