(A) Effect of IL-7 on CXCR4 expression at the surface of primary CD4⁺ T cells. PBMCs were cultured for 7 days in the absence (line with bold dots) or presence of various concentrations (dashed line, 100 pg/mL; thin solid line, 500 pg/mL; bold solid line, 10 ng/mL) of IL-7 (R&D Systems) indirectly labeled with the anti-CXCR4 antibody 12G5 (BD PharMingen) and directly labeled with a fluorescent anti-CD4 antibody as described.²  CXCR4 expression was analyzed by flow cytometry after gating on CD4⁺ lymphocytes. An irrelevant isotype-matched antibody was used as a negative control (line with thin dots). (B) One million PBMCs were cultured in triplicate in RPMI supplemented with 10% fetal calf serum in presence (solid line) or absence (dashed line) of 10 ng/mL of IL-7. Five days later the cells were exposed for 4 hours to 10 ng of p24 equivalent of the R5 strain AD8, washed, and further cultured in presence or absence of IL-7. Once a week, fresh PBMCs were added to the culture. The mean expression over time of CCR5 (squares) and CXCR4 (circles) at the surface of CD4⁺ T cells treated (closed symbol) or not (open symbol) was monitored. (C) In the same experiment, p24 concentration was measured in the cell supernatant. Thrice a week, 200 μL of culture supernatant were added to CD4⁺CXCR4⁺CCR5⁻ MT2 cells to detect the presence of X4 virions as previously described.²  The emergence of an X4 strain at day 67 in IL-7–treated PBMC culture is indicated. R5-to-X4 switch occurred at day 65 in a similar, independent experiment. (D) Alignment of V3 loop sequences from the input virus and the mutant clone obtained at day 67. Predicted amino acid sequences are shown, and similarities are indicated with dashes.