Figure 5. Superior immunity of HA-ferritin is not driven by ferritin-specific T_FH cells.

(A) C57BL/6 (n = 10 mice per group) mice were immunized with HA-ferritin (5 or 0.5 μg) or a molar equivalent of soluble HA (3.8 or 0.38 μg), or 1.2 μg ferritin alone, adjuvanted with AddaVax. Fourteen days following vaccination, draining iliac (left) and inguinal (right) LNs were harvested. The proportion of T_FH cells (identified as CXCR5⁺PD-1⁺) within the CD4⁺ T cell population was determined by flow cytometry. Data shown are combined from 2 independent experiments. Fourteen days after mice were vaccinated as in A, inguinal and iliac LNs were harvested and pooled for each mouse (n = 5 mice per group). Lymph node suspensions were cultured for 18 hours with pools of overlapping peptides spanning HA or ferritin proteins (final concentration of each peptide, 2 μg/mL) or DMSO alone. Antigen specificity was determined by the proportion of T_FH cells positive for ligand CD154 (B) or activation markers (CD25⁺OX40⁺) (C) following subtraction of DMSO background. Data represent mean ± SD. *P < 0.05, determined by a Mann-Whitney U test.