The type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and... Show moreThe type and strength of effector functions mediated by immunoglobulin G (IgG) antibodies rely on the subclass and the composition of the N297 glycan. Glycosylation analysis of both bulk and antigen-specific human IgG has revealed a marked diversity of the glycosylation signatures, including highly dynamic patterns as well as long-term stability of profiles, yet information on how individual B cell clones would contribute to this diversity has hitherto been lacking. Here, we assessed whether clonally related B cells share N297 glycosylation patterns of their secreted IgG. We differentiated single antigen-specific peripheral IgG+ memory B cells into antibody-secreting cells and analysed Fc glycosylation of secreted IgG. Furthermore, we sequenced the variable region of their heavy chain, which allowed the grouping of the clones into clonotypes. We found highly diverse glycosylation patterns of culture-derived IgG, which, to some degree, mimicked the glycosylation of plasma IgG. Each B cell clone secreted IgG with a mixture of different Fc glycosylation patterns. The majority of clones produced fully fucosylated IgG. B cells producing afucosylated IgG were scattered across different clonotypes. In contrast, the remaining glycosylation traits were, in general, more uniform. These results indicate IgG-Fc fucosylation to be regulated at the single-clone level, whereas the regulation of other glycosylation traits most likely occurs at a clonotypic or systemic level. The discrepancies between plasma IgG and culture-derived IgG, could be caused by the origin of the B cells analysed, clonal dominance or factors from the culture system, which need to be addressed in future studies. Show less
Koers, J.; Pollastro, S.; Tol, S.; Niewold, I.T.G.; Schouwenburg, P.A. van; Vries, N. de; Rispens, T. 2022
In past years ex vivo and in vivo experimental approaches involving human naive B cells have proven fundamental for elucidation of mechanisms promoting B cell differentiation in both health and... Show moreIn past years ex vivo and in vivo experimental approaches involving human naive B cells have proven fundamental for elucidation of mechanisms promoting B cell differentiation in both health and disease. For such studies, it is paramount that isolation strategies yield a population of bona fide naive B cells, i.e., B cells that are phenotypically and functionally naive, clonally non-expanded, and have non-mutated BCR variable regions. In this study different combinations of common as well as recently identified B cell markers were compared to isolate naive B cells from human peripheral blood. High-throughput BCR sequencing was performed to analyze levels of somatic hypermutation and clonal expansion. Additionally, contamination from mature mutated B cells intrinsic to each cell-sorting strategy was evaluated and how this impacts the purity of obtained populations. Our results show that current naive B cell isolation strategies harbor contamination from non-naive B cells, and use of CD27-IgD(+) is adequate but can be improved by including markers for CD45RB glycosylation and IgM. The finetuning of naive B cell classification provided herein will harmonize research lines using naive B cells, and will improve B cell profiling during health and disease, e.g. during diagnosis, treatment, and vaccination strategies. Show less
Background: Drawing genotype-to-phenotype maps in tumors is of paramount importance for understanding tumor heterogeneity. Assignment of single cells to their tumor clones of origin can be... Show moreBackground: Drawing genotype-to-phenotype maps in tumors is of paramount importance for understanding tumor heterogeneity. Assignment of single cells to their tumor clones of origin can be approached by matching the genotypes of the clones to the mutations found in RNA sequencing of the cells. The confidence of the cell-to-clone mapping can be increased by accounting for additional measurements. Follicular lymphoma, a malignancy of mature B cells that continuously acquire mutations in parallel in the exome and in B cell receptor loci, presents a unique opportunity to join exome-derived mutations with B cell receptor sequences as independent sources of evidence for clonal evolution.Methods: Here, we propose CACTUS, a probabilistic model that leverages the information from an independent genomic clustering of cells and exploits the scarce single cell RNA sequencing data to map single cells to given imperfect genotypes of tumor clones.Results: We apply CACTUS to two follicular lymphoma patient samples, integrating three measurements: whole exome, single-cell RNA, and B cell receptor sequencing. CACTUS outperforms a predecessor model by confidently assigning cells and B cell receptor-based clusters to the tumor clones.Conclusions: The integration of independent measurements increases model certainty and is the key to improving model performance in the challenging task of charting the genotype-to-phenotype maps in tumors. CACTUS opens the avenue to study the functional implications of tumor heterogeneity, and origins of resistance to targeted therapies. CACTUS is written in R and source code, along with all supporting files, are available on GitHub (https://github.com/LUMC/CACTUS). Show less
