Mutational patterns along different evolution paths of follicular lymphoma.

Follicular lymphoma (FL) is an indolent disease, characterized by a median life expectancy of 18-20 years and by intermittent periods of relapse and remission. FL frequently transforms into the more aggressive diffuse large B cell lymphoma (t-FL). In previous studies, the analysis of immunoglobulin heavy chain variable region (IgHV) genes in sequential biopsies from the same patient revealed two different patterns of tumor clonal evolution: direct evolution, through acquisition of additional IgHV mutations over time, or divergent evolution, in which lymphoma clones from serial biopsies independently develop from a less-mutated common progenitor cell (CPC). Our goal in this study was to characterize the somatic hypermutation (SHM) patterns of IgHV genes in sequential FL samples from the same patients, and address the question of whether the mutation mechanisms (SHM targeting, DNA repair or both), or selection forces acting on the tumor clones, were different in FL samples compared to healthy control samples, or in late relapsed/transformed FL samples compared to earlier ones. Our analysis revealed differences in the distribution of mutations from each of the nucleotides when tumor and non-tumor clones were compared, while FL and transformed FL (t-FL) tumor clones displayed similar mutation distributions. Lineage tree measurements suggested that either initial clone affinity or selection thresholds were lower in FL samples compared to controls, but similar between FL and t-FL samples. Finally, we observed that both FL and t-FL tumor clones tend to accumulate larger numbers of potential N-glycosylation sites due to the introduction of new SHM. Taken together, these results suggest that transformation into t-FL, in contrast to initial FL development, is not associated with any major changes in DNA targeting or repair, or the selection threshold of the tumor clone.

Long-lived antigen-induced IgM plasma cells demonstrate somatic mutations and contribute to long-term protection.

Long-lived plasma cells are critical to humoral immunity as a lifelong source of protective antibodies. Antigen-activated B cells-with T-cell help-undergo affinity maturation within germinal centres and persist as long-lived IgG plasma cells in the bone marrow. Here we show that antigen-specific, induced IgM plasma cells also persist for a lifetime. Unlike long-lived IgG plasma cells, which develop in germinal centres and then home to the bone marrow, IgM plasma cells are primarily retained within the spleen and can develop even in the absence of germinal centres. Interestingly, their expressed IgV loci exhibit somatic mutations introduced by the activation-induced cytidine deaminase (AID). However, these IgM plasma cells are probably not antigen-selected, as replacement mutations are spread through the variable segment and not enriched within the CDRs. Finally, antibodies from long-lived IgM plasma cells provide protective host immunity against a lethal virus challenge.

Aging affects B-cell antigen receptor repertoire diversity in primary and secondary lymphoid tissues.

The elderly immune system is characterized by reduced responses to infections and vaccines, and an increase in the incidence of autoimmune diseases and cancer. Age-related deficits in the immune system may be caused by peripheral homeostatic pressures that limit bone marrow B-cell production or migration to the peripheral lymphoid tissues. Studies of peripheral blood B-cell receptor spectratypes have shown that those of the elderly are characterized by reduced diversity, which is correlated with poor health status. In the present study, we performed for the first time high-throughput sequencing of immunoglobulin genes from archived biopsy samples of primary and secondary lymphoid tissues in old (74 ± 7 years old, range 61-89) versus young (24 ± 5 years old, range 18-45) individuals, analyzed repertoire diversities and compared these to results in peripheral blood. We found reduced repertoire diversity in peripheral blood and lymph node repertoires from old people, while in the old spleen samples the diversity was larger than in the young. There were no differences in somatic hypermutation characteristics between age groups. These results support the hypothesis that age-related immune frailty stems from altered B-cell homeostasis leading to narrower memory B-cell repertoires, rather than changes in somatic hypermutation mechanisms.

High Throughput Sequencing Analysis of the Immunoglobulin Heavy Chain Gene from Flow-Sorted B Cell Sub-Populations Define the Dynamics of Follicular Lymphoma Clonal Evolution.

Understanding the dynamics of evolution of Follicular Lymphoma (FL) clones during disease progression is important for monitoring and targeting this tumor effectively. Genetic profiling of serial FL biopsies and examples of FL transmission following bone marrow transplant suggest that this disease may evolve by divergent evolution from a common ancestor cell. However where this ancestor cell resides and how it evolves is still unclear. The analysis of the pattern of somatic hypermutation of the immunoglobulin gene (Ig) is traditionally used for tracking the physiological clonal evolution of B cells within the germinal center and allows to discriminate those cells that have just entered the germinal center and display features of ancestor cells from those B cells that keep re-circulating across different lymphoid organs. Here we investigated the pattern of somatic hypermutation of the heavy chain of the immunoglobulin gene (IgH-VH) in 4 flow-sorted B cells subpopulations belonging to different stages of differentiation, from sequential lymph node biopsies of cases displaying diverse patterns of evolution, using the GS-FLX Titanium sequencing platform. We observed an unexpectedly high level of clonality, with hundreds of distinct tumor subclones in the different subpopulations from the same sample, the majority detected at a frequency <10-2. By using a lineage trees analysis we observed in all our FL and t-FL cases that the oligoclonal FL population was trapped in a narrow intermediate stage of maturation that maintains the capacity to undergo SHM, but was unable to further differentiate. The presence of such a complex architecture highlights challenges currently encountered in finding a cure for this disease.

Immunoglobulin gene repertoire diversification and selection in the stomach - from gastritis to gastric lymphomas.

Chronic gastritis is characterized by gastric mucosal inflammation due to autoimmune responses or infection, frequently with Helicobacter pylori. Gastritis with H. pylori background can cause gastric mucosa-associated lymphoid tissue lymphoma (MALT-L), which sometimes further transforms into diffuse large B-cell lymphoma (DLBCL). However, gastric DLBCL can also be initiated de novo. The mechanisms underlying transformation into DLBCL are not completely understood. We analyzed immunoglobulin repertoires and clonal trees to investigate whether and how immunoglobulin gene repertoires, clonal diversification, and selection in gastritis, gastric MALT-L, and DLBCL differ from each other and from normal responses. The two gastritis types (positive or negative for H. pylori) had similarly diverse repertoires. MALT-L dominant clones (defined as the largest clones in each sample) presented higher diversification and longer mutational histories compared with all other conditions. DLBCL dominant clones displayed lower clonal diversification, suggesting the transforming events are triggered by similar responses in different patients. These results are surprising, as we expected to find similarities between the dominant clones of gastritis and MALT-L and between those of MALT-L and DLBCL.

Automated analysis of immunoglobulin genes from high-throughput sequencing: life without a template.

BACKGROUND: Immunoglobulin (that is, antibody) and T cell receptor genes are created through somatic gene rearrangement from gene segment libraries. Immunoglobulin genes are further diversified by somatic hypermutation and selection during the immune response. Studying the repertoires of these genes yields valuable insights into immune system function in infections, aging, autoimmune diseases and cancers. The introduction of high throughput sequencing has generated unprecedented amounts of repertoire and mutation data from immunoglobulin genes. However, common analysis programs are not appropriate for pre-processing and analyzing these data due to the lack of a template or reference for the whole gene. RESULTS: We present here the automated analysis pipeline we created for this purpose, which integrates various software packages of our own development and others', and demonstrate its performance. CONCLUSIONS: Our analysis pipeline presented here is highly modular, and makes it possible to analyze the data resulting from high-throughput sequencing of immunoglobulin genes, in spite of the lack of a template gene. An executable version of the Automation program (and its source code) is freely available for downloading from our website: http://immsilico2.lnx.biu.ac.il/Software.html.

[B lymphocyte clonal evolution of human reactive lymph nodes revealed by lineage tree analysis].

INTRODUCTION: Hypermutation and selection processes, characterizing T-dependent B cell responses taking place in germinal centers of lymph nodes, lead to B cell receptor affinity maturation. Those immune responses lead to the development of memory B cells and plasma cells that secrete high amounts of antibody molecules. The dynamics of B cell clonal evolution during affinity maturation has significant importance in infectious and autoimmune diseases, malignancies and aging. Immunoglobulin (Ig) gene mutational Lineage tree construction by comparing variable regions of Ig-gene sequences to the Ig germline gene is an interesting approach for studying B cell cLonal evolution. Lineage tree shapes and Ig gene mutations can be evaluated not only qualitatively and intuitively, but also quantitatively, and thus reveal important information related to hypermutation and selection. AIM: In this paper we describe the experimental protocols that we used for PCR amplification of Igvariable region genes from human formalin fixed paraffin embedded reactive lymph node tissues and the subsequent bioinformatical analyses of sequencing data using Ig mutational lineage trees. RESULTS: B cell populations of three out of four reactive Lymph node tissues were composed of several clones. Most of the Ig gene mutational lineage trees were small and narrow. Significant differences were not detected by quantification of Lineage trees. SUMMARY: B lymphocyte clones that were detected in human reactive lymph node tissues represent major responding clones in normal polyclonal immune response. This result is in line with the polyclonal profile of B Lymphocyte populations that reside in reactive lymph node tissues.

PCR amplification and high throughput sequencing of immunoglobulin heavy chain genes from formalin-fixed paraffin-embedded human biopsies.

The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives.

Models and methods for analysis of lymphocyte repertoire generation, development, selection and evolution.

T and B cell receptor repertoires are diversified by variable region gene rearrangement and selected based on functionality and lack of self-reactivity. Repertoires can also be defined based on phenotype and function rather than receptor specificity - such as the diversity of T helper cell subsets. Natural killer (NK) cell repertoires, in which each cell expresses a randomly chosen subset of its inhibitory receptor genes, and is educated based on self-MHC recognition by yet unknown mechanisms, are also phenotypic repertoires. Studying the generation, development and selection of lymphocyte repertoires, and their functions during immune responses, is essential for understanding the function of the immune system in healthy individuals and in immune deficient, autoimmune or cancer patients. The study of lymphocyte repertoires will enable clinical immunologists to develop better therapeutic monoclonal antibodies, vaccines, transplantation donor-recipient matching protocols, and other immune intervention strategies. The recent development of high-throughput methods for repertoire data collection - from multicolor flow cytometry through single-cell imaging to deep sequencing - presents us now, for the first time, with the ability to analyze and compare large samples of lymphocyte repertoires in health, aging and disease. The exponential growth of these datasets, however, challenges the theoretical immunology community to develop methods for data organization and analysis. Furthermore, the need to test hypotheses regarding immune function, and generate predictions regarding the outcomes of medical interventions, necessitates the development of complex mathematical and computational models, covering processes on multiple scales, from the genetic and molecular to the cellular and system scales.

Automated cleaning and pre-processing of immunoglobulin gene sequences from high-throughput sequencing.

High-throughput sequencing (HTS) yields tens of thousands to millions of sequences that require a large amount of pre-processing work to clean various artifacts. Such cleaning cannot be performed manually. Existing programs are not suitable for immunoglobulin (Ig) genes, which are variable and often highly mutated. This paper describes Ig High-Throughput Sequencing Cleaner (Ig-HTS-Cleaner), a program containing a simple cleaning procedure that successfully deals with pre-processing of Ig sequences derived from HTS, and Ig Insertion-Deletion Identifier (Ig-Indel-Identifier), a program for identifying legitimate and artifact insertions and/or deletions (indels). Our programs were designed for analyzing Ig gene sequences obtained by 454 sequencing, but they are applicable to all types of sequences and sequencing platforms. Ig-HTS-Cleaner and Ig-Indel-Identifier have been implemented in Java and saved as executable JAR files, supported on Linux and MS Windows. No special requirements are needed in order to run the programs, except for correctly constructing the input files as explained in the text. The programs' performance has been tested and validated on real and simulated data sets.