Figure 4.

TCR and CD80 are segregated in the IS of CD11c⁺ splenic DCs. (A-B) Image of T cell-DC IS. Overnight LPS/αCD40 matured (A) WT and (B) CD80-eCFP DC pulsed with 5μM OVA were fixed (non-permeabilizing conditions) 30 minutes after adding OT-II T cells and stained with non-blocking antibodies to TCR (H57) and LFA-1 (H155). The distribution of molecules was analyzed in at least 2 independent experiments in WT and CD80-eCFP. CD80 is shown in green, TCR in red, and LFA-1 in blue. Images were cross-section of a 3-D plane rotated in an en face view. Dotted white line depicts the interface of the IS. Solid yellow arrow depicts cluster of CD80 accumulations, and solid white arrow depicts cluster of TCR accumulations. The transmitted light image depicts a side view of T cell – DC conjugate. The scale bar is 2 m. (C) Quantification of CD80 accumulation patterns with respect to the TCR of (A) versus (B) in the IS over a 30 min time point. WT is shown in black and CD80-eCFP in grey. CD80 clusters segregated from TCR clusters were scored as “segregated” and CD80 clusters colocalized with the TCR clusters partially or entirely were scored as “colocalized”. When there were no CD80 clusters in the interface these were scored as “none”. The difference in molecular accumulation between segregated and colocalized is extremely statistically significant with a p-value of 0.0001 and 0.0005 for WT and CD80-eCFP, respectively, using multinomial test for equal probabilities. (*) The patterns of molecular segregation were not significantly different between WT and CD80-eCFP expressing DCs, p-value = 0.850 using Fisher exact test.