Phenotypic and Functional Characterization Of a Novel Immature Hematopoietic Myeloid Suppressive Progenitor Cells Mobilized By G-CSF. Application To Gvhd Treatment

Myeloid derived suppressive cells (MDSCs) represent a heterogeneous population of cells endowed with immunosuppressive properties. They have been first described in the tumor microenvironment. Some mature MDSCs either induced by GM-CSF and IL-13 (Highfill et al., Blood 2010) or mobilized by G-CSF (Joo et al., Immunology 2009) have been reported to control experimental GVHD by inhibiting alloreactive T cell proliferation. We describe here the existence in mice and humans of a not yet characterized population of GCSF-mobilized hematopoïetic cells with phenotypic characteristics of immature MDSCs (called therefore pro-MDSCs) that can inhibit GVHD by a distinct mechanism than those described with classical mature MDSCs.

In the C57BL6 mouse and human, G-CSF mobilized MDSCs were collected and analyzed in the spleen and PBSC using several antibodies directed against various markers of maturity, lineage specific antigens and chemokine receptors. Depending on the expression of maturity antigens various population were sorted. In vitro, functions of sorted MDSC were analyzed by co-cultures with T cells activated either by anti-CD3 and CD28 mAbs or allogeneic dendritic cells. In vivo, the effect of various population of MDSCs on GVHD was assessed either by the transfer of murine C57BL6 (H-2^b) cells (2x10⁶ splenic T cells + 5x10⁶ T depleted bone marrow cells +/- 0.5x10⁶ MDSC subtypes) into lethally irradiated BALB/c (H-2^d) recipients or by injecting 2x10⁵ human pro-MDSCs with 2.5x10⁶ human PBMC into 2 Gy irradiated Nod/SCID/gammac^-/- mice. In 19 allografted patients, proportions of MDSC subpopulations contained in the peripheral stem cell graft were correlated to the occurrence of acute GVHD and to the post-transplant peripheral blood levels of conventional proliferating T cells and CD4⁺ CD25⁺ CD127^low reguatory T cells (T regs).

In the G-CSF mobilized cells, immature Lin^- Sca1^high cKit^high CD34⁺ CX3CR1⁺ CD16/32⁺ CD11b⁺ Ly6C+ and Lin^- CD34⁺ HLA-DR^- CD33^high CD11b^low CD14⁺ cell populations were identified in mice spleen and human PBSC, respectively. Because the pattern of maturity antigen expression, these populations were named pro-MDSCs. The mature MDSC counterparts shared the same differentiation phenotype without the markers of maturity.

In vitro, both murine and human pro-MDSCs, but not the corresponding mature MDSCs, could inhibit the proliferation and induced the apoptosis of activated T cells (p<0,001). The inhibition of T cell activation by pro-MDSCs required IFN-gamma produced by activated T-cells and the production of NO by pro-MDSCs in response to IFN-gamma. NO suppressed T-cell functions through impaired responses to IL2 and induction of apoptosis. In vivo, in the C57BL6 to BALB/c GVHD model, the administration of murine pro-MDSCs significantly reduced the development of clinical and histological GVHD signs as compared to allografted mice without pro-MDSCs or with GCSF-mobilized mature MDSCs (p=0,03). Murine pro-MDSCs could migrate to site of allo-priming and induced the apoptosis of allogeneic T cells when compared to mice allografted without pro-MDSCs (p<0,01). In mice that had received pro-MDSCs, we observed that apoptotic T cells could be engulfed by phagocytes and that those phagocytes produced high levels of cytokines (IL-10, TGF-beta), which was associated with increased induced CD4⁺CD25⁺Foxp3⁺ T regs leading to the induction of tolerance. These observations were not seen in mice allografted without pro-MDSCs (p<0,05). Human pro-MDSCs could protect all xeno-grafted Nod/SCID/gamma c^-/- mice from GVHD mortality as compared to 100% GVHD lethality in controlled xeno-grafted mice without pro-MDSCs (p<0,001). Allografted patients having received a stem cell graft containing levels of Pro-MDSCs >10% of the CD34⁺ fraction had a significantly reduced risk of developing grade II-IV acute GVHD (p= 0,04) and reduced numbers of proliferating conventional T cells but higher numbers of T regs in the peripheral blood on days 15 and 30 post-HSCT (p<0.05). No correlation between the occurrence of acute GVHD and the proportions of mature MDSCs contained in the graft was observed.

We have characterized a new homogeneous population of G-CSF mobilized immature MDSCs, which has been named pro-MDSC that can regulate alloreactive T cell activation in vitro and in vivo by inducing tolerance with potential therapeutic application in allogeneic HSCT.

No relevant conflicts of interest to declare.