Fig. 4: Fungal-specific CD4 T cells have limited TCR engagement in the brain.

a Fungal burdens in brain and lung at day 7 post-infection of mice (n = 6 mice) intravenously infected with C. neoformans and adoptively transferred with CnT.II cells as outlined in Fig. 2a. Data pooled from two independent experiments and analysed by Mann Whitney U-test (*P = 0.0189). Whiskers refer to the maximum/minimum values, the box refers to interquartile ranges, the centre line refers to the mean. b Frequency of CnT.II cells expressing Nr4a3-Tocky (blue, red or both forms) in the indicated organs at day 7 post-infection (n = 6 mice). Data pooled from two independent experiments, presented as mean +/- SEM and analysed by two-way ANOVA (comparing total Nr4a3-Tocky⁺ cells), ****P < 0.0001. c Frequency of Nr4a3-Tocky⁺ CnT.II cells that were cultured in vitro either with splenocytes alone (unstimulated), splenocytes loaded with Cda2 peptide or cultured onto plates coated with anti-CD3/28 antibody. Each point represents a technical replicate (n = 3 wells). Data is a representative experiment from two independent repeats (each with at least 3 technical replicates). d Mice were treated as in Fig. 2a, and then injected with indicated amounts of Cda2 peptide intraperitoneally 4 h prior to analysis (n = 3 mice for 80 µg [closed symbol], n = 4 mice for 200 µg [open symbol], with line representing the mean). Each point represents an individual animal. Data analysed by unpaired t-test. *P = 0.0479, ****P < 0.0001. e Nr4a3-Tocky mice were infected intravenously with C. neoformans and the proportion of infiltrating CD4 T cells in the brain (closed symbol), lung (grey symbol) and spleen (open symbol) expressing Nr4a3-Tocky (all forms) determined at days 3, 7 and 9 post-infection. Example plots are gated on total CD3⁺CD4⁺ cells in the brain. Data is pooled from two independent experiments, n = 3 mice per time point, shown as mean +/- SEM. f Mean fluorescence intensity (MFI) of TCRβ expression of CD4 T cells in the brain, lung and spleen of mice (n = 10 mice analysed, with line representing the mean) at day 7 post-infection. Data pooled from two independent experiments; each point represents an individual animal and analysed by one-way ANOVA. ****P < 0.0001.