Phenotype of young and aged CD4 T cells. (A) Young (bold gray line) and aged (thin black line) donor T cells from the experiment in (day 14 after immunization) were prepared for flow cytometry and stained for expression of CD4, Vβ3, and CXCR5. The flow cytometry dot plots are gated on CD4+Vβ3+ T cells and show the percent positive for CXCR5 expression. (B) Naive young and aged Tg CD4 T cells were stimulated with Ag/APC for 4 d. Effector populations were harvested and stained for CD4, Vβ3, and CD134 expression; histogram is gated on Vβ3+CD4+ cells. (C) 4 d young and aged Tg effectors were also stained for CD4, Vβ3, and CD28 expression; histogram is gated on Vβ3+CD4+ cells. (D) Young (filled squares) and aged (open squares) Tg CD4 T cells were stimulated in vitro with Ag/APC. At 6, 24, 48, 72, and 96 h, cultures were harvested, stained for CD4, Vβ3, and CD154 expression and analyzed by flow cytometry. T cell populations were gated on CD4+ Vβ3+ T cells and the percent CD154 positive in young and aged populations were determined. Graph shows the mean ± SE; *P < 0.05. Representative of three experiments.