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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 isolated directly from the pancreatic lesion protect from diabetes, whereas CD4+CD25loCD69+ and CD25−CD69− cells are pathogenic. (A) Cells from the pancreatic lesion of 3–4-wk-old BDC2.5 mice were sorted into CD4+CD25+CD69− (▴CD25+CD69−), CD4+CD25loCD69+ (▪CD25loCD69+), and CD4+CD25−CD69−(♦CD25−CD69−) subsets by flow cytometry. Sorted cells were transferred i.p. separately into NOD.scid recipients at two doses as follows: 50,000 cells (closed symbols) or 10–20,000 cells (open symbols). Mice were monitored every other day after 7 d for the onset of hyperglycemia by urine, and diabetes was confirmed by blood glucose levels >250 mg/dl. Data represent pooled results of three separate experiments with similar results. (B) CD4+CD25+CD69− cells were sorted as in A. Either 10–20,000 (▴), 5,000 (▪), or no (♦) CD4+CD25+CD69− cells were transferred i.p. into NOD.scid recipient animals in combination with 5 × 105 diabetogenic BDC2.5 splenocytes. Mice were monitored as described in A. Data represent pooled results of three separate experiments with similar results. (C) Histological sections were prepared from pancreata of transferred NOD.scid animals. Hematoxylin and eosin–stained sections from recipients that did not develop diabetes (protected by Treg) by the end of 30 d after receiving the 10–20,000 cell dose of CD4+CD25+CD69− cells and 5 × 105 diabetogenic BDC2.5 splenocytes are shown.
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fig3: CD4+CD25+CD69− cells isolated directly from the pancreatic lesion protect from diabetes, whereas CD4+CD25loCD69+ and CD25−CD69− cells are pathogenic. (A) Cells from the pancreatic lesion of 3–4-wk-old BDC2.5 mice were sorted into CD4+CD25+CD69− (▴CD25+CD69−), CD4+CD25loCD69+ (▪CD25loCD69+), and CD4+CD25−CD69−(♦CD25−CD69−) subsets by flow cytometry. Sorted cells were transferred i.p. separately into NOD.scid recipients at two doses as follows: 50,000 cells (closed symbols) or 10–20,000 cells (open symbols). Mice were monitored every other day after 7 d for the onset of hyperglycemia by urine, and diabetes was confirmed by blood glucose levels >250 mg/dl. Data represent pooled results of three separate experiments with similar results. (B) CD4+CD25+CD69− cells were sorted as in A. Either 10–20,000 (▴), 5,000 (▪), or no (♦) CD4+CD25+CD69− cells were transferred i.p. into NOD.scid recipient animals in combination with 5 × 105 diabetogenic BDC2.5 splenocytes. Mice were monitored as described in A. Data represent pooled results of three separate experiments with similar results. (C) Histological sections were prepared from pancreata of transferred NOD.scid animals. Hematoxylin and eosin–stained sections from recipients that did not develop diabetes (protected by Treg) by the end of 30 d after receiving the 10–20,000 cell dose of CD4+CD25+CD69− cells and 5 × 105 diabetogenic BDC2.5 splenocytes are shown.

Mentions: These data are suggestive of an active balance of regulatory and pathogenic CD4+ T cells in the pancreatic lesion. To evaluate function, we sorted CD25+CD69−, CD25loCD69+, and CD25−CD69− cells from BDC2.5/NOD pancreata were transferred into NOD.scid recipients. Mice that received CD25−CD69− cells or CD25loCD69+ cells developed diabetes shortly after transfer (Fig. 3 A). As few as 10–20,000 cells of these phenotypes were enough to provoke diabetes in 50–80% of recipients within 1 mo after transfer. All mice that received CD25−CD69− cells or CD25loCD69+ cells, at the doses tested, developed insulitis (unpublished data). In contrast, CD25+CD69− cells did not cause diabetes or develop insulitis after transfer. We asked whether the CD25+CD69− cells residing in the pancreas did, in fact, have protective capacity by transferring 10–20,000 CD25+CD69− cells isolated from the lesion with 5 × 105 diabetogenic BDC2.5/NOD splenocytes into NOD.scid recipients. This small dose of cells, 10–100-fold lower than the dose used in other models of regulation using polyclonal populations (20, 42), provided significant protection from diabetes in this cotransfer context (Fig. 3 B). BDC2.5 LN Tregs (CD4+CD25+CD69−) were also effective at this dose (unpublished data). In vivo regulation was dose dependent because protection from diabetes was lost if only 5,000 Tregs were cotransferred (Fig. 3 B). In protected mice that had received 20,000 CD25+CD69− Tregs and 5 × 105 diabetogenic BDC2.5/NOD splenocytes, we observed formidable, but respectful, insulitis in the islet after 30 d (Fig. 3 C), similar to that seen in islets of unmanipulated BDC2.5/NOD mice (compare with Fig. 1 A). Interestingly, Tregs taken directly from the pancreatic lesion were unable to set up a presence there in the absence of other T cells in the NOD.SCID host, suggesting that they may require signals from Teffs to access the pancreas or persist there. These data demonstrate that Teff and Treg subsets coexist in the pancreatic lesion, and that T effectors capable of disease transfer are being actively suppressed by CD25+CD69− Tregs during the nondestructive period of insulitis.


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 isolated directly from the pancreatic lesion protect from diabetes, whereas CD4+CD25loCD69+ and CD25−CD69− cells are pathogenic. (A) Cells from the pancreatic lesion of 3–4-wk-old BDC2.5 mice were sorted into CD4+CD25+CD69− (▴CD25+CD69−), CD4+CD25loCD69+ (▪CD25loCD69+), and CD4+CD25−CD69−(♦CD25−CD69−) subsets by flow cytometry. Sorted cells were transferred i.p. separately into NOD.scid recipients at two doses as follows: 50,000 cells (closed symbols) or 10–20,000 cells (open symbols). Mice were monitored every other day after 7 d for the onset of hyperglycemia by urine, and diabetes was confirmed by blood glucose levels >250 mg/dl. Data represent pooled results of three separate experiments with similar results. (B) CD4+CD25+CD69− cells were sorted as in A. Either 10–20,000 (▴), 5,000 (▪), or no (♦) CD4+CD25+CD69− cells were transferred i.p. into NOD.scid recipient animals in combination with 5 × 105 diabetogenic BDC2.5 splenocytes. Mice were monitored as described in A. Data represent pooled results of three separate experiments with similar results. (C) Histological sections were prepared from pancreata of transferred NOD.scid animals. Hematoxylin and eosin–stained sections from recipients that did not develop diabetes (protected by Treg) by the end of 30 d after receiving the 10–20,000 cell dose of CD4+CD25+CD69− cells and 5 × 105 diabetogenic BDC2.5 splenocytes are shown.
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fig3: CD4+CD25+CD69− cells isolated directly from the pancreatic lesion protect from diabetes, whereas CD4+CD25loCD69+ and CD25−CD69− cells are pathogenic. (A) Cells from the pancreatic lesion of 3–4-wk-old BDC2.5 mice were sorted into CD4+CD25+CD69− (▴CD25+CD69−), CD4+CD25loCD69+ (▪CD25loCD69+), and CD4+CD25−CD69−(♦CD25−CD69−) subsets by flow cytometry. Sorted cells were transferred i.p. separately into NOD.scid recipients at two doses as follows: 50,000 cells (closed symbols) or 10–20,000 cells (open symbols). Mice were monitored every other day after 7 d for the onset of hyperglycemia by urine, and diabetes was confirmed by blood glucose levels >250 mg/dl. Data represent pooled results of three separate experiments with similar results. (B) CD4+CD25+CD69− cells were sorted as in A. Either 10–20,000 (▴), 5,000 (▪), or no (♦) CD4+CD25+CD69− cells were transferred i.p. into NOD.scid recipient animals in combination with 5 × 105 diabetogenic BDC2.5 splenocytes. Mice were monitored as described in A. Data represent pooled results of three separate experiments with similar results. (C) Histological sections were prepared from pancreata of transferred NOD.scid animals. Hematoxylin and eosin–stained sections from recipients that did not develop diabetes (protected by Treg) by the end of 30 d after receiving the 10–20,000 cell dose of CD4+CD25+CD69− cells and 5 × 105 diabetogenic BDC2.5 splenocytes are shown.
Mentions: These data are suggestive of an active balance of regulatory and pathogenic CD4+ T cells in the pancreatic lesion. To evaluate function, we sorted CD25+CD69−, CD25loCD69+, and CD25−CD69− cells from BDC2.5/NOD pancreata were transferred into NOD.scid recipients. Mice that received CD25−CD69− cells or CD25loCD69+ cells developed diabetes shortly after transfer (Fig. 3 A). As few as 10–20,000 cells of these phenotypes were enough to provoke diabetes in 50–80% of recipients within 1 mo after transfer. All mice that received CD25−CD69− cells or CD25loCD69+ cells, at the doses tested, developed insulitis (unpublished data). In contrast, CD25+CD69− cells did not cause diabetes or develop insulitis after transfer. We asked whether the CD25+CD69− cells residing in the pancreas did, in fact, have protective capacity by transferring 10–20,000 CD25+CD69− cells isolated from the lesion with 5 × 105 diabetogenic BDC2.5/NOD splenocytes into NOD.scid recipients. This small dose of cells, 10–100-fold lower than the dose used in other models of regulation using polyclonal populations (20, 42), provided significant protection from diabetes in this cotransfer context (Fig. 3 B). BDC2.5 LN Tregs (CD4+CD25+CD69−) were also effective at this dose (unpublished data). In vivo regulation was dose dependent because protection from diabetes was lost if only 5,000 Tregs were cotransferred (Fig. 3 B). In protected mice that had received 20,000 CD25+CD69− Tregs and 5 × 105 diabetogenic BDC2.5/NOD splenocytes, we observed formidable, but respectful, insulitis in the islet after 30 d (Fig. 3 C), similar to that seen in islets of unmanipulated BDC2.5/NOD mice (compare with Fig. 1 A). Interestingly, Tregs taken directly from the pancreatic lesion were unable to set up a presence there in the absence of other T cells in the NOD.SCID host, suggesting that they may require signals from Teffs to access the pancreas or persist there. These data demonstrate that Teff and Treg subsets coexist in the pancreatic lesion, and that T effectors capable of disease transfer are being actively suppressed by CD25+CD69− Tregs during the nondestructive period of insulitis.

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