Prevention Of GVHD By Donor MDSCs In Mouse Haploidentical HSCT Model

Objective:The aim of this study is to investigate the prevention of GVHD by donor MDSCs in mouse haploidentical hematopoietic stem cell transplantation model based on busulfan and fludarabine as a conditioning regimen, as well as to explore the mechanism of prevention of GVHD by MDSCs mobilized with methylprednisolone or rh G-CSF.Methods:B6D2F1 mice were conditioned with busulfan and fludarabine and transplanted with splenocytes and bone marrow from C57BL/6 mice.B6D2F1 mice were assigned into four groups: MP group(6*106methylprednisolone mobilized MDSCs + 75*106 splenocytes+10*106 bone marrow cells), G-CSF group(6*106 G-CSF mobilized MDSCs+ 75*106 splenocytes+10*106 bone marrow cells), Resting MDSCs group(6*106resting MDSCs+ 75*106 splenocytes+10*106 bone marrow cells), SPL control group(75*106 splenocytes+10*106 bone marrow cells without MDSCs), Flow cytometric was applied to confirm engraftment. GVHD was monitored by the lost of body weight, GVHD clinical scores and the survival of each group. Histology of the skin, small intestine and lung were used to analysis the servity of GVHD. quantitative RT-PCR assay was applied to compare the expression of micro RNA155, micro RNA21, AGR1, i NOS in MDSCs before and after mobilization. Mixed lymphocyte reaction was applied to detect the inhibition function of donor MDSCs.Results:①Establishment of Haplo-HSCT a GVHD mouse model: we found busulfan 20ug/g and fludarabine 10ug/g twice a day for four days as a conditioning regimen followed by transplantation of 75*106 SPL and 10 *106BM cells, lethal GVHD can be induced, showing lost of body weight, hunch back, diarrhea and hair lost. Engraftment was confirmed by flow cytometry, measuring donor-derived CD4-Positive lymphocytes,CD8-positive lymphocytes,DCs and B cells.②Optimized dose of methylprednisolone and hn G-CSF mobilization: By comparing the expression of MDSCs( CD11 b and GR – 1),we determined the optimized dose as follows: methylprednisolone 4mg/per mouse qd*3 and G-CSF 250 ug/kg qd*5. ③Prevention of GVHD by methylprednisolone or hn G-CSF mobilized MDSCs: the survival in MP and G-CSF group was longer than SPL group. Furthermore, we observed that MP and G-CSF group markedly reduced lymphocyte infiltration and tissue damage in GVHD target organs. ④ The mechanism of prevention of GVHD by donor MDSCs: we observed that the expression of mi R-155, mi R-21 significantly increased in MP and G-CSF group; Also, the expression of i NOS significantly increased in G-CSF group. There was no significant difference in the expression of PD-L1.Conclusion:1. Donor MDSCs mobilized by methylprednisolone and hn G-CSF can prevent GVHD effectively in the established mouse haplo-HSCT model. 2. The prevention of GVHD by donor MDSCs mobilized by methylprednisolone and hn G-CSF is associated with up-regulation of micro RNA155, micro RNA21 and i NOS.