YY1 is required for antigen-specific germinal center development and for generation of antigen-specific IgG1.

<p><b>(A)</b> Splenocytes from NP-CGG immunized <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were harvested at 14 days after immunization and stained with various antibodies, as well as PNA to detect GC B cells. We gated on CD4⁻CD8⁻F4/80⁻Gr1⁻(DUMP⁻) IgD^- cells that were subdivided into PNA⁺B220⁺ GC-B cells. GC-B cells were gated and further subsetted into NP-specific (NP⁺B220⁺) GC-B cells. Representative results are from three independent experiments. <b>(B)</b> Numbers of NP-specific (NP⁺B220⁺) GC-B cells per spleen of immunized mice (<i>n</i> = 3). <b>(C)</b> <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were immunized with NP-CGG, and 14 days later spleen sections were stained with anti-GL7, anti-IgD and anti-TCRβ antibody. GL7-rich regions demarcate germinal center B cells. <b>(D, E)</b> Serum from NP-CGG immunized <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were collected at 14 days after immunization and NP-specific serum Igs were analyzed using ELISA. <i>D</i>. The concentration of low affinity (NP26, left panel) and high affinity (NP4, right panel) IgG1 in the serum. <i>E</i>. Titer of NP-specific total IgM in the sera of immunized mice. Data are derived from sera samples that were obtained from three experiments. Asterisks indicate p<0.001.</p

YY1 is required for germinal center B cell development and immunoglobulin class switching.

<p><b>(A)</b> Spleen cells from non-immunized <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice were stained with various antibodies to identify total B cells (CD19⁺AA4.1⁺, upper panel) and germinal center B cells (GC-B, DUMP^-IgD^-GL7^hiCD95^hi, lower panel). Percentages and number of <b>(B)</b> total B cells, and <b>(C)</b> GC-B cells per spleen of <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice. Fig A-C are from three independent experiments (<i>n</i> = 3 mice for each genotype). <b>(D)</b> We used ELISA to detect various isotypes of serum immunoglobulins from <i>YY1</i>^<i>f/</i>, <i>γ1CRE</i> and <i>YY1</i>^<i>f/</i>f <i>γ1CRE</i> mice. The concentration of IgM, IgA, total IgG, as well as IgG subclasses, IgG1, IgG2 and IgG3 were measured from sera samples that were obtained from four experiments (<i>n</i> ≥ 4 mice for each genotype). Asterisks indicate p<0.001.</p

LAG-3-deficient mice produce higher amounts of cytokine following mercury treatment.

<p>Wild-type and <i>Lag-3^−/−</i> mice were either given a challenge dose of HgCl₂ (30 µg/mouse s.c. on days 0, 2 and 4) or were left untreated. Splenocytes were harvested on day 8 and then stimulated in vitro with plate bound anti-CD3 and anti-CD28 antibodies. Supernatant were collected after 72 hrs to determine the levels of cytokine by ELISA (n = 3 or 4). Two way ANOVA was used to determine the statistical significance between the groups where *indicates <i>p</i><0.05 and ***indicates <i>p</i><0.0001.</p

Anti-LAG-3 monoclonal antibody breaks established tolerance against mercury-induced autoimmunity.

<p>A.SW mice were tolerized to Hg and 7 days later they received a challenge dose of Hg in conjunction with anti-LAG-3 or control antibody as depicted in the Figure. Serum IgG1 and IgE levels were measured by ELISA and autoantibody titers were determined by IF assay (n = 5). The IgG and IgE concentrations depicted at wk -1 represent the baseline levels of antibodies in serum of untreated mice and no autoantibodies were detectable in untreated mice. To determine statistical significance between the groups two way ANOVA was used where *indicates <i>p</i><0.05.</p

A short course of HgCl₂ injections is sufficient to induce mercury-induced autoimmunity in LAG-3-deficient B6.SJL mice.

<p>(A) Wild-type or <i>Lag-3^−/−</i> B6.SJL mice were challenged with HgCl₂ (30 µg/mouse s.c.) on days 0, 2 and 4. Serum was analyzed by ELISA to determine IgG1 and IgE polyclonal antibodies and ANoA titers were measured by IF assay (n = 5 or 6). (B) Kidneys of mice from this experiment were harvested after 4 weeks, sectioned and stained with goat anti-mouse IgG-FITC to detect IgG deposit. The extent of kidney disease was scored depending on the amount and intensity of IgG staining as described in details in Material and Method section. To determine statistical significance between the groups of fig. 2A and 2B two way ANOVA was used where *indicates <i>p</i><0.05, **indicates <i>p</i><0.005 and ***indicates <i>p</i><0.0001.</p

Exposure to Hg results in higher expression of LAG-3 on CD4⁺T cells and in an increased amount of soluble LAG-3 in serum.

<p>(A) B6.SJL mice were left untreated or given 3 injections of HgCl₂ s.c. on days 0, 2 and 4. Splenocytes were harvested on day 8 and analyzed by flow cytometry. Figure depicts a representative dot plot of total splenocytes gated for LAG-3⁺ CD4⁺ T cells. (B) Results are expressed as frequency of LAG-3⁺ CD4⁺ T cells in each group (n = 3 or 4). (C) A.SW mice were challenged with 3 injections of HgCl₂ (30 µg/mouse) on days 0, 2 and 4. Concentration of soluble LAG-3 (sLAG-3) in serum was measured by ELISA (n = 4 or 5). Unpaired t test was used for statistical analysis where *indicates <i>p</i><0.05.</p

Transfer of wild-type CD4⁺ T cells decreases autoimmunity in LAG-3-deficient mice.

<p>CD4⁺ T cells were sorted using FACS and then transferred to LAG-3-deficient mice as depicted in the Figure. Autoantibody titers were measured by IF assay (n = 6). To determine statistical significance between the groups two way ANOVA was used where *indicates <i>p</i><0.05.</p

LAG-3-deficient mice cannot be tolerized to mercury.

<p>Wild-type or LAG-3-deficient mice were given a tolerogenic dose of HgCl₂ (3 µg/mouse given i.p.) on day -7. Mice were then exposed to 3 injections of HgCl₂ (30 µg/mouse given s.c) on days 0, 2 and 4. Serum Ig were measured by ELISA and ANoA titers were determined by IF assay (n = 5 or 6). To determine statistical significance between the groups two way ANOVA was used where *indicates <i>p</i><0.05.</p