Fig. 2: Robust GC-T_FH cell help and Env-specific T cell responses detected after priming immunization.

a Flow cytometry gating of GC-T_FH cells (PD-1^hiCXCR5⁺). b Longitudinal analysis of GC-T_FH cell frequency as a percentage of CD4⁺ T cells. c AUC of GC-T_FH cells post-priming immunization. d B_GC to GC-T_FH cell ratio at week 3. e B_GC to GC-T_FH cell ratio at week 13. f Representative flow plot of an AIM assay performed at week 2 to detect Env-specific CD4+ T cells. Stimulation conditions include: negative, unstimulated DMSO control (“Neg”), 15-mer overlapping MD39-Env peptide megapool (“Env”), and positive control with superantigen staphylococcal enterotoxin (“Pos”). AIM assays were also performed at week 10. g AIM⁺ (CD40L⁺OX40⁺) T cell frequency as a percentage of CD4⁺ T cells at week 2. h Representative flow plots of an intracellular cytokine assay (ICS) to assess cytokine function in Env-specific CD4⁺ T cells. CD40L⁺IL-2⁺ is shown with corresponding Env-stimulated sample and unstimulated control sample. i, AIM⁺ICS⁺ (CD40L⁺IL-2⁺) T cells as a percentage of CD4⁺ T cells at week 2. j AIM⁺ICS⁺ (CD40L⁺IL-2⁺) T cells as a percentage of CD4⁺ T cells at week 10. k Representative flow plots of an intracellular cytokine assay (ICS) to assess cytokine function in Env-specific CD4⁺ T cells. CD40L⁺IL-21⁺ is shown with corresponding Env-stimulated sample and unstimulated control sample. l AIM⁺ICS⁺ (CD40L⁺IL-21⁺) T cells as a percentage of CD4⁺ T cells at week 2. m Representative flow plot of an AIM assay to detect Env-specific circulating T_FH (cT_FH) cells. n AIM⁺ (CD40L⁺OX40⁺) cT_FH frequency as a percentage of CD4⁺ T cells at week 2. o, AIM⁺ (CD40L⁺OX40⁺) cT_FH frequency as a percentage of CD4⁺ T cells at week 10. Black dotted lines indicate the limit of quantification. Black triangles represent time of immunization. Mean and SEM or geometric mean and geometric SD are plotted depending on the scale in all figures unless otherwise stated. For Fig. 2b–e, n = 12 samples (left and right LN, 6 animals per group). For Fig. 2f–o, n = 6 per group. Statistical significance for GC-T_FH cell frequencies (a-e) was tested using unpaired two-tailed Mann-Whitney test or Kruskal-Wallis test with Dunn’s multiple comparisons test, depending on the objectives of the study. Statistical significance for Env-specific AIM assays (f-o) were tested using Kruskal-Wallis test with uncorrected Dunn’s multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data File.