*Purpose: We previously demonstrated that donor-derived regulatory dendritic cells (DCreg), contrary to recipient derived DCreg, promote antigen-specific allograft protection. As myeloid-derived suppressor cells (MDSC) show an enhanced immune suppressive capability, we hypothesize that donor-type MDSC suppress alloimmunity by regulating donor-specific T cell clones.

*Methods: MDSC were generated from BALB/c bone marrow cells. Allogeneic mix lymphocyte reaction (alloMLR) and regulatory T cells (Treg) induction assays were performed in vitro. B6 recipients were pre-treated with BALB/c MDSCs or conventional DCs (cDC) control. Subsequently, these mice were transplanted with either donor-type (BALB/c) or third-party (C3H) cardiac grafts. We then examined graft survival, allo-rejection/tolerance associated immunophenotype and graft mRNA expression.

*Results: Flowcytometry showed increased level of CX3CR3, PD1, IL4 and decreased level CD40/80/86, MHC class-II in MDSC when compared to cDC (fig-A). RNA-seq showed an upregulation of Tgfbi and s100A8/A9 (fig-B). MDSC significantly suppressed the proliferation of alloreactive CD4⁺ and CD8⁺ T cells in alloMLR.

MDSC treated group (MG) had a prolonged BALB/c cardiac graft survival when compared to other groups (fig-C). HE staining revealed a significant decrease of allograft infiltrating lymphocytes (GIL) in MG (fig-D). The proportion of CD4⁺ effector memory T cells (T_EM) within GIL and draining lymph nodes (dLN) was significantly decreased in MG (fig-E). The mRNA expression analysis revealed an interesting observation of FoxP3 upregulation along with IFNg and IRF1 in MG’s allografts (fig-F).

To better understand the mechanisms involved, we simulated allorejection by stimulating MDSC with IFNg in vitro and performed RNA-seq. When we analyzed upregulated genes by using STRING database, we found an enrichment of type I IFN related genes that showed an active interaction pattern (fig-I). Interestingly, stimulating MDSC with IFNg resulted in upregulation of secretion of IFNa by MDSC (fig-G). Moreover, we found that IFNa preferentially induce Treg and suppress the proliferation of non-Treg both in vitro (fig-H) and in vivo.

*Conclusions: MDSC protect cardiac allograft from acute rejection by suppressing T_EM, and inducing Treg in a donor antigen specific manner. MDSC induce immune regulation by transforming inflammatory IFNg into regulatory IFNa signaling that preferentially promote Treg.

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