Phenotypic Myeloid-Derived Suppressor Cells (MDSCs) are expanded after Post-Transplantation Cyclophosphamide (PTCy), but are not necessary for Graft-Versus-Host Disease Prevention by PTCy

The use of PTCy substantially lowers the rates of severe acute and chronic graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT), but the mechanisms of its action are incompletely understood. Recently, we showed in a T-cell–replete, MHC-haploidentical, murine HCT model (B6C3F1→B6D2F1) that PTCy prevents GVHD not by eliminating alloreactive T cells but instead by inducing alloreactive T-cell dysfunction and suppression. Depletion of CD4+regulatory T cells (Tregs) early post-transplant did not result in substantially worsening GVHD for ∼4 weeks. Surprisingly, we found that alloreactive T cells could be infused in extremely high numbers early after PTCy but not cause GVHD. Here, we used this same MHC-haploidentical murine HCT model to test the role of Tregs in preventing GVHD induced by infusion of new splenocytes after PTCy; depleting Tregs in the initial graft but not the reinfused splenocytes did not result in worsening GVHD in these mice. However, when Tregs were depleted both in the initial graft and in the reinfused splenocytes, universally fatal GVHD ensued. These results suggested that Tregs were necessary to prevent GVHD but a direct effect of PTCy on Tregs was not required. Thus, we hypothesized that other suppressive cell populations may be playing a critical role early after PTCy. We examined the frequency of MDSCs after PTCy in our model and found that both granulocytic and monocytic MDSCs were increased at post-transplant days +7 and +21 compared with vehicle-treated mice. Next we examined the role of MDSCs in GVHD prevention after PTCy by using a Gr-1-depleting antibody. Gr-1 depletion immediately post-transplant led to decreased percentages of Tregs and inferior survival, clinical scores, and weights in PTCy-treated mice. Conversely, Gr-1 depletion at days +28 or +150 had a transient effect only on weights but no impact on clinical scores or survival. Yet, histopathologic evaluation revealed that mice depleted of MDSCs early post-transplant did not have worse GVHD; instead the worse survival in those mice appeared related to infection presumably as a result of neutrophil depletion by the anti-Gr-1 antibody in mice with mucosal disruption by radiation and GVHD. We also tested the suppressive functionality of MDSCs on alloreactive T cells in in vitro mixed lymphocyte cultures. We found neither population of MDSCs from either vehicle-treated or PTCy-treated mice suppressed the proliferation of vehicle-treated T cells nor was inflammatory cytokine secretion reduced. Overall, these findings confirm that Tregs are required for GVHD prevention by PTCy but a direct effect of PTCy on Tregs is not required; although phenotypic MDSCs are expanded after PTCy and may contribute to Treg expansion, these MDSCs do not appear functionally capable of suppressing alloreactive T-cell response to alloantigen in vitro or decreasing GVHD in vivo.