Limits...
Expansion of Th17 cells and functional defects in T regulatory cells are key features of the pancreatic lymph nodes in patients with type 1 diabetes.

Ferraro A, Socci C, Stabilini A, Valle A, Monti P, Piemonti L, Nano R, Olek S, Maffi P, Scavini M, Secchi A, Staudacher C, Bonifacio E, Battaglia M - Diabetes (2011)

Bottom Line: We found upregulation of Th17 immunity and functional defects in CD4(+)CD25(bright) Tregs in the PLNs of type 1 diabetic subjects but not in their peripheral blood.The dysfunctional Tregs isolated from diabetic subjects did not contain contaminant effector T cells and were all epigenetically imprinted to be suppressive, as defined by analysis of the Treg-specific demethylated region within the forkhead box P3 (FOXP3) locus.These data provide evidence for an unbalanced immune status in the PLNs of type 1 diabetic subjects, and treatments restoring the immune homeostasis in the target organ of these patients represent a potential therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Institute, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT

Objective: Autoimmune diseases, including type 1 diabetes, are thought to have a Th17-cell bias and/or a T-regulatory cell (Treg) defect. Understanding whether this is a hallmark of patients with type 1 diabetes is a crucial question that is still unsolved, largely due to the difficulties of accessing tissues targeted by the disease.

Research design and methods: We phenotypically and functionally characterized Th17 cells and Tregs residing in the pancreatic-draining lymph nodes (PLNs) of 19 patients with type 1 diabetes and 63 nondiabetic donors and those circulating in the peripheral blood of 14 type 1 diabetic patients and 11 healthy subjects.

Results: We found upregulation of Th17 immunity and functional defects in CD4(+)CD25(bright) Tregs in the PLNs of type 1 diabetic subjects but not in their peripheral blood. In addition, the proinsulin-specific Treg-mediated control was altered in the PLNs of diabetic patients. The dysfunctional Tregs isolated from diabetic subjects did not contain contaminant effector T cells and were all epigenetically imprinted to be suppressive, as defined by analysis of the Treg-specific demethylated region within the forkhead box P3 (FOXP3) locus.

Conclusions: These data provide evidence for an unbalanced immune status in the PLNs of type 1 diabetic subjects, and treatments restoring the immune homeostasis in the target organ of these patients represent a potential therapeutic strategy.

Show MeSH

Related in: MedlinePlus

Purity of the flow cytometry–based sorted CD25bright T cells tested in the functional assays. A and B: The amount of transcript expression (relative to an internal control) of the indicated genes in the sorted CD25bright T cells was assessed by RT-PCR. Relative expression of the mRNA of interest was determined by normalizing to hypoxanthine phosphoribosyltransferase expression and calculating the fold-change vs. the expression values measured in total PBMC isolated from normal donors. C: The frequency of Tregs within the sorted CD25bright T cells was measured by the TSDR assay. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. ND, nondiabetic donors.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3198077&req=5

Figure 5: Purity of the flow cytometry–based sorted CD25bright T cells tested in the functional assays. A and B: The amount of transcript expression (relative to an internal control) of the indicated genes in the sorted CD25bright T cells was assessed by RT-PCR. Relative expression of the mRNA of interest was determined by normalizing to hypoxanthine phosphoribosyltransferase expression and calculating the fold-change vs. the expression values measured in total PBMC isolated from normal donors. C: The frequency of Tregs within the sorted CD25bright T cells was measured by the TSDR assay. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. ND, nondiabetic donors.

Mentions: Overall, the just-mentioned functional assays posed the question of whether the CD25bright T cells isolated from PLNs of diabetic patients were contaminated by activated cytokine-producing effector T cells. We exclude such a possibility because the CD25bright T cells sorted from PLNs of diabetic and nondiabetic donors contained the same amount of FOXP3 mRNA (Fig. 5A) and expressed the same low transcript levels of the master regulators of Th1 (T-box expressed in T cells [T-bet]), Th2 (GATA-binding protein 3 [GATA3]), and Th17 cells (retinoic acid–related orphan receptor C [RORC]) and of IL-2 and IFN-γ (Fig. 5B). Most importantly, the sorted CD25bright T cells were all demethylated at the FOXP3 locus, proving their epigenetic Treg imprinting (Fig. 5C). These data demonstrate that the sorted CD25bright T cells within the PLNs of diabetic patients do not contain effector T cells, and despite being epigenetically determined to be Tregs, they do not exert consistent in vitro regulatory function.


Expansion of Th17 cells and functional defects in T regulatory cells are key features of the pancreatic lymph nodes in patients with type 1 diabetes.

Ferraro A, Socci C, Stabilini A, Valle A, Monti P, Piemonti L, Nano R, Olek S, Maffi P, Scavini M, Secchi A, Staudacher C, Bonifacio E, Battaglia M - Diabetes (2011)

Purity of the flow cytometry–based sorted CD25bright T cells tested in the functional assays. A and B: The amount of transcript expression (relative to an internal control) of the indicated genes in the sorted CD25bright T cells was assessed by RT-PCR. Relative expression of the mRNA of interest was determined by normalizing to hypoxanthine phosphoribosyltransferase expression and calculating the fold-change vs. the expression values measured in total PBMC isolated from normal donors. C: The frequency of Tregs within the sorted CD25bright T cells was measured by the TSDR assay. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. ND, nondiabetic donors.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198077&req=5

Figure 5: Purity of the flow cytometry–based sorted CD25bright T cells tested in the functional assays. A and B: The amount of transcript expression (relative to an internal control) of the indicated genes in the sorted CD25bright T cells was assessed by RT-PCR. Relative expression of the mRNA of interest was determined by normalizing to hypoxanthine phosphoribosyltransferase expression and calculating the fold-change vs. the expression values measured in total PBMC isolated from normal donors. C: The frequency of Tregs within the sorted CD25bright T cells was measured by the TSDR assay. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. ND, nondiabetic donors.
Mentions: Overall, the just-mentioned functional assays posed the question of whether the CD25bright T cells isolated from PLNs of diabetic patients were contaminated by activated cytokine-producing effector T cells. We exclude such a possibility because the CD25bright T cells sorted from PLNs of diabetic and nondiabetic donors contained the same amount of FOXP3 mRNA (Fig. 5A) and expressed the same low transcript levels of the master regulators of Th1 (T-box expressed in T cells [T-bet]), Th2 (GATA-binding protein 3 [GATA3]), and Th17 cells (retinoic acid–related orphan receptor C [RORC]) and of IL-2 and IFN-γ (Fig. 5B). Most importantly, the sorted CD25bright T cells were all demethylated at the FOXP3 locus, proving their epigenetic Treg imprinting (Fig. 5C). These data demonstrate that the sorted CD25bright T cells within the PLNs of diabetic patients do not contain effector T cells, and despite being epigenetically determined to be Tregs, they do not exert consistent in vitro regulatory function.

Bottom Line: We found upregulation of Th17 immunity and functional defects in CD4(+)CD25(bright) Tregs in the PLNs of type 1 diabetic subjects but not in their peripheral blood.The dysfunctional Tregs isolated from diabetic subjects did not contain contaminant effector T cells and were all epigenetically imprinted to be suppressive, as defined by analysis of the Treg-specific demethylated region within the forkhead box P3 (FOXP3) locus.These data provide evidence for an unbalanced immune status in the PLNs of type 1 diabetic subjects, and treatments restoring the immune homeostasis in the target organ of these patients represent a potential therapeutic strategy.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Institute, San Raffaele Scientific Institute, Milan, Italy.

ABSTRACT

Objective: Autoimmune diseases, including type 1 diabetes, are thought to have a Th17-cell bias and/or a T-regulatory cell (Treg) defect. Understanding whether this is a hallmark of patients with type 1 diabetes is a crucial question that is still unsolved, largely due to the difficulties of accessing tissues targeted by the disease.

Research design and methods: We phenotypically and functionally characterized Th17 cells and Tregs residing in the pancreatic-draining lymph nodes (PLNs) of 19 patients with type 1 diabetes and 63 nondiabetic donors and those circulating in the peripheral blood of 14 type 1 diabetic patients and 11 healthy subjects.

Results: We found upregulation of Th17 immunity and functional defects in CD4(+)CD25(bright) Tregs in the PLNs of type 1 diabetic subjects but not in their peripheral blood. In addition, the proinsulin-specific Treg-mediated control was altered in the PLNs of diabetic patients. The dysfunctional Tregs isolated from diabetic subjects did not contain contaminant effector T cells and were all epigenetically imprinted to be suppressive, as defined by analysis of the Treg-specific demethylated region within the forkhead box P3 (FOXP3) locus.

Conclusions: These data provide evidence for an unbalanced immune status in the PLNs of type 1 diabetic subjects, and treatments restoring the immune homeostasis in the target organ of these patients represent a potential therapeutic strategy.

Show MeSH
Related in: MedlinePlus