TNFα Mediated Facilitating Cell Enhancement of Hematopoietic Stem Cell Function in Vivo and In Vitro Involves Bcl-3.

Approaches to enhance engraftment of hematopoietic stem cells (HSC) are of primary interest in BM transplantation. CD8⁺TCR⁻ facilitating cells (FC) improve HSC engraftment in allogeneic recipients without causing graft versus host disease. FC also significantly enhance the engraftment of limiting numbers of HSC in syngeneic recipients, suggesting that FC act directly on HSC. We therefore analyzed the mechanism for FC-mediated effects on HSC. We found that FC increased the ability of purified HSC to generate colonies in both the CFC and CAFC/LTC-IC assays, confirming a direct effect of FC on different HSC compartments. Co-incubation of HSC with FC for 18 or 40 hours significantly increased the survival of HSC and their subsequent ability to generate CFC at these same time points. We determined that the anti-apoptotic effect of FC on HSC was associated with the up regulation of anti-apoptotic Bcl-3 transcripts. We postulated here that the effect of FC on HSC was due to cytokine secretion. As FC produce TNFα after CpG ODN stimulation and TNFα has various activities on HSC, we evaluated the role of TNFα on FC function. FC were sorted from TNFα deficient mice and the facilitative activity of FC on HSC engraftment was assessed. FC from TNFα deficient mice were impaired in facilitating HSC engraftment in both the syngeneic and allogeneic models, suggesting a role for TNFα in FC function. Notably, TNFα transcripts were present in FC by 16 hours of co-incubation of FC + HSC and FC produce TNFα (surface and intra-cellular) when in contact with HSC. Furthermore, when TNFα was blocked (using anti-TNFα mAb), FC from wild type mice lost the ability to increase HSC clonogenicity (from 38.2±13.6 CFC/1000 HSC in HSC alone to 65.7 ± 22 for HSC + FC and 38.2 ± 19.6 for FC pre-incubated 1 hour with anti-TNFα mAb before incubation with HSC). Moreover, anti-TNFα mAb also blocked the ability of FC to up-regulate Bcl-3 transcripts in HSC. In conclusion, FC act directly on HSC via several mechanisms to maintain the balance between proliferation/differentiation/survival of HSC. One central mechanism implicates TNFα production by FC, which may protect HSC from undergoing apoptosis by up-regulating anti-apoptotic transcripts (e.g. Bcl-3). These findings may have great impact for the use of accessory cells in HSC transplantation, especially when numbers of HSC are limiting.