IGSF4 enhances T cell–T cell and T cell–APC adhesions by ectodomain interaction. (A) J-GFP or J-IGSF4_GFP cells (2 × 105 cells/well) were cultured in the presence or absence of anti-CD3/28 for 3 h and photographed. Quantitation of cell aggregation was determined as described in Materials and methods. The results are the mean ± SD of six experiments. *, P < 0.05 versus J-GFP cells without stimulation. **, P < 0.05 versus J-GFP cells with anti-CD3/28 stimulation. DIC, differential interference contrast; EV, empty vector. (B) A representative conjugate formation profile with T cells and Raji B cells and the percentage of DP cells (blue) are shown in the flow cytometric plot and bar graph, respectively. The results are the mean ± SD of triplicate experiments. *, P < 0.05 versus J-GFP cells without stimulation. **, P < 0.05 versus J-GFP cells with SEE-pulsed Raji B cells. (C and D) Domain swapping from the IGSF4 ectodomain to ICAM-1 D3–5 in IGSF4-mediated T cell activation. (C, top) Arrows indicate the relocation of IGSF4 at the cell–cell contact regions in Jurkat T cells expressing WT-IGSF4_GFP but not IC1_IGSF4_GFP. (bottom) Schematic illustration showing swapped region of IGSF4/EXTD with ICAM1/D3-5. Quantitation of IGSF4 or IC1_IGSF4 relocation was performed as described in Fig. 2 A. The results are the mean ± SD of four experiments. IG4, IGSF4; IC1, ICAM-1 D3–5. (D) These cells were stimulated with SEE-pulsed Raji B cells, and the IL-2 mRNA levels were assessed by real-time quantitative PCR. The results are the mean ± SD of triplicate experiments. *, P < 0.05, as compared with cells expressing WT-IGSF4. Bars: (A) 100 µm; (C) 10 µm.
