CD26 Inhibition Preferentially Enhances In Vitro Migration of G-CSF + Plerixafor (AMD3100) Mobilized PB as Compared to G-CSF Mobilized PB In Multiple Myeloma Autografts.

Abstract 3714

Hematopoietic stem and progenitor cell (HSC/HPC) trafficking into the bone marrow during hematopoietic stem cell transplantation (HSCT) is thought to be an important biological step that can be targeted to improve transplant efficiency and subsequent immune reconstitution. It has been reported that loss/inhibition of the peptidase CD26, also known as DPPIV (dipeptidylpeptidase IV), increases the transplant efficiency of mouse HSC/HPC (Christopherson, KW 2^nd, et al. Science 2004). In a similar manner, inhibition of CD26 on CD34⁺ or lin⁻ cord blood (CB) cells improves in vitro cell migration and in vivo engraftment into immunodeficient mice (Christopherson, KW 2^nd, et al. Stem Cells and Dev 2007). There is currently controversy as to whether inhibition of CD26 on mobilized peripheral blood (mobPB) has benefit since it has been reported that inhibition of CD26 does not improve migration of G-CSF mobPB CD34⁺ cells in response to SDF-1 (Kawai K, et al. Stem Cells and Dev 2007. 16:361–70) but does improve the migration of G-CSF mobPB mononuclear cells (MNC) (Frank R, et al. ASH Abstract #372, 2009). Additionally, it has been reported that AMD3100 mobPB c-kit⁺ mouse cells have high CD26 expression similar to BM in contrast to G-CSF mobPB c-kit⁺ mouse cells which have very little CD26 expression (Bonig H, Exp Hem, 2009). The combination G-CSF + AMD3100 (a CXCR4 antagonist also known as plerixafor or Mozobil ®) has been recently introduced into the clinic for use in obtaining mobPB from Multiple Myeloma patients. We therefore investigated whether CD26 on human mobPB MNC obtained from Multiple Myeloma autografts mobilized with either G-CSF or G-CSF + plerixafor would have an effect on SDF-1 induced cell migration. To do this, non-adherent MNC from mobPB were obtained from discarded bags following the completion of stem cell infusion of previously frozen autografts. CD26 expression was detected by flow cytometery. CD26 activity was determined using the substrate Gly-Pro P-nitroanilide (Gly-Pro-pNA). To assess migration, chemotaxis assays were performed by loading 5×10⁴ cells into the upper chamber of a 96 well transwell plate, adding human SDF-1α (0–400 ng/mL) in the lower chamber, and incubating for 2 hours at 37°C, 5% CO₂. By flow cytometry, CD26 expression was 21.61±2.19% and 21.65±3.68% of the CD45⁺ cell population for G-CSF and G-CSF + plerixafor mobPB MNCs respectively. CD26 activity was 15.4±1.2 pmol pNA/minute and 16.3±0.4 pmol pNA/minute for G-CSF and G-CSF + AMD3100 MNCs. During functional in vitro assays, cells were cultured with or without a 5mM concentration of the CD26 inhibitor Diprotin A (DpA). DpA treatment of G-CSF + plerixafor mobPB resulted in a significant increase in cell migration as compared to untreated cells. Specifically, the percent migration at 400ng/mL SDF-1α was 21.3±2.4% and 31.6±3.0% for untreated and DpA treated G-CSF + plerixafor mobPB cells respectively (p=<0.01, n=10). The percent migration in response to 400ng/mL SDF-1α was 19.8±2.0% and 25.3±2.6% for untreated and DpA treated G-CSF mobPB cells respectively (p=0.21, n=7). These data indicate that CD26 inhibition, by treatment with DpA, results in a preferential increase in SDF-1 induced migration of G-CSF + plerixafor mobPB cells as compared to G-CSF mobPB cells. The preferential response does not appear to correlate to any change in CD26 expression or activity since both types of mobPB have equivalent expression and activity prior to the migration assay. Our data suggest that un-fractionated mobPB collected following mobilization using the combination of G-CSF + plerixafor (AMD3100) may have inherently different cell trafficking regulatory properties when compared to mobPB collected following G-CSF alone and that additional regulatory mechanisms allow the G-CSF + plerixafor mobPB to have an enhanced response to SDF-1 in the absence of CD26 suppression. Additionally, our data suggest that inhibition of CD26 peptidase activity may be more clinically relevant in the G-CSF + plerixafor mobilized patient population with regard to achieving improved stem cell trafficking.