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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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Suppression of polyclonally activated PLN T cells by CD25bright T cells. A: The inhibition of proliferation of PLN responder T cells by autologous CD25bright–sorted T cells at the indicated ratios (3:1 and 1:1) was tested. A negative percentage of inhibition of T-cell proliferation indicates that the presence of Tregs in the culture led to improved proliferation. The Mann-Whitney U test was used for group comparison, and P ≤ 0.05 was considered significant. B: T-cell proliferation after 6 days of culture. C: The inhibition of proliferation of responder PLN T cells of diabetic (T1D) patients by CD25bright T cells sorted from autologous PLNs or allogeneic PLNs of nondiabetic donors (ND) was tested. The dotted lines link experiments in which the same responder T cells were used. D: The inhibition of proliferation of responder PLN T cells of diabetic patients by CD25bright T cells sorted from autologous PLN, autologous PB, or allogeneic PB of healthy control subjects (HC) was tested. The same symbol corresponds to the same diabetic patient. The Wilcoxon test was used for comparison of data shown in C and D, and P ≤ 0.05 was considered significant. Each dot represents one donor, and lines indicate median values. In A, C, and D, one representative histogram for each responder/CD25bright T-cell pairs with relative percentage of suppression is shown on the right. Dotted black lines represent responder PLN cells activated in the absence of CD25bright T cells, and solid gray lines represent responder PLN cells activated in the presence of sorted CD25bright T cells.
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Figure 3: Suppression of polyclonally activated PLN T cells by CD25bright T cells. A: The inhibition of proliferation of PLN responder T cells by autologous CD25bright–sorted T cells at the indicated ratios (3:1 and 1:1) was tested. A negative percentage of inhibition of T-cell proliferation indicates that the presence of Tregs in the culture led to improved proliferation. The Mann-Whitney U test was used for group comparison, and P ≤ 0.05 was considered significant. B: T-cell proliferation after 6 days of culture. C: The inhibition of proliferation of responder PLN T cells of diabetic (T1D) patients by CD25bright T cells sorted from autologous PLNs or allogeneic PLNs of nondiabetic donors (ND) was tested. The dotted lines link experiments in which the same responder T cells were used. D: The inhibition of proliferation of responder PLN T cells of diabetic patients by CD25bright T cells sorted from autologous PLN, autologous PB, or allogeneic PB of healthy control subjects (HC) was tested. The same symbol corresponds to the same diabetic patient. The Wilcoxon test was used for comparison of data shown in C and D, and P ≤ 0.05 was considered significant. Each dot represents one donor, and lines indicate median values. In A, C, and D, one representative histogram for each responder/CD25bright T-cell pairs with relative percentage of suppression is shown on the right. Dotted black lines represent responder PLN cells activated in the absence of CD25bright T cells, and solid gray lines represent responder PLN cells activated in the presence of sorted CD25bright T cells.

Mentions: The most efficient way to isolate viable and pure human Tregs devoid of contaminating effector T cells remains the flow cytometry–based cell sorting. Thus, functional experiments were performed in this study by using FACS to isolate Tregs, and this led to a highly purified population of Tregs (Supplementary Fig. 2). The limited CD25bright T cells that could be sorted from PLNs of diabetic subjects forced us to perform the in vitro functional assay with a low number of Tregs (i.e., 7,000 cells) in the presence of responder PLN cells that were three times higher. At this Treg/responder cell ratio, the CD25bright T cells isolated from nondiabetic subjects had an average suppressive function of 30%, which is consistent with published data (36,37). In addition, the same cells had an average suppressive activity of 80% when the assay was performed at the commonly used 1:1 ratio, confirming the solidity of our assay (Fig. 3A). CD25bright T cells isolated from PLNs of three diabetic patients did not inhibit proliferation of polyclonally activated autologous PLN T cells, but rather, they had a helper function because their presence in the culture led to increased proliferation of the responder T cells. The CD25bright T cells purified from the PLNs of a further three diabetic patients had a reduced suppressive activity that fell into the low range of those isolated from nondiabetic donors (Fig. 3A). The statistical analysis performed on all the samples tested showed that Tregs isolated from the PLNs of diabetic patients have an impaired suppressive capability compared with those of nondiabetic donors (P = 0.0066).


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)

Suppression of polyclonally activated PLN T cells by CD25bright T cells. A: The inhibition of proliferation of PLN responder T cells by autologous CD25bright–sorted T cells at the indicated ratios (3:1 and 1:1) was tested. A negative percentage of inhibition of T-cell proliferation indicates that the presence of Tregs in the culture led to improved proliferation. The Mann-Whitney U test was used for group comparison, and P ≤ 0.05 was considered significant. B: T-cell proliferation after 6 days of culture. C: The inhibition of proliferation of responder PLN T cells of diabetic (T1D) patients by CD25bright T cells sorted from autologous PLNs or allogeneic PLNs of nondiabetic donors (ND) was tested. The dotted lines link experiments in which the same responder T cells were used. D: The inhibition of proliferation of responder PLN T cells of diabetic patients by CD25bright T cells sorted from autologous PLN, autologous PB, or allogeneic PB of healthy control subjects (HC) was tested. The same symbol corresponds to the same diabetic patient. The Wilcoxon test was used for comparison of data shown in C and D, and P ≤ 0.05 was considered significant. Each dot represents one donor, and lines indicate median values. In A, C, and D, one representative histogram for each responder/CD25bright T-cell pairs with relative percentage of suppression is shown on the right. Dotted black lines represent responder PLN cells activated in the absence of CD25bright T cells, and solid gray lines represent responder PLN cells activated in the presence of sorted CD25bright T cells.
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Related In: Results  -  Collection

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Figure 3: Suppression of polyclonally activated PLN T cells by CD25bright T cells. A: The inhibition of proliferation of PLN responder T cells by autologous CD25bright–sorted T cells at the indicated ratios (3:1 and 1:1) was tested. A negative percentage of inhibition of T-cell proliferation indicates that the presence of Tregs in the culture led to improved proliferation. The Mann-Whitney U test was used for group comparison, and P ≤ 0.05 was considered significant. B: T-cell proliferation after 6 days of culture. C: The inhibition of proliferation of responder PLN T cells of diabetic (T1D) patients by CD25bright T cells sorted from autologous PLNs or allogeneic PLNs of nondiabetic donors (ND) was tested. The dotted lines link experiments in which the same responder T cells were used. D: The inhibition of proliferation of responder PLN T cells of diabetic patients by CD25bright T cells sorted from autologous PLN, autologous PB, or allogeneic PB of healthy control subjects (HC) was tested. The same symbol corresponds to the same diabetic patient. The Wilcoxon test was used for comparison of data shown in C and D, and P ≤ 0.05 was considered significant. Each dot represents one donor, and lines indicate median values. In A, C, and D, one representative histogram for each responder/CD25bright T-cell pairs with relative percentage of suppression is shown on the right. Dotted black lines represent responder PLN cells activated in the absence of CD25bright T cells, and solid gray lines represent responder PLN cells activated in the presence of sorted CD25bright T cells.
Mentions: The most efficient way to isolate viable and pure human Tregs devoid of contaminating effector T cells remains the flow cytometry–based cell sorting. Thus, functional experiments were performed in this study by using FACS to isolate Tregs, and this led to a highly purified population of Tregs (Supplementary Fig. 2). The limited CD25bright T cells that could be sorted from PLNs of diabetic subjects forced us to perform the in vitro functional assay with a low number of Tregs (i.e., 7,000 cells) in the presence of responder PLN cells that were three times higher. At this Treg/responder cell ratio, the CD25bright T cells isolated from nondiabetic subjects had an average suppressive function of 30%, which is consistent with published data (36,37). In addition, the same cells had an average suppressive activity of 80% when the assay was performed at the commonly used 1:1 ratio, confirming the solidity of our assay (Fig. 3A). CD25bright T cells isolated from PLNs of three diabetic patients did not inhibit proliferation of polyclonally activated autologous PLN T cells, but rather, they had a helper function because their presence in the culture led to increased proliferation of the responder T cells. The CD25bright T cells purified from the PLNs of a further three diabetic patients had a reduced suppressive activity that fell into the low range of those isolated from nondiabetic donors (Fig. 3A). The statistical analysis performed on all the samples tested showed that Tregs isolated from the PLNs of diabetic patients have an impaired suppressive capability compared with those of nondiabetic donors (P = 0.0066).

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