Limits...
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.

Show MeSH

Related in: MedlinePlus

IL-10–producing islet-specific CD4+ T-cells express cytotoxic molecules directly ex vivo. PBMCs from donors M.H. and R.A. were stimulated with islet peptides, and after 48 h single CD4+ IL-10+ or CD4+ IL-10− T-cells isolated by flow cytometry were deposited into 96-well plates containing 8 μl lysis buffer. RT and first-round PCR (30 cycles) were performed using gene-specific primers and the One Step RT-PCR kit following the manufacturer's instructions (Qiagen, Crawley, U.K.). After multiplex RT-PCR, 1 μl of a 1:100 dilution of the reaction was used in individual gene, second round, nested PCRs (30 cycles) using internal primers and HotStar Taq Polymerase kit (Qiagen). Products were analyzed by agarose gel electrophoresis. Data were considered for analysis only from cells that yielded a CD3ε product. Each lane represents products amplified from a single cell. As a positive control, pools of five cells of the clones RAR5.3 and MHB10.3 were used after peptide stimulation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2874706&req=5

Figure 8: IL-10–producing islet-specific CD4+ T-cells express cytotoxic molecules directly ex vivo. PBMCs from donors M.H. and R.A. were stimulated with islet peptides, and after 48 h single CD4+ IL-10+ or CD4+ IL-10− T-cells isolated by flow cytometry were deposited into 96-well plates containing 8 μl lysis buffer. RT and first-round PCR (30 cycles) were performed using gene-specific primers and the One Step RT-PCR kit following the manufacturer's instructions (Qiagen, Crawley, U.K.). After multiplex RT-PCR, 1 μl of a 1:100 dilution of the reaction was used in individual gene, second round, nested PCRs (30 cycles) using internal primers and HotStar Taq Polymerase kit (Qiagen). Products were analyzed by agarose gel electrophoresis. Data were considered for analysis only from cells that yielded a CD3ε product. Each lane represents products amplified from a single cell. As a positive control, pools of five cells of the clones RAR5.3 and MHB10.3 were used after peptide stimulation.

Mentions: To establish whether IL-10–secreting islet-specific Tregs express cytotoxic molecules directly ex vivo, or gain this expression as a consequence of the cloning process in vitro, we performed a multiplex RT-PCR on single IL-10+ CD4+ T-cells freshly isolated from islet peptide–stimulated PBMC cultures (Fig. 8). These experiments were performed with the original donors used to clone MHB10.3 and RAR5.3 Tregs. All sorted T-cells were examined for CD3 expression as a template quality control and also for IL-10 expression. Only IL-10+ sorted T-cells that were also IL-10 transcript–positive were included in the analysis. These studies demonstrated that 67% (6/9) of the IL-10 transcript–positive T-cells expressed at least one cytotoxic molecule, compared with 5.6% (1/18) of the IL-10 transcript–negative T-cells (P = 0.0006).


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)

IL-10–producing islet-specific CD4+ T-cells express cytotoxic molecules directly ex vivo. PBMCs from donors M.H. and R.A. were stimulated with islet peptides, and after 48 h single CD4+ IL-10+ or CD4+ IL-10− T-cells isolated by flow cytometry were deposited into 96-well plates containing 8 μl lysis buffer. RT and first-round PCR (30 cycles) were performed using gene-specific primers and the One Step RT-PCR kit following the manufacturer's instructions (Qiagen, Crawley, U.K.). After multiplex RT-PCR, 1 μl of a 1:100 dilution of the reaction was used in individual gene, second round, nested PCRs (30 cycles) using internal primers and HotStar Taq Polymerase kit (Qiagen). Products were analyzed by agarose gel electrophoresis. Data were considered for analysis only from cells that yielded a CD3ε product. Each lane represents products amplified from a single cell. As a positive control, pools of five cells of the clones RAR5.3 and MHB10.3 were used after peptide stimulation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: IL-10–producing islet-specific CD4+ T-cells express cytotoxic molecules directly ex vivo. PBMCs from donors M.H. and R.A. were stimulated with islet peptides, and after 48 h single CD4+ IL-10+ or CD4+ IL-10− T-cells isolated by flow cytometry were deposited into 96-well plates containing 8 μl lysis buffer. RT and first-round PCR (30 cycles) were performed using gene-specific primers and the One Step RT-PCR kit following the manufacturer's instructions (Qiagen, Crawley, U.K.). After multiplex RT-PCR, 1 μl of a 1:100 dilution of the reaction was used in individual gene, second round, nested PCRs (30 cycles) using internal primers and HotStar Taq Polymerase kit (Qiagen). Products were analyzed by agarose gel electrophoresis. Data were considered for analysis only from cells that yielded a CD3ε product. Each lane represents products amplified from a single cell. As a positive control, pools of five cells of the clones RAR5.3 and MHB10.3 were used after peptide stimulation.
Mentions: To establish whether IL-10–secreting islet-specific Tregs express cytotoxic molecules directly ex vivo, or gain this expression as a consequence of the cloning process in vitro, we performed a multiplex RT-PCR on single IL-10+ CD4+ T-cells freshly isolated from islet peptide–stimulated PBMC cultures (Fig. 8). These experiments were performed with the original donors used to clone MHB10.3 and RAR5.3 Tregs. All sorted T-cells were examined for CD3 expression as a template quality control and also for IL-10 expression. Only IL-10+ sorted T-cells that were also IL-10 transcript–positive were included in the analysis. These studies demonstrated that 67% (6/9) of the IL-10 transcript–positive T-cells expressed at least one cytotoxic molecule, compared with 5.6% (1/18) of the IL-10 transcript–negative T-cells (P = 0.0006).

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