The SH2 domain of Itk is required for recruitment of HS1 to the IS. A, Jurkat cells transfected with Itk suppression or control vectors were conjugated to CMAC-stained, staphylococcal enterotoxin E-loaded Raji B cells (blue) for 15 min. Conjugates were fixed and stained for HS1 (red) or F-actin (green). B, CD4+ T cells from HS1+/− or HS1−/− AND TCR transgenic mice were conjugated to CMAC-stained, MCC peptide-loaded CH27 B cells for 3 min and stained for Itk (red). C, Itk-suppressed or control Jurkat T cells conjugated to Raji B cells were scored for recruitment of HS1 to the cell-cell contact site. D, Jurkat cells were transfected with expression vectors for wild-type, SH2 domain mutant (SH2m), or kinase-dead (KD) Itk. Conjugates were prepared and analyzed, as in C. Data in C and D represent analysis of 50–100 conjugates obtained from two independent experiments.