Cells from spinal cord were restimulated in vitro with MOG peptide and stained intracellularly for IL-17 and IFN-γ. As shown in Fig. 4, MOG-specific T cells from inflamed click here spinal cords belonged to Th1, Th17, and Th17/Th1 subsets. However, the percentage of CD4+ T cells from LFA-1+/+ and LFA-1−/− mice producing these cytokines was absolutely comparable on the level of antigen-specific cells. In addition, the amount of cytokines

produced did not differ (MFI for IL-17: 20 020±1457 (LFA-1+/+) versus 21 460±1080 (LFA-1−/−), p=0.436; MFI for IFN-γ: 15 436±2127 (LFA-1+/+) versus 14 940±804 (LFA-1−/−), p=0.832). The same results were obtained for IL-2 and TNF-α. However, it is important to note that the increased total number of antigen-specific cells finally results in a higher absolute number of cytokine-producing CD4+ T cells. Interestingly, there was also no correlation between EAE score of an individual animal and cytokine production on the single cell level. Again, only the number of infiltrating CD4+ T cells correlates with disease severity (see above). Polyfunctional Th1 cells producing multiple effector cytokines at the same time are believed to be particularly

destructive in inflammation 12. Therefore, we also analyzed whether the frequency of IL-2, IL-17, IFN-γ double JNK inhibitor or triple producers was altered between WT and KO mice, but did not find any significant differences (data not shown). Alternatively, a change in Th2 or anti-inflammatory cytokines could influence the severity of disease. Therefore, we tested for the production of IL-4 and IL-10. Only

very few (<2%) antigen-specific CD4+ T cells in the spinal cord produced these two cytokines. However, for we did not observe any significant differences between LFA-1+/+ and LFA-1−/− T cells (data not shown). To analyze the general capacity of T cells to produce certain cytokines, we additionally used an antigen-independent stimulation with PMA and ionomycin. Also, with this kind of stimulation, none of the analyzed cytokines differed between KO and WT mice (data not shown). Taken together, these results clearly show that loss of LFA-1 does not alter the cytokine pattern of autoreactive CD4+ T cells. Therefore, only the increased total number of antigen-specific, cytokine-producing cells in LFA-1−/− mice can be accounted for the increased severity of EAE. Treg play an important role for the suppression of chronic inflammation 8, 13. They control the expansion as well as the function of autoreactive effector T cells. Utilizing intracellular staining for the lineage-specific transcription factor FoxP3, we analyzed Treg in the spinal cord of LFA-1−/− and LFA-1+/+ mice after EAE induction. The absolute number of Treg was the same in both groups (Fig. 5).