HJ1 T cells are autoreactive and restricted to CD1b. (A) Generation of HJ1Tg mice. The presence of rearranged Vα8.5-Jα14 TCRα chain was examined by PCR from genomic DNA of HJ1Tg⁺, HJ1Tg⁻ littermates, and HJ1 T-cell hybridoma (left). Histograms depict FACS staining for Vβ2 of blood lymphocytes isolated from HJ1Tg⁺ (open histogram) and HJ1Tg⁻ littermates (filled histogram; right). Results are representative of 4 individual experiments. (B) CD1b is required for the development of HJ1 thymocytes. Thymocytes were isolated from HJ1Tg/Rag^−/− and HJ1Tg/hCD1Tg/Rag^−/− mice, stained with mAb against CD4, CD8, and TCRβ, and analyzed by flow cytometry. Dot plots depict CD4 and CD8 staining; numbers indicate cell percentages in each quadrant (left). Histograms represent TCRβ expression (open histogram) on thymocytes isolated from HJ1Tg/hCD1Tg/Rag^−/− mice in DP, CD8^SP and DN gates (right). Isotype control staining is depicted as filled histograms. Results are representative of 3 independent experiments. (C) HJ1 T cells are restricted to CD1b. ConA-blasts generated from the splenocytes of HJ1Tg/hCD1Tg/Rag^−/− mice were tested for the cytotoxic activity against C1R and CD1b-transfected C1R at an E/T ratio of 30:1. Results are representative of 2 experiments. (D) HJ1 T cells are autoreactive. HJ1 T cells were enriched from the liver leukocytes of HJ1Tg/hCD1Tg/Rag^−/− mice and were cultured with WT or hCD1Tg BMDC. Cytokines in the supernatant were measured by Th1/Th2/Th17 CBA kit. Results are representative of 3 experiments.