ITK-SYK mimics a TCR signal in vitro. (A) Schematic representations of ITK, SYK, and ITK-SYK. Kinase, tyrosine kinase domain. See text for details. (B) Constitutive ITK-SYK signaling in T cell lipid rafts. Jurkat cells were infected with retroviruses carrying ITK-SYK, ITK-SYK^KD, ITK-SYK^PHmut together with GFP (ITK-SYK, ITK-SYK^KD, or ITK-SYK^PHmut), or GFP only as a control (GFP). Lipid raft fractions from unstimulated or anti-CD3– (5 µg/ml) and anti-CD28 (1 µg/ml)–stimulated cells were prepared and subjected to Western blot analysis with antibodies against ITK (top), phospho-tyrosine (p-Tyr), or phosphorylated PLCγ1 (p-PLCγ1). Dot blots with the lipid raft marker GM1 confirm equal loading. Data shown are representative of at least two independent experiments. (C) Phosflow analysis of ITK-SYK signaling. Jurkat cells were infected with ITK-SYK together with GFP or GFP only expressing retrovirus and intracellularly stained with activation-specific anti-phospho antibodies directed against the indicated T cell signaling molecules. Signaling was analyzed within the infected viable GFP⁺ populations. Data shown are representative of four independent experiments. (D) ITK-SYK triggers kinase and PH domain–dependent T cell activation. Jurkat cells were infected as in B, left unstimulated, stimulated with 10 µg/ml anti-CD3 and 2 µg/ml anti-CD28, or treated with 2 µM R406 as indicated and analyzed by FACS. The percentage of infected GFP⁺ CD69-expressing cells from unstimulated, stimulated, or R406-treated cells is indicated. Data shown are representative of five independent experiments. (E) ITK-SYK induces kinase and PH domain–dependent IL-2 production. Jurkat cells were infected as in B. Cells were left unstimulated, stimulated with 10 µg/ml anti-CD3 and 2 µg/ml anti-CD28, or treated with 2 µM R406 as indicated. IL-2 concentrations in the cell supernatants were determined by ELISA. Shown are the mean ± SD from triplicate samples. Data shown are representative of four independent experiments.