Fig 1.
Overview of experimental setup.
Anti-TG2 was administered by intravenous injection at the indicated time points. Either a mix of IgG2b and IgG2c (100 μg each) or IgA (400 μg) or PBS was given each time. Blood was collected at indicated time points prior to injections. At the end of the experiment, samples of the small intestine were fixed in formalin or embedded in OCT and frozen. The figure was created using elements from Servier medical arts (www.smarts.servier.com).
Fig 2.
Serum was obtained on day 0, 10 and 20 and analyzed for anti-TG2 antibodies by ELISA. Absolute concentrations of IgG2b (a), IgG2c (b) and IgA (c) were estimated by interpolating from standard curves. For four IgA-injected mice, the third serum sample was taken either on day 16 (n = 2) or day 17 (n = 2) and a fourth sample was taken on day 20 by postmortem cardiac puncture. Arrows indicate time of antibody injections. Dots and bars represent mean +/- SD. Data represent all mice from the two independent experiments.
Fig 3.
Injected anti-TG2 antibodies reach the intestinal tissue.
(a) Immunofluorescence staining of frozen sections of small intestine obtained on day 20 show deposition of mouse IgG (cyan) in the basement membrane of IgG injected WT mice that co-localizes with ECM staining for endogenous TG2 (magenta). No IgG deposits were detected in PBS injected WT mice and no clear ECM deposits were observed in IgG-injected Tgm2-/- mice. Nuclei counterstained with DAPI are shown in blue. (b) Distribution of IgG2b and IgG2c in the small intestine of WT mice injected with IgG (top panels). Weak antibody signal is detected also in the intestine of Tgm2-/- mice while no signal is seen in PBS-injected mice, which indicates that antibody presence in tissue does not per se depend on presence of cognate antigen. (c) Quantification of subepithelial fluoresence signal intensity from staining for IgG2b and IgG2c together (top) or separately (bottom). Each dot represents mean fluorescence intensity calculated from one image as described in materials and methods. Bar graphs show the group mean fluorescence intensity with standard error of mean. Scale bars represent 100 μm.
Fig 4.
Injection of anti-TG2 antibodies does not impair weight gain.
The graphs report weight as % change from baseline for WT mice and Tgm2 deficient mice receiving injections of IgG. Dots and bars represent mean +/- SD. Data represent all mice from the two independent experiments.
Fig 5.
No difference in mucosal architecture between groups.
(a) Examples of Vh and Cd measurements. Formalin-fixed paraffin-embedded samples stained with hematoxylin/eosin. Representative images show well-oriented pairs of villus and crypt from duodenum and ileum. Numbers indicate length of corresponding bars. (b, c) Vh, Cd and Vh/Cd ratio in duodenum (b) and ileum (c) of the different experimental groups. The WT IgG group was compared to each control group by individual Mann-Whitney tests. Data are pooled from two independent experiments. Bars represent mean +/- SD. *P ≤ 0.05. n.s.: Not significant.
Fig 6.
IEL count in duodenum and ileum.
(a) Examples of staining for anti-CD3. Formalin-fixed paraffin-embedded sections stained with anti-CD3 and counterstained with hematoxylin. Representative images show CD3+ cells in duodenum and ileum. (b) CD3+ IELs per 100 epithelial cells in well-oriented villi in duodenum and ileum. The WT IgG group was compared to each control group by individual Mann-Whitney tests. Data represent all mice from the two independent experiments. Bars represent mean +/- SD. **P ≤ 0.01. n.s.: Not significant.