FIGURE 7.

Effect of CD3ϵ mutations on TCR triggering. Jurkat T cells expressing a luciferase reporter construct for NFAT/IL-2 promoter activation, and either wild-type or mutant HA-tagged CD3ϵ was activated with plate-bound anti-CD2 and anti-CD28 antibodies, plus either an anti-HA antibody, an anti-CD3 antibody (OKT3), or an irrelevant antibody (OX7, anti-Thy-1). A, example of data obtained for cells expressing wild-type or Pro⁷⁹, Leu⁶⁹ or Asp⁵⁰-mutated HA-tagged CD3ϵ. Anti-HA IgG (left panel) and OKT3 (right panel) responses were normalized against the responses obtained with OX7. Responses for wild-type CD3ϵ (open bar) or the CD3ϵ mutants (colored by expression level as in Fig. 5) are shown. B, normalized IL-2 promoter activity plotted against the normalized cell surface expression level of wild-type or mutant HA-tagged CD3ϵ. Normalized IL-2 promoter activity was calculated as the specific response (that is, following subtraction of the response to OX7) of each cell line to anti-HA antibody, divided by the specific response to OKT3, expressed as a percentage. Cell surface expression levels were normalized using the geometric mean of anti-HA antibody staining expressed as percentages of that obtained for wild-type HA-tagged CD3ϵ. Circles corresponding to the mutant CD3ϵ responses are colored by surface expression level (as in Fig. 5) with wild-type shown as an open circle. C and D, similar results were obtained for wild-type and mutant forms of TCRα (C) and TCRβ (D). The data shown are representative of two separate experiments; the means of triplicates and standard deviations are shown.