Biologic activity of scFv28.3-HaaT in a binding assay and MLRs. (A) CD28⁺ Jurkat T cells stained with calcein am dye were incubated on a monolayer of CD80-expressing fibroblasts, as in Figure 2B, in the presence of dilutions of supernatant from Cos cells transfected with pSecTag2B-scFv28.3-HaaT (▪) or pSecTag2B-scFv28.3 (⋄), in which recombinant protein was quantified by ELISA. Control-transfected supernatant (○) was used at similar dilutions. The percentage of inhibition of adhesion after washing was evaluated by luminescence. (B) CD4⁺ T cells from healthy volunteers were incubated in microtiter plates with irradiated allogeneic PBMCs for 5 days in the presence of dilutions of purified sc28.3-HaaT. Proliferation was measured by incorporation of ³H-thymidine for the last 16 hours of culture; m indicates proliferation obtained after addition of a control mAb (mouse IgG1) to the cultures. Data shown here are means ± SDs of triplicates and are representative of 5 experiments. (C-E) MLRs similar to panel B were performed with (filled bars and symbols) or without (empty bars and symbols) saturating amounts of sc28.3-HaaT. After 5 days an aliquot of cells was assessed for proliferation (“primary” in panel C) and for frequency of IFN-γ secretion (“primary” in panel E). The remaining cells were washed and cultured for a resting period of 7 days, after which time cells were collected and restimulated (“secondary”) with the same (C, circles in panels D and E) or third-party (triangles in panel D) stimulatory cells. Proliferation was measured 2 (C-D), 3, and 4 (D) days later. The frequency of IFN-γ–secreting cells during secondary stimulation was measured 24 hours after restimulation (“secondary” in panel E).