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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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Related in: MedlinePlus

Cytokine production profiles of islet-specific T-cell clones. A: MHB10.3 (open symbols) and RAR5.3 (closed symbols) were stimulated with insulin B11–30 and IA-2 709–736, respectively, in the presence of autologous irradiated PBMCs, and IL-10 production was measured by enzyme-linked immunosorbent assay after 3 days. B: Cytokine production by MHB10.3 (open bars) and RAR5.3 (closed bars) clones cultured as above with suboptimal doses of peptide (1 μg/ml) and by (C) MHB10.3 stimulated with whole recombinant insulin (100 μg/ml) in the presence of autologous irradiated dendritic cells. Cytokines were measured in 3-day supernatants by multiplex bead technology. D: Cytokine production by MHB10.3 was assessed by flow cytometry for intracellular synthesis of IL-10, IL-13, and IFN-γ after stimulation with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 2 days.
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Figure 2: Cytokine production profiles of islet-specific T-cell clones. A: MHB10.3 (open symbols) and RAR5.3 (closed symbols) were stimulated with insulin B11–30 and IA-2 709–736, respectively, in the presence of autologous irradiated PBMCs, and IL-10 production was measured by enzyme-linked immunosorbent assay after 3 days. B: Cytokine production by MHB10.3 (open bars) and RAR5.3 (closed bars) clones cultured as above with suboptimal doses of peptide (1 μg/ml) and by (C) MHB10.3 stimulated with whole recombinant insulin (100 μg/ml) in the presence of autologous irradiated dendritic cells. Cytokines were measured in 3-day supernatants by multiplex bead technology. D: Cytokine production by MHB10.3 was assessed by flow cytometry for intracellular synthesis of IL-10, IL-13, and IFN-γ after stimulation with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 2 days.

Mentions: We first examined IL-10 responses to a panel of naturally processed IA-2 and proinsulin peptides and a set of overlapping peptides encompassing the insulin molecule in a group of healthy nondiabetic individuals (n = 11). Using a highly sensitive and specific cytokine ELISpot, we found that the majority of individuals (8/11, 72%) mounted a significant IL-10 response (insulin sensitivity index ≥3) to at least one peptide (data not shown), consistent with our previous work (16). Individuals with positive responses by ELISpot were recalled a minimum of 1 month later, and IL-10–secreting CD4 T-cells were detected, isolated by sensitive cytokine secretion assay, and immediately single-cell sorted by flow cytometry. The cytokine secretion assay confirmed the reproducible detection of IL-10–secreting, islet peptide–specific CD3+CD4+ T-cells in nondiabetic individuals (Fig. 1). Cloning was initiated after brief (48 h) culture with antigen and was carried out in the absence of any IL-10 biasing culture conditions. T-cell clones specific for insulin B11–30 (clone MHB10.3) and IA-2 709–736 (clone RAR5.3) were isolated from nondiabetic individuals, M.H. (age 27 years; HLA-DRB1*0301-DRB1*0404) and R.A. (age 27 years; HLA-DRB1*0101-DRB1*0407), respectively. Both clones produced large amounts of IL-10 (>1 ng/ml), in response to submicrogram doses of the peptide used in their isolation (Fig. 2A).


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)

Cytokine production profiles of islet-specific T-cell clones. A: MHB10.3 (open symbols) and RAR5.3 (closed symbols) were stimulated with insulin B11–30 and IA-2 709–736, respectively, in the presence of autologous irradiated PBMCs, and IL-10 production was measured by enzyme-linked immunosorbent assay after 3 days. B: Cytokine production by MHB10.3 (open bars) and RAR5.3 (closed bars) clones cultured as above with suboptimal doses of peptide (1 μg/ml) and by (C) MHB10.3 stimulated with whole recombinant insulin (100 μg/ml) in the presence of autologous irradiated dendritic cells. Cytokines were measured in 3-day supernatants by multiplex bead technology. D: Cytokine production by MHB10.3 was assessed by flow cytometry for intracellular synthesis of IL-10, IL-13, and IFN-γ after stimulation with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 2 days.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2874706&req=5

Figure 2: Cytokine production profiles of islet-specific T-cell clones. A: MHB10.3 (open symbols) and RAR5.3 (closed symbols) were stimulated with insulin B11–30 and IA-2 709–736, respectively, in the presence of autologous irradiated PBMCs, and IL-10 production was measured by enzyme-linked immunosorbent assay after 3 days. B: Cytokine production by MHB10.3 (open bars) and RAR5.3 (closed bars) clones cultured as above with suboptimal doses of peptide (1 μg/ml) and by (C) MHB10.3 stimulated with whole recombinant insulin (100 μg/ml) in the presence of autologous irradiated dendritic cells. Cytokines were measured in 3-day supernatants by multiplex bead technology. D: Cytokine production by MHB10.3 was assessed by flow cytometry for intracellular synthesis of IL-10, IL-13, and IFN-γ after stimulation with plate-bound anti-CD3 and soluble anti-CD28 antibodies for 2 days.
Mentions: We first examined IL-10 responses to a panel of naturally processed IA-2 and proinsulin peptides and a set of overlapping peptides encompassing the insulin molecule in a group of healthy nondiabetic individuals (n = 11). Using a highly sensitive and specific cytokine ELISpot, we found that the majority of individuals (8/11, 72%) mounted a significant IL-10 response (insulin sensitivity index ≥3) to at least one peptide (data not shown), consistent with our previous work (16). Individuals with positive responses by ELISpot were recalled a minimum of 1 month later, and IL-10–secreting CD4 T-cells were detected, isolated by sensitive cytokine secretion assay, and immediately single-cell sorted by flow cytometry. The cytokine secretion assay confirmed the reproducible detection of IL-10–secreting, islet peptide–specific CD3+CD4+ T-cells in nondiabetic individuals (Fig. 1). Cloning was initiated after brief (48 h) culture with antigen and was carried out in the absence of any IL-10 biasing culture conditions. T-cell clones specific for insulin B11–30 (clone MHB10.3) and IA-2 709–736 (clone RAR5.3) were isolated from nondiabetic individuals, M.H. (age 27 years; HLA-DRB1*0301-DRB1*0404) and R.A. (age 27 years; HLA-DRB1*0101-DRB1*0407), respectively. Both clones produced large amounts of IL-10 (>1 ng/ml), in response to submicrogram doses of the peptide used in their isolation (Fig. 2A).

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