CCR2 chemokine receptor promotes hematopoietic stem and progenitor cell mobilization Free

Hematopoietic stem and progenitor cell (HSPC) transplantation offers a lifesaving therapy for many hematological and non-hematological disorders. Success of this treatment largely relies upon adequate numbers of HSPCs being mobilized from bone marrow (BM) to peripheral blood (PB). Granulocyte-colony stimulating factor (G-CSF) is the most widely used mobilizing agent. Targeted inhibition of CXCR4, a chemokine receptor involved in HSPC migration and BM adhesion, by plerixafor is frequently used to enhance HSPC mobilization. While current mobilization strategies are effective for most healthy donors, a significant number of patients undergoing autologous HSPC collection, particularly those with prior chemotherapy or advanced disease, fail to collect optimal numbers of donor cells for transplantation. Consequences of suboptimal cell collection include delayed transplantation, risk of disease progression, multiple apheresis sessions that add donor discomfort and healthcare cost, and poorer transplant outcomes. These challenges underscore the need for novel, targeted strategies to optimize HSPC mobilization and reduce the occurrence of poor mobilizers.

Recently, we uncovered a functional interaction between the chemokine receptors CXCR4 and CCR2 that guides BM homing and occupancy. We hypothesize that CCR2-CXCR4 interactions also govern HSPC mobilization and may be harnessed to improve graft collections. To explore the role of CCR2 in PB trafficking, we enriched and analyzed HSPCs from mobilized human donors who received G-CSF alone (G; HSPC^G) or G-CSF in combination with plerixafor (GP; HSPC^GP).

GP-mobilized donor HSCs (CD19^-CD34⁺CD38^-CD45RA^-CD49f⁺CD90⁺) and granulocyte monocyte progenitors (GMPs; CD19^-CD34⁺CD38⁺CD7^-CD10^-CD45RA⁺) showed higher levels of CXCR4 and CCR2 surface expression compared to G-mobilized cells. In transwell migration assays, HSPC^GP showed higher baseline chemotaxis compared to HSPC^G (1.9 fold-change; p<0.05); however, in both cohorts, the CCR2 ligand CCL7 synergized with CXCL12 to enhance CCR2 HSPC chemotaxis (>2 fold-change; p=0.05). To more broadly understand transcriptional differences across mobilized precursors, we performed single-cell RNA sequencing (scRNA-seq) of lineage-depleted HSPC^G and HSPC^GP(n=8 patients). For the first time to our knowledge, we provide a reference dataset for both CD34^- and CD34⁺ mobilized HSPCs to enable analysis of a broad spectrum of precursors in donor-derived grafts. These data revealed shifts in enriched precursor clusters and transcriptional programs of mobilized HSPCs across mobilization regimens and among patients with variable PB CD34⁺ cell yields. Corroborating our initial studies, CCR2 expression was noted to be enriched in HSPC^GP. Integration of scRNA-seq data and serum protein profiling of donors revealed several additional cytokine/receptor axes to be co-regulated with CCR2 in GP-mobilized HSPCs, suggesting possible additional receptor targets to enhance mobilization.

Finally, to investigate the utility of targeting CCR2 in vivo, CCL7 (100 ng i.v.) was added to G or GP mobilizing regimens in C57BL/6 mice. CCL7 increased white blood cell count (WBC; p<0.01), mobilized Lin⁻Sca-1⁺c-Kit⁺ (LSK) cells (2 fold-change, p<0.05), and PB colony-forming units (1.6 fold-change, p=0.06) compared to G alone. Surprisingly, when combined with GP, CCL7 injection resulted in fewer circulating LSK cells, but an increase in CFU output (1.9 fold-change, p=0.08), suggesting improved graft potency. Moreover, we demonstrate that a single dose of G combined with CCL7 increased WBCs, mobilized lineage-negative and LSK cells, and serial CFU output to levels comparable to those achieved with four daily doses of G.

Overall, our findings identify CCR2 as a key regulator of HSPC trafficking and graft potency, and support further investigation into targeting CCR2 to improve the yield and potency of mobilized donor HSPCs.