Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis - PubMed (original) (raw)

Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: a novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis

Peter McGuirk et al. J Exp Med. 2002.

Abstract

Antigen-specific T helper type 1 (Th1) cells mediate protective immunity against a range of infectious diseases, including that caused by Bordetella pertussis. Distinct T cell subtypes that secrete interleukin (IL)-10 or tumor growth factor (TGF)-beta are considered to play a role in the maintenance of self-tolerance. However, the antigens recognized by these regulatory T cells in vivo have not been defined. Here we provide the first demonstration of pathogen-specific T regulatory type 1 (Tr1) cells at the clonal level and demonstrate that these cells are induced at a mucosal surface during an infection where local Th1 responses are suppressed. Tr1 clones specific for filamentous hemagglutinin (FHA) and pertactin were generated from the lungs of mice during acute infection with B. pertussis. The Tr1 clones expressed T1/ST2 and CC chemokine receptor 5, secreted high levels of IL-10, but not IL-4 or interferon (IFN)-gamma, and suppressed Th1 responses against B. pertussis or an unrelated pathogen. Furthermore, FHA inhibited IL-12 and stimulated IL-10 production by dendritic cells (DCs), and these DCs directed naive T cells into the regulatory subtype. The induction of Tr1 cells after interaction of a pathogen-derived molecule with cells of the innate immune system represents a novel strategy exploited by an infectious pathogen to subvert protective immune responses in vivo.

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Figures

Figure 1.

Figure 1.

FHA inhibits DC IL-12 and CCL3 production by inducing IL-10. (A) Bone marrow–derived DCs were stimulated with FHA alone or with FHA after 2 h with LPS (1 μg/ml) and IFN-γ (20 ng/ml). After 24 h of culture, supernatants were removed and IL-12p70 and IL-10 were determined by specific immunoassays. (B) DCs were preincubated with FHA (5 μg/ml) with or without anti–IL-10, followed 2 h later with LPS (1 μg/ml) and IFN-γ (20 ng/ml). After 24 h of culture, supernatants were removed and IL-12p70, IL-12p40, CCL3, and IL-10 determined by specific immunoassays. Results are representative of six experiments.

Figure 2.

Figure 2.

Influence of FHA on DC maturation. Bone marrow–derived DCs were stimulated with LPS (1 μg/ml) and IFN-γ (20 ng/ml), FHA (5 μg/ml), or medium. After 24 h of culture, cells were washed and immunofluorescence analysis performed for CD80, CD86, CD40, MHC class II, and CCR5 (black histograms) or isotype-matched control antibodies (gray histograms). Profiles are shown for a single experiment, and are representative from four experiments.

Figure 3.

Figure 3.

Cytokine production and phenotypic analysis of Tr1 clones generated from the respiratory tract of mice infected with B. pertussis. (A) Tr1 clones were generated from lungs of mice 21 d after aerosol challenge with B. pertussis. TEK.1, specific for PRN was cloned from a T cell line generated by stimulation with antigen, APCs, and IL-10 (200 pg/ml). FHA1.1, specific for FHA, was generated by stimulation with antigen and APCs in the absence of exogenous IL-10. Th1 clones, HDS2.6 and 1A.12, specific for influenza virus HA and B. pertussis, respectively, and Th2 clones PT1.1 and PRN2, specific for pertussis toxin and PRN, respectively, were generated from spleens of influenza virus or _B. pertussis_–infected mice. T cell clones were stimulated with antigen and APCs. IL-2 concentrations were determined in 24 h supernatants by CTLL bioassay and IFN-γ, IL-4, IL-5, and IL-10 concentrations in 3-d supernatants were determined by immunoassay. (B) Th1 clone HDS2.6, Th2 clone 2PT2 (specific for pertussis toxin), and Tr1 clone FHA1.1 were labeled with antibodies specific for CD4, CD25, CD28, CTLA4, CCR5, and T1/ST2 and immunofluoresence analysis performed on a FACScan™. The dotted line indicated staining with isotype matched control antibody. T cell clones were used at the end of the resting stage of the growth cycle, 7 d after addition of IL-2. Results are representative of three experiments and results for B are shown for one Tr1 clone only, but were essentially identical to that found for the second Tr1 clone.

Figure 3.

Figure 3.

Cytokine production and phenotypic analysis of Tr1 clones generated from the respiratory tract of mice infected with B. pertussis. (A) Tr1 clones were generated from lungs of mice 21 d after aerosol challenge with B. pertussis. TEK.1, specific for PRN was cloned from a T cell line generated by stimulation with antigen, APCs, and IL-10 (200 pg/ml). FHA1.1, specific for FHA, was generated by stimulation with antigen and APCs in the absence of exogenous IL-10. Th1 clones, HDS2.6 and 1A.12, specific for influenza virus HA and B. pertussis, respectively, and Th2 clones PT1.1 and PRN2, specific for pertussis toxin and PRN, respectively, were generated from spleens of influenza virus or _B. pertussis_–infected mice. T cell clones were stimulated with antigen and APCs. IL-2 concentrations were determined in 24 h supernatants by CTLL bioassay and IFN-γ, IL-4, IL-5, and IL-10 concentrations in 3-d supernatants were determined by immunoassay. (B) Th1 clone HDS2.6, Th2 clone 2PT2 (specific for pertussis toxin), and Tr1 clone FHA1.1 were labeled with antibodies specific for CD4, CD25, CD28, CTLA4, CCR5, and T1/ST2 and immunofluoresence analysis performed on a FACScan™. The dotted line indicated staining with isotype matched control antibody. T cell clones were used at the end of the resting stage of the growth cycle, 7 d after addition of IL-2. Results are representative of three experiments and results for B are shown for one Tr1 clone only, but were essentially identical to that found for the second Tr1 clone.

Figure 4.

Figure 4.

Tr1 clones inhibit cytokine production by Th1 but not Th2 clones. (A) PRN-specific Tr1 clone TEK.1 (105/ml) and influenza virus HA-specific Th1 clone HDS2.6 (105/ml) were cultured alone or together with PRN (5 μg/ml) and/or influenza virus (10 HAU/ml) and splenic APCs (2 × 106/ml). Supernatants were removed after 2–3 d and concentrations of IL-4, IL-5, IL-10, and IFN-γ determined by immunoassays. In the cocultures, Tr1 and Th1 cells were cultured in the same well, HDS2.6 plus TEK.1, or separated by a semi-permeable membrane, where supernatants were sampled from the Th1 side, HDS2.6 (TEK.1) or the Tr1 side, TEK.1 (HDS2.6). *P < 0.05, **P < 0.01, ***P < 0.001 HDS2.6 alone versus cocultured with TEK.1. (B) PRN-specific Tr1 clone TEK.1 was cultured with antigen alone or with PT-specific Th2 clone PT1.1 or PRN-specificTh2 clone PRN2 and cytokine production assessed by immunoassay. Results are representative of two experiments.

Figure 5.

Figure 5.

Suppression of Th1 cell proliferation and IFN-γ production by Tr1 clone is mediated by IL-10. FHA-specific Tr1 clone FHA 1.1 and influenza virus HA-specific Th1 clone HDS2.6 (105/ml) were cultured alone (105/ml) or together at a ratio of 1:1 (105/ml) or 3:1 (3 × 105/ml: 105/ml) with FHA (5 μg/ml) and/or influenza virus (10 HAU/ml) and splenic APCs (2 × 106/ml). Cocultures were also performed in the presence a neutralizing anti-IL-10 antibody (10 μg/ml). Supernatants were removed after 3 d and concentrations of IFN-γ determined by immunoassays. (A) Cells were harvested after 4 d and proliferation assessed by [3H]thymidine incorporation (B). In the cocultures Tr1 and Th1 cells were cultured in the same well, HDS.6 plus FHA1.1, or separated by a semi-permeable membrane, where supernatants was sampled from the Th1 side, HDS2.6 (FHA1.1) or the Tr1 side, FHA1.1 (HDS2.6). Proliferation could only be assessed on the Th1 side of the membrane. *P < 0.05, **P < 0.01, ***P < 0.001 HDS2.6 alone versus cocultured with FHA1.1. Results are representative of two experiments.

Figure 5.

Figure 5.

Suppression of Th1 cell proliferation and IFN-γ production by Tr1 clone is mediated by IL-10. FHA-specific Tr1 clone FHA 1.1 and influenza virus HA-specific Th1 clone HDS2.6 (105/ml) were cultured alone (105/ml) or together at a ratio of 1:1 (105/ml) or 3:1 (3 × 105/ml: 105/ml) with FHA (5 μg/ml) and/or influenza virus (10 HAU/ml) and splenic APCs (2 × 106/ml). Cocultures were also performed in the presence a neutralizing anti-IL-10 antibody (10 μg/ml). Supernatants were removed after 3 d and concentrations of IFN-γ determined by immunoassays. (A) Cells were harvested after 4 d and proliferation assessed by [3H]thymidine incorporation (B). In the cocultures Tr1 and Th1 cells were cultured in the same well, HDS.6 plus FHA1.1, or separated by a semi-permeable membrane, where supernatants was sampled from the Th1 side, HDS2.6 (FHA1.1) or the Tr1 side, FHA1.1 (HDS2.6). Proliferation could only be assessed on the Th1 side of the membrane. *P < 0.05, **P < 0.01, ***P < 0.001 HDS2.6 alone versus cocultured with FHA1.1. Results are representative of two experiments.

Figure 6.

Figure 6.

Tr1 clones suppress _B. pertussis_–specific IFN-γ production in vitro and in vivo. (A) Spleen cells from B. pertussis convalescent mice 6 wk after challenge (designated Th1) were cultured at 2 × 106/ml with antigen (B. pertussis sonic extract, 5 μg/ml) alone or with Tr1 clone TEK.1 (105/ml) and PRN (5 μg/ml). Spleen cells from naive mice served as a control. Supernatants were removed after 3 d and tested for IFN-γ and IL-10 by immunoassay. (B and C) Spleen cells from naive or convalescent mice (designated Th1) were transferred into sublethally irradiated BALB/c mice (2 × 107 per mouse) alone or with Tr1 clone TEK.1 (2 × 105 per mouse). Mice were challenged with B. pertussis and killed 7, 14, and 21 d later. (B) Spleen cells were isolated after 7 d and stimulated with B. pertussis antigen and IFN-γ concentrations determined in supernatants 3 d later. (C) Lungs were removed and the number of viable B. pertussis determined by performing CFU counts. Results are mean (SD) for four mice per group at each time point. *P < 0.05, Th1 vs. Th1 plus Tr1. The results shown in A are representative of two experiments and due to the large numbers of cells required the experiment shown in B and C were performed once.

Figure 6.

Figure 6.

Tr1 clones suppress _B. pertussis_–specific IFN-γ production in vitro and in vivo. (A) Spleen cells from B. pertussis convalescent mice 6 wk after challenge (designated Th1) were cultured at 2 × 106/ml with antigen (B. pertussis sonic extract, 5 μg/ml) alone or with Tr1 clone TEK.1 (105/ml) and PRN (5 μg/ml). Spleen cells from naive mice served as a control. Supernatants were removed after 3 d and tested for IFN-γ and IL-10 by immunoassay. (B and C) Spleen cells from naive or convalescent mice (designated Th1) were transferred into sublethally irradiated BALB/c mice (2 × 107 per mouse) alone or with Tr1 clone TEK.1 (2 × 105 per mouse). Mice were challenged with B. pertussis and killed 7, 14, and 21 d later. (B) Spleen cells were isolated after 7 d and stimulated with B. pertussis antigen and IFN-γ concentrations determined in supernatants 3 d later. (C) Lungs were removed and the number of viable B. pertussis determined by performing CFU counts. Results are mean (SD) for four mice per group at each time point. *P < 0.05, Th1 vs. Th1 plus Tr1. The results shown in A are representative of two experiments and due to the large numbers of cells required the experiment shown in B and C were performed once.

Figure 6.

Figure 6.

Tr1 clones suppress _B. pertussis_–specific IFN-γ production in vitro and in vivo. (A) Spleen cells from B. pertussis convalescent mice 6 wk after challenge (designated Th1) were cultured at 2 × 106/ml with antigen (B. pertussis sonic extract, 5 μg/ml) alone or with Tr1 clone TEK.1 (105/ml) and PRN (5 μg/ml). Spleen cells from naive mice served as a control. Supernatants were removed after 3 d and tested for IFN-γ and IL-10 by immunoassay. (B and C) Spleen cells from naive or convalescent mice (designated Th1) were transferred into sublethally irradiated BALB/c mice (2 × 107 per mouse) alone or with Tr1 clone TEK.1 (2 × 105 per mouse). Mice were challenged with B. pertussis and killed 7, 14, and 21 d later. (B) Spleen cells were isolated after 7 d and stimulated with B. pertussis antigen and IFN-γ concentrations determined in supernatants 3 d later. (C) Lungs were removed and the number of viable B. pertussis determined by performing CFU counts. Results are mean (SD) for four mice per group at each time point. *P < 0.05, Th1 vs. Th1 plus Tr1. The results shown in A are representative of two experiments and due to the large numbers of cells required the experiment shown in B and C were performed once.

Figure 7.

Figure 7.

Deletion of FHA attenuates local immunosuppression and enhances B. pertussis clearance form the lungs. Groups of 24 BALB/c mice were challenged by exposure to an aerosol of B. pertussis BP338 or FHA− mutant BPM409. (A) The course of infection was followed by performing CFU counts on the lungs of four mice per group at 3 h, 7, 14, 21, and 28 d after challenge. (B) Spleen, lymph nodes, and lung mononuclear cells were prepared from mice 21 d after challenge and antigen-specific IFN-γ production was tested by culturing with B. pertussis antigen and assessing cytokine levels in supernatants 3 d later. (C) IL-10 concentrations in BAL fluid from mice 3–21 d after infection with B. pertussis BP338 or FHA− mutant BPM409. Results are representative of four experiments.

Figure 7.

Figure 7.

Deletion of FHA attenuates local immunosuppression and enhances B. pertussis clearance form the lungs. Groups of 24 BALB/c mice were challenged by exposure to an aerosol of B. pertussis BP338 or FHA− mutant BPM409. (A) The course of infection was followed by performing CFU counts on the lungs of four mice per group at 3 h, 7, 14, 21, and 28 d after challenge. (B) Spleen, lymph nodes, and lung mononuclear cells were prepared from mice 21 d after challenge and antigen-specific IFN-γ production was tested by culturing with B. pertussis antigen and assessing cytokine levels in supernatants 3 d later. (C) IL-10 concentrations in BAL fluid from mice 3–21 d after infection with B. pertussis BP338 or FHA− mutant BPM409. Results are representative of four experiments.

Figure 7.

Figure 7.

Deletion of FHA attenuates local immunosuppression and enhances B. pertussis clearance form the lungs. Groups of 24 BALB/c mice were challenged by exposure to an aerosol of B. pertussis BP338 or FHA− mutant BPM409. (A) The course of infection was followed by performing CFU counts on the lungs of four mice per group at 3 h, 7, 14, 21, and 28 d after challenge. (B) Spleen, lymph nodes, and lung mononuclear cells were prepared from mice 21 d after challenge and antigen-specific IFN-γ production was tested by culturing with B. pertussis antigen and assessing cytokine levels in supernatants 3 d later. (C) IL-10 concentrations in BAL fluid from mice 3–21 d after infection with B. pertussis BP338 or FHA− mutant BPM409. Results are representative of four experiments.

Figure 8.

Figure 8.

FHA stimulates DCs to direct naive T cells to a Tr1 subtype. OVA-specific CD4+ T cell lines were generated in vitro by stimulating purified naive T cells from DO11.10 TCR Tg mice with DCs preincubated for 24 h with OVA peptide (0, 0.5, 5, 50 μg/ml) and FHA (5 μg/ml), CpG-ODN (1 μg/ml), FHA, and anti–IL-10 (10 μg/ml) or medium only. After two rounds of antigen stimulation supernatants were removed and concentrations of cytokines determined by immunoassay. Results are representative of two experiments.

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