Epitope-specific CD4⁺ T cells express CXCR5 at both acute and memory time points after infection. (A) Representative flow plots of antigen-specific CD4⁺ T cells isolated from secondary lymphoid organs at naive or acute time points after LCMV or Py-GP66 infection. Plots are gated on dump⁻ CD3⁺ CD8⁻ CD4⁺ CD44⁺ GP66⁺ cells. (B) Summary data of the percentage of CXCR5⁺ GP66⁺ cells shown in A (denoted by red gates). This quantification method for total CXCR5⁺ cells, including Tfh and GC Tfh at acute time points and CXCR5⁺ T_CM at later time points, is used throughout the paper. Data are pooled from four to eight mice per time point from at least two independent experiments and were analyzed by two-way ANOVA. (C) Representative flow plots of GP66-specific CD4⁺ T cells at memory time points after LCMV (L) or Py-GP66 (P) infection. Frequencies of CXCR5⁺ cells are indicated in the plots. (D) Summary data of the percentage of CXCR5⁺ GP66⁺ cells shown in C. Data are pooled from four to six mice per time point from at least two independent experiments and were analyzed by two-way ANOVA. (E) Representative flow plots from GP66⁺ CD4⁺ T cells at memory time points after Py-GP66. CCR7⁺ T_CM cells and CCR7⁻ T_EM populations are shown. GC Tfh cells are included in the CCR7⁻ CXCR5⁺ population (Haynes et al., 2007). (F) Summary data of the percentage of the T_CM and the T_EM populations from E. Data are pooled from 3 to 10 mice per time point from at least two independent experiments and were analyzed by unpaired t test. For B, D, and F, data are shown as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. dpi, days post-infection.