Colonic epithelial cell mediated suppression of CD4 T cell activation - PubMed (original) (raw)
Colonic epithelial cell mediated suppression of CD4 T cell activation
S M Cruickshank et al. Gut. 2004 May.
Abstract
Background and aims: As the first point of contact with enteric antigens, intestinal epithelial cells (IEC) may be key in regulating mucosal immune responses. We determined therefore if murine colonic epithelial cells (CEC) have tolerogenic or activating effects on CD4 T cells.
Methods: Using a novel CEC, macrophages, and CD4 T cell coculture system, mitogen and antigen specific responses of naïve and antigen primed CD4 T cells were assessed.
Results: Although a proportion of CEC express the costimulatory molecules B7.1, B7.2, CD40, and CD54, they were unable to promote mitogen or antigen driven activation of CD4 T cells, even in the presence of exogenous costimulatory signals. CD4 T cells cocultured with CEC were CD25lo and CD45RBlo and remained in the G1 phase of the cell cycle. CEC were also able to prevent CD4 T cell activation by professional antigen presenting cells. CEC mediated suppression of T cell activation was cell contact dependent and transforming growth factor beta independent.
Conclusions: These observations suggest that CEC contribute to the maintenance of T cell tolerance in the gut by preventing inappropriate activation of CD4 T cells.
Figures
Figure 1
Colonic epithelial cells (CEC) inhibit the response of T cells to conventional mitogens. Splenic CD4 T cells (A, B, D, F, G) or colonic lamina propria CD4 T cells (C, E) were cultured with freshly isolated C57BL/6 CEC or macrophages (Mφ) in the presence of mitogenic CD3 antibody for 72 hours prior to flow cytometric analysis for expression of CD25 on splenic (A) and lamina propria T cells (C), CD45RB on splenic CD4 T cells (B), cell cycle progression of splenic T cells (D) and lamina propria T cells (E), and cytokine production of splenic T cells using cytokine bead assays (F, G). In (A), (B), and (C), the filled in and solid line profiles represent levels of antibody staining of T cells after coculture with Mφ and CEC, respectively, and the broken lines represent the isotype matched control antibodies. (D, E) Proportion of splenic and lamina propria CD4 T cells in the G1, S, and G2 phases of the cell cycle. (F, G) Cytokines produced by splenic T cells after culture with CEC and Mφ in the presence of mitogen, respectively. Results are representative of those obtained from at least six independent experiments. IFN-γ, interferon γ; IL, interleukin; TNF-α, tumour necrosis factor α.
Figure 2
Responses of antigen specific T cells are inhibited by colonic epithelial cells (CEC). I-Ad-restricted DO11.10 CD4 T cells were cocultured with OVA primed DO11.10 CEC or macrophages (Mφ) in the presence or absence of OVA for 72 hours prior to flow cytometric analysis of T cells for expression of CD25 (A), CD45RB (B), cell cycle progression (C), and cytokine production (D). (A, B) Cells cultured in the absence or presence of OVA: **p<0.005 (A) and **p<0.005 (B) comparing CD4 T cell activation in the presence of BALB/C (I-Ad) CEC or Mφ. (C) Proportion of CD4 T cells in the G1, S, and G2 phases of the cell cycle, respectively, with the plus and minus symbols referring to the presence or absence of OVA in the coculture. (D) Cytokine production by CD4 T cells cocultured with CEC and Mφ, respectively. Results are representative of those obtained from at least eight independent experiments. IFN-γ, interferon γ; IL, interleukin; TNF-α, tumour necrosis factor α. (E) C57BL/6 (I-Ab) CEC can present OVA to an OVA specific I-Ab restricted CD4 T cell hybridoma KZO. Cultured CEC were pulsed with OVA, washed, and incubated for 24 hours with KZO, which had been transfected with an NFAT-lacZ reporter gene construct. T cell hybridoma activation and lacZ activity were determined spectrophotometrically using the chromogenic substrate and measuring absorbance at 595 nm (OD 595).
Figure 3
Colonic epithelial cells (CEC) express costimulatory molecules. Freshly isolated CEC were analysed by flow cytometry using antibodies specific for the epithelial cell specific marker cytokeratin and the costimulatory molecules B7.1 (CD80), B7.2 (CD86), CD40, and CD54. The quadrants were drawn based on isotype and single stained controls. Per cent values refer to the proportion of cells that were stained positive. Results are representative of those obtained from more than 10 independent experiments.
Figure 4
T cells cultured with colonic epithelial cells (CEC) are not anergised. CD4 T cells were cultured with freshly isolated murine CEC or macrophages (Mφ) and mitogenic CD3 antibody in the presence (+) or absence (−) of 200 U/ml interleukin 2 (IL-2) (A, B) or 10 μg/ml CD28 cross linking antibody (C, D). Whole spleen cells (Spl) were cultured in the presence or absence of CEC and a mitogenic anti-CD3 antibody (E, F). Activation was assessed by cell cycle analysis (A, C, E) and expression of the activation marker CD25 (B, D, F). (A, C, E) Proportion of CD4 T cells in the G1, S, and G2 phases of the cell cycle, respectively. The positive and minus symbols refer to the presence or absence of IL-2 (A), anti-CD28 cross linking antibody (C), and anti-CD3 antibody (E), respectively. (B, D, F) Absence or presence of IL-2 (B), anti-CD28 antibody (D), and anti-CD3 antibody (F). Results are representative of those obtained from at least five independent experiments.
Figure 5
Colonic epithelial cells (CEC) mediated suppression of T cell responses is transforming growth factor β (TGF-β) independent. Freshly isolated CEC were analysed by flow cytometry for expression of cell surface TGF-β (A) and latency associated peptide (LAP) of TGF-β (B). Filled histograms show antibody labelling compared with the isotype control (black line). (C) Effect of neutralising TGF-β antibody (20 μg/ml) on cell cycle progression of CD4 cells in the presence of CEC or macrophages (Mφ); proportion of CD4 T cells in the G1, S, and G2 phases of the cell cycle, and the plus and minus symbols refer to the presence or absence of the neutralising TGF-β antibody in the coculture. (D) Effect of the neutralising antibody on proliferation of fibroblasts grown in Matrigel in the presence or absence of TGF-β1 (100 ng/ml). Results are representative of those obtained from at least four independent experiments.
Figure 6
CD4 cells previously cocultured with colonic epithelial cells (CEC) responded normally to restimulation. Naïve (A, B) or antigen primed (C, D) CD4 cells were cocultured with CEC or macrophages (Mφ) for 72 hours in the presence of mitogenic anti-CD3 antibody or OVA, after which they were isolated from cocultures and stimulated with phorbol-12-myristate 13-acetate (PMA)/ionomycin plus fresh Mφ (A, B) or OVA pulsed Mφ (C, D). T cell activation was assessed by cell cycle analysis (A, C) and expression of the activation marker CD25 (B, D). (A, C) Proportion of CD4 T cells in the G1, S, and G2 phases of the cell cycle, respectively. (B, D) Expression of CD25 on CD4 T cells cultured with CEC or Mφ, respectively. Results shown are representative of those obtained from at least four independent experiments.
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