FIGURE 7.

TCR Vβ5.1 chains from CD4⁺ T cell populations require strict Vα chain pairing to maintain Be specificity. (A) Hybridomas expressing Vβ5.1⁺ AQGG or GQGG CDR3β chains were paired with multiple Vα chains on the surface of a murine T cell hybridoma and were stained with soluble Be-loaded HLA-DP2-mimotope-2 (Mim2) and HLA-DP2-plexin A4 (plexin) tetramers. An HLA-DP2-mimotope-2 tetramer without Be loading (Control) was used as a negative control. The mean fluorescence intensity (MFI) for each subset is shown. Positive tetramer staining was observed for the AQGG⁺ Vβ5.1 chain when paired with all Vα chains tested, whereas the GQGG⁺ chain required Vα8. With the exception of the Vβ5.1⁺ AQGG/Vα22⁺ T cell hybridoma, the plexin A4 tetramer stained all hybridomas with lower intensity compared to the mimotope-2 tetramer. (B) The hybridomas from (A) were stimulated overnight with 500 nM mimotope-2 peptide, varying BeSO₄ concentrations and HLA-DP2-transfected fibroblasts as antigen-presenting cells. Data are plotted as % maximal IL-2 secretion against concentration of Be and are representative of three independent experiments. The mean ± SEM EC₅₀ values for each of the hybridomas are shown. (C) Stimulation of T cell hybridomas expressing a Vα8⁺ CDR3α-CAASSYSGA⁺ chain with varying Vβ5.1 chains isolated from the tet^hi and tet^lo T cell populations. T cell hybridomas were stimulated as in (B). Data are plotted as % maximal IL-2 secretion against concentration of Be and are representative of three independent experiments. The mean ± SEM EC₅₀ values for each of the hybridomas are shown.