Hematopoietic Multipotent Progenitor Cells Mobilized by G-CSF Can Inhibit Gvhd

Acute graft-versus-host-disease (aGVHD) is a frequent life threatening complication of allogeneic hematopoietic stem cell transplantation (HSCT). Despite the infusion of higher doses of T cells with the use of G-CSF-mobilized HSC grafts, the incidence of aGVHD is not increased. The mechanisms by which G-CSF-mobilized HSC can control GVHD are imperfectly elucidated. We previously described the mobilization of murin hematopoïetic progenitor cells (HPCs) by G-CSF and FLT3 ligand capable of inducing tolerance against autoimmune diabetes in the nude mice (Kared, Immunity 2006). We now show that G-CSF can mobilize murin HPCs with immunoregulatory functions in the allogeneic immune response and describe their mechanisms of action.

Mobilization of HPCs is performed by subcutaneous administration of human recombinant G-CSF at 200μg/kg per day, for 4 consecutive days in the C57BL6 (H-2^b) mouse. HPCs are collected in the spleen by FACS sorting according to their phenotype: Lin- Sca1^high cKit^high FLT3^low CD34⁺ CD106⁺ CD127⁻. In vitro, functions and mechanisms of action were analyzed by co-cultures with i) T cells (from C57BL6) activated by anti-CD28 and -CD3 mAbs or activated by BALB/c (H-2^d) allogeneic splenic LPS matured dendritic cells, ii) C57BL6 splenic selected CD4⁺CD25^high T regulatory T cells activated by anti-CD28 and -CD3 mAbs iii) activated antitumor specific CD8 T cells (C57BL6 ovalbumin specific TCR transgenic T cells). These different cultures were performed in the presence or absence of inhibitors of selective cytokines or other regulatory molecules. In vivo, we assessed the effect of donor HPCs on GVHD development by injecting C57BL6 derived HPCs (0.5×10⁶/mouse), splenic T cells (1×10⁶/mouse) and T depleted bone marrow cells (5×10⁶/mouse) into lethally irradiated (8 Gy) Balb/c recipients.

In vitro, as compared to controls without HPCs, after 3 days of culture, HPCs: 1) promote the proliferation of natural T regs activated by anti-CD3 and anti-CD28 (>80% at 3 days of culture compared to control <50%), 2) inhibit the proliferation of activated T cells (>80% T cells blocked before 4 divisions as compared to control-T cells alone >80% after 4 divisions- p<0, 001) and 3) induce the apoptosis of activated T cells (30% increased, p=0, 01). The proliferation of T regs was cell contact dependant and required the presence of TGF-b. The inhibition of T cell activation required IFN γ produced by activated T-cells and some contact-dependent stimuli. In such pro-inflammatory conditions, HPCs differentiate after 4 days in myeloid derived suppressor cells (MDSC). These cells could then produce NO in response to IFN γ and suppress the proliferation of activated T cell. However, T cell suppression was not dependant on L-arginine depletion. Induction of apoptosis of T cells was Fas/Fas-L dependant. Although in the presence of HPCs the proliferation of CD8⁺ T TCR transgenic against the dominant ovalbumin epitope SIINFEKL was reduced, the cytotoxic response against the SIINFEKL-pulsed EL4 cell line was enhanced (cytotoxicity >90% with HPCs versus <90% w/o HPCs, p<0, 001). In addition, HPCs express CCR7 and CD62L, which should allow their migration to the sites of allopriming. In vivo, none of the mice that had received allogeneic HSCT with HPCs developed clinical or histological GVHD signs as compared to 50% of the control allografted mice without HPCs.

Hematopoietic progenitor cells acquire an immunosuppressive potential after G-CSF mobilization. These cells can be isolated from mobilized peripheral stem cells and suppress GVHD while possibly preserving the GVL effect. Work is underway in humans to identify and amplify this population ex vivo for potential therapeutic application in allogeneic HSCT.

No relevant conflicts of interest to declare.