Fig. 4: Effects of TG3 binding by TG3-specific BCRs.

a Flow cytometry histograms showing staining of BCR-transduced A20 B cell lines targeting three different TG3 epitopes (Ep.1-3). The cells were assessed for surface IgD expression or binding to monomeric TG3 by staining with recombinant biotinylated TG3 followed by PE-conjugated streptavidin (SA-PE). b Western blot showing detection of IgD in A20 cell lysates. The cells were incubated with active (cleaved) or zymogen (uncleaved) TG3 prior to lysis and reducing SDS-PAGE. Two monomeric δ heavy chain bands are visible, representing the fully glycosylated form with both O-linked and N-linked glycans (open triangle) and a partially glycosylated form with N-linked glycans only (closed triangle). The upper band disappears or is diminished after incubation with cleaved TG3 due to incorporation into high-MW complexes, some of which are too large to enter the gel. One of three experiments is shown. c-e, Calcium flux assay showing representative flow cytometry signals (c) and quantification of peak heights (d) and peak time points (e). After recording baseline signal for 30 s, BCR-transduced A20 cells were stimulated with cleaved or uncleaved TG3 (indicated by arrows). A crosslinking anti-IgD F(ab’)2 was included as positive control for BCR-induced Ca²⁺ release. Bar heights indicate means of two experiments. f, g Uptake of fluorescently labeled gluten peptide by BCR-transduced A20 cells. Representative histograms of A20 cells pre-incubated in the presence or absence of cleaved or uncleaved TG3 are shown in (f). Peptide uptake relative to the amount of BCR-bound TG3 (g) was assessed as the ratio between median fluorescence intensity (MFI) of cell-associated peptide in presence of cleaved TG3 (f) and MFI of BCR-bound TG3 (a). Bar heights indicate means of two experiments. h, Stimulation of DQ2.5-glia-ω2-specific BW58 T cells by BCR-transduced A20 B cells expressing HLA-DQ2.5. The cells were pulsed with native gluten peptide in combination with cleaved or uncleaved TG3 prior to co-culture with T cells. Symbols represent means of culture triplicates, and error bars indicate SD. Results from one of two experiments are shown. Source data are provided as a Source Data file.