Figure 1.
DR3 surface expression in B cells.
(A) Representative flow cytometry histograms of surface DR3 expression in purified B cells, in resting conditions or following stimulation with anti-IgM (n = 10). Analyses were gated on lymphocytes (based on forward and side scatter), living cells (7AAD-negative), and B cells (CD19-positive). (B) Surface DR3 expression in resting and BCR-stimulated B cells (n = 10). Data are expressed as DR3-expression median fluorescence intensity (MFI) divided by isotype-matched control (relative median fluorescence intensity = RMFI). Comparison between resting and anti-IgM-stimulated B cells was performed by the two-sample Wilcoxon signed rank sum test. (C) Surface DR3 expression in IgM-negative (IgM-) and IgM-positive (IgM+) B cells (n = 10). Data are expressed as difference in DR3-expression median fluorescence intensity (MFI) divided by isotype-matched control (relative median fluorescence intensity = RMFI). Comparison between IgM-negative and IgM-positive B cells was performed by the Mann Whitney test. Data are represented as mean ± SEM. (D) Western blot analysis of cell lysates of purified B cells (n = 4), in resting conditions or following stimulation with anti-IgM. The level of DR3 induction after anti-IgM stimulation is reported as fold change.
Figure 2.
Expression of DR3 in tissue tonsil in vivo.
Immunofluorescence analysis of DR3 in a representative tissue tonsil section (n = 4). Pseudocolour images of DR3 200x (A) and 1000x (B), CD3 (1000x) (C), CD20 (1000x) (D). (E–F) Merged pseudocolour images of CD20 (blue), CD3 (yellow), DR3 (red) and DNA (green); (200x) (E), (1000x) (F). GC: germinal center; M: mantle zone.
Figure 3.
Expression of DR3 in tissue spleen in vivo.
Immunofluorescence analysis of DR3 in a representative tissue spleen section (n = 3). Pseudocolour images of CD20 (1000x) (A) and DR3 (1000x) (B). (C) Merged pseudocolour image of CD20 (green), DR3 (red) and DNA (blue) (200x) (1000x).
Figure 4.
B-cell proliferation induced by increasing doses of anti-IgM antibodies in the presence of IL-2.
CFSE-labeled purified B cells were activated with different doses of anti-IgM antibodies (1, 2, 5, 10, 20 µg/ml) in the presence or absence of 20 U/ml IL-2 for 96 h, and analyzed for CFSE dilution. Three parameters were calculated (division index, percentage (%) divided and number of divisions) and represented in distinct graphs.
Figure 5.
TL1A reduces proliferation of activated B-cells.
(A) Representative flow cytometry histograms of CFSE-labeled B cells stimulated (empty curve) or not (grey curve) with 2 µg/ml (n = 8) or 20 µg/ml (n = 3) anti-IgM and 20 U/ml IL-2 for 96 h, in the presence or absence of 100 ng/ml TL1A. Bold numbers indicate percentage of proliferating cells. Analyses are gated on lymphocytes (based on forward and side scatter), and living (7AAD-negative) B cells (CD19-positive). (B) Proliferating B cells were stimulated with 2 µg/ml (n = 8) or 20 µg/ml (n = 3) anti-IgM and 20 U/ml IL-2 for 96 h, in the presence or absence of 100 ng/ml TL1A. Three parameters were calculated (division index, % divided and number of divisions) and represented as distinct histograms. Data are represented as mean±SEM. Comparison between treatments was performed by the two-sample Wilcoxon signed rank sum test. (C) Proliferation of B cells stimulated with 2 µg/ml anti-IgM, 20 U/ml IL-2 for 96 h, in the presence of different doses of TL1A (n = 3). Three parameters were calculated (division index, % divided and number of divisions) and represented as separated histograms. Data are represented as mean±SEM. * = p<0.05. (D) Time-course experiment (n = 2) of CFSE-labeled purified B cells stimulated with 2 µg/ml anti-IgM, 20 U/ml IL-2; 2 µg/ml anti-IgM, 20 U/ml IL-2, 100 ng/ml TL1A; 100 ng/ml TL1A; or medium. Three parameters were calculated (division index, % divided and number of divisions) and represented as separated graphs.
Figure 6.
TL1A induces similar reduction extents of proliferation in the CD27+ and CD27− B-cell subsets.
CFSE-labeled purified B cells (n = 2) were activated with 2 µg/ml anti IgM, 20 U/ml IL-2 for 96 h, in presence or absence of 100 ng/ml TL1A. Analyses were gated on lymphocytes (based on forward and side scatter), living (7AAD-negative) and CD19-positive cells and discriminating CD27+ and CD27− cells. Three parameters were calculated (division index, % divided and number of divisions) and represented as separated histograms. Data are represented as mean±SEM. * = p<0.05.
Figure 7.
TL1A does not modulate surface expression of CD19, CD20, CD38, and CD138 in B cells.
Data from a representative experiment (n = 2) on CD19, CD20, CD38, and CD138 expression in B cells activated with 2 µg/ml anti-IgM, 20 U/ml IL-2 in the presence or absence of 100 ng/ml TL1A at different time point (24, 48, 72, 96 h).
Figure 8.
TL1A does not modulate B-cell proliferation induced by anti-IgM and CpG-ODN or CD40L.
(A) Proliferating B cells were stimulated with different doses of anti-IgM antibodies (1, 2, 5, 10, 20 µg/ml) and of 2.5 µg/ml CpG-ODN for 96 h, in the presence or absence of 100 ng/ml TL1A (n = 4). Three parameters were calculated (division index, % divided and number of divisions) and represented as separated graphs. (B) Proliferating B cells were stimulated with different doses of anti-IgM antibodies (1, 2, 5, 10, 20 µg/ml) in the presence of 50 ng/ml CD40L for 96 h, in the presence or absence of 100 ng/ml TL1A (n = 4). Three parameters were calculated (division index, % divided and number of divisions) and represented as separated graphs.
Figure 9.
TL1A does not affect B-cell survival.
Purified B cells were activated with 2 µg/ml anti-IgM, 20 U/ml IL-2 for 24, 48,72 and 96 h, in presence or absence of 100 ng/ml TL1A, stained with annexinV/PI and analyzed by flow cytometry. Analyses were gated on CD19-positive cells. (A) Dot plots representatives of three independent experiments. Bold numbers indicate percentage of cells in each quadrant. (B) Percentage of viable cells (annexinV-negative/PI-negative) following incubation with 2 µg/ml anti-IgM, 20 U/ml IL-2; 2 µg/ml anti-IgM, 20 U/ml IL-2, 100 ng/ml TL1A; medium. Data are represented as mean±SEM. * = p<0.05; ns = not significant.