Heavy and light chain gene features of influenza-specific antibodies from SLE patients and controls.

<p>(A) Pie charts show the proportion of clonally related sequences in the influenza-positive antibodies from SLE patients and controls. (B) Number of somatic mutations in VH and VK chain gene sequences of influenza-positive antibodies is plotted. For each graph, in the left panel, each symbol represents an individual antibody, while in the right panel; each symbol is the average number of mutations of all the antibodies for each individual. (C) Proportion of influenza-positive antibodies with different variable heavy (VH) CDR3 lengths (number of amino acids). (D) Proportion of pooled antibodies with different joining region JH, VH, VK and JK gene identities. (E) Isoelectric points of VH chain CDR3 gene segments of influenza-positive antibodies from SLE patients and controls. The left panel shows each symbol representing an individual antibody, while in the right, each symbol is the average of all the antibodies for each individual. Means were compared in B and E using unpaired t-test. Chi-squared test was used to compare the frequency of the individual categories between SLE and controls (* p < 0.05) in C and D.</p

Distinct serum cytokine profiles seen in subjects with high avidity antibodies and models discussing the production of high affinity antibodies in context of autoimmunity.

<p>(A) The levels of various cytokines in the serum of subjects, measured by multiplex bead assay, are shown on a graded color scale. Cytokines are grouped according to their major roles in immune responses and subjects are ranked by the median avidity of each subject as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125618#pone.0125618.g001" target="_blank">Fig 1D</a>. Serum cytokine levels were normalized by the minimum (blue) and maximum (red) values within each dataset. (B) The affinity-risk model postulates that individuals who have the propensity to make high affinity responses will have higher risk for autoimmunity, especially if primary B cell tolerance mechanisms are also disrupted and the naïve B cell population is more self-reactive than normal. Thicker red arrows indicate greater probability of the event. (C) The autoreactivity-risk model proposes that anergy (or other factors) may be contribute to the increased activation threshold of self-reactive B cells in SLE patients so that only B cells with high enough affinity for the antigen are activated, thus skewing the response towards a higher affinity B cell response.</p

Higher avidities and neutralization capacities observed for monoclonal antibodies from SLE patients.

<p>(A) Monoclonal antibodies generated from plasmablasts isolated from subjects were tested for virus binding by ELISA. Binding avidities (K_D) were estimated by Scatchard plot analyses of ELISA data. K_D values of pooled antibodies are shown. Medians were compared using Mann-Whitney test. (B) The distribution of K_D values is shown by subject. Each bar graph represents the median of the avidities of antibodies (represented by symbol) for each subject. The red dotted line represents the global median avidity of all SLE and control influenza-positive antibodies. (C) Avidities of 53 influenza-positive antibodies from SLE patients and 27 from controls were measured by SPR. Correlation between approximated K_D values from virus ELISA data and K_D values measured by SPR is shown. Black symbols are antibodies from SLE patients while blue are those from controls. Correlation was determined by Spearman’s correlation test. (D-E) Influenza-positive antibodies were serially diluted and tested in the standard HAI assay (D) and microneutralization (E) for functional capability. Medians were compared using Mann-Whitney test. (D) Minimum antibody concentration effective at inhibiting hemagglutination is plotted for each HAI-positive antibody. (E) Same as (D), except for microneutralization. Results are representative of at least three independent replicates.</p

Poly- and self-reactivity of influenza-positive antibodies.

<p>Antibodies were tested for polyreactivity against dsDNA, LPS and insulin by ELISA and for HEp-2 reactivity by ELISA and IFA. (A) Pie charts represent the frequency of polyreactive and non-polyreactive influenza-positive antibodies for each individual. Numbers in the center indicate number of influenza-positive antibodies for each individual. Dot plot (right) summarizes the percent of polyreactive antibodies from the pie charts; each symbol represents an individual. (B) Similar to (A), except for HEp-2 ELISA reactivity. (C) Distribution of the HEp-2 IFA scores for each individual is shown and the dot plot summarizes the percent of antibodies with a score of 2 and above for each individual. (D) Distribution of antibodies with HEp-2- only, poly- only and both reactivities is shown for pooled influenza-positive antibodies in the pie charts. Values in pie chart segments are percentages. Statistical significance of the difference between the two cohorts was determined by the Chi-squared test (*p<0.05; **p<0.001; ***p<0.0001). Dot plots show the percent of each type of reactivity for each individual. (E) Average HEp-2 IFA score of all influenza-positive antibodies for each individual is plotted. Medians were compared by Mann-Whitney test in A-C, D (dot plot), E; and data are representative of at least three independent replicates.</p