Human B-cell development at the single-cell level

Abstract

B cells play an integral part of our immune system. In adults they develop in the bone marrow in a regulated process in which the specificity of their B-cell antigen receptor (BCR), a membrane bound antibody, is established. This is generated by somatic recombination of immunoglobulin (Ig) gene segments that encode the antigen binding region of Ig heavy and light chains. Most studies of B-cell development have been carried out in mouse models, while those in humans have mainly been performed at the population level. This thesis aimed to delineate early B-cell development in human bone marrow and investigate how the BCR repertoire is shaped, at the single-cell level. To this end, we analysed human bone marrow early B-lineage cells using flow cytometry and 5’ single-cell RNA- and VDJ sequencing. In Paper I, we investigated the influence of BCR and T-cell antigen receptor genes on unsupervised clustering and downstream analyses in 5’ single-cell RNA sequencing data from B and T cells. In Paper II, we aimed to delineate the stages of B-cell development in the adult human bone marrow at the single-cell level and investigate how the BCR repertoire is shaped throughout development. In Paper III, we analysed the expression of Ig light chain transcripts during B-cell development, to infer recombination status of the kappa and lambda light chain loci. In conclusion, we have developed a protocol to analyse 5’ single-cell RNA sequencing data which generates biologically relevant clusters of B and T cells. By using flow cytometry and the protocol developed in Paper I to analyse 5’ single-cell RNA and VDJ sequencing data, we can put forward a novel model of human B-cell development, which includes shaping of the BCR repertoire from the earliest pro-B cells to mature naïve B cells. For instance, we show a step-wise selection of the BCR repertoire, mainly exerted on the H-CDR3 region. Moreover, our data indicate that recombination of Ig lambda light chain is independent of that of kappa, a model that challenges the current dogma.