Affinity maturation selects B cells expressing somatically mutated antibody variants with improved antigen-binding properties to protect from invading pathogens. We determined the molecular mechanism underlying the clonal selection and affinity maturation of human B cells expressing protective antibodies against the circumsporozoite protein of the malaria parasite (PfCSP). We show in molecular detail that the repetitive nature of PfCSP facilitates direct homotypic interactions between two PfCSP repeat-bound monoclonal antibodies, thereby improving antigen affinity and B cell activation. These data provide a mechanistic explanation for the strong selection of somatic mutations that mediate homotypic antibody interactions after repeated parasite exposure in humans. Our findings demonstrate a different mode of antigen-mediated affinity maturation to improve antibody responses to PfCSP and presumably other repetitive antigens.

Affinity maturation, the clonal selection and expansion of antigen-activated B cells expressing somatically mutated antibody variants that develop during T cell-dependent germinal center reactions, is considered pivotal for efficient development of protective B cell memory responses to infection and vaccination. Repeated antigen exposure promotes affinity maturation but each time also recruits antigen-reactive naïve B cells into the response. Here, we determined the relative impact of affinity maturation versus antigen-mediated clonal selection of naïve B cells to mount potent B cell memory responses in humans after repeated exposure to a complex pathogen, the malaria parasite (Pf). Using single-cell immunoglobulin (Ig) gene sequencing and production of recombinant monoclonal antibodies, we analyzed the origin, development, and quality of memory B cell responses to Pf circumsporozoite protein (PfCSP), the major sporozoite surface protein. We show that after repeated immunization of Pf-naïve volunteers with infectious Pf sporozoites (PfSPZ Challenge) under chloroquine prophylaxis (PfSPZ-CVac), the clonal selection of potent germline and memory B cell precursors against the central PfCSP NANP repeat outpaces affinity maturation because the majority of Ig gene mutations are affinity-neutral. Mathematical modeling explains how the efficiency of affinity maturation decreases strongly with antigen complexity. Thus, in the absence of long-term exposure, the frequency of antigen-reactive precursors and likelihood of their activation rather than affinity maturation will determine the quality of anti-PfCSP memory B cell responses. These findings have wide implications for the design of vaccination strategies to induce potent B cell memory responses against PfCSP and presumably other structurally complex antigens.

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21–derived heavy chains (HCs) with IGLV3-21–derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110–expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR–BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01–expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110–expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

The circadian clock coordinates daily physiological, metabolic and behavioural rhythms. These endogenous oscillations are synchronized with external cues ('zeitgebers'), such as daily light and temperature cycles. When the circadian clock is entrained by a zeitgeber, the phase difference c between the phase of a clock-controlled rhythm and the phase of the zeitgeber is of fundamental importance for the fitness of the organism. The phase of entrainment c depends on the mismatch between the intrinsic period t and the zeitgeber period T and on the ratio of the zeitgeber strength to oscillator amplitude. Motivated by the intriguing complexity of empirical data and by our own experiments on temperature entrainment of mouse suprachiasmatic nucleus (SCN) slices, we present a theory on how clock and zeitgeber properties determine the phase of entrainment. The wide applicability of the theory is demonstrated using mathematical models of different complexity as well as by experimental data. Predictions of the theory are confirmed by published data on Neurospora crassa strains for different period mismatches t 2 T and varying photoperiods. We apply a novel regression technique to analyse entrainment of SCN slices by temperature cycles. We find that mathematical models can explain not only the stable asymptotic phase of entrainment, but also transient phase dynamics. Our theory provides the potential to explore seasonal variations of circadian rhythms, jet lag and shift work in forthcoming studies.

Pooled CRISPR screens are a powerful tool to probe genotype-phenotype relationships at genome-wide scale. However, criteria for optimal design are missing, and it remains unclear how experimental parameters affect results. Here, we report that random decreases in gRNA abundance are more likely than increases due to bottleneck effects during the cell proliferation phase. Failure to consider this asymmetry leads to loss of detection power. We provide a new statistical test that addresses this problem and improves hit detection at reduced experiment size.

Background: cAMP research in intracellular parasites remains underappreciated, and it requires a specific method for cyclic nucleotide regulation. Results: Optogenetic induction of cAMP in T. gondii affects host-cell invasion, stage-specific expression, and parasite differentiation. The underlying method allows a versatile control of parasite cAMP. Conclusions: Optogenetic parasite strains offer valuable tools for dissecting cAMP-mediated processes. Significance: The method is applicable to other gene-tractable intertwined systems.

A hallmark of the adaptive immune system is the specificity of B cell and T cell responses. Mechanistically, this feature relies on the fact that the two genes that encode B cell and T cell antigen receptors are not germline encoded and instead are assembled from a large number of small gene segments during lymphocyte development. The underlying somatic gene recombination process can generate a quasi-unlimited repertoire of antigen receptors. The high degree of diversity is essential to guarantee recognition of nearly any antigenic structure to protect from the large variety of potential invading pathogens and to keep the balance with commensals. Due to the enormous complexity of the antigen receptor repertoire, our understanding of its actual size and functional convergence at the level of the individual and the population is still limited. A better understanding of the actual degree of diversity could help to predict adaptive immune responses and would have wide implications for the development of preventive and therapeutic measures against infectious and autoimmune diseases as well as cancer. Here, we discuss the recent advances in the field with a specific focus on B cells and the function of antibodies.

BackgroundThe sequencing of immunoglobulin (Ig) transcripts from single B cells yields essential information about Ig heavy:light chain pairing, which is lost in conventional bulk sequencing experiments. The previously limited throughput of single-cell approaches has recently been overcome by the introduction of multiple next-generation sequencing (NGS)-based platforms. Furthermore, single-cell techniques allow the assignment of additional data types (e.g. cell surface marker expression), which are crucial for biological interpretation. However, the currently available computational tools are not designed to handle single-cell data and do not provide integral solutions for linking of sequence data to other biological data.ResultsHere we introduce sciReptor, a flexible toolkit for the processing and analysis of antigen receptor repertoire sequencing data at single-cell level. The software combines bioinformatics tools for immunoglobulin sequence annotation with a relational database, where raw data and analysis results are stored and linked. sciReptor supports attribution of additional data categories such as cell surface marker expression or immunological metadata. Furthermore, it comprises a quality control module as well as basic repertoire visualization tools.ConclusionsciReptor is a flexible framework for standardized sequence analysis of antigen receptor repertoires on single-cell level. The relational database allows easy data sharing and downstream analyses as well as immediate comparisons between different data sets.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-0920-1) contains supplementary material, which is available to authorized users.