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

Suppression of proinsulin-activated PLN T cells by CD25bright T cells. CD14+ cells isolated from the spleen of nondiabetic donors (ND) or PB of diabetic (T1D) patients were cultured for 7 days with 10 ng/mL IL-4 (R&D Systems, Minneapolis, MN) and 50 ng/mL granulocyte microphage colony-stimulating factor (R&D Systems) to generate DCs. On day 6, DCs were incubated for 6 h with 10 μg of rhu-proinsulin or rhu-GAD65 or left unpulsed and then activated with 100 units/mL of IFN-γ and 10 ng/mL of lipopolysaccharide overnight. On day 7, PLNs depleted of CD25bright T cells were stained with CFSE and cultured with autologous DC unloaded or loaded with antigen at a ratio of 1:40 (DC/PLN). PLN ΔTregs, lymph-node cells depleted of CD25bright T cells by flow cytometry–based sorting. A: The percentages of CD3+ T-cell proliferation in response to proinsulin upon culture of CD25bright T-cell–depleted PLN and total PLN are shown (left). The same data are presented to illustrate inhibition of T-cell proliferation in response to proinsulin by CD25bright T cells (middle). Representative dot plots of CD25bright T-cell–depleted PLN and total PLN proliferating in response to proinsulin are shown (right). In the dot plots, proliferating T cells are gated on CD3+ T cells and percentages of proinsulin-specific T cells are indicated without background subtraction. SSC, side scatter. B: The IFN-γ produced by CD25bright T-cell–depleted PLN and that produced by total PLN in response to proinsulin was tested (left). The same data are presented to show inhibition of IFN-γ-production in response to proinsulin by CD25bright T cells (right). The bracket highlights the “low-responder” nondiabetic donors. PLN cell proliferation observed in the presence of proinsulin-loaded DC was measured upon subtraction of PLN cell proliferation observed in the presence of unloaded control DC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Suppression of proinsulin-activated PLN T cells by CD25bright T cells. CD14+ cells isolated from the spleen of nondiabetic donors (ND) or PB of diabetic (T1D) patients were cultured for 7 days with 10 ng/mL IL-4 (R&D Systems, Minneapolis, MN) and 50 ng/mL granulocyte microphage colony-stimulating factor (R&D Systems) to generate DCs. On day 6, DCs were incubated for 6 h with 10 μg of rhu-proinsulin or rhu-GAD65 or left unpulsed and then activated with 100 units/mL of IFN-γ and 10 ng/mL of lipopolysaccharide overnight. On day 7, PLNs depleted of CD25bright T cells were stained with CFSE and cultured with autologous DC unloaded or loaded with antigen at a ratio of 1:40 (DC/PLN). PLN ΔTregs, lymph-node cells depleted of CD25bright T cells by flow cytometry–based sorting. A: The percentages of CD3+ T-cell proliferation in response to proinsulin upon culture of CD25bright T-cell–depleted PLN and total PLN are shown (left). The same data are presented to illustrate inhibition of T-cell proliferation in response to proinsulin by CD25bright T cells (middle). Representative dot plots of CD25bright T-cell–depleted PLN and total PLN proliferating in response to proinsulin are shown (right). In the dot plots, proliferating T cells are gated on CD3+ T cells and percentages of proinsulin-specific T cells are indicated without background subtraction. SSC, side scatter. B: The IFN-γ produced by CD25bright T-cell–depleted PLN and that produced by total PLN in response to proinsulin was tested (left). The same data are presented to show inhibition of IFN-γ-production in response to proinsulin by CD25bright T cells (right). The bracket highlights the “low-responder” nondiabetic donors. PLN cell proliferation observed in the presence of proinsulin-loaded DC was measured upon subtraction of PLN cell proliferation observed in the presence of unloaded control DC.

Mentions: We then tested the ability of the CD25bright T cells to inhibit diabetes-related antigen-specific T-cell proliferation, maintaining the physiologic CD25bright/responder cell ratio present within the PLN, which was estimated to be ∼1/50. To accomplish this, we compared the frequency of T cells proliferating in response to proinsulin within the lymph nodes depleted of CD25bright T cells by flow cytometry (as shown in Supplementary Fig. 2) with that within the total PLN. The CD25bright T cells present in the PLNs of diabetic patients did not inhibit proinsulin-specific T-cell proliferation but, rather, had a helper activity. By contrast, CD25bright T cells present in the PLNs of nondiabetic subjects exerted an average suppressive function of 38% in response to proinsulin in inhibition of T-cell proliferation and IFN-γ production, and this is in line with the low Treg/responder cell ratio (Fig. 4A). Moreover, we found that CD25bright T cells from PLNs of patients with diabetes did not inhibit proinsulin-specific IFN-γ production, contrary to those isolated from nondiabetic subjects. Removal of CD25bright T cells from PLNs of patients with diabetes led to a dramatic fall in IFN-γ release (Fig. 4B). Thus, depletion of Tregs in the PLNs of diabetic patients leads to a reduced proinsulin-specific cell response rather than to an increased one (as observed in the PLNs of nondiabetic donors). These data further strengthen those shown in the polyclonal suppressive assays and demonstrate that Tregs isolated from the PLNs of diabetic donors have a defective suppressive activity and also acquire a helper phenotype.


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 proinsulin-activated PLN T cells by CD25bright T cells. CD14+ cells isolated from the spleen of nondiabetic donors (ND) or PB of diabetic (T1D) patients were cultured for 7 days with 10 ng/mL IL-4 (R&D Systems, Minneapolis, MN) and 50 ng/mL granulocyte microphage colony-stimulating factor (R&D Systems) to generate DCs. On day 6, DCs were incubated for 6 h with 10 μg of rhu-proinsulin or rhu-GAD65 or left unpulsed and then activated with 100 units/mL of IFN-γ and 10 ng/mL of lipopolysaccharide overnight. On day 7, PLNs depleted of CD25bright T cells were stained with CFSE and cultured with autologous DC unloaded or loaded with antigen at a ratio of 1:40 (DC/PLN). PLN ΔTregs, lymph-node cells depleted of CD25bright T cells by flow cytometry–based sorting. A: The percentages of CD3+ T-cell proliferation in response to proinsulin upon culture of CD25bright T-cell–depleted PLN and total PLN are shown (left). The same data are presented to illustrate inhibition of T-cell proliferation in response to proinsulin by CD25bright T cells (middle). Representative dot plots of CD25bright T-cell–depleted PLN and total PLN proliferating in response to proinsulin are shown (right). In the dot plots, proliferating T cells are gated on CD3+ T cells and percentages of proinsulin-specific T cells are indicated without background subtraction. SSC, side scatter. B: The IFN-γ produced by CD25bright T-cell–depleted PLN and that produced by total PLN in response to proinsulin was tested (left). The same data are presented to show inhibition of IFN-γ-production in response to proinsulin by CD25bright T cells (right). The bracket highlights the “low-responder” nondiabetic donors. PLN cell proliferation observed in the presence of proinsulin-loaded DC was measured upon subtraction of PLN cell proliferation observed in the presence of unloaded control DC.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Suppression of proinsulin-activated PLN T cells by CD25bright T cells. CD14+ cells isolated from the spleen of nondiabetic donors (ND) or PB of diabetic (T1D) patients were cultured for 7 days with 10 ng/mL IL-4 (R&D Systems, Minneapolis, MN) and 50 ng/mL granulocyte microphage colony-stimulating factor (R&D Systems) to generate DCs. On day 6, DCs were incubated for 6 h with 10 μg of rhu-proinsulin or rhu-GAD65 or left unpulsed and then activated with 100 units/mL of IFN-γ and 10 ng/mL of lipopolysaccharide overnight. On day 7, PLNs depleted of CD25bright T cells were stained with CFSE and cultured with autologous DC unloaded or loaded with antigen at a ratio of 1:40 (DC/PLN). PLN ΔTregs, lymph-node cells depleted of CD25bright T cells by flow cytometry–based sorting. A: The percentages of CD3+ T-cell proliferation in response to proinsulin upon culture of CD25bright T-cell–depleted PLN and total PLN are shown (left). The same data are presented to illustrate inhibition of T-cell proliferation in response to proinsulin by CD25bright T cells (middle). Representative dot plots of CD25bright T-cell–depleted PLN and total PLN proliferating in response to proinsulin are shown (right). In the dot plots, proliferating T cells are gated on CD3+ T cells and percentages of proinsulin-specific T cells are indicated without background subtraction. SSC, side scatter. B: The IFN-γ produced by CD25bright T-cell–depleted PLN and that produced by total PLN in response to proinsulin was tested (left). The same data are presented to show inhibition of IFN-γ-production in response to proinsulin by CD25bright T cells (right). The bracket highlights the “low-responder” nondiabetic donors. PLN cell proliferation observed in the presence of proinsulin-loaded DC was measured upon subtraction of PLN cell proliferation observed in the presence of unloaded control DC.
Mentions: We then tested the ability of the CD25bright T cells to inhibit diabetes-related antigen-specific T-cell proliferation, maintaining the physiologic CD25bright/responder cell ratio present within the PLN, which was estimated to be ∼1/50. To accomplish this, we compared the frequency of T cells proliferating in response to proinsulin within the lymph nodes depleted of CD25bright T cells by flow cytometry (as shown in Supplementary Fig. 2) with that within the total PLN. The CD25bright T cells present in the PLNs of diabetic patients did not inhibit proinsulin-specific T-cell proliferation but, rather, had a helper activity. By contrast, CD25bright T cells present in the PLNs of nondiabetic subjects exerted an average suppressive function of 38% in response to proinsulin in inhibition of T-cell proliferation and IFN-γ production, and this is in line with the low Treg/responder cell ratio (Fig. 4A). Moreover, we found that CD25bright T cells from PLNs of patients with diabetes did not inhibit proinsulin-specific IFN-γ production, contrary to those isolated from nondiabetic subjects. Removal of CD25bright T cells from PLNs of patients with diabetes led to a dramatic fall in IFN-γ release (Fig. 4B). Thus, depletion of Tregs in the PLNs of diabetic patients leads to a reduced proinsulin-specific cell response rather than to an increased one (as observed in the PLNs of nondiabetic donors). These data further strengthen those shown in the polyclonal suppressive assays and demonstrate that Tregs isolated from the PLNs of diabetic donors have a defective suppressive activity and also acquire a helper phenotype.

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