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Phenotypic and functional properties of Helios+ regulatory T cells.

Zabransky DJ, Nirschl CJ, Durham NM, Park BV, Ceccato CM, Bruno TC, Tam AJ, Getnet D, Drake CG - PLoS ONE (2012)

Bottom Line: In tumor-bearing mice, we found that Helios(+) Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios(+) Treg proliferated more than Helios(-) Treg.We hypothesized that Helios-enriched Treg might exert increased suppressive effects.Taken together, these data show that Helios(+) Treg represent a functional subset with associated CD103 and GITR expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
Helios, an Ikaros family transcription factor, is preferentially expressed at the mRNA and protein level in regulatory T cells. Helios expression previously appeared to be restricted to thymic-derived Treg. Consistent with recent data, we show here that Helios expression is inducible in vitro under certain conditions. To understand phenotypic and functional differences between Helios(+) and Helios(-) Treg, we profiled cell-surface markers of FoxP3(+) Treg using unmanipulated splenocytes. We found that CD103 and GITR are expressed at high levels on a subset of Helios(+) Treg and that a Helios(+) Treg population could be significantly enriched by FACS sorting using these two markers. Quantitative real-time PCR (qPCR) analysis revealed increased TGF-β message in Helios(+) Treg, consistent with the possibility that this population possesses enhanced regulatory potential. In tumor-bearing mice, we found that Helios(+) Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios(+) Treg proliferated more than Helios(-) Treg. We hypothesized that Helios-enriched Treg might exert increased suppressive effects. Using in vitro suppression assays, we show that Treg function correlates with the absolute number of Helios(+) cells in culture. Taken together, these data show that Helios(+) Treg represent a functional subset with associated CD103 and GITR expression.

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Increased suppressive function of Helios-enriched Treg populations.Standard in vitro suppression assays were performed using either bulk CD4+ CD25+ Treg, CD4+CD25+GITR+CD103+ Treg, CD4+CD25+GITRlow CD103− Treg or CD4+CD25+CD103−GITR+ Treg. Data shown are representative of 2 independent experiments, n = 4. A) Relative proliferation of effectors by H3 incorporation. B) Analysis of Treg function at 1∶10 suppressor∶effector ratio. C) Relative proliferation of effectors by H3 incorporation. D) Analysis of Treg fuction at 1∶10 suppressor∶effector ratio. E) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+ Treg admixed. F) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+Helios+ Treg admixed.
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pone-0034547-g005: Increased suppressive function of Helios-enriched Treg populations.Standard in vitro suppression assays were performed using either bulk CD4+ CD25+ Treg, CD4+CD25+GITR+CD103+ Treg, CD4+CD25+GITRlow CD103− Treg or CD4+CD25+CD103−GITR+ Treg. Data shown are representative of 2 independent experiments, n = 4. A) Relative proliferation of effectors by H3 incorporation. B) Analysis of Treg function at 1∶10 suppressor∶effector ratio. C) Relative proliferation of effectors by H3 incorporation. D) Analysis of Treg fuction at 1∶10 suppressor∶effector ratio. E) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+ Treg admixed. F) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+Helios+ Treg admixed.

Mentions: Based on the finding that TGF-β is relatively over-expressed at the message level in Helios enriched (CD103+ GITR+) Treg, we hypothesized that these Helios-enriched Treg might demonstrate a greater level of in vitro suppressive capacity than a non-enriched population. To test this hypothesis, we first directly compared the suppressive capacity of Helios enriched (GITR+, CD103+) Treg to CD4+ CD25+ bulk Treg.(Figure 5A–B). As expected, at most of the suppressor to effector ratios examined, the CD4+CD25+ bulk Treg population showed a titratable suppression phenotype. However, the Helios/FoxP3 enriched CD4+CD25+CD103+GITR+ Treg population demonstrated significantly increased suppressive capabilities at most ratios examined. Furthermore, the CD4+CD25+CD103+GITR+ Tregs still showed moderate suppressive function even at a 1∶25 ratio (Figure 5A). In order to further examine the differences in the suppressive capabilities of these subpopulations, we assayed the three populations shown in Figure 3A, sorting CD4+CD25+ bulk Treg by GITR and CD103. As show in Figure 5C–D, the CD4+CD25+CD103+GITR+ Treg displayed significantly increased suppressive ability over the CD4+CD25+CD103−GITRlow population at all ratios assayed. Additionally, the CD4+CD25+CD103+GITR+ Tregs showed a small increase in suppressive ability over the CD4+CD25+CD103−GITR+ Tregs. These differences in the suppressive capacity of the sorted Treg populations could be due to increases in the number of cells in each population expressing FoxP3, Helios, or both. To examine this question, we compared the percent suppression observed to the number of FoxP3+ singly positive (Figure 5E) or FoxP3+Helios+ doubly positive (Figure 5F) Tregs in culture (based on post sort staining percentages). We were surprised to find very little correlation (R2 = 0.40) between the number of FoxP3+ Tregs in culture and percentage suppression (Figure 5E). However, percentage suppression correlated strongly with the number of FoxP3+, Helios+ cells (R2 = 0.89, Figure 5F). Taken together, these data show that several suppressor populations exist within the bulk CD4+CD25+ Treg population, and that enriching for FoxP3+Helios+ Tregs results in an increased in vitro suppressive capability.


Phenotypic and functional properties of Helios+ regulatory T cells.

Zabransky DJ, Nirschl CJ, Durham NM, Park BV, Ceccato CM, Bruno TC, Tam AJ, Getnet D, Drake CG - PLoS ONE (2012)

Increased suppressive function of Helios-enriched Treg populations.Standard in vitro suppression assays were performed using either bulk CD4+ CD25+ Treg, CD4+CD25+GITR+CD103+ Treg, CD4+CD25+GITRlow CD103− Treg or CD4+CD25+CD103−GITR+ Treg. Data shown are representative of 2 independent experiments, n = 4. A) Relative proliferation of effectors by H3 incorporation. B) Analysis of Treg function at 1∶10 suppressor∶effector ratio. C) Relative proliferation of effectors by H3 incorporation. D) Analysis of Treg fuction at 1∶10 suppressor∶effector ratio. E) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+ Treg admixed. F) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+Helios+ Treg admixed.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316700&req=5

pone-0034547-g005: Increased suppressive function of Helios-enriched Treg populations.Standard in vitro suppression assays were performed using either bulk CD4+ CD25+ Treg, CD4+CD25+GITR+CD103+ Treg, CD4+CD25+GITRlow CD103− Treg or CD4+CD25+CD103−GITR+ Treg. Data shown are representative of 2 independent experiments, n = 4. A) Relative proliferation of effectors by H3 incorporation. B) Analysis of Treg function at 1∶10 suppressor∶effector ratio. C) Relative proliferation of effectors by H3 incorporation. D) Analysis of Treg fuction at 1∶10 suppressor∶effector ratio. E) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+ Treg admixed. F) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3+Helios+ Treg admixed.
Mentions: Based on the finding that TGF-β is relatively over-expressed at the message level in Helios enriched (CD103+ GITR+) Treg, we hypothesized that these Helios-enriched Treg might demonstrate a greater level of in vitro suppressive capacity than a non-enriched population. To test this hypothesis, we first directly compared the suppressive capacity of Helios enriched (GITR+, CD103+) Treg to CD4+ CD25+ bulk Treg.(Figure 5A–B). As expected, at most of the suppressor to effector ratios examined, the CD4+CD25+ bulk Treg population showed a titratable suppression phenotype. However, the Helios/FoxP3 enriched CD4+CD25+CD103+GITR+ Treg population demonstrated significantly increased suppressive capabilities at most ratios examined. Furthermore, the CD4+CD25+CD103+GITR+ Tregs still showed moderate suppressive function even at a 1∶25 ratio (Figure 5A). In order to further examine the differences in the suppressive capabilities of these subpopulations, we assayed the three populations shown in Figure 3A, sorting CD4+CD25+ bulk Treg by GITR and CD103. As show in Figure 5C–D, the CD4+CD25+CD103+GITR+ Treg displayed significantly increased suppressive ability over the CD4+CD25+CD103−GITRlow population at all ratios assayed. Additionally, the CD4+CD25+CD103+GITR+ Tregs showed a small increase in suppressive ability over the CD4+CD25+CD103−GITR+ Tregs. These differences in the suppressive capacity of the sorted Treg populations could be due to increases in the number of cells in each population expressing FoxP3, Helios, or both. To examine this question, we compared the percent suppression observed to the number of FoxP3+ singly positive (Figure 5E) or FoxP3+Helios+ doubly positive (Figure 5F) Tregs in culture (based on post sort staining percentages). We were surprised to find very little correlation (R2 = 0.40) between the number of FoxP3+ Tregs in culture and percentage suppression (Figure 5E). However, percentage suppression correlated strongly with the number of FoxP3+, Helios+ cells (R2 = 0.89, Figure 5F). Taken together, these data show that several suppressor populations exist within the bulk CD4+CD25+ Treg population, and that enriching for FoxP3+Helios+ Tregs results in an increased in vitro suppressive capability.

Bottom Line: In tumor-bearing mice, we found that Helios(+) Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios(+) Treg proliferated more than Helios(-) Treg.We hypothesized that Helios-enriched Treg might exert increased suppressive effects.Taken together, these data show that Helios(+) Treg represent a functional subset with associated CD103 and GITR expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Johns Hopkins University, Baltimore, Maryland, United States of America.

ABSTRACT
Helios, an Ikaros family transcription factor, is preferentially expressed at the mRNA and protein level in regulatory T cells. Helios expression previously appeared to be restricted to thymic-derived Treg. Consistent with recent data, we show here that Helios expression is inducible in vitro under certain conditions. To understand phenotypic and functional differences between Helios(+) and Helios(-) Treg, we profiled cell-surface markers of FoxP3(+) Treg using unmanipulated splenocytes. We found that CD103 and GITR are expressed at high levels on a subset of Helios(+) Treg and that a Helios(+) Treg population could be significantly enriched by FACS sorting using these two markers. Quantitative real-time PCR (qPCR) analysis revealed increased TGF-β message in Helios(+) Treg, consistent with the possibility that this population possesses enhanced regulatory potential. In tumor-bearing mice, we found that Helios(+) Treg were relatively over-represented in the tumor-mass, and BrdU studies showed that, in vivo, Helios(+) Treg proliferated more than Helios(-) Treg. We hypothesized that Helios-enriched Treg might exert increased suppressive effects. Using in vitro suppression assays, we show that Treg function correlates with the absolute number of Helios(+) cells in culture. Taken together, these data show that Helios(+) Treg represent a functional subset with associated CD103 and GITR expression.

Show MeSH
Related in: MedlinePlus