Figure 4.
Figure 4. IGSF4 recruitment to the immunological synapse is independent off receptor–ligand interaction but is mediated by the TM domain. (A, left) J-IGSF4_GFP cells were placed on chambered coverslips coated with PLL or anti-CD3. Confocal images were obtained and reconstituted to the three-dimensional images by the FLUOVIEW program. The fluorescent intensity caused by the accumulation at the PLL or anti-CD3–coated surface was quantified. Note, a = contact region and b = opposite region. Each dot represents a single measurement, and at least 50 cells were examined. The data are representative of two independent experiments. (right) J-IGSF4_GFP cells were incubated with either BSA or anti-CD3/28–coated beads (top) or SEE-pulsed Raji B cells in the presence or absence of 1 µg/ml colchicine (Col) for 30 min (bottom), and live-cell imaging was performed. Arrowheads indicate IGSF4 accumulation at the synapse sites. Boxed areas (green) are shown as magnified images in the micrographs below. The fluorescent intensity caused by the accumulation at the T cell–APC contact site was quantified. Data analysis was performed as described in A (left). The data are representative of two independent experiments. DIC, differential interference contrast. (B, top left) Schematic diagram showing deletion and swapping mutants of IGSF4 (top). T cell–B cell conjugates were stained with anti-CD43 (FITC) and anti-CD3 (cy3). The arrowhead reveals the exclusion of CD43 from the immunological synapse (bottom). (top right) Localization pattern of each mutant of IGSF4 in HEK293T and Jurkat T cells. (bottom left and middle) Jurkat T cells expressing IGSF4_GFP or mutants (IGSF4ΔPDZ, IGSF4ΔCT, IGSF4/CD43TM, IGSF4/ΔEXT, or IGSF4/ΔEXT/CD43TM) were either incubated with SEE-loaded Raji B cells (bottom left) or placed on coverslips coated with PLL or anti-CD3 (bottom middle), and confocal analysis was performed. At least 20 z-stack images were reconstituted into a three-dimensional image for the bottom middle panel. Intensity represents accumulation from low (blue) to high (red). Quantitation and data analysis were performed as described in A (left). The data are representative of four independent experiments. (bottom right) Real-time quantitative PCR analysis of IL-2 mRNA expression in response to anti-CD3/28. *, P < 0.05; and +, P < 0.01, as compared with cells expressing WT-IGSF4. The results are the mean ± SD of triplicate experiments. EV, empty vector. (A and B) Horizontal bars indicate the mean. Bars, 10 µm.

IGSF4 recruitment to the immunological synapse is independent off receptor–ligand interaction but is mediated by the TM domain. (A, left) J-IGSF4_GFP cells were placed on chambered coverslips coated with PLL or anti-CD3. Confocal images were obtained and reconstituted to the three-dimensional images by the FLUOVIEW program. The fluorescent intensity caused by the accumulation at the PLL or anti-CD3–coated surface was quantified. Note, a = contact region and b = opposite region. Each dot represents a single measurement, and at least 50 cells were examined. The data are representative of two independent experiments. (right) J-IGSF4_GFP cells were incubated with either BSA or anti-CD3/28–coated beads (top) or SEE-pulsed Raji B cells in the presence or absence of 1 µg/ml colchicine (Col) for 30 min (bottom), and live-cell imaging was performed. Arrowheads indicate IGSF4 accumulation at the synapse sites. Boxed areas (green) are shown as magnified images in the micrographs below. The fluorescent intensity caused by the accumulation at the T cell–APC contact site was quantified. Data analysis was performed as described in A (left). The data are representative of two independent experiments. DIC, differential interference contrast. (B, top left) Schematic diagram showing deletion and swapping mutants of IGSF4 (top). T cell–B cell conjugates were stained with anti-CD43 (FITC) and anti-CD3 (cy3). The arrowhead reveals the exclusion of CD43 from the immunological synapse (bottom). (top right) Localization pattern of each mutant of IGSF4 in HEK293T and Jurkat T cells. (bottom left and middle) Jurkat T cells expressing IGSF4_GFP or mutants (IGSF4ΔPDZ, IGSF4ΔCT, IGSF4/CD43TM, IGSF4/ΔEXT, or IGSF4/ΔEXT/CD43TM) were either incubated with SEE-loaded Raji B cells (bottom left) or placed on coverslips coated with PLL or anti-CD3 (bottom middle), and confocal analysis was performed. At least 20 z-stack images were reconstituted into a three-dimensional image for the bottom middle panel. Intensity represents accumulation from low (blue) to high (red). Quantitation and data analysis were performed as described in A (left). The data are representative of four independent experiments. (bottom right) Real-time quantitative PCR analysis of IL-2 mRNA expression in response to anti-CD3/28. *, P < 0.05; and +, P < 0.01, as compared with cells expressing WT-IGSF4. The results are the mean ± SD of triplicate experiments. EV, empty vector. (A and B) Horizontal bars indicate the mean. Bars, 10 µm.

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