Figure 4

Endogenous MDSCs (CD11b⁺Gr1⁺) are essential to donor-derived MDSCs mediated alloimmune suppression. (A,B) Flow cytometry analysis of BALB/c MDSCs treated C57BL/6 recipients’ splenocytes on POD7 (n = 8–9 per group). (A) The proportion of CD11b⁺Gr1⁺ significantly increased in donor-derived the MDSCs treated group. (B) Cells were gated on CD11b⁺Gr1⁺, PDL1 expression was up-regulated in the MDSCs treated group. Mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, one-way ANOVA and Tukey’s test. Data represents one of 4 separate experiments. (C) Flow cytometry analysis of BALB/c MDSCs treated C57BL/6 recipients’ graft infiltration lymphocytes (GILs) on POD7. CD11b⁺Gr1⁺ population was significantly increased in MDSCs treated group. n = 3–6 per group. Mean ± SEM. Data represents one of 3 separate experiments. *p < 0.05, two-tailed unpaired t test. (D) Schematic illustration of the experimental design to test the ex vivo immunosuppressive function of BALB/c MDSCs induced endogenous MDSCs (CD11b⁺Gr1⁺) in recipients. Naïve C57BL/6 T cells were stimulated with BALB/c APCs for 3 days. CD11b⁺Gr1⁺ cells were isolated by fluorescence activated cell sorting (FACS) from C57BL/6 recipient splenocytes at POD7 (n = 3–4 per group) and were added as modulator. (E) Cells were gated on CD4⁺FoxP3^-, and the Ki67 expression was measured. Graphs showing significant decrease of the proportion of Ki67⁺FoxP3^- in CD4⁺ T cells in the presence of CD11b⁺Gr1⁺ cells induced with MDSCs compared to those induced with cMDCs. Mean ± SEM, * p < 0.05, two-tailed unpaired t test. Data represents one of 3 separate experiments. (F) Schematic illustration of the treatment protocol. C57BL/6 recipients received a single-dose intravenous injection of 1 × 10⁶ BALB/c 7 days prior to the transplantation. Recipients were treated with anti-Gr1 mAb on POD 0, 2, 4, 6. (G) Anti-Gr1 mAb administration attenuates MDSCs induced immune suppression. Kaplan–Meier cumulative survival of allograft showing the shortened allograft survival in the anti-Gr1 Ab treated group compared to the IgG treated control group (n = 7 per group). **p < 0.01, log-rank test.