Figure 3. HIV‐1 NEF interferes with B‐cell effector functions via an N‐terminal protein interaction motif.

• A
  Linear representation of HIV‐1_SF2 NEF. To identify a motif of NEF critical for suppression of the humoral immune response, highlighted regions were either deleted or mutated.
• B
  Production of anti‐HEL IgG at the indicated day post‐immunization with HEL‐OVA/CFA following adoptive transfer of B_HEL and CD4_OT‐II cells expressing NEF or various NEF mutants. Shown are relative levels of HEL IgG production with the median of IgG levels in mice receiving control cells on day 14 post‐immunization arbitrarily set to 1. Each dot represents one animal. Shown are mean values with SD from 5 to 12 mice from 4 experiments.
• C
  HEL‐OVA pulsed‐ B_HEL cells (blue) and CD4_OT‐II cells (green) were placed on PLL‐coated cover glasses and subjected to live‐cell time‐lapse imaging. Shown are representative still images. B_HEL cells were loaded with SiRAct to visualize actin polymerization in B cells (F‐actin displayed in red). Filled and empty arrowheads indicate the presence or absence of F‐actin accumulation at the IS, respectively. Scale bars, 5 µm.
• D
  Quantification of B‐cell actin polymerization as shown in C with frequency of actin polymerization in B cells co‐cultured with control T cells arbitrarily set to 100%. Shown are mean values with SD from three independent experiments with at least 30 conjugates evaluated per condition.
• E–G
  Effects of NEF expression in CD4_OT‐II cells on gene expression in B_HEL cells. After 24 h of B–T co‐culture, B_HEL cells were separated from CD4_OT‐II using magnetic beads before RNA isolation for microarray analysis. The top 235 deregulated genes (see Dataset EV2) were grouped into genes that were highly induced (E), moderately induced (F), or repressed (G) by Ag stimulation in control cells and the effects of NEF WT or NEF Δ12–39 displayed as heat maps. Heat map values are plotted as average Log₂ FC (n = 3 biological repeats) relative to unstimulated B_HEL. Red and green represent higher and lower expression, respectively.
• H
  Relative surface expression of CD4, CD28, OX40, ICOS, CD45, and CD54 on T cells expressing NEF WT or the indicated NEF mutants. Analysis of surface expression was performed using flow cytometry 3–7 days post‐transduction of T cells. Depicted are mean values with SD from 3 to 6 independent experiments, with control T cells arbitrarily set to 100% as indicated by the dashed line.
• I
  Representative confocal images of the subcellular distribution of IL‐4 in CD4_OT‐II cells expressing the indicated proteins engaged in an IS with B_HEL cells. B cells and T cells were placed on PLL‐coated cover glasses for 60–90 min, fixed, and stained for intracellular IL‐4. Filled and empty arrowheads indicate presence or absence of IL‐4 accumulation at the IS, respectively. Scale bars, 5 µm.
• J
  Quantification of IL‐4 accumulation at the IS as depicted in I. Shown are mean values with SD from 3 independent experiments relative to control cells set to 100%.

Data information: Statistical significance of bar graphs was assessed by a Kruskal–Wallis test with Dunn’s multiple comparison test. ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05.