Increased frequency of intestinal CD4+ T cells reactive with mycobacteria in patients with Crohn's disease

Abstract

Objective: The aim of this study was to assess the frequency of mycobacteria and Escherichia coli reactive T cells in intestinal biopsies from patients with Crohn's disease (CD) and ulcerative colitis (UC).

Materials and methods: The biopsies were obtained by colonoscopy from adult patients with active CD (n = 5) and active UC (n = 4). The number of CD4+ T cell clones expanded and screened from each patient varied from 383 to 3972 giving a total of 16639 individual clones. The T cell clones were tested for responses to Mycobacterium avium subspecies paratuberculosis (MAP) and E. coli. The cytokine profile of 42 individual T cell clones from four CD patients was assessed.

Results: The frequency of mycobacteria reactive T cell clones in CD patients ranged from 0.17 to 1.63% and was higher (p = 0.038) than the frequency of E. coli reactive T cells ranging from 0 to 0.18%. No or very low numbers of mycobacteria reactive clones were detected in three UC patients while the fourth UC patient had a frequency similar to what was observed in CD patients. The frequencies of E. coli reactive T cell clones in UC patients ranged from 0 to 0.52%. T cell clones (n = 42) from CD patients all produced IL-17 and/or IFN-γ. Several clones were also able to produce IL-10.

Conclusions: The high frequency of intestinal tissue resident T cells reactive to mycobacteria suggests that an adaptive immune response have taken place and argues that these bacteria may contribute to the chronic inflammation in CD.

Figure 1.

Screening of 3972 clones from one CD patient (IBD55). CD4 T cells were isolated from intestinal biopsies and expanded in vitro. The responses to MAP and E. coli was tested in a ³H thymidine incorporation assay using autologous adherent cells as APC. Each symbol represents one tested well. The line indicates the cut-off of a stimulation index >10 (response in antigen stimulated well/response in unstimulated wells). The response to the two bacterial antigens was compared using a binominal test (p = 0.032).

Figure 2.

Cytokine secretion in CD4 T cell clones. Production of IFN-γ, IL-10, IL-17 and IL-4 by CD4 T cell clones (n = 20) from two different patients after stimulation with and anti-CD3/anti CD28 beads for IL-10 and IL-4, and PMA/Ionomycin for IL-17 and IFN-γ. The cells were stimulated in duplicates for 24 h. The cytokine production in the supernatant was measured in the Bio-Plex™ assay.

Figure 3.

Expression of surface markers in a Th1 clone (left panel) and a Th17/Th1 clone (right panel). The clones were stained with anti-CD4 and then mixed with unstained PBMC to be used as an internal control. The mix was subsequently stained with antibodies against the surface markers β7 integrin, CCR6 and CD161 and analysed by flow cytometry. The negative population of the PBMC for the respective markers were used as an internal control (open histograms) while gating on the CD4+ T cells gave the expression of the various surface markers on the tested clone (grey histograms). Left panels depicts TCC955.M.1 and the right panel TCC958.B.M.1.