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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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Helios+ Treg proliferate within tumors.Lymphocytes were isolated from the spleens, irrelevant lymph nodes, tumor draining lymph nodes, and tumor masses of mice bearing 4T1 tumors 10 days post implantation. Data shown are representative of 2 independent experiments, n = 5/group. A) Intracellular staining: lymphocytes were obtained from the indicated tissues and stained for CD4, followed by intracellular staining for Helios, FoxP3, and BrdU. B) Quantification of Helios+ versus Helios− Treg. Absolute number of Helios+ Treg divided by absolute number of Helios− Treg in given tissues, i.e. numerical ration of Helios+ versus Helios− Treg. Data plotted are +/− SEM. C) Brdu incorporation into Helios+ (solid line) versus Helios− (shaded histogram) Treg. Percentages denote the percentage of Treg from each population that are Brdu+. D) Relative proliferation of Helios+ Treg versus Helios− Treg. MFI of BrdU staining in Helios+ was divided by MFI of BrdU staining in Helios− Treg in given tissues. Data plotted are +/− SEM.
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pone-0034547-g004: Helios+ Treg proliferate within tumors.Lymphocytes were isolated from the spleens, irrelevant lymph nodes, tumor draining lymph nodes, and tumor masses of mice bearing 4T1 tumors 10 days post implantation. Data shown are representative of 2 independent experiments, n = 5/group. A) Intracellular staining: lymphocytes were obtained from the indicated tissues and stained for CD4, followed by intracellular staining for Helios, FoxP3, and BrdU. B) Quantification of Helios+ versus Helios− Treg. Absolute number of Helios+ Treg divided by absolute number of Helios− Treg in given tissues, i.e. numerical ration of Helios+ versus Helios− Treg. Data plotted are +/− SEM. C) Brdu incorporation into Helios+ (solid line) versus Helios− (shaded histogram) Treg. Percentages denote the percentage of Treg from each population that are Brdu+. D) Relative proliferation of Helios+ Treg versus Helios− Treg. MFI of BrdU staining in Helios+ was divided by MFI of BrdU staining in Helios− Treg in given tissues. Data plotted are +/− SEM.

Mentions: Given the well-documented role of Treg in attenuating an anti-tumor immune response [5], [7], we next examined the number and relative proliferation of Helios+ versus Helios− Treg in tumor-bearing mice. To perform these studies, wildtype BALB/c mice were inoculated with 4T1 mammary tumors, and harvested 10 days after implantation. Interestingly, CD4+ FoxP3+ Helios+ Treg appeared to be relatively enriched in the tumor parenchyma as compared to corresponding spleens (Figure 4A). Quantitative analyses verified these observations by supporting the concept that Helios+ Treg are significantly more prevalent in the tumor parenchyma than are Helios− Treg (Figure 4B). In non-tumor bearing mice, the ratio of Helios+ to Helios− Treg in the spleen and axillary lymph nodes was approximately the same as in tumor-bearing mice (data not shown). More significantly, the Helios+ Treg in the tumor showed a greater extent of BrdU incorporation than Helios− Treg in the same site (Figure 4C). This observation was not limited to the spleen; in all tissues examined Helios+ Treg showed greater BrdU incorporation than their Helios− counterparts (Figure 4D). Interestingly, we further found that in Treg from tumors CD103 no longer distinguished between Helios− and Helios+ Treg (data not shown), a finding consistent with recently published data [22], but which compromised our ability to perform functional analyses of Helios+ versus Helios− Treg derived from the tumor-infiltrating population. In total, these data show that the predominant FoxP3+ population found within tumor parenchyma expresses Helios and proliferates more robustly in comparison to their Helios− counterparts.


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)

Helios+ Treg proliferate within tumors.Lymphocytes were isolated from the spleens, irrelevant lymph nodes, tumor draining lymph nodes, and tumor masses of mice bearing 4T1 tumors 10 days post implantation. Data shown are representative of 2 independent experiments, n = 5/group. A) Intracellular staining: lymphocytes were obtained from the indicated tissues and stained for CD4, followed by intracellular staining for Helios, FoxP3, and BrdU. B) Quantification of Helios+ versus Helios− Treg. Absolute number of Helios+ Treg divided by absolute number of Helios− Treg in given tissues, i.e. numerical ration of Helios+ versus Helios− Treg. Data plotted are +/− SEM. C) Brdu incorporation into Helios+ (solid line) versus Helios− (shaded histogram) Treg. Percentages denote the percentage of Treg from each population that are Brdu+. D) Relative proliferation of Helios+ Treg versus Helios− Treg. MFI of BrdU staining in Helios+ was divided by MFI of BrdU staining in Helios− Treg in given tissues. Data plotted are +/− SEM.
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Related In: Results  -  Collection

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pone-0034547-g004: Helios+ Treg proliferate within tumors.Lymphocytes were isolated from the spleens, irrelevant lymph nodes, tumor draining lymph nodes, and tumor masses of mice bearing 4T1 tumors 10 days post implantation. Data shown are representative of 2 independent experiments, n = 5/group. A) Intracellular staining: lymphocytes were obtained from the indicated tissues and stained for CD4, followed by intracellular staining for Helios, FoxP3, and BrdU. B) Quantification of Helios+ versus Helios− Treg. Absolute number of Helios+ Treg divided by absolute number of Helios− Treg in given tissues, i.e. numerical ration of Helios+ versus Helios− Treg. Data plotted are +/− SEM. C) Brdu incorporation into Helios+ (solid line) versus Helios− (shaded histogram) Treg. Percentages denote the percentage of Treg from each population that are Brdu+. D) Relative proliferation of Helios+ Treg versus Helios− Treg. MFI of BrdU staining in Helios+ was divided by MFI of BrdU staining in Helios− Treg in given tissues. Data plotted are +/− SEM.
Mentions: Given the well-documented role of Treg in attenuating an anti-tumor immune response [5], [7], we next examined the number and relative proliferation of Helios+ versus Helios− Treg in tumor-bearing mice. To perform these studies, wildtype BALB/c mice were inoculated with 4T1 mammary tumors, and harvested 10 days after implantation. Interestingly, CD4+ FoxP3+ Helios+ Treg appeared to be relatively enriched in the tumor parenchyma as compared to corresponding spleens (Figure 4A). Quantitative analyses verified these observations by supporting the concept that Helios+ Treg are significantly more prevalent in the tumor parenchyma than are Helios− Treg (Figure 4B). In non-tumor bearing mice, the ratio of Helios+ to Helios− Treg in the spleen and axillary lymph nodes was approximately the same as in tumor-bearing mice (data not shown). More significantly, the Helios+ Treg in the tumor showed a greater extent of BrdU incorporation than Helios− Treg in the same site (Figure 4C). This observation was not limited to the spleen; in all tissues examined Helios+ Treg showed greater BrdU incorporation than their Helios− counterparts (Figure 4D). Interestingly, we further found that in Treg from tumors CD103 no longer distinguished between Helios− and Helios+ Treg (data not shown), a finding consistent with recently published data [22], but which compromised our ability to perform functional analyses of Helios+ versus Helios− Treg derived from the tumor-infiltrating population. In total, these data show that the predominant FoxP3+ population found within tumor parenchyma expresses Helios and proliferates more robustly in comparison to their Helios− counterparts.

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