Characterization of Human CD8(+)TCR(-) Facilitating Cells In Vitro and In Vivo in a NOD/SCID/IL2rγ(null) Mouse Model.

CD8(+)/TCR(-) facilitating cells (FCs) in mouse bone marrow (BM) significantly enhance engraftment of hematopoietic stem/progenitor cells (HSPCs). Human FC phenotype and mechanism of action remain to be defined. We report, for the first time, the phenotypic characterization of human FCs and correlation of phenotype with function. Approximately half of human FCs are CD8(+)/TCR(-)/CD56 negative (CD56(neg)); the remainder are CD8(+)/TCR(-)/CD56 bright (CD56(bright)). The CD56(neg) FC subpopulation significantly promotes homing of HSPCs to BM in nonobese diabetic/severe combined immunodeficiency/IL-2 receptor γ-chain knockout mouse recipients and enhances hematopoietic colony formation in vitro. The CD56(neg) FC subpopulation promotes rapid reconstitution of donor HSPCs without graft-versus-host disease (GVHD); recipients of CD56(bright) FCs plus HSPCs exhibit low donor chimerism early after transplantation, but the level of chimerism significantly increases with time. Recipients of HSPCs plus CD56(neg) or CD56(bright) FCs showed durable donor chimerism at significantly higher levels in BM. The majority of both FC subpopulations express CXCR4. Coculture of CD56(bright) FCs with HSPCs upregulates cathelicidin and ß-defensin 2, factors that prime responsiveness of HSPCs to stromal cell-derived factor 1. Both FC subpopulations significantly upregulated mRNA expression of the HSPC growth factors and Flt3 ligand. These results indicate that human FCs exert a direct effect on HSPCs to enhance engraftment. Human FCs offer a potential regulatory cell-based therapy for enhancement of engraftment and prevention of GVHD.

Sensitization to minor antigens is a significant barrier in bone marrow transplantation and is prevented by CD154:CD40 blockade.

Sensitization to major histocompatibility complex (MHC) alloantigens is critical in transplantation rejection. The mechanism of sensitization to minor histocompatibility antigens (Mi-HAg) has not been thoroughly explored. We used a mouse model of allosensitization to Mi-HAg to study the Mi-HAg sensitization barrier in bone marrow transplantation (BMT). AKR mice were sensitized with MHC congenic Mi-HAg disparate B10.BR skin grafts. Adaptive humoral (B-cells) and cellular (T cells) responses to Mi-HAg are elicited. In subsequent BMT, only 20% of sensitized mice engrafted, while 100% of unsensitized mice did. In vivo cytotoxicity assays showed that Mi-HAg sensitized AKR mice eliminated CFSE labeled donor splenocytes significantly more rapidly than naïve AKR mice but less rapidly than MHC-sensitized recipients. Sera from Mi-HAg sensitized mice also reacted with cells from other mouse strains, suggesting that Mi-HAg peptides were broadly shared between mouse strains. The production of anti-donor-Mi-HAg antibodies was totally prevented in mice treated with anti-CD154 during skin grafting, suggesting a critical role for the CD154:CD40 pathway in B-cell reactivity to Mi-HAg. Moreover, anti-CD154 treatment promoted BM engraftment to 100% in recipients previously sensitized to donor Mi-HAg. Taken together, Mi-HAg sensitization poses a significant barrier in BMT and can be overcome with CD154:CD40 costimulatory blockade.

Effects of plasma membrane Ca(2+) -ATPase tyrosine phosphorylation on human platelet function.

BACKGROUND: The plasma membrane Ca(2+)-ATPase (PMCA) plays an essential role in maintaining low intracellular Ca(2+) ([Ca(2+)](i)) in resting platelets. Earlier studies demonstrated that platelet activation by thrombin results in tyrosine phosphorylation of PMCA, which inhibits pump activity. OBJECTIVES: The objective was to determine the functional consequences of PMCA tyrosine phosphorylation. METHODS: A decapeptide including the tyrosine phosphorylation site of PMCA and a scrambled version were synthesized and introduced into human platelets using saponin. Fura-2 calcium monitoring and aggregometry were used to characterize the effects of inhibition of tyrosine phosphorylation. RESULTS: Western blot analysis of immunoprecipitates showed that introduction of the inhibitory peptide decreased tyrosine phosphorylation of PMCA by nearly 60% in saponin-permeabilized, thrombin-treated platelets as compared with the scrambled control peptide. Concomitant with inhibition of PMCA tyrosine phosphorylation was a significant decrease in [Ca(2+)](i) during thrombin-mediated platelet activation. The functional consequence of reduced PMCA tyrosine phosphorylation and decreased [Ca(2+)](i) was a significant delay in the onset of thrombin-mediated platelet aggregation. CONCLUSIONS: The results demonstrate that PMCA tyrosine phosphorylation regulates [Ca(2+)](i) during platelet activation, which affects downstream events in the activation process. Moreover, PMCA tyrosine phosphorylation and resultant inhibition of PMCA activity produces a positive feedback loop mechanism by enhancing the increase in [Ca(2+)](i) accompanying platelet activation.