Fig. 2.

MHC class II-specific expression of transgenic MBP-IA^u in the two founder lines and in vitro activation of Tg4 cells by bone marrow-derived dendritic cells. (A) MHC class II-specific expression of transgenic MBP-IA^u on LN B cells. FACS analysis of LN cells from B10.PL (H-2^u) in comparison to LN cells from transgenic and non-transgenic littermate on an H-2^b background, stained with antibodies against I-A^u (MKS4-bio/SA-PE) and CD45R (B cells; B220-FITC). Dead cells are excluded from the analysis by propidium iodide (PI) staining. Numbers in the quadrants represent the percentage of cells in the latter. (B) Transgene expression on BMDCs from ${\mathrm{MBP}-\mathrm{IA}}_{35}^{\mathrm{u}}$ mice. Dendritic cells were acquired after differentiation of bone marrow cells with GM-CSF. The histograms show FACS analyzes of PI-negative BMDCs after activation (empty) for 24 h with LPS (1 μg ml^-1) in comparison to non-activated BMDCs (filled). Transgenic and non-transgenic control mice for the BMDC experiments were on an H-2^dxb background. After activation, BMDCs showed different activation makers as size increases, up-regulation of endogenous MHC class II and CD86 (data not shown). (C) LPS-stimulated BMDCs from ${\mathrm{MBP}-\mathrm{IA}}_{35}^{\mathrm{u}}$ mice activate Tg4 T cells in vitro. Tg4 LN and spleen cells were depleted from B cells and other MHC class II-positive cells. For each well, 2 × 10⁵ Tg4 Tcells were co-incubated with titrated cell numbers of LPS-activated, irradiated BMDCs (0, 13, 64, 320, 1600, 8000, 40 000, 80 000 and 120 000 BMDCs). After 36 h of co-incubation, [³H]TdR was added and proliferation was measured 24 h later. As positive control BMDCs from B10.PL mice were pulsed 4 h before LPS activation with Ac1-10 (4Y) at 5 μg ml^-1 and washed before the proliferation assay to remove residual peptide. The data show mean values of triplicates and the corresponding standard deviations. The experiment was performed twice with similar results.