Delayed germinal center formation and decreased accumulation of CD4⁺ T cells and CD19⁺ B cells in DLN after WNV infection of old mice.

<p>Mice were infected with 10² PFU of WNV (New York strain) in the footpad. <b>A-D</b>. Draining popliteal LN cells were analyzed by flow cytometry at the indicated days after infection. Numbers of GC B cells (CD19⁺ Fas⁺ GL7⁺ cells) (<b>A</b>) and T_FH cells (CD4⁺ PD1⁺ CXCR5⁺ cells) were quantified (<b>C</b>). <b>B and D.</b> Representative flow cytometry plots of GC B cells (<b>B</b>, on gated CD19⁺ cells) and T_FH cells (<b>D</b>, on gated CD4⁺ cells) from adult and old mice at 8 days post infection. <b>E</b>. Immunofluorescence staining of germinal centers in DLN from 6 days post WNV-infected adult and old mice. Slides were stained with GL7-PE, and IgD-FITC. Arrows point to sites of active GCs indicated by GL7 staining in the red channel. In the graph, the number of GCs per section is recorded with each point representing a section taken from the center of draining popliteal lymph nodes from adult or old mice from two independent experiments. <b>F.</b> Hematoxylin and eosin stained sections of the draining popliteal LN from adult and old mice 4 days post infection. <b>G-M</b>. At the indicated days post infection, the DLN was harvested, and total cells were counted (<b>G</b>). Antibody staining detected specific lymphocyte subsets including CD4⁺ T cells (<b>H</b>) and CD19⁺ B cells (<b>K</b>). CD4⁺ T cells (<b>I, J</b>) and CD19⁺ B cells (<b>L, M</b>) were co-stained with antibodies to CD69 (<b>I, L</b>) or Ki-67 (<b>J, M</b>) to establish their state of activation or proliferation. The results are pooled from at least three independent experiments with a total of 6 to 14 mice per group. Data is expressed as the mean ± the standard error of the mean (SEM). Asterisks indicate statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001; Mann-Whitney test).