Unaltered response to αGalCer by CD8 expressing Vα14i NKT cell hybridomas. (A) CD8 expression in Vα14i NKT cell hybridomas 1.2 (left) and 1.4 (right) after transduction with CD8α and CD8β retroviral constructs and selection for expression of CD8 and TCR. Top, staining with anti-CD8α; bottom, staining with anti-CD8β. Solid black lines, hybridoma transduced with CD8α and CD8β retroviral constructs; gray lines, TCR-β⁺ CD8SP splenocytes; dotted lines, hybridoma transduced with empty vector retroviral construct. (B–D) Hybridomas were cultured in the presence of thymocytes (B) or A20.CD1d cells (C) pulsed with 2, 20, or 200 ng/ml of αGalCer, or plates coated with recombinant mouse CD1d with 2, 20, or 200 ng/well of αGalCer (D), and supernatants collected after overnight culture were assayed for IL-2 by ELISA. The amounts of IL-2 produced by hybridomas stimulated by APCs or recombinant CD1d not incubated with αGalCer were below the detectable levels in these experiments (not depicted). Data are representative of at least two independent experiments.