TCR-Induced Transmembrane Signaling by Peptide/MHC Class II Via Associated Ig-a/b Dimers Paul Lang, John C. Stolpa, Benjamin A. Freiberg, Frances Crawford, John Kappler, Abraham Kupfer, and John C. Cambier

Supplementary Material

Note 1. Digital microscopy and capping were performed as described [C. R. Monks, H. Kupfer, I. Tamir, A. Barlow, A. Kupfer, Nature 385, 83 (1997); C. R. Monks, B. A. Freiberg, H. Kupfer, N. Sciaky, A. Kupfer, Nature 395, 82 (1998)]. For TCR analyses, cells were pulsed overnight with 100 mg ml^-1 ovalbumin (OVA), and M280 streptavidin beads (Dynal) were coupled overnight with saturating concentrations of soluble, biotinylated DO.11.10 monomers. Both cells and beads were washed extensively before incubating them together for 3 hours (6 hours for late time point) at 37°C to allow conjugate formation. Conjugates were then fixed, permeabilized, and stained. Soluble TCR from the OVA/I-A^d-reactive T cell hybridoma DO.11.10 was produced in baculovirus and purified as previously described [J. L. Seibel, N. Wilson, H. Kozono, P. Marrack, J. W. Kappler, J. Exp. Med. 185, 1919 (1997)], except that a peptide tag was added to the COOH-terminus of the b chain that was a substrate for biotinylation by the Escherichia coli enzyme BirA. The biotinylation of the soluble TCR was carried out as previously described for biotinylation of soluble MHC molecules [F. Crawford, H. Kozono, J. White, P. Marrack, J. Kappler, Immunity 8, 675 (1998)].

Note 2. For surface biotinylation and cross-linking, cells were washed three times in cold PBS, resuspended at 1 × 10⁷ ml^-1 in PBS containing 30 mg ml^-1 sulfo-NHS-LC-biotin (Pierce), and incubated for 10 min at room temperature. After two PBS washes, cells were resuspended (3 × 10⁷ ml^-1) in PBS containing 2 mM sulfo-EGS (Pierce) and incubated 30 min at 4°C. The cells were then spun down, lysed in 1% NP-40 lysis buffer, and subjected to immunoprecipitation as above. Before nonreducing SDS-PAGE analysis, the cross-linker was reduced by incubating immunoprecipitates for 3 hours at 37°C in a solution of 1 M hydroxylamine in PBS (pH 8.5). Lysis of cells in 0.33% CHAPS buffer and streptavidin blotting were carried out as described [B. J. Vilen, T. Nakamura, J. C. Cambier, Immunity 10, 239 (1999)].

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Supplemental Figure 1. (A) Antigen induces coupling of MHC class II to calcium mobilization. 3-83 md splenic B lymphocytes were cultured 9 hours without stimulus (resting) or with K^b₇-Dex. Cells were then washed, loaded with Indo1-AM, and stimulated through their BCRs (antibodies against immunogloblin) or MHC class II (antibodies against I-A^d, D3.137). Intracellular free calcium levels were monitored by flow cytometry before and during stimulation. Shown is intracelluar free Ca²⁺ as a function of time and relative cell number. The time of addition of stimuli is illustrated by the arrows. (B) Cocapping of Ig-a with MHC class II occurs in primed-phenotype K46m lymphoma cells (see note 1). Capping of MHC class II (I-E) on K46m (upper panels) and M12.g3r (lower panels) cells was achieved using biotinylated 14-4-4 (antibodies against I-E) and FITC streptavidin (in green). Alternatively, BCR were capped with biotinylated b-7-6 (antibodies against m) and FITC streptavidin. The cells were then fixed, permeabilized, and labeled with rabbit polyclonal antibody specific for Ig-a (in red), with the overlaid images shown in the third column. Note that although Ig-a coclusters with IgM in both cell lines, it only colocalizes with MHC class II in the K46m cells. (C) MHC class II is associated with Ig-a/b in K46m but not M12.g3r lymphoma cells. M12.g3r and K46m?cells were stimulated through their BCRs (antibodies against immunogloblin), MHC class II (I-A^d, D3.137, or MKD6), or MHC class I (H-2K) and lysed in CHAPS buffer. Postnuclear lysates were subjected to immunoprecipitation using antibodies specific for I-A^d/b (D3.137) coupled to Sepharose. Beads were eluted and eluates subjected to SDS-PAGE fractionation and immunoblotting with antibodies specific for phosphotyrosine or Ig-a. (D) K46 and K46 membrane immunoglobulin-negative cells express comparable levels of MHC class II. Cells were stained with FITC-conjugated antibodies specific for I-A^d/b, membrane immunoglobulin, Ig-b, or a control monoclonal antibody, as indicated, and were analyzed on a fluorescence-activated cell sorter (FACScan, Becton Dickinson). Results are representative of multiple analyses.

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