CCR2 Cooperatively Regulates Hematopoietic Stem Cell Homing to the Bone Marrow

Hematopoietic stem and progenitor cell transplantation (HSPCT) is a lifesaving therapy for otherwise incurable hematopoietic malignancies or bone marrow (BM) failure. Graft success after intravenous infusion requires donor HSC migration to specialized, highly vascularized BM niches for durable occupancy and sustained hematopoietic output. Homing to the BM occurs in part along the CXCL12-CXCR4 axis, and relies upon interaction with sinusoidal endothelial cells (ECs), where adhesion and tethering allows subsequent transmigration through the endothelium to the niche. However, beyond canonical cytokines including stem cell factor (SCF) and CXCL12, the scope and specificity of essential paracrine mediators in EC:HSPC crosstalk in the BM is not well understood.

Here we leverage ex vivo culture systems to characterize the human and murine HSPC extracellular vesicle (HSPC-EV) secretome. We demonstrate that enrichment of integrin α4β7 on HSPC-EV (6.5-fold compared to plasma EV, p<0.05) targets uptake into human and murine BM ECs through interaction with its ligand, vascular cell adhesion molecule-1 (VCAM-1). HSPC-EV uptake into ECs results in canonical NF-kB activation and downstream transcriptional upregulation of CC motif chemokine receptor 2 ligands (CCR2L; including Ccl2, Ccl7, and Ccl12) (>8-fold, p<0.05). Ongoing investigations of small RNA (sRNA) content in HSPC-EV have revealed Klf4, a negative regulator of NF-kB, to be an enriched cellular target of EV miRNA, and transcriptionally suppressed in ECs after EV uptake (0.7-fold, p<0.01). These data support a role for HSPC-EV cargo in shaping the EC secretome.

Functionally, CCR2L synergistically enhance transwell migration in vitro in combination with CXCL12 (up to 2-fold, p<0.05). Confocal immunofluorescence imaging and immunoblot analysis reveal increased co-localization and co-precipitation of CCR2 and CXCR4 in EC-activated HSPCs (>3-fold, p<0.05), supporting a model of CCR2L-induced receptor cooperativity. CCR2 also showed dynamic upregulation on homed HSPCs within 20 hours of transplantation. In competitive transplantation studies, deficits in overall homing from Ccr2^-/- grafts were noted compared to wild type (WT) (0.3-fold, p<0.001). However, significant specificity in CCR2 dependency for BM homing was seen between HSCs and more differentiated multipotent progenitors (MPPs), resulting in impaired homing of Ccr2^-/- HSC and MPP2, and relative enrichment of Ccr2^-/- MPP3/4. Subpopulation-specific differences in homing were reflected in diminished myeloid output (0.7-fold, p<0.05) from Ccr2^-/- grafts at 12 weeks post-transplantation, consistent with prior evidence supporting a myeloid differentiation bias of CCR2+ HSPC. Furthermore, BM retention of Ccr2^-/- HSPCs was seen (2.2-fold, p<0.001), with coincident relative enrichment of CCR2+ cells in the PB and spleen (0.2-0.5-fold, p<0.001), supporting later roles of CCR2 in regulating niche occupancy and marrow egress. Finally, to test whether transient activation of the CCR2-ligand signaling axis could enhance early engraftment, mice were conditioned intrafemorally with HSPC-EV or recombinant CCR2L prior to transplantation. Conditioned femurs showed improved graft homing and HSPC engraftment at 20 hours and 4 weeks post-transplantation, respectively (>1.4-fold, p<0.05).

Despite the curative potential and expanding access of HSPCT, patient morbidity and mortality remains a major concern, in part attributable to delayed or insufficient levels of engraftment. Understanding and refining mechanisms of HSPC homing and niche retention has promising potential to minimize toxicity and maximize early engraftment. Our findings identify a therapeutically accessible axis of HSPC:EC vesicle crosstalk and newly define a role for the highly dynamic, niche-dependent regulation of CCR2 in HSPC BM occupancy. Altogether, these studies inform novel translational conditioning strategies to improve HSPCT.