Incidence of Ex Vivo Expansion on the Capacity of Cord Blood Graft To Generate Immune Cells: Rational for Co-Infusion of Expanded and Non Expanded Fractions?.

The aim of our study was to determine whether ex vivo expansion of umbilical cord blood (UCB) progenitor cells induces changes in their capacity to generate immune cells. CD34⁺ CB cells were cultured for 14 days with SCF, FLT3-l, TPO and G-CSF, inducing a total cells, CD34⁺ and LTC-ICs increase of 1500, 120 and 8 fold respectively. Non expanded (d0) and 14-day expanded (d14) CD34⁺ cells were compared for their capacity to produce T lymphocytes (TLs) using the fetal thymus organ culture system and DCs generated from d0 and d14 CD34⁺ cells were compared for their differentiation, phenotype and function. Total percentages of CD4⁺, CD4⁺CD8⁻ and CD4⁺CD8⁺ TLs obtained from d0 and d14 CD34⁺ cells were comparable. In both fractions, most of the CD4⁺ T cells co-expressed iCD3 but a lower proportion of d14 derived TLs expressed sCD3. However, there was no significant difference between d0 and d14 derived TLs in term of Vb chain representation, all TCR-Vb chains examined being represented in each case. These data indicate that d0 and d14 CD34⁺ cells have a similar capacity to generate TLs and that expansion does not induced any skewing of the TCR-Vb repertoire. D0 and d14 CD34⁺ cells were next cultured with SCF, FL, GM-CSF and TNF-a to compare their capacity to differentiate into DCs. Similar percentages of CD1a⁺ DCs expressing the same levels of HLA-DR and co stimulatory molecules were obtained. DCs derived from d14 CD34⁺ cells were less potent to stimulate allogeneic TLs, but the pattern of cytokines produced by stimulated TLs was similar and no shift towards a predominant Th1 or Th2 response was observed. Moreover, in spite of a quantitative increase (15 fold) related to the CD34 pool amplification, we observed a decreased capacity (13-fold) of d14 cells to generate DCs compared to d0 CD34⁺ cells.

Overall, these results indicate that ex vivo expansion of CD34⁺ cells doesn’t induce any major modification in T Lymphopoiesis capacity while alters somehow the capacity of the graft to generate DCs. We discuss in the context of UCB transplantation, the putative interest of co-infusion of expanded and non expanded fractions in view of improving myelopoiesis in the graft without subverting the immune reconstitution.