HJ1 T cells found in HJ1Tg/hCD1Tg mice exhibit an activated phenotype. Cells were isolated from the thymus, spleen, and liver of HJ1Tg/Rag^−/− and HJ1Tg/hCD1Tg/Rag^−/− mice, stained with mAb against various cell surface markers, and analyzed by flow cytometry. Results are representative of 3 independent experiments. (A) NK1.1⁺ HJ1 T cells require CD1b molecules for proper development. Cells were stained with mAb against CD3 and NK1.1. The percentages of CD3⁺NK1.1⁺ and CD3⁺NK1.1⁻ T cells in each organ are indicated. (B) The majority of the HJ1 T cells developed in the presence of CD1b molecules are DN. Liver leukocytes were stained with mAb against CD3, NK1.1, CD4 and CD8α. Dot plots depict the co-receptor usage of HJ1 T cells in the CD3⁺NK1.1⁺ and CD3⁺NK1.1⁻ gates. The percentage of cells in each quadrant is indicated. (C) HJ1 T cells isolated from HJ1Tg/hCD1Tg/Rag^−/− mice express activation markers. Cells from HJ1Tg/hCD1Tg/Rag^−/− and Vα14Tg mice were stained with CD3 together with CD44, CD69, or CD122. Histograms depict the surface expression of CD44, CD69 and CD122 on the CD3⁺ cells. Isotype control staining is depicted as filled histograms and the staining of Vα14Tg T cells and HJ1 T cells is depicted as dotted line and solid line, respectively. (D) HJ1 thymocytes express the transcription factor PLZF. Thymocytes from HJ1Tg/hCD1Tg/Rag^−/− mice were surface stained with CD3 and intracellularly stained for PLZF. The specificity of the anti-PLZF antibody was confirmed by staining conventional T cells (TCRβ⁺CD1d/α-GalCer tetramer⁻ gate) and iNKT cells (TCRβ⁺CD1d/α-GalCer tetramer⁺ gate) isolated from WT mice.