Induction of colitis by a CD4+ T cell clone specific for a bacterial epitope

Abstract

It is now well established that the intestinal flora plays an important role in the pathogenesis of inflammatory bowel disease (IBD). However, whether bacteria serve as the sole target of the immune response in this process or whether they act indirectly by triggering an anti-self response is still unclear. We have previously shown that specific pathogen-free IL-10-deficient (IL-10 KO) mice develop a T helper (Th1)-cytokine associated colitis after experimental infection with Helicobacter hepaticus. We here show that H. hepaticus Ag (SHelAg)-specific CD4+ Th1 clones transfer disease to H. hepaticus-infected T cell-deficient RAG KO hosts. Importantly, uninfected recipients of the SHelAg-specific clones did not develop intestinal inflammation, and a control Schistosoma mansoni-specific Th1 clone did not induce colitis upon transfer to infected RAG KO mice. The disease-inducing T cell clones recognized antigen(s) (Ag) specifically expressed by certain Helicobacter species as they responded when stimulated in vitro with H. hepaticus and Helicobacter typhlonius Ag, but not when cultured with Ag preparations from Helicobacter pylori, various non-helicobacter bacteria, or with cecal bacterial lysate from uninfected mice. Characterization of the Ag specificity of one of the clones showed that it reacts uniquely with a 15-mer peptide epitope on the flagellar hook protein (FlgE) of H. hepaticus presented by I-Ab. Together, our results demonstrate that colitis can be induced by clonal T cell populations that are highly specific for target Ag on intestinal bacteria, suggesting that an aberrant T cell response directed against gut flora is sufficient to trigger IBD.

Fig. 1.

Characterization of SHelAg-reactive T cell clones. (A) T cell clones B1 and B2 were cultured with APC and medium or boiled SHelAg in the absence or presence of anti-CD4, anti-CD8, or anti-class II mAb. IFN-γ was measured after 48 h. *, P < 0.05 compared with cells stimulated with SHelAg alone. No IL-4 or IL-5 was detected in cultures stimulated with SHelAg; however, both clones produced tumor necrosis factor α in response to this Ag (not shown). (B) TCR Vβ expression was analyzed by flow cytometry using anti-Vβ chain-specific or anti-pan-Vβ mAb (thick line) or isotype control (filled histograms). Histograms shown were gated on CD4⁺ cells. By PCR, clone B1 tested positive for Vβ15 (not shown).

Fig. 2.

SHelAg-specific T cell clones induce colitis when transferred to H. hepaticus-infected RAG KO mice. (A) Uninfected (open bars) or infected (filled bars) RAG KO mice were reconstituted with SHelAg-specific clones B1 or B2, or with S. mansoni-specific clone B as indicated. Shown is cecal inflammation (typhlitis) 7 wk after cell transfer. Bars represent mean histology scores ± SD of three to four mice per group from one representative experiment of three performed. Similar results were observed for the colon (data not shown). *, P < 0.05 compared with infected mice receiving no cells or S. mansoni clone B. (B-G) Histology of representative cecum and colon sections from mice in the experiment shown in A. (B and E) Uninfected RAG KO plus clone B1. (C and F) Infected RAG KO plus S. mansoni clone B. (D and G) Infected RAG KO plus clone B1. (B-D) Hematoxylin/eosin-stained cecal sections. (Bar = 100 μm.) (E-G) Immunohistochemical staining for CD3⁺ cells in ascending colon (≈1 cm from the cecum). (Bar = 500 μm.)

Fig. 3.

SHelAg-specific T cell clones fail to recognize Ag from non-helicobacter bacteria, but respond to other murine helicobacters. Clones B1 and B2 were cultured with APC in medium or 0.625-10 μg/ml soluble Ag from the bacteria indicated (A) or APC pulsed overnight with medium, SHelAg, or CBL (B). IFN-γ was measured after 72 h. Bars represent means ± SD of culture duplicates.

Fig. 4.

Clone B2 recognizes a defined peptide epitope on H. hepaticus FlgE. (A) Clones B1 and B2 were cultured with APC in medium or 10 μg/ml SHelAg or 0.1-10 μg/ml recombinant H. hepaticus FlgE. (B) Schematic map of H. hepaticus FlgE with fragments 1-4 and peptides a-e. (C and D) Clone B2 was cultured with APC and 1 μg/ml recombinant FlgE fragments (C) or 1 μg/ml synthetic peptides as indicated (D). IFN-γ was measured after 48 h. Bars represent means ± SD of culture duplicates.

Fig. 5.

Amino acid sequence of H. hepaticus FlgE. The flgE gene was cloned from H. hepaticus NCI-Frederick isolate 1A and sequenced (GenBank accession no. AJ583505). Shown is the amino acid sequence in one-letter code aligned to FlgE of H. pylori strain 26695 (GenBank accession no. NP 207664, H. pylori 26695 database accession no. HP0870). The first 20 underlined amino acids represent the sequence from the initial N-terminal sequencing of OMP from SHelAg. Recombinant FlgE fragments 1-4 correspond to amino acids 1-233, 194-393, 355-550, and 507-718, respectively. The boxed sequence represents peptide FlgE_532-546 that has stimulatory activity for T cell clone B2.