Simultaneous in situ measurements of B cell clonality and single cell transcriptomes

Abstract

The B cell response to antigen is carried out within various compartments of different tissues. B cell pathologies often coincide with a malformation of one or many of these tissue compartments, highlighting a direct connection between tissue organization and dysregulation. To better interrogate this connection, spatial transcriptomics has evolved to encompass a suite of new tools that have promised an unprecedented molecular description of whole tissue architecture. A breakout technique in this field is Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH). MERFISH is an image-based single-cell transcriptomic tool that leverages optical barcodes to extend the scalability of traditional single-molecule FISH approaches to near transcriptome-level. MERFISH has been used to atlas a diverse range of cell types and states in the brain, liver, and gastrointestinal tract. However, as a probe-based approach, MERFISH was unable to distinguish between highly homologous gene targets such as the components of the B cell receptor, ultimately prohibiting MERFISH from tracking the interactions of specific B cell clones. Here, I present a technical extension to MERFISH, hereby termed B Cell Receptor MERFISH (BCR-MERFISH), that equips the standard MERFISH measurement with the ability to assign clone IDs to plasma B cells. BCR-MERFISH uses an orthogonal optical barcoding set to detect and label IGHV, IGKV, IGLV, and Fc usage within single cells. Importantly, BCR-MERFISH is co-stainable with standard MERFISH libraries and places unique plasma B cell clones in their defined, native tissue contexts – allowing the user to identify key cellular interactions that occur between unique clones and their neighboring cells. I have validated the accuracy of BCR-MERFISH through transient transfections of specific IGHV and IGKV genes in HEK293 cells, a transgenic mouse with a biased expression of a known IGHV/IGKV pair, and by comparing its results to BCR-sequencing of matched ileal slices from wild-type mice. I have used BCR-MERFISH in germ-free mice to reveal an enrichment of recurrent plasma cell clones within the lamina propria compared to that of specific pathogen-free mice – a phenomenon that was suggested by an enrichment of the same recurrent clonotypes found in the Peyer’s Patch germinal centers of germ-free mice. I show that BCR-MERFISH can capture the spatial distribution of specific clones across both the mucosal and intestinal axes. Regarding the mucosa, the lamina propria of the villi recruits Ccr9+ plasma cells via the secretion of Ccl25. I demonstrate that in wild-type specific pathogen-free mice, that the distribution of unique plasma B cell clones is not dependent on the sequence of the BCR. However, despite the uniformity within the layers of the villus, certain plasma cell clones show significant enrichment within proximal, medial, and distal ileum. Furthermore, certain clones even exhibit a common co-occurrence with either their sisters or other specific plasma B cell clones. Together, I believe that BCR-MERFISH offers a path to many new insights into a wide range of immunological questions that require a more comprehensive description of the immune tissue environment compared to other existing technologies.