Figure 2

TSAd affects polarization of actin, TCR and polarity complex proteins at the IS. Murine Id-specific TCR-transgenic CD4+ T cells were incubated for 30 minutes in the presence of Id negative (A20 cells) or Id positive (F9 cells) APC, prior to staining with the indicated markers and analysis of T cell:APC conjugates by IFC. (A) Conjugates are identified as a single SNARF⁺ T cell in association with an unlabeled APC. A T cell mask is applied together with a synapse mask. The polarization feature is determined as the signal intensity in the synapse area relative to that of the entire T cell. A value >1.5 implies that the marker is polarized towards the APC. Conjugates where F-actin in T cells is polarized towards the APC is defined as IS positive. IS-positive conjugates with polarized GB113⁺ (Id-specific TCR) are defined as maturing IS-positive. Conjugates with a maturing IS are then analyzed for polarization of other markers, as indicated in the text and figures.) Masking strategy was employed to identify the T cell synapse in T cell:APC conjugates. Sample images show absence (−) and presence (+) of F-actin polarization towards IS in Id-specific T:APC conjugates. (C) Histogram shows ratio of F-actin intensity values at synapse of individual Sh2d2a+/+ or Sh2d2a−/− Id-specific TCR CD4+ T cells interacting with Id negative A20 cells or Id positive F9 cells (all analyzed conjugates from one representative experiment). The polarization ratio was calculated by dividing the F-actin signal in the synapse mask with the F-actin signal in the entire cell. The stippled line indicates the threshold value of 1.5. Conjugates with polarization ratio >1.5 were defined as having F-actin polarized to the IS. (D) Graph shows frequency of Id-specific T:APC conjugates displaying F-actin polarization ratio >1.5. (E) Graph shows median F-actin intensity ratio values in Id-specific T:APC conjugates in the presence of Id positive F9. Data in C and D represents median values (+95% CI) of three independent experiments, paired student’s t-test. (F) Polarization of Id-specific TCR (staining with TCR clonotype-specific GB113 mAb) in conjugates displaying F-actin polarization. Image gallery shows conjugate without (−) and with (+) Id-specific TCR polarization. (G) Histogram shows Id-specific TCR intensity values (calculated as for F-actin above) at synapse of individual Sh2d2a+/+ or Sh2d2a−/− Id-specific TCR CD4+ T cells in conjugate with Id negative or positive APC as in (B) and gated for polarization of F-actin (all analyzed conjugates from one representative experiment). Stippled line defines threshold for identification of conjugates with Id-specific TCR polarized to IS. Non-parametric Mann-Whitney test. (H) Graph shows frequency of Id-specific T:APC conjugates with Id-specific TCR polarized to the synapse. (I) Graph shows median Id-specific TCR polarization ratios towards synapse in Id-specific T:APC conjugates. Data in G and H represent median values (+95% CI) of three independent experiments, analyzed using paired student’s t-test. (J) Image gallery showing polarization of PKCξ towards synapse upon engagement of TCR. (K) Graph shows relative frequencies of Id-specific T: -APC conjugates with polarization of indicated polarity complex molecules towards IS relative to Sh2d2a+/+. (L) Graph show relative values of the intensity ratios in Id-specific T:-APC conjugates of the indicated polarity complex molecules. (j) and (K) Data represents the median values (+95% CI) normalized to those observed in Sh2d2a+/+ Id-specific T: APC conjugates of three independent experiments, n >50 gated events in each experiment. Statistical significance was assessed using paired student’s t-test.