Research ArticleImmunology

The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development

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Science Signaling  01 Sep 2015:
Vol. 8, Issue 392, pp. ra88
DOI: 10.1126/scisignal.aaa5157
  • Fig. 1 TRAF3 inhibits the development of PCs.

    (A) Representative plots from the flow cytometric analysis of cells from the spleen (SP) and bone marrow (BM) of B-Traf3−/− mice and littermate control (LMC) mice. Outlined areas and numbers indicate the percentages of CD138+B220low PCs. Data are representative of four experiments. (B) Percentages (left) and numbers (right) of CD138+B220low PCs in the spleens and bone marrow of littermate control mice and B-Traf3−/− mice based on data as identified in (A). Each symbol represents a single mouse, and the horizontal line indicates the mean value of each group. (C) Left: Representative wells from the enzyme-linked immunospot (ELISPOT) analysis of ASCs in the spleen and bone marrow of littermate control mice and B-Traf3−/− mice. Right: The numbers of ASCs from the spleen and bone marrow of mice of each strain. Each symbol represents the mean of technical triplicate samples from a single mouse, and the horizontal lines indicate mean values of six mice per group. ***P < 0.001, **P < 0.01, *P < 0.05 by Student’s t test.

  • Fig. 2 The enhanced development of PCs in B-Traf3−/− mice is IL-6–dependent.

    (A) Representative plots from the flow cytometric analysis of spleen and bone marrow cells from littermate control mice, B-Traf3−/−, IL6−/−, and B-Traf3−/−IL6−/− mice. Outlined areas and numbers indicate the percentages of CD138+B220low PCs. (B) Quantification of the percentages of PCs in the bone marrow (left) and spleens (right) of the indicated mice from the experiments depicted in (A). Data are means ± SEM of values from at least four mice from each group. *P < 0.05; ns, not statistically significant. (C) ELISPOT analysis of splenocytes and bone marrow cells from the indicated mouse strains. The graph shows the numbers of ASCs; each symbol represents a technical triplicate, whereas the lines indicate the mean values from three mice per group. **P < 0.01 by one-way analysis of variance (ANOVA). (D) Sorted PCs from the bone marrow of the indicated strains of mice were cultured in the presence or absence of IL-6 (50 ng/ml) for the indicated times. The percentage cell viability in each sample was determined by flow cytometric analysis of the percentage of propidium iodide (PI)–negative cells. Data are means ± SEM from three independent experiments. **P < 0.01, *P < 0.05 by one-way ANOVA. (E) Splenic B cells from the indicated mice were stimulated with LPS (10 μg/ml) in the presence or absence of IL-6 (20 ng/ml) for 3 days before they were analyzed by flow cytometry to detect CD138+B220low PCs. Numbers in the plots represent the percentages of CD138+B220low PCs. (F) Quantification of the percentages of CD138+B220low PCs in the experiments shown in (E). Data are means ± SEM values from three independent experiments. *P < 0.05 by Student’s t test.

  • Fig. 3 TRAF3 inhibits the IL-6–dependent activation of STAT3.

    (A) Top: TRAF3-sufficient and TRAF3-deficient subclones of the mouse B cell line A20.2J (A20 and A20.T3−/− cells, respectively) were left untreated or were stimulated with IL-6 for the indicated times. Whole-cell lysates were then subjected to Western blotting analysis with antibodies against the indicated proteins. Western blots are representative of three independent experiments. Bottom: Densitometric analysis of the ratio of the abundance of pSTAT3 protein to that of total STAT3 protein, setting the maximum ratio in TRAF3-sufficient B cells at 1. Data are means ± SD of three independent experiments. (B) A20.T3−/− cells were stably transfected with a plasmid encoding IPTG-inducible Traf3. Cells were cultured in the presence or absence of IPTG for 12 hours before being stimulated with IL-6 for the indicated times and then analyzed by Western blotting with antibodies against the indicated proteins. Western blots are from one experiment and are representative of three independent experiments. (C) Top: Splenic B cells from the indicated mice were left unstimulated or were stimulated with IL-6 for the indicated times before being analyzed by Western blotting as described in (A). Western blots are representative of five individual experiments. Bottom: Densitometric analysis of the ratio of the abundance of pSTAT3 protein to that of total STAT3 protein, setting the maximum ratio in TRAF3-sufficient B cells at 1. Data are means ± SD of five independent experiments. (D) Left: Normal human peripheral B cells isolated as described in Materials and Methods were transfected with the indicated siRNAs before being left untreated or treated with recombinant human IL-6 for 30 min. Samples were then analyzed by Western blotting as described in (A). Western blots are from a single experiment and are representative of three experiments. Right: Data are means ± SEM of the ratio of the abundances of pSTAT3 protein to total STAT3 protein from three experiments, setting the maximum ratio in cells transfected with control siRNA (sictrl) at 1. *P < 0.05 by Student’s t test. (E and F) Splenic B cells from the indicated mice were left untreated or were stimulated with IL-6 for 30 min before being subjected to immunoprecipitation (IP) of (E) Jak1 or (F) gp130. Samples were then analyzed by Western blotting with antibodies specific for (E) pJak1 and total Jak1 or (F) phosphotyrosines (pY) and gp130. Western blots are from a single experiment and are representative of four experiments. (G to I) A20.2J cell lines (G) or splenic B cells (H and I) were left untreated or were treated with IL-6 for the indicated times. Cell lysates were then subjected to immunoprecipitation with antibodies against the indicated proteins before being analyzed by Western blotting. Blots are from a single experiment and are representative of two independent experiments. (J) Human B cells were left untreated or were treated with recombinant human IL-6 for 30 min. Cell lysates were then subjected to immunoprecipitation with antibody against TRAF3, and the samples were then analyzed by Western blotting with antibodies against the indicated proteins. Western blots are from a single experiment and are representative of two independent experiments. hc, heavy chain.

  • Fig. 4 TRAF3 interacts with PTPN22 to inhibit IL-6R signaling and PC development.

    (A and B) Splenic B cells (A) and the indicated A20.2J cell lines (B) were left untreated or were treated with IL-6 for 30 min before being subjected to immunoprecipitation with an antibody specific for Jak1. An IgG control was included to control for the specificity of the anti-Jak1 antibody. Immunoprecipitates were then analyzed by Western blotting with antibodies specific for the indicated proteins. Blots are from a single experiment and are representative of three independent experiments. (C) Splenic B cells from the indicated mice were left untreated or were treated with IL-6 for 30 min. Whole-cell lysates were subjected to the immunoprecipitation of Jak1, and samples were then analyzed by Western blotting to detect PTPN22. As a control, whole-cell lysates (input) were also analyzed. Blots are from a single experiment and are representative of three independent experiments. (D) Left: Splenic B cells from the indicated mice were left untreated or were treated with IL-6 for the indicated times. Whole-cell lysates were then analyzed by Western blotting with antibodies against the indicated targets. Western blots are representative of three individual experiments. Right: Densitometric analysis of the ratio of the abundance of pSTAT3 protein to that of total STAT3 protein, setting the maximum ratio in PTPN22-sufficient B cells at 1. Data are means ± SD of five independent experiments. (E) Representative plots from the flow cytometric analysis of CD138+B220low PCs in the spleen and bone marrow of the indicated mice. The outlined areas and numbers indicate the percentages of the CD138+B220low PCs. (F) Quantification of the percentages of CD138+B220low PCs in the bone marrow (top) and spleens (bottom) of the indicated mouse strains from the experiments depicted in (E). Data are means ± SEM of six (Ptpn22−/−), four [wild type (WT)], and three (B-Traf3−/−) mice. (G) ELISPOT analysis of IgM-secreting (left) and IgG-secreting (right) ASCs in the spleen and bone marrow of the indicated mice. The graph represents the numbers of ASCs from the bone marrow and spleen. Each symbol represents the mean of technical triplicates from a single mouse, whereas the horizontal lines indicate means from three mice per group. *P < 0.05, **P < 0.01 by one-way ANOVA.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/392/ra88/DC1

    Fig. S1. The increase in the number of B cells caused by TRAF3 deficiency is independent of IL-6.

    Fig. S2. Loss of TRAF3 in B cells does not alter the amounts of serum IL-6 in mice.

    Fig. S3. Loss of TRAF3 does not affect IL-6R abundance on B cells.

    Fig. S4. TRAF3 is not required for the IL-21–dependent activation of STAT3.

    Fig. S5. TCPTP does not associate with Jak1 in B cells in response to IL-6.

    Fig. S6. PTPN22-deficient B cells do not show enhanced PC differentiation in vitro in response to LPS and IL-6.

  • Supplementary Materials for:

    The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development

    Wai W. Lin, Zuoan Yi, Laura L. Stunz, Christian J. Maine, Linda A. Sherman, Gail A. Bishop*

    *Corresponding author. E-mail: gail-bishop{at}uiowa.edu

    This PDF file includes:

    • Fig. S1. The increase in the number of B cells caused by TRAF3 deficiency is independent of IL-6.
    • Fig. S2. Loss of TRAF3 in B cells does not alter the amounts of serum IL-6 in mice.
    • Fig. S3. Loss of TRAF3 does not affect IL-6R abundance on B cells.
    • Fig. S4. TRAF3 is not required for the IL-21–dependent activation of STAT3.
    • Fig. S5. TCPTP does not associate with Jak1 in B cells in response to IL-6.
    • Fig. S6. PTPN22-deficient B cells do not show enhanced PC differentiation in vitro in response to LPS and IL-6.

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    Citation: W. W. Lin, Z. Yi, L. L. Stunz, C. J. Maine, L. A. Sherman, G. A. Bishop, The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development. Sci. Signal. 8, ra88 (2015).

    © 2015 American Association for the Advancement of Science

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