CD4+ T cells reactive to enteric bacterial antigens in spontaneously colitic C3H/HeJBir mice: increased T helper cell type 1 response and ability to transfer disease

Abstract

C3H/HeJBir mice are a new substrain that spontaneously develop colitis early in life. This study was done to determine the T cell reactivity of C3H/HeJBir mice to candidate antigens that might be involved in their disease. C3H/HeJBir CD4+ T cells were strongly reactive to antigens of the enteric bacterial flora, but not to epithelial or food antigens. The stimulatory material in the enteric bacteria was trypsin sensitive and restricted by class II major histocompatibility complex molecules, but did not have the properties of a superantigen. The precursor frequency of interleuken (IL)-2-producing, bacterial-reactive CD4+ T cells in colitic mice was 1 out of 2,000 compared to 1 out of 20,000-25,000 in noncolitic control mice. These T cells produced predominately IL-2 and interferon gamma, consistent with a T helper type 1 cell response and were present at 3-4 wk, the age of onset of the colitis. Adoptive transfer of bacterial-antigen-activated CD4+ T cells from colitic C3H/HeJBir but not from control C3H/HeJ mice into C3H/HeSnJ scid/scid recipients induced colitis. These data represent a direct demonstration that T cells reactive with conventional antigens of the enteric bacterial flora can mediate chronic inflammatory bowel disease.

Figure 1

CD4⁺ T cells from C3H/HeJBir mice responded to cecal bacterial antigens but not food or epithelial cell antigens. Cecal bacterial antigens from C3H/HeJ mice, food antigens, and epithelial cell antigens were prepared as described in Materials and Methods. APCs from C3H/HeJ mice were incubated with 200 μg/ml of antigen for 18 h, then 4 × 10⁵ antigen pulsed APCs were added to 4 × 10⁵ splenic and MLN CD4⁺ T cells of C3H/HeJBir mice. 0.5 μCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures and representative of three experiments with similar results.

Figure 2

Responses of C3H/HeJBir and C3H/HeJ CD4⁺ T cells to enteric bacterial antigens. APCs from C3H/HeJ mice were prepared as in Fig. 1, then 4 × 10⁵ CD4⁺ T cells from spleen and MLN of 12-wk-old mice were cultured with 4 × 10⁵ of antigen pulsed APCs per well. (A) Proliferative response. 0.5 mCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures and representative of three experiments with similar results. (B) IL-3 cytokine response. Supernatants were collected at day 3 of triplicate cultures and pooled together for IL-3 bioassay by using the IL-3–dependent cell line FDC-1 (see Materials and Methods). These results are representative of three experiments.

Figure 3

Dose–response of C3H/HeJBir CD4⁺ T cells (A) and C3H/ HeJ CD4⁺ T cells (B) to cecal bacterial antigens. 4 × 10⁵ CD4⁺ T cells from spleen and MLN of 12-wk-old C3H/HeJBir mice were cultured with 4 × 10⁵ per well of APCs pulsed overnight concentrations of antigen ranging between 0.2 and 1,000 μg/ml. 0.5 μCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures and are representative of three experiments.

Figure 4

C3H/HeJBir CD4⁺ T cell response to enteric bacterial antigens is MHC class II– dependent. 4 × 10⁵ C3H/HeJBir spleen and MLN CD4⁺ T cells from 12-wk-old C3H/HeJBir mice were cultured with 4 × 10⁵ cecal bacterial antigen pulsed APCs from C3H/HeJ mice per well prepared as in Fig. 1, in the presence or absence of 10 μg/ml monoclonal anti–I-A^b or anti–I-A^k. 0.5 μCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures and are representative of three experiments.

Figure 5

The cecal bacterial lysate component that stimulates C3H/ HeJBir CD4⁺ T cells is a protein. APCs from C3H/HeJ mice were pulsed with cecal bacterial antigens from C3H/HeJ mice that had been pretreated with trypsin and then dialyzed, or dialyzed but not trypsin-treated, or stored in a tube for an equivalent amount of time. Control APCs were pulsed overnight with freshly obtained cecal bacterial antigens. Each of these antigen-pulsed APCs was added to aliquots of the same pool of C3H/HeJBir CD4⁺ T cells and cultured as described in the legend to Fig. 1. 0.5 μCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures and are representative of three experiments.

Figure 6

The cecal bacterial protein that stimulates C3H/HeJBir CD4⁺ T cells is not a superantigen. C3H/HeJBir CD4⁺ T cells were cultured with APCs from C3H/HeJ mice that were pulsed with SEB or CBA from C3H/HeJ mice for 30 min, or fixed with paraformadehyde and then pulsed with SEB or CBA overnight, or pulsed with SEB or CBA overnight. 0.5 μCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures and are representative of three experiments.

Figure 7

Kinetics of the C3H/HeJBir CD4⁺ T cell response to enteric bacterial antigens. 4 × 10⁵ spleen and MLN CD4⁺ T cells from C3H/ HeJBir mice of various ages from 2 to 25 wk were cultured with 4 × 10⁵ cecal bacterial (closed circles) or E. coli (open circles) antigen pulsed APCs from C3H/HeJ mice per well, prepared as in Fig. 1. 0.5 μCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures.

Figure 8

C3H/HeJBir CD4⁺ T cell responses to different enteric bacterial species. 4 × 10⁵ spleen and MLN CD4⁺ T cells from 12-wk-old C3H/HeJBir, C3H/HeJ or C57Bl/6 mice were cultured with 4 × 10⁵ syngeneic APCs that were pulsed with lysates of E. coli (A), or with Bacteroides vulgatus, Eubacterium species, or Proteus mirabilis (B). C3H/HeJ APCs were used for both C3H/HeJBir and C3H/HeJ cultures. 0.5 μCi of [³H]TdR was added to the wells for the last 18 h of a 5-d culture. Results are expressed as mean cpm ± SD of triplicate cultures.

Figure 9

Histopathology of the colon of C3H/HeSnJ scid/scid mice that had been injected with CD4⁺ T cells from either C3H/HeJBir or C3H/HeJ mice 3 mo before. Before the transfer, CD4⁺ T cells from both sources were activated for 4 d with cecal bacterial antigen-pulsed APCs. Three months after transfer, the colon of the recipient of the activated C3H/HeJ T cells (left panel) shows normal mucosa overlying a lymphoid follicle, whereas the colon of the recipient of activated C3H/HeJBir T cells (right panel) shows inflammation and a focal ulcer.

Figure 9

Histopathology of the colon of C3H/HeSnJ scid/scid mice that had been injected with CD4⁺ T cells from either C3H/HeJBir or C3H/HeJ mice 3 mo before. Before the transfer, CD4⁺ T cells from both sources were activated for 4 d with cecal bacterial antigen-pulsed APCs. Three months after transfer, the colon of the recipient of the activated C3H/HeJ T cells (left panel) shows normal mucosa overlying a lymphoid follicle, whereas the colon of the recipient of activated C3H/HeJBir T cells (right panel) shows inflammation and a focal ulcer.