Rutella S, Zavala F, Danese S, Kared H, Leone G. mobilization influences CD4+ T cells through ICAM-1/LFA-1 conversation. CD4+ T cells were isolated from healthy volunteers (control group) and G-CSF mobilized HSC donors (G-CSF-mobilization group). The purity of the CD4+ T cells was greater than 96% in the two groups (Physique ?(Figure2A).2A). There was no difference in the purity of CD4+ T cells between the control group and the G-CSF-mobilization group (Physique ?(Figure2B).2B). The culture supernatants were collected after stimulation of purified CD4+ T cells in different conditions for 72h, and the levels of the cytokines, including IL-2, IL-4, IL-10, IFN- and TNF-, were detected. The results showed that incubation of CD4+ T cells with either ICAM-1 or anti-CD3 increased the IL-2 level (Physique ?(Figure2C).2C). This increase in IL-2 secretion was also observedwhen the cells were stimulated withboth ICAM-1 and anti-CD3. The increase of IL-2 was abrogated when anti-LFA-1 blocking antibody was used (Physique ?(Figure2C).2C). Compared with the control group, G-CSF mobilization inhibited IL-2 production by > 50% when the cells were stimulated with both ICAM-1 and anti-CD3 (Physique ?(Figure2C).2C). Furthermore, LFA-1/ICAM-1 signaling in CD4+ T cells increased the anti-CD3-mediated production of IFN- and TNF-, which was abrogated in thepresence of anti-LFA-1 blocking antibody (Physique ?(Physique2D2D and ?and2E).2E). G-CSF mobilization also significantly inhibited the release of IFN- and TNF- when the cells were stimulated with both ICAM-1 and anti-CD3 (Physique ?(Physique2D2D and ?and2E).2E). In contrast, LFA-1/ICAM-1 stimulation in CD4+ T cells significantly decreased anti-CD3-mediated secretion of IL-4 (Physique Digoxin ?(Figure2F)2F) and IL-10 (Figure ?(Figure2G).2G). In addition, the ICAM-1- and anti-CD3-stimulated secretion of IL-4 and IL-10 in the control group showed~2-fold than the G-CSF-mobilization group (Physique ?(Physique2F2F and ?and2G).2G). We further analyzed the cellular expression of TH1/TH2 cytokines in LFA-1/ICAM-1- and anti-CD3-stimulated CD4+ T cells using flow cytometry (Physique ?(Physique2H).2H). CD4+ T cells from the G-CSFCmobilization group exhibited significant different in cytokine expression. In comparison to the control group, G-CSF mobilization decreased IFN- and IL-4 production by more than 50% (Physique ?(Physique2I),2I), indicating that G-CSF mobilization suppressed the percentage of TH1 and TH2 cells. However, there was no statistically significant difference in the TH1/TH2 ratio between the G-CSF mobilization group and the control group (data not shown). Collectively, these data supported the critical involvement of LFA-1 signaling in CD4+ T cell cytokine secretion and suggested that G-CSF mobilization decreased the release of inflammatory cytokines from CD4+ T cells through the LFA-1/ICAM-1 conversation but did not alter the balance of the TH1/TH2 subsets. Open in a separate window Physique 2 G-CSF mobilization inhibited the secretion of inflammatory cytokines from CD4+ T cellsCD4+ T cells were purified from the peripheral blood of healthy volunteers and G-CSF-mobilized donors. CD4+ T cells were isolated using magnetic beads conjugated to a human CD4 antibody. Digoxin (A) The purity of the isolated cells was then Digoxin analyzed by flow cytometry. (B) The purified cells from the two groups were quantified (n=15 per group). Unstimulated, and ICAM-1-stimulated CD4+ T cells from G-CSF-mobilized donors and healthy volunteers were cultured with or without anti-CD3 or ICAM-1 plus anti-CD3 for an additional 72 h. To inhibit LFA-1-mediated signalings, the cells were treated with anti-LFA-1 blocking Ab. At the end of the incubation period, the culture supernatants were harvested, and the secreted levels of IL-2 (C), IFN- (D), TNF- (E), IL-4 (F) and IL-10 (G) were analyzed by the ProcartaPlex? Multiplex Immunoassay (n=13 per group). (H) IFN- and IL-4 production by CD4+ T cells, evaluated by intracellular IFN- and IL-4 staining. (I) The percentage of IFN- and IL-4 expression is summarized in the graph (n=8 per group). The data are shown as the mean SD. ns: no significant difference; * < 0.05; ** < 0.01; *** < 0.001. G-CSF mobilization decreased the polarization and LAMA4 antibody migration of CD4+ T cells Given the crucial role of LFA-1/ICAM-1 in T cell attachment, polarization and migration, we examined whether G-CSF mobilization could affect LFA-1-mediated CD4+ T cell polarization and migration. Purified CD4+ T cells were.