Studying lymphocyte differentiation in the spleen via spatial transcriptomics

Abstract Immune cell positioning within secondary lymphoid tissues likely affects cell-cell interaction and subsequent immune responses. Techniques such as intra-vital imaging and multiplex immunohistochemistry provide insight into this, but require specialist reagents. To examine cell-cell interactions in dense tissue at whole-genome scale, we tested the feasibility of a single-cell spatial transcriptomics method, Slide-seq2. We first confirmed using murine gut tissue that small tertiary lymphoid structures, particularly rich in B cells, could be identified and examined at a cellular level in the small intestine. We then hypothesized that microanatomical alterations could be detected. To test this, we compared mouse spleens before and 7 days after infection with blood-stage malaria parasites. To increase the molecular resolution of our data, we integrated Slide-seq2 data with high-depth, droplet-based, scRNA-seq data, generated via Chromium controller from 10× Genomics. We found that Slide-seq2 produced sufficiently rich data to map cell types from an scRNA-seq reference. Some spatially defined transcriptomes appeared to derive from mixtures of cell types, indicating that further deconvolution of spatially resolved transcriptomic data was required. Via unsupervised clustering of whole transcriptomes, we confirmed that T and B cell zones within naïve mice became less ordered at the peak of malaria infection, reflecting T and B cell interaction. Ongoing analyses aim to define novel splenic T and B cell interactions during malaria, both in extra follicular areas as well as within the germinal center.