CD26/Dipeptidylpeptidase IV Regulates Potency of Selected Hematopoietic Growth Factors through Truncation, and Recovery In Vivo After Cytotoxic Stress.

Abstract

Abstract 3602Poster Board III-539CD26 is a dipeptidylpeptidase IV (DPPIV) that cleaves dipeptides from the N-terminus after a proline or alanine. An amino acid sequence search identified putative CD26/DPPIV truncation sites in a number of colony stimulating factors (CSFs), including human (hu) and mouse (mu) GM-CSF and G-CSF, hu IL-3, and hu and mu EPO. These truncation sites were not apparent in mu IL-3, hu and mu M-CSF, or in hu and mu stem cell factor (SCF) or Flt3-ligand (FL). We hypothesized that CD26/DPPIV served as a regulator of the potency of the selected CSFs that contain this putative truncation site, and that hematopoietic recovery after stress would be enhanced and accelerated in CD26 −/− mice. We first used Diprotin A (Ile-Pro-Ile), a known CD26/DPPIV inhibitor for mu and hu cells. Mu cytokines were assessed for activity on mu BM, and hu cytokines on hu cord blood (CB), all in dose-response fashion. Hu EPO was tested on mu and hu cells. One hour pre-treatment of mu BM cells with Diprotin A, or use of CD26 −/− mu BM cells, resulted in a two-fold or greater enhancement of CFU-GM-, CFU-G-, and BFU-E- colony formation of cells respectively stimulated by mu GM-CSF, mu G-CSF, and hu EPO. The CSF activities of mu M-CSF for CFU-M, and mu IL-3 for CFU-GM were not enhanced by inhibition/deletion of CD26/DPPIV. Also, pretreatment of hu CB cells with Diprotin A, enhanced colony formation of CFU-GM stimulated by hu GM-CSF or hu IL-3, and of BFU-E stimulated by hu EPO, but did not influence stimulation of CFU-G or CFU-M by hu M-CSF. Stimulation of cells with two CSFs results in additive to greater than additive hematopoietic progenitor cell (HPC) colony formation compared to that of each CSF alone. When both CSFs had putative CD26/DPPIV truncation sites, colony formation by HPC was even further increased by pretreatment of target cells with Diprotin A. Pretreatment of cells with Diprotin A did not enhance colony formation of mu BM or hu CB cells each respectively stimulated with SCF or FL alone, nor did it enhance the synergistic effects noted when SCF or FL was used in combination with CSFs. To assess truncation and activity directly, CSFs were pretreated with purified soluble DPPIV. Truncation was detected by Mass Spectrometry for CSFs with the putative truncation site, but not for those without this site. Truncated CSFs manifested greatly reduced activity against target cells, effects that were more apparent when the cells were pretreated with Diprotin A. Moreover, mixture of a truncated CSF with a non-truncated form of that CSF reduced colony formation to the level of the truncated CSF, suggesting that the truncated CSF interfered with or blocked activity of the full-length CSF. To evaluate effects of CD26/DPPIV on hematopoietic recovery, CD26 −/− and +/+ mice were treated with sublethal dosages of irradiation, or either cycle-specific or non-cycle specific drugs. In all cases, enhanced and accelerated recovery of hematopoietic progenitor cells was noted in the CD26 −/− mice, compared to the control CD26 +/+ mice. These results demonstrate that CD26/DPPIV regulates the activity of selected CSFs, and inhibition/deletion of CD26/DPPIV allows for enhanced in vitro potency of selected CSFs, and in vivo recovery of hematopoiesis in mice stressed with irradiation and cytotoxic drugs. These results may have practical relevance for understanding, and manipulating hematopoietic recovery after cytotoxic treatment or hematopoietic stem cell transplantation. Disclosures:No relevant conflicts of interest to declare.