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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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Enrichment of Helios+ Treg by sorting on CD103 and GITR.CD4+ enriched cells were FACS sorted by gating on CD4+ CD25+ cells, then sorted into three populations based on relative CD103 and GITR staining levels. Data shown are representative of 2 independent experiments, n = 5. A) Pre and Post sort analysis: Expression of FoxP3 and Helios is shown in both bulk CD4+CD25+ cells and sorted cells after intracellular staining. B) qPCR analysis: mRNA was extracted from the FACS sorted populations and reverse transcribed into cDNA. Expression of each mRNA of interest was quantified as compared to an internal control18S rRNA. Data shown are relative expression as compared to a CD4+ CD25− reference sample. Mean values are plotted +/− SEM.
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pone-0034547-g003: Enrichment of Helios+ Treg by sorting on CD103 and GITR.CD4+ enriched cells were FACS sorted by gating on CD4+ CD25+ cells, then sorted into three populations based on relative CD103 and GITR staining levels. Data shown are representative of 2 independent experiments, n = 5. A) Pre and Post sort analysis: Expression of FoxP3 and Helios is shown in both bulk CD4+CD25+ cells and sorted cells after intracellular staining. B) qPCR analysis: mRNA was extracted from the FACS sorted populations and reverse transcribed into cDNA. Expression of each mRNA of interest was quantified as compared to an internal control18S rRNA. Data shown are relative expression as compared to a CD4+ CD25− reference sample. Mean values are plotted +/− SEM.

Mentions: We used this difference in the expression of GITR and CD103 to enrich for Helios+ Treg by FACS sorting using only extracellular markers. The sorting strategy, shown in Figure 3A, was to gate on CD4+ CD25+ and then sort these cells into distinct populations based on CD103 and GITR expression. By sorting CD4+ CD25+ cells into a CD103+ GITR+ population, we were able to enrich for Helios+ Treg cells by approximately 2.5 fold compared to sorting for the CD4+ CD25+ CD103− GITRlow population and by approximately 1.5 fold compared to the bulk CD4+CD25+ population. Interestingly, sorting CD4+ CD25+ cells on GITR alone provided a modest enrichment for Helios expression compared to sorting on CD4 and CD25, but not as much as by sorting on both GITR and CD103. We next explored differences in the expression of several Treg associated transcripts, at the mRNA level, in the populations obtained through sorting, comparing mRNA expression levels in the post-sort Treg populations using qPCR. For these studies, relative mRNA expression was compared to that in naïve CD4+ T cells (Figure 3B). The increased level of expression of Helios seen at the protein level in the CD103+ GITR+ population compared to the CD103− GITRlow population was also observed at the mRNA level: Helios mRNA expression was tenfold higher in Helios+ enriched CD103+ GITR+ Treg compared to CD103− GITRlow Treg. We found a decreased expression of FoxP3 mRNA in the CD103− GITRlow population, which supports the finding that the CD4+ CD25+ CD103− GITRlow population shows slightly decreased levels of FoxP3 protein expression by FACS analysis (Figure 3A). Interestingly, the Helios+ enriched CD103+ GITR+ population showed relatively increased expression of LAG-3, which has been suggested to be a marker of functional Treg [20], [21] at the mRNA level, but cell-surface protein levels of LAG-3 were not significantly different between Helios+ and Helios− Treg (data not shown). Increased levels of IL-2 might reflect that Helios+ Treg represent a set of actively dividing population of Treg. In addition, we found that the CD103+ GITR+ population had a tenfold increase in TGF-β mRNA expression compared to CD103− GITRlow Treg. This relative up-regulation of TGF-β message appeared to be associated with CD103, as opposed to GITR expression, as it was not noted in the CD103− GITR+ population. Taken together, these data led us to hypothesize that Helios+ Treg might exhibit more suppressive function in vitro than Helios− Treg.


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)

Enrichment of Helios+ Treg by sorting on CD103 and GITR.CD4+ enriched cells were FACS sorted by gating on CD4+ CD25+ cells, then sorted into three populations based on relative CD103 and GITR staining levels. Data shown are representative of 2 independent experiments, n = 5. A) Pre and Post sort analysis: Expression of FoxP3 and Helios is shown in both bulk CD4+CD25+ cells and sorted cells after intracellular staining. B) qPCR analysis: mRNA was extracted from the FACS sorted populations and reverse transcribed into cDNA. Expression of each mRNA of interest was quantified as compared to an internal control18S rRNA. Data shown are relative expression as compared to a CD4+ CD25− reference sample. Mean values are plotted +/− SEM.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3316700&req=5

pone-0034547-g003: Enrichment of Helios+ Treg by sorting on CD103 and GITR.CD4+ enriched cells were FACS sorted by gating on CD4+ CD25+ cells, then sorted into three populations based on relative CD103 and GITR staining levels. Data shown are representative of 2 independent experiments, n = 5. A) Pre and Post sort analysis: Expression of FoxP3 and Helios is shown in both bulk CD4+CD25+ cells and sorted cells after intracellular staining. B) qPCR analysis: mRNA was extracted from the FACS sorted populations and reverse transcribed into cDNA. Expression of each mRNA of interest was quantified as compared to an internal control18S rRNA. Data shown are relative expression as compared to a CD4+ CD25− reference sample. Mean values are plotted +/− SEM.
Mentions: We used this difference in the expression of GITR and CD103 to enrich for Helios+ Treg by FACS sorting using only extracellular markers. The sorting strategy, shown in Figure 3A, was to gate on CD4+ CD25+ and then sort these cells into distinct populations based on CD103 and GITR expression. By sorting CD4+ CD25+ cells into a CD103+ GITR+ population, we were able to enrich for Helios+ Treg cells by approximately 2.5 fold compared to sorting for the CD4+ CD25+ CD103− GITRlow population and by approximately 1.5 fold compared to the bulk CD4+CD25+ population. Interestingly, sorting CD4+ CD25+ cells on GITR alone provided a modest enrichment for Helios expression compared to sorting on CD4 and CD25, but not as much as by sorting on both GITR and CD103. We next explored differences in the expression of several Treg associated transcripts, at the mRNA level, in the populations obtained through sorting, comparing mRNA expression levels in the post-sort Treg populations using qPCR. For these studies, relative mRNA expression was compared to that in naïve CD4+ T cells (Figure 3B). The increased level of expression of Helios seen at the protein level in the CD103+ GITR+ population compared to the CD103− GITRlow population was also observed at the mRNA level: Helios mRNA expression was tenfold higher in Helios+ enriched CD103+ GITR+ Treg compared to CD103− GITRlow Treg. We found a decreased expression of FoxP3 mRNA in the CD103− GITRlow population, which supports the finding that the CD4+ CD25+ CD103− GITRlow population shows slightly decreased levels of FoxP3 protein expression by FACS analysis (Figure 3A). Interestingly, the Helios+ enriched CD103+ GITR+ population showed relatively increased expression of LAG-3, which has been suggested to be a marker of functional Treg [20], [21] at the mRNA level, but cell-surface protein levels of LAG-3 were not significantly different between Helios+ and Helios− Treg (data not shown). Increased levels of IL-2 might reflect that Helios+ Treg represent a set of actively dividing population of Treg. In addition, we found that the CD103+ GITR+ population had a tenfold increase in TGF-β mRNA expression compared to CD103− GITRlow Treg. This relative up-regulation of TGF-β message appeared to be associated with CD103, as opposed to GITR expression, as it was not noted in the CD103− GITR+ population. Taken together, these data led us to hypothesize that Helios+ Treg might exhibit more suppressive function in vitro than Helios− Treg.

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