IL-9R deficiency ameliorates the severity of EAE. (A) WT (n = 20) and KO (n = 21) mice were immunized for EAE and scored to generate graphs of mean disease score ± SEM. Results were pooled from three independent experiments, and a Mann-Whitney U test was performed to assess the p-value shown. (B) Transverse spinal cord sections of WT (n = 8) and KO (n = 8) mice from two independent experiments were taken to perform the hematoxylin and eosin staining shown. Bar, 200 µm. (C) On day 7 after EAE immunization, draining lymph nodes of WT and KO mice were taken and CD4 T cells were assessed for CD44 and CCR6 expression (percentages are shown). Results are representative of three independent experiments with four to six mice from each group per experiment.

Treatment with neutralizing anti–IL-9 antibody slightly delays EAE. (A) Mice were immunized for EAE and were treated with control Ig (n = 6) or αIL-9 antibody (n = 6) i.p. every other day starting with day −1. Mice were scored for disease severity, and the mean ± SEM for each time point is indicated. Results are representative of three independent experiments. The p-value obtained by a Mann-Whitney U test is indicated. (B and C) CD4 T cells from the peripheral lymph nodes of EAE-immunized mice were taken from each group at day 12 (score = 0 or 1 for all mice used) and stimulated with antigen-presenting cells and MOG for (B) IL-17A spot-forming units (SFU) in ELISPOTs or (C) IL-9 concentration in ELISA. Means ± SD are shown for both graphs. *, P < 0.05 from the control sample by a Student’s t test. Results shown are pooled from three independent experiments. (D) RNA was made from spinal cord samples of EAE-immunized mice from each group at the peak of disease severity in control mice, and the mean expression relative to β-actin is shown. Results are representative of four naive, eight control, and eight αIL-9–treated mice taken from three independent experiments.

Th17 cells produce IL-9. (A) T cells were stimulated in vitro with the indicated concentration of TGF-β for 4 d. Cells were washed, counted, and restimulated in the presence of αCD3/αCD28 for 24 h before supernatants were collected for ELISA. Results show means ± SD of two independent experiments. (B) Effector T cells were generated in vitro under the indicated conditions for 4 d and were restimulated as in A. Results are representative of at least four independent experiments. (C) Cells from TGF-β + IL-2 cultures were sorted based into FoxP3-GFP–positive and –negative subsets and restimulated as in A. Results are representative of four independent experiments. (D) T cells cultured under Th17 conditions were separated into IL-17F–positive and –negative subsets based on Thy1.1 expression using MACS columns (percentages are shown). Representative purity of both populations is shown. Cells were restimulated as in A for IL-9 production. Results were pooled from two independent experiments, and a Student’s t test was performed to compare the samples. Means ± SD are shown. (E) RORC-GFP mice were immunized with MOG emulsified in CFA, and splenic T cells were harvested 7 d later for sorting into the indicated populations (percentages are shown). Cells were restimulated with PMA/ionomycin for 4 h before supernatant was collected for ELISA. Results show means ± SD of two independent experiments, and a Student’s t test was performed to compare the samples. (F) qRT-PCR from Th1, Th2, and Th17 cells and BM-MCs was performed for IL-9R expression. All samples were standardized to the expression of IL-9R by BM-MCs. Results are representative of three independent samples from each experimental group.

IL-9R expression by both T cells and other cell types contributes to adoptively transferred EAE. (A and B) Lymphocytes from the brains and spinal cords of WT (n = 4) and KO (n = 4) mice were isolated, counted, and stained for (A) CD4 T cells and IL-17A and (B) macrophages and IL-6 after restimulation. The frequency of cytokine-producing cells by FACS was used to calculate the absolute numbers of cells shown. *, P < 0.01 by the Student’s t test. Results show means ± SD and are representative of three independent experiments. (C) Peripheral lymph nodes of mice were pooled and enriched for FCϵRI expression and stained for c-kit to identify MCs. Representative FACS plots are shown of WT and KO mice. Absolute numbers of MCs in lymph nodes were determined for WT naive (n = 5), KO naive (n = 5), WT EAE (n = 12), and KO EAE (n = 14) mice pooled from four independent experiments. A Student’s t test between the WT naive group and each other group was performed to assess statistical significance. Horizontal bars represent means. (D) T cells from WT and KO mice with EAE were transferred to either WT or KO mice to cause disease. Mice were scored and the mean disease scores ± SEM are shown. A Mann-Whitney U test was done to compare the WT T cell → WT group with each of the indicated groups to obtain the p-values shown. Results were pooled from two independent experiments, and the numbers of mice used are as follows: WT T cell → WT, n = 16; KO T cell → WT, n = 15; WT T cell → KO, n = 14; and KO T cell → KO, n = 12.