Adoptively transferred GVHD-regulatory MDSCs enhance donor nTreg recovery through generation of PD-L1–expressing MDCs. (A) Representative FACS plots of peripheral blood in a WT B6 adoptive recipient at day 7 following infusion of 2 × 10⁶ sorted CD45.1⁺ MDSCs and WT BALB/c BMT, as shown in Figure 5A. Left panel, gated live CD45.1⁺ cells demonstrating gating of CD11b⁺Gr-1^low cells; right panel, gated live CD45.1⁺CD11b⁺Gr-1^low cells demonstrating gating of side scatter (SSc) and CD11c to determine percentage MDCs. (B) Mean ± SEM absolute number (log 10) CD45.1⁺ recipient MDSC-derived MDCs expressing PD-L1 (PD-L1⁺ MDC) at day 7 post-BMT in spleens of adoptive recipients in treatment groups shown in Figure 5B. (C) Mean ± SEM absolute number (log 10) recipient MDSC-derived MDCs (CD45.1⁺ MDC) (left) and H-2K^d+CD4⁺CD25⁺Foxp3⁺ donor-derived Treg (H-2K^d+ Treg) (right) per 10⁶ PBMC at day 7 following secondary BMT in adoptive recipients shown in Figure 5B. Data are cumulative (n = 5 experiments). (D) Protocol for depletion of MDSC-derived MDCs following MDSC adoptive transfer: (1) CD11c-hDTR transgenic (CD45.2) B6 mice received TLI/ATS/CTX followed by (2) CD45.1 BALB/c BMT. (3) At day 7 following BMT, H-2K^d-negB220^negCD11b⁺Gr-1^highCD11c^neg MDSCs were sorted from spleens of recipients in (2) and 2 × 10⁶ MDSCs infused IV (3) into adoptive CD45.1 B6 recipients of 1000 cGy TBI (4), followed by (5) CD45.1 BALB/c BMT. (5) Adoptive recipients of CD11c-hDTR transgenic MDSCs received either PBS vehicle control or 8 ng/g DT at 72 hours (day 3) post-BMT (6). Adoptive hosts were followed for 80 days for survival and signs of GVHD, with peripheral blood collected on days 7 and 28 to assess key immune subsets by FACS. BMT at day −7 = 50 × 10⁶ bone marrow cells + 60 × 10⁶ spleen cells from BALB/c donors; BMT at day 0 = 10 × 10⁶ bone marrow cells + 10 × 10⁶ spleen cells from BALB/c donors. (E) Representative FACS plots (left) and mean ± SEM absolute number (log 10) (right) CD45.2⁺ MDSC-derived MDCs (CD45.2⁺ MDC) per 10⁶ PBMC in adoptive recipients shown in Figure 6D. Data represent n = 2 experiments. (F) Representative FACS plots (left) and mean ± SEM absolute number (log 10) H-2K^d+CD4⁺CD25⁺Foxp3⁺ donor-derived Treg (H-2K^d+ Treg) (right) in adoptive recipients shown in Figure 6D. Data represent n = 2 experiments. (G) Representative proliferation plots for CD4⁺Foxp3⁺ donor Treg, CD4⁺Foxp3^neg donor CD4 effector, and CD8⁺ effector T cells among gated H-2K^d+TCRβ⁺ cells at day 7 in spleens of WT recipients of TLI/ATS/CTX conditioning and BMT from WT (top) or PD-1^−/− donors (bottom). Data represent a total of n = 10-15 individual mice over n = 2 separate experiments. (H) Mean ± SEM proliferation for CD4⁺Foxp3⁺ donor Treg, CD4⁺Foxp3^neg donor CD4 effector, and CD8⁺ effector T cells among gated H-2K^d+TCRβ⁺ cells at day 7 in spleens of WT recipients of TLI/ATS/CTX conditioning and BMT from WT (top) or PD-1^−/− donors (bottom). Data represent pooled samples from 2 to 3 animals per data point (n = 10 WT and n = 15 PD-1^−/−) over n = 2 experiments. (I) Mean ± SEM absolute number (log 10) CD4⁺Foxp3⁺ donor Treg among colonic IELs at day 7 in mice represented in panel H. Data represent a total of n = 10-15 individual mice per group over n = 2 separate experiments.