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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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Related in: MedlinePlus

Frequency of T-cell subsets. The PLNs and PB isolated from diabetic (T1D) and nondiabetic donors (ND) had comparable percentages of isolated live cells (all >90%), and the PB contained similar cell numbers in the two donor groups (data not shown). The number of cells within the PLN was difficult to compare due to the manual isolation of the lymphocytes and the variability in the size of the PLNs. A: Box plots represent percentages of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells all within CD45+ cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 18; ND PLN, n = 58; T1D PB, n = 16; and healthy control subjects (HC) PB, n = 10. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively; and the whiskers mark the minimum and maximum. One representative dot plot of CD4 and CD8 cells (within CD45+ cells) with relative percentages is shown. B: Box plots represent the frequency of CD45RO+ T cells within CD4+ and CD8+ T cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 13; ND PLN, n = 37; T1D PB, n = 14; and HC PB, n = 10. One representative dot plot of CD45RO/CD45RA (within CD4+ and CD8+ T cells) with relative percentages is shown. C: The frequency of Th1 cells was measured by flow cytometry (left), and by IFN-γ+–producing CD4+ T cells (right). One representative dot plot of IFN-γ+ cells (within CD4+ T cells) with relative percentages is shown. D: The frequency of Th17 cells was measured by surface phenotype (left) and by IL-17–producing CD4+ T cells (right). One representative dot plot of IL-17+ cells (within CD4+ T cells) with relative percentages is shown. E: The IL-17 (left) and IFN-γ (right) produced by total PLN in response to proinsulin and GAD65 were tested. Dotted lines indicate the detection level of the assay. 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 1: Frequency of T-cell subsets. The PLNs and PB isolated from diabetic (T1D) and nondiabetic donors (ND) had comparable percentages of isolated live cells (all >90%), and the PB contained similar cell numbers in the two donor groups (data not shown). The number of cells within the PLN was difficult to compare due to the manual isolation of the lymphocytes and the variability in the size of the PLNs. A: Box plots represent percentages of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells all within CD45+ cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 18; ND PLN, n = 58; T1D PB, n = 16; and healthy control subjects (HC) PB, n = 10. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively; and the whiskers mark the minimum and maximum. One representative dot plot of CD4 and CD8 cells (within CD45+ cells) with relative percentages is shown. B: Box plots represent the frequency of CD45RO+ T cells within CD4+ and CD8+ T cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 13; ND PLN, n = 37; T1D PB, n = 14; and HC PB, n = 10. One representative dot plot of CD45RO/CD45RA (within CD4+ and CD8+ T cells) with relative percentages is shown. C: The frequency of Th1 cells was measured by flow cytometry (left), and by IFN-γ+–producing CD4+ T cells (right). One representative dot plot of IFN-γ+ cells (within CD4+ T cells) with relative percentages is shown. D: The frequency of Th17 cells was measured by surface phenotype (left) and by IL-17–producing CD4+ T cells (right). One representative dot plot of IL-17+ cells (within CD4+ T cells) with relative percentages is shown. E: The IL-17 (left) and IFN-γ (right) produced by total PLN in response to proinsulin and GAD65 were tested. Dotted lines indicate the detection level of the assay. 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 percentages of total CD3+ T cells and of CD3+CD4+ T cells were similar in diabetic patients and nondiabetic donors in the PLNs and PB (Fig. 1A). Conversely, the frequency of CD3+CD8+ T cells was higher in the PLNs of diabetic patients (Fig. 1A), and this led to a lower CD4/CD8 T-cell ratio in diabetic donors compared with nondiabetic subjects (Supplementary Table 2). The antigen-experienced T cells, defined by the expression of CD45RO, were enriched within the lymph node and circulating CD4+ T cells, but not within the CD8+ T cells, of diabetic subjects (Fig. 1B). The expansion of CD4+CD45RO+ T cells observed in diabetic patients was independent of donor age (data not shown).


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 T-cell subsets. The PLNs and PB isolated from diabetic (T1D) and nondiabetic donors (ND) had comparable percentages of isolated live cells (all >90%), and the PB contained similar cell numbers in the two donor groups (data not shown). The number of cells within the PLN was difficult to compare due to the manual isolation of the lymphocytes and the variability in the size of the PLNs. A: Box plots represent percentages of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells all within CD45+ cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 18; ND PLN, n = 58; T1D PB, n = 16; and healthy control subjects (HC) PB, n = 10. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively; and the whiskers mark the minimum and maximum. One representative dot plot of CD4 and CD8 cells (within CD45+ cells) with relative percentages is shown. B: Box plots represent the frequency of CD45RO+ T cells within CD4+ and CD8+ T cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 13; ND PLN, n = 37; T1D PB, n = 14; and HC PB, n = 10. One representative dot plot of CD45RO/CD45RA (within CD4+ and CD8+ T cells) with relative percentages is shown. C: The frequency of Th1 cells was measured by flow cytometry (left), and by IFN-γ+–producing CD4+ T cells (right). One representative dot plot of IFN-γ+ cells (within CD4+ T cells) with relative percentages is shown. D: The frequency of Th17 cells was measured by surface phenotype (left) and by IL-17–producing CD4+ T cells (right). One representative dot plot of IL-17+ cells (within CD4+ T cells) with relative percentages is shown. E: The IL-17 (left) and IFN-γ (right) produced by total PLN in response to proinsulin and GAD65 were tested. Dotted lines indicate the detection level of the assay. 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 1: Frequency of T-cell subsets. The PLNs and PB isolated from diabetic (T1D) and nondiabetic donors (ND) had comparable percentages of isolated live cells (all >90%), and the PB contained similar cell numbers in the two donor groups (data not shown). The number of cells within the PLN was difficult to compare due to the manual isolation of the lymphocytes and the variability in the size of the PLNs. A: Box plots represent percentages of CD3+ T cells, CD3+CD4+ T cells, and CD3+CD8+ T cells all within CD45+ cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 18; ND PLN, n = 58; T1D PB, n = 16; and healthy control subjects (HC) PB, n = 10. The horizontal line in the middle of each box indicates the median; the top and bottom borders of the box mark the 75th and 25th percentiles, respectively; and the whiskers mark the minimum and maximum. One representative dot plot of CD4 and CD8 cells (within CD45+ cells) with relative percentages is shown. B: Box plots represent the frequency of CD45RO+ T cells within CD4+ and CD8+ T cells determined by flow cytometry–based analysis. The number of donors in each group is as follows: T1D PLN, n = 13; ND PLN, n = 37; T1D PB, n = 14; and HC PB, n = 10. One representative dot plot of CD45RO/CD45RA (within CD4+ and CD8+ T cells) with relative percentages is shown. C: The frequency of Th1 cells was measured by flow cytometry (left), and by IFN-γ+–producing CD4+ T cells (right). One representative dot plot of IFN-γ+ cells (within CD4+ T cells) with relative percentages is shown. D: The frequency of Th17 cells was measured by surface phenotype (left) and by IL-17–producing CD4+ T cells (right). One representative dot plot of IL-17+ cells (within CD4+ T cells) with relative percentages is shown. E: The IL-17 (left) and IFN-γ (right) produced by total PLN in response to proinsulin and GAD65 were tested. Dotted lines indicate the detection level of the assay. 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 percentages of total CD3+ T cells and of CD3+CD4+ T cells were similar in diabetic patients and nondiabetic donors in the PLNs and PB (Fig. 1A). Conversely, the frequency of CD3+CD8+ T cells was higher in the PLNs of diabetic patients (Fig. 1A), and this led to a lower CD4/CD8 T-cell ratio in diabetic donors compared with nondiabetic subjects (Supplementary Table 2). The antigen-experienced T cells, defined by the expression of CD45RO, were enriched within the lymph node and circulating CD4+ T cells, but not within the CD8+ T cells, of diabetic subjects (Fig. 1B). The expansion of CD4+CD45RO+ T cells observed in diabetic patients was independent of donor age (data not shown).

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