Figure 4.

Combining selected CDR3 variants further improves tetramer binding and TCR activation. a, the most improved α-chain variant (α2) was transfected into CHO-T cells in pairwise combinations with the WT and selected β-chain variants (β1, β4, β7, and β8). After 2 days of expression, cells were stained and analyzed by flow cytometry, as in Fig. 3. b, activation of human Jurkat T cells expressing RA14 and selected TCR variants was measured by CD69 up-regulation. Selected TCR variable regions were cloned into expression vectors with mouse constant domains, native TCR transmembrane, and signaling sequences and transfected into Jurkat cells. After 24 h of co-culture with peptide-pulsed human T2 antigen-presenting cells, TCR-positive cells (NLV-tetramer-binding and Vβ-positive) were further monitored for CD69 up-regulation using an anti-CD69-FITC antibody. Data shown are the results, average and standard deviation of three independent experiments, each performed in duplicate for every treatment condition. Analysis of variance was used to compare the anti-CD69 mean fluorescence intensity for each clone combination (****, p < 0.001).