Ig Sequences Research Articles

A systematic approach for peptide characterization of B-cell receptor in chronic lymphocytic leukemia cells.

A wide variety of immunoglobulins (Ig) is produced by the immune system thanks to different mechanisms (V(D)J recombination, somatic hypermutation, and antigen selection). The profiling of Ig sequences (at both DNA and peptide levels) are of great relevance to developing targeted vaccines or treatments for specific diseases or infections. Thus, genomics and proteomics techniques (such as Next-Generation Sequencing (NGS) and mass spectrometry (MS)) have notably increased the knowledge in Ig sequencing and serum Ig peptide profiling in a high-throughput manner. However, the peptide characterization of membrane-bound Ig (e.g., B-cell receptors, BCR) is still a challenge mainly due to the poor recovery of mentioned Ig.Herein, we have evaluated three different sample processing methods for peptide sequencing of BCR belonging to chronic lymphocytic leukemia (CLL) B cells identifying up to 426 different peptide sequences (MS/MS data are available via ProteomeXchange with identifier PXD004466). Moreover, as a consequence of the results here obtained, recommended guidelines have been described for BCR-sequencing of B-CLL samples by MS approaches.For this purpose, an in–house algorithm has been designed and developed to compare the MS/MS results with those obtained by molecular biology in order to integrate both proteomics and genomics results and establish the steps to follow when sequencing membrane-bound Ig by MS/MS.

Dissemination and genetic diversity of chlamydial agents in Polish wildfowl: Isolation and molecular characterisation of avian Chlamydia abortus strains

Wild birds are considered as a reservoir for avian chlamydiosis posing a potential infectious threat to domestic poultry and humans. Analysis of 894 cloacal or fecal swabs from free-living birds in Poland revealed an overall Chlamydiaceae prevalence of 14.8% (n = 132) with the highest prevalence noted in Anatidae (19.7%) and Corvidae (13.4%). Further testing conducted with species-specific real-time PCR showed that 65 samples (49.2%) were positive for C. psittaci whereas only one was positive for C. avium. To classify the non-identified chlamydial agents and to genotype the C. psittaci and C. avium-positive samples, specimens were subjected to ompA-PCR and sequencing (n = 83). The ompA-based NJ dendrogram revealed that only 23 out of 83 sequences were assigned to C. psittaci, in particular to four clades representing the previously described C. psittaci genotypes B, C, Mat116 and 1V. Whereas the 59 remaining sequences were assigned to two new clades named G1 and G2, each one including sequences recently obtained from chlamydiae detected in Swedish wetland birds. G1 (18 samples from Anatidae and Rallidae) grouped closely together with genotype 1V and in relative proximity to several C. abortus isolates, and G2 (41 samples from Anatidae and Corvidae) grouped closely to C. psittaci strains of the classical ABE cluster, Matt116 and M56. Finally, deep molecular analysis of four representative isolates of genotypes 1V, G1 and G2 based on 16S rRNA, IGS and partial 23S rRNA sequences as well as MLST clearly classify these isolates within the C. abortus species. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.

Evolutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna

BackgroundPolyploid hybrids represent a rich natural resource to study molecular evolution of plant genes and genomes. Here, we applied a combination of karyological and molecular methods to investigate chromosomal structure, molecular organization and evolution of ribosomal DNA (rDNA) in nightshade, Atropa belladonna (fam. Solanaceae), one of the oldest known allohexaploids among flowering plants. Because of their abundance and specific molecular organization (evolutionarily conserved coding regions linked to variable intergenic spacers, IGS), 45S and 5S rDNA are widely used in plant taxonomic and evolutionary studies.ResultsMolecular cloning and nucleotide sequencing of A. belladonna 45S rDNA repeats revealed a general structure characteristic of other Solanaceae species, and a very high sequence similarity of two length variants, with the only difference in number of short IGS subrepeats. These results combined with the detection of three pairs of 45S rDNA loci on separate chromosomes, presumably inherited from both tetraploid and diploid ancestor species, example intensive sequence homogenization that led to substitution/elimination of rDNA repeats of one parent. Chromosome silver-staining revealed that only four out of six 45S rDNA sites are frequently transcriptionally active, demonstrating nucleolar dominance. For 5S rDNA, three size variants of repeats were detected, with the major class represented by repeats containing all functional IGS elements required for transcription, the intermediate size repeats containing partially deleted IGS sequences, and the short 5S repeats containing severe defects both in the IGS and coding sequences. While shorter variants demonstrate increased rate of based substitution, probably in their transition into pseudogenes, the functional 5S rDNA variants are nearly identical at the sequence level, pointing to their origin from a single parental species. Localization of the 5S rDNA genes on two chromosome pairs further supports uniparental inheritance from the tetraploid progenitor.ConclusionsThe obtained molecular, cytogenetic and phylogenetic data demonstrate complex evolutionary dynamics of rDNA loci in allohexaploid species of Atropa belladonna. The high level of sequence unification revealed in 45S and 5S rDNA loci of this ancient hybrid species have been seemingly achieved by different molecular mechanisms.

Sequence and phylogenetic analysis of Intergenic spacer (IGS) region of ten microsporian isolates infecting Indian vanya silkworms (Samia cynthia ricini and Antheraea assamensis).

Ten microsporidian isolates from Samia cynthia ricini, and Antheraea assamensis in India along with a Nosema reference strain (NIK-1s_mys) from B. mori India were characterised morphologically and molecular based tools. The test isolates observed elongated oval in shape while reference strain was oval and ranging from 3.80 to 4.90 <mu>m in length and 2.60 to 3.05 <mu>m in width. The ribosomal DNA region 'IGS' of test isolates assessed by PCR amplification, followed by cloning and sequencing. IGS sequence and phylogenetic analysis of test microsporidian isolates showed very close relationship with three Nosema references species: N. philosamia, N. antheraea isolated from Philosamia cynthia ricini and Antheraea perny in China respectively and N. disstriae from Malacosma disstriae in Canada. The clustering pattern of dendogram reveals all test isolates appear distinct from Nosema std. (NIK-1s_mys) India used as reference strain in the study. The result suggests IGS indeed a suitable and highly applicable molecular tool for identifying and characterise the microsporidian isolates in similar population.

Molecular organization of 5S rDNA in two ukrainian populations of sycamore (Acer pseudoplatanus)

Aim. The genetic variability of tree species has still not been studied enough at the molecular level. Considering the relatively low rate of molecular evolution in perennial tree species, it is necessary to use only those regions of genome, which demonstrate a high level of variability, such as 5S rDNA. Accordingly, to estimate the potential of 5S rDNA as a molecular marker for tree species, the organization of this genomic region was compared between samples from two geographically remote Ukrainian populations of sycamore, Acer pseudoplatanus. Methods. PCR amplification, cloning and sequencing of the 5S rDNA IGS of A.pseudoplatanus. Results. It was shown that only one variant of 5S rDNA repeat with a length of 475 bp is present in the genome of A. pseudoplatanus. Also, it was found that the elements typical for the angiosperm RNA polymerase III promoter, which are localized in IGS, appear to be different from those previously described for species of other families. The level of IGS sequence similarity within the populations exceeds 99%, while the level of IGS sequence similarity between various populations is only 93,3-94,3%. Conclusions. High rate of molecular evolution of the 5S rDNA IGS makes them a convenient molecular marker for evaluation of intraspecific variation in populations of A. pseudoplatanus.Key words: 5S rDNA, molecular markers, intraspecific variability, Acer

Automated Clustering Analysis of Immunoglobulin Sequences in Chronic Lymphocytic Leukemia Based on 3D Structural Descriptors

Immunoglobulins (Igs) are crucial for the defense against pathogens, but they are also important in many clinical and biotechnological applications. Their characteristics, and ultimately their function, depend on their three-dimensional (3D) structure; however, the procedures to experimentally determine it are extremely laborious and demanding. Hence, the ability to gain insight into the structure of Igs at large relies on the availability of tools and algorithms for producing accurate Ig structural models based on their primary sequence alone. These models can then be used to determine structural and eventually functional similarities between different Igs. An example of such a task is the clustering of Igs based on their structure to determine meaningful common features such as the possible existence of common molecular targets (antigens). Several approaches have been proposed in order to achieve an optimal solution to this task yet their results were hindered mainly due to the lack of efficient clustering methods based on the similarity of 3D structure descriptors. Here, we present a novel workflow for robust Ig 3D modeling and automated clustering. We validated our protocol in chronic lymphocytic leukemia (CLL), where the clonotypic Igs are critically implicated in the disease ontogeny and evolution. Indeed, immunogenetic studies on the clonotypic Igs have strongly implicated antigen selection in the pathogenesis of CLL, while also providing robust prognostic information. In the present study, we used the structure prediction tools PIGS and I-TASSER for creating the 3D models and the TM-align algorithm to superpose them. The innovation of the current methodology resides in the usage of methods adapted from 3D content-based search methodologies to determine the local structural similarity between the 3D models. The Fast Point Feature Histograms descriptors derived from the structurally aligned parts are used to compute a distance matrix, which is then used as input for the clustering procedure. Clustering analysis on the data is performed through the application of the agglomerative and density-based clustering approaches. The first method is unsupervised whereas the second belongs to the semi-supervised type, i.e. requires a predefined number of clusters. To evaluate the quality of the herein described workflow, we performed a supervised analysis of 125 Ig 3D models originating from 5 CLL stereotyped subsets i.e. subgroups sharing (quasi) identical IGs, namely subsets #1, #2, #4, #6, #8. The reasoning behind this choice was that (i) homologous Ig primary sequences can be reasonably anticipated to be reflected in overall similar 3D structures, hence providing a reference for evaluating the developed workflow; and, (ii) these subsets are well characterized at both the clinical and biological levels. Subset size distribution was as follows: subset #1 (IGHV clan I/IGKV1(D)-39), n=37; subset #2 (IGHV3-21/IGLV3-21), n=43; subset #4 (IGHV4-34/IGKV2-30), n=22; subset #6 (IGHV1-69/IGKV3-20), n=12; and, subset #8 (IGHV4-39/IGKV1(D)-39), n=11. Overall, we obtained a high level of clustering accuracy i.e. Ig 3D model clusters matched to a very high degree the subsets defined by Ig primary sequence similarity. In detail, 5 Ig 3D model clusters were produced by: (i) cluster 1 containing 37/37 (100%) subset #1 models and one (8.3%) subset #6 model, (ii) cluster 2 containing 43/43 (100%) subset #2 models, (iii) cluster 3 containing 21/22 (95.5%) subset #4 models, (iv) cluster 4 containing 11/12 (91.7%) #6 models, and, (v) cluster 5 containing 11/11 (100%) subset #8 models along with a single (4.5%) subset #4 model (subsets #4 and #8 concern IgG CLL, in itself a rarity for CLL). These findings support that the innovative workflow described here enables robust clustering of 3D models produced from Ig sequences from patients with CLL. Furthermore, they indicate that CLL classification based on stereotypy of Ig primary sequences is likely also verified at the Ig 3D structural level. Studies are ongoing for both addressing the minor discrepancies observed here and producing the unsupervised 3D clustering of the IGs from a large series of both stereotyped and non-stereotyped CLL cases. DisclosuresRosenquist:Gilead Sciences: Speakers Bureau. Stamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Janssen: Honoraria, Other: Travel expenses, Research Funding; Novartis: Honoraria, Research Funding.

Reappraising Immunoglobulin Repertoire Restrictions in Chronic Lymphocytic Leukemia: Focus on Major Stereotyped Subsets and Closely Related Satellites

Reappraising Immunoglobulin Repertoire Restrictions in Chronic Lymphocytic Leukemia: Focus on Major Stereotyped Subsets and Closely Related Satellites

A Novel Evolutionary Pattern Revealed Using Deep Sequencing of Immunoglobulin Loci at Diagnosis and over the Course of Treatment in Multiple Myeloma Patients

Introduction: Immunoglobulin (Ig) gene rearrangement is a hallmark of early B-cell development. As multiple myeloma is considered a clonal disease originating from the transformation of a single plasma cell, myeloma cells are traditionally thought to have one clonal Ig gene sequence that remains stable throughout the course of the disease. We previously observed that multiple Ig sequences related by somatic hypermutation (SHM) may be present in some MM patients at diagnosis. Here we provide an expanded observation in a very large cohort of the patients, and perform mutational analysis of the oligoclonal myeloma clonotypes observed at diagnosis and post-treatment, revealing changes in the relative frequency of the MM clonotypes and emergence of new Ig clones.Methods: 620 MM patientsenrolled in IFM/DFCI and Hospital 12 de Octubre trials were included in this analysis. The next-generation sequencing (NGS)-based immunosequencing platform was used to detect evidence of oligoclonality at the Ig heavy chain loci. Using universal primer sets, we amplified IGH variable, diversity, and joining gene segments from DNA and/or RNA isolated from purified CD138+ MM cells collected at the time of diagnosis. MM-specific clonotypes were identified for each patient based on their high frequency (5%) within the B-cell repertoire in the diagnostic (dx) sample. The highest frequency MM clonotype in a dx sample is termed the "index clonotype." DNA and/or RNA isolated from dx AND post-treatment bone marrow samples were assessed for evidence of evolved MM clonotypes.Results: We identified Ig clones in 367 RNA samples and 430 DNA samples from the cohort. We first looked for cases with evidence that myeloma cells have two unrelated origins. We found 11/620 (1.8%) cases at diagnosis, which had evidence of unrelated clones as evident by having three IgH or two functional sequences. In 8 of the 11 cases (72.6%), we had multiple samples to analyze, including two samples at diagnosis or diagnosis/post-treatment pairs. In 4 of the 8 samples, we saw dramatically different relative frequencies of unrelated clones in these samples suggesting that these unrelated clones are likely to be present in two distinct cells. We then considered cases where we found two IgH sequences that are related to each other by SHM at diagnosis. Overall 79 (12.7%) of 620 samples had more than one evolved clone; of these 63/367 (17.2%) of RNA dx samples showed evidence of evolved clones via SHM, while 22/430 patients (5.1%) showed evidence of evolved clones related to the index clone via SHM in DNA samples from diagnosis. Mutant clonotypes had an average of 3.9 to 4.5 mutations in the CDR3 region. We also noted mixed isotypes in 13 clones from 13 patients at diagnosis. The majority of related clones observed in the RNA samples are present at very low frequencies (<10-4), as the greater sequencing depth in RNA allows for identification of low frequency clones. 304 post-treatment samples from 206 patients were MRD positive and were assessed for the presence of clonal evolution. In 27/304 follow-up samples (8.8%) and 7/206 patients (3.4%), an evolved clone related to the index clone was observed even though the period between diagnosis and post-treatment samples was only 6 months. In 6 patients, a substantial change in the relative index and unrelated clone frequencies was observed from the dx to post-treatment time points suggesting a differential sensitivity to treatment.Conclusions: We confirm presence of multiple evolved clonotypes in a substantial percentage of diagnostic MM samples in a large cohort of patients. The evolution of multiple clones related by SHM indicates that SHM remains active after myeloma development and may also impact other non-Ig sites. These findings shed light on the biology and pathogenesis of MM and may provide prognostic information. The very high depth of our sequencing also indicates that the emergence of new IgH clones may be newly acquired mutations in the Ig gene, driven by some ongoing genomic mutation process. Thus, these evolved myeloma clonotypes may be useful as surrogate markers for other oncogenic mutations providing resistance to therapy. DisclosuresMunshi:Takeda: Consultancy; Pfizer: Consultancy; Merck: Consultancy; Celgene Corporation: Consultancy; Oncopep: Consultancy, Equity Ownership. Carlton:Adaptive Biotechnologies: Employment, Equity Ownership. Richardson:Jazz Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Attal:amgen: Consultancy, Research Funding; celgene: Consultancy, Research Funding; janssen: Consultancy, Research Funding; sanofi: Consultancy. Moreau:Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria, Speakers Bureau; Novartis: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Onyx: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sonofi Aventis: Membership on an entity's Board of Directors or advisory committees; Acetylon: Other: Scientific Founder; Oncopep: Other: Scientific Founder. Faham:Adaptive Biotechnologies Corp: Employment. Avet-Loiseau:janssen: Consultancy; sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy.

RNA-Seq De Novo Assembly of Clonal Immunoglobulin Rearrangements Identifies Interesting Biology and Uncovers Prognostic Features in Multiple Myeloma

IntroductionAlthough monoclonal immunoglobulin (Ig) production by myeloma cells is one of the central features of the disease, genotypic identification of the clonal Ig sequence remains understudied in multiple myeloma (MM). Here, using extensive RNA-seq data, we study molecular features of clonal Ig rearrangements, as well as their association with other MM markers and patient outcome.MethodsWe performed deep RNA-seq on purified CD138+ MM cells from 429 newly-diagnosed uniformly-treated patients with long clinical follow-up. For each sample, we performed de novo assembly using sequences that appeared in the library with a frequency of at least one in a million. Germline V and J genes were then BLASTed against the assembled contigs to determine the clonal germline genes and pinpoint mutations. Using the sequences reconstructed from the Ig contigs and the BLAST output, we ran IgBLAST to fully characterize the predominant Ig V(D)J sequence.ResultsWe tested the accuracy of our approach by looking at 24 technical duplicates and one triplicate. In all cases, the predicted gene and gene allele were consistent across replicates. Next, we evaluated our large patient cohort, identifying IGHV3 as the most common clonal VH gene subgroup (53.3%), followed by IGHV4 (17.8%) and IGHV1 (15.6%). Importantly, we observed a significant association between poorer prognosis and IGHV3, both for progression-free survival (PFS) (p=0.0019) and overall survival (OS) (p=0.012). IGHV3-30 (11%, the most commonly rearranged VH gene) and IGHV3-9 (4.8%) were the drivers behind this poor prognosis (IGHV3-30: PFS p=0.021; OS p=0.013) (IGHV3-9: PFS p=0.002). IGHV3-30 was even more preferentially rearranged than in normal B-cell VH repertoires from previous studies (8.5%, 6.3%) and ours (2%). Remarkably, these results sharply contrast with what has been observed in CLL. In this malignancy, IGHV3-30 use has been seen to be underrepresented and usually characterizes an indolent clinical course, while IGHV3-21 and possibly IGHV3-23 carry poor prognosis.We predicted light chain usage through the presence of clonal VL sequences. The most frequent VL genes were from the κ locus (69.4% total): IGKV1-33 (12.4%), IGKV1-5 (11.3%), IGKV3-20 (9.9%) and IGKV1-39 (8.0%). Del(22q) was observed more frequently in patients with IGλ (OR=10.0, p=6e-15) and, within this group, del(22q) was more frequent if Vλ belonged to the more centromeric V-clusters C or B, in contrast to cluster A (OR=8.4, p=5e-4). Remarkably, patients with Vλ gene from cluster A presented worse OS (vs. Vk: p=0.0079; vs. Vλ B,C: p=0.067).The proportion of mutated bases was higher in the heavy chain than in the light chain (mean 7.0% vs. 4.8%, max 14.6% vs. 14.3%), and it was associated with OS (heavy p=0.0020, light p=0.036, both=0.0056), but not PFS. Interestingly, mutated Ig in CLL results in a more benign clinical course. We further found that 24.9% and 22.7% of the mutations lay within WRCY or RGYW AID motifs in the light and heavy chains respectively (enrichment p<1e-16), while AID mutations in a TW or WA context accounted for 22.9% and 25.7% (p=0.14, p=0.64). Higher ratios of mutations in WRCY vs. RGYW motifs within the light chain were highly predictive of poor prognosis (PFS p=0.0019, OS p=6.3e-4). Strikingly, IGλ usage was linked to higher ratios (p=3e-6), an association not explained by germline sequence variability (p=0.24).The usage of IGHV3 genes and the AID WRCY/RGYW motif ratio were independent markers of each other (p=1) and of other markers of poor prognosis in MM, such as presence of either t(4;14) or del(17p) (IGHV3 p=0.10; motif ratio p=0.49). In conclusion, de novo Ig heavy and light chain assembly using RNA-seq identifies interesting biology, may provide MM markers and highlights a novel application of high-throughput genomics. DisclosuresAnderson:OncoPep Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Avet-Loiseau:sanofi: Consultancy; celgene: Consultancy; amgen: Consultancy; janssen: Consultancy.

Aligning, analyzing, and visualizing sequences for antibody engineering: Automated recognition of immunoglobulin variable region features.

The analysis and comparison of large numbers of immunoglobulin (Ig) sequences that arise during an antibody selection campaign can be time-consuming and tedious. Typically, the identification and annotation of framework as well as complementarity-determining regions (CDRs) is based on multiple sequence alignments using standardized numbering schemes, which allow identification of equivalent residues among different family members but often necessitate expert knowledge and manual intervention. Moreover, due to the enormous length variability of some CDRs the benefit of conventional Ig numbering schemes is limited and the calculation of correct sequence alignments can become challenging. Whereas, in principle, a well established set of rules permits the assignment of CDRs from the amino acid sequence alone, no currently available sequence alignment editor provides an algorithm to annotate new Ig sequences accordingly. Here we present a unique pattern matching method implemented into our recently developed ANTICALIgN editor that automatically identifies all hypervariable and framework regions in experimentally elucidated antibody sequences using so-called "regular expressions." By combination of this widely supported software syntax with the unique capabilities of real-time aligning, editing and analyzing extended sets of amino acid and/or nucleotide sequences simultaneously on a local workstation, ANTICALIgN provides a powerful utility for antibody engineering. Proteins 2016; 85:65-71. © 2016 Wiley Periodicals, Inc.

Molecular and antifungal susceptibility study on trichosporonemia and emergence of Trichosporon mycotoxinivorans as a bloodstream pathogen.

A total of 21 Trichosporon spp. isolates from blood over a period of 5 years (January 2009 to December 2013) were included in the study. The most common underlying diseases found were pancreatitis (33.3%) and cancer (33.3%). Trichosporon asahii (80.9%) was the commonest species followed by Trichosporon mycotoxinivorans (14.2%) and Trichosporon faecale (4.7%). On IGS1 region sequencing the most predominant T. asahii type in our region was genotype 1 (16/17 isolates; 94.1%) and one isolate belonged to genotype 4. Following the interpretative breakpoints for Candida albicans according to CLSI guidelines amphotericin B minimum inhibitory concentrations (MICs) were ≤1 μg/ml for 38% of isolates. Fluconazole MICs were ≤4 μg/ml for 33.3% of the isolates. Itraconazole MICs were ≤0.5 μg/ml for 52.3% of the isolates. However, the MICs to posaconazole and voriconazole were ≤0.5 μg/ml for all the isolates. The MICs to caspofungin and micafungin were ≤0.5 μg/ml for only 0.09% of the isolates. This study reemphasizes that IGS1 sequencing is the most reliable technique for accurate identification of Trichosporon spp. and also to identify the newer species like T. mycotoxinivorans, which still remains rare. Surveillance of antifungal susceptibility patterns can provide the local drug resistance data to the clinicians which can further aid better management of patients.

A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data.

Analyses of somatic hypermutation (SHM) patterns in B cell Ig sequences have important basic science and clinical applications, but they are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot and cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 H chain transgenic mice with nitrophenyl to stimulate nitrophenyl-specific λ+ germinal center B cells and sequenced the unexpressed κ L chains using next-generation methods. Most of these κ sequences had out-of-frame junctions and were presumably uninfluenced by selection. Despite being nonfunctionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot and cold spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and nonfunctional sequences, suggesting similar biological processes operate at both loci. However, we observed species- and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns.

An update on IgBLAST, a tool for Ig and TCR sequence analysis

Abstract The variable domain of B and T cell receptor is encoded by multiple genes, including the variable (V) gene, the diversity (D) gene and the joining (J) gene. Previously we developed an analysis tool, IgBLAST, for immunoglobulin sequences (http://www.ncbi.nlm.nih.gov/igblast/). Here we describe our recent update on this tool. The new functionalities include capability to analyze T cell receptor sequences (TR), annotating the CDR3 region, extending alignment to 5′ end of the V region, more flexibility in search criteria and providing summarized information to aid large-scale sequence analysis. We use examples to demonstrate these new functionalities and their implications in Ig and TCR sequence analysis.

New insights into rheumatoid arthritis by whole transcriptome profiling of autoreactive B cells

Abstract B cells are important in the pathogenesis of rheumatoid arthritis (RA). By utilizing a flow-cytometric assay to reliably detect the low frequency of autoreactive B cells (ABC) in the blood of RA patients, we performed RNA sequencing of citrulline-specific ABCs and compared them with hemaglutinin (HA) specific B cells. To the best of our knowledge, this is the first complete transcriptome map of human ABCs in autoimmunity, and our data shows a specific up regulation of amphiregulin (AREG) (q-value = 3.1×10−12, fold-change = 250); a member of the EGF growth factor of ligands. Also, the receptor signaling pathways associated with AREG including EGF mediated signaling (FDR q-value = 0.06) and the NRG1 mediated signaling (FDR q-value = 0.03) were enriched, supporting an important role for AREG. Consistent with the well-recognized role of TNF in RA, our RNA-seq data revealed highly significant enrichment of both the TNF pathway (FDR q-value = 0.02) and genes that are targets of TNF (FDR q-value = 0.08) in ABCs from RA patients. Comparisons of our differential expression gene analyses with known risk loci of RA (SNPs) identified genes with decreased expression including TNFAIP3 (leading to inflammatory responses), and REL (anti-apoptotic); and increased expression including CCR6 (maturation and differentiation), and ETS1 (development and maturation) within ABCs than HA specific B cells. Ig sequencing of ABC show normal mutational targeting frequencies but increased somatic hymermutation. In aggregate, our results indicate that the pro-inflammatory nature of IL15 and TNF mediated signaling might promote a feed-forward loop promoting the altered differentiation of ABCs through the AREG-TNF signaling axis, which in turn promotes inflammation.

Emerging Functions of Natural IgM and Its Fc Receptor FCMR in Immune Homeostasis.

Most natural IgM antibodies are encoded by germline Ig sequences and are produced in large quantities by both mice and humans in the absence of intentional immunization. Natural IgM are reactive with many conserved epitopes, including those shared by microorganisms and autoantigens. As a result, these antibodies play important roles in clearing intruding pathogens, as well as apoptotic/necrotic cells and otherwise damaged tissues. While natural IgM binds to target structures with low affinity due to a lack of significant selection by somatic hypermutation, its pentameric structure with 10 antigen-binding sites enables these antibodies to bind multivalent target antigens with high avidity. Opsonization of antigen complexed with IgM is mediated by cell surface Fc receptors. While the existence of Fc alpha/mu receptor has been known for some time, only recently has the Fc receptor specific for IgM (FCMR) been identified. In this review, we focus on our current understandings of how natural IgM and FCMR regulate the immune system and maintain homeostasis under physiological and pathological conditions.

Rhizobial diversity, symbiotic effectiveness and structure of nodules of Vachellia macracantha

The diversity and symbiotic effectiveness of rhizobia isolated from root nodules of native American acacia Vachellia macracantha (formerly Acacia macracantha) grown in soils from different locations in Peru was investigated. Thirteen bacterial isolates were characterized by RFLP analysis of the IGS region, and sequencing and phylogenetic analyses of the 16S rRNA and the symbiotic nodC and nifH genes. Isolates were ascribed to the genera Ensifer or Rhizobium, and phylogenetic analyses of the symbiotic genes allowed relating them to rhizobial strains of American origin, probably comprising a new symbiovar. Most strains formed highly effective nitrogen-fixing indeterminate nodules with V. macracantha. Nodules presented distinctive structural characteristics, including tannin deposits in the inner cortex and a cap of cells with dense vacuole content over an area of intermingled meristematic and freshly infected cells in the apical central zone. Typical zones I, II and interzone II/III of indeterminate nodules were not clearly defined. Unusually complex mitochondrial structures were observed in mature and senescent infected cells of nitrogen-fixing nodules. The effect of inoculation on seedling growth and physiology was evaluated in both V. macracantha and the often co-existing non-nodulating tree legume Caesalpinia spinosa. Inoculation had an overall positive effect on the growth of V. macracantha seedlings, which correlated with the symbiotic effectiveness of isolates, and some strains significantly improved the photochemical efficiency of C. spinosa. To our knowledge, this is the first report on V. macracantha nodulating rhizobia and nodule structure and ultrastructure. This work provides the basis for the formulation of effective inoculants to be used in restoration programmes in the Andean region.

A case of canine borreliosis in Iran caused by Borrelia persica

Tick-borne relapsing fever is an endemic disease in Iran, with most cases attributed to infection by Borrelia persica, which is transmitted by Ornithodoros tholozani soft ticks. Here, we report spirochetemia in blood of a puppy residing in Tehran, Iran. The causative species was identified by use of highly discriminative IGS sequencing; the 489bp IGS sequence obtained in our study showed 99% identity (100% coverage) when compared with B. persica sequences derived from clinical cases or from O. tholozani ticks. Our IGS sequence also showed 99% similarity over 414bp (85% coverage) with a strain from a domestic dog, and 96% over 328bp (69% coverage) with a strain from a domestic cat. Pet-keeping in cosmopolitan cities like Tehran has become increasingly popular in recent years. Animals are often transported into the city in cages or cardboard boxes that might also harbor minute tick larvae and/or early stages of the nymphs bringing them into the urban environment. This may pose a threat to household members who buy and keep these puppies and as a result may come into close contact with infected ticks.

Genotype, antifungal susceptibility, and biofilm formation of Trichosporon asahii isolated from the urine of hospitalized patients

In this study, the culture analysis of urine samples from patients hospitalized in the Central-West region of Brazil was performed, and the isolated microorganisms were phylogenetically identified as Trichosporon asahii. Maximum parsimony analysis of the IGS1 sequences revealed three novel genotypes that have not been described. The minimum inhibitory concentrations of the nine isolates identified were in the range of 0.06–1μg/ml for amphotericin B, 0.25–4μg/ml for fluconazole, and 0.03–0.06μg/ml for itraconazole. Approximately 6/9 of the T. asahii isolates could form biofilms on the surface of polystyrene microplates. This study reports that the microorganisms isolated here as T. asahii are agents of nosocomial urinary tract infections. Furthermore, the IGS1 region can be considered a new epidemiological tool for genotyping T. asahii isolates. The least common genotypes reported in this study can be related to local epidemiological trends.

Deep Igh Sequencing Identifies an Ongoing Somatic Hypermutation Process with Complex and Evolving Clonal Architecture in Myeloma

Deep Igh Sequencing Identifies an Ongoing Somatic Hypermutation Process with Complex and Evolving Clonal Architecture in Myeloma

Unique Versus Common: Disease-Biased Immunoglobulin Gene Repertoires Along with Public Antigen Receptor Stereotypes in Marginal Zone B-Cell Lymphoproliferations

B cells residing the marginal zone (MZ) provide a first line of defense against blood borne pathogens, producing the greater part of circulating natural antibodies conferring protection against infection. Dysregulated homeostasis and function of MZ B cells has been implicated in a wide range of B lymphoproliferations, encompassing the distinct MZ lymphomas recognized by the WHO Classification, the related provisional entities and even chronic lymphocytic leukemia (CLL), for which a MZ derivation has been proposed. Here, taking advantage of a large multi-institutional series, we aimed at obtaining insight into the ontogenetic relationship of MZ lymphoproliferations, related entities and CLL through cross-comparison of their B cell receptor immunoglobulin (BcR IG) gene repertoires. Our sequence dataset included 3660 unique IGHV-IGHD-IGHJ gene rearrangement sequences from our collaborative centers and/or public databases derived from: (1) MZ lymphomas: splenic (SMZL), n=379; nodal (NMZL), n=37; extranodal (ENMZL), n=95; (2) provisional entities of postulated MZ origin, including splenic diffuse red pulp lymphoma (SDRL, n=16) and clonal B cell lymphocytosis of MZ origin (CBL-MZ, n=60); (3) persistent polyclonal B cell lymphocytosis (PPBL), n=286 (from 2 cases); (4) MZ cells isolated from six spleen specimens free of neoplastic cells at histological inspection (non-malignant MZ), obtained at surgery for cancer, n=489; (5) autoimmune conditions, n=1243; (6) various types of normal B cells, n=1055. The most pronounced IG gene repertoire skewing was observed in SMZL with the IGHV1-2*04 gene accounting for 26% of cases. Restrictions, though less striking, were also identified in the other MZ lymphomas as well: (i) the IGHV4-34 gene predominated in NMZL (14.3%); and, (ii) the IGHV1-69 gene predominated in ENMZL (14.6%), albeit with significantly different distribution depending on the primary site of involvement, ranging from 38% in salivary ENMZL to 11% in gastric ENMZL to 4% in ocular adnexa ENMZL (p<0.01). The vast majority of MZL cases showed at least some impact of somatic hypermutation (SHM), with the proportion of cases lacking any SHM ranging from 0% in salivary ENMZ to only 13% in SMZL. Following established bioinformatics approaches, we searched for stereotyped BcR IG sequences i.e. IGHV-IGHD-IGHJ gene rearrangements with restricted antigen-binding site sequence motifs. For the purposes of this analysis, the present sequence dataset was cross-compared to a large dataset of 20451 IGHV-IGHD-IGHJ gene rearrangement sequences from CLL patients from the IMGT/CLL-DB. Overall, 6437 different clusters with stereotyped BcR IG sequences were identified in the merged dataset, including from only 2 to more than 350 sequences. Two categories of clusters with stereotyped BcR IG were identified: disease-specific (n=4813) and 'mixed' (n=1624) i.e. comprised of cases with different diagnosis. The great majority of clusters in the former category concerned exclusively CLL and corresponded to well-established CLL stereotyped subsets, while only a small minority concerned exclusively MZ lymphomas, all with a diagnosis of SMZL. Mixed clusters were relatively small in size, with only 4 populated by more than 10 cases; of these, 2 utilized the IGHV1-69 gene, while the remaining 2 utilized the IGHV3-7 and IGHV4-59 gene, respectively. They comprised rearrangements from various entities, including SMZL, ENMZL (gastric, salivary gland, ocular adnexa), CLL, hepatitis C virus-associated diffuse large B cell lymphoma (DLBCL), but also rheumatoid factors and non-malignant spleen MZ cells. Notably, shared (recurrent) amino acid changes introduced by SHM (i.e. the same amino acid replacement at the same position) were identified in each mixed cluster. In conclusion, we document different immunogenetic signatures for MZ lymphomas, with limited overlap both amongst the various distinct and provisional WHO entities but also versus CLL. These findings indicate distinct antigen exposure histories and/or different (micro)environments underlying the ontogeny of MZ lymphomas. That said, the existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms, may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. DisclosuresGhia:Janssen Pharmaceuticals: Research Funding.