Local Clonal Diversification and Dissemination of B Lymphocytes in the Human Bronchial Mucosa

The efficacy of the adaptive humoral immune response likely requires diverse, yet focused regional B cell antibody production throughout the body. Here we address, in the first study of its kind, the B cell repertoire in the bronchial mucosa, an important barrier to antigens inhaled from the atmosphere. To accomplish this, we have applied high-throughput Adaptive Immune Receptor Repertoire Sequencing (AIRR-Seq) to 10 bronchial biopsies from altogether four different sites in the right lungs from an asthmatic patient and a healthy subject. While the majority of identified B cell clones were restricted to a single site, many were disseminated in multiple sites. Members of a clone were shared more between adjacent biopsies than between distal biopsies, suggesting local mucosal migration and/or a homing mechanism for B cells through the blood or lymph. A smaller fraction of clones spanned the bronchial mucosa and peripheral blood, suggesting ongoing trafficking between these compartments. The bronchial mucosal B cell repertoire in the asthmatic patient was geographically more variable but less diverse compared to that of the healthy subject, suggesting an ongoing, antigen-driven humoral immune response in atopic asthma. Whether this is a feature of atopy or disease status remains to be clarified in future studies. We observed a subset of highly mutated and antigen-selected IgD-only cells in the bronchial mucosa. These cells were found in relative high abundance in the asthmatic individual but also, albeit at lower abundance, in the healthy subject. This novel finding merits further exploration using a larger cohort of subjects

Icing: Large-scale inference of immunoglobulin clonotypes

Immunoglobulin (IG) clonotype identification is a fundamental open question in modern immunology. An accurate description of the IG repertoire is crucial to understand the variety within the immune system of an individual, potentially shedding light on the pathogenetic process. Intrinsic IG heterogeneity makes clonotype inference an extremely challenging task, both from a computational and a biological point of view. Here we present icing, a framework that allows to reconstruct clonal families also in case of highly mutated sequences. icing has a modular structure, and it is designed to be used with large next generation sequencing (NGS) datasets, a technology which allows the characterisation of large-scale IG repertoires. We extensively validated the framework with clustering performance metrics on the results in a simulated case. icing is implemented in Python, and it is publicly available under FreeBSD licence at https://github.com/slipguru/icing

Integrating B Cell Lineage Information into Statistical Tests for Detecting Selection in Ig Sequences

Detecting selection in B cell immunoglobulin (Ig) sequences is critical to understanding affinity maturation, and can provide insights into antigen-driven selection in normal and pathologic immune responses. The most common sequence-based methods for detecting selection analyze the ratio of replacement (R) and silent (S) mutations using a binomial statistical analysis. However, these approaches have been criticized for low sensitivity. An alternative method is based on the analysis of lineage trees constructed from sets of clonally-related Ig sequences. Several tree shape measures have been proposed as indicators of selection that can be statistically compared across cohorts. However, we show that tree shape analysis is confounded by underlying experimental factors that are difficult to control for in practice, including the sequencing depth and number of generations in each clone. Thus, though lineage tree shapes may reflect selection, their analysis alone is an unreliable measure of in vivo selection. To usefully capture the information provided by lineage trees, we propose a new method that applies the binomial statistical method to mutations identified based on lineage tree structure. This hybrid method is able to detect selection with increased sensitivity in both simulated and experimental data sets. We anticipate that this approach will be especially useful in the analysis of large-scale Ig sequencing data sets generated by high-throughput sequencing technologies

Salmonella Infection Drives Promiscuous B Cell Activation Followed by Extrafollicular Affinity Maturation

SummaryThe B cell response to Salmonella typhimurium (STm) occurs massively at extrafollicular sites, without notable germinal centers (GCs). Little is known in terms of its specificity. To expand the knowledge of antigen targets, we screened plasmablast (PB)-derived monoclonal antibodies (mAbs) for Salmonella specificity, using ELISA, flow cytometry, and antigen microarray. Only a small fraction (0.5%–2%) of the response appeared to be Salmonella-specific. Yet, infection of mice with limited B cell receptor (BCR) repertoires impaired the response, suggesting that BCR specificity was important. We showed, using laser microdissection, that somatic hypermutation (SHM) occurred efficiently at extrafollicular sites leading to affinity maturation that in turn led to detectable STm Ag-binding. These results suggest a revised vision of how clonal selection and affinity maturation operate in response to Salmonella. Clonal selection initially is promiscuous, activating cells with virtually undetectable affinity, yet SHM and selection occur during the extrafollicular response yielding higher affinity, detectable antibodies

Optimality of mutation and selection in germinal centers

The population dynamics theory of B cells in a typical germinal center could play an important role in revealing how affinity maturation is achieved. However, the existing models encountered some conflicts with experiments. To resolve these conflicts, we present a coarse-grained model to calculate the B cell population development in affinity maturation, which allows a comprehensive analysis of its parameter space to look for optimal values of mutation rate, selection strength, and initial antibody-antigen binding level that maximize the affinity improvement. With these optimized parameters, the model is compatible with the experimental observations such as the ~100-fold affinity improvements, the number of mutations, the hypermutation rate, and the "all or none" phenomenon. Moreover, we study the reasons behind the optimal parameters. The optimal mutation rate, in agreement with the hypermutation rate in vivo, results from a tradeoff between accumulating enough beneficial mutations and avoiding too many deleterious or lethal mutations. The optimal selection strength evolves as a balance between the need for affinity improvement and the requirement to pass the population bottleneck. These findings point to the conclusion that germinal centers have been optimized by evolution to generate strong affinity antibodies effectively and rapidly. In addition, we study the enhancement of affinity improvement due to B cell migration between germinal centers. These results could enhance our understandings to the functions of germinal centers.Comment: 5 figures in main text, and 4 figures in Supplementary Informatio

Antigen-specific, antibody-coated, exosome-like nanovesicles deliver suppressor T-cell microRNA-150 to effector T cells to inhibit contact sensitivity

Background: T-cell tolerance of allergic cutaneous contact sensitivity (CS) induced in mice by high doses of reactive hapten is mediated by suppressor cells that release antigen-specific suppressive nanovesicles. Objective: We sought to determine the mechanism or mechanisms of immune suppression mediated by the nanovesicles. Methods: T-cell tolerance was induced by means of intravenous injection of hapten conjugated to self-antigens of syngeneic erythrocytes and subsequent contact immunization with the same hapten. Lymph node and spleen cells from tolerized or control donors were harvested and cultured to produce a supernatant containing suppressive nanovesicles that were isolated from the tolerized mice for testing in active and adoptive cell-transfer models of CS. Results: Tolerance was shown due to exosome-like nanovesicles in the supernatants of CD81 suppressor T cells that were not regulatory T cells. Antigen specificity of the suppressive nanovesicles was conferred by a surface coat of antibody light chains or possibly whole antibody, allowing targeted delivery of selected inhibitory microRNA (miRNA)–150 to CS effector T cells. Nanovesicles also inhibited CS in actively sensitized mice after systemic injection at the peak of the responses. The role of antibody and miRNA-150 was established by tolerizing either panimmunoglobulin-deficient JH2/2 or miRNA-1502/2 mice that produced nonsuppressive nanovesicles. These nanovesicles could be made suppressive by adding antigen-specific antibody light chains or miRNA-150, respectively. Conclusions: This is the first example of T-cell regulation through systemic transit of exosome-like nanovesicles delivering a chosen inhibitory miRNA to target effector T cells in an antigen-specific manner by a surface coating of antibody light chains