GLaMST: grow lineages along minimum spanning tree for b cell receptor sequencing data.

BACKGROUND: B cell affinity maturation enables B cells to generate high-affinity antibodies. This process involves somatic hypermutation of B cell immunoglobulin receptor (BCR) genes and selection by their ability to bind antigens. Lineage trees are used to describe this microevolution of B cell immunoglobulin genes. In a lineage tree, each node is one BCR sequence that mutated from the germinal center and each directed edge represents a single base mutation, insertion or deletion. In BCR sequencing data, the observed data only contains a subset of BCR sequences in this microevolution process. Therefore, reconstructing the lineage tree from experimental data requires algorithms to build the tree based on partially observed tree nodes. RESULTS: We developed a new algorithm named Grow Lineages along Minimum Spanning Tree (GLaMST), which efficiently reconstruct the lineage tree given observed BCR sequences that correspond to a subset of the tree nodes. Through comparison using simulated and real data, GLaMST outperforms existing algorithms in simulations with high rates of mutation, insertion and deletion, and generates lineage trees with smaller size and closer to ground truth according to tree features that highly correlated with selection pressure. CONCLUSIONS: GLaMST outperforms state-of-art in reconstruction of the BCR lineage tree in both efficiency and accuracy. Integrating it into existing BCR sequencing analysis frameworks can significant improve lineage tree reconstruction aspect of the analysis.

Isolation of Tailor-Made Antibody Fragments from Yeast-Displayed B-Cell Receptor Repertoires by Multiparameter Fluorescence-Activated Cell Sorting.

In the past decades, monoclonal antibodies have made an unprecedented transformation from research tools to a rapidly growing class of therapeutics. Advancements in the yeast surface display platform enable the selection of favorable mouse or human antibody variants from large B-cell receptor (BCR) gene repertoires that are derived from immunized normal or transgenic animals. Application of high-throughput fluorescence-activated cell sorting (FACS) screening along with well-chosen selection settings can be utilized to identify variants with desired affinities and predefined epitope binding properties. In the following chapter, we describe in detail a multiparameter screening protocol for the selection of antibody variants from yeast libraries generated from BCR gene repertoires from immunized transgenic rats. The procedure provides guidance for the selection of antigen-specific, high-affinity binding, and species cross-reactive human antibodies with a broad epitope coverage. Essentially, this can accelerate target-specific antibody characterization as multiple desirable antibody features can be easily integrated into the selection procedure. In addition, we provide information on how to validate binding behavior of selected candidates after expression as soluble, full-length IgG molecules.

Isolation of Anti-Hapten Antibodies by Fluorescence-Activated Cell Sorting of Yeast-Displayed B-Cell Receptor Gene Repertoires.

Anti-hapten antibodies are widely used as specific immunochemical detection tools in a variety of clinical and environmental analyses. The sensitivity, however, is limited due to the resulting antibody affinities to the haptens which, in turn, leads to a high demand for specific affinity reagents. A well-established path for the generation of high-affinity antibodies is the immunization of animals with the target antigen. However, the generation of anti-hapten antibodies via immunization remains challenging as small molecule haptens usually possess low immunogenicity and, therefore, must be coupled to an immunogenic and high molecular weight carrier to provoke an immune response.Consequently, antibodies are primarily raised against the carrier molecule or structural features of the hapten-linker fused to the carrier protein. This turns the generation of antibodies which bind exclusively to the hapten structure into a search for the needle in a haystack. In the following chapter, we describe how yeast surface display and high-throughput fluorescence-activated cell sorting can be used to isolate anti-hapten antibodies from a large, yeast-displayed B-cell receptor gene library derived from immunized animals. For this, we describe in detail the preparation of protein-hapten conjugates, the immunization procedure, and the subsequent screening process. Moreover, we provide a simple flow cytometry protocol that allows for a rapid analysis of the enriched clones toward free hapten binding.

The utility of B-cell receptor gene rearrangement studies in diagnosing diffuse large B-cell lymphoma with plasmacytic differentiation.

We present the case of a 50-year-old man with nodal diffuse large B-cell lymphoma characterized by CD10, BCL-6, and BCL-2 expression and a complex karyotype, including t(14;18)(q32;q21) and del6q, suggesting transformation from an antecedent follicular lymphoma. Following rituximab-based chemotherapy, residual nodal disease was characterized by a proliferation of plasmacytoid cells positive for CD138, MUM-1, and cytoplasmic kappa light chain. Immunoglobulin heavy chain and kappa light chain gene rearrangement studies detected the same clone in the diagnostic and post-therapy lymph node specimens. This case illustrates the diagnostic utility of B-cell receptor gene rearrangement studies in detecting a clonal relationship in lymphoma cases with extensive morphologic and immunophenotypic variation following chemotherapy.