Fv-receptor expression and triggering.

(A) Expression of Fv-receptor transgenes leads to surface expression of chimeric Fv-ζ and Fv-ε receptors. Lymph node cell suspensions of nontransgenic littermates (wt), Fv-ζ– (Fv-ζ), and Fv-ε– (Fv-ε) transgenic mice were analyzed by 3-color flow cytometry using CD4-PE (not shown), CD8-FITC (not shown), and anti-Fv-biotin plus Streptavidin-Red 613. Histograms show CD4⁺ cells (thin line) and CD8⁺ cells (bold line). (B, upper panel) Triggering of Fv-receptors is not sufficient to induce proliferation in resting T lymphocytes. T cells of lymph nodes from nontransgenic littermates (■, ▪), or Fv-εζ (○, ●) double transgenic mice were incubated for 20 hours at 2 × 10⁴ cells per well. [³H]-thymidine was then added and the radioactivity incorporation measured after another 12 hours. Cultures were performed in 96-well plates previously coated with mAb2C11 (■, ○), specific for TCR/CD3 or anti-Fv–specific mAb (▪, ●) at the indicated concentrations. (B, lower panel) Preactivated T cells from Fv-ζ–transgenic mice were stimulated with TCR- (■, ▪) or Fv-specific mAb (○, ●). Before addition of [³H]-thymidine for measurements of the proliferative response (■, ○), culture supernatant was removed from each well and tested for its content of IL-2. Results for proliferation (left y-axis, ■, ○) and cytokine measurements (right y-axis, ▪, ●) were overlayed within the same graph. (C) Analysis of cytokine mRNA in differentially stimulated transgenic T cells. Preactivated Fv-ζ⁺ T cells were restimulated in presence of plastic-coated mAb specific for TCR (TCR), chimeric receptor (Fv), or medium alone (no Ab) for 4 or 24 hours. Then cDNA was analyzed by RT-PCR, equal amounts of 10-, 100-, or 1000-fold dilutions (10 ×, 100 ×, and 1000 ×) of cDNA were amplified with oligonucleotides specific for IL-2 or HPRT (H). (D) Differential production and secretion of IL-2 in Fv-ζ–expressing T cells. 2 × 10⁶preactivated T cells were cultured with optimal concentrations of anti-Fv (Fv; 8 μg/mL, lanes 2, 5), anti-TCR/CD3 (TCR; 5 μg/mL, lanes 3, 6) mAb, or no mAb (lanes 1, 4) in the presence of [³⁵S]-methionine for 4 hours. Culture supernatants (lanes 4, 5, 6) and activated cells (lanes 1, 2, 3) were processed separately. Cell lysates and culture supernatants were submitted to immunoprecipitations with IL-2–specific mAb and analyzed on 11% SDS-PAGE under reducing conditions. The gels were dried and autoradiography on x-ray film revealed the radioactively labeled cytokines.