Donor myeloid derived suppressor cells (MDSCs) prolong allogeneic cardiac graft survival through programming of recipient myeloid cells in vivo

Abstract

Solid organ transplantation is a lifesaving therapy for patients with end-organ disease. Current immunosuppression protocols are not designed to target antigen-specific alloimmunity and are uncapable of preventing chronic allograft injury. As myeloid-derived suppressor cells (MDSCs) are potent immunoregulatory cells, we tested whether donor-derived MDSCs can protect heart transplant allografts in an antigen-specific manner. C57BL/6 (H2K^b, I-A^b) recipients pre-treated with BALB/c MDSCs were transplanted with either donor-type (BALB/c, H2K^d, I-A^d) or third-party (C3H, H2K^k, I-A^k) cardiac grafts. Spleens and allografts from C57BL/6 recipients were harvested for immune phenotyping, transcriptomic profiling and functional assays. Single injection of donor-derived MDSCs significantly prolonged the fully MHC mismatched allogeneic cardiac graft survival in a donor-specific fashion. Transcriptomic analysis of allografts harvested from donor-derived MDSCs treated recipients showed down-regulated proinflammatory cytokines. Immune phenotyping showed that the donor MDSCs administration suppressed effector T cells in recipients. Interestingly, significant increase in recipient endogenous CD11b⁺Gr1⁺ MDSC population was observed in the group treated with donor-derived MDSCs compared to the control groups. Depletion of this endogenous MDSCs with anti-Gr1 antibody reversed donor MDSCs-mediated allograft protection. Furthermore, we observed that the allogeneic mixed lymphocytes reaction was suppressed in the presence of CD11b⁺Gr1⁺ MDSCs in a donor-specific manner. Donor-derived MDSCs prolong cardiac allograft survival in a donor-specific manner via induction of recipient's endogenous MDSCs.

Figure 1

Donor-derived MDSCs suppress alloimmune reaction in vitro and in vivo. (A) Naïve C57BL/6T cells were stimulated with BALB/c antigen presenting cells (APCs: BALB/c bone marrow derived cDCs). MDSCs or cMDCs were added as modulator. CD4⁺ and CD8⁺ T cell proliferation in response to allogeneic cDCs was analyzed by CellTrace violet dye dilution (n = 5 per group). (B) Graphs showing the proliferation of CD4⁺ and CD8⁺ T cells in the presence of MDSCs compared to cMDCs. Mean ± SEM, *p < 0.05, **p < 0.01, two-tailed unpaired t test. Data represents one of 4 separate experiments. (C) Schematic diagram of the experimental design. C57BL/6 recipients received a single-dose intravenous injection of 1 × 10⁶ BALB/c MDSCs or cMDCs 7 days prior to the cardiac transplantation. (D) Kaplan–Meier cumulative survival of allograft shows the prolonged survival in MDSCs treated group compared to control groups (n = 8–9 per group). **p < 0.01, ***p < 0.001, log-rank test.

Figure 2

Allografts from donor-derived MDSCs treated recipients exhibit ameliorated T cell mediated inflammation and increased recipient-derived endogenous MDSCs. (A) Representative example of the allograft histology (H&E staining) from the MDSCs treated group and the cMDCs treated control on POD7. Scale bar represent 500 μm (left) and 100 μm (right). Data represents the one of 3 separate experiments. (B) Allografts were harvested on POD7 and stained with anti-CD3, anti-CD11b (green) and DAPI (blue). Scale bars represent 200 μm. Positive cells were qualitied by ImageJ and expressed as mean ± SEM. ****p < 0.0001, two-tailed unpaired t test. (C) Flow cytometry analysis of BALB/c MDSCs treated C57BL/6 recipients’ graft infiltration lymphocytes (GILs) on POD7. Graphs showing significant decrease of the proportion of CD44⁺CD62L^lo and Ki67⁺ in CD4⁺FoxP3^- T cells in the MDSCs treated group. (D) qRT-PCR analysis of allografts (whole tissues) on POD3 (n = 3 per group). Graph represented as RQ (relative quantification) = 2^-ΔΔCt. Naïve BALB/c heart serve as basic ΔCt (n = 3, not shown in graph). Mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, two-tailed unpaired t test.

Figure 3

Donor-derived MDSCs suppress effector T cell activation. (A) Schematic diagram of the experimental design. T cells were isolated from recipient splenocytes on POD7 and were stimulated with BALB/c cDCs for 4 days (n = 4 per group). (B) CD4⁺ and CD8⁺ effector T cells induction was analyzed by flow cytometry. Cells were gated on CD4⁺ Teff (CD4⁺FoxP3^-CD44⁺CD62^lo) and CD8⁺ Teff (CD8⁺FoxP3^-CD44⁺CD62^lo). Graphs showing the percentage of activated Teff (Ki67⁺Teff). Mean ± SEM, *p < 0.05, **p < 0.01, two-tailed unpaired t test. Data represents one of 4 separate experiments.

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.

Figure 5

Donor-derived MDSCs prolong allogeneic cardiac graft survival in a donor-specific manner. (A) Schematic diagram of the experimental design. C57BL/6 mice received a single-dose intravenous injection of 1 × 10⁶ BALB/c or C3H MDSCs. Cardiac grafts from C3H or BALB/c mice were transplanted into pre-sensitized C57BL/6 recipients. (B) Kaplan–Meier cumulative survival of allograft showing that the BALB/c MDSCs administration successfully prolong BALB/c allograft survival but fail to prolong C3H allograft survival, while C3H MDSCs administration prolong C3H allograft survival but fail to prolong BALB/c allograft survival. *p < 0.05, **p < 0.01, ***p < 0.001, log-rank test. (C) Schematic illustration of the experimental design to study the ex vivo donor-specific immunosuppressive function of donor MDSCs induced endogenous MDSCs (CD11b⁺Gr1⁺). Naïve C57BL/6 spleen T cells were labeled with CellTrace violet and were stimulated with BALB/c cDCs or C3H cDCs (third-party). CD11b⁺Gr1⁺ cells were isolated by FACS from BALB/c MDSCs treated C57BL/6 recipient splenocytes at POD7 (n = 3–4 per group) and were added as modulator. (D) CD4⁺ and CD8⁺ T cell proliferation in response to primary donor-type BALB/c cDCs or third-party C3H cDCs was analyzed by CellTrace violet dye dilution. Graphs showed the attenuated suppression efficiency (SE) of CD11b⁺Gr1⁺ cells in third-party compared to donor-type allo MLR setting. $SE=\frac{p\left(w/oMDSC\right)-p\left(\mathit{MDSC}\right)}{p\left(w/oMDSC\right)}\times 100\%$. Mean ± SEM, *p < 0.05, **p < 0.01, two-tailed unpaired t test. Data represents one of 3 separate experiments.

Figure 6

CD11b⁺Gr1⁺ population in donor-derived MDSCs is the effective suppressor subset. (A) Schematic illustration of the experimental design to study which subset in donor-derived MDSCs impart the immune suppressive function. C57BL/6 recipients received a single-dose intravenous injection of 1 × 10⁶ CD11b⁺Gr1⁺ or CD11b⁺Gr1^- or CD11b^- cells FACS isolated from BALB/c derived MDSCs 7 days prior to BALB/c derived cardiac graft transplantation. Whole MDSCs infusion and PBS administration groups served as positive and negative control. (B) Kaplan–Meier cumulative survival of allograft showing only CD11b⁺Gr1⁺ subset rather than CD11b⁺Gr1^- and CD11b^- subsets, reproduces similar allograft prolongation to the whole BALB/c MDSC infusion (p = 0.2459, no significant difference). Log-rank test. (C) Schematic diagram of the experimental design to study whether CD11b⁺Gr1⁺ population from MDSCs and cMDCs have the same immune suppressive function. C57BL/6 recipients received a single-dose intravenous injection of 1 × 10⁶ CD11b⁺Gr1⁺ cells FACS isolated from BALB/c MDSC or cMDC 7 days prior to the cardiac transplantation. (D) Kaplan–Meier cumulative survival of allograft showing CD11b⁺Gr1⁺ cells from MDSCs prolong allograft survival, while C57BL/6 mice treated with CD11b⁺Gr1⁺ cells from cMDC slightly accelerate the rejection. *p < 0.05, **p < 0.01, ***p < 0.001, log-rank test.