Pegylated Murine GM-CSF Increases Myeloid Derived Suppressor Cells In Vivo

Abstract 2967

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells initially identified in tumor bearing mice that have potent immunosuppressive capabilities. Recent evidence suggests that graft-versus-host disease (GvHD) can be abrogated by ex vivo expanded, bone marrow derived, MDSCs generated in the presence of GM-CSF, G-CSF and IL-13 (Highfill et al. Blood 2010 116 :5738). It remains to be shown whether phenotypic MDSCs identified in non-tumor bearing mice are capable of immune suppression. In addition, the mechanism by which an immature myeloid cell becomes a functional MDSC remains unknown.

We hypothesized that pegylated murine GM-CSF (peg-mGM) may be protective from acute GvHD in MHC mismatched murine models by increasing regulatory T-cells (Treg) and MDSCs. Previously, we reported that peg-mGM increased circulating and splenic Tregs by 2–3 fold and they were functional in mixed leukocyte reactions (MLRs). We have also reported on the in vivo potential of mobilized splenocytes to abrogate murine GvHD. B6D2F1 mice receiving C57/Bl6 GM treated splenocytes had improved survival and less weight loss compared to G-CSF and PBS controls (3 independent experiments, n=15-19/group, GM vs. G p = 0.0005, GM vs. PBS p = 0.0005, G vs. PBS p = 0.5 (Log rank test)).

In an attempt to identify cellular mediators of the reduced incidence of GvHD we investigated the impact of peg-mGM on putative MDSCs. We have observed an ∼8 fold increase in putative monocytic MDSCs (monoMDSCs) (CD11b+Ly6C+Ly6G-) and an ∼18 fold increase in putative granulocytic MDSCs (granMDSCs) (CD11b+Ly6C+Ly6G+) in the spleens and blood of mice mobilized with peg-mGM. To investigate the function of MDSCs we performed bead stimulated tritiated thymidine and CFSE based proliferation assays. We observed that granMDSCs and monoMDSCs isolated from spleens of mice treated with peg-mGM have potent suppressive function on bead stimulated T-cell proliferation exceeding that of na•ve Tregs at equal suppressor :Tcell ratios (Fold suppression of CD4+ T-cells: granMDSCs = 4.5, monoMDSCs = 2.3, Tregs = 1.08. Fold suppression of CD8+ T-cells: granMDSCs = 2.26, monoMDSCs = 1.4, Tregs = 1.05). To investigate mechanism we performed a transwell experiment using bead stimulated T-cells separated from MDSCs by a permeable membrane. Sorted monoMDSCs and granMDSCs were not suppressive in this assay suggesting the dependence on contact for inhibition of T-cell proliferation. In addition, we observed that in bead stimulated proliferation assays wells containing putative MDSCs had more dispersed beads suggesting possible sequestration of beads by the suppressor cells. To determine if all subsets were suppressive in an alternative non-bead based proliferation assay we coated plates with CD3/CD28 antibodies. Only the putative monoMDSCs were suppressive in this assay. We observed that suppression of bead stimulated T-cells was abrogated by adding an arginase-1 inhibitor, nor-NOHA, to cultures containing putative monoMDSCs. When attempting to validate these results in a MLR using MHC mismatched antigen-presenting cell (APC) stimulation, the suppressive effect was decreased or lost suggesting that the magnitude of stimulation by APC, bead or antibodies may affect activation and function of MDSCs; or, a critical factor produced in bead and antibody stimulated T-cell proliferation assays is lacking in the APC setting. We are currently functionally characterizing the monoMDSCs generated by treatment with peg-mGM and investigating potential secondary factors critical to the development of MDSCs such as IL-13 and IFN-gamma. In addition, future studies will evaluate the in vivo function of monoMDSCs generated by peg-mGM mobilization on GvHD and GVL outcomes.

In summary, treatment with peg-mGM results in enrichment in functional MDSCs in the spleens of non-tumor bearing mice. The mechanism by which immature myeloid cells generated by peg-mGM become MDSCs is under investigation but appears to be contact dependent. This work is currently being translated in a clinical trial investigating the combination of GM-CSF and plerixafor for the mobilization of peripheral blood stem cells for allogeneic stem cell transplantation from matched sibling donors. Correlative studies to characterize stem cell subsets and evaluate the content of Tregs and MDSCs in the blood and apheresis product are ongoing.