Journal:

Article Title: Natural Alpha Interferon-Producing Cells Respond to Human Immunodeficiency Virus Type 1 with Alpha Interferon Production and Maturation into Dendritic Cells

doi: 10.1128/JVI.77.6.3777-3784.2003

Figure Lengend Snippet: Purification of blood DCs by cell sorting. Total PBMCs were depleted of lymphocytes and monocytes with a mixture of anti-CD3, anti-CD14, anti-CD16, anti-CD19, and anti-CD56 MAbs and with magnetic beads. Subsequently, CD4+ (or HLA− DR+) CD11c+ lineage-negative cells and CD4+ (or HLA-DR+) CD11c− lineage-negative cells were isolated as CD11c+ DCs and IPCs, respectively, by cell sorting.

Article Snippet: In some experiments, HIV-1 was added together with 2 μg/ml of anti-CD4 MAb (Leu-3a [Becton Dickinson]; PRA-T4 [eBioscience, San Diego, Calif.]) or control IgG to examine the involvement of CD4 in HIV-1-induced IFN-α production.

Techniques: Purification, FACS, Magnetic Beads, Isolation

Journal:

Article Title: Natural Alpha Interferon-Producing Cells Respond to Human Immunodeficiency Virus Type 1 with Alpha Interferon Production and Maturation into Dendritic Cells

doi: 10.1128/JVI.77.6.3777-3784.2003

Figure Lengend Snippet: (A) IFN-α production by IPCs cultured with HIV-1 or HSV. IPCs were stimulated with HIV-1 or HSV for 48 h, and the concentrations of IFN-α in the supernatants were measured by ELISA. (B) Effect of anti-CD4 MAbs on HIV-1-induced IFN-α production by IPCs. (C) Effect of anti-IFN-α MAb on HIV-1 production.

Article Snippet: In some experiments, HIV-1 was added together with 2 μg/ml of anti-CD4 MAb (Leu-3a [Becton Dickinson]; PRA-T4 [eBioscience, San Diego, Calif.]) or control IgG to examine the involvement of CD4 in HIV-1-induced IFN-α production.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: The murine Microenvironment Cell Population counter method to estimate abundance of tissue-infiltrating immune and stromal cell populations in murine samples using gene expression

doi: 10.1101/2020.03.10.985176

Figure Lengend Snippet: a Correlation graphs between the flow cytometry estimates (logarithmic scale, expressed in percent of the total of living cells) and the mMCP-counter scores for populations for which the signature was accepted. Each graph corresponds to a different population. The dotted line shows the linear regression model. Correlations are estimated with the Pearson correlation. b Correlation graph for canonical CD4 + regulatory T cells, presented as in a . Following this validation step, this signature was discarded.

Article Snippet: For flow cytometry, cells were stained with the following monoclonal antibodies: PE-conjugated anti-CD4, Fitc-conjugated anti-CD8, BV786-conjugated anti-CD11c, PE-Cy7 conjugated anti-CD45, BV605 conjugated anti-NK-1.1 (all from BD Biosciences), eFluor 450-conjugated anti-CD11b, Alexa Fluor 700-conjugated anti-CD19, APC-eFluor 780-conjugated anti-CD19, PerCP-eFluor 710-conjugated anti-CD49b, PE-CF594-conjugated anti-Siglec-F, (all from eBioscience), Brilliant Violet 785-conjugated anti-CD11b, APC/Fire 750 conjugated anti-TCR-β, Pacific Blue conjugated anti-GL7, Fitc-conjugated anti-FcεRIa, Alexa Fluor 700-conjugated anti-F4/80, Brilliant Violet 605-conjugated anti-Ly-6C and Brilliant Violet 650-conjugated anti-Ly-6G (all from BioLegend).

Techniques: Flow Cytometry

Journal: Arthritis Research & Therapy

Article Title: Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

doi: 10.1186/s13075-020-02397-4

Figure Lengend Snippet: TIGIT+CD226+ CD4 T cell frequencies were highly correlated with DM disease activity. a Multi-parameter strategy for assessing the cell surface expression of TIGIT and CD226 on T cells. b Frequency of TIGIT+CD226+ CD4 T cell subset was positively associated with disease activity and closely related to ILD involvement. DM patients with ILD ( n = 16), DM patients without ILD ( n = 14), and healthy controls ( n = 26). Spearman’s correlation analysis was used to test for correlation; two groups were compared using unpaired two-tailed t test. Data are shown as the mean ± SD. c Representative FACS plots showing the percentages of TIGIT+CD226+ T cell/CD4+ T cells in DM patient with ILD and DM patient without ILD. d Representative FACS plots and a scatter plot showing decreased percentages of TIGIT+CD226+ CD4 T cells following treatment with medium dose glucocorticoids and disease-modifying anti-rheumatic drugs ( n = 5). Comparison between two groups was carried out using paired two-tailed t test. Data are shown as the mean ± SD. e One-way ANOVA test was used to compare the means of TIGIT+CD226+ CD4 T cells levels between MSAs specific subtypes. p values < 0.05 were considered significant, * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following anti-human monoclonal antibodies were used to stain cells: allophycocyanin (APC)-H7-labeled monoclonal anti-CD3, peridin-chlorophyll protein (PerCP)-CY5.5-labeled monoclonal anti-CD4, phycoerythrin (PE)-CY7-labeled monoclonal anti-CD8, APC-labeled monoclonal anti-CD25, and fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD226, purchased from BD Bioscience (Franklin Lakes, NJ, USA), and PE-labeled monoclonal anti-TIGIT, purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Activity Assay, Expressing, Two Tailed Test

Journal: Arthritis Research & Therapy

Article Title: Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

doi: 10.1186/s13075-020-02397-4

Figure Lengend Snippet: Clinical and laboratory features of enrolled individuals

Article Snippet: The following anti-human monoclonal antibodies were used to stain cells: allophycocyanin (APC)-H7-labeled monoclonal anti-CD3, peridin-chlorophyll protein (PerCP)-CY5.5-labeled monoclonal anti-CD4, phycoerythrin (PE)-CY7-labeled monoclonal anti-CD8, APC-labeled monoclonal anti-CD25, and fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD226, purchased from BD Bioscience (Franklin Lakes, NJ, USA), and PE-labeled monoclonal anti-TIGIT, purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

doi: 10.1186/s13075-020-02397-4

Figure Lengend Snippet: TIGIT and CD226 expression on T cells of patients with DM and healthy controls

Article Snippet: The following anti-human monoclonal antibodies were used to stain cells: allophycocyanin (APC)-H7-labeled monoclonal anti-CD3, peridin-chlorophyll protein (PerCP)-CY5.5-labeled monoclonal anti-CD4, phycoerythrin (PE)-CY7-labeled monoclonal anti-CD8, APC-labeled monoclonal anti-CD25, and fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD226, purchased from BD Bioscience (Franklin Lakes, NJ, USA), and PE-labeled monoclonal anti-TIGIT, purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Expressing

Journal: Arthritis Research & Therapy

Article Title: Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

doi: 10.1186/s13075-020-02397-4

Figure Lengend Snippet: CD4 T cell subset function varied by TIGIT/CD226 phenotype. a Representative FACS plots showing the multi-parameter strategy for assessing the proliferation and cytokine production from CD4 T cells with different TIGIT/CD226 phenotypes. b , c Both in patients with DM ( b ) and HCs ( c ), the proliferation and cytokine production potential were the highest in the TIGIT–CD226+ subset, followed by in the TIGIT+CD226+, TIGIT+CD226−, and TIGIT−CD226− subsets. For the proliferation assay, 5 independent experiments evaluating a total of 9 patients with DM and 5 HCs were carried out; for intracellular cytokine staining, 5 independent experiments evaluating a total 10 patients with DM and 5 HCs were carried out. Data are shown as the mean ± SD, comparisons between DM and HCs were carried out using unpaired two-tailed t tests. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following anti-human monoclonal antibodies were used to stain cells: allophycocyanin (APC)-H7-labeled monoclonal anti-CD3, peridin-chlorophyll protein (PerCP)-CY5.5-labeled monoclonal anti-CD4, phycoerythrin (PE)-CY7-labeled monoclonal anti-CD8, APC-labeled monoclonal anti-CD25, and fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD226, purchased from BD Bioscience (Franklin Lakes, NJ, USA), and PE-labeled monoclonal anti-TIGIT, purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Proliferation Assay, Staining, Two Tailed Test

Journal: Arthritis Research & Therapy

Article Title: Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

doi: 10.1186/s13075-020-02397-4

Figure Lengend Snippet: Effector function of TIGIT+CD226+ CD4 T cells was altered in patients with DM. a The functional comparisons between DM and healthy controls showed that expression of HLA-DR, the percentage of proliferating cells, and TNF-α production potential of TIGIT+CD226+ subset in DM were significantly higher than that of healthy controls. b In DM patients with ILD, the expression of HLA-DR, the percentage of proliferating cells, and cytokine production potential of TIGIT+CD226+ CD4 T cells were increased compared to in DM patients without ILD. c Representative FACS plots of different groups. Data are shown as the mean ± SD. Comparisons between two groups were carried out using unpaired two-tailed t tests. Bar graphs showed summary of 5 independent experiments with total 5 DM with ILD, 5 DM without ILD, and 5 HCs. Samples from all groups were included in each run. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following anti-human monoclonal antibodies were used to stain cells: allophycocyanin (APC)-H7-labeled monoclonal anti-CD3, peridin-chlorophyll protein (PerCP)-CY5.5-labeled monoclonal anti-CD4, phycoerythrin (PE)-CY7-labeled monoclonal anti-CD8, APC-labeled monoclonal anti-CD25, and fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD226, purchased from BD Bioscience (Franklin Lakes, NJ, USA), and PE-labeled monoclonal anti-TIGIT, purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Functional Assay, Expressing, Two Tailed Test

Journal: Arthritis Research & Therapy

Article Title: Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

doi: 10.1186/s13075-020-02397-4

Figure Lengend Snippet: TIGIT expression in purified TIGIT-negative CD4 T cells after prolonged stimulation. Purified CD4+CD25−TIGIT− T cells were stimulated by anti-CD3/CD28 and were re-analyzed 72 h later. TIGIT-positive CD4 T cells were obviously generated from TIGIT-negative cells after 72-h stimulation. The graph was representative of 2 independent experiments with 3 DM and 1 HC

Article Snippet: The following anti-human monoclonal antibodies were used to stain cells: allophycocyanin (APC)-H7-labeled monoclonal anti-CD3, peridin-chlorophyll protein (PerCP)-CY5.5-labeled monoclonal anti-CD4, phycoerythrin (PE)-CY7-labeled monoclonal anti-CD8, APC-labeled monoclonal anti-CD25, and fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD226, purchased from BD Bioscience (Franklin Lakes, NJ, USA), and PE-labeled monoclonal anti-TIGIT, purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Expressing, Purification, Generated

Journal: Arthritis Research & Therapy

Article Title: Expansion of circulating peripheral TIGIT+CD226+ CD4 T cells with enhanced effector functions in dermatomyositis

doi: 10.1186/s13075-020-02397-4

Figure Lengend Snippet: Anti-CD226 antibodies downregulated TIGIT+CD226+ CD4 T cell function. Representative FACS plots showing IFN-γ ( a ) and TNF-α ( b ) expression in TIGIT+CD226+ CD4 T cells after stimulation with or without anti-CD226 treatment (left panel). Graphs showing remarkably reduced production of IFN-γ ( a ) and TNF-α ( b ) from TIGIT+CD226+ CD4 T cells following CD226 blockade in both patients with DM and HCs (right panel). Comparisons between two groups were carried out using paired two-tailed t tests. Five independent experiments evaluating a total 5 patients with DM and 5 HCs were carried out. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The following anti-human monoclonal antibodies were used to stain cells: allophycocyanin (APC)-H7-labeled monoclonal anti-CD3, peridin-chlorophyll protein (PerCP)-CY5.5-labeled monoclonal anti-CD4, phycoerythrin (PE)-CY7-labeled monoclonal anti-CD8, APC-labeled monoclonal anti-CD25, and fluorescein isothiocyanate (FITC)-labeled monoclonal anti-CD226, purchased from BD Bioscience (Franklin Lakes, NJ, USA), and PE-labeled monoclonal anti-TIGIT, purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Techniques: Cell Function Assay, Expressing, Two Tailed Test

Journal: Journal of autoimmunity

Article Title: Sex-specific Tau methylation patterns and synaptic transcriptional alterations are associated with neural vulnerability during chronic neuroinflammation

doi: 10.1016/j.jaut.2019.04.003

Figure Lengend Snippet: (A) Splenocytes from Mapt−/− and wildtype male mice primed with MOG35–55 peptide for 11 days were re-stimulated in vitro with increasing concentrations of MOG35–55 peptide and their proliferation was assessed after 3 days by thymidine incorporation. The differences are expressed as fold-increases to the proliferation rate of non-stimulated cells. (B) Splenocytes from Mapt−/− and wildtype male mice primed with MOG peptide were re-stimulated in vitro with 10 μg/mL MOG35–55 peptide in polarizing conditions toward Th1, Th17 and Treg lineages. After 3 days, the percentage of CD4+ T cells acquiring the different phenotypes was assessed by flow cytometry via intracellular cytokine staining (for Th1/Th17) or FOXP3 staining (for Treg). (C) Splenocytes from MOG peptide-primed Mapt−/− and wildtype male mice were re-stimulated with 5 or 25 μg/mL MOG35–55 peptide for 3 days and the surface expression of the activation markers CD44 and CD80 was measured by flow cytometry in CD3+, CD4+ and CD8+ T cells, B cells and monocytes. (D) Splenocytes from MOG peptide-primed Mapt−/− and wildtype male mice were re-stimulated with 10 μg/mL MOG35–55 peptide for 24 hours and the activation status of 7 phosphoproteins (p38, ERK1/2, STAT1, STAT3–6) was assessed by phosphoflow cytometry in CD4+ and CD8+ T cells, and in B cells. For panels C and D, the fluorescence values were converted in Z-scores and plotted as heatmaps where each column represents a different mouse and each row depicts the levels of a surface marker or a phosphoprotein in a specific cell population. 4 mice per genotype were used in the different analyses. NS; non-significant.

Article Snippet: The following antibodies were used in the study: TAU-5 mouse monoclonal antibody (577801, Millipore); anti-FLAG rabbit polyclonal antibody (2368, Cell Signaling); anti-α-tubulin mouse monoclonal antibody (3863, Cell Signaling); anti-β-Actin rabbit monoclonal antibody (8457, Cell Signaling); anti-rabbit IgG F(ab’)2 Fragment Alexa Fluor 555 Conjugate (4413, Cell Signaling); anti-mouse IgG F(ab’)2 Fragment Alexa Fluor 488 Conjugate (4408, Cell Signaling); anti-p38 MAPK (pT180/pY182) mouse monoclonal antibody (36/p38, BD Biosciences); anti-ERK1/2 (pT202/pY204) mouse monoclonal antibody (20A, BD Biosciences); anti-STAT1 (pY701) mouse monoclonal antibody (14/P-STAT1, BD Biosciences); anti-STAT3 (pY705) mouse monoclonal antibody (4/P-STAT3, BD Biosciences); anti-STAT4 (pY693) mouse monoclonal antibody (38/p-STAT4, BD Biosciences); anti-STAT5 (pY694) mouse monoclonal antibody (47/STAT5(pY694), BD Biosciences); anti-STAT6 (pY641) mouse monoclonal antibody (18/P-STAT6, BD Biosciences); anti-CD3e hamster monoclonal antibody (145–2C11, BD Biosciences); anti-CD19 rat monoclonal antibody (ID3, BD Biosciences); anti-CD4 rat monoclonal antibody (GK1.5, BD Biosciences); anti-CD8a rat monoclonal antibody (53–6.7, BD Biosciences); anti-IL-17A rat monoclonal antibody (TC11H10, BD Biosciences); anti-IFNγ rat monoclonal antibody (XMG1.2, BD Biosciences); anti-FOXP3 rat monoclonal antibody (MF23, BD Biosciences); anti-CD44 rat monoclonal antibody (IM7, BD Biosciences); anti-CD80 armenian hamster monoclonal antibody (16–10A1, BioLegend).

Techniques: In Vitro, Flow Cytometry, Staining, Expressing, Activation Assay, Cytometry, Fluorescence, Marker