Fig. 4: SEMA3A negatively regulates CD8⁺ T cell immunological synapse formation.

a Live-cell imaging visualizing surface interface using IRM of stimulated CD8⁺ T cells dropped onto an activating surface with immobilized ICAM-1 and CD3 and SEMA3A_S-P or IgG present in medium (left). Cell contour of representative cells from either condition (right). Color of contour indicates time from 0 to 200 sec as denoted on colorbar. Scale bar = 10 μm. b Quantification of maximum size of cell contact area (top) and spreading speed from initial contact to maximum contact area (bottom) (n = 25 cells per group) in same experiment as (a). c Live-cell imaging of activated T cells pre-treated with SEMA3A_S-P-I-AF647 and allowed to form synapses on supported lipid bilayers with ICAM-1, CD80 and H-2Kb-SIINFEKL. Arrows in merged image indicate cells that have bound SEMA3A and do not form immunological synapses. Scale bar = 10 μm. Experiment performed once. d Representative image from high-throughput analysis of immunological synapses on supported lipid bilayers as in (c) with OT-I T cells pre-treated with SEMA3A or not. Scale bar = 30 μm. e Quantification of immunological synapses with or without SEMA3A_S-P-I pre-treated OT-I T cells. (n = 90–1100 cells per mouse per group). Abbreviations: IRM, interference reflection microscopy. Sec seconds. Error bars are means ± SD combined from 3 (b) and 6 (e) independent experiments. **P = 0.0011 (b), P = 0.0042 (E, left), P = 0.0039 (E, right), ***P = 0.0002 (B) by two-tailed Mann–Whitney test (b) or two-tailed paired t-test (e). Source data are provided as a Source Data file.