A disconnect between precursor frequency, expansion potential, and site-specific CD4+ T cell responses in aged mice
Adult and old C57Bl/6 mice were immunized subcutaneously with 20μg peptide and 10μg LPS on both sides of the base of the tail and mice were kept on BrdU drinking water during the course of the challenge. Day 6 post immunization, pooled spleen and lymph node cells were incubated with E641:I-Ab, OVA:I-Ab and MCC:I-Ek tetramers, enriched using anti-His magnetic beads, and enumerated by flow cytometry after antibody staining and gating on: lymphocytes via forward and side scatter; then CD3+ CD19−, CD11c−, F4/80−, CD8− T-cells; then CD3+ CD4+ T-cells; and finally E641:I-Ab+, OVA:I-Ab−, MCC:I-Ek− T-cells in the tetramer enrichment bound fraction from adult and old mice . (A) Absolute number of E641:I-Ab+, OVA:I-Ab−, MCC:I-Ek− cells from adult and old mice after immunization with E641+LPS. Bars represent median values (ns p>0.05; Mann-Whitney). The average number per group (±SEM) is also shown below the respective label. (B) Concatenated contour plots showing BrdU incorporation by E641:I-Ab+, OVA:I-Ab−, MCC:I-Ek− cells in adult and old mice (gated as above). E641:I-Ab+ cells are divided into BrdU negative (neg), intermediate (int) and high (hi) subsets. Numbers indicate percent (±SEM) E641:I-Ab+ CD4+ T cells in the respective gates. Shown are the aggregate results of two experiments with 4 mice/group.