Fig. 3: Ferritin-specific CD4 T cells participate in the germinal center response to HA-nanoparticle immunization.
BALB/c and CBA/J mice were immunized with equimolar quantities of HA in the form of soluble HA trimers or HA-nanoparticles. Draining lymph nodes were harvested D15 post immunization. Antigen-specific CD4 T cell responses were quantified by IL-2 ELISpot for A BALB/c mice and B CBA/J mice following stimulation with total HA or total ferritin peptide pools. Absolute numbers of CD4 TFH per lymph node were quantified for C BALB/c and D CBA/J. Antigen specificity of the TFH response was quantified by activation induced marker (AIM) assay by scoring upregulation of CD154 and CD69 in response to peptide stimulation for E BALB/c and F CBA/J. The fraction of the AIM+ TFH specific for ferritin (black) or HA (white) is shown for G BALB/c and H CBA/J. Quantification of antigen experienced non-TFH that upregulate CD154 and CD69 in response to peptide stimulation for I BALB/c and J CBA/J. The fraction of the AIM+ antigen experienced CD4 T cell responses specific for ferritin (black) or HA (white) is shown for K BALB/c and L CBA/J. Data are shown as the mean and SD three individual mice per group from three independent experiments, for a total of 9 individual mice per group. In A, B, E, F, I, J, significant differences between HA-trimer and HA-nanoparticle immunized mice were determined by two-way ANOVA with Tukey’s correction for multiple comparisons. In C, D, significant differences between HA-trimer and HA-nanoparticle immunized mice were determined by unpaired, two-tailed Mann-Whitney test.
