Surface phenotype of CD4⁺ γδ T cells from WT and Itk^−/− mice. (A) Thymocytes from WT and Itk^−/− mice were gated on either CD3⁺γδTCR⁺ cells (top) or CD4⁺CD3⁺γδTCR⁺ cells and CD4⁻CD3⁺γδTCR⁺ cells (bottom), and analyzed for the expression of CD44 and CD24. (B) Splenocytes from WT and Itk^−/− mice were gated on either CD3⁺γδTCR⁺ (top) or CD4⁺CD3⁺γδTCR⁺ cells and CD4⁻CD3⁺γδTCR⁺ cells (bottom) and analyzed for the expression of CD62L and CD44. NK1.1 is expressed but is not required for the development of CD4⁺ γδ T cells in WT and Itk^−/− mice. (C) Thymocytes and splenocytes from WT and Itk^−/− mice were analyzed for the percentage of NK1.1⁺γδ T cells by flow cytometry (n = 7 or 8 mice). *P < .01. (D) γδTCR⁺CD3⁺ cells in the thymus and spleens of WT, Itk^−/−, T-bet^−/−, or T-bet/Itk DKO mice were analyzed for the expression of CD4 and NK1.1 by flow cytometry. Data are representative of 2 independent experiments.