CXCR4 endocytosis and recycling in ICL CD4⁺ T cells. PBMCs from 3 independent healthy donors (H1, H3, and H4; □) and 3 ICL patients (P1, P3, and P4; ○) were cultured overnight in complete medium supplemented with 10% FCS, allowing CXCR4 re-expression at the surface of ICL cells, then incubated for 40 minutes with 200nM CXCL12 (treatment, t40/0, CXCR4 endocytosis), and further cultured for up to 120 minutes in the absence of CXCL12 (chase, t40/10 and above, CXCR4 recycling). The protein synthesis inhibitor CHX (50 μg/mL) was present throughout the experiment. Levels of membrane CXCR4 expression were assessed by flow cytometry in CD3⁺CD4⁺–gated T cells. Effects of IL-2 on CXCR4 endocytosis and recycling were evaluated in CD4⁺ T cells from healthy (■) and ICL (●) subjects. The kinetic of CXCR4 down-modulation in PBMCs recovered from P1 immediately after the course of IL-2 treatment was compared with that obtained in cells from H1 recovered the same day and left untreated (). Displayed data are means of duplicate determinations and are expressed as percentages of CXCR4 expression (100% corresponding to CXCR4 expression at the surface of CD4⁺ T cells incubated in medium alone).