Figure 5: Comparison of gut-resident and CNS-infiltrating CD4⁺2D2-IEL-T_HIGH cells.

(a) CD4⁺2D2-IEL-T_HIGH cells were sorted from the IELs of 2D2 mice. 2D2-IEL-T_HIGH cells (5.0 × 10⁵) were adoptively transferred to WT mice (day −1) and EAE was induced (day 0). Eighteen or twenty days later, CD4⁺2D2-TCR⁺ cells were recovered from the CNS of WT recipients (CNS CD4⁺2D2-IEL-T_HIGH). Spleen CD4⁺2D2-T cells were also sorted for comparison (Sp-T). mRNA was obtained from each sample, and quantitative RT–PCR of each indicated gene was performed. Gene expression levels were normalized to that of Gapdh. Fold-induction of the indicated gene expression in CD4⁺2D2-IEL-T_HIGH and CNS-CD4⁺2D2-IEL-T_HIGH cells was compared. (b) The expression levels of selected genes in CD4⁺2D2-IEL-T_HIGH, CNS-CD4⁺2D2-IEL-T_HIGH and SpCD4T cells were compared. Each sample was pooled from four mice. The mean expression of duplicate samples was calculated. (c) In total, 2.0 × 10⁶ 2D2-IEL-T_HIGH cells were transferred (day −1) to WT recipients and EAE was induced (day 0). Nineteen days later, the surface and intracellular expression of LAG-3 in CD4⁺2D2-IEL-T_HIGH (left) or the CNS-CD4⁺2D2-IEL-T_HIGH (right) cells were assessed. (d) In total, 7.5 × 10⁵ 2D2-IEL-T_HIGH cells were transferred to WT recipients (day −1) and EAE was induced (day 0). Eighteen days later, Foxp3 expression in CNS-CD4⁺2D2-IEL-T_HIGH cells was analysed. Data are representative of three (c) or two experiments (d). RT–PCR, reverse transcription PCR.