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, 8 (4), e60136

The TNF-family Cytokine TL1A Inhibits Proliferation of Human Activated B Cells

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The TNF-family Cytokine TL1A Inhibits Proliferation of Human Activated B Cells

Chiara Cavallini et al. PLoS One.

Abstract

Death receptor (DR3) 3 is a member of the TNFR superfamily. Its ligand is TNF-like ligand 1A (TL1A), a member of the TNF superfamily. TL1A/DR3 interactions have been reported to modulate the functions of T cells, NK, and NKT cells and play a crucial role in driving inflammatory processes in several T-cell-dependent autoimmune diseases. However, TL1A expression and effects on B cells remain largely unknown. In this study, we described for the first time that B cells from human blood express significant amounts of DR3 in response to B cell receptor polyclonal stimulation. The relevance of these results has been confirmed by immunofluorescence analysis in tonsil and spleen tissue specimens, which showed the in situ expression of DR3 in antigen-stimulated B cells in vivo. Remarkably, we demonstrated that TL1A reduces B-cell proliferation induced by anti-IgM-antibodies and IL-2 but did not affect B-cell survival, suggesting that TL1A inhibits the signal(s) important for B-cell proliferation. These results revealed a novel function of TL1A in modulating B-cell proliferation in vitro and suggest that TL1A may contribute to homeostasis of effector B-cell functions in immune response and host defense, thus supporting the role of the TL1A/DR3 functional axis in modulating the adaptive immune response.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
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
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
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
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
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
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
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
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
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.

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This study was granted by Fondazione Cassa di Risparmio di Verona, Vicenza, Belluno e Ancona and Associazione Italiana Ricerca sul Cancro (AIRC) (grant #6599) and Fondazione Cassa di Risparmio di Verona, Vicenza, Belluno, e Ancona (grant #2008.14.44). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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