Fig 2: Screening for cell surface receptor modulations in HIV-1–infected primary CD4+ T cells.(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.

Fig 3: CD96-associated cell surface receptor expression profile of primary CD4+ T cells.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 log2-fold modulation of receptors was quantified. Receptors with a log2-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 CD96Hi- and CD96Lo-gated CD4+ T cells (see exemplary FACS plot), and the log2-fold modulation of receptors was quantified. Receptors which are >1.00 log2-fold higher expressed on CD96Hi cells compared to CD96Lo 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.

Fig 4: 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 5: Functionality of CD96 in CD4+ T cells and HIV-1 infection.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) CD96Hi cells was compared to CD96Lo 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.