Impaired B cell anergy is not sufficient to breach tolerance to nuclear antigen in Vκ8/3H9 lupus-prone mice.

BackgroundSystemic lupus erythematosus (SLE) is a severe autoimmune disease in which immune tolerance defects drive production of pathogenic anti-nuclear autoantibodies. Anergic B cells are considered a potential source of these autoantibodies due to their autoreactivity and overrepresentation in SLE patients. Studies of lupus-prone mice have shown that genetic defects mediating autoimmunity can breach B cell anergy, but how this breach occurs with regards to endogenous nuclear antigen remains unclear. We investigated whether B and T cell defects in congenic mice (c1) derived from the lupus-prone New Zealand Black strain can breach tolerance to nuclear self-antigen in the presence of knock-in genes (Vκ8/3H9; dKI) that generate a ssDNA-reactive, anergic B cell population.MethodsFlow cytometry was used to assess splenic B and T cells from 8-month-old c1 dKI mice and serum autoantibodies were measured by ELISA. dKI B cells stimulated in vitro with anti-IgM were assessed for proliferation and activation by examining CFSE decay and CD86. Cytokine-producing T cells were identified by flow cytometry following culture of dKI splenocytes with PMA and ionomycin. dKI B cells from 6-8-week-old mice were adoptively transferred into 4-month-old wild type recipients and assessed after 7 days via flow cytometry and immunofluorescence microscopy.Resultsc1 dKI mice exhibited B cell proliferation indicative of impaired anergy, but had attenuated autoantibodies and germinal centres compared to wild type littermates. This attenuation appeared to stem from a decrease in PD-1hi T helper cells in the dKI strains, as c1 dKI B cells were recruited to germinal centres when adoptively transferred into c1 wild type mice.ConclusionAnergic, DNA-specific autoreactive B cells only seem to drive profound autoimmunity in the presence of concomitant defects in the T cell subsets that support high-affinity plasma cell production

Multiple tolerance defects contribute to the breach of B cell tolerance in New Zealand Black chromosome 1 congenic mice

<div><p>Lupus is characterized by a loss of B cell tolerance leading to autoantibody production. In this study, we explored the mechanisms underlying this loss of tolerance using B6 congenic mice with an interval from New Zealand Black chromosome 1 (denoted c1(96–100)) sufficient for anti-nuclear antibody production. Transgenes for soluble hen egg white lysozyme (sHEL) and anti-HEL immunoglobulin were crossed onto this background and various tolerance mechanisms examined. We found that c1(96–100) mice produced increased levels of IgM and IgG anti-HEL antibodies compared to B6 mice and had higher proportions of germinal center B cells and long-lived plasma cells, suggesting a germinal center-dependent breach of B cell anergy. Consistent with impaired anergy induction, c1(96–100) double transgenic B cells showed enhanced survival and CD86 upregulation. Hematopoietic chimeric sHEL mice with a mixture of B6 and c1(96–100) HEL transgenic B cells recapitulated these results, suggesting the presence of a B cell autonomous defect. Surprisingly, however, there was equivalent recruitment of B6 and c1(96–100) B cells into germinal centers and differentiation to splenic plasmablasts in these mice. In contrast, there were increased proportions of c1(96–100) T follicular helper cells and long-lived plasma cells as compared to their B6 counterparts, suggesting that both B and T cell defects are required to breach germinal center tolerance in this model. This possibility was further supported by experiments showing an enhanced breach of anergy in double transgenic mice with a longer chromosome 1 interval with additional T cell defects.</p></div

IL-10 Production Is Critical for Sustaining the Expansion of CD5⁺ B and NKT Cells and Restraining Autoantibody Production in Congenic Lupus-Prone Mice

<div><p>The development and progression of systemic lupus erythematosus is mediated by the complex interaction of genetic and environmental factors. To decipher the genetics that contribute to pathogenesis and the production of pathogenic autoantibodies, our lab has focused on the generation of congenic lupus-prone mice derived from the New Zealand Black (NZB) strain. Previous work has shown that an NZB-derived chromosome 4 interval spanning 32 to 151 Mb led to expansion of CD5⁺ B and Natural Killer T (NKT) cells, and could suppress autoimmunity when crossed with a lupus-prone mouse strain. Subsequently, it was shown that CD5⁺ B cells but not NKT cells derived from these mice could suppress the development of pro-inflammatory T cells. In this paper, we aimed to further resolve the genetics that leads to expansion of these two innate-like populations through the creation of additional sub-congenic mice and to characterize the role of IL-10 in the suppression of autoimmunity through the generation of IL-10 knockout mice. We show that expansion of CD5⁺ B cells and NKT cells localizes to a chromosome 4 interval spanning 91 to 123 Mb, which is distinct from the region that mediates the majority of the suppressive phenotype. We also demonstrate that IL-10 is critical to restraining autoantibody production and surprisingly plays a vital role in supporting the expansion of innate-like populations.</p></div

Knockout of IL-10 in B6.NZBc4 but not B6.NZBc4m mice results in a breach of tolerance to ssDNA, dsDNA, and chromatin.

<p>Levels of anti-ssDNA, -dsDNA, and–chromatin antibodies were measured in 4 month old mice by ELISA. Each point represents a single mouse, with the lines for each group representing the median. Statistical analyses were carried out using a Mann-Whitney <i>U</i> test between homozygous and IL-10 knockout animals of the same genetic background. * P < 0.05, ** P < 0.01.</p

Knockout of IL-10 in full-length B6.NZBc4 but not B6.NZBc4m mice resulted in a loss of transitional B cells and an expansion of marginal zone B cells.

<p>(A) Representative gating of transitional (CD21^loCD23^-), follicular (CD21^intCD23⁺), and marginal zone/marginal zone precursor (CD21^hiCD23^-) B cells from 4 month old mice. Frequencies of splenic B cell subsets were measured by flow cytometry. (B,C,D) The frequency of follicular, transitional and marginal zone B cells, respectively, was measured by flow cytometry as gated in (A). Each point represents a single mouse, with the lines for each group representing the median. Statistical analyses were carried out using a Mann-Whitney <i>U</i> test between homozygous and IL-10 knockout animals of the same genetic background. * P < 0.05, ** P < 0.01, ***P < 0.001.</p

The expansion of peritoneal B1a B cells, splenic CD5⁺ B cells, and NKT cells localizes to an NZB-derived interval spanning 91 to 123 Mb on chromosome 4.

<p>(A) Figure illustrating the NZB chromosome 4 congenic strains used in these studies. D4Mit markers demarcate the known boundaries of introgression. Splenic and peritoneal immune cell frequencies were measured by flow cytometry from 4 month old mice. (B) Splenic CD5⁺ B cells were measured based on FSC, SSC, CD19, and CD5 staining. (C) Frequencies of peritoneal cells were identified by granularity and size and gated as CD19⁺ and CD5⁺. (D) Splenic NKT cells were gated based on size and granularity using FSC, SSC, and gated as PBS57 Tetramer⁺ NK1.1^-. (E) Splenic NK cells were measured based on FSC, SSC, and gated as NK1.1⁺ and PBS57 Tetramer^-. Each point represents a single mouse, with the lines indicating the median of each group. Statistical analyses were carried out using a Kruskal-Wallis test with select Dunn multiple comparison posttests to control B6 mice. * P < 0.05, ** P < 0.01, ***P <0.001.</p

c1(96–100) DTg B cells show altered function consistent with impaired anergy induction.

<p>(A) Surface levels of IgM^a on splenic B (B220⁺) cells from 4-month-old B6 (open circles) and c1(96–100) (filled circles) IgTg or DTg mice were measured using flow cytometry (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179506#pone.0179506.s001" target="_blank">S1 Fig</a> for representative plot showing typical levels of IgM^a on B220⁺ cells). (B) Scatter plots showing the proportions of T1 (CD24^hiCD21^-), T2 (CD24^hi, CD21^int), follicular (CD24^lo, CD21⁺; Fo) and marginal zone/precursor (CD24^int, CD21^hi; MZ/P) B cells in B6 (open circles) and c1(96–100) (filled circles) IgTg and DTg mice. Cells were gated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179506#pone.0179506.s002" target="_blank">S2A Fig</a>. (C) Purified B cells from B6 (open circles) and c1(96–100) (filled circles) DTg mice were stimulated in vitro with HEL (0 or 100 ng/ml) together with a submitogenic concentration of LPS (50 ng/mL). B cell proliferation was measured by [³H]-thymidine incorporation at 36 hours by pulsing the cells overnight with 1 μCi/well. Uptake of [³H]-thymidine was quantified using a scintillation counter and expressed as the mean CPM of triplicate wells. The stimulation index (SI), the ratio of stimulated (HEL+LPS) to un-stimulated (LPS only) cells, was calculated for each experiment. Results shown are from three independent experiments. (D) Splenocytes from B6 (open circles) or c1(96–100) DTg (filled circles) mice were stimulated in media alone, with anti-IgM F(ab’)₂ (10 μg/ml), or with HEL (100 ng/ml) for 18 hours. Cultured cells were stained with anti-IgM^a, -CD86, and -B220, and analyzed by flow cytometry. CD86 levels were gated on IgM^a+B220⁺ cells, or for the anti-IgM stimulation for IgTg mice on all B220⁺ cells, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179506#pone.0179506.s002" target="_blank">S2B Fig</a>. (E) Representative histograms showing the CD86 expression on B6 DTg (thin line, filled histograms) vs c1(96–100) DTg (bold line) IgM^a+B220⁺ B cells for unstimulated and stimulated conditions. (F) To quantify phosphatidylinositol 3,4,5,-triphosphate (PIP₃) production, splenocytes were stimulated with anti-IgM F(ab)’₂ Ab for 5 min, fixed with 1% paraformaldehyde, stained with anti-B220, permeabilized, and then stained intracellularly with an anti-PIP₃ Ab. Scatter plots show the percentage of PIP₃⁺ cells from DTg mice from B6 (open circles) or c1 (96–100) (filled circles), calculated as the expression of PIP₃ on stimulated B220⁺ B cells above the media control. (G) Representative histograms showing PIP₃ expression in B6 DTg (thin line) vs c1(96–100) DTg (bold line) B220⁺ B cells for unstimulated and stimulation conditions. For scatterplots, each symbol represents the determination for an individual mouse. Horizontal lines represent the mean. The asterisks indicate p values <0.05 (*) or <0.001 (**). Statistical significance was determined by Mann-Whitney U test.</p

Comparison of splenic HEL⁺ and IgM^a+ B cell subsets in B6 and c1(96–100) DTg mice.

<p>Comparison of splenic HEL⁺ and IgM^a+ B cell subsets in B6 and c1(96–100) DTg mice.</p

Increased levels of IgM and IgG anti-HEL Abs in c1(96–100) DTg mice indicate a breach of B cell anergy.

<p>(A) Scatter plots showing serum levels of anti-HEL and anti-ssDNA Abs from B6 (open circles) and c1(96–100) (filled circles) mice. IgM^a, IgM^b, or IgG anti-HEL and IgG anti-ssDNA Abs from 3- to 6-month-old mice (mean age: B6 DTg = 4.17 ± 0.77 months; c1(96–100) DTg = 4.64 +/- 0.77 months) were measured by ELISA. (B) Immunofluorescent imaging of HEL-binding plasmablasts within the B cell follicle, bridging channels, red pulp, and marginal zones of B6 and c1(96–100) DTg mice. Spleen sections (5 μm) were stained with anti-IgD (green), anti-CD4 (purple), and biotinylated-HEL, with streptavidin-AMCA as the secondary stain (blue). Magnification x10. (C) Flow plots showing the regions used to gate antibody-secreting cells (CD138^hiCD21^-) in the bone marrow. Scatter plots show the proportions of long-lived plasma cells in the bone marrow of c1(96–100) DTg mice (filled circles) compared to B6 DTg mice (open circles). (D) Flow plots showing the regions used to gate germinal center (PNA⁺GL7⁺) B cells in the spleen. Scatter plots show the proportions of germinal center B cells in c1(96–100) DTg mice (filled circles) compared to B6 DTg mice (open circles). (E) Immunofluorescent imaging of GC (indicated by arrows) in the spleens of two representative DTg mice per strain. Spleen sections have been stained with biotinylated-PNA followed by streptavidin-AMCA (blue). Magnification x10. (F) A representative GC at higher magnification (x20) from a c1(96–100) DTg mouse stained as in (E) together with FITC anti-IgD (green) and PE-anti-IgM^a (red). For scatterplots, each symbol represents the determination for an individual mouse. Horizontal lines represent the mean. The asterisks indicate p values <0.05 (*) or <0.001 (**). Statistical significance was determined by the Mann-Whitney U test.</p

c1(70–100) DTg mice have an enhanced breach of B cell tolerance.

<p>(A) Scatter plots showing serum levels of IgM^a anti-HEL, IgG anti-HEL and IgG anti-ssDNA Abs from 8 week-old to 4 month-old B6 (open circles), c1 (96–100) (filled circles) and c1(70–100) (filled triangles) mice, as measured by ELISA. (B) Shown on the left are flow plots gated upon B220⁺ cells indicating the regions used to gate GC B cells. Scatter plots show the proportion of GC cells within the B220⁺ subset of B6 DTg (open circles), c1(96–100) DTg (filled circles) or c1(70–100) DTg (filled triangles) mice. (C) Flow plots gated on CD4⁺ T cells showing the regions used to gated CXCR5^hi cells (regions were established as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0179506#pone.0179506.g004" target="_blank">Fig 4G</a>). Scatter plots show the proportion of these cells within the CD4+ T cell subset in B6 DTg (open circles), c1(96–100) DTg (filled circles), or c1(70–100) DTg (filled triangles) mice. (D) Representative flow cytometry contour plots showing the gating used to identify the proportion of IFN-γ-, IL-17-, and IL-21-producing CD3⁺CD4⁺ T cells in c1(70–100) DTg mice. The quadrants used to define positively and negatively stained cells are indicated. For each condition, the upper plots show unstimulated cells and the lower plots show cells stimulated with PMA and ionomycin. Scatter plots showing the proportion of cytokine-producing T cells in B6 (open circles), c1(96–100) (filled circles) and c1(70–100) (filled triangles) mice. Each symbol represents an individual mouse. Horizontal lines represent the mean. The asterisks indicate p values <0.05 (*) or <0.001 (**). Statistical analyses were performed using a Mann-Whitney <i>U</i> test.</p