Isoproterenol- and forskolin-induced phosphoprotein signaling in the presence of the PKA inhibitor H89.

<p><b>A</b>, MEFs were pretreated with H89 (10 μM) for 1 hour and then were stimulated with isoproterenol (Iso) (10 μM) for various times (0, 5, 10, 20, 30, or 60 minutes). The heat map shows phosphorylation (yellow) and dephosphorylation (blue) events after isoproterenol stimulation. The color scale shows fold change as compared with unstimulated MEFs. Data are means of three independent stimulation experiments. * denotes pathways that are significantly altered by pretreatment with H89 as assessed using the SAM algorithm. A two-class unpaired time course analysis was performed comparing signed-areas under the curve for each pathway. <b>B</b>, MEFs were pretreated with H89 as in (A) but were stimulated with forskolin (50 μM). Data are means of three independent stimulation experiments. * denotes pathways that are significantly altered by pretreatment with H89 as assessed using the SAM algorithm. <b>C</b>, The phosphorylation kinetics of ERK1/2(T202/Y204), PP1α(T320), GSK3β(S9) in response to isoproterenol (10 μM) stimulation in the presence (dashed line) or absence (solid line) of H89. The phosphorylation of ERK1/2(T202/Y204) is blocked by H89, dephosphorylation of PP1α(T320) is unchanged by H89, and dephosphorylation of GSK3β(S9) is enhanced by H89. Graphs show means ± SEM for three independent experiments. Values in the graph represent fold change as compared with unstimulated MEFs. D, Rapid phosphorylation kinetics of GSK3β(S9) by isoproterenol (10 μM) in the presence (dashed line) or absence (solid line) of H89. The graphs show means ± SEM of three independent experiments. * indicates a significant difference (P<0.05) from baseline phosphorylation level as determined using a Student’s T-Test.</p

Isoproterenol-induced dephosphorylation events in the presence of okadaic acid.

<p>MEFs were pretreated with okadaic acid (1 μM) for 30 minutes and then were stimulated with isoproterenol (10 μM) for various times (0, 5, 10, 20, 30, or 60 minutes). Graphs show dephosphorylation of GSK3β(S9), P70S6K(T389), PP1α(T320), and AKT(T308) after the addition of isoproterenol in the presence (dashed line) or absence (solid line) of okadaic acid. The basal levels of phosphorylation of GSK3β(S9), P70S6K(T389), and PKA substrate were increased by okadaic acid at the unstimulated (0 minute) time point. Values in graphs are median ± S.D fluorescence intensity (MFI). of lysate microarray features. ** denotes a significant difference (P<0.05) from okadaic acid treatment as determined using a one-way ANOVA with repeated measures. * denotes significant difference (P<0.05) from the baseline time point as determined using a Dunnett post-hoc test.</p

Isoproterenol induced phosphoprotein signaling in the presence of selective β-AR antagonists.

<p>MEFs were pretreated with the β1-AR antagonist CGP 20712A (CGP) and β2-AR antagonist ICI 118,551 (ICI) prior to stimulation with isoproterenol. CGP and ICI were used at final concentrations of 10 μM. MEFs were then stimulated with isoproterenol (Iso) (100 nM) for various times (0, 5, 10, 20, 30, or 60 minutes). The heat map shows phosphorylation (yellow) and dephosphorylation (blue) events after isoproterenol stimulation. The color scale shows fold change as compared with unstimulated MEFs. Data are means of three independent stimulation experiments. * denotes pathways with reduced phosphorylation in the presence of ICI 118,551 as determined using the SAM algorithm (q value < 0.05).</p

Inhibition of PI3K/AKT results in dephosphorylation of downstream signaling molecules.

<p><b>A</b>, In vitro AKT kinase assay. MEFs were treated with vehicle control (C), LY294002 (LY) (10 μM), wortmannin (Wort) (100 nM) or forskolin (Fsk) (50 μM) for 30 minutes. MEF lysates were collected and were assayed for AKT activity using an in vitro kinase assay. As a readout for AKT activity, phosphorylated GSK3β fusion protein was detected by western blotting. A representative blot is shown. B, The quantification of the AKT kinase assay. The graph shows mean ± SEM values for three independent experiments. * indicates a significant difference (P<0.05) as compared with the control phosphorylation level of GSK3β fusion protein. <b>C</b>, Phosphoprotein signaling in the presence of PI3K inhibitors. MEFs were treated with LY294002 (LY) (10 μM) or wortmannin (Wort) (100 nM) for various times (0, 5, 30, and 60 minutes). The heat map shows phosphorylation (yellow) and dephosphorylation (blue) events after the various treatments. Data are representative of two independent experiments.</p

Characterization of Influenza Vaccine Immunogenicity Using Influenza Antigen Microarrays

<div><p>Background</p><p>Existing methods to measure influenza vaccine immunogenicity prohibit detailed analysis of epitope determinants recognized by immunoglobulins. The development of highly multiplex proteomics platforms capable of capturing a high level of antibody binding information will enable researchers and clinicians to generate rapid and meaningful readouts of influenza-specific antibody reactivity.</p><p>Methods</p><p>We developed influenza hemagglutinin (HA) whole-protein and peptide microarrays and validated that the arrays allow detection of specific antibody reactivity across a broad dynamic range using commercially available antibodies targeted to linear and conformational HA epitopes. We derived serum from blood draws taken from 76 young and elderly subjects immediately before and 28±7 days post-vaccination with the 2008/2009 trivalent influenza vaccine and determined the antibody reactivity of these sera to influenza array antigens.</p><p>Results</p><p>Using linear regression and correcting for multiple hypothesis testing by the Benjamini and Hochberg method of permutations over 1000 resamplings, we identified antibody reactivity to influenza whole-protein and peptide array features that correlated significantly with age, H1N1, and B-strain post-vaccine titer as assessed through a standard microneutralization assay (p<0.05, <i>q</i> <0.2). Notably, we identified several peptide epitopes that were inversely correlated with regard to age and seasonal H1N1 and B-strain neutralization titer (p<0.05, <i>q</i> <0.2), implicating reactivity to these epitopes in age-related defects in response to H1N1 influenza. We also employed multivariate linear regression with cross-validation to build models based on age and pre-vaccine peptide reactivity that predicted vaccine-induced neutralization of seasonal H1N1 and H3N2 influenza strains with a high level of accuracy (84.7% and 74.0%, respectively).</p><p>Conclusion</p><p>Our methods provide powerful tools for rapid and accurate measurement of broad antibody-based immune responses to influenza, and may be useful in measuring response to other vaccines and infectious agents.</p></div

Table_4.csv

<p>Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (T_reg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.</p

Table_2.xlsx

<p>Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (T_reg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.</p

Image_2.PDF

<p>Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (T_reg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.</p

Table_3.xlsx

<p>Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (T_reg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.</p

Table_1.PDF

<p>Anti-cytokine autoantibodies (ACAAs) have been described in a growing number of primary immunodeficiencies with autoimmune features, including autoimmune polyendocrine syndrome type I (APS-1), a prototypical disease of defective T cell-mediated central tolerance. Whether defects in peripheral tolerance lead to similar ACAAs is unknown. Immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) is caused by mutations in FOXP3, a master regulator of T regulatory cells (T_reg), and consequently results in defective T cell-mediated peripheral tolerance. Unique autoantibodies have previously been described in IPEX. To test the hypothesis that ACAAs are present in IPEX, we designed and fabricated antigen microarrays. We discovered elevated levels of IgG ACAAs against interferon-α (IFN-α) in a cohort of IPEX patients. Serum from IPEX patients blocked IFN-α signaling in vitro and blocking activity was tightly correlated with ACAA titer. To show that blocking activity was mediated by IgG and not other serum factors, we purified IgG and showed that blocking activity was contained entirely in the immunoglobulin fraction. We also screened for ACAAs against IFN-α in a second geographically distinct cohort. In these samples, ACAAs against IFN-α were elevated in a post hoc analysis. In summary, we report the discovery of ACAAs against IFN-α in IPEX, an experiment of nature demonstrating the important role of peripheral T cell tolerance.</p