The Role of CD26 in Human Umbilical Cord Blood CD34+ Cell Engraftment of NOD/SCID Mice.

Abstract CD26 is a surface serine protease expressed on many different cell types, including human and mouse hematopoietic cells. CD26 cleaves the N-terminal dipeptide from some chemokines, including Stromal-Derived Factor 1 (SDF-1/CXCL12). SDF-1 plays an important role in hematopoietic cell homing and mobilization into and out of the bone marrow. The cleavage of SDF-1 by CD26 produces an inactive form which negatively affects human CD34+ cord blood cell chemotaxis in vitro, and inhibiting the CD26 protease activity using the small peptide Diprotin A causes an enhancement of in vitro chemotaxis to SDF-1 (Christopherson II et al ( 2002). The Journal of Immunology 169: 7000–7008). In addition, this treatment causes an enhancement of in vivo homing, engraftment and competitive as well as non-competitive repopulation of murine hematopoietic stem cells in lethally irradiated congenic recipients (Christopherson II et al ( 2004). Science 305:1000–1003). The goal of our current studies was to determine what role CD26 plays in the in vivo engraftment of hematopoietic stem cells from human umbilical cord blood (hUCB). In order to answer this question, we used the sublethally irradiated NOD/SCID mouse model, which is one of the best in vivo tools available for studying human hematopoietic stem cell function. We isolated CD34+ cells from hUCB and transplanted these cells into NOD/SCID animals after treating the CD34+ cells with or without Diprotin A or a polyclonal goat anti-human CD26 antibody with isotype as control. In preliminary experiments, we showed that pretreatment of CD34+ cells with either Diprotin A or a polyclonal goat anti-human CD26 antibody resulted in a greater percentage of animals with detectable engraftment compared to control as measured by flow cytometry and PCR (83% in pretreated group versus 25% in control group). In a further experiment, we found that pretreating purified CD34+ human cord blood cells with Diprotin A caused a significant enhancement (> 3 fold) of engraftment in NOD/SCID mice at 6 weeks post-transplant as measured by the percentage of human CD45+ cells in the mouse bone marrow (average chimerism of 6.5% in control group versus 23.9% in Diprotin A group). The relative proportion of human engrafted cells expressing four different blood cell markers (CD33, CD38, CD19 and CD34) did not significantly differ between the control and Diprotin A groups, suggesting that Diprotin A treatment does not significantly alter hematopoiesis towards one lineage at the expense of another lineage. We also investigated the nature of the CD26+ population contained within the human CD34+ cells by staining these cells with CD34, CD26 and CD38 antibodies and analyzing by flow cytometry. The CD34+CD38lo/− population represents a more immature population of hematopoietic stem/progenitor cells. The percentage of CD26+ cells within this population is 2-fold higher than the percentage of CD26+ cells in the CD34+CD38+ population (12.1% compared to 5.7%, respectively, average of 4 experiments). This may suggest that immature stem/progenitor cells express CD26 preferentially. These results suggest that inhibiting CD26 on immature subsets of CD34+ cells enhances the engrafting capability of these cells in sublethally irradiated NOD/SCID mice. Clearly, further studies are needed to determine the full extent of these effects, information that may be of use for enhancement of the clinical engrafting capability of limiting numbers of stem cells.