Blockade of alpha6-integrin reveals diversity in homing patterns among human, baboon, and murine cells

Abstract

Our understanding of the mechanisms by which intravenously transplanted hematopoietic stem/progenitor cells (HSPCs) home to and engraft the bone marrow (BM) remains incomplete, but participation of adhesion molecules has been documented. We here demonstrate that blockade of the alpha6-integrin enhanced BM homing of human and nonhuman primate BM-derived HSPCs by >60% in the xenogeneic transplant model and led to significantly improved engraftment. The effect was limited to BM-derived HSPCs, as granulocyte-colony-stimulating factor mobilized peripheral blood or cord blood HSPCs express little or no alpha6 integrin. By contrast, despite high alpha6 integrin expression, no effect of alpha6 blockade on murine BM-HSPCs homing/engraftment was observed.

FIG. 1.

Blockade of α6-integrin enhances marrow homing of human and baboon BM CFU-C. (A) Expression of α6-integrin on human BM (dark gray) or human MPB (light gray) CD34+ cells (unfilled black line: isotype control). (B) Expression of α6-integrin on human CB (unfilled black line) CD34+ cells (light gray: isotype control). (C) Saturation of α6-integrin-binding sites under the conditions used for α6-blockade. In contrast to control-Ab-incubated HSPCs, anti-α6-Ab-treated HSPCs (here, baboon CD34+ BM cells) did not bind PE-conjugated anti-α6-Ab (dark gray: anti-α6-Ab-incubated HSPCs; unfilled black line: control incubated HSPCs; light gray: isotype control). (D) No effect of incubation of HSPCs (here, human CD34+ cells) with antifunctional α6-Ab on CFU-C formation, suggesting the absence of direct effects of anti-α6-Ab on proliferation. (E) Marrow homing of human BM CFU-C in lethally irradiated (1,250 cGy) NOD/SCIDβ2-m^−/− recipients, enumerated 20 h after transplantation, was >75% increased after α6-integrin blockade (three experiments, each with 3–5 mice/group, one representative experiment shown here). Concomitantly, CFU-C content in spleens was significantly reduced. Average CFU-C content in lungs of recipients of anti-α6-Ab-treated HSPCs was also reduced >50%, but because of great variability this did not reach statistical significance. (F) Marrow homing of human MPB CFU-C in lethally irradiated (1,250 cGy) NOD/SCIDβ2m^−/− recipients, enumerated 20 h after transplantation, was neither significantly increased nor was spleen homing different from controls (one experiment, 4 mice/group). (G) 20-h marrow homing of baboon BM CFU-C in lethally irradiated (1,250 cGy) NOD/SCIDβ2m^−/− hosts was almost 70% increased, and spleen homing 50% reduced, after α6-blockade (two experiments, each with 4 recipients/group). (H) The probability of human engraftment, measured 8 weeks after transplantation, was significantly increased in sublethally (350 cGy) radioconditioned NOD/SCIDγc^−/− recipients of anti-α6-Ab-treated human BM-CD34+ cells (7/9 mice) compared to recipients of control-treated cells (4/10 mice). Black diamonds indicate engraftment (i.e., chimerism >1%), empty diamonds indicate nonengraftment (P = 0.027).

FIG. 2.

Blockade of α6-integrin has no effect on marrow homing of murine CFU-C. (A) α6-Integrin is expressed on almost all c-kit+ murine BM cells. (B) The anti-α6-Ab concentration/incubation time was sufficient to completely block binding of directly fluorescence-conjugated anti-α6-Ab, indicating saturating conditions (solid black line histogram: anti-α6-Ab-treated c-kit+ cells; gray histogram: control-Ab-treated c-kit+ cells; fine gray line: isotype control). (C and D) Marrow homing of murine BM CFU-C in lethally irradiated (1,250 cGy) isogeneic hosts, measured at 3 h (C), or 20 h (D) was not affected by α6-blockade. This outcome was observed irrespectively of whether whole BM cells (C and D, left) or c-kit-enriched BM cells (D, middle, right) were used, in normal (C; D, left, middle) or in splenectomized (D, right) recipients (one experiment each, with five recipients/group). The number of circulating CFU-C, a sensitive measure of altered marrow homing, also did not differ between recipients of anti-α6-treated and control cells (C). Spleen homing was also not different between the groups in nonsplenectomized recipients of whole BM or c-kit+ BM cells (not shown). (E) The efficiency of marrow and spleen homing of murine BM CFU-C in lethally irradiated NOD/SCIDβ2m^−/− recipients instead of isogeneic ones also did not differ between anti-α6-Ab-incubated and control incubated CFU-C (one experiment, three mice/group). (F) Short-term engraftment of CFU-C 8 days after transplantation did not differ between recipients of control treated and anti-α6-Ab-treated cells.