CD8+/TCR- Graft Facilitating Cells Enhance Homing of Hematopoietic Stem Cells

Abstract

Establishment of mixed chimerism by hematopoietic stem cell (HSC) infusion is a promising approach to induce immune tolerance for organ transplantation. CD8+/TCR- bone marrow facilitating cells (FC) facilitate engraftment of HSC in both allogeneic and syngeneic recipients. The mechanisms by which FC promote HSC engraftment have not been fully elucidated. Homing of HSC to the bone marrow niche is believed to be a crucial prerequisite for engraftment. Therefore, we evaluated whether FC enhance functional HSC homing and lodgment in the hematopoietic niche using the in vivo syngeneic homing model followed by colonyforming cell (CFC) assay. Recipient B6 mice were conditioned with a supralethal dose (1200 cGy) of total body irradiation and transplanted with 75,000 B6 FC alone; 25,000 B6 HSC alone; or FC plus HSC 24 hours after irradiation. At 18 hours post-transplantation, bone marrow was harvested from recipient's femurs and tibias and placed in CFC assay using methylcellulose-based media. Bone marrow cells harvested from the mice transplanted FC alone or received conditioning alone did not generate colonies, which confirmed that there was no recipient's HSC left after a supralethal dose of total body irradiation, and FC themselves did not have repopulation capacity in vivo. Notably, the bone marrow cells harvested from the mice transplanted with HSC and FC formed significantly higher numbers of colonies compared to that from the mice transplanted with HSC alone. The chemokine receptor CXCR4 plays a pivotal role in HSC homing. We then determined whether the effect of FC on HSC homing was mediated by FC increasing CXCR4 expression in HSC. CXCR4 expression in HSC was measured by flow cytometery 18 hours after co-culture of HSC and FC in vitro. Incubation of FC with HSC did not lead to any alteration of CXCR4 expression in HSC compared with HSC cultured alone. Our previous studies suggested that cell:cell interaction between FC and HSC was necessary for FC facilitation. We thus investigated whether the migration function of FC was important for FC to the augmentation of HSC homing. DOCK2 activates the small GTPase Rac and is indispensable for migration of neutrophils, lymphocytes and plasmacytoid dendritic cells (DC). FC is a heterogeneous cell population, with a predominant subpopulation resembling plasmacytoid precursor DC. Cell migration-compromised FC (sorted from the bone marrow of DOCK2 knock-out mice) were co-transplanted with HSC. FC augmentation of HSC colony formation after in vivo syngeneic homing was abrogated in the cell migration-compromised DOCK2 -/- FC plus HSC group compared to wild-type FC plus HSC group. In summary, our results suggested FC might have a tropic effect on HSC stem cell homing and retention in the bone marrow environment. This effect was not mediated by increasing CXCR4 expression in HSC. Migration function of FC might be critical for FC to enhance HSC homing. These data suggest that FC may be a critical component of the hematopoietic niche.