The Th17 cell lineage dominates during inflammation. IL-17A fate-reporter mice (IL-17A^WT) were subjected to MOG/CFA administration to induce EAE or given H. hepaticus (Hh) plus anti–IL-10R mAb to induce typhlocolitis, and the cytokine-secreting phenotype of Th17 lineage-positive (RFP⁺) CD4⁺ cells was assessed at different time points. (A) Representative intracellular flow cytometry plots for IFN-γ and IL-17 of gated RFP⁺CD4⁺ T cells during EAE (day 17) in iLN and CNS, or during H. hepaticus colitis (day 14) in mLN and large intestinal LP. (B) Dynamics of Th17 cell–derived populations as a proportion of RFP⁺CD4⁺ T cells (upper panels) and their absolute numbers (lower panels) in indicated tissues during EAE induction (PEC, peritoneal exudate cells). Naive = prior to MOG/CFA administration, presym = presymptomatic (day 6), and peak = peak of clinical score (day 17). Values represent average ± SEM, n = 4/time point. (C) Representative staining for I-Ab/MOG_38–49 (top flow panels) and average distribution (bottom panel) in indicated T cell populations as proportion of CD4⁺CD44^hi T cells harvested from the CNS of IL-17A^WTIFN-γ^eYFP mice at day 17 after EAE induction. Data are representative of two independent experiments (average ± SEM, n = 6), ***p < 0.001.

Th17 to Th1 conversion is not required for H. hepaticus-induced intestinal pathology. IL-17A^WT mice were crossed with floxed Tbx21 mice and their naive T cell polarization potential assessed in vitro (A and B), and IL-17A^WT, IL-17A^ΔTbet, Rag1^WT, and Rag1^ΔTbet mice were inoculated with H. hepaticus (Hh) plus anti–IL-10R, and, 2 wk later, ceca, colons, and mLN were collected and processed for histology (C) and/or intracellular staining for cytokines and Tbet (D–H). (A) In vitro differentiation of naive CD4⁺ T cells from IL-17A^WT controls and IL-17A^ΔTbet mice toward Th1 and Th17 lineages. (B) Proliferation profile of naive CD4⁺ T cells cultured under Th17-polarizing conditions from indicated mouse lines. (C) Histology scores of ascending colon from indicated mouse lines 2 wk post-Hh/anti–IL-10R administration (n = 4 per group except for Rag1^WT where only two mice were examined). Data for IL-17A^WT and IL-17A^ΔTbet mice are representative of two independent experiments. (D and E) Proportions (D) and numbers (E) of LP RFP⁺CD4⁺ and RFP⁻CD4⁺ T cells expressing IFN-γ alone, IL-17 alone, or both IFN-γ and IL-17 from pooled cecum and colon from indicated mouse lines 2 wk post-Hh/anti–IL-10R administration. Data for IL-17A^WT and IL-17A^ΔTbet mice are representative of two independent experiments. (F) Dot plots show the proportions of IL-17 single-positive, IL-17/IFN-γ double-positive, and IFN-γ single-positive cells within the mLN and LP RFP⁺CD4⁺ T cell population from indicated mouse lines 2 wk post-Hh/anti–IL-10R administration. (G) Assessment of Tbet expression by flow cytometry staining in RFP⁺ (top row) or RFP⁻ (bottom row) LP CD4⁺ T cells positive for either IL-17 alone (filled gray), IL-17 and IFN-γ (thin line), or IFN-γ alone (bold line) from indicated mouse lines 2 wk post-Hh/anti–IL-10R administration. (H) Average mean fluorescence intensity (MFI) of Tbet staining of indicated RFP⁺ CD4⁺ T cell subsets derived from indicated mouse lines. Data are from the mice shown in (D) and (E) (averages ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001.

Tbet is required for Th17 to Th1 conversion in EAE. IL-17A fate-reporter mice (IL-17A^WT) were crossed with floxed Tbx21 or Eomes mice. T cells were sourced from the iLN or CNS of IL-17A^WT controls, IL-17A^ΔTbet, and IL-17A^ΔEomes mice at the onset of EAE symptoms (day 17) and characterized for cytokine production (A–C), or from the Peyer’s patches of nonchallenged mice (D). (A) Flow cytometry for IFN-γ and IL-17 in RFP⁺ Th17 lineage–positive cells in indicated mouse lines and tissues 17 d after EAE induction. (B) Representative dot plots of RFP⁺ (top row) or RFP⁻ (bottom row) CD4⁺ T cells harvested from the CNS at day 17 post-EAE induction from IL-17A^WT controls (left panels) and IL-17A^ΔTbet mice (right panels) and stained for IFN-γ and IL-17. (C) RFP⁺ Th17 lineage-positive (top panels) and RFP⁻ lineage-negative (bottom panels) cells from IL-17A^WT controls (open bars) or IL-17A^ΔTbet mice (black bars) were stained for IL-17 and IFN-γ, and proportions (left panels) and cell numbers (right panels) of cells expressing IL-17 and/or IFN-γ are shown. (D) Staining for PD-1 and CXCR5 in Peyer’s patches of indicated mouse lines. Dot plots are gated on RFP⁺CD4⁺ cells. Data are from two independent experiments with n = 4–5 per experiment (averages ± SEM). *p < 0.05, ***p < 0.001.

Characterization of Tbet-deficient Th17 cells in EAE. T cells were sourced from the CNS of IL-17A^WT controls and IL-17A^ΔTbet mice at the peak of EAE symptoms (day 15–17) and characterized for their cytokine production (A–D). RFP⁺ Th17 lineage-positive (top panels) and RFP⁻ lineage-negative (bottom panels) cells from IL-17A^WT controls (white bars) or IL-17A^ΔTbet mice (black bars) were stained for IL-17, GM-CSF, and TNF, and proportions (left panels) and cell numbers (right panels) of cells expressing IL-17 and/or IL-17F (A), GM-CSF (B), and TNF (C) are shown. (D) Total proportion and numbers of IL-17F–, GM-CSF–, and TNF-producing CD4⁺ T cells present in the CNS of indicated mouse lines (averages ± SEM, n = 4–5). *p < 0.05, **p < 0.01, ***p < 0.001.

Th17 to Th1 conversion is not required for EAE pathogenesis. (A) Clinical EAE scores of four indicated mouse lines immunized with MOG/CFA (n = 8–9/group, two biological repeats). (B) Relative distribution (top panels) and number of cells (lower panels) expressing indicated cytokines in the CNS of IL-17A^WT controls (white bars) or IL-17A^ΔTbet (black bars) during EAE (day 17). (C) Clinical EAE scores of Rag2^−/− mice, upon adoptive transfer of flow-sorted CD4⁺RFP⁺ T cells obtained from indicated mouse lines, immunized with MOG/CFA (n = 6/group pooled from two biological repeats). (D) Flow cytometry for indicated cytokines on cells obtained from mice undergoing EAE, as shown in (C). (E) Staining for I-Ab/MOG_38–49 in indicated T cell populations (left panels) harvested from the CNS and proportional distribution (right panel) as proportion of CD4⁺CD44^hi T cells (n = 8). Data are pooled from two independent experiments (average ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001.

Th17 cell maintenance is not required for EAE immunopathology. (A) Flow cytometry for IFN-γ and IL-17 from naive T cells from Rag1^WT and Rag1^ΔRORa mouse lines polarized in vitro toward Th1 or Th17 cells. (B) Clinical EAE scores of Rag1^WT and Rag1^ΔRORa mouse lines immunized with MOG/CFA (n = 6–7). (C) Clinical EAE scores of IL-17A^WT and IL-17A^ΔRORγt mouse lines immunized with MOG/CFA (n = 8/group). (D) Numbers of total CD4⁺ T cells, RFP⁺ Th17 lineage-positive, and RFP⁻ Th17 lineage-negative cells present in the CNS of IL-17A^WT controls (white bars) or IL-17A^ΔRORγt mice (gray bars) upon EAE induction at day 17 (averages ± SEM., n = 6). (E and F) Flow cytometry for indicated cytokines on RFP⁺ Th17 lineage-positive (E) or RFP⁻ lineage-negative (F) cells obtained from IL-17A^WT and IL-17A^ΔRORγt mice undergoing EAE, as shown in (C). (G) Numeric presence of total CD4⁺ T cells expressing indicated cytokines in the CNS of IL-17A^WT controls (white bars) or IL-17A^ΔRORγt (gray bars) during EAE at day 17 (averages ± SEM, n = 6). *p < 0.05, **p < 0.01, ***p < 0.001.