Figure 8: Microarray analysis of larger and smaller sized T cells from influenza-infected mice.

RNA samples were prepared from sorted populations of larger or smaller sized cells from spleens of influenza virus PR8-OVA-infected mice on day 7 p.i. or from in vitro 7 days culture after stimulation with plate-bound anti-CD3ε (1.0 μg ml⁻¹) and anti-CD28 mAb (0.5 μg ml⁻¹). Effector T-cell control samples were prepared from SIINFEKL (100 ng ml⁻¹) stimulated OT-I cells after 4 days of in vitro culture and sorted as CD8⁺CD44^hiCD62L^lo. Control bona fide effector memory and central memory T cells were sorted from the spleens of PR8-OVA-infected mice on day 42 p.i. Naive cells were sorted as CD8⁺CD44^loCD62L^hi cells from uninfected C57BL/6 mice. Duplicate samples were prepared from independent experiments. (a) Clustering analysis and heatmap of gene expression values to depict the similarity of gene profiles between samples for the 934 significant genes (P<0.01). The colour key shown on the top illustrates the relative expression level across all samples: blue represents expression above the mean and yellow represents expression lower than the mean. The in vivo samples of interest are labelled with a double underline and in vitro samples are labelled with a single underline. (b) The effector or memory phenotype-associated genes were compared between duplicate samples of larger and smaller sized cells sorted from the spleens of infected mice on day 7 p.i. The log fold change of the expression value (the larger cells/the smaller cells) is shown as black bars. The means with s.d. are shown. (c) Gene set enrichment analysis (GSEA) plot shows that the duplicated samples of sorted smaller cells from spleens on day 7 p.i. are enriched for previously reported gene sets for memory CD8⁺ cells (P<0.001, false discovery rate (FDR)<0.25). (d) GSEA plots showing the enrichment of cell cycle checkpoint or DNA repair gene sets in the duplicate samples of sorted larger cells from spleens on day 7 p.i. (P<0.001, FDR<0.25).