Fig. 5: Impact on local and systemic effects on ileitis of deleting Lta or Ltb in B cells.

Chimeras were studied at 30–32 weeks after BMT, wherein WT recipient mice received μMT-TNF^ΔARE/+ BM at 90% and 10% marrow from WT, Lta^−/− or Ltb^−/− mice to resupply the B cell compartment with these respective genotypes. a, Quantification of the number of TLSs in the branch of the mesentery that drains the distal ileum in these chimeras where B cells were WT, Lta^−/− or Ltb^−/−. Three independent experiments, WT B cells (n = 13), Lta^−/− B cells (n = 8) and Ltb^−/− B cells (n = 5). Statistical significance was evaluated using Kruskal–Wallis test. b, Representative whole-mount images (LYVE-1 red, CSFR1 green, B220 blue) of lymphoid aggregates in the ileal associated mesentery of WT/μMT-TNF^ΔARE/+ or Ltb^−/−/μMT-TNF^ΔARE/+ chimeric mice. Scale bar: 500 μm. c, TLS area was assessed in whole-mount images and the area of each TLS quantified depicted in the graph, with each symbol representing one TLS from WT/μMT-TNF^ΔARE/+ (n = 4 mice evaluated) or Ltb^−/−/μMT-TNF^ΔARE/+ (n = 5 mice evaluated) chimeric mice. Statistical significance used unpaired t test with Welch’s correction for nonparametric distribution. d, Flow cytometry on single-cell suspensions of the distal ileum of mice that received WT/μMT-TNF^ΔARE/+, Lta^−/−/μMT-TNF^ΔARE/+ or Ltb^−/−/μMT-TNF^ΔARE/+ BM to quantify neutrophils and T cells. Four independent experiments were done, WT/μMT-TNF^ΔARE/+ (n = 19), Lta^−/−/μMT-TNF^ΔARE/+ (n = 9), Ltb^−/−/μMT-TNF^ΔARE/+ (n = 10). e, Eight weeks after TNF^ΔARE/+ BMT, mice were dosed weekly with isotype control or LTβR-Fc for an additional 8 weeks in one experiment, isotype and LTβR-Fc (n = 3 mice in each cohort). TLSs were quantified in TNF^ΔARE/+ BM recipients given LTβR-Fc in the distal mesentery or isotype control. Flow cytometry on the ileum of mice given TNF^ΔARE/+ BM and treated with LTβR-Fc or isotype control to quantify neutrophils. f, Weekly percent weight change in μMT-TNF^ΔARE/+ BM chimeras that had WT (n = 13), LTα^−/− (n = 12) or LTβ^−/− (n = 10) B cells. Data are normalized to mouse weight 2 weeks post-BMT. Statistical analysis was carried out using two-way ANOVA with Tukey’s multiple comparisons post hoc test. Actual body weights at the endpoint are graphed separately on the right, with statistical analysis carried out using one-way ANOVA, with Tukey’s test for multiple comparisons. g, Relative intestinal permeability to FITC–dextran administered by gavage and assessed 2 h later (two independent experiments, WT B cells (n = 5), Lta^−/− B cells and Ltb^−/− B cells (n = 5)). h,i, Plasma (h) and fecal (i) IgA levels measured on mice given WT BM or mice lacking Lta^−/− or Ltb^−/− (two independent experiments, WT B cells (n = 8), Lta^−/− B cells (n = 4), Ltb^−/− B cells (n = 5)). j, Flow cytometry on single cell-digests of the ileum to quantify IgA⁺ plasma cells from the same mice used to generate data in d. k,l, Representative images of the mid-villus region in the ileum viewed in whole-mount specimens from en face after staining for IgA (red), smooth-muscle actin (white) and DAPI to identify nuclei (blue) (k) and quantification of their frequency (l). Two independent experiments, WT B cells (n = 5), Lta^−/− B cells and Ltb^−/− B cells (n = 5). Statistical comparisons used one-way ANOVA with Tukeyʼs test for multiple comparisons. Gating strategy for flow cytometry shown in Supplementary Fig. 1. Graphic plots show one mouse per symbol and depict mean ± s.e.m.

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