c-Abl is required for actin polymerization in response to TCR engagement. (A) Jurkat T cells were transfected with control vector or shAbl-3. At 48 hours after transfection, cells were treated with or without imatinib mesylate for 1 hour, and conjugate formation with SEE-pulsed (or unpulsed) Epstein-Barr virus (EBV) B cells was analyzed by flow cytometry. (B) Jurkat T cells were mock treated or treated with 10 μM imatinib mesylate for 1 hour and allowed to conjugate with SEE-pulsed Raji B cells for 15 minutes. Conjugates were fixed, and F-actin was labeled with bodipy-phallicidin (green). (C) Jurkat T cells were transfected with control vector or shAbl-3 and allowed to conjugate with SEE-pulsed Raji B cells as in panel B. F-actin was labeled with rhodamine-phalloidin (red). B cells are labeled blue with CMAC; T cells expressing the suppression vectors are green due to GFP expression. (D) Conjugates prepared as in panels B and C were scored for the frequency of F-actin at the IS. Data are averages from 3 independent experiments, each with 50 randomly selected conjugates. **P < .01; *P < .05. (E) Purified primary lymph node CD4⁺ T cells from abl^flox/floxCD4-Cre⁺ mice and control littermates were stimulated with 10 μg/mL anti-CD3 for the indicated times. Cells were fixed, labeled with bodipy-phallicidin, and analyzed by flow cytometry. Red indicates unstimulated; blue, anti-CD3–stimulated. Representative data from one of 3 independent experiments performed in duplicate is shown. (F) Purified CD4⁺ T cells from AND TCR transgenic mice were blasted and treated with 10 μM imatinib mesylate for 1 hour. Cells were conjugated with CH12 B cells prepulsed with or without 10 μM moth cytochrome c 88-103 peptide. Conjugates were fixed, and F-actin was stained with rhodamine-phalloidin. (G) Conjugates prepared as in panel F were scored for the frequency of F-actin at the IS. Data are averages plus or minus 1 SD from 3 independent experiments, each with 50 randomly selected conjugates; **P < .01.