Supernumerary and mature tLTs induced by DSS-mediated colitis. (A) Quantification of tLTs in the colon of wild-type mice before and after multiple cycles of DSS treatment. During each cycle, mice were treated with DSS for a period of 7 d, followed by a 10-d recovery period without DSS. Data are shown for one representative experiment out of two with three mice per group. Statistical significance was assessed by the paired Student’s t test. *, P < 0.05. Error bars are SD. (B) Structure of colonic tLTs in sections of colons from wild-type mice after two cycles of DSS treatment. Sections were stained with the indicated antibodies and with DAPI for nuclear staining (shown in gray). RORγt is visualized through GFP expression in Rorc(gt)-Gfp^TG reporter mice. Bars, 200 µm.

The LTi function of B cells. (A) Quantification of tLTs in the colons of LTβR-deficient and wild-type control mice that were either untreated or treated with two cycles of 2.5% DSS. Data are shown for one representative experiment out of two with three mice per group. (B) Quantification of colonic tLTs in RORγt^−/− mice that were treated with two cycles of 2.5% DSS and by weekly i.p. injections of LTβR-Ig fusion protein or control Ig. Data are shown for one representative experiment out of two with six mice per group. *, P < 0.005. (C) Quantification of tLTs in the colon of wild-type mice and mixed bone marrow chimeras after two cycles with 2% DSS. For mixed bone marrow chimeras, irradiated adult C57BL/6 wild-type mice received bone marrow from LTβ^−/− mice that was mixed 1:1 with bone marrow from μM^−/−, CD3ε^−/−, or RAG^−/− mice to create mice that lack LTβ expression in B cells, T cells, or both B and T cells. DSS treatment was initiated 6 wk after transfer. Data are shown for one representative experiment out of three with five mice per group. *, P < 0.001. Statistical significance was assessed by the paired Student’s t test. Error bars are SD.

tLTs aggravate DSS-mediated colitis. (A) Histological disease score in RORγt^−/− and wild-type control mice exposed to two cycles of DSS. Scores are shown for eight mice per group from three independent experiments. *, P < 0.01. H&E staining of representative sections from distal colon of RORγt^−/− and wild-type control mice after exposure to two cycles of DSS is shown. Bars, 200 µm. (B) Wasting disease. RORγt^−/− and wild-type control mice were exposed to DSS cycles as indicated, and mouse body weight was assessed at the beginning of every cycle. Shown are weights relative to the starting weight. Data derive from eight mice per group from three independent experiments. *, P < 0.01. (C and D) LTβR blockade ameliorates DSS colitis. Wild-type and RORγt-deficient mice were exposed to two cycles of DSS. RORγt^−/− mice were either treated by weekly i.p. injections of LTβR-Ig protein or with a control Ig. One group of RORγt^−/− mice was treated from birth with a cocktail of antibiotics. Data are shown for six mice per group from two to three independent experiments. *, P < 0.01. Shown is mouse weight at the end of the second DSS cycle in percentage of the starting weight (C) and histological disease score (D). Abx, antibiotic treated. (E) Quantitative real-time PCR on whole colon tissue from untreated wild-type controls and RORγt^−/−, as well as wild-type control mice after exposure to DSS. Ct values were normalized to Gapdh expression. Data shown are for one representative experiment out of three with two mice per group. *, P < 0.05. (F) RORγt^−/− mice were treated with two i.p. injections of 250 µg of neutralizing anti–IFN-γ antibody before the first and the second DSS cycle. Data are shown for one representative experiment out of two with three mice per group. ns, not specific. P > 0.05. Statistical significance was assessed by the paired Student’s t test. Error bars are SD.

tLTs form in the absence of LTi cells. (A) Quantification of influenza virus A–induced iBALT. RORγt^−/− or wild-type control mice were infected intranasally with 50 pfu influenza virus A and sacrificed 3 wk later. Histograms show mean numbers of lymphoid follicles in sections of whole lungs. Data shown are the mean for seven mice per group from two independent experiments. *, P < 0.05. Sections were stained with the indicated antibodies. Bars, 200 µm. (B and C) Quantification of tLTs in the colon of RORγt^−/− and wild-type control mice. The number of colonic tLTs was assessed as described in Materials and methods and is indicated as the mean number of tLTs per whole colon section. Data shown for adult mice that were either treated with two cycles of 2.5% DSS (B) or left untreated (C). Figures show data compiled from 10–15 mice per group from three independent experiments. *, P < 0.001. Histology shows representative colon samples from RORγt^−/− or wild-type control mice either treated with two cycles of 2.5% DSS or left untreated. Sections were stained with anti-CD45R/B220 antibodies to visualize tLTs and DAPI for nuclear staining. Single pictures of 100× magnified sections were assembled to generate an integral view of a colon. Bars, 0.5 cm. Statistical significance was assessed by the paired Student’s t test. Error bars are SD.

Containment of microbiota in the absence of RORγt⁺ cells. (A) RORγt^−/− and wild-type control mice were treated from birth with a cocktail of antibiotics. 8-wk-old mice were exposed or not to two cycles of DSS before quantification of tLTs. Data are shown for one representative experiment out of two with five mice per group. *, P < 0.0001. Statistical significance was assessed by the paired Student’s t test. n.s., not significant; Abx, antibiotic treated. (B) The bacterial content in the spleen of RORγt^−/− and wild-type control mice was determined by plating spleen extracts from three individual mice exposed or not to DSS on blood agar plates. CFU, colony forming unit. Data are shown for one representative experiment out of two. (C) The IgG response specific for intestinal bacteria was determined in the sera from the peripheral blood of RORγt^−/− and wild-type control mice exposed or not to DSS. Data from one representative experiment out of three and three mice per group show the percentage of bacteria positive for IgG binding. *, P < 0.05. As a baseline control, bacteria were directly stained with anti–mouse IgG without the addition of serum (not depicted). (D) Colon biofilms were collected from 8-wk-old RORγt^−/− and wild-type control mice and the bacterial content was determined by quantitative PCR. Data are shown for one representative experiment out of two with five mice per group. SFB, segmented filamentous bacteria; Lactob, Lactobacillaceae; Clostr, Clostridiales; Bact, Bacteroides; Enterob, Enterobacteriaceae. Error bars are SD.

Hyperactive tLTs and B cells as a cause of aggravated colonic pathology. RORγt^−/− and wild-type control mice were treated with two cycles of 2.5% DSS. (A) The expression of AID was assessed by quantitative RT-PCR in laser-captured tLTs from DSS-treated RORγt^−/− and wild-type control mice. Data are shown for one representative experiment out of two with two mice per group. Ct values were normalized to Gapdh expression. (B) Colon sections were stained for IgG, B220, and DAPI for nuclear staining (shown in gray). IgG⁺ B plasma cells express low levels of B220. Bars, 200 µm. Data are shown for one representative experiment out of two with three mice per group. (C) RORγt^−/− mice were treated with two i.p. injections of IVIG. The histological disease score was assessed as described in Materials and methods. Data are shown for one representative experiment out of two with two to three mice per group. Statistical significance was assessed by the paired Student’s t test. *, P < 0.05. Error bars are SD.