Human Facilitating Cells (FCs): Two Distinct, Complementary, and Synergistic CD56 Subpopulations That Uniquely Define FC Function

Abstract

▪Hematopoietic stem/progenitor cell (HSPC) transplantation is increasingly applied for treatment of hematologic malignancies and autoimmune diseases. Despite significant clinical advances of HSPC transplantation, the morbidity and mortality of ablative conditioning and graft-versus-host disease (GVHD) remain a challenge. These risks can be overcome through less toxic nonmyeloablative conditioning and approaches that selectively remove GVHD-causing cells while retaining engraftment-enhancing, tolerogenic cells. We were the first to discover CD8+ T cell receptor (TCR)- graft facilitating cells (FCs) in mouse bone marrow (BM). The addition of as few as 30,000 FCs to 10,000 HSPCs significantly enhances engraftment of hematopoietic stem cells (HSCs) in allogeneic recipients without causing GVHD. FCs also potently enhance engraftment of limiting numbers of syngeneic HSPCs. FCs are distinct from HSPCs since they do not generate multilineage engraftment when infused alone, nor do they function as a stem cell in vitro. The phenotype and mechanism of action of human FCs remains to be defined. We report here for the first time that two distinct FC subpopulations are critical to overall human FC function: CD8+TCR-CD56bright (CD56bright FCs) and CD8+TCR-CD56dim (CD56dim FCs). FACS analysis showed that the majority of CD56bright FCs are CD11c+CD11b+ and C-X-C motif chemokine receptor 4 (CXCR4)bright and keep a dendritic morphology after stimulation with CpG oligodeoxynucleotides (CpG ODN) (TLR9). The majority of CD56dim FCs express CD3ε and exhibit a lymphoid morphology. The CD56dim FC subpopulation significantly promotes homing of HSPCs to BM in non-obese diabetic/severe combined immunodeficiency/interleukin-2 receptor gnull (NSG) recipients and enhances hematopoietic colony formation. The CD56dim FC subpopulation produces rapid reconstitution of donor hematopoietic cells without causing GVHD. In contrast, recipients of CD56bright FCs plus HSPCs show low donor chimerism early after transplantation. However, the level of donor chimerism significantly increases with time. Both recipients of HSPCs plus CD56dim or CD56bright FC subpopulations showed durable donor chimerism at significantly higher levels in BM compared to recipients of HSPCs alone. Co-culture of CD56bright FCs with HSPCs up-regulates small innate immunity-related peptides such as cathelicidin or beta defensin-2, factors that prime responsiveness of HSCs to a low stromal cell-derived factor-1 (SDF-1) gradient. Both CD56bright and CD56dim FC subpopulations highly up-regulate mRNA expression of the HSC growth factors, stem cell factors and fms-related tyrosine kinase 3 (FLT3) Ligand (3,000 fold and 14 fold respectively). Taken together, our data indicate that: 1) human CD8+TCR- FCs are two distinct, complementary and synergistic subpopulations with multifactorial effects on HSCs; 2) CD56bright FCs significantly enhance durable chimerism in the BM of NSG mice and produce significantly increased levels of factors that prime HSC homing; 3) CD56dim FCs significantly enhance homing of human HSC to the BM in NSG recipients and promote HSC clonogenicity; and 4) recipients of HSCs plus CD56dim FCs or CD56bright FCs show durable donor human chimerism in peripheral blood, BM and spleen. DisclosuresBozulic:Regenerex, LLC, a start-up biotech company: Employment. Ildstad:Regenerex, LLC, a start-up biotech company: CEO Other.