TAGLN2 is necessary for effector T cell function. (A) CD3⁺ T cells from TAGLN2^+/+ and TAGLN2^−/− mice were stimulated with anti-CD3/28 or incubated with SEB-pulsed CD19⁺ B cells for 24 h, and the levels of IL-2 production were measured using ELISA. CON, control. (B) OTII TCR⁺ CD4⁺ or OTI TCR⁺ CD8⁺ T cells from TAGLN2^+/+ and TAGLN2^−/− mice were stimulated with or without OVA-loaded DCs for 24 h. IFN-γ and IL-2 in the supernatant were measured using ELISA. (A and B) All data represent the means ± SD of three independent experiments. *, P < 0.05 versus WT. (C) After 72 h, cell division was monitored by flow cytometry of CFSE-labeled T cells. Data are one representative of three individual experiments, each with three mice per group. (D) After 4-h coincubation of OTI TCR⁺ CD8⁺ T cells with OVA-loaded or -unloaded EL4 cells, ex vivo cytotoxicity was tested at various ratios of effector/target cells. The data represent the mean ± SD of three independent experiments. *, P < 0.05 versus WT. (E) OTI TCR⁺ CD8⁺ T cells were stimulated with 200 nM PMA and 1 µM ionomycin, and cellular expression of granzyme B and perforin was determined by flow cytometry. The boxed areas indicate the CD8⁺ granzyme B^high or CD8⁺ perforin^high population. Data are one representative of three individual experiments, each with three mice per group. MFI, mean fluorescence intensity. (F) Under the condition as described in D, secretion levels of granzyme B were measured using ELISA. (G) Conjugate formation was also quantitated after 30-min incubation of OTI TCR⁺ CD8⁺ T cells with OVA-loaded EL4 cells. (H) TAGLN2^+/+ or TAGLN2^−/− CD3⁺ T cells were retrovirally transduced with empty vector (EV), WT-TG2, or TG2ΔABM. Reconstitution was confirmed by Western blotting. Cells were loaded on anti-CD3/28 coverslips for 5 min and stained for TG2 and F-actin. Cell spreading was determined. Images are representative of >50 cells examined. Bar, 5 µm. (I) Cells were also incubated with SEB-pulsed CD19⁺ B cells for 24 h, and IL-2 levels were measured using ELISA. NT, not treated. (F–I) Data are means ± SD of triplicate experiments. *, P < 0.05 versus empty vector–expressing T cells. ⁺, P < 0.05 versus WT-expressing T cells. (J) Cells were loaded on anti-CD3/28 coverslips, with or without ICAM-1(±ICAM-1). After 10 min, the cells were lysed, and Western blotting was performed, as shown in Fig. 8 (D and E). The data are representative of two independent experiments. KO, knockout; M, molecular mass.