CD8⁺/TCR^– facilitating cells (FC) in mouse BM significantly enhance engraftment of hematopoietic stem cells (HSC) in syngeneic and allogeneic recipients. Human FC phenotype and mechanism of action remain to be defined. Approximately half of human FC are CD56^neg, with the remainder CD56^bright. The majority of CD56^neg FC express CD3ε, while CD56^bright FC express CD11c and CD11b. The CD56^neg FC subpopulation significantly promotes homing of HSC to BM in NSG mouse recipients and enhances hematopoietic colony formation. The CD56^neg FC subpopulation produces rapid reconstitution of donor hematopoietic stem cells without GVHD. Recipients of CD56^bright FC plus HSC exhibit low donor chimerism early after transplantation but the level of chimerism significantly increases with time. Recipients of HSC plus CD56^neg or CD56^bright FC showed durable donor chimerism at significantly higher levels in BM compared to recipients of HSC alone. Here, we evaluated whether FC protect HSC from apoptosis in vitro. Human CD45⁺/CD34⁺ HSC, CD8⁺/TCR^– FC total, CD56^neg or CD56^bright FC were sorted from human mobilized PBMC. 15,000 HSC were cultured alone or with 30,000 FC total or CD56^neg or CD56^bright FC subpopulations in a 96-well round-bottom plate for 18 hours. After 18 hours, cells were stained with CD34 PE-CF594, CD45 PE-Cy7, CD8α PE, αβTCR FITC, γδTCR FITC and CD56 APC and then stained with annexin V Am Cyan and 7-aminoactinomycin D (7-AAD). Dead and live cells were measured by flow cytometry. FC total significantly prevented apoptosis of HSC in vitro (Figure 1A; n = 10; P = 0.007), compared to HSC alone. The full anti-apoptotic effect of FC total did not occur when HSC were cultured with CD56^neg or CD56^bright FC alone (Figure 1B-C), suggesting that both subpopulations are required for the anti-apoptotic effect. These results indicate that human FC exert a direct effect on HSC to enhance engraftment. Human FC offer a potential regulatory cell-based therapy for enhancement of engraftment and prevention of GVHD.

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