Separation of B cells into different subsets has been useful to understand their different functions in various immune scenarios. In some instances, the subsets defined by phenotypic FACS separation are relatively homogeneous and so establishing the functions associated with them is straightforward. Other subsets, such as the “Double negative” (DN, CD19+CD27-IgD-) population, are more complex with reports of differing functionality which could indicate a heterogeneous population. Recent advances in single-cell techniques enable an alternative route to characterize cells based on their transcriptome. To maximize immunological insight, we need to match prior data from phenotype-based studies with the finer granularity of the single-cell transcriptomic signatures. We also need to be able to define meaningful B cell subsets from single cell analyses performed on PBMCs, where the relative paucity of a B cell signature means that defining B cell subsets within the whole is challenging. Here we provide a reference single-cell dataset based on phenotypically sorted B cells and an unbiased procedure to better classify functional B cell subsets in the peripheral blood, particularly useful in establishing a baseline cellular landscape and in extracting significant changes with respect to this baseline from single-cell datasets. We find 10 different clusters of B cells and applied a novel, geometry-inspired, method to RNA velocity estimates in order to evaluate the dynamic transitions between B cell clusters. This indicated the presence of two main developmental branches of memory B cells. A T-independent branch that involves IgM memory cells and two DN subpopulations, culminating in a population thought to be associated with Age related B cells and the extrafollicular response. The other, T-dependent, branch involves a third DN cluster which appears to be a precursor of classical memory cells. In addition, we identify a novel DN4 population, which is IgE rich and closely linked to the classical/precursor memory branch suggesting an IgE specific T-dependent cell population.

In this work we have microdissected lamina propria plasma cells and used polymerase chain reaction and sequencing to investigate immunoglobulin (Ig) gene rearrangements and mutations in human intestine. In addition, specific primers were designed for individual Ig gene rearrangements to analyze the distribution of related B cell and plasma cell clones at different sites along the bowel. Confirming our earlier work, intestinal IgVH genes were highly mutated in plasma cells from older individuals (> 30 years). IgVH genes were significantly less mutated in samples taken from patients aged 11-30 years, and there were fewer mutations again in samples from young children (< 11 years). In age-matched specimens the number of mutations was equivalent in the duodenum and colon. Using complementarity-determining region 3 primers to amplify specific Ig gene rearrangements, evidence was also found for the existence of related lamina propria plasma cells along the small bowel and colon, although these were quite scarce. In addition, analysis of the numbers of related clones in a random sampling from discrete areas of lamina propria indicates that the local population is diverse. These results suggest that the highly mutated IgVH genes in adult intestinal plasma cells are a consequence of chronic antigen exposure with age. Duodenal plasma cells are as highly mutated as colonic plasma cells, despite the fact that the upper bowel has no indigenous microbial flora (the stimulus for intestinal plasma cells). They also show that the plasma cell population is diverse and can be widely disseminated along the bowel.

IgE is the least abundant circulating antibody class but is constitutively present in healthy tissues bound to resident cells via its high-affinity receptor, FcεRI. The physiological role of endogenous IgE antibodies is unclear but it has been suggested that they provide host protection against a variety of noxious environmental substances and parasitic infections at epithelial barrier surfaces. Here we show, in mice, that skin inflammation enhances levels of IgE antibodies that have natural specificities and a repertoire, VDJ rearrangements and CDRH3 characteristics similar to those of IgE antibodies in healthy tissue. IgE-bearing basophils are recruited to inflamed skin via CXCL12 and thymic stromal lymphopoietin (TSLP)/IL-3-dependent upregulation of CXCR4. In the inflamed skin, IgE/FcεRI-signalling in basophils promotes epithelial cell growth and differentiation, partly through histamine engagement of H1R and H4R. Furthermore, this IgE response strongly drives tumour outgrowth of epithelial cells harbouring oncogenic mutation. These findings indicate that natural IgE antibodies support skin barrier defences, but that during chronic tissue inflammation this role may be subverted to promote tumour growth.

Intestinal homeostasis relies on a continuous dialogue between the commensal bacteria and the immune system. Natural killer T (NKT) cells, which recognize CD1d‐restricted microbial lipids and self‐lipids, contribute to the regulation of mucosal immunity, yet the mechanisms underlying their functions remain poorly understood. Here, we demonstrate that NKT cells respond to intestinal lipids and CD11c+ cells (including dendritic cells (DCs) and macrophages) are essential to mediate lipid presentation within the gut ultimately controlling intestinal NKT cell homeostasis and activation. Conversely, CD1d and NKT cells participate in the control of the intestinal bacteria composition and compartmentalization, in the regulation of the IgA repertoire and in the induction of regulatory T cells within the gut. These changes in intestinal homeostasis require CD1d expression on DC/macrophage populations as mice with conditional deletion of CD1d on CD11c+ cells exhibit dysbiosis and altered immune homeostasis. These results unveil the importance of CD11c+ cells in controlling lipid‐dependent immunity in the intestinal compartment and reveal an NKT cell–DC crosstalk as a key mechanism for the regulation of gut homeostasis.

SummaryB cells undergo a number of different developmental stages, from initial formation of their B cell receptor (BCR) genes to differentiation into antibody-secreting plasma cells. Because the BCR is vital in these differentiation steps, autoreactive and exogenous antigen binding to the BCR exert critical selection pressures to shape the B cell repertoire. Older people are more prone to infectious disease, less able to respond well to vaccination and more likely to have autoreactive antibodies. Here we review evidence of changes in B cell repertoires in older people, which may be a reflection of age-related changes in B cell selection processes.