Fig 1: Downmodulation of cell surface CD96 from HIV-1–infected primary CD4+ T cells by Nef and Vpu.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.
Fig 2: CD96 ligation induces release of antiviral and proinflammatory cytokines.(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.
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