Figure 6

Th2 derived TCRαβ has low avidity. (A) ELISPOT assays for Th1 (open squares; n = 4), Th2 (open triangles; n = 3) and Th17 (open diamonds; n = 6) polarized, non-transgenic cell lines cultured for eight days detect IFNγ, IL-4 and IL-17 producing cells, respectively, in response to increasing concentration of PLP 56-70 peptide. (B) Th1 (open squares; n = 6), Th2 (open triangles; n = 6) or Th17 (open diamonds; n = 2) polarized, non-transgenic cell lines incubated with H2-A^g7/PLP 56-70 or control (H2-A^g7/CLIP 103-117) tetramers on Day 8 after primed draining lymph nodes (DLN) were incubated with peptide. Percentages shown are the difference between staining with H2-A^g7/PLP 56-70 and control (H2-A^g7/CLIP 103-117) tetramers. Data are representative of three separate experiments. (C) Th1 and Th2 polarized cell lines (A, B) have similar levels of CD3 expression. (D) ELISPOT assays for unpolarized, Th2 cytokine producing TCRαβ lines (filled triangles; n = 3) and IFNγ producing littermate control lines (filled squares; n = 5) cultured in the absence of polarizing cytokines through two successive 10-day cycles of re-stimulation with peptide, detect IL-4 and IFN-γ producing cells, respectively, in response to increasing concentration of PLP 56-70 peptide. (E) Unpolarized Th2 cytokine producing TCRαβ lines (filled triangles; n = 6), TCRα lines (filled circles; n = 6) and IFNγ producing littermate control lines (filled squares; n = 5) cultured through two successive 10-day cycles of re-stimulation with peptide, incubated with H2-A^g7/PLP 56-70 or control (H2-A^g7/CLIP 103-117) tetramers at the concentrations shown. Percentages shown are the difference between staining with H2-A^g7/PLP 56-70 and control (H2-A^g7/CLIP 103-117) tetramers. (F) Functional binding of tetramers determined by cytokine production from unpolarized TCRαβ lines and littermate controls following incubation with plate bound H2-A^g7/PLP 56-70 tetramer. Cytokine concentration shown is a total minus background cytokine production for cells incubated with phosphate-buffered saline (PBS) only. The TCRαβ transgenic line (black bars) does not make IFNγ while the littermate control line (white bars) does. An IFNγ absent cytokine profile is elicited from the TCRαβ transgenic line.