Supplementary Figure 8: pMHC-restricted TCR signaling in SKW3.SP3.4 and SKW3.HA1.7 T cells
From: T cell receptor reversed polarity recognition of a self-antigen major histocompatibility complex
2x105 BLCL9031 APCs were incubated with or without 50 μg/ml of (a) DQ8-glia-α1 or (b) hemagglutinin (HA)-peptide for 1 hour at 37°C. BLCL9031 cells were washed twice to remove excess amounts of peptide and antibody and incubated with 1x105 (a) SKW3.SP3.4 T cells or (b) SKW3.HA1.7 T cells. Cells were briefly spun down at 4°C to allow for cell-cell conjugation. T cells were the incubated with or without peptide-pulsed BLCL9031 APCs for the indicated times at 37°C. Un-stimulated cells were left on ice (untreated) in the presence of peptide-free BLCL9031 cells. Cells were fixed and permeabilized and stained with fluorochrome-conjugated antibodies for p-(Y418) SFK, p-(Y142) CD3ζ and p-(T202/Y204) ERK1/2 and analyzed by flow cytometry. Representative histograms of two independent experiments are shown.
