Antibody repertoire and gene expression dynamics of diverse human B cell states during affinity maturation

Abstract

15In response to antigen challenge, B cells clonally expand, undergo selection and differentiate to produce 16 mature B cell subsets and high affinity antibodies. However, the interplay between dynamic B cell states 17 and their antibody-based selection is challenging to decipher in primary human tissue. We have applied 18 an integrated analysis of bulk and single-cell antibody repertoires paired with single-cell transcriptomics 19 of human B cells undergoing affinity maturation. We define unique gene expression and antibody 20 repertoires of known and novel B cell states, including a pre-germinal centre state primed to undergo 21 class switch recombination. We dissect antibody class-dependent gene expression of germinal centre 22 and memory B cells to find that class switching prior to germinal centre entry dictates the capacity of B 23 cells to undergo antibody-based selection and differentiate. Together, our analyses provide 24 unprecedented resolution into the gene expression and selection dynamics that shape B cell-mediated 25 immunity. 26 27 121 122 157 sampling of IgH sequences from bulk repertoires to enhance genotype correction, identification of 158

Comprehensive mapping of tissue cell architecture via integrated single cell and spatial transcriptomics

“…Beyond confirming known biology, the NMF analysis enabled the fine-grained spatial mapping of a rare and putatively novel secondary activation B cell state, which has been recently reported as primed to undergo class switch recombination preceding the GC entry 37,40 . Our data indicate that this population (termed here “preGC”) occupies a different tissue compartment from the upstream activated cells and downstream GC cells (Fig 4E, panel 4), suggesting it is more likely to initiate new GC reactions rather than joining existing ones.…”

“…Unlike the mouse brain, the human lymph node is characterised by dynamic micro-environments with many spatially interlaced cell populations. We analysed a publicly available Visium dataset of the human lymph node from 10X Genomics 34 , spatially mapping a comprehensive atlas of 34 reference cell types derived by integration of scRNA-seq datasets from human secondary lymphoid organs composed of 73,260 cells [35][36][37] (Fig 4A-B; Methods).…”

“…We, therefore, included scRNA-seq datasets spanning lymph nodes, spleen and tonsils in our single-cell reference to ensure that we captured the full diversity of immune cell states likely to exist in the spatial transcriptomic dataset (Fig 4A-B). The scRNA-seq reference was combined from 3 studies [35][36][37] . Integration was performed using Scanpy 55 including removal of doublets with scrublet 53 , regressing out batch from PC space and performing BBKNN.…”

Single-Cell Transcriptomic Analyses Define Distinct Peripheral B Cell Subsets and Discrete Development Pathways

“…Curated reference databases, such as the Human Cell Atlas ( 21 ), and baseline transcriptomic profiles of particular immune sites ( 22 ) are important for comparison to experimental datasets as well as providing new insights into already well studied biological systems. A frequently occurring problem in single-cell datasets, however, is the relative lack of B cells, given that most human experiments are run on readily available PBMCs; where B cells only make up between 5-10% of the total.…”

Platypus: an open-access software for integrating lymphocyte single-cell immune repertoires with transcriptomes

“…In another study, clonal expansion and activation signatures of tumor-infiltrating T cells were profiled 12 . Other studies have leveraged this technology to answer fundamental questions across a variety of areas in immunology such as bacterial infection responses 13 , tumor-immune microenvironment 14 , clonal expansion in Alzheimer’s disease 9 , and B cell differentiation 15 .…”