Th40 cells (CD4+CD40+ Tcells) drive a more severe form of Experimental Autoimmune Encephalomyelitis than conventional CD4 T cells
Fig 1
EAE induction causes expansion of the Th40 cell subset and infiltration of Th40 cells into brain and spinal cord tissues.
Female C57BL/6 mice were EAE induced. (A) Representative plots of CD4 and CD40 stained cells (blue) in peripheral draining lymph nodes (dLN) and spleens from age- and sex-matched, wild type, control (without injection of any EAE induction component) and EAE induced mice (disease score of at least 2). Gate was set from isotype (red) and FMO (separate plots) controls. CD3 expression in CD4+CD40+ quadrant was confirmed (histograms). (B) Graph depicting percent Th40 cells from cumulative data in B (Control, n = 11; EAE, n = 8). Data are represented as mean ± SEM. Significance was calculated by two-tailed t-test. (C) Graph depicting absolute number of T cells recovered from brains and spinal cords of EAE (n = 9; disease score of at least 2) and control (n = 5) mice. Cells were MHCII and CD11b depleted to remove B cells, macrophages and contaminating microglial cells. Data is represented as mean +/- SEM. Significance was calculated using one-tailed t-test. (D) CD40 and CD3 western blots on T cells recovered from brains and spinal cords of control and EAE induced mice as in D. Disease scores are noted below each lane. Control samples from both brain and spinal cord were pooled from 3 mice to achieve enough cells for western blot. EAE samples from spinal cord were pooled from 3 mice 10 (10 d), 14 (14 d), 21 (21 d), and 28 (28 d) days post induction. As an internal control, membranes were stripped and stained with Coomassie blue and a representative band is shown below each blot (Standard). Graphs depict densitometry for CD40 and CD3. It should be noted that the exposure times in the western blots are different between brain and spinal cord samples in order to better visualize spinal cord samples. However, densitometry was done on the same exposures for brain and spinal cord. Data in figure represent at least 3 experiments.