Figure 6.

Expression of a rearranged V_α14-J_α18 TCR transgene restores iNKT cell development in the absence of HEB. (a) Expression of CD1d-tet and TCRβ by V_α14tg⁺ wild-type, V_α14tg⁺ HEB-TKO and HEB-TKO lymphocytes from thymus, liver and spleen. Numbers adjacent to outlined areas indicate percent CD1d-tet⁺TCRβ⁺ cells in gate. Data are representative of five independent experiments. (b) Frequency (left) and absolute number (right) of CD1d-tet⁺TCRβ⁺ iNKT cells in thymus, liver and spleen of V_α14tg⁺ wild-type, V_α14tg⁺ HEB-TKO and HEB-TKO mice. Data are representative of two experiments (average and s.e.m.). (c) Surface expression of CD44 and NK1.1 by iNKT cells in thymus, liver and spleen of V_α14tg⁺ wild-type and V_α14tg⁺ HEB-TKO mice (left); numbers in quadrants indicate percent cells in each. Right, frequency of iNKT cells at developmental stages 1–3. *P < 0.05, **P < 0.005, ***P < 0.0005 (unpaired two-tailed t-test). Data are representative of three experiments (right, average and s.e.m. of four mice). (d) Expression of CD1d-tet and TCRβ (top left) by infected donor (CD45.2⁺Thy-1.1⁺) thymocytes in irradiated CD45.1⁺ hosts reconstituted with wild-type or HEB-TKO bone marrow infected with empty vector (EV; control) or HEB-TKO bone marrow infected with retrovirus encoding Bcl-x_L (bottom). Right, annexin V expression by DP thymocytes obtained from the recipients described above and cultured for 0–72 h. Below, quantitative PCR analysis of transcripts of V_α14 rearrangements to J_α56, J_α18 or J_α9 segments by sorted thymocytes, presented relative to C_α transcripts. Data are representative of two experiments (top left), two independent experiments (top right) or two experiments (below; average and s.e.m. of two individual samples).