Fig. 3.

tpCD4⁺ T cells establish immune synapses with naive CD8⁺ T cells. H-2K^b (a) or CD86 (b) expression on CD4⁺ T cells before (blue line) and after Listeria-OVA transphagocytosis (green line). Antibody negative controls are shown (grey). c H-2K^b/OVA expression on Listeria-OVA tpCD4⁺ T cells, 24 (blue) or 48 h (green) post-transphagocytosis and on non-transphagocytic T cells (grey). Confocal images of Listeria-OVA (d) or Listeria-WT tpCD4⁺ T cells (e) incubated 1 h with OT-I naive CD8⁺ T cells (blue). CD3 and actin fluorescence is shown on a “fire” scale. Merged images show actin (cyan) and CD3 (red). Bars = 10 μm. f Quantification of actin accumulation at the IS analyzed using Synapse measures software³¹. Naive OT-I CD8⁺ T cells were conjugated with Listeria-OVA or Listeria WT tpCD4⁺ T cells and fluorescence microscopy was performed as in d and e. Each dot represents the fold increase of actin accumulation in the IS in an individual CD8⁺/tpCD4⁺ T cell contact from three independent experiments, the mean and the s.e.m. are shown in red. ***P < 0.001 (analyzed by Mann–Whitney U-test). g Relative specific cytotoxicity of effector CD8⁺ T cells (CTL) activated by Listeria-OVA tpCD4⁺ T cells (solid line) or by OVAp-I-loaded splenocytes (dashed line). Various EL-4 (target cell):CTL ratios were measured