To analyze the contribution of individual T cell antigen receptor (CD3/Ti) components, CD3/Ti variants were generated. MA 5.8, a subclone of the pigeon cytochrome c-specific T cell hybridoma 2B4.11, was produced by mutagenesis with EMS followed by selection in the presence of antigen and antigen-presenting cells. MA 5.8 failed to express any cell surface or internal CD3-zeta chain. Pulse-chase studies found that MA 5.8 did not synthesize detectable CD3-zeta. Furthermore, the half-life of other CD3 subunits, notably delta and epsilon, was considerably shortened. Survival was dramatically increased by the addition of ammonium chloride, implying that the degradation of these subunits was enhanced in the absence of CD3-zeta. Fluorescent antibody staining found that MA 5.8 expressed approximately 20-fold less of the other CD3/Ti components on its surface. In spite of this, MA 5.8 did respond to plastic-adherent anti-CD3 or anti-Ti monoclonal antibodies (mAb) by producing IL-2. However, when compared to 2B4.11 or 2B4.3.12 (another 2B4 subclone that expressed 20-fold less cell surface CD3/Ti but that synthesed CD3-zeta), MA 5.8 responded poorly. In fact, MA 5.8 made barely detectable IL-2 when stimulated with antigen, and no IL-2 when stimulated with a mitogenic anti-Thy-1 mAb. Another 2B4.11 variant, 21.2.2, spontaneously lost 3 or 4 Ti chains, and consequently failed to express CD3/Ti. When 2 of these chains were transfected back into this cell (yielding the cell T 1.2), CD3/Ti expression was regained. 2B4.11 and T 1.2, but not 21.2.2, produced IL-2 in response to stimulation with an anti-Thy-1 or an anti-Ly-6 mAb. Despite this, neither 21.2.2 nor T alpha beta 1.2 manifested increases in intracellular Ca2+ or phosphatidylinositol metabolites. These results suggest that products of phosphatidylinositol metabolism are either not involved in IL-2 production, or one effective at levels below the limits of detection. Attempts will be made to generate other CD3/Ti variants that have modified or missing CD3/Ti components, and to analyze the expression and function of the remaining subunits.
