In-Vitro and In-Vivo Effects of Autologous and Allogeneic Lymphocytes on Human Cord Blood CD34+ Cell Function.

T cell depletion is known to prolong the time of engraftment after hematopoietic stem cell (HSC) transplantation. In this study, we tested whether autologous and allogeneic mononuclear cells (MNC) can differentially affect human cord blood (CB) HSC function. Initially, CB immunomagnetically purified CD34+ or CD133+ cells were incubated with autologous, or allogeneic blood MNC for 1 h or 24 h before initiation of cultures in methylcellulose medium supplemented with GM-CSF, Flt3L, SCF, IL3 and Epo. As compared to cultures of CD34+ or CD133+ cells alone, the addition of auto- or allo-MNC did not affect the numbers of CFU-GM, BFU-E or CFU-Mix generated from CD34+ cells, while they both increased the number of CD133-derived BFU-E (p=0.01). In order to evaluate the effect of alloantigen-specific activated T cells on CD34+ CB cells, autologous or allogeneic MNC were initially stimulated with irradiated CD34+ cells (ratio 2:1) for 6 d, then responders were harvested and incubated overnight (ratio 50:1) with the same non-irradiated progenitors, that had been previously frozen, before CFC assay. In this setting, the addition of allogeneic activated T cells, as opposed to autologous T cells, reduced the number of CFU-GM (12±6 vs 23±3 colonies/10³ CD34+ cells plated), whereas they did not affect the number of assayable BFU-E or CFU-Mix. Finally, to assess the effect of autologous and allogeneic lymphocytes on CD34+ cell marrow repopulating potential, 10 NOD/SCID mice were irradiated (300 cGy) and injected with 0.25 x 10⁶ CD34+ CB cells and autologous (CD34/auto) or allogeneic (CD34/allo) MNC (ratio 1:2), or with autologous or allogeneic cells alone as control. At 6 weeks after transplant, marrow cells were harvested and triple stained with human anti-CD45 mAb and different lineage markers. Human CD45+ cells were on average 1.8% in CD34/auto and 3.2% in CD34/allo. While similar proportions of B cells (CD19+) and monocytes (CD14+) were detected in the two groups, a greater number of myeloid dendritic cells (CD45+BDCA-1+) was observed in CD34/allo than in CD34/auto (0.45±0.30% vs 0.04±0.04%). Control mice injected only with allo-MNC did not engraft. In conclusion, while both autologous and allogeneic lymphocytes sustain CB CD34+ cell clonogenicity, alloantigen activated T cells may target committed myeloid progenitors, consistent with our previous data on immunogenic HSC subsets (Exp Hematol 31:31,2003). In addition, autologous and allogeneic lymphocytes facilitated the engraftment of CD34+ CB cells in NOD/SCID mice in a similar fashion, although allogeneic effectors induced a greater number of dendritic cells, which might play a role in the development of GVHD or graft rejection following the transplantation of adult allogeneic HSC. Future studies will address whether different patterns of cytokines are released in-vivo by autologous or allogeneic lymphocytes. Also, this model will allow us to study the role of different cell subsets in HSC engraftment after transplant.