Abstract

It has been previously described that CD34+ blood or marrow progenitors expressing costimulatory ligands are committed to the dendritic cell (DC) lineage. In this study we addressed the question of whether initial contact with allogeneic lymphocytes may differently affect hemopoietic stem cells (HSC) with alloantigen presenting capacity. Human CD34+ and CD133+ cord blood (CB) cells were obtained after immunomagnetic purification. Initially, both HSC fractions were irradiated and tested with allogeneic blood mononuclear cells (MNC) (ratio 1:2) in primary mixed leukocyte culture (MLC) for 6 d. CD34+ and CD133+ CB cells induced similar activation of allo-responders, with increased numbers of CD3+CD69+, CD3+CD25+ and CD8+CD134+ (OX-40+) cells, as well as allogeneic T cell proliferative response (mean Stimulation Index: 19±5 and 16±5, respectively) (n=6). Then, to evaluate the effect of allogeneic effectors on HSC, we tested non-irradiated CD34+ or CD133+ cells in co-culture with irradiated allogeneic MNC or purified T-cells for 6 d, or with medium alone as control, and observed a brisk proliferation of both HSC fractions only upon stimulation with allogeneic cells (mean Stimulation Index: 17±2; n=3). Interestingly, costimulatory blockade with CTLA4-Ig resulted in 70% inhibition of HSC proliferation, suggesting that B7:CD28 signaling is necessary to the T cell-mediated HSC growth. Finally, CD34+ or CD133+ cells that had been cocultured with irradiated allogeneic T cells for 6 d, were harvested, irradiated and rechallenged as stimulators in MLC (S:R ratio=1:2). Progenitors derived from both CD34+ and CD133+ cells induced a significantly greater T cell alloresponse as compared to the same freshly isolated HSC (overall mean Stimulation Index: 134±54 vs 24±8, p=0.03; n=4 exp). The phenotypic kinetics of CD34+ and CD133+ CB cells upon contact with irradiated T cells, showed a rapid upregulation of CD86 within 24 h (on average from 2±1% to 7±2%, and from <1% to 4±1%, respectively, n=3), and at 3 and 6 d of culture both CD34+ and CD133+ CB cells progressively became larger and included a population of HLA-DR+CD14+CD86+CD11c+ progenitors, on average 16% of cultured progenitors (n=3), consistent with the phenotype of DC precursors. In addition, irradiated allogeneic lymphocytes induced the generation of CD1a+ DC after 8 d of culture with CD34+ cells or CD133+ cells (7% and 9% CD1a+ cells, respectively), in the absence of exogenous cytokines. Therefore, we show here that CB CD34+ and CD133+ are similarly enriched in progenitors capable of stimulating allogeneic T cell responses in-vitro. Moreover, we demonstrate that an early crosstalk of cord blood HSC (CD34+ and CD133+ cells) and allogeneic T cells leads to the reciprocal activation and to the rapid proliferation and differentiation of DC precursors, that elicit potent alloimmune responses. Future studies aimed at either selectively abrogating this subset of progenitors, or blocking HSC-induced T cell activation, will be developed in the attempt of facilitating the induction of tolerance in allogeneic HSC transplantation.

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