Be-specific CD4⁺ T cells require Be and a specific peptide for antigen recognition. (A) TCR gene segment usage and junctional region amino acid sequence of the Be-specific T cell hybridomas AV22 and AV9. (B) Be-specific (AV22 and AV9) and dengue virus–specific (DV-13) T cell hybridomas were stimulated with the following antigen-presenting cell lines: HLA-DP2–transfected mouse DAP3.L cells (DP8302), fibroblasts (B6 DK10) derived from MHCII- and invariant chain-deficient mice and transfected with native HLA-DP2 (DP2.21), and B6 DK10 cells transfected with HLA-DP2 with a transferrin receptor peptide (DP2-pTf, EPLSYTRFSLAR) or a dengue virus NS3–derived peptide (DP2-pDV, REIVDLMCHATF) covalently attached to the N terminus of the DP2 β-chain. 200 µM BeSO₄ was added to all antigen-presenting cells except DP2-pDV–expressing fibroblasts, and the IL-2 response (mean ± SEM pg/ml) by the hybridomas was measured by ELISA. (C) AV22 and AV9 T cell hybridomas were stimulated with 200 µM BeSO₄ presented by HLA-DP2–transfected fibroblasts grown in either FBS or protein-free (PF) medium. IL-2 secretion (mean ± SEM pg/ml) was measured by ELISA. (B and C) Data shown are representative of three experiments performed in triplicate. (D) AV22 hybridoma cells were stimulated over a range of BeSO₄ concentrations (0.3–1,000 µM) using DP8302 cells grown in FBS-containing medium compared with DP2.21 cells grown in FBS and protein-free conditions. Data shown (mean IL-2 ± SD pg/ml) are representative of three experiments performed in triplicate.