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: PE anti-human CD155 , Flow cytometry , 1:50 in 1% FBS in PBS , Miltenyi Biotec , 130-105-905.

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: 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: PE anti-human CD155 , Flow cytometry , 1:50 in 1% FBS in PBS , Miltenyi Biotec , 130-105-905.

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: PE anti-human CD155 , Flow cytometry , 1:50 in 1% FBS in PBS , Miltenyi Biotec , 130-105-905.

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

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD155 , REA1081 , 50 , 130-118-998 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD155 , REA1081 , 50 , 130-118-998 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Cellular neighborhood analysis of PD1 high/low T cells in the tumor margin and core. (A–D) Topology of PD1 high (left) and PD1 low (right) T cells and their cellular neighborhood within a 5 µm range. (A, B) represent tumor margin and (C, D) show tumor core areas. Cell types showing different distribution patterns around PD1 high and PD1 low T cells (mDCs, M1-like M, M2-like M, MDSCs, Fibroblasts, vessels, tumor cells) are highlighted by arrowheads. (E, F) Quantification of cells in a 5 µm range around of PD1 high/low T cells for tumor margin and tumor core, (E) represents immune cells and (F) stroma/tumor cells. (G) Violin plots for expression levels of eight immune-modulating markers (CD112, CD155, CD276, CD39, CD73, IDO, PD-L1, and VISTA) for the most important immune and tumor cells around PD1 high/low T cells in the tumor core area. Violin plots for tumor margin are shown in Supplementary Figure S5E . Depicted markers and annotated cell types as indicated by the color code. ROI sizes: Tumor margin (ROI15) and tumor core (ROI16): 975 x 769 µm.

Article Snippet: CD155 , REA1081 , 50 , 130-118-998 , PE , Miltenyi Biotec.

Techniques: Expressing