Neta S. Zuckerman

IgTree © : Creating Immunoglobulin variable region gene lineage trees

Lineage trees describe the microevolution of cells within an organism. They have been useful in the study of B cell affinity maturation, which is based on somatic hypermutation of immunoglobulin genes in germinal centers and selection of the resulting mutants. Our aim was to create and implement an algorithm that can generate lineage trees from immunoglobulin variable region gene sequences. The IgTree program implements the algorithm we developed, and generates lineage trees. Original sequences found in experiments are assigned to either leaves or internal nodes of the tree. Each tree node represents a single mutation separating the sequences. The mutations that separate the sequences from each other can be point mutations, deletions or insertions. The program can deal with gaps and find potential reversion mutations. The program also enumerates mutation frequencies and sequence motifs around each mutation, on a per-tree basis. The algorithm has proven useful in several studies of immunoglobulin variable region gene mutations.

Somatic hypermutation and antigen-driven selection of B cells are altered in autoimmune diseases

B cells have been found to play a critical role in the pathogenesis of several autoimmune (AI) diseases. A common feature amongst many AI diseases is the formation of ectopic germinal centers (GC) within the afflicted tissue or organ, in which activated B cells expand and undergo somatic hypermutation (SHM) and antigen-driven selection on their immunoglobulin variable region (IgV) genes. However, it is not yet clear whether these processes occurring in ectopic GCs are identical to those in normal GCs. The analysis of IgV mutations has aided in revealing many aspects concerning B cell expansion, mutation and selection in GC reactions. We have applied several mutation analysis methods, based on lineage tree construction, to a large set of data, containing IgV productive and non-productive heavy and light chain sequences from several different tissues, to examine three of the most profoundly studied AI diseases - Rheumatoid Arthritis (RA), Multiple Sclerosis (MS) and Sjögrens Syndrome (SS). We have found that RA and MS sequences exhibited normal mutation spectra and targeting motifs, but a stricter selection compared to normal controls, which was more apparent in RA. SS sequence analysis results deviated from normal controls in both mutation spectra and indications of selection, also showing differences between light and heavy chain IgV and between different tissues. The differences revealed between AI diseases and normal control mutation patterns may result from the different microenvironmental influences to which ectopic GCs are exposed, relative to those in normal secondary lymphoid tissues.

Ectopic GC in the thymus of myasthenia gravis patients show characteristics of normal GC.

Young patients with myasthenia gravis (MG) frequently have ectopic GC in their thymus. We investigated these ectopic GC by microdissection of GC B cells and analysis of their Ig gene characteristics, in comparison to normal GC. CDR3 length distribution, a measure of clonal variability, and Ig gene family usage were similar in MG and normal tonsil samples. Lineage tree analysis demonstrated similar diversification and mutations per cell compared with normal control trees. Mutations were observed in the framework regions, responsible for the structural integrity of the BCR; however, these mutations were mostly conservative or neutral, confirming that a functional BCR is conserved in MG. In the CDR, responsible for Ag binding, selection against replacement mutations was revealed. This may indicate that the MG clones analyzed are already highly Ag‐specific, and therefore potential affinity‐reducing replacement mutations in the CDR3 are not propagated, due to Ag‐driven selection. Somatic hypermutation (SHM) targeting motifs and aa substitution preferences in MG were similar to those of normal controls. Overall, these results suggest that B cells in the ectopic GC in MG appear to undergo normal diversification and selection, in spite of the chronic nature and different environment of the response.

Ig gene diversification and selection in follicular lymphoma, diffuse large B cell lymphoma and primary central nervous system lymphoma revealed by lineage tree and mutation analyses

Follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL) and primary central nervous system lymphoma are B cell malignancies. FL and DLBCL have a germinal center origin. We have applied mutational analyses and a novel algorithm for quantifying shape properties of mutational lineage trees to investigate the nature of the diversification, somatic hypermutation and selection processes that affect B cell clones in these malignancies and reveal whether they differ from normal responses. Lineage tree analysis demonstrated higher diversification and mutations per cell in the lymphoma clones. This was caused solely by the longer diversification times of the malignant clones, as their recent diversification processes were similar to those of normal responses, implying similar mutation frequencies. Since previous analyses of antigen-driven selection were shown to yield false positives, we performed a corrected analysis of replacement and silent mutation patterns, which revealed selection against replacement mutations in the framework regions, responsible for the structural integrity of the B cell receptor, but not for positive selection for replacements in the complementary determining regions. Most replacements, however, were neutral or conservative, suggesting that if at all selection operates in these malignancies it is for structural B cell receptor integrity but not for antigen binding.

B‐cell clonal diversification and gut‐lymph node trafficking in ulcerative colitis revealed using lineage tree analysis

In studies of inflammatory bowel diseases (IBD), research has so far focused mainly on the role of T cells. Despite evidence suggesting that B cells and the production of autoantibodies may play a significant role in IBD pathogenesis, the role of B cells in gut inflammation has not yet been thoroughly investigated. In the present study we used the new approach of lineage tree analysis for studying immunoglobulin variable region gene diversification in B cells found in the inflamed intestinal tissue of two ulcerative colitis patients as well as B cells from mucosa‐associated lymph nodes (LN) in the same patients. Healthy intestinal tissue of three patients with carcinoma of the colon was used as normal control. Lineage tree shapes revealed active immune clonal diversification processes occurring in ulcerative colitis patients, which were quantitatively similar to those in healthy controls. B cells from intestinal tissues and the associated LN are shown here to be clonally related, thus supplying the first direct evidence supporting B‐cell trafficking between gut and associated LN in IBD and control tissues.

Novel Analysis of Clonal Diversification in Blood B Cell and Bone Marrow Plasma Cell Clones in Immunoglobulin Light Chain Amyloidosis

Immunoglobulin light chain amyloidosis (AL) is characterized by a limited clonal expansion of plasma cells and amyloid formation. Here, we report restriction in the diversity of VL gene usage with a dominance of clonally related B cells in the peripheral blood (PB) isotype-specific repertoire of AL patients. A rigorous quantification of lineage trees reveals presence of intraclonal variations in the PB clones compared to the bone marrow (BM) clones, which suggests a common precursor that is still subject to somatic mutation. When compared to normal BM and PB B cells, AL clones showed significant but incomplete impairment of antigenic selection, which could not be detected by conventional R and S mutation analysis. Therefore, graphical analysis of B cell lineage trees and mathematical quantification of tree properties provide novel insights into the process of B cell clonal evolution in AL.

The V(H) repertoire and clonal diversification of B cells in inflammatory myopathies.

The contribution of antigen‐driven B‐cell adaptive immune responses within the inflamed muscle of inflammatory myopathies (IMs) is largely unknown. In this study, we investigated the immunoglobulin VH gene repertoire, somatic hypermutation, clonal diversification, and selection of infiltrating B cells in muscle biopsies from IM patients (dermatomyositis and polymyositis), to determine whether B cells and/or plasma cells contribute to the associated pathologies of these diseases. The data reveal that Ig VH gene repertoires of muscle‐infiltrating B cells deviate from the normal VH gene repertoire in individual patients, and differ between different types of IMs. Analysis of somatic mutations revealed clonal diversification of muscle‐infiltrating B cells and evidence for a chronic B‐cell response within the inflamed muscle. We conclude that muscle‐infiltrating B cells undergo selection, somatic hypermutation and clonal diversification in situ during antigen‐driven immune responses in patients with IMs, providing insight into the contribution of B cells to the pathological mechanisms of these disorders.