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T helper 1 and T helper 2 cells are pathogenic in an antigen-specific model of colitis.

Iqbal N, Oliver JR, Wagner FH, Lazenby AS, Elson CO, Weaver CT - J. Exp. Med. (2002)

Bottom Line: Transfer of antigen-naive DO11.RAG-2(-/-) T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease.The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable.Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35233, USA.

ABSTRACT
Dysregulated T cell responses to enteric bacteria have been implicated as a common mechanism underlying pathogenesis in rodent models of colitis. However, the bacterial species and T cell specificities that induce disease have been poorly defined. We have developed a model system in which target antigen, bacterial host, and corresponding T cell specificity are defined. OVA-specific T cells from DO11.RAG-2(-/-) TCR transgenic mice were transferred into RAG-2(-/-) recipients whose intestinal tracts were colonized with OVA-expressing or control Escherichia coli. Transfer of antigen-naive DO11.RAG-2(-/-) T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease. In contrast, transfer of polarized T helper (Th) 1 and Th2 populations resulted in severe wasting and colitis in recipients colonized with OVA-expressing but not control E. coli. The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable. Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA. These results establish that a single bacterially associated antigen can drive the progression of colitis mediated by both Th1 and Th2 cells and provide a new model for understanding the immunoregulatory interactions between T cells responsive to gut floral antigens.

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Inducible expression of immunogenic OVA by pnir15.OVA-E. coli. (A) Schematic of the pnir15.OVA plasmid. Restriction enzyme sites are: Bgl, Bgl II; RV, Eco RV; B, Bam H1. The ampicillin resistance gene (Ampr) and origin of replication (pBR322ORI) are indicated. (B) Western blot analysis of DH5α E. coli transformed with pnir15.OVA or a control plasmid that codes for expression of the C fragment of tetanus toxoid (pnir15.TET). Cytosolic fractions of lysates of bacteria cultured for 12 h under aerobic (−; noninduced) or anaerobic (+; induced) conditions were prepared and analyzed by Western blotting (see Materials and Methods). The indicated lanes were loaded with 6 × 105 bacterial equivalents or 500 ng purified native OVA. The antibody used for detection was a murine IgG2b monoclonal (OVA-3) that is specific for OVA. Note that the bacterially expressed OVA runs at a lower MW than native OVA due to absent glycosylation. (C) Splenic adherent cells isolated from BALB/c mice were pulsed for 18 h with the indicated number of gentamycin-killed, anaerobically induced pnir15.TET-E. coli (white circles) or pnir15.OVA-E. coli (black circles) then irradiated (2,500 rads) before culture with CD4+ T cells isolated for DO11.10 mice (left). The response of the same cells to APCs pulsed with nothing or an optimal dose of native OVA (100 μg/ml) is shown for comparison (right). Data are the mean ± SD of day 3 proliferative responses determined by 3[H]-TdR incorporation.
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fig1: Inducible expression of immunogenic OVA by pnir15.OVA-E. coli. (A) Schematic of the pnir15.OVA plasmid. Restriction enzyme sites are: Bgl, Bgl II; RV, Eco RV; B, Bam H1. The ampicillin resistance gene (Ampr) and origin of replication (pBR322ORI) are indicated. (B) Western blot analysis of DH5α E. coli transformed with pnir15.OVA or a control plasmid that codes for expression of the C fragment of tetanus toxoid (pnir15.TET). Cytosolic fractions of lysates of bacteria cultured for 12 h under aerobic (−; noninduced) or anaerobic (+; induced) conditions were prepared and analyzed by Western blotting (see Materials and Methods). The indicated lanes were loaded with 6 × 105 bacterial equivalents or 500 ng purified native OVA. The antibody used for detection was a murine IgG2b monoclonal (OVA-3) that is specific for OVA. Note that the bacterially expressed OVA runs at a lower MW than native OVA due to absent glycosylation. (C) Splenic adherent cells isolated from BALB/c mice were pulsed for 18 h with the indicated number of gentamycin-killed, anaerobically induced pnir15.TET-E. coli (white circles) or pnir15.OVA-E. coli (black circles) then irradiated (2,500 rads) before culture with CD4+ T cells isolated for DO11.10 mice (left). The response of the same cells to APCs pulsed with nothing or an optimal dose of native OVA (100 μg/ml) is shown for comparison (right). Data are the mean ± SD of day 3 proliferative responses determined by 3[H]-TdR incorporation.

Mentions: To generate bacteria that express the antigen recognized by the DO11.10 TCR, a plasmid encoding the full-length OVA cDNA (pnir15.OVA) was introduced into DH5α E. coli (Fig. 1 A). E. coli was chosen as the bacterial host because it is a normal commensal and because it is an immune stimulatory organism in spontaneously colitic mice (28). Western blot analyses of lysates of DH5α E. coli transformed with the pnir15.OVA plasmid indicate that this construct directs excellent cytosolic expression of OVA protein under anaerobic conditions (Fig. 1 B). E. coli transformed with a plasmid in which the coding sequence of OVA was replaced with that of the C fragment of tetanus toxoid (pnir15.TET-E. coli) served as a negative control. Based on densitometric comparisons between immunoblots performed with bacterial lysates of pnir15.OVA-E. coli and purified OVA, the anaerobically induced plasmid expressed ∼1 μg OVA per 106 bacteria under in vitro conditions. The immunogenicity of pnir15.OVA-E. coli was determined in vitro by T cell proliferation assays (Fig. 1 C). Splenic adherent cells pulsed with anaerobically induced, gentamycin-killed pnir15.OVA-E. coli–stimulated proliferation of naive DO11.10 T cells, whereas splenic adherent cells pulsed with pnir15.TET-E. coli did not. The dose of bacteria that stimulated a maximal response (107 organisms) expressed ∼10 μg OVA; the dose of free OVA that was required for a maximal response was 100 μg/ml. Thus, induced pnir15.OVA-E. coli are a potent immunogen for stimulation of DO11.10 cells in vitro.


T helper 1 and T helper 2 cells are pathogenic in an antigen-specific model of colitis.

Iqbal N, Oliver JR, Wagner FH, Lazenby AS, Elson CO, Weaver CT - J. Exp. Med. (2002)

Inducible expression of immunogenic OVA by pnir15.OVA-E. coli. (A) Schematic of the pnir15.OVA plasmid. Restriction enzyme sites are: Bgl, Bgl II; RV, Eco RV; B, Bam H1. The ampicillin resistance gene (Ampr) and origin of replication (pBR322ORI) are indicated. (B) Western blot analysis of DH5α E. coli transformed with pnir15.OVA or a control plasmid that codes for expression of the C fragment of tetanus toxoid (pnir15.TET). Cytosolic fractions of lysates of bacteria cultured for 12 h under aerobic (−; noninduced) or anaerobic (+; induced) conditions were prepared and analyzed by Western blotting (see Materials and Methods). The indicated lanes were loaded with 6 × 105 bacterial equivalents or 500 ng purified native OVA. The antibody used for detection was a murine IgG2b monoclonal (OVA-3) that is specific for OVA. Note that the bacterially expressed OVA runs at a lower MW than native OVA due to absent glycosylation. (C) Splenic adherent cells isolated from BALB/c mice were pulsed for 18 h with the indicated number of gentamycin-killed, anaerobically induced pnir15.TET-E. coli (white circles) or pnir15.OVA-E. coli (black circles) then irradiated (2,500 rads) before culture with CD4+ T cells isolated for DO11.10 mice (left). The response of the same cells to APCs pulsed with nothing or an optimal dose of native OVA (100 μg/ml) is shown for comparison (right). Data are the mean ± SD of day 3 proliferative responses determined by 3[H]-TdR incorporation.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196021&req=5

fig1: Inducible expression of immunogenic OVA by pnir15.OVA-E. coli. (A) Schematic of the pnir15.OVA plasmid. Restriction enzyme sites are: Bgl, Bgl II; RV, Eco RV; B, Bam H1. The ampicillin resistance gene (Ampr) and origin of replication (pBR322ORI) are indicated. (B) Western blot analysis of DH5α E. coli transformed with pnir15.OVA or a control plasmid that codes for expression of the C fragment of tetanus toxoid (pnir15.TET). Cytosolic fractions of lysates of bacteria cultured for 12 h under aerobic (−; noninduced) or anaerobic (+; induced) conditions were prepared and analyzed by Western blotting (see Materials and Methods). The indicated lanes were loaded with 6 × 105 bacterial equivalents or 500 ng purified native OVA. The antibody used for detection was a murine IgG2b monoclonal (OVA-3) that is specific for OVA. Note that the bacterially expressed OVA runs at a lower MW than native OVA due to absent glycosylation. (C) Splenic adherent cells isolated from BALB/c mice were pulsed for 18 h with the indicated number of gentamycin-killed, anaerobically induced pnir15.TET-E. coli (white circles) or pnir15.OVA-E. coli (black circles) then irradiated (2,500 rads) before culture with CD4+ T cells isolated for DO11.10 mice (left). The response of the same cells to APCs pulsed with nothing or an optimal dose of native OVA (100 μg/ml) is shown for comparison (right). Data are the mean ± SD of day 3 proliferative responses determined by 3[H]-TdR incorporation.
Mentions: To generate bacteria that express the antigen recognized by the DO11.10 TCR, a plasmid encoding the full-length OVA cDNA (pnir15.OVA) was introduced into DH5α E. coli (Fig. 1 A). E. coli was chosen as the bacterial host because it is a normal commensal and because it is an immune stimulatory organism in spontaneously colitic mice (28). Western blot analyses of lysates of DH5α E. coli transformed with the pnir15.OVA plasmid indicate that this construct directs excellent cytosolic expression of OVA protein under anaerobic conditions (Fig. 1 B). E. coli transformed with a plasmid in which the coding sequence of OVA was replaced with that of the C fragment of tetanus toxoid (pnir15.TET-E. coli) served as a negative control. Based on densitometric comparisons between immunoblots performed with bacterial lysates of pnir15.OVA-E. coli and purified OVA, the anaerobically induced plasmid expressed ∼1 μg OVA per 106 bacteria under in vitro conditions. The immunogenicity of pnir15.OVA-E. coli was determined in vitro by T cell proliferation assays (Fig. 1 C). Splenic adherent cells pulsed with anaerobically induced, gentamycin-killed pnir15.OVA-E. coli–stimulated proliferation of naive DO11.10 T cells, whereas splenic adherent cells pulsed with pnir15.TET-E. coli did not. The dose of bacteria that stimulated a maximal response (107 organisms) expressed ∼10 μg OVA; the dose of free OVA that was required for a maximal response was 100 μg/ml. Thus, induced pnir15.OVA-E. coli are a potent immunogen for stimulation of DO11.10 cells in vitro.

Bottom Line: Transfer of antigen-naive DO11.RAG-2(-/-) T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease.The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable.Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Alabama at Birmingham, Birmingham, AL 35233, USA.

ABSTRACT
Dysregulated T cell responses to enteric bacteria have been implicated as a common mechanism underlying pathogenesis in rodent models of colitis. However, the bacterial species and T cell specificities that induce disease have been poorly defined. We have developed a model system in which target antigen, bacterial host, and corresponding T cell specificity are defined. OVA-specific T cells from DO11.RAG-2(-/-) TCR transgenic mice were transferred into RAG-2(-/-) recipients whose intestinal tracts were colonized with OVA-expressing or control Escherichia coli. Transfer of antigen-naive DO11.RAG-2(-/-) T cells into recipients colonized with OVA-E. coli resulted in enhanced intestinal recruitment and cell cycling of OVA-specific T cells; however, there was no development of disease. In contrast, transfer of polarized T helper (Th) 1 and Th2 populations resulted in severe wasting and colitis in recipients colonized with OVA-expressing but not control E. coli. The histopathologic features of disease induced by Th1 and Th2 transfers were distinct, but disease severity was comparable. Induction of disease by both Th1 and Th2 transfers was dependent on bacterially associated OVA. These results establish that a single bacterially associated antigen can drive the progression of colitis mediated by both Th1 and Th2 cells and provide a new model for understanding the immunoregulatory interactions between T cells responsive to gut floral antigens.

Show MeSH
Related in: MedlinePlus