Fig. 3.

PTPN22 negatively regulates ICAM-1-LFA-1 dependent T-cell synapse formation and IFNγ secretion. (A–C) WT and Ptpn22^−/− CD4⁺ T-cells were stimulated via plate bound anti-CD3 and ICAM-1-Fc in the presence of IL-12 and anti-IL-4. Cell free supernatants at (A) day 3 and (B) day 5 were assessed for IFNγ by ELISA. (C) Day 5 T-cells were restimulated for 6 h with PMA, ionomycin and monensin and the proportion of CD3⁺ CD4⁺ IFNγ⁺ cells determined by intracellular flow cytometry. (D–F) WT and Ptpn22^−/− CD4⁺ T-cells were stimulated via plate bound anti-CD3 and ICAM-1-Fc in the presence of anti-IL-4 but the absence of IL-12. Cell free supernatants at (D) day 3 and (E) day 5 were assessed for IFNγ by ELISA. (F) Day 5 T-cells were restimulated for 6 h with PMA, ionomycin and monensin and the proportion of CD3⁺ CD4⁺ IFNγ⁺ cells determined by intracellular flow cytometry. (G, H) Immune synapse formation by WT and Ptpn22^−/− CD4⁺ T-cells following incubation on planar supported lipid bilayers containing anti-CD3 and ICAM-1. (G) representative example of CD3 and ICAM-1 distribution over time within a WT T-cell immune synapse. (H) For each cell the mean intensity of CD3 and ICAM-1 was calculated by dividing the total fluorescence intensity of the region of interest (ROI, shown by yellow outline) by the area of the ROI. >50 cells/group, representative of >3 independent experiments. Bars represent mean ****p < 0.0001 by one-way ANOVA. (I, J) Conjugates were formed between activated CTV labelled WT and Ptpn22^−/− OT-II CD4⁺ T-cells and CFTR labelled OVA_323-339 pulsed WT BMDC. CTV⁺CFTR⁺ conjugates were determined by flow cytometry at 0 and 10 min at 37 °C. (I) representative flow cytometry plots, quantified in (J) percentage of CD4⁺ T-cells in conjugates. N = 4–5 independent experiments per group. Bars represent mean ± s.d; NS = not significant, *p < 0.05, **p < 0.01 by two-way ANOVA, applying Sidak's multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)