image_1_V(D)J Rearrangement Is Dispensable for Producing CDR-H3 Sequence Diversity in a Gene Converting Species.PDF

<p>An important characteristic of chickens is that the antibody repertoire is based on a single framework, with diversity found mainly in the CDRs of the light and heavy chain variable regions. Despite this apparent limitation in the antibody repertoire, high-affinity antibodies can be raised to a wide variety of targets, including those that are highly conserved. Transgenic chickens have previously been generated that express a humanized antibody repertoire, with a single framework that incorporates diversity by the process of gene conversion, as in wild-type chickens. Here, we compare the sequences and antibodies that are generated purely by gene conversion/somatic hypermutation of a pre-rearranged heavy chain, with the diversity obtained by V(D)J rearrangement followed by gene conversion and somatic hypermutation. In a gene converting species, CDR-H3 lengths are more variable with V(D)J rearrangement, but similar levels of amino acid diversity are obtainable with gene conversion/somatic hypermutation alone.</p

image_2_V(D)J Rearrangement Is Dispensable for Producing CDR-H3 Sequence Diversity in a Gene Converting Species.PDF

<p>An important characteristic of chickens is that the antibody repertoire is based on a single framework, with diversity found mainly in the CDRs of the light and heavy chain variable regions. Despite this apparent limitation in the antibody repertoire, high-affinity antibodies can be raised to a wide variety of targets, including those that are highly conserved. Transgenic chickens have previously been generated that express a humanized antibody repertoire, with a single framework that incorporates diversity by the process of gene conversion, as in wild-type chickens. Here, we compare the sequences and antibodies that are generated purely by gene conversion/somatic hypermutation of a pre-rearranged heavy chain, with the diversity obtained by V(D)J rearrangement followed by gene conversion and somatic hypermutation. In a gene converting species, CDR-H3 lengths are more variable with V(D)J rearrangement, but similar levels of amino acid diversity are obtainable with gene conversion/somatic hypermutation alone.</p

Percentage of seropositive patients by age group.

<p>Significant differences in the frequency of autoantibody titers:</p><p>*<20 v. 20–40;</p><p>**20–40 v. >40.</p

Percentage of patients in each cluster with specific clinical manifestations and autoantibodies.

<p>Percentage of patients in each cluster with specific clinical manifestations and autoantibodies.</p

Validation of two major autoantibody clusters in SLE.

<p>A second SLE cohort was analyzed for autoantibody titers. A heatmap was again constructed and antibody clusters were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032001#pone-0032001-g001" target="_blank">Figure 1</a>. Similar to the prevalence seen in the first cohort, 47% of the SLE samples showed a Sm/RNP cluster phenotype and 51% showed a Ro/La cluster phenotype.</p

Comparison of LIPS mixture test with the sum of the individual tests for SLE diagnosis.

<p>Antibody titer data from the LIPS mixture test was plotted against the sum of the titer of the individual Ro52, Ro60, La, Sm-D3, RNP-A and RNP-70k autoantibody titers. Each circle represents an individual patient. Using the Spearman rank test, the correlation between the two tests was <i>R</i> = 0.95.</p

Percentage of seropositive patients by ethnicity.

<p><b>C</b>: Caucasian; <b>AA</b>: African American; <b>A</b>: Asian. Significant differences in the frequency of autoantibody titers:</p><p>*White v. African American;</p><p>**African American v Asian.</p

SLE autoantibody clusters in the pilot cohort.

<p>A colored heat map was used to visualize the autoantibody titers in the SLE patients. Autoantibody titers were transformed to <i>Z</i> scores as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032001#s2" target="_blank">Materials and Methods</a> and color coded as indicated by the scale at right, in which signal intensities from green to black indicate high and low titers, respectively. To segregate the SLE patients into clusters, the relative ratio (RR) of the sum titer of Sm-D3, RNP-A and RNP-70k divided by the sum titer of Ro52, Ro60 and La autoantibodies was calculated for each patient. Patients with a RR≥1 were assigned to the Sm/RNP cluster (top panel) while patients with a RR<1 were assigned to the Ro/La cluster (bottom panel). Patients with a pure Sm/RNP or pure Ro/La phenotype are indicated.</p

LIPS diagnostic autoantibody characteristics in SLE.

<p>*Excluding Histone 2B.</p>†<p>Statistically significant, <i>P</i><0.05.</p

LIPS detection of antibodies to a C-terminal capsid fragment of HMOAstV-C.

<p>Antibodies to the N- and C-terminal capsid protein of HMOAstV-C and the C-terminal capsid protein of HMOAstV-CA capsid protein fragments were analyzed in 45 adult serum samples. Each symbol represents individual serum samples tested with each protein fragments and LU values were adjusted by subtracting background binding to protein A/G beads. The short solid line represents the mean antibody titer for each group.</p