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: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: ISR pathway activation induces PD-L1 and CD155 protein. A, Western blot analysis of human KRAS -mutant H441 or EGFR -mutant PC9 cells treated for 24 or 48 hours with 100 or 200 μmol/L salubrinal or DMSO vehicle (Veh) control. Vinculin served as a loading control for this and subsequent Western blots. B, Western blot analysis of human H441, H358, PC9, and HCC827 cells after 24 hours in RPMI 1640 media supplemented with or without amino acids. C, Western blot analysis of human H441, H358, PC9, and HCC827 cells treated for 24 hours with 5 μmol/L thapsigargin (ER Ca+ ATPase pump inhibitor) to induce ER stress or DMSO vehicle control. D, Western blot analysis of H358 cells with control or UROD sgRNA. E, Western blot analysis of H358 or HCC827 cells with control or UROD siRNA. F, Flow cytometric analysis of cell-surface CD155 and PD-L1 protein in DMSO vehicle control and thapsigargin-treated (5 μmol/L for 24 hours) H358 or HCC827 NSCLC cells. G, Western blot analysis in eIF2α WT (S/S) or mutant (Ser51Ala A/A) MEFs treated with DMSO vehicle control or 100 μmol/L salubrinal for 24 hours. H, Western blot analysis of thapsigargin- and ISRIB-treated human NSCLC cells. Data from a single experiment are shown and are representative of at least three independent experiments. p-eIF2α, phosphorylated eIF2α.

Article Snippet: The tissue was incubated with a peroxidase block and then with the antibodies CD155 (1:100 for 20 minutes, CST, 81254S, RRID: AB_2799970) and PD-L1 (1:100 for 30 minutes, CST, 13684S, RRID: AB_2687655).

Techniques: Activation Assay, Western Blot, Mutagenesis, Control

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: ISR pathway activation enhances PD-L1 and CD155 translation. A, Polysome profiling of H1944 vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). B and C, qRT-PCR analysis of PD-L1(CD274) ( B ) and CD155(PVR) ( C ) mRNA in ribosomal fractions from A . qRT-PCR analysis for each gene shown was performed with 1 primer set spanning an exon–exon junction (primer set 1). Data for primers set 2 are available in Supplementary Fig. S2. Fractions associated with <3 ribosomes were grouped to represent poorly translated mRNAs, and fractions associated with >3 ribosomes were grouped as efficiently translated mRNAs. PD-L1 and CD155 mRNA expression in each fraction was normalized to luciferase , and mRNA abundance was calculated as the percent of total in all fractions. Luciferase control mRNA was added to each fraction prior to RNA extraction to control for variability. Error bars represent the SD from the mean from three independent fractions (<3 or >3 ribosomes). D, Diagram of the WT human CD155 5′ UTR with 6 CTGs and mutant constructs with CTGs mutated to CTCs cloned upstream of a luciferase reporter. E, Dual luciferase assay of MEFs transfected with indicated CD155 -5′ UTR firefly luciferase reporter constructs normalized to cotransfected control Renilla luciferase. Luciferase activity was monitored after 48 hours. Error bars represent the SD from the mean from n = 3 biological replicates. Data from a single experiment are shown and are representative of three independent experiments. F, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in E . Error bars represent the SD from the mean from n = 3 biological replicates. G, Dual luciferase assay of the CD155 5′ UTR in MEF vehicle- or salubrinal-treated cells (100 μmol/L for 24 hours). Error bars represent the SD from the mean from n = 3 biological replicates. H, qRT-PCR analysis of the mean luciferase mRNA normalized to actin in MEFs shown in G . n = 3 biological replicates. A Student t test was used to determine statistical significance. *, P < 0.05; **, P < 0.005.

Article Snippet: The tissue was incubated with a peroxidase block and then with the antibodies CD155 (1:100 for 20 minutes, CST, 81254S, RRID: AB_2799970) and PD-L1 (1:100 for 30 minutes, CST, 13684S, RRID: AB_2687655).

Techniques: Activation Assay, Quantitative RT-PCR, Expressing, Luciferase, Control, RNA Extraction, Mutagenesis, Construct, Clone Assay, Transfection, Activity Assay

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: CD155 and PD-L1 are positively correlated in primary human lung adenocarcinoma tumors. A, Representative images of IHC for PD-L1 and CD155 of 33 primary human lung adenocarcinomas. Shown are 20× representative images. Scale bar, 100 μm. B, Correlation of CD155 H-score and PD-L1 H-score of tissues in A (Pearson correlation r = 0.506, P = 0.0031). C, Microphotographs of CD155 and PD-L1 IHC analysis of primary lung adenocarcinomas displaying different combinations of expression in malignant cells (CD155 + /PD-L1+, CD155 − /PD-L1−, CD155 + /PD-L1−, and CD155 − /PD-L1+). Shown are 20× representative images. Scale bar, 100 μm. Red arrows, positive expression of PD-L1 or CD155 in MCs. D, Association of CD155 H-score expression in all primary lung adenocarcinomas with PD-L1 status (cutoff for positive PD-L1 expression: tumor proportion score ≥1%; P = 0.0004). A Mann–Whitney test was used to determine statistical significance. Graph represents the median, and error bars represent the 95% confidence interval. E, Association of CD155 H-score expression in all primary lung adenocarcinoma with pathologic stage ( P = 0.001). A Mann–Whitney test was used to determine statistical significance. Graph represents the median, and error bars represent the 95% confidence interval. F, Correlation of CD155 H-score expression with tumor size (Spearman correlation r = 0.1946, P < 0.0001). ***, P < 0.0005; ****, P < 0.00005.

Article Snippet: The tissue was incubated with a peroxidase block and then with the antibodies CD155 (1:100 for 20 minutes, CST, 81254S, RRID: AB_2799970) and PD-L1 (1:100 for 30 minutes, CST, 13684S, RRID: AB_2687655).

Techniques: Expressing, MANN-WHITNEY

Journal: Cancer Research

Article Title: The Integrated Stress Response Pathway Coordinates Translational Control of Multiple Immune Checkpoints in Lung Cancer

doi: 10.1158/0008-5472.CAN-24-3844

Figure Lengend Snippet: Model of translational control of CD155 and PD-L1 in response to ISR activation. Stresses commonly present in the TME activate the ISR pathway in tumor cells. This results in enhanced translation of CD155 and PD-L1 , which suppresses T-cell function by binding the inhibitory T-cell receptors PD-1 and TIGIT, respectively, thereby leading to enhanced tumorigenesis. Created in BioRender. O’Donnell, K. (2025) https://BioRender.com/vphxffz .

Article Snippet: The tissue was incubated with a peroxidase block and then with the antibodies CD155 (1:100 for 20 minutes, CST, 81254S, RRID: AB_2799970) and PD-L1 (1:100 for 30 minutes, CST, 13684S, RRID: AB_2687655).

Techniques: Control, Activation Assay, Cell Function Assay, Binding Assay