anti mouse cd96  (Bio X Cell)

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: ( A ) Scheme of the experimental setup of the surface screen. Analysis used PE MFI values from both infected (eCFP + cells) and uninfected cells (eCFP − cells). Ab, antibody. ( B ) Representative dot plot and histograms from analysis of one donor showing the down-regulation of CD4, CD317 (Tetherin), and MHCI (HLA-ABC). FI, fluorescence intensity. ( C ) Water fall plot displaying fold receptor modulations at the plasma membrane of productively HIV-1–infected CD4 + T cells directly related to uninfected cells. Selected down-regulated genes that were confirmed by this screen are labeled in red, and CD96 down-regulation is shown in green. The P value for all receptors shown is <0.05 calculated with a paired t test from the 13 donors sampled here and corrected for multiple hypothesis testing. ( D ) Representative histograms of selected receptor modulations in HIV-1–infected versus uninfected CD4 + T cells. ( E ) Representative histograms and dot plots of cell surface receptors that identify CD4 + T cells, which are not permissive for productive HIV-1 infection. The underlying raw data are included in dataset S1.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: Infection, Fluorescence, Clinical Proteomics, Membrane, Labeling

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: Primary CD4 + T cells were infected with HIV-1 NL4-3–IRES–eGFP reporter virus containing the indicated deletions in vpr , nef , and vpu . At 48 hours postinfection, cells were harvested and stained for the indicated cell surface receptors with specific antibodies and analyzed by flow cytometry. ( A ) Representative dot plots of HIV-1–infected primary CD4 + T cells that were stained for CD96 cell surface expression. GFP was used to identify HIV-1–infected cells (green box), and modulation was directly compared to uninfected cells (blue box). ( B ) CD96, ( C ) CD226, ( D ) CD155, and ( E ) NTB-A MFIs were used to calculate the respective surface expression of HIV-1–infected cells compared to uninfected cells. ( F ) Infection of primary CD4 + T cells with HIV-1 NL4-3 and two HIV-1 strains isolated from PLWH during the chronic stage (CH058 and CH077) harboring inactivating mutations in vpu and nef and assessment of cell surface CD96 levels via flow cytometry at 3 days postinfection. Values from (B) nine (NL4-3, Nef − , Vpu − , and N − U − ), six (Vpr − ), and four (N − U − R − ) and for [(C) to (E)] four and two (N − U − R − ) independent experiments were plotted (means ± SD). (F) Data from three to five independent donors (means ± SD), statistical differences were assessed using a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: Infection, Virus, Staining, Flow Cytometry, Expressing, Isolation, Comparison

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: 293T cells were transfected to express CD96v1, CD96v2, and either NL4-3 Vpu, NA7 Nef, GFP, or the Nef-G2A mutant. At 36 hours, cells were ( A ) harvested to measure cell surface CD96 via flow cytometry ( n = 3) or ( B ) lysed for Western blot (WB) analysis with the indicated antibodies. ( C ) 293T cells were transfected to express HA-tagged CD96 and AU1-tagged Nef or Vpu. At 24 hours, cells were lysed in 0.5% Brij 98 immunoprecipitation buffer. CD96-HA and AU1-tagged Nef and Vpu were analyzed in input lysates and immunoprecipitates (IP) by anti-HA (a-HA)– and anti-AU1–specific antibodies. ( D ) Primary CD4 + T cells were infected with HIV-1 NL4-3–IRES–eGFP containing the indicated deletions in vpr , nef , and vpu . Five hours postinfection, cells were treated with 100 nM bafilomycin A1 (BafA1), 10 μM lactacystin (LC), or dimethyl sulfoxide (DMSO). Twenty-four hours postinfection, cells were harvested and analyzed for cell surface CD96 by flow cytometry. The CD96 MFI was used to calculate CD96 surface expression of HIV-1–infected cells compared to uninfected cells. ( E ) HeLa cells were transfected to express CD96v2–yellow fluorescent protein (YFP) and the indicated pmScarlet vectors to label cellular compartments. Twenty-four hours after transfection, cells were infected with vesicular stomatitis virus glycoprotein–pseudotyped HIV-1 NL4-3–IRES–mTagBFP reporter virus with deletions in nef and vpu . Twenty-four hours postinfection, cells were imaged via confocal microscopy. ( F ) CD96 colocalization within the indicated compartments was quantified using Pearson’s correlation coefficient. Representative confocal images of n = 17 to 48 analyzed cells were plotted (means ± SEM). Significance was tested using a one-way ANOVA with Tukey’s multiple comparison test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: Transfection, Mutagenesis, Flow Cytometry, Western Blot, Immunoprecipitation, Infection, Expressing, Virus, Confocal Microscopy, Comparison

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: ( A to C ) Jurkat-TAG cells stably expressing CD96v2 were electroporated to express the indicated Nef (A), Vpu (B), and WITO Vpu (C) variants. Twenty-four hours postelectroporation, cells were harvested and stained for cell surface CD96 and analyzed by flow cytometry. The CD96 receptor expression was calculated relative to the GFP-only–expressing negative control, which is marked in gray. Reference NA7 and NL4-3 Nef and Vpu proteins are marked in red. ( D to F ) Primary CD4 + T cells were infected with nef -defective HIV-1 NL4-3 Gag-iGFP expressing the indicated Vpu variants. Forty-eight hours postinfection, cells were harvested and stained for cell surface CD96 (D), CD4 (E), and CD317 (F) and analyzed by flow cytometry. The receptor expression of infected cells was calculated relative to the negative control GFP (Vpu − ). Values from three independent experiments were plotted (means ± SD). Significance was tested using a one-way ANOVA with Dunnett’s multiple comparison test. **** P < 0.0001, *** P < 0.001, ** P < 0.01, and * P < 0.05. n.s., not significant.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: Stable Transfection, Expressing, Staining, Flow Cytometry, Negative Control, Infection, Comparison

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: CD96 was knocked out in stimulated primary CD4 + T cells by CRISPR-Cas9. ( A ) Three days later, the KO was confirmed by CD96 cell surface staining, and ( B to D ) cells were infected with equal p24 amounts of HIV-1 NL4-3–IRES–eGFP with or without deletions in nef and vpu . (B) Forty-eight hours postinfection, cells were analyzed for the rate of productive infection by their GFP expression. (C) In addition, supernatant was collected to quantify for HIV-1 production and release via HIV-1 p24 ELISA and (D) determine the infectivity of released HIV-1 particles via reinfection of Jurkat cells with supernatants normalized for HIV-1 p24. Values from four independent experiments were plotted (means ± SD). Significance was tested using a two-way ANOVA with Sidak’s multiple comparison test. ( E ) CD96 KO CD4 + T cells were used as target cells and cocultured with PKH67-stained PBMCs, which served as effector cells for the cellular cytotoxicity assay. The coculture was incubated for 16 hours at an effector:target cell ratio of 40:1. Cell count of target cells was analyzed by flow cytometry. Killing efficiency was calculated by dividing the cell counts of target cells in the absence of effector cells though the cell counts in the presence of effectors relative to mock. Values from four independent experiments were plotted.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: CRISPR, Staining, Infection, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Cytotoxicity Assay, Incubation, Cell Counting, Flow Cytometry

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: Primary CD4 + T cells from two donors were stimulated for 3 days with IL-2 and PHA ( A ) before CRISPR-Cas9–mediated KO of CD96. After confirmation of the CD96 KO by flow cytometry, CD96 KO cells and scr-RNA control cells were stained with 332 PE-conjugated antibodies of the LegendScreen kit and analyzed by flow cytometry. MFIs of all receptors were compared between CD96 KO and scr-RNA control cells, and the log 2 -fold modulation of receptors was quantified. Receptors with a log 2 -fold modulation of <−0.58 or >0.58 on CD96 KO cells compared to scr-RNA cells are summarized in the table. These receptors were analyzed via Enrichr and are linked to the shown biological pathways (adjusted P value for all pathways < 0.05). The underlying raw data are included in dataset S2. GO, gene ontology. ( B ) Stimulated primary CD4 + T cells were stained with anti-CD96–PE/Cy7–specific antibody and 332 PE-conjugated antibodies of the LegendScreen kit and analyzed via flow cytometry. MFIs of all receptors were compared between CD96 Hi - and CD96 Lo -gated CD4 + T cells (see exemplary FACS plot), and the log 2 -fold modulation of receptors was quantified. Receptors which are >1.00 log 2 -fold higher expressed on CD96 Hi cells compared to CD96 Lo cells are summarized in the table. These receptors were analyzed via Enrichr and are linked to the shown biological pathways (adjusted P value for all pathways < 0.05). The underlying raw data are included in dataset S4.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: CRISPR, Flow Cytometry, Control, Staining

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: Primary CD4 + T cells were IL-2/PHA stimulated for 3 days and left untreated ( A ) or subjected to CRISPR-Cas9–mediated CD96 KO ( B ). Twenty-four hours later, cells were restimulated with anti-CD3/anti-CD28, and, 48 hours later, cells were analyzed for cytokine secretion and costained for CD96. Cytokine secretion of (A) CD96 Hi cells was compared to CD96 Lo cells and (B) that of CD96 KO cells to scr-RNA control cells ( n = 8, means ± SEM, two-way ANOVA with Sidak’s multiple comparison). ** P < 0.01 and * P < 0.05. ( C ) An hCD96-GFP fusion protein is predominantly localized on the cell membrane of medaka thymic T cells (movie S1) compared with GFP-only–expressing controls (movie S2). Scale bars, 30 μm. Average cell speed ( D ) and straightness ( E ) of GFP + cells in the thymus. N = total number of cells examined from >3 samples per condition (means ± SEM, two-tailed Mann-Whitney test). **** P < 0.0001. The asterisk (*) marks an autofluorescent pigment cell cluster. ( F ) Primary CD4 + T cells prestimulated with CD3/CD28 for 3 days were infected with HIV-1 NL4-3–IRES–eGFP or the N − /U − variant. Forty-eight hours postinfection, cells were seeded onto 96-well plates coated with CD96 ligands Nectin-1, CD155, and anti-CD96 or an isotype control. After 45 min at 37°C, nonadherent and adherent cells were collected separately and quantified by flow cytometry. Adhesion was calculated as the percentage of adherent cells relative to the total number of cells ( n = 5, means ± SEM, paired two-way ANOVA with Fisher’s least significant difference). ** P < 0.01 and * P < 0.05.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: CRISPR, Control, Comparison, Membrane, Expressing, Two Tailed Test, MANN-WHITNEY, Infection, Variant Assay, Flow Cytometry

Journal: Science Advances

Article Title: HIV-1 manipulates CD96 on CD4 + T cells to subvert antiviral immunity

doi: 10.1126/sciadv.adx7485

Figure Lengend Snippet: ( A ) PBMCs were stimulated with HCMV pp65 peptide and costimulated either alone or in combination with CD155 or anti-CD96 (each 5 μg/ml) and stained for IFN-γ 21 hours later. IFN-γ secretion was calculated as fold change relative to pp65 stimulation only (mock). ( n = 10, Kruskal-Wallis test with uncorrected Dunn’s test). * P < 0.05 and ** P < 0.01. Depicted are primary data from two donors. ( B ) Resting primary CD4 + T cells were infected with HIV-1 NL4-3–IRES–eGFP or the N − /U − variant and stimulated at 6 hours postinfection with CD3/CD28 antibodies. Two days postinfection, cells were restimulated with CD3/CD28 antibodies either alone or in combination with anti-CD96 or an isotype control. Three days later, IFN-γ secretion was assessed via flow cytometry ( n = 5, paired one-tailed t test). ( C ) Primary CD4 + T cells were stimulated with CD3/CD28 antibodies (0.5 μg/ml each) and mock treated or costimulated with coated CD96 antibody (2.5 μg/ml) or isotype (2.5 μg/ml). Three days poststimulation, supernatants were harvested for a cytokine array ( n = 3, one representative array shown). GM-CSF, granulocyte-macrophage colony-stimulating factor. ( D ) Heatmap illustrating relative abundance of analyzed cytokines normalized to the respective controls. [Mean values, n = 3; n = 2 for IL-6, macrophage colony-stimulating factor (M-CSF), and fibroblast growth factor 6 (FGF-6)]. Only cytokines with >2% relative abundance in at least one treatment condition were included. LIF, leukemia inhibitory factor; BDNF, brain-derived neurotrophic factor; SCF, stem cell factor; MIF, migration inhibition factor; EGF, epidermal growth factor; GDNF, glial cell line–derived neurotrophic factor. ( E ) X-fold change in cytokine secretion between CD96 antibody versus isotype-treated CD4 + T cells was calculated, and the waterfall plot depicts changes of >2 ( n = 3, ±SD, multiple unpaired t test with individual P values). ( F ) Pathway analyses using Enrichr (Kyoto Encyclopedia of Genes and Genomes 2021 database) and as input the nine significantly regulated genes from (E). Bar length and brightness correlates with significance ( q > 0.0001). For “IL-17 signaling,” key cytokines from (E) are indicated.

Article Snippet: Coating was performed by incubating wells with 70 μl of PBS containing either recombinant human Nectin-1 (5 μg/ml; BioLegend), CD155 (5 μg/ml; BioLegend), anti-CD96 antibody (2.5 μg/ml; clone REA195, Miltenyi Biotec), or an isotype control antibody (2.5 μg/ml; REA control human IgG1, Miltenyi Biotec) for 2 hours at 37°C.

Techniques: Staining, Infection, Variant Assay, Control, Flow Cytometry, One-tailed Test, Derivative Assay, Migration, Inhibition