Fig 1.
TG2-specific B cells and gluten-specific CD4+ T cells interact in vitro.
B cells isolated from Tgm2-/- DQ2.5.14E06 or HLA-DQ2.5 mice, and CD4+ T cells isolated from DQ2.5.TCR-glia-α2 or DQ2.5.TCR-glia-ω2 mice were labeled with different proliferation-tracking dyes and co-cultured in vitro for three days stimulated with indicated antigens. (A) Representative histogram plots gated on CD4+ T cells showing proliferation in response to TG2-α33merEEE fusion protein at 0,2 μg/mL. (B) Dose response curve of T-cell proliferation to TG2-α33merEEE fusion protein. (C) Representative histogram plots gated on B220+ B cells showing proliferation in response to TG2-α33merEEE fusion or TG2 at 5μg/mL. (D) Dose response curve of B-cell proliferation to titrations of TG2-α33merEEE fusion protein. Asterisk (*) indicates the undivided peak. Graphs report proliferation as % proliferated CD4+ T or B220+ B cells. Points and bars represent mean +/- SEM. Data is representative of three independent experiments set in duplicates. CFSE: Cell Trace Carboxyfluorescein Succinimidyl Ester CTV: Cell Trace Violet.
Fig 2.
TG2-gluten multimers are superior to monomers at activating gluten-specific T cells and TG2-specific B cells.
B cells isolated from Tgm2-/- DQ2.5.14E06 mice and CD4+ T cells isolated from DQ2.5.TCR-glia-α2 mice were labeled with different proliferation-tracking dyes and co-cultured in vitro for three days stimulated with indicated antigens. (A,B) Dose response curve of proliferative response to TG2-α33merEEE fusion monomers and multimers for CD4+ T cells (A) and B cells (B). (C,D) Dose response curve of proliferative response to TG2-α33mer monomers and multimers for CD4+ T cells (C) and B cells (D). Graphs report proliferation as % proliferated when gated on dye-labeled CD4+ or B220+ cells. Points and bars represent mean +/- SEM. Data is representative of three independent experiments set in duplicates.
Fig 3.
TG2-specific B cells expand in vivo upon help from gluten-specific CD4+ T cells.
(A) Experimental setup: CD4+ T cells isolated from DQ2.5.TCR-glia-α2 or DQ2.5.TCR-glia-ω2 mice were administered intravenously to HLA-DQ2.5 expressing recipient mice and primed with cognate deamidated 33mer α-gliadin or cognate deamidated 34mer ω-gliadin peptides emulsified in CFA. TG2-specific B cells were isolated from DQ2.5.14E06 mice, labeled with CTV and administered intravenously 4 days after T-cell priming. Antigen was administered intramuscularly the following day. B cell proliferation was assayed by flow cytometry 6 days after immunization. (B) Representative histogram plots gated on B220+ 14E06+ cells show B cell proliferation in draining popliteal and inguinal LNs. The panel shows response to TG2-α33merEEE fusion protein, TG2, and to pre-formed monomeric and multimeric TG2-α33mer complexes. Data are representative of in total n = 6 (TG2-α33merEEE fusion protein, TG2) or n = 4 (TG2-α33mer monomers, multimers) from at least 3 independent experiments). CTV: Cell Trace Violet.
Fig 4.
DGP-specific B cells efficiently bind deamidated gliadin peptides.
(A) Binding of deamidated ω34mer and α33mer gluten peptide by A20 transfectant B cells expressing either HLA-DQ2.5 alone (black line, no fill), HLA-DQ2.5 and an irrelevant IgD BCR (693-2-F02; blue) or HLA-DQ2.5 and the gluten-specific IgD BCR 1130-3-B01 (red). Cells were stained with biotinylated deamidated ω34mer or α33mer that was multimerized with APC-conjugated streptavidin. (B). Presentation of deamidated gluten peptides by irradiated transfectant A20 B cells expressing HLA-DQ2.5 and the DGP-specific 3B01 IgD BCR or the 2F02 control BCR. A20 B cells were either pulsed with indicated concentrations of deamidated ω34mer or α33mer peptide for 5 min on ice, or peptides were present continuously during the assay. A20 B cells were cultured overnight at 37°C before gluten-specific T cells specific for the DQ2.5-glia-ω1 (TCC 1383.P.A.6), DQ2.5-glia-ω2 (TCC 737.30), DQ2.5-glia-α1 (TCC 430.1.57) and DQ2.5-glia-α2 (TCC 436.5.3) epitopes were added. T-cell proliferation was assessed by [3H] thymidine incorporation 72h later. Symbols represent mean counts per minute (CPM) +/- SEM of culture duplicates or triplicates and are representative of at least 3 independent experiments.
Fig 5.
TG2-gluten complexes are efficient antigens for both DGP-specific and TG2-specific B cells and allow for help of T cells with many different specificities.
(A) Amino acid sequences of the rhodamine-labeled α33mer and the FITC-labeled ω34mer peptides. Glutamine residues expected to be targeted by TG2 are enlarged and given in bold. (B) Gradient SDS-PAGE (4–20%) showing incorporation of FITC-conjugated ω-34mer and Rhodamine-conjugated α33mer into both TG2 monomers, dimers and multimers. The gel shows TG2-gluten complexes separated into monomers (lanes 1, 4), dimers/ trimers (lanes 2, 5) and multimers (lanes 3, 6) made using a 2:1 (lanes 1–3) or 17:1 (lanes 4–6) molar ratio of α33mer: ω34mer peptide. Arrow indicates monomeric TG2 (78 kDa). (C) The ω34mer peptide is crosslinked to TG2 to a higher degree than the α33mer peptide. Protein concentration and fluorescent labels were quantified using a Nanodrop UV-Vis spectrophotometer, and the number of fluorescent peptides per TG2 molecule were calculated. The table shows the average values of monomers, dimers/trimers and multimers of one representative experiment. (D) Irradiated A20 B cells expressing HLA-DQ2.5 and a TG2-specific BCR (14E06), gluten-specific BCR (3B01) or non-specific BCR (2F02) were incubated with complexes of TG2 and FITC-labeled native ω34mer gliadin peptide and rhodamine B-labeled α33mer gliadin peptide, and their ability to present deamidated peptides to TCC specific for the DQ2.5-glia-ω1 (TCC1383.P.A.6), DQ2.5-glia-ω2 (TCC737.30), DQ2.5-glia-α1 epitope (TCC430.1.57) or DQ2.5-glia-α2 epitope (TCC436.5.3) was assessed by [3H] thymidine incorporation. Symbols represent mean counts per minute (CPM) +/- SEM of culture triplicates. Data are representative of 3 independent experiments.
Fig 6.
Activation of gluten-specific T cells following pulsing of A20 B cells with active TG2 and native gluten peptides.
(A) Irradiated A20 cells expressing HLA-DQ2.5 and a TG2-specific BCR (14E06), gluten-specific BCR (3B01) or non-specific BCR (2F02) were incubated with different concentrations of native ω34mer or native α33mer gliadin peptide in the presence of 50 nM TG2 and 2 mM CaCl2 for 5 min at 37°C, then washed and cultured at 37°C. The next day, gluten-specific TCC specific for the DQ2.5-glia-ω1 (TCC1383.P.A.6), DQ2.5-glia-ω2 (TCC737.30), DQ2.5-glia-α1 epitope (TCC387.9) or DQ2.5-glia-α2 epitope (TCC436.5.3) were added. T-cell proliferation was assessed after 72h by [3H] thymidine incorporation. Symbols represent mean counts per minute (CPM) +/- SEM of culture duplicates and are representative of 2–3 independent experiments.