Figure 2.

The two distinct TCRβ chains are neighbors in the plasma membrane of live T cells bearing dual αβTCRs. Spleen T cell lines from V_β2×V_β8 double transgenic mice were stained (Upper) with mAb to V_β2 conjugated with FITC alone or followed by PE-labeled mAb against either V_β8, CD3, CD4, or CD8. Alternatively (Lower), cells were stained with FITC-conjugated anti-V_β8 mAb alone or followed by PE-labeled mAb against either V_β2, CD3, CD11a, or CD45. Flow cytometry analyses of average donor quenching (10, 31) measure the putative reduction in the green FITC fluorescence emission on the surface of viable T cells promoted by neighbor PE-acceptor molecules (10, 31). In cells stained with the two anti-TCRV_β mAbs (A and D), the FITC fluorescence distribution was reduced as indicated by the shift to the left in the presence of the quenching, reciprocal anti-TCRV_β mAb (shaded histograms). Both TCRβ chains were also in the vicinity of CD3 subunits (shaded histograms in C and F). Histograms for TCRV_β staining in the presence and absence of the PE-labeled mAb against CD4, CD8, CD11a, and CD45 instead are superimposed. The ratio of V_β2 to V_β8 mean fluorescence intensity ranged from 1:1 to 2:3 in samples from several mice. Similar results were observed for tetraplicate samples in three independent experiments as well as in analyses of freshly isolated spleen and thymus cells (data not shown).