Human CD8+TCR- Facilitating Cells (FC) Upregulate Priming Factors That Enhance Proliferation of HSPC and Increase Responsiveness of HSPC to an SDF-1 Homing Gradient.

Abstract

CD8+TCR- graft facilitating cells (FC) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPC) in syngeneic and allogeneic recipients. We report for the first time the phenotypic and functional characterization of the predominant human CD8+TCR- FC subpopulations. Half of FC are CD56dim/- and half are CD56bright. The majority of CD56dim/- FC coexpress CD3ε+, while CD56bright FC coexpress CD11c+ and CD11b+. Both FC subpopulations significantly promote clonogenicity of HSPC in vitro and enhance their homing to BM after transplantation into NSG mice. Recipients of HSPC combined with CD56dim/- or CD56bright FC showed durable donor chimerism in BM. To test if co-culture of the FC subpopulations with HSPC induces growth factors that promote expansion of HSPC, both CD56dim/- and CD56bright FC were sorted from mobilized PBMC and each subpopulation co-cultured with HSPC for 18 hours. Real time quantitative PCR showed significant upregulation of transcripts for stem cell factor (SCF), fibroblast growth factor (FGF)-2, and FLT-3 ligand (FL) by both FC subpopulations. Upregulation was more pronounced in the CD56bright FC cocultures (3000 fold for FL). To address the effect of FC on homing and engraftment in vivo, we examined the effect of FC on the SDF-1 and CXCR4 axis that plays an important role in homing and retention of HSPC in the hematopoietic niche. Several small innate immunity-related peptides such cathelicidin (LL-37) or β2-defensin (BD-2) enhance responsiveness of HSPC to low SDF-1 gradients. To test if FC subpopulations cultured with HSPC express LL-37 and BD-2, CD56dim/- FC and CD56bright FC were sorted from mobilized PBMC and cultured for 18 hours with HSPC. There was a significant increase in mRNA expression for LL-37 and BD-2 in the group of CD56bright FC cocultured with HSPC. In conclusion, we report here for the first time that FC upregulate several factors that promote expansion of HSPC (SCF, FGF-2, FL) and enhance their homing responsiveness to SDF-1 gradients (LL-37, BD-2) after contact with HSPC. Taken together these data suggest that FC exert an effect on HSPC to promote homing and migration of HSPC. DISCLOSURES:Bozulic, L.: Employee, Regenerex, LLC, a start-up biotech company. Ildstad, S.: Other, Regenerex, LLC, a start-up biotech company, CEO.