Enrichment of Helios⁺ Treg by sorting on CD103 and GITR.

<p>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. <b>A</b>) 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>B</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.</p

Helios⁺ and Helios⁻ FoxP3⁺ Treg cells differ in their cell surface protein expression of CD103 and GITR.

<p><b>A</b>) Gating strategy: Unstimulated, CD4⁺ FoxP3⁺ cells were obtained from BALB/cJ splenocytes, stained and gated on the Helios⁺ and Helios⁻ populations. <b>B</b>) Direct <i>ex vivo</i> Treg: Top row: Expression of indicated cell surface molecules on CD4⁺ FoxP3⁺ Helios⁺ Treg (blue) overlaid with expression on naïve CD4 T cells (green). Bottom row: Expression on CD4⁺ FoxP3⁺ Helios⁻ Treg (red), overlaid with expression on naïve CD4⁺ cells. Percentages indicate the percentage of Treg that are positive for expression of the surface marker compared to naïve control cells. <b>C</b>) Mean Fluorescence Intensity (MFI) values for each cell surface protein in the Helios⁺ (blue) and Helios⁻ (red) Treg populations. For comparison purposes, values were normalized to the MFI of naïve, control CD4⁺ T cells (green); i.e. relative MFI values are shown. Values are +/− SEM, * (p<0.05), ** (p<0.01) comparing MFI of Helios⁺ versus Helios⁻ Treg. Data shown are representative of 3 independent experiments, n = 3/group. <b>D</b>) <i>In vitro</i> induced Treg: Top row: Expression of indicated cell surface molecules on <i>in vitro</i> induced CD4⁺ FoxP3⁺ Helios⁺ Treg (blue) overlaid with expression on naïve CD4 T cells (green). Bottom row: Expression on <i>in vitro</i> induced CD4⁺ FoxP3⁺ Helios⁻ Treg (red), overlaid with expression on naïve CD4⁺ cells. Percentages given indicate the percentage of Treg that are positive for expression of the surface marker compared to control cells. <b>E</b>) Mean Fluorescence Intensity (MFI) values for each cell surface protein in the <i>in vitro</i> induced Helios⁺ (blue) and Helios⁻ (red) Treg populations. For comparison purposes, values were normalized to the MFI of naïve, control CD4⁺ T cells (green); i.e. relative MFI values are shown. Values are +/− SEM, * (p<0.05), ** (p<0.01), *** (p<0.001) comparing MFI of Helios⁺ versus Helios⁻<i>in vitro</i> induced Treg. Data shown are representative of 3 independent experiments, n = 3/group.</p

Helios up-regulation in induced Treg is determined by TCR signal.

<p><b>A</b>) Wildtype mice: Naïve CD4⁺ T cells were purfied by sorting, and stimulated <i>in vitro</i> in the presence of TGF-β and αCD3/αCD28 microbeads. 48 hours post stimulation, Helios and FoxP3 expression was assayed by intracellular staining. Data shown are representative of 2 independent experiments, n = 5. <b>B</b>) FoxP3-GFP mice: Naïve, GFP⁻ cells were obtained by sorting and stimulated <i>in vitro</i> with either monoclonal antibodies or αCD3/αCD28 beads. As above, Helios and FoxP3 expression was assayed after 48 hours. Data shown are representative of 2 independent experiments, n = 5.</p

Increased suppressive function of Helios-enriched Treg populations.

<p>Standard <i>in vitro</i> suppression assays were performed using either bulk CD4⁺ CD25⁺ Treg, CD4⁺CD25⁺GITR⁺CD103⁺ Treg, CD4⁺CD25⁺GITR^low CD103⁻ Treg or CD4⁺CD25⁺CD103⁻GITR⁺ Treg. Data shown are representative of 2 independent experiments, n = 4. <b>A</b>) Relative proliferation of effectors by H³ incorporation. <b>B</b>) Analysis of Treg function at 1∶10 suppressor∶effector ratio. <b>C</b>) Relative proliferation of effectors by H³ incorporation. <b>D</b>) Analysis of Treg fuction at 1∶10 suppressor∶effector ratio. <b>E</b>) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3⁺ Treg admixed. <b>F</b>) Percent suppression (as compared to no suppressor control) versus the absolute number of FoxP3⁺Helios⁺ Treg admixed.</p

Helios+ Treg proliferate within tumors.

<p>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. <b>A</b>) Intracellular staining: lymphocytes were obtained from the indicated tissues and stained for CD4, followed by intracellular staining for Helios, FoxP3, and BrdU. <b>B</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. <b>C</b>) Brdu incorporation into Helios⁺ (solid line) versus Helios⁻ (shaded histogram) Treg. Percentages denote the percentage of Treg from each population that are Brdu⁺. <b>D</b>) 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.</p

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems

Regulation of Lipid Metabolism by Dicer Revealed through SILAC Mice

Dicer is a ribonuclease whose major role is to generate mature microRNAs, although additional functions have been proposed. Deletion of Dicer leads to embryonic lethality in mice. To study the role of Dicer in adults, we generated mice in which administration of tamoxifen induces deletion of Dicer. Surprisingly, disruption of Dicer in adult mice induced lipid accumulation in the small intestine. To dissect the underlying mechanisms, we carried out miRNA, mRNA, and proteomic profiling of the small intestine. The proteomic analysis was done using mice metabolically labeled with heavy lysine (SILAC mice) for an in vivo readout. We identified 646 proteins, of which 80 were up-regulated >2-fold and 75 were down-regulated. Consistent with the accumulation of lipids, Dicer disruption caused a marked decrease of microsomal triglyceride transfer protein, long-chain fatty acyl-CoA ligase 5, fatty acid binding protein, and very-long-chain fatty acyl-CoA dehydrogenase, among others. We validated these results using multiple reaction monitoring (MRM) experiments by targeting proteotypic peptides. Our data reveal a previously unappreciated role of Dicer in lipid metabolism. These studies demonstrate that a systems biology approach by integrating mouse models, metabolic labeling, gene expression profiling, and quantitative proteomics can be a powerful tool for understanding complex biological systems