Figure 2(A) Schematic experimental flow of the analysis of IS formation and function between NEF-expressing CD4OT-II cells and antigen-specific BHEL cells. BHEL cells were stimulated with LPS in the presence (+Ag) or absence (-Ag) of HEL-OVA antigen. B cells and T cells were labelled with different CellTrackerTM dyes before seeding on PLL-coated cover glasses and analyzed by microscopy. (B) Representative confocal micrographs of F-actin (grey signal) polarization at B-T cell contacts after 1-2h of co-culture. Cell conjugates were stained with Phalloidin to visualize F-actin. Scale bar, 5µm. (C, D) Quantification of the frequency of B-T conjugate formation (C) and F-actin accumulation at the B-T interface (D). Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment. (E) Representative confocal micrographs of p-tyrosine (p-Tyr) polarization at B-T cell contacts after 1-2h of co-culture. Cell conjugates were stained with anti-pTyr antibody. Scale bar, 5µm. The top panels display only the p-Tyr signal in grey, the bottom panel depicts p-Tyr (red), BHEL cells (blue) and CD4OT-II cells (green). (F) Quantification of p-Tyr accumulation at the B-T interface as shown in E. Shown are mean values with SD from three independent experiments with at least 50 conjugates analyzed per experiment. (G) NEF prevents induction of F-actin remodeling in BHEL during IS. BHEL cells (blue) and CD4OT-II cells (green) were placed on PLL-coated slides and subjected to live-cell time-lapse imaging. Shown are representative still images from the indicated time points post contact formation (see Movies EV1 and EV2). Red signals indicate actin polymerization specifically in BHEL cells. Scale bars, 5µm. Filled and empty arrowheads indicate examples of accumulation of polarized actin or p-Tyr signal at B-T interface during IS formation or lack thereof, respectively. Asterisks mark pairs of interacting T and B cell. (H) Quantification of B cell actin polymerization as shown in G with the 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 50 conjugates counted per experiment. (I) Volcano plot of NEF-induced changes to BHEL cell transcription. NEF-expressing or control CD4OT-II cells were co-cultured with BHEL cells in the presence of HEL-OVA for 4h before isolation of BHEL cells and transcriptome analysis by microarray. Shown are mRNAs with log2fold change against -log10 p-value. Out of around 20,000 genes investigated, 564 genes were induced and 977 genes were suppressed by NEF (see Dataset EV1), with 187 genes induced and 407 suppressed by NEF in a statistically significant manner. (J) Pathways involved in cell proliferation were negatively modulated by NEF, while lipid metabolism pathways were induced by NEF. 1541 genes (see Dataset EV1) were included for pathway analysis. (K) Ex vivo BHEL proliferation after 3d of co-culture with control or NEF-expressing CD4OT-II cells. BHEL cells were labelled with the cell proliferation dye eFluor670 and co-cultured with NGFR-enriched CD4OT-II cells expressing NEF in the presence or absence of HEL-OVA Ag. T cell-dependent B cell proliferation was assessed by quantification of the dilution of eFluor670 using flow cytometry. (L) Relative B cell proliferation (quantified by gating on the last 3-4 generations) is plotted as mean values with SD from four independent experiments. Control T cells +Ag were arbitrarily set to 1.