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Naturally arising human CD4 T-cells that recognize islet autoantigens and secrete interleukin-10 regulate proinflammatory T-cell responses via linked suppression.

Tree TI, Lawson J, Edwards H, Skowera A, Arif S, Roep BO, Peakman M - Diabetes (2010)

Bottom Line: Islet-specific IL-10(+) CD4 T-cells are potent suppressors of Th1 effector cells, operating through a linked suppression mechanism in which there is an absolute requirement for the cognate antigen of both the regulatory and effector T-cells to be presented by the same antigen-presenting cell (APC).The regulatory T-cells secrete perforin and granzymes, and suppression is associated with the specific killing of APCs presenting antigen to effector T-cells.This hitherto undescribed population of islet autoantigen-specific Tregs displays unique characteristics that offer exquisite specificity and control over the potential for pathological autoreactivity and may provide a suitable target with which to strengthen beta-cell-specific tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, King's College London, Guy's Hospital, London, UK. timothy.tree@kcl.ac.uk

ABSTRACT

Objective: Regulatory T-cells (Tregs) recognizing islet autoantigens are proposed as a key mechanism in the maintenance of self-tolerance and protection from type 1 diabetes. To date, however, detailed information on such cells in humans, and insight into their mechanisms of action, has been lacking. We previously reported that a subset of CD4 T-cells secreting high levels of the immunosuppressive cytokine interleukin-10 (IL-10) is significantly associated with late onset of type 1 diabetes and is constitutively present in a majority of nondiabetic individuals. Here, we test the hypothesis that these T-cells represent a naturally generated population of Tregs capable of suppressing proinflammatory T-cell responses.

Research design and methods: We isolated and cloned islet-specific IL-10-secreting CD4(+) T-cells from nondiabetic individuals after brief ex vivo exposure to islet autoantigens using cytokine capture technology and examined their phenotype and regulatory potential.

Results: Islet-specific IL-10(+) CD4 T-cells are potent suppressors of Th1 effector cells, operating through a linked suppression mechanism in which there is an absolute requirement for the cognate antigen of both the regulatory and effector T-cells to be presented by the same antigen-presenting cell (APC). The regulatory T-cells secrete perforin and granzymes, and suppression is associated with the specific killing of APCs presenting antigen to effector T-cells.

Conclusions: This hitherto undescribed population of islet autoantigen-specific Tregs displays unique characteristics that offer exquisite specificity and control over the potential for pathological autoreactivity and may provide a suitable target with which to strengthen beta-cell-specific tolerance.

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Regulation of clonal T-cell responses by MHB10.3 and RAR5.3. A–C: The tetanus toxoid–specific Th1 clone RATT6 was labeled with CFSE and stimulated with combinations of tetanus toxoid (100 ng/ml) and insulin B11–30 (10 μg/ml) in the presence or absence of DDAO-labeled MHB10.3 as indicated. D–F: The hemagglutinin-specific Th1 clone RAHA5 was labeled with CFSE and stimulated with combinations of recombinant hemagglutinin (45 ng/ml) and IA-2 709–736 (25 μg/ml) in the presence or absence of DDAO-labeled RAR5.3 as indicated. HLA-matched DDAO-labeled irradiated PBMCs were used as a source of antigen-presenting cells. Proliferation of Th1 clones (DDAO− cells) was assessed after 3 days by flow cytometry, and the gated regions represent the percentage of live clone cells that have undergone division. Proliferation of the Th1 clones RATT6 and RAHA5 is suppressed by activated MHB10.3 and RAR5.3 Treg clones. Data are representative of a minimum of three independent experiments.
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Figure 4: Regulation of clonal T-cell responses by MHB10.3 and RAR5.3. A–C: The tetanus toxoid–specific Th1 clone RATT6 was labeled with CFSE and stimulated with combinations of tetanus toxoid (100 ng/ml) and insulin B11–30 (10 μg/ml) in the presence or absence of DDAO-labeled MHB10.3 as indicated. D–F: The hemagglutinin-specific Th1 clone RAHA5 was labeled with CFSE and stimulated with combinations of recombinant hemagglutinin (45 ng/ml) and IA-2 709–736 (25 μg/ml) in the presence or absence of DDAO-labeled RAR5.3 as indicated. HLA-matched DDAO-labeled irradiated PBMCs were used as a source of antigen-presenting cells. Proliferation of Th1 clones (DDAO− cells) was assessed after 3 days by flow cytometry, and the gated regions represent the percentage of live clone cells that have undergone division. Proliferation of the Th1 clones RATT6 and RAHA5 is suppressed by activated MHB10.3 and RAR5.3 Treg clones. Data are representative of a minimum of three independent experiments.

Mentions: To investigate the regulatory potential of IL-10–secreting islet-specific T-cells, a dual fluorescence–based suppression assay was used. Regulation was first assessed in an autologous assay system, measuring the response of PBMCs to recall antigen HA. Individual M.H. exhibited a robust proliferative response to HA (9.6% of CD3+ PBMCs; Fig. 3B). Addition of either T-cell clone MHB10.3 or its epitope insulin B11–30 had little effect on proliferation (Fig. 3C and D), whereas addition of MHB10.3 and its epitope insulin B11–30 together resulted in complete suppression of the HA-specific proliferative response (Fig. 3E). These data demonstrate that IL-10–secreting islet-specific T-cells are potent regulators, and that regulation is dependent on activation of Tregs by APC presentation of cognate peptide. To pursue further analysis of the mechanisms of suppression, we used a panel of CD4+ Th1 clones specific for recall antigens TT and HA (RATT6 and RAHA5, respectively). Both clones proliferate rapidly in response to cognate antigen with 68 and 52% of cells divided after 3 days, respectively (Fig. 4B and E). However, when cultured with MHB10.3 (Fig. 4C) or RAR5.3 (Fig. 4F) in the presence of the cognate islet peptide, proliferation was dramatically reduced (33 and 16%), resulting in suppression rates of 51 and 69%, respectively. Blocking IL-10 and TGF-β either individually or in combination had no effect on the ability of MHB10.3 or RAR5.3 to suppress proliferation of RATT6 (74–80% suppression, Fig. 5A–H) or RAHA5 (51–59% suppression, Fig. 5I). This absence of IL-10 dependency of regulation was also present in studies in which Treg number and peptide concentration were titrated to extinction of activity (supplementary Fig. 3). Furthermore, blockade of IL-10 during the activation and expansion of IL-10–secreting islet-specific T-cell clones also did not affect the ability of the cells to regulate (supplementary Fig. 4).


Naturally arising human CD4 T-cells that recognize islet autoantigens and secrete interleukin-10 regulate proinflammatory T-cell responses via linked suppression.

Tree TI, Lawson J, Edwards H, Skowera A, Arif S, Roep BO, Peakman M - Diabetes (2010)

Regulation of clonal T-cell responses by MHB10.3 and RAR5.3. A–C: The tetanus toxoid–specific Th1 clone RATT6 was labeled with CFSE and stimulated with combinations of tetanus toxoid (100 ng/ml) and insulin B11–30 (10 μg/ml) in the presence or absence of DDAO-labeled MHB10.3 as indicated. D–F: The hemagglutinin-specific Th1 clone RAHA5 was labeled with CFSE and stimulated with combinations of recombinant hemagglutinin (45 ng/ml) and IA-2 709–736 (25 μg/ml) in the presence or absence of DDAO-labeled RAR5.3 as indicated. HLA-matched DDAO-labeled irradiated PBMCs were used as a source of antigen-presenting cells. Proliferation of Th1 clones (DDAO− cells) was assessed after 3 days by flow cytometry, and the gated regions represent the percentage of live clone cells that have undergone division. Proliferation of the Th1 clones RATT6 and RAHA5 is suppressed by activated MHB10.3 and RAR5.3 Treg clones. Data are representative of a minimum of three independent experiments.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Regulation of clonal T-cell responses by MHB10.3 and RAR5.3. A–C: The tetanus toxoid–specific Th1 clone RATT6 was labeled with CFSE and stimulated with combinations of tetanus toxoid (100 ng/ml) and insulin B11–30 (10 μg/ml) in the presence or absence of DDAO-labeled MHB10.3 as indicated. D–F: The hemagglutinin-specific Th1 clone RAHA5 was labeled with CFSE and stimulated with combinations of recombinant hemagglutinin (45 ng/ml) and IA-2 709–736 (25 μg/ml) in the presence or absence of DDAO-labeled RAR5.3 as indicated. HLA-matched DDAO-labeled irradiated PBMCs were used as a source of antigen-presenting cells. Proliferation of Th1 clones (DDAO− cells) was assessed after 3 days by flow cytometry, and the gated regions represent the percentage of live clone cells that have undergone division. Proliferation of the Th1 clones RATT6 and RAHA5 is suppressed by activated MHB10.3 and RAR5.3 Treg clones. Data are representative of a minimum of three independent experiments.
Mentions: To investigate the regulatory potential of IL-10–secreting islet-specific T-cells, a dual fluorescence–based suppression assay was used. Regulation was first assessed in an autologous assay system, measuring the response of PBMCs to recall antigen HA. Individual M.H. exhibited a robust proliferative response to HA (9.6% of CD3+ PBMCs; Fig. 3B). Addition of either T-cell clone MHB10.3 or its epitope insulin B11–30 had little effect on proliferation (Fig. 3C and D), whereas addition of MHB10.3 and its epitope insulin B11–30 together resulted in complete suppression of the HA-specific proliferative response (Fig. 3E). These data demonstrate that IL-10–secreting islet-specific T-cells are potent regulators, and that regulation is dependent on activation of Tregs by APC presentation of cognate peptide. To pursue further analysis of the mechanisms of suppression, we used a panel of CD4+ Th1 clones specific for recall antigens TT and HA (RATT6 and RAHA5, respectively). Both clones proliferate rapidly in response to cognate antigen with 68 and 52% of cells divided after 3 days, respectively (Fig. 4B and E). However, when cultured with MHB10.3 (Fig. 4C) or RAR5.3 (Fig. 4F) in the presence of the cognate islet peptide, proliferation was dramatically reduced (33 and 16%), resulting in suppression rates of 51 and 69%, respectively. Blocking IL-10 and TGF-β either individually or in combination had no effect on the ability of MHB10.3 or RAR5.3 to suppress proliferation of RATT6 (74–80% suppression, Fig. 5A–H) or RAHA5 (51–59% suppression, Fig. 5I). This absence of IL-10 dependency of regulation was also present in studies in which Treg number and peptide concentration were titrated to extinction of activity (supplementary Fig. 3). Furthermore, blockade of IL-10 during the activation and expansion of IL-10–secreting islet-specific T-cell clones also did not affect the ability of the cells to regulate (supplementary Fig. 4).

Bottom Line: Islet-specific IL-10(+) CD4 T-cells are potent suppressors of Th1 effector cells, operating through a linked suppression mechanism in which there is an absolute requirement for the cognate antigen of both the regulatory and effector T-cells to be presented by the same antigen-presenting cell (APC).The regulatory T-cells secrete perforin and granzymes, and suppression is associated with the specific killing of APCs presenting antigen to effector T-cells.This hitherto undescribed population of islet autoantigen-specific Tregs displays unique characteristics that offer exquisite specificity and control over the potential for pathological autoreactivity and may provide a suitable target with which to strengthen beta-cell-specific tolerance.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, King's College London, Guy's Hospital, London, UK. timothy.tree@kcl.ac.uk

ABSTRACT

Objective: Regulatory T-cells (Tregs) recognizing islet autoantigens are proposed as a key mechanism in the maintenance of self-tolerance and protection from type 1 diabetes. To date, however, detailed information on such cells in humans, and insight into their mechanisms of action, has been lacking. We previously reported that a subset of CD4 T-cells secreting high levels of the immunosuppressive cytokine interleukin-10 (IL-10) is significantly associated with late onset of type 1 diabetes and is constitutively present in a majority of nondiabetic individuals. Here, we test the hypothesis that these T-cells represent a naturally generated population of Tregs capable of suppressing proinflammatory T-cell responses.

Research design and methods: We isolated and cloned islet-specific IL-10-secreting CD4(+) T-cells from nondiabetic individuals after brief ex vivo exposure to islet autoantigens using cytokine capture technology and examined their phenotype and regulatory potential.

Results: Islet-specific IL-10(+) CD4 T-cells are potent suppressors of Th1 effector cells, operating through a linked suppression mechanism in which there is an absolute requirement for the cognate antigen of both the regulatory and effector T-cells to be presented by the same antigen-presenting cell (APC). The regulatory T-cells secrete perforin and granzymes, and suppression is associated with the specific killing of APCs presenting antigen to effector T-cells.

Conclusions: This hitherto undescribed population of islet autoantigen-specific Tregs displays unique characteristics that offer exquisite specificity and control over the potential for pathological autoreactivity and may provide a suitable target with which to strengthen beta-cell-specific tolerance.

Show MeSH
Related in: MedlinePlus