Figure 3: Evaluation of early Tfh cell and GC responses in the dLNs (Day 13).

Mice were immunized i.m. with 1 μg Pfs25-EPA or Pfs25-TT in Alhydrogel or GLA-LSQ. dLNs were harvested on day 13 and examined by flow cytometry (inguinal LN) or confocal microscopy (popliteal LN). (a) Representative flow cytometry plots showing, within the activated CD44^hiFoxp3⁻CD4⁺ T cell population, the frequency of ICOS⁺PD-1⁺ cells (top row) and within this population the frequency of Bcl6⁺CXCR5⁺ cells (bottom row). (b) Frequency of ICOS⁺PD-1⁺CXCR5⁺Bcl6⁺ Tfh cells within the CD44^hiFoxp3⁻CD4⁺ T cell population. (c–e) Popliteal lymph nodes (dLN) were harvested 13 days post-immunization, stained with the indicated antibodies, and examined by confocal microscopy. (c) Representative dLN sections from 5–6 dLNs per mouse with germinal centers denoted by GC and plasma cells denoted by PC. Colors of the word labels correspond to the colors of the stains here and throughout. Scale bar corresponds to 100 μm. (d) Number of GC reactions per dLN. Each symbol represents a dLN. (e) Area of GC reactions per dLN section. Area was calculated from the widest part of each GC. Each symbol represents one GC per 5–6 whole dLNs. Shown is the mean ± SEM. (*P < 0.05, ***P < 0.001; One-way ANOVA with Tukey post-test). Data are pooled from 2 similar experiments.