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B cells drive lymphocyte activation and expansion in mice with the CD45 wedge mutation and Fas deficiency.

Gupta VA, Hermiston ML, Cassafer G, Daikh DI, Weiss A - J. Exp. Med. (2008)

Bottom Line: T cell activation required signaling in response to endogenous or commensal antigens, demonstrated by the introduction of a transgenic T cell receptor.Genetic deletion of B cells also prevented T cell activation.Similarly, T cells were necessary for B cell autoantibody production.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Biomedical Sciences Graduate Program, Medical Scientist Training Program, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
CD45 and Fas regulate tyrosine phosphorylation and apoptotic signaling pathways, respectively. Mutation of an inhibitory wedge motif in CD45 (E613R) results in hyperresponsive thymocytes and B cells on the C57BL/6 background, but no overt autoimmunity, whereas Fas deletion results in a mild autoimmune disease on the same genetic background. In this study, we show that these two mutations cooperate in mice, causing early lethality, autoantibody production, and substantial lymphoproliferation. In double-mutant mice, this phenotype was dependent on both T and B cells. T cell activation required signaling in response to endogenous or commensal antigens, demonstrated by the introduction of a transgenic T cell receptor. Genetic deletion of B cells also prevented T cell activation. Similarly, T cells were necessary for B cell autoantibody production. However, B cells appeared to be intrinsically activated even in the absence of T cells, suggesting that they may drive the phenotype of these mice. These results reveal a requirement for careful control of B cell signaling and cell death in preventing inappropriate lymphocyte activation and autoimmunity.

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TCR signaling and T cells contribute to lymphocyte activation and expansion. (A) Splenic weight as an indicator of lymphoproliferation in normal, OT2 TCR transgenic (OT2), T cell-deficient (TCRα−/−), and B cell–deficient mice (Ig−/−) comparing 2-mo-old WT and wedge/lpr. Each point represents a single mouse, five to six mice per genotype. Bars represent the mean of the group. ANOVA, P < 0.0001. (B) Representative flow cytometric analysis of CD4 memory phenotype cells in 2-mo-old OT2 TCR transgenic mice. OT2 Tg+ cells were gated as CD3+ CD4+ Vα2+ Vβ5+. Tg− cells are CD3+ CD4+ Vα2− Vβ5−. Memory phenotype cells are CD44high, whereas naive cells are CD44low, CD62Lhigh. Numbers represent percentage of CD4+ cells. Data are representative of two to four mice from three independent experiments. (C) MHC class II and CD86 expression on CD19+ B cells and CD11c+ DCs from 2-mo-old TCRα−/− mice, shown as the mean MFI for 4 mice per genotype ± SEM. Data are shown as relative MFI to correct for different antibody clones and fluorophores. Data are representative of three independent experiments. Unpaired Student's t test, P < 0.05 compared with WT, *; wedge, **; and lpr, ***.
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fig4: TCR signaling and T cells contribute to lymphocyte activation and expansion. (A) Splenic weight as an indicator of lymphoproliferation in normal, OT2 TCR transgenic (OT2), T cell-deficient (TCRα−/−), and B cell–deficient mice (Ig−/−) comparing 2-mo-old WT and wedge/lpr. Each point represents a single mouse, five to six mice per genotype. Bars represent the mean of the group. ANOVA, P < 0.0001. (B) Representative flow cytometric analysis of CD4 memory phenotype cells in 2-mo-old OT2 TCR transgenic mice. OT2 Tg+ cells were gated as CD3+ CD4+ Vα2+ Vβ5+. Tg− cells are CD3+ CD4+ Vα2− Vβ5−. Memory phenotype cells are CD44high, whereas naive cells are CD44low, CD62Lhigh. Numbers represent percentage of CD4+ cells. Data are representative of two to four mice from three independent experiments. (C) MHC class II and CD86 expression on CD19+ B cells and CD11c+ DCs from 2-mo-old TCRα−/− mice, shown as the mean MFI for 4 mice per genotype ± SEM. Data are shown as relative MFI to correct for different antibody clones and fluorophores. Data are representative of three independent experiments. Unpaired Student's t test, P < 0.05 compared with WT, *; wedge, **; and lpr, ***.

Mentions: To examine the role of endogenous signaling through the TCR, we restricted the T cell repertoire to a single TCR using the ovalbumin peptide–reactive OT2 TCR transgene. The OT2 TCR transgene completely eliminated the presence of memory phenotype T cells in wedge/lpr mice (Fig. 4 B). In these mice, the small fraction of T cells escaping OT2 TCR restriction showed a slight increase in the percentage of memory cells compared with WT OT2. The degree of activation in these cells likely depends on the specific TCR transgene, the efficiency of restriction, and the strain background (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20081204/DC1). Restricting the TCR repertoire also corrected the lymphadenopathy and splenomegaly, as well as the survival defect seen in wedge/lpr mice (Fig. 4 A). None of the mice in a cohort of 6 wedge/lpr OT2 mice had died at 6 mo compared with the 50% mortality seen at this time point in nontransgenic wedge/lpr mice (unpublished data). Genetic elimination of T cells using TCRα−/− mice also prevented lymphadenopathy and splenomegaly (Fig. 4 A). Furthermore, wedge/lpr TCRα−/− mice failed to produce autoantibodies by 5 mo of age (unpublished data). Wedge/lpr B cells must therefore require T cell help to expand and secrete autoantibodies. Interestingly, B cells and DCs from wedge and wedge/lpr TCRα−/− mice still expressed higher levels of MHC class II and CD86, suggesting that B cell and DC activation is a T cell–independent effect that results mainly from the wedge mutation alone (Fig. 4 C).


B cells drive lymphocyte activation and expansion in mice with the CD45 wedge mutation and Fas deficiency.

Gupta VA, Hermiston ML, Cassafer G, Daikh DI, Weiss A - J. Exp. Med. (2008)

TCR signaling and T cells contribute to lymphocyte activation and expansion. (A) Splenic weight as an indicator of lymphoproliferation in normal, OT2 TCR transgenic (OT2), T cell-deficient (TCRα−/−), and B cell–deficient mice (Ig−/−) comparing 2-mo-old WT and wedge/lpr. Each point represents a single mouse, five to six mice per genotype. Bars represent the mean of the group. ANOVA, P < 0.0001. (B) Representative flow cytometric analysis of CD4 memory phenotype cells in 2-mo-old OT2 TCR transgenic mice. OT2 Tg+ cells were gated as CD3+ CD4+ Vα2+ Vβ5+. Tg− cells are CD3+ CD4+ Vα2− Vβ5−. Memory phenotype cells are CD44high, whereas naive cells are CD44low, CD62Lhigh. Numbers represent percentage of CD4+ cells. Data are representative of two to four mice from three independent experiments. (C) MHC class II and CD86 expression on CD19+ B cells and CD11c+ DCs from 2-mo-old TCRα−/− mice, shown as the mean MFI for 4 mice per genotype ± SEM. Data are shown as relative MFI to correct for different antibody clones and fluorophores. Data are representative of three independent experiments. Unpaired Student's t test, P < 0.05 compared with WT, *; wedge, **; and lpr, ***.
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fig4: TCR signaling and T cells contribute to lymphocyte activation and expansion. (A) Splenic weight as an indicator of lymphoproliferation in normal, OT2 TCR transgenic (OT2), T cell-deficient (TCRα−/−), and B cell–deficient mice (Ig−/−) comparing 2-mo-old WT and wedge/lpr. Each point represents a single mouse, five to six mice per genotype. Bars represent the mean of the group. ANOVA, P < 0.0001. (B) Representative flow cytometric analysis of CD4 memory phenotype cells in 2-mo-old OT2 TCR transgenic mice. OT2 Tg+ cells were gated as CD3+ CD4+ Vα2+ Vβ5+. Tg− cells are CD3+ CD4+ Vα2− Vβ5−. Memory phenotype cells are CD44high, whereas naive cells are CD44low, CD62Lhigh. Numbers represent percentage of CD4+ cells. Data are representative of two to four mice from three independent experiments. (C) MHC class II and CD86 expression on CD19+ B cells and CD11c+ DCs from 2-mo-old TCRα−/− mice, shown as the mean MFI for 4 mice per genotype ± SEM. Data are shown as relative MFI to correct for different antibody clones and fluorophores. Data are representative of three independent experiments. Unpaired Student's t test, P < 0.05 compared with WT, *; wedge, **; and lpr, ***.
Mentions: To examine the role of endogenous signaling through the TCR, we restricted the T cell repertoire to a single TCR using the ovalbumin peptide–reactive OT2 TCR transgene. The OT2 TCR transgene completely eliminated the presence of memory phenotype T cells in wedge/lpr mice (Fig. 4 B). In these mice, the small fraction of T cells escaping OT2 TCR restriction showed a slight increase in the percentage of memory cells compared with WT OT2. The degree of activation in these cells likely depends on the specific TCR transgene, the efficiency of restriction, and the strain background (Fig. S4, available at http://www.jem.org/cgi/content/full/jem.20081204/DC1). Restricting the TCR repertoire also corrected the lymphadenopathy and splenomegaly, as well as the survival defect seen in wedge/lpr mice (Fig. 4 A). None of the mice in a cohort of 6 wedge/lpr OT2 mice had died at 6 mo compared with the 50% mortality seen at this time point in nontransgenic wedge/lpr mice (unpublished data). Genetic elimination of T cells using TCRα−/− mice also prevented lymphadenopathy and splenomegaly (Fig. 4 A). Furthermore, wedge/lpr TCRα−/− mice failed to produce autoantibodies by 5 mo of age (unpublished data). Wedge/lpr B cells must therefore require T cell help to expand and secrete autoantibodies. Interestingly, B cells and DCs from wedge and wedge/lpr TCRα−/− mice still expressed higher levels of MHC class II and CD86, suggesting that B cell and DC activation is a T cell–independent effect that results mainly from the wedge mutation alone (Fig. 4 C).

Bottom Line: T cell activation required signaling in response to endogenous or commensal antigens, demonstrated by the introduction of a transgenic T cell receptor.Genetic deletion of B cells also prevented T cell activation.Similarly, T cells were necessary for B cell autoantibody production.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Biomedical Sciences Graduate Program, Medical Scientist Training Program, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
CD45 and Fas regulate tyrosine phosphorylation and apoptotic signaling pathways, respectively. Mutation of an inhibitory wedge motif in CD45 (E613R) results in hyperresponsive thymocytes and B cells on the C57BL/6 background, but no overt autoimmunity, whereas Fas deletion results in a mild autoimmune disease on the same genetic background. In this study, we show that these two mutations cooperate in mice, causing early lethality, autoantibody production, and substantial lymphoproliferation. In double-mutant mice, this phenotype was dependent on both T and B cells. T cell activation required signaling in response to endogenous or commensal antigens, demonstrated by the introduction of a transgenic T cell receptor. Genetic deletion of B cells also prevented T cell activation. Similarly, T cells were necessary for B cell autoantibody production. However, B cells appeared to be intrinsically activated even in the absence of T cells, suggesting that they may drive the phenotype of these mice. These results reveal a requirement for careful control of B cell signaling and cell death in preventing inappropriate lymphocyte activation and autoimmunity.

Show MeSH
Related in: MedlinePlus