Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: Characterization of human CD4+ T helper cell-derived extracellular vesicles. A, FlowJo proliferation tool was used to calculate the percentage of divided cells (left panel) and the proliferation index (right panel; n = 6) of CD4+ T cell subsets (as indicated) analyzed for Cell Trace Violet fluorescence by flow cytometry upon 72 h of aCD3-aCD28 stimulation. B, a representative NTA-size distribution of CD4+ T cell-derived EVs. C, whisker plots showing EV size (mode, n = 6) determined by NTA for the indicated subset derived EVs. D, representative image by transmission electron microscopy of CD4+ T cell-derived EVs. E, Western blot from protein lysates from either CD4+ T cells or derived EVs for the indicated proteins. F, FACS analysis of purified EVs released by either unstimulated or stimulated CD4+ T cells previously stained with SYTO RNASelect green fluorescent. Isolated EVs were subsequently stained with Pacific Blue Cell Trace for assessing membrane integrity. G, bioanalyzer qualitative analysis of CD4+ T cell intracellular (left panel) and EV-associated (right panel) RNA. One representative sample is reported. H, correlation of Log10 relative (to miRNA global mean) Quantity values (evaluated by RT-qPCR) for 122 miRNAs (each circle is a single miRNA) coexpressed (Ct < 35) in extracellular vesicles isolated from an expanded Treg cell line conditioned medium by ultracentrifugation (final speed, 100000 × g) versus ExoSpin protocol (mean of 4 samples/group). The p value and Pearson r value are reported.

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: Derivative Assay, Fluorescence, Flow Cytometry, Whisker Assay, Transmission Assay, Electron Microscopy, Western Blot, Purification, Staining, Isolation, Membrane, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: miRNAs of CD4 + T cell-derived EVs Shown is a list of miRNAs ( n = 26) found invariably detectable (Ct <35) in all samples (commonly in discovery and validation phase) of CD4 + T cell-derived EVs independently of the subset evaluated/vesicle isolation procedure.

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: EV-associated miRNAs contribute to classify Treg and Th1/Th17 cell subsets. A, bi-dimensional score plot resulting by the PCA performed on the miRNA profiling dataset from the entire in vitro CD4+ T cell subset experiment. Each dot represents a sample, and each color represents one experimental group as reported. For each T cell subset (Th1, Th17, and Treg) three conditions are reported: intracellular miRNA profile at time 0 and 72 h after anti-CD3/anti-CD28 stimulation; and vesicle miRNA profile at 72 h (EVs). The percentage of variation explained by each principal component is reported close to the axes. The 95% confidence ellipse is drawn for each experimental group. B, the average correlation between miRNA NRQs values of each in vitro CD4+ T cell subset experimental group is calculated by Pearson Correlation algorithm, and it is shown in the matrix of correlation, where the coefficient of correlation r is reported both numerically and through color code. In the red boxes, the correlation between Treg cells and the other subsets for ex vivo isolated resting cells (upper left), in vitro stimulated cells (middle), and EVs (lower right).

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: In Vitro, Ex Vivo, Isolation

Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: CD4+ T cell subset-specific EV-associated miRNA signature does not mirror differences among subsets at the intracellular level. A, histogram graphs (means with S.E.) showing the fold change (evaluated by RT-qPCR and normalized relative to global mean) of the indicated extracellular miRNAs when comparing Treg with either Th1 (left panel) or Th17 (right panel) at the intracellular level (in ex vivo purified resting cells, white) compared with EV-associated level (gray). B, column bars (mean with S.E.) showing the relative quantities (evaluated by RT-qPCR and normalized relative to global mean) of the indicated extracellular miRNAs in smaller (<200 nanometers, left panel) versus larger (>200 nanometers, right panel) vesicles. The results were obtained by nine biological replicates.

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: Quantitative RT-PCR, Ex Vivo, Purification

Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: Extracellular T cell-miRNA signature RT-qPCR plate to profile psoriasis patient sera. A, scheme of miRNA representation in the RT-qPCR custom plate used to profile serum circulating miRNome of psoriasis patients. B, unsupervised hierarchical clustering of log-transformed normalized relative quantities of either all detectable serum circulating miRNAs (upper panel) or the 26 miRNAs belonging to the in vitro CD4+ T cell-derived miRNA signature (lower panel). The distance was determined by Pearson correlation with average linkage. C, Ct values variability among 8 samples (four psoriasis patients and four healthy donors) for the exogenous spike-ins added before extraction (in blue: UniSp4 and UniSp5) and before retro-transcription (in green: UniSp6), miRNA global mean (red), and the endogenous normalization factor (in orange: miR-18a-5p).

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: Quantitative RT-PCR, Transformation Assay, In Vitro, Derivative Assay, Extraction

Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: Significant modulation of miRNAs of the in vitro identified CD4+ T cell subset extracellular signature in serum of psoriasis patients. For each of the six validated serum miRNAs, two panels are reported. Left panels, whisker plots showing the fold change in 39 psoriasis patients (gray) versus 38 healthy donors (white) with p values referring to discovery and validation phase data together. Right panels, ROC curves (showing an area under the curve ≥ 0.75) with the associated p values.

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: In Vitro, Whisker Assay

Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: Gene Set Enrichment Analysis. A, schematic model of CD4+ T cell in vitro stimulation. B, Venn diagram showing the overlap between the genes expressed in all samples of CD4+ T naïve and in vitro stimulated cells (n = 5512), the genes that are validated targets of Th1/Th17-derived miRNA signature (n = 142) and Treg-derived miRNA signature (n = 172) (see supplemental Table S1 for gene lists). C, the gene sets obtained by the list of targets of Treg- (left panels) and Th1/Th17-derived (right panels) EV miRNA signature were imposed on the CD4+ T cell in vitro activation gene expression data by GSEA. Genes whose expression levels are modulated the most by CD4+ T cell stimulation get the highest metric scores with positive or negative sign and are located at the left or right edge of the list. At the top, the enrichment score shows a significant enrichment of genes that get up-regulated by CD4+ T cell stimulation in the list of targets of Treg- (but not Th1/Th17)-derived EV miRNA signature.

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: In Vitro, Derivative Assay, Activation Assay, Expressing, Cell Stimulation

Journal: The Journal of Biological Chemistry

Article Title: Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity *

doi: 10.1074/jbc.M116.769893

Figure Lengend Snippet: Biological effect of Treg-derived EVs. A, FACS analysis showing the uptake of Treg-derived EVs (transfected with positive control siRNA TexRed) by HuH-7 cells upon 20 h of incubation. B, histogram graph (means with S.E.) showing the fold change (evaluated by RT-qPCR and normalized relative to RNA 18S) of c-Myb when comparing Mock and mimic mir-150-5p transfected HuH-7 cells (left panel, n = 4) or PBS and EVs-treated cells (right panel, n = 6). *, p value < 0.05; ***, p value < 0.001. C, Cell Trace Violet fluorescence CD4+ T cells analyzed by flow cytometry upon 96 h of stimulation with CD3/CD28 Dynabeads in presence of either Th17 or Treg-derived EVs. For each group one representative sample is reported. D, FlowJo proliferation tool was used to calculate the percentage of dividing cells, the division index cells and the proliferation index of Cell Trace Violet Fluorescent CD4+ T cells upon 96 h of stimulation with CD3/CD28 Dynabeads in the presence of either Th17- or Treg-derived EVs (n = 4). PBS only was used as negative controls. E, histogram graph (means with S.E.) showing the fold change of miR-146a-5p and miR-146a validated targets STAT1 and IRAK2 in CD4+ T cells stimulated for 96h with CD3/CD28 Dynabeads in the presence of either Th17- (set to 1) or Treg-derived EVs (n = 4). All parameters were evaluated by RT-qPCR and normalized relative to EV commonly expressed miR-15a-5p (miR-146a-5p) and RNA 18S (all mRNAs). *, p value ≤ 0.05.

Article Snippet: Human CD4 + T cells have been isolated from peripheral blood mononuclear cells using CD4 MultiSort kit (Miltenyi Biotec), and then specific CD4 + T cell subsets (Th1, Th17, and Treg) were purified by sorting on a FACSAria (BD) by various combinations of surface markers: Th1 (CD127 + , CXCR3 + , CCR6 − ), Th17 (CD127 + , CCR6 + , CXCR3 − ), and Treg (CD127 dim , CD25 + ).

Techniques: Derivative Assay, Transfection, Positive Control, Incubation, Quantitative RT-PCR, Fluorescence, Flow Cytometry