S245: ALTERATION OF BONE MARROW NICHE BY ALLOGENEIC IMMUNE REACTION AFTER HSCT

Abstract

Background: Allogeneic stem cell transplantation (allo-SCT) has been considered as a curable therapy for hematologic diseases. Post-transplant myelosuppression is one of the life-threatening complications. Hematopoietic stem cell (HSC) functions are tightly regulated by a specialized microenvironment called “niche” in the bone marrow (BM) and we have identified differential contributions of perivascular stromal cells as HSC niche (Asada et al. Nat Cell Biol 2017). Recent studies indicate that BM niche can be targeted by allo-immune reaction but it remains unclear how the perivascular niche cells are involved in the mechanism of HSC dysfunction after allo-SCT. Aims: In this study, we aim to investigate the mechanisms of the impairment of the niche cells by allo-immune reactions, and to explore a targetable pathway for myelosuppression after allo-SCT. Methods: To evaluate the alterations in niche cells after allo-SCT, we utilized allo-SCT mice models (C3H/HeJ into C57BL/6, C57BL/6 into B6D2F1). Hematopoietic cells, including HSCs and mature cells, and perivascular stromal cells in BM were analyzed by flow cytometry (FACS) after transplantation. In imaging studies, we visualized perivascular stromal cells using Nestin-GFP (Nes-GFP), Myh11-CreER/tdTomato, LeptinR-Cre/tdTomato transgenic mice and observed them with confocal laser scanning microscopy. The gene expression analysis of niche factors was performed by real-time PCR. To assess the landscape of transcriptional remodeling in BM after allo-SCT, we performed single-cell RNA sequencing (scRNAseq) of BM cells. Results: FACS analyses showed that Nes-GFP+ stromal cells were dramatically reduced, and HSC recovery was severely impaired in allo-SCT mice compared to control mice at day 21 after SCT. Imaging studies also revealed that perivascular stromal cells are morphologically damaged in allo-SCT mice. Nes-GFP+ stromal cells in allo-SCT mice presented a decreased expression of niche factors, including Cxcl12 and Scf, which are essential for HSC maintenance, indicating the dysfunction of niche cells. We confirmed the impaired niche function in allo-SCT mice by tandem transplantation analysis in vivo, in which the recovery of syngeneic HSCs was also impaired in damaged microenvironment in allo-SCT mice. Prevention of allo-reactive T cell expansion by post-transplant cyclophosphamide ameliorated the niche cell reduction along with improved recovery of HSCs. These results indicate that niche impairment caused by allo-immune reaction leads to HSC dysfunction. The scRNAseq of BM highlighted the elevated expression levels of inflammation response genes, including Cxcl9 and Cxcl10, and MHC classⅡ, together with the deceased expression of niche factors in niche cells in allo-SCT mice. These alterations seemed to be induced by proinflammatory cytokines such as interferon-gamma (INF-γ) derived from allo-T cells, as the increased number of T cells and high levels of INF-γ in BM were observed in allo-SCT mice. Additionally, evaluation of T cell localization by using imaging techniques of BM revealed that allo-T cells reside significant closer to LepR+ niche cells in allo-SCT mice. Since activation of CXCR3, which is a receptor for chemokines Cxcl9 and Cxcl10, has been shown to induce chemotaxis of activated T cells, we treated allo-SCT mice with AMG487, an antagonist of CXCR3, resulted in an improved recovery of both perivascular niche cells and hematopoiesis in allo-SCT mice. Summary/Conclusion: Collectively, allo-immune reaction severely damages perivascular HSC niche cells, leading to impaired recovery of hematopoiesis after allo-SCT.