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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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CD4+CD25+CD69− cells have a gene expression profile of Tregs, whereas CD25loCD69+ and CD25−CD69− cells are distinct. (A) Cells were sorted to high purity directly from the pancreas lesion of 3–4-wk-old BDC2.5 mice for the CD4+CD25+CD69− population, or the CD25loCD69+ and CD25−CD69− combined populations. RNA was prepared and hybridized to Affymetrix U74Av2 array GeneChips® as described in Materials and Methods. RMA analysis was used to compare relative expression of data from the CD4+CD25+CD69− population to the CD25loCD69+ and CD25−CD69− combined populations. The cutoff for significant differences was determined using a random dataset to identify a 10% FPR for the real data, in this case a 2.1-fold increase (or decrease) in gene expression (highlighted in blue). For each cell type, three to five separate experiments were performed. (B) The gene list of 77 from McHugh et al. (reference 45) is highlighted on our relative gene expression graph in blue (up in LN CD25+) and red (up in LN CD25−).
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fig4: CD4+CD25+CD69− cells have a gene expression profile of Tregs, whereas CD25loCD69+ and CD25−CD69− cells are distinct. (A) Cells were sorted to high purity directly from the pancreas lesion of 3–4-wk-old BDC2.5 mice for the CD4+CD25+CD69− population, or the CD25loCD69+ and CD25−CD69− combined populations. RNA was prepared and hybridized to Affymetrix U74Av2 array GeneChips® as described in Materials and Methods. RMA analysis was used to compare relative expression of data from the CD4+CD25+CD69− population to the CD25loCD69+ and CD25−CD69− combined populations. The cutoff for significant differences was determined using a random dataset to identify a 10% FPR for the real data, in this case a 2.1-fold increase (or decrease) in gene expression (highlighted in blue). For each cell type, three to five separate experiments were performed. (B) The gene list of 77 from McHugh et al. (reference 45) is highlighted on our relative gene expression graph in blue (up in LN CD25+) and red (up in LN CD25−).

Mentions: To better understand the function and phenotype of effector and regulatory cells residing in the target organ during autoimmunity, we compared the gene expression profiles of CD25+CD69− Tregs to the combined Teff populations of CD25loCD69+ and CD25−CD69− cells. We sorted cells from the lesion, isolated RNA, prepared double amplified biotinylated aRNA, and hybridized each sample to an Affymetrix murine genome U74Av2 array GeneChip® for at least three replicates of each population. A false positive rate (FPR) was estimated from a randomized dataset generated by shuffling expression values between samples (Fig. 4 A, randomized control). The CD25+CD69− Tregs and Teff cell populations from the prediabetic lesion could be distinguished by the expression of a limited set of genes; with a 10% FPR cutoff, 96 genes were overexpressed in CD25+CD69− cells, and 73 were underrepresented, significantly more than in the randomized dataset (Fig. 4 A). Several of the genes overexpressed in CD25+CD69− cells such as GITR, CD103, Nrp-1, IL-10, and CTLA-4 are diagnostic of Tregs with suppressive activity (Table SI, available at http://www.jem.org/cgi/content/full/jem.20040179/DC1; references 14–16, 43–46). Overall, this group was dominated by cell surface or cytokine genes. Conversely, the genes underrepresented in Tregs seemed dominated by transcription or signaling factors (OBF-1, Tcf7, Eomesodermin, and Smad1). We compared our data to 77 genes shown previously to be overrepresented in LN CD4+CD25+ cells from normal mice or from in vitro–activated cultures (45); as illustrated in Fig. 4 B, many of these were also overrepresented in pancreatic CD25+CD69− Tregs (21 out of 77 overlap with our list at 10% FPR; Table SI). However, many of the genes on our list have not been identified previously as particularly expressed by Tregs. They might represent a unique subset of genes either induced in a subset of Tregs to make them capable of accessing the tissues, or in Tregs once within the lesion.


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)

CD4+CD25+CD69− cells have a gene expression profile of Tregs, whereas CD25loCD69+ and CD25−CD69− cells are distinct. (A) Cells were sorted to high purity directly from the pancreas lesion of 3–4-wk-old BDC2.5 mice for the CD4+CD25+CD69− population, or the CD25loCD69+ and CD25−CD69− combined populations. RNA was prepared and hybridized to Affymetrix U74Av2 array GeneChips® as described in Materials and Methods. RMA analysis was used to compare relative expression of data from the CD4+CD25+CD69− population to the CD25loCD69+ and CD25−CD69− combined populations. The cutoff for significant differences was determined using a random dataset to identify a 10% FPR for the real data, in this case a 2.1-fold increase (or decrease) in gene expression (highlighted in blue). For each cell type, three to five separate experiments were performed. (B) The gene list of 77 from McHugh et al. (reference 45) is highlighted on our relative gene expression graph in blue (up in LN CD25+) and red (up in LN CD25−).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211778&req=5

fig4: CD4+CD25+CD69− cells have a gene expression profile of Tregs, whereas CD25loCD69+ and CD25−CD69− cells are distinct. (A) Cells were sorted to high purity directly from the pancreas lesion of 3–4-wk-old BDC2.5 mice for the CD4+CD25+CD69− population, or the CD25loCD69+ and CD25−CD69− combined populations. RNA was prepared and hybridized to Affymetrix U74Av2 array GeneChips® as described in Materials and Methods. RMA analysis was used to compare relative expression of data from the CD4+CD25+CD69− population to the CD25loCD69+ and CD25−CD69− combined populations. The cutoff for significant differences was determined using a random dataset to identify a 10% FPR for the real data, in this case a 2.1-fold increase (or decrease) in gene expression (highlighted in blue). For each cell type, three to five separate experiments were performed. (B) The gene list of 77 from McHugh et al. (reference 45) is highlighted on our relative gene expression graph in blue (up in LN CD25+) and red (up in LN CD25−).
Mentions: To better understand the function and phenotype of effector and regulatory cells residing in the target organ during autoimmunity, we compared the gene expression profiles of CD25+CD69− Tregs to the combined Teff populations of CD25loCD69+ and CD25−CD69− cells. We sorted cells from the lesion, isolated RNA, prepared double amplified biotinylated aRNA, and hybridized each sample to an Affymetrix murine genome U74Av2 array GeneChip® for at least three replicates of each population. A false positive rate (FPR) was estimated from a randomized dataset generated by shuffling expression values between samples (Fig. 4 A, randomized control). The CD25+CD69− Tregs and Teff cell populations from the prediabetic lesion could be distinguished by the expression of a limited set of genes; with a 10% FPR cutoff, 96 genes were overexpressed in CD25+CD69− cells, and 73 were underrepresented, significantly more than in the randomized dataset (Fig. 4 A). Several of the genes overexpressed in CD25+CD69− cells such as GITR, CD103, Nrp-1, IL-10, and CTLA-4 are diagnostic of Tregs with suppressive activity (Table SI, available at http://www.jem.org/cgi/content/full/jem.20040179/DC1; references 14–16, 43–46). Overall, this group was dominated by cell surface or cytokine genes. Conversely, the genes underrepresented in Tregs seemed dominated by transcription or signaling factors (OBF-1, Tcf7, Eomesodermin, and Smad1). We compared our data to 77 genes shown previously to be overrepresented in LN CD4+CD25+ cells from normal mice or from in vitro–activated cultures (45); as illustrated in Fig. 4 B, many of these were also overrepresented in pancreatic CD25+CD69− Tregs (21 out of 77 overlap with our list at 10% FPR; Table SI). However, many of the genes on our list have not been identified previously as particularly expressed by Tregs. They might represent a unique subset of genes either induced in a subset of Tregs to make them capable of accessing the tissues, or in Tregs once within the lesion.

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