ITK-SYK drives constitutive TCR and Jak/Stat signaling in CD4+TCLs and induces positive autoregulatory loops in between TCLs and granulocytes via IFN-γ and IL-6. ITK-SYK/GFP⁺ CD4⁺ T cells and granulocytes were sorted from the spleens of diseased ITK-SYK⁺ mice, total RNA was isolated, and subsequently transcriptome analysis using microarrays was performed. (A,B) Volcano plots show the top genes upregulated or downregulated for GFP⁺ CD4⁺ malignant T cells (A) and GFP^– CD11b⁺ Ly6G⁺ splenic neutrophils (B). Bars show highest upregulated pathways by GESA using gene sets for hallmarks of cancer. A heatmap was generated using Kyoto Encyclopedia of Genes and Genomes gene sets analysis and shows genes involved in the Jak/Stat signaling pathway in malignant T cells (A) and neutrophils (B). (C-G) TaqMan qPCRs were performed using complementary DNA generated from messenger RNA isolated from primary murine ITK-SYK/GFP⁺CD4⁺ T cells (C,D) and GFP^–CD11b⁺Ly6G⁺ granulocytes (E-G) for IFN-γ, IL-6, STAT1, and IRF7 and compared with β-Actin as a control (n = 3 per group). Cells were extracted from the spleens on day 35 after transplantation. (H) Western blot of the murine CD4⁺ T-cell line D10.G4.1 expressing pMIG (control) or ITK-SYK/GFP and stained with antibodies against phosphorylated and total JAK2, phosphorylated SYK³²³, ITK, and β-Actin as loading control. (I,J) Western blot of D10.G4.1 expressing pMIG, pMIG/SYK WT, pMIG/myr-SYK, or pMIG/ITK-SYK and stained with antibodies against total SYK, ITK (I) as well as phosphorylated (I) and total (J) JAK1, JAK3, TYK2, STAT6, STAT5, and STAT3, with β-actin as the loading control. (C-G) Bars represent mean values with error bars showing the SEM. Statistical significance was calculated using Student unpaired t test. Asterisks represent the significance of the difference between GFP⁺ and GFP⁻ populations within the same mice. ∗P < .05, ∗∗∗P < .001. GESA, GSE222050; N.D, not detected; qPCR, quantitative PCR.