Systematic evaluation of B-cell clonal family inference approaches.

The reconstruction of clonal families (CFs) in B-cell receptor (BCR) repertoire analysis is a crucial step to understand the adaptive immune system and how it responds to antigens. The BCR repertoire of an individual is formed throughout life and is diverse due to several factors such as gene recombination and somatic hypermutation. The use of Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) using next generation sequencing enabled the generation of full BCR repertoires that also include rare CFs. The reconstruction of CFs from AIRR-seq data is challenging and several approaches have been developed to solve this problem. Currently, most methods use the heavy chain (HC) only, as it is more variable than the light chain (LC). CF reconstruction options include the definition of appropriate sequence similarity measures, the use of shared mutations among sequences, and the possibility of reconstruction without preliminary clustering based on V- and J-gene annotation. In this study, we aimed to systematically evaluate different approaches for CF reconstruction and to determine their impact on various outcome measures such as the number of CFs derived, the size of the CFs, and the accuracy of the reconstruction. The methods were compared to each other and to a method that groups sequences based on identical junction sequences and another method that only determines subclones. We found that after accounting for data set variability, in particular sequencing depth and mutation load, the reconstruction approach has an impact on part of the outcome measures, including the number of CFs. Simulations indicate that unique junctions and subclones should not be used as substitutes for CF and that more complex methods do not outperform simpler methods. Also, we conclude that different approaches differ in their ability to correctly reconstruct CFs when not considering the LC and to identify shared CFs. The results showed the effect of different approaches on the reconstruction of CFs and highlighted the importance of choosing an appropriate method.

Convergent evolution and B-cell recirculation in germinal centers in a human lymph node.

Germinal centers (GCs) play a central role in generating an effective immune response against infectious pathogens, and failures in their regulating mechanisms can lead to the development of autoimmune diseases and cancer. Although previous works study experimental systems of the immune response with mouse models that are immunized with specific antigens, our study focused on a real-life situation, with an ongoing GC response in a human lymph node (LN) involving multiple asynchronized GCs reacting simultaneously to unknown antigens. We combined laser capture microdissection of individual GCs from human LN with next-generation repertoire sequencing to characterize individual GCs as distinct evolutionary spaces. In line with well-characterized GC responses in mice, elicited by immunization with model antigens, we observe a heterogeneous clonal diversity across individual GCs from the same human LN. Still, we identify shared clones in several individual GCs, and phylogenetic tree analysis combined with paratope modeling suggest the re-engagement and rediversification of B-cell clones across GCs and expanded clones exhibiting shared antigen responses across distinct GCs, indicating convergent evolution of the GCs.

Exploring the impact of clonal definition on B-cell diversity: implications for the analysis of immune repertoires.

The adaptive immune system has the extraordinary ability to produce a broad range of immunoglobulins that can bind a wide variety of antigens. During adaptive immune responses, activated B cells duplicate and undergo somatic hypermutation in their B-cell receptor (BCR) genes, resulting in clonal families of diversified B cells that can be related back to a common ancestor. Advances in high-throughput sequencing technologies have enabled the high-throughput characterization of B-cell repertoires, however, the accurate identification of clonally related BCR sequences remains a major challenge. In this study, we compare three different clone identification methods on both simulated and experimental data, and investigate their impact on the characterization of B-cell diversity. We observe that different methods lead to different clonal definitions, which affects the quantification of clonal diversity in repertoire data. Our analyses show that direct comparisons between clonal clusterings and clonal diversity of different repertoires should be avoided if different clone identification methods were used to define the clones. Despite this variability, the diversity indices inferred from the repertoires' clonal characterization across samples show similar patterns of variation regardless of the clonal identification method used. We find the Shannon entropy to be the most robust in terms of the variability of diversity rank across samples. Our analysis also suggests that the traditional germline gene alignment-based method for clonal identification remains the most accurate when the complete information about the sequence is known, but that alignment-free methods may be preferred for shorter sequencing read lengths. We make our implementation freely available as a Python library cdiversity.

Understanding repertoire sequencing data through a multiscale computational model of the germinal center.

Sequencing of B-cell and T-cell immune receptor repertoires helps us to understand the adaptive immune response, although it only provides information about the clonotypes (lineages) and their frequencies and not about, for example, their affinity or antigen (Ag) specificity. To further characterize the identified clones, usually with special attention to the particularly abundant ones (dominant), additional time-consuming or expensive experiments are generally required. Here, we present an extension of a multiscale model of the germinal center (GC) that we previously developed to gain more insight in B-cell repertoires. We compare the extent that these simulated repertoires deviate from experimental repertoires established from single GCs, blood, or tissue. Our simulations show that there is a limited correlation between clonal abundance and affinity and that there is large affinity variability among same-ancestor (same-clone) subclones. Our simulations suggest that low-abundance clones and subclones, might also be of interest since they may have high affinity for the Ag. We show that the fraction of plasma cells (PCs) with high B-cell receptor (BcR) mRNA content in the GC does not significantly affect the number of dominant clones derived from single GCs by sequencing BcR mRNAs. Results from these simulations guide data interpretation and the design of follow-up experiments.

Huwe1 supports B-cell development, B-cell-dependent immunity, somatic hypermutation and class switch recombination by regulating proliferation.

The development and differentiation of B cells is intimately linked to cell proliferation and the generation of diverse immunoglobulin gene (Ig) repertoires. The ubiquitin E3 ligase HUWE1 controls proliferation, DNA damage responses, and DNA repair, including the base excision repair (BER) pathway. These processes are of crucial importance for B-cell development in the bone marrow, and the germinal center (GC) response, which results in the clonal expansion and differentiation of B cells expressing high affinity immunoglobulins. Here, we re-examined the role of HUWE1 in B-cell proliferation and Ig gene diversification, focusing on its involvement in somatic hypermutation (SHM) and class switch recombination (CSR). B-cell-specific deletion of Huwe1 resulted in impaired development, differentiation and maturation of B cells in the bone marrow and peripheral lymphoid organs. HUWE1 deficiency diminished SHM and CSR by impairing B-cell proliferation and AID expression upon activation in vitro and in vivo, and was unrelated to the HUWE1-dependent regulation of the BER pathway. Interestingly, we found that HUWE1-deficient B cells showed increased mRNA expression of Myc target genes upon in vitro activation despite diminished proliferation. Our results confirm that the E3 ligase HUWE1 is an important contributor in coordinating the rapid transition of antigen naïve, resting B cells into antigen-activated B cells and regulates mutagenic processes in B cells by controlling AID expression and the post-transcriptional output of Myc target genes.

Base Excision Repair in the Immune System: Small DNA Lesions With Big Consequences.

The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring at an estimated rate of 10,000-30,000 per cell/per day, and mostly involves chemical DNA base lesions caused by oxidation, depurination, alkylation, and deamination. The base excision repair (BER) pathway is primary responsible for removing and repairing these small base lesions that would otherwise lead to mutations or DNA breaks during replication. Next to preventing DNA mutations and damage, the BER pathway is also involved in mutagenic processes in B cells during immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM), which are instigated by uracil (U) lesions derived from activation-induced cytidine deaminase (AID) activity. BER is required for the processing of AID-induced lesions into DNA double strand breaks (DSB) that are required for CSR, and is of pivotal importance for determining the mutagenic outcome of uracil lesions during SHM. Although uracils are generally efficiently repaired by error-free BER, this process is surprisingly error-prone at the Ig loci in proliferating B cells. Breakdown of this high-fidelity process outside of the Ig loci has been linked to mutations observed in B-cell tumors and DNA breaks and chromosomal translocations in activated B cells. Next to its role in preventing cancer, BER has also been implicated in immune tolerance. Several defects in BER components have been associated with autoimmune diseases, and animal models have shown that BER defects can cause autoimmunity in a B-cell intrinsic and extrinsic fashion. In this review we discuss the contribution of BER to genomic integrity in the context of immune receptor diversification, cancer and autoimmune diseases.