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CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion.

Herman AE, Freeman GJ, Mathis D, Benoist C - J. Exp. Med. (2004)

Bottom Line: Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset.We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity.Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile.

View Article: PubMed Central - PubMed

Affiliation: Section on Immunology and Immunogenetics, Joslin Diabetes Center, Boston, MA 02215, USA.

ABSTRACT
CD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69- cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile. Thus, CD4+CD25+69- Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.

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Infiltrate in the pancreatic lesion contains subsets of CD4+ cells with activated or regulatory phenotypes. (A) Islets of BDC2.5 mice at 4 wk of age. (B) Cells from pancreatic infiltrate of 3–4-wk-old BDC2.5 mice were isolated as aforementioned and analyzed for CD4, B220, live cell dye, CD25, and CD69 markers. Plots are gated on CD4+B220− live lymphocytes, and expression of activation markers CD25 and CD69 is shown. Data represent 10 separate experiments. (C) Pancreatic cells gated for CD4+ expression, live lymphocytes, and B220− cells were sorted (whole CD4+) or sorted separately into the distinct CD25/CD69 subsets observed in B. Whole BDC2.5 LNs, or LN sorted into CD4+CD25+ and CD4+CD25− subsets were also prepared. cDNA was prepared from each indicated group and assessed for expression of FoxP3 cDNA by real-time PCR analysis. Data are presented as relative expression of FoxP3 compared with the value in whole LNs (set to 1). Averaged results of three experiments are shown.
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fig1: Infiltrate in the pancreatic lesion contains subsets of CD4+ cells with activated or regulatory phenotypes. (A) Islets of BDC2.5 mice at 4 wk of age. (B) Cells from pancreatic infiltrate of 3–4-wk-old BDC2.5 mice were isolated as aforementioned and analyzed for CD4, B220, live cell dye, CD25, and CD69 markers. Plots are gated on CD4+B220− live lymphocytes, and expression of activation markers CD25 and CD69 is shown. Data represent 10 separate experiments. (C) Pancreatic cells gated for CD4+ expression, live lymphocytes, and B220− cells were sorted (whole CD4+) or sorted separately into the distinct CD25/CD69 subsets observed in B. Whole BDC2.5 LNs, or LN sorted into CD4+CD25+ and CD4+CD25− subsets were also prepared. cDNA was prepared from each indicated group and assessed for expression of FoxP3 cDNA by real-time PCR analysis. Data are presented as relative expression of FoxP3 compared with the value in whole LNs (set to 1). Averaged results of three experiments are shown.

Mentions: Initially, we asked whether cells in the prediabetes pancreatic lesion of BDC2.5 mice (Fig. 1 A) are simply incompetent to cause disease, or whether there is an active regulatory process occurring in the islets. Preparation of islets followed by isolation of lymphocytes proved detrimental to identifying all T cells because heavily infiltrated islets fell apart during isolation and were not included in the final yield. Therefore, infiltrating T cells were prepared by mechanical disruption of whole pancreata of young 3–4-wk-old BDC2.5/NOD mice after careful removal of any LNs. The cells obtained (routinely 3–5 × 106 per BDC2.5/NOD pancreas) were a true component of the infiltrate in that lymphocytes could not be isolated from pancreata of strains with no islet infiltration (unpublished data). We divided the CD4+ population into three subsets on the basis of the early activation marker CD69 and the activation/regulatory marker CD25: CD4+CD25+CD69−, CD4+CD25loCD69+, and CD4+ CD25−CD69− (Fig. 1 B). Although CD4+ cells in the irrelevant inguinal LN (ILN) were mainly naive, those from the pancreatic LN (PLN) or from the pancreatic lesion were highly activated, with 15–40% of CD4+ cells displaying a CD25loCD69+ profile consistent with recent activation. 3–11% of pancreatic infiltrating cells had a CD4+ CD25+CD69− phenotype, consistent with Tregs.


CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion.

Herman AE, Freeman GJ, Mathis D, Benoist C - J. Exp. Med. (2004)

Infiltrate in the pancreatic lesion contains subsets of CD4+ cells with activated or regulatory phenotypes. (A) Islets of BDC2.5 mice at 4 wk of age. (B) Cells from pancreatic infiltrate of 3–4-wk-old BDC2.5 mice were isolated as aforementioned and analyzed for CD4, B220, live cell dye, CD25, and CD69 markers. Plots are gated on CD4+B220− live lymphocytes, and expression of activation markers CD25 and CD69 is shown. Data represent 10 separate experiments. (C) Pancreatic cells gated for CD4+ expression, live lymphocytes, and B220− cells were sorted (whole CD4+) or sorted separately into the distinct CD25/CD69 subsets observed in B. Whole BDC2.5 LNs, or LN sorted into CD4+CD25+ and CD4+CD25− subsets were also prepared. cDNA was prepared from each indicated group and assessed for expression of FoxP3 cDNA by real-time PCR analysis. Data are presented as relative expression of FoxP3 compared with the value in whole LNs (set to 1). Averaged results of three experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211778&req=5

fig1: Infiltrate in the pancreatic lesion contains subsets of CD4+ cells with activated or regulatory phenotypes. (A) Islets of BDC2.5 mice at 4 wk of age. (B) Cells from pancreatic infiltrate of 3–4-wk-old BDC2.5 mice were isolated as aforementioned and analyzed for CD4, B220, live cell dye, CD25, and CD69 markers. Plots are gated on CD4+B220− live lymphocytes, and expression of activation markers CD25 and CD69 is shown. Data represent 10 separate experiments. (C) Pancreatic cells gated for CD4+ expression, live lymphocytes, and B220− cells were sorted (whole CD4+) or sorted separately into the distinct CD25/CD69 subsets observed in B. Whole BDC2.5 LNs, or LN sorted into CD4+CD25+ and CD4+CD25− subsets were also prepared. cDNA was prepared from each indicated group and assessed for expression of FoxP3 cDNA by real-time PCR analysis. Data are presented as relative expression of FoxP3 compared with the value in whole LNs (set to 1). Averaged results of three experiments are shown.
Mentions: Initially, we asked whether cells in the prediabetes pancreatic lesion of BDC2.5 mice (Fig. 1 A) are simply incompetent to cause disease, or whether there is an active regulatory process occurring in the islets. Preparation of islets followed by isolation of lymphocytes proved detrimental to identifying all T cells because heavily infiltrated islets fell apart during isolation and were not included in the final yield. Therefore, infiltrating T cells were prepared by mechanical disruption of whole pancreata of young 3–4-wk-old BDC2.5/NOD mice after careful removal of any LNs. The cells obtained (routinely 3–5 × 106 per BDC2.5/NOD pancreas) were a true component of the infiltrate in that lymphocytes could not be isolated from pancreata of strains with no islet infiltration (unpublished data). We divided the CD4+ population into three subsets on the basis of the early activation marker CD69 and the activation/regulatory marker CD25: CD4+CD25+CD69−, CD4+CD25loCD69+, and CD4+ CD25−CD69− (Fig. 1 B). Although CD4+ cells in the irrelevant inguinal LN (ILN) were mainly naive, those from the pancreatic LN (PLN) or from the pancreatic lesion were highly activated, with 15–40% of CD4+ cells displaying a CD25loCD69+ profile consistent with recent activation. 3–11% of pancreatic infiltrating cells had a CD4+ CD25+CD69− phenotype, consistent with Tregs.

Bottom Line: Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset.We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity.Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile.

View Article: PubMed Central - PubMed

Affiliation: Section on Immunology and Immunogenetics, Joslin Diabetes Center, Boston, MA 02215, USA.

ABSTRACT
CD4+CD25+ T regulatory cells (Tregs) prevent autoimmune disease, yet little is known about precisely where they exert their influence naturally in a spontaneous autoimmune disorder. Here, we report that Tregs and T effector cells (Teffs) coexist within the pancreatic lesion before type 1 diabetes onset. We find that BDC2.5 T cell receptor transgenic animals contain a small subset of FoxP3 positive CD4+CD25+CD69- cells in the pancreas, actively turning over, expressing the clonotypic receptor, and containing functional regulatory activity. Gene expression profiling confirms that the CD4+CD25+CD69- cells in pancreatic tissue express transcripts diagnostic of regulatory cells, but with significantly higher levels of interleukin 10 and inducible costimulator (ICOS) than their lymph node counterparts. Blockade of ICOS rapidly converts early insulitis to diabetes, which disrupts the balance of Teffs and Tregs and promotes a very broad shift in the expression of the T regulatory-specific profile. Thus, CD4+CD25+69- Tregs operate directly in the autoimmune lesion and are dependent on ICOS to keep it in a nondestructive state.

Show MeSH
Related in: MedlinePlus