Supplementary Materials1. T cells (Tregs) in the draining lymph nodes and CNS of mice with EAE . Through the production of IL-6 and engagement of OX40L, mast cells counteract effector Th cell N-(p-Coumaroyl) Serotonin suppression by Tregs, thus amplifying the autoreactive response. In this statement, we demonstrate that in the absence of mast cells, Th cells do not accumulate in the meninges nor do they generate a strong GM-CSF response. Mast cell-T cell co-culture experiments and selective meningeal reconstitution of mast cell-deficient mice reveal that resident meningeal mast cells are an early source of caspase-1-dependent IL-1, which in turn licenses Th cells to produce GM-CSF and become encephalitogenic. We also provide evidence of mast cell-T cell co-localization in the meninges in a subset of acute MS patients with prominent meningeal inflammation, suggesting that interactions between these cell types occur in human disease. These data have implications for developing meningeal mast cell targeted therapies that inhibit IL-1 production in early MS. 2.?Methods 2.1. Mice C57BL/6; WBB6F1-and transferred to Thy1.2+ recipients. Thy1.1+ cells were examined in the meninges (Fig. 1a,c) and the CNS (Fig. 1b,c) at days 3 and 6 post transfer. At both time points significantly more MOG-specific Th cells than OVA-specific Th cells were detected in the meninges and the CNS (Fig. 1c). This selective accumulation of MOG-specific Th cells likely reflects their conversation with myelin-bearing APCs in the meninges that serve to activate and maintain MOG-specific T cells as previously reported [6,7,14]. Open in a separate N-(p-Coumaroyl) Serotonin windows Fig. 1. MOG35C55-primed, but not OVA323C339-primed, Th cells accumulate in the meninges and CNS early in EAE.Four x106 MOG35C55- or OVA323C339-primed T cell blasts from Thy1.1+ donor mice were restimulated with peptide and transferred to congenic Thy1.2+ recipients. The frequency and numbers of Thy1.1+CD45+CD4+ cells in the meninges (a,c) and CNS (b,c) was determined 3 or 6 days post transfer. (a) Representative analysis of MOG35C55 or OVA323C339-primed CD4+Thy1.1+ T cells detected in the pooled meninges of Thy1.2+ recipients at day 6 EIF4G1 post transfer. (b) Representative analysis of MOG35C55 or OVA323C339-primed CD4+Thy1.1+ T cells detected in the CNS (pooled brain and spinal cord) of Thy1.2+ recipients 6 days post transfer. Percentages of the CD45+/hi parent gate are shown. (c) Numbers of CD45+CD4+Thy1.1+ MOG35C55 or OVA323C339-primed T cells in the meninges and CNS at N-(p-Coumaroyl) Serotonin indicated days post T cell transfer. For meninges samples, each point represents the analysis of a N-(p-Coumaroyl) Serotonin pool of tissues from 3 to 5 5 mice and is expressed as figures/mouse. CNS data points represent the analysis of individual mice. *p 0.05 and **p 0.01 by Students for 4 days before transfer of 4 106 blasts to Thy1.2+ wild type (WT), mast cell-deficient and expressed as fold induction over na?ve. n = 2 pooled samples of 5 mice each for each time point. 2 independent experiments. (h) Meningeal mast cells recognized by toluidine blue staining N-(p-Coumaroyl) Serotonin and (i) mast cell figures in na?ve (N) and T cell recipient mice (AT) at 24 h post transfer, n = 9 mice. (jCm) RT-PCR analysis of pooled meninges samples as explained in (dCg) 24 h after transfer of T cell blasts [0 (N), 2, 4, and 8 106] to wild type recipients. *p 0.05 by Students and (Fig. 2dCf), genes that.