CXCL9 and IL32 Regulate Progenitor Expansion and Protect Hematopoietic Progenitor Cells From Chemotherapy

Abstract

Abstract 1316 Background:We have reported that the cytokines CXCL9 and IL32 regulate murine bone marrow regeneration post chemotherapy, but the reasons for this effect and whether they work directly on progenitor cells remain unclear. Methods:Human CD34+ cells from cord blood were incubated with CXCL9 or IL32. Cell numbers were determined on a weekly basis, and one-week expanded cells were seeded on top of a confluent MS-5 stroma cell layer to determine the number of cobblestone-area forming and long-term culture initiating cells (LTC-IC). Apoptosis rates after incubation with CXCL9/IL32 and SCF/G-CSF/IL3 prior to Ara-C treatment (300mM, 1 h) were assessed by Annexin V detection. Subsequently, signaling pathways after stimulation with IL32 or CXCL9 were examined using the luminex map technology. Results:CXCL9 did not influence CD34+ cell expansion, while IL32 enhanced the expansion rates significantly [6.69±1.38 versus to 3.57±0.70 fold in the control group, p<0.05]. However, more LTC-ICs after CXCL9 treatment (1357±123 of CXCL9 group versus 1081±119 of control group, p<0.05) were found, while IL32 reduced the number of LTC-ICs (78±8 of IL32 group, p<0.005). That suggests that CXCL9 kept more primitive LTC-ICs quiescent instead of entering expansion during the one-week incubation, while IL32 rather stimulated the differentiation of LTC-ICs. Since SCF, G-CSF and IL-3 are the most widely used hematopoietic growing factors (HGF) in stem cell expansion, we detected their roles during chemotherapeutical treatment in vitro. We observed enhanced apoptosis during Ara-C treatment when the cells were incubated with SCF or IL3. But when CXCL9 or IL32 were added, results were different. Both CXCL9 and IL-32 reduced the apoptosis rate resulting from Ara-C treatment, when SCF is present (26.37±1.12% of CXCL9+SCF group, 29.97±0.72% of IL32+SCF group, versus 35.52±1.21% of SCF alone, p <0.005 and p<0.05), but none of them affected IL-3 related apoptosis. Especially the effect of CXCL9 was inhibited using antibodies to its receptor CXCR3 (37.97±1.50% with anti-CXCR3 versus 35.52±1.21% of SCF alone, p= 0.09, versus 26.37±1.12% of CXCL9+SCF group p<0.05). G-CSF alone did not influence Ara-C induced apoptosis, but in combination with IL32 the apoptosis rate increased (23.37±0.09% of IL32+G-CSF versus 19.59±0.79% of G-CSF alone, p<0.005). That suggests that IL32 could regulate stem cell expansion differently through various pathways in collaboration with SCF and G-CSF. In fact, IL32 reduced STAT5 and p38 activity, while CXCL9 activated p38 and JNK pathways of CD34+ cells in combination with SCF. Conclusions:Our results demonstrate that both CXCL9 and IL32 can regulate stem cell expansion in vitro; CXCL9 could protect HPCs from chemotherapy and therefore support the following recovery, IL32 could help progenitor cells to expand, differentiate rapidly and thereby enhance the regeneration of the hematologic system. Disclosures:No relevant conflicts of interest to declare.