Organized B cell sites in cartilaginous fishes reveal the evolutionary foundation of germinal centers

Abstract

The absence of germinal centers (GCs) in cartilaginous fishes lies at odds with data showing that nurse sharks can produce robust antigen-specific responses and affinity mature their B cell repertoires. To investigate this apparent incongruity, we performed RNA sequencing on single nuclei, allowing us to characterize the cell types present in the nurse shark spleen, and RNAscope to provide in situ cellular resolution of key marker gene expression following immunization with R-phycoerythrin (PE). We tracked PE to the splenic follicles where it co-localizes with CXCR5^high centrocyte-like B cells and a population of putative T follicular helper (Tfh) cells, surrounded by a peripheral ring of Ki67⁺ AID⁺ CXCR4⁺ centroblast-like B cells. Further, we reveal selection of mutations in B cell clones dissected from these follicles. We propose that the B cell sites identified here represent the evolutionary foundation of GCs, dating back to the jawed vertebrate ancestor.

Keywords: B cells; CP: Immunology; T follicular helper cells; antibody; cartilaginous fish; evolution; germinal center; shark.

Figure 1.. Single-cell transcriptome of the nurse shark spleen post-immunization

(A) Integrated UMAP plot of 22 transcriptionally distinct cell clusters gained using snRNA-seq from splenic tissue of two immunized nurse sharks. (B) Violin plots showing the expression of key marker genes used to determine the identity of cell clusters.

Figure 2.. Characterization of B cell clusters identified in nurse shark spleen and expression of AID in the splenic follicles

(A) Violin plots showing the expression of key marker genes used to characterize B cell clusters. (B) RNA FISH microscopy images showing transcript patterns of Ignar (green), Aicda (red), and Mki67 (yellow) in B cell follicles of nurse shark spleen at day 30 post-immunization. (C) RNA FISH microscopy images showing transcript patterns of Cxcr4 (green) and Cxcr5 (red) in nurse shark spleen at days 40 and 50 post-immunization. Cell nuclei are stained with DAPI (blue). Scale bar, 100 μm in all images.

Figure 3.. Characterization of T cell clusters and a population of T follicular helper (Tfh)-like cells in the nurse shark spleen

(A) Violin plots showing the expression of key marker genes used to characterize T cell clusters. (B) RNA FISH microscopy images showing transcript patterns of Ignar (green) and Cd3e (red) in nurse shark spleen at days 10–50 post-immunization. Cell nuclei are stained with DAPI (blue). Scale bar,100 μm. (C) UMAP performed after subclustering of T cells extracted from the global dataset. (D) Violin plots showing the expression of marker genes corresponding to Tfh cell function in mammals in T cell subclusters. (E) UMAP plot of the original T cell clusters (Figure 1A), demonstrating that Tsc6 is largely found within T cell cluster T1.

Figure 4.. Presentation of non-degraded antigen in nurse shark spleen permits B cell selection

(A) Violin plots showing the expression of key marker genes used to characterize myeloid cell clusters. (B) Fluorescence microscopy images showing phycoerythrin (PE; magenta) moving into IgNAR⁺ (green) B cell follicles and accumulating in the follicular center around day 40 post-immunization. Cell nuclei are stained with DAPI (blue). Scale bar, 100 μm. (C) Wu-Kabat amino acid variability across all distinct NAR V region sequences isolated by LMD, showing comparison of the FRs compared with the CDR/HVs (left). p values shown are for Wilcoxon rank-sum test of all CDR/HV sites vs. all FR sites. R/S ratios of the CDR/HVs and FRs of all distinct NAR V region sequences (middle) and the top 100 most abundant distinct NAR V region sequences (right).