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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.

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Frequency of Tregs. A: The frequency of CD25bright T cells within CD45+ cells (upper), of FOXP3+CD127− cells within CD25bright T cells (middle), and of CD25brightFOXP3+CD127− T cells within CD45+ cells (lower) were determined by flow cytometry. One representative dot plot for each staining is shown on the right. B: The frequency of Tregs was determined by TSDR analysis within total PLNs and PB. C: The frequency of Tregs, determined by FACS as CD4+FOXP3+ cells and by the TSDR assay, within total PB (left) and total PLN (right) of type 1 diabetic (T1D) patients, healthy control subjects (HC), and nondiabetic donors (ND) is shown. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. The Mann-Whitney–Wilcoxon test and the t test with unequal variances were used for group comparisons.
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Figure 2: Frequency of Tregs. A: The frequency of CD25bright T cells within CD45+ cells (upper), of FOXP3+CD127− cells within CD25bright T cells (middle), and of CD25brightFOXP3+CD127− T cells within CD45+ cells (lower) were determined by flow cytometry. One representative dot plot for each staining is shown on the right. B: The frequency of Tregs was determined by TSDR analysis within total PLNs and PB. C: The frequency of Tregs, determined by FACS as CD4+FOXP3+ cells and by the TSDR assay, within total PB (left) and total PLN (right) of type 1 diabetic (T1D) patients, healthy control subjects (HC), and nondiabetic donors (ND) is shown. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. The Mann-Whitney–Wilcoxon test and the t test with unequal variances were used for group comparisons.

Mentions: The frequency of Tregs was first determined by flow cytometry through analysis of CD25bright within the CD45+ cells, of FOXP3+CD127− within the CD25bright T cells, and of CD25bright FOXP3+CD127− within the CD45+ cells. All three analyses revealed a significant Treg reduction only in the PLNs of patients with type 1 diabetes and not, as already largely demonstrated by us and others (23,24,26–28), in their PB (Fig. 2A). The amount of FOXP3 assessed by median fluorescence intensity (MFI) expressed in CD4+ T cells and in CD25bright T cells was similar within the PLNs of diabetic and nondiabetic donors (Supplementary Table 3). Because CD25 and FOXP3 are markers that can be altered by the environment (13), we performed the Treg-specific demethylated region (TSDR) assay on the total cells isolated from the PLNs and PB. Unlike the phenotype analysis, this readout identifies epigenetically imprinted Tregs (14–16) and demonstrated that the Treg frequency was similar among diabetic and nondiabetic donors, both in the PLNs and PB (Fig. 2B). Thus, the frequency of T cells epigenetically marked to be Tregs is similar in the PLNs and PB of diabetic and nondiabetic individuals, whereas the frequency of T cells that express the FOXP3 protein is reduced only in the target organ of diabetic patients.


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)

Frequency of Tregs. A: The frequency of CD25bright T cells within CD45+ cells (upper), of FOXP3+CD127− cells within CD25bright T cells (middle), and of CD25brightFOXP3+CD127− T cells within CD45+ cells (lower) were determined by flow cytometry. One representative dot plot for each staining is shown on the right. B: The frequency of Tregs was determined by TSDR analysis within total PLNs and PB. C: The frequency of Tregs, determined by FACS as CD4+FOXP3+ cells and by the TSDR assay, within total PB (left) and total PLN (right) of type 1 diabetic (T1D) patients, healthy control subjects (HC), and nondiabetic donors (ND) is shown. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. The Mann-Whitney–Wilcoxon test and the t test with unequal variances were used for group comparisons.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Frequency of Tregs. A: The frequency of CD25bright T cells within CD45+ cells (upper), of FOXP3+CD127− cells within CD25bright T cells (middle), and of CD25brightFOXP3+CD127− T cells within CD45+ cells (lower) were determined by flow cytometry. One representative dot plot for each staining is shown on the right. B: The frequency of Tregs was determined by TSDR analysis within total PLNs and PB. C: The frequency of Tregs, determined by FACS as CD4+FOXP3+ cells and by the TSDR assay, within total PB (left) and total PLN (right) of type 1 diabetic (T1D) patients, healthy control subjects (HC), and nondiabetic donors (ND) is shown. Each symbol identifies a patient (as described in Supplementary Table 1), and lines indicate median values. The Mann-Whitney–Wilcoxon test and the t test with unequal variances were used for group comparisons.
Mentions: The frequency of Tregs was first determined by flow cytometry through analysis of CD25bright within the CD45+ cells, of FOXP3+CD127− within the CD25bright T cells, and of CD25bright FOXP3+CD127− within the CD45+ cells. All three analyses revealed a significant Treg reduction only in the PLNs of patients with type 1 diabetes and not, as already largely demonstrated by us and others (23,24,26–28), in their PB (Fig. 2A). The amount of FOXP3 assessed by median fluorescence intensity (MFI) expressed in CD4+ T cells and in CD25bright T cells was similar within the PLNs of diabetic and nondiabetic donors (Supplementary Table 3). Because CD25 and FOXP3 are markers that can be altered by the environment (13), we performed the Treg-specific demethylated region (TSDR) assay on the total cells isolated from the PLNs and PB. Unlike the phenotype analysis, this readout identifies epigenetically imprinted Tregs (14–16) and demonstrated that the Treg frequency was similar among diabetic and nondiabetic donors, both in the PLNs and PB (Fig. 2B). Thus, the frequency of T cells epigenetically marked to be Tregs is similar in the PLNs and PB of diabetic and nondiabetic individuals, whereas the frequency of T cells that express the FOXP3 protein is reduced only in the target organ of diabetic patients.

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