Immune cell architecture of cartilaginous and bony fishTo reveal the cellular architecture of the cartilaginous and bony fish immune systems, we performed scRNA-seq of the immune organs of the white-spotted bamboo shark, zebrafish, and Chinese tongue sole (Supplementary Fig. 1a, b). The spleen, liver, and kidney (in the case of the white-spotted bamboo shark) or the head kidney (specific to zebrafish and Chinese tongue sole) play crucial roles in immune responses and harbor a wide array of immune cells. We constructed a total of 60 single-cell libraries (Supplementary Data 1). Following stringent quality control, we obtained transcriptome data of 58,866 cells (Supplementary Fig. 1c and Supplementary Data 1).Next, we integrated the cells from the different immune organs of each species (Fig. 1a–c). Our results showed that organ-specific cell types were mainly tissue parenchymal cells. In contrast, immune cells were present in all organs with only quantitative differences (Supplementary Fig. 1c). Cell types were identified through the analysis of marker gene expression (Fig. 1d–f and Supplementary Data 2), revealing 12, 17, and 16 cell types in bamboo shark, zebrafish, and Chinese tongue sole, respectively. We observed shared cell types among the three fish species, including hematopoietic stem cells (HSCs), B cells, T cells, and erythrocytes. The HSCs were defined by the transcription factor meis1b and the stem cell regulatory factor ahnak) (ahnak-like in bamboo shark). In the bamboo shark, HSCs were also marked by lmo2, a crucial factor in hematopoietic development25. HSCs were identified by the transcription factors mef2aa in zebrafish and zeb2 in Chinese tongue sole. We also identified hematopoietic progenitor cells (HPCs) by the stem cell-related genes myb, npm1a, and csf1rb in zebrafish, and npm1a, myb, and kita in Chinese tongue sole. B cells were characterized by blnk, btk, flt3, and igll1-like in bamboo shark, cd79a and pax5 in the two bony fishes. T cells were defined by tcf7, cd247, zap70, and ccr7 in the bamboo shark and zap70 in the bony fishes. We further defined bamboo shark myeloid cells from the expression of myeloid genes such as mpeg1-like, mmp9, and fam133b. In zebrafish, we also identified different myeloid cells, such as M1 macrophages (cd74a, irf8, and mpeg1.1), M2 macrophages (grn1, marco, and mfap4), neutrophils-I (mmp9, mmp13a, and adam8a), neutrophils-II (adam8a, lect2l, and cpa5) and eosinophils cells (gata2a, gyg1a, and viml). In Chinese tongue sole, we identified M1 macrophages (cd74a, irf8, and cd74b-like), M2 macrophages (marco, csf3r, and ctss2.1), neutrophils-I (mmp9 and cebpb), neutrophils-II (cpa1, mmp9, and cebpb) and dendritic cells (flt3, s1pr4, and spi1). Furthermore, we defined erythrocytes by the marker genes including blvrb, cahz-like, and rhag-like in bamboo shark, and cahz and alas2 in two bony fishes. And we defined a cluster of proliferating cells with cell cycle related genes including top2a, ube2c, and mki67-like in bamboo shark, and pcna and mki67 in zebrafish, and top2a and ube2c in Chinese tongue sole. Other non-immune cells identified were hepatocytes (fabp10a), endothelial cells (kdrl), acinar cells (prss1), and goblet cells (gata3, agr2, and cldn7-like) (Supplementary Fig. 1d–f). We also generated expression profiles of additional marker genes for each cell type in the three species (Supplementary Fig. 2a–c). We analyzed the proportion of immune cell types from the three species, revealing a higher proportion of B and T cells in the bamboo shark (Supplementary Fig. 2d). Our results indicated that the two bony fishes share more marker genes than they do with the cartilaginous bamboo shark. This indicates a high conservation of immune cells in the two bony fish species examined.Fig. 1: Single-cell atlas of immune organs from white-spotted bamboo shark, zebrafish, and Chinese tongue sole.a–c UMAP visualization of all cells. Bamboo shark (a), zebrafish (b), and Chinese tongue sole (c). Each dot represents one cell, with labeled cell types being the predominant cell types in each cluster. d–f Dot plots showing the expression of key marker genes (x-axis) of major cell types (y-axis) in bamboo shark (d), zebrafish (e), and Chinese tongue sole (f). The depth of the color from light to dark and the size of the dots represent the average expression from low to high and the percentage of cells expressing the gene.Evolution of immune cells in cartilaginous and bony fishThe expression patterns of one-to-one orthologous genes allow the study of immune system evolution26,27. In this study, we analyzed the transcriptional similarity of immune cells from three species and the expression of 7337 one-to-one orthologous genes (Fig. 2a and Supplementary Data 3). Same cell types across different species exhibited similar transcriptional profiles (Fig. 2a). For example, all T cells from the three species clustered together and then clustered with B and NK cells (Fig. 2a). This pattern indicates a conserved expression pattern of orthologs in the lymphocyte lineages from cartilaginous to bony fishes. Furthermore, although previous studies have shown that erythrocytes and myeloid cells differentiated from common myeloid progenitors28, we found a higher similarity between myeloid and lymphoid cells than between erythrocytes and myeloid cells (Fig. 2a). Similar results were observed comparing all immune cells from different organs of the three fish species (Supplementary Fig. 3).Fig. 2: Analysis of the cell-type evolution of three species.a Heatmap displays the ‘one versus best MetaNeighbour’ scores for immune cell types across three distinct species. AUROCs are determined by computing between the two closest neighbors in the test dataset, with the assumption that proximity equates to a higher scoring relationship. b, c Cross-species pairwise cell type similarities between bamboo shark and zebrafish (b), bamboo shark and Chinese tongue sole (c), based on Kullback-Leibler divergence (KLD). The top 5% highest values are depicted as arches linking different cell types, where the breadth of each arch corresponds to the degree of KLD-based similarity. d–f Violin plot showing the normalized expression of top shared orthologous genes (FC > 1.3, P-value < 0.001) in highly similar cell types across species, compared to the average expression of the same genes in all other cell types. Significance was calculated using paired Wilcoxon test between in- and outgroup cell types, Wilcoxon signed rank test adjusted P-value < 0.001 for all comparisons. d HSCs. e B cells in bamboo shark and zebrafish. f T cells in bamboo shark and Chinese tongue sole.Next, we constructed cell-type phylogenies with single-cell transcriptomes and performed pairwise comparisons (Supplementary Fig. 4a). Generally, we found that the broad immune cell types were in the same branch across species. We also found a one-to-one connection between cell types such as erythrocytes, HSCs, and B cells, demonstrating the higher similarity of these cells (Supplementary Fig. 4a). We proceeded to examine the similarities among cell types across species using the Kullback-Leibler divergence (KLD)29,30 (Fig. 2b, c, and Supplementary Fig. 4b). We found that some cell types are similar to their counterparts across species (Fig. 2b, c, and Supplementary Fig. 4b). For example, HSCs were highly conserved among the three species, characterized by co-expression of 32 orthologs of stemness maintenance-related genes and transcription factors such as meis1b, gfi1b, and tal1 (Fig. 2d, Supplementary Fig. 4c, and Supplementary Data 4). The proliferating cells co-expressed 104 orthologs (e.g., mki67, cdk1, pcna, and mcm7) involved in DNA replication and cell cycle regulation (Supplementary Fig. 4c, d and Supplementary Data 4). Interestingly, proliferating cells also expressed epigenetic enzymes such as dnmt1, dek, and smarca5. In addition, erythrocytes were also highly conserved among the three fish species, manifested as the co-expression of 36 orthologs, including the transcription factors gata2, zfpm1, and lmo2 and erythrocyte marker genes (e.g., epor, urod, and fech) (Supplementary Fig. 4c, d and Supplementary Data 4).We found that the species-specific orthologs in the same cell type contribute to a similar function. For example, there were 154, 130, and 146 orthologs specifically expressed in the proliferating cells of bamboo shark, zebrafish, and Chinese tongue sole, respectively (Supplementary Fig. 4c and Supplementary Data 4). Gene enrichment analysis revealed an intersection of GO terms or pathways such as ‘DNA Replication’, ‘Cell cycle’, and ‘Metabolism of RNA’. For ‘DNA Replication’, we detected topbp1, orc1, and parp1 in bamboo shark proliferating cells; prim1, orc4, dscc1, and chrac1 in zebrafish; and pole, polq, and pold3 in Chinese tongue sole. This indicates a similar functional gene expression pattern of these cell types among the three species. We also found some species-specific characteristics. For example, genes responsible for cartilage morphogenesis (e.g., chsy1, ids, and furina) were specifically expressed in the HSCs of bamboo sharks (Supplementary Data 4).Bamboo shark immune cell types showed the greatest differences in transcriptomic profiles relative to the two bony fish species based on the number of connections between immune cells of different species, consistent with its more distant phylogenetic position (Fig. 2b, c, and Supplementary Fig. 4b). For example, HPCs, B cells, T cells, neutrophils, and M1 and M2 macrophages showed high similarity between zebrafish and Chinese tongue sole, along with a large number of shared orthologous genes (Supplementary Fig. 4e and Supplementary Data 4). We also observed partial similarity of immune cells between the cartilaginous fish and teleost fishes. A high similarity of B cells between the bamboo shark and zebrafish was characterized by 123 co-expressed orthologs (Fig. 2e). Furthermore, T cells were similar (199 co-expressed orthologs) between the bamboo shark and Chinese tongue sole (Fig. 2f). Surprisingly, T cells from the bamboo shark and B cells from zebrafish shared 136 co-expressed orthologs (Supplementary Fig. 4f). This indicates a close linkage of B and T cells in the bamboo shark, revealing its divergent adaptive immune system.Conserved cell function with divergent gene sets in B cells of cartilaginous and bony fishBoth cartilaginous and bony fishes possess Ig-based adaptive immune systems5. The shared orthologs of DEGs between cartilaginous and bony fish indicate conservation of B cells (Fig. 2e and Supplementary Data 4). To better understand the conservation and diversification of B cells between cartilaginous and bony fish, we compared the B cells from the three species in our dataset. The transcriptional profiles of 7,184 orthologous genes from 900 B cells from each species were integrated and visualized using UMAP (Fig. 3a).Fig. 3: Conservation and divergence of B cell sub-types.a UMAP plot showing the clustering of B cell subclusters from three species. b Dot plot showing the expression of most significant DEGs in each sub-cluster of B cells. c Heatmap showing the conserved and teleost-specific differentially expressed genes in B cell sub-clusters. Enriched Gene Ontology categories for DEGs of B-3 (colored green) and B-6 (colored purple), respectively. The enrichment analysis was generated using Fisher’s exact test on the Metasape web server, with Bonferroni correction for multiple hypotheses testing. P-values are indicated on the x-axis. d, e UpSet plots showing shared orthologs of three species in B-3 (d) and B-6 (e). f Heatmap showing the expression of conserved DEGs in B-6 sub-cluster of three species.Generally, B cells from the three species studied were evenly distributed in the integration results, indicating a high similarity of B cells among species (Supplementary Fig. 5a). We identified six B cell subsets (Supplementary Fig. 5b and Supplementary Data 5). The B-5 cluster was characterized by the expression of rag1 and rag2 and transcription factors such as zeb2b and rbx1, indicating that they are B cell precursors (pre-B cells) (Fig. 3b). DNA replication and cell cycle genes, including lig1, pcna, mcm7, slbp, and dnmt1, were highly expressed in the B-3 cluster, indicating that B-3 are plasmablasts (i.e., plasma cell precursors). The B-6 cluster expressed plasma cells marker genes including pdia4, ppib, ostc, and ddost. The B-1 cluster was characterized by a high expression of mef2cb, sf3b1, kmt2a, and smc5, indicating that they are immature B cells. Genes including cd74a, rpl37, and rps14 characterized the B-2 cluster. Interestingly, the B-4 cluster expressed phagocytosis-related genes such as mmp9, wbp4, prdx6, and sod1 (Fig. 3b). Although the same cell types were identified in the three species, the proportion of cells in the six clusters differed for each species (Supplementary Fig. 5c). For example, the proportion of B-1 cells was about 25% in the two bony fishes but more than 52% in the cartilaginous fish.We further analyzed the similarity of B cells (Supplementary Fig. 5d). B-3 were the most similar among the three species, followed by the B-6 sub-cluster (Supplementary Fig. 5d). Analysis of shared DEGs among the three species also showed a higher similarity of B-3 and B-6 (Fig. 3c and Supplementary Data 6). The 217 shared DEGs among the three species included 144 DEGs in B-3 (Fig. 3d) and 32 in B-6 (Fig. 3e). The 144 shared B-3 DEGs in three species involved cell cycle proteins and transcription factors, including dkc1, ybx1, and ncl (Supplementary Fig. 5e).Previous studies have revealed that cartilaginous fish possess three types of antibodies, IgM, IgW, and IgNAR, different from those in teleost fish. We annotated six Ig heavy chain constant region encoding genes in the bamboo shark genome. IgNAR (LOC122543616) was highly expressed in the B cells of the white-spotted bamboo sharks. While IgNAR (LOC122543617 and LOC122543620), IgM (LOC122552345), and IgW (LOC122547843 and LOC122547844) were relatively lowly expressed (Supplementary Fig. 5f and Supplementary Data 7). Interestingly, the ostensible immunoglobin-secreting plasma cell cluster B-6 shared 32 DEGs including protein folding-related genes (e.g., pdia4, ppib, and hspa5) and genes related to protein localization in the endoplasmic reticulum (e.g., erp44, hyou1, and derl2) (Fig. 3e and Supplementary Data 6). Shared high expression of protein glycosylation modification-related genes (e.g., ost4, ddost, and rpn2) was also observed in B-6 (Fig. 3f). Meanwhile, we observed high expression of RNA splicing-related genes snw1, snrpf, and thoc5 and mitochondrial translation elongation related genes mrpl3, gfm1, oxa1l, and mrpl2 in B-6 of bamboo shark, indicating the unusual regulation of plasma cells in cartilaginous fish (Supplementary Fig. 5g and Supplementary Data 6).We also analyzed 269 teleost-specific (zebrafish and Chinese tongue sole) DEGs in the six B cell sub-clusters (Fig. 3c). Interestingly, the shared DEGs and teleost-specific DEGs of the B-3 cluster showed enrichment for the gene ontology categories ‘Metabolism of RNA’, ‘Proteasome’ and cell cycle-related processes. ‘Protein processing in endoplasmic reticulum’ and ‘protein glycosylation’ were enriched by the shared DEGs and ‘protein localization to endoplasmic reticulum’ was enriched by the teleost-specific DEGs of the B-6 cluster. Species-specific DEGs (786 in bamboo shark, 234 in zebrafish, and 1008 in tongue sole) revealed the gene expression diversity in each species (Supplementary Fig. 5g). The enrichment of pathways and ontologies by different gene sets reflect that gene adaptations in one species can be substituted with other genes in different species while performing similar cellular functions (i.e., functional convergence).Characterization of phagocytosis of B cells in cartilaginous fishPrevious studies have demonstrated that B cells in teleost fish are involved in innate immune functions such as phagocytosis10. In agreement, many innate immunity genes were found in the signature genes of B cells in zebrafish and Chinese tongue soles (Supplementary Fig. 6a, b). Notably, the genes specifically expressed in bamboo shark B cells are also associated with innate immunity (Supplementary Fig. 6c). Pathway enrichment analysis revealed that ‘Fc-gamma receptor (FCGR) dependent phagocytosis’ and ‘Regulation of actin dynamics for phagocytic cup formation’ signaling pathways were enriched in B cell of bamboo sharks (Supplementary Fig. 6d). Furthermore, compared with bony fish, the bamboo shark B cells exhibit upregulation of genes involved in phagocytosis (such as rhoh, met, pikfyve, and tmem175) as well as myeloid cell differentiation (kmt2a, sf3b1, ncor1, ddx46, pdcd2, etc.) (Supplementary Fig. 7). These results suggest that the potential phagocytic function of B cells present in cartilaginous fishes.We generated a phagocytosis-related gene set using the GO terms of the zebrafish, mouse, and human31 (Supplementary Data 8). Many phagocytic genes showed high expression in the B cells of the bamboo shark, including pecam1, nckap1l, and rubcn(Fig. 4a). Both B cells and myeloid cells expressed plcg2, irf8, ncf2, il1b, elmo3, etc. The phagocytic genes plcg2, tlr2, and ncf2 were co-expressed with B cell marker genes such as blnk and flt3 (Fig. 4b and Supplementary Fig. 8a). Interestingly, bamboo shark B cells highly expressed several pattern recognition receptors. Fc-gamma receptors (e.g., fcgr1a-like, fcrl3, and fcrl5) and toll-like receptors (e.g., tlr1, tlr2, tlr7, and tlr9) were highly expressed in B cells (Fig. 4c). Considering the different types of Ig genes, we analyzed the co-expression of Ig genes and phagocytic genes (plcg2, tlr2, and ncf2), and found a high co-expression of IgNAR (LOC122543616) with phagocytic genes (Supplementary Fig. 8b–f).Fig. 4: Phagocytic function of B cells in bamboo shark.a Heatmap showing the expression of phagocytosis gene set in B cells of bamboo shark. b UMAP plots showing co-expression of blnk with plcg2, ncf2, and tlr2, respectively, in B cells of bamboo shark. The co-expression color threshold for all genes is based on the same standard of 0.5. c Heatmap showing the expression of pattern recognition receptors in B cell. d Confocal laser scanning microscopic images of phagocytosis of fluorescence-labeled microbeads by B cells (n = 3 biological replicates), B cells were detected by fluorescence in situ hybridization (FISH) with B cell markers (blnk and ignar). Cell membranes were stained with Dil. Scale bar = 10 μm. e Flow cytometry identification of phagocytic B cells in bamboo shark spleen cells by the combination of fluorescence-labeled microbeads and anti-IgNAR antibody. LL, lower left: indicates IgNAR- non-phagocytic cells; UL, upper left: indicates IgNAR- phagocytic cells; LR, lower right: indicates IgNAR+ non-phagocytic cells; UR, upper right: indicates IgNAR+ non-phagocytic cells. f, RT-qPCR analysis of the expression of phagocytotic gene in FACS-sorted cells in Fig. 4e. n = 3 independent biological replicates. Bars represent mean values + /− SEM. P-values were determined using the two-tailed Mann–Whitney test.Next, we investigated the phagocytosis of B cells in the bamboo shark by flow cytometry. Forward and side scatter parameters (FSC/SSC) were used to gate lymphocytes. After incubating with microbeads, 3.27% of lymphocyte cells gave a positive result. An increased microsphere-cell ratio increased the proportion of fluorescence-positive lymphocytes to 6.23% (Supplementary Fig. 9a). Next, we observed the phagocytosis of B cells by confocal microscopy. Microspheres with blue fluorescence were found in the cytoplasm of blnk-positive or IgNAR-positive cells, indicating that B cells had ingested microbeads (Fig. 4d). Next, we sorted both IgNAR+ phagocytotic, IgNAR- phagocytotic cells, and non-phagocytotic cells by using a combination of fluorescence-labeled microbeads and anti-IgNAR antibody by FACS (Fig. 4e and Supplementary Fig. 9b). The increased expression of phagosome maturation genes including rab5b, indicated the activation of phagocytosis (Fig. 4f).We also detected the expression of these pattern recognition receptors in B cells from zebrafish and Chinese tongue soles (Supplementary Fig. 9c, d). Many receptors were expressed in B cells in the two bony fishes. Flow cytometry showed that only about 0.36% of lymphocyte cells in the Chinese tongue sole were fluorescence-positive after microbead incubation (Supplementary Fig. 9e). We also observed the phagocytosis of Chinese tongue sole B cells by confocal microscopy (Supplementary Fig. 9f). These results hint at an enhanced phagocytosis function of cartilaginous fish B cells compared to bony fish.Identification of T cell subsets in cartilaginous fishMuch effort has been expended to identify CD4 genes and related cytokines in cartilaginous fishes to understand the function of T cell subtypes better12,21. We explored the main functions of T cells by KEGG pathway enrichment. DEGs of T cells in the bamboo shark were enriched in pathways related to CD4+ T cell subsets such as ‘Th17 cell differentiation’ and ‘Th1 and Th2 cell differentiation’ (Fig. 5a).Fig. 5: Characterization of T cell sub-types in the bamboo shark.a Heatmap showing the expression of all DEGs of T cells in bamboo shark. Gene expression levels utilize a Z score, which depicts variance from the mean, as defined on the color key in the right top corner (left). Histogram showing KEGG pathways enriched by these genes (right). The enrichment analysis was generated using Fisher’s exact test, with Benjamini-Hochberg (BH) corrected for multiple hypotheses testing. The P-values is represented by the depth of red. b UMAP plot showing 6 T cell subsets of bamboo shark. c Dot plot showing expression levels of selected signature genes in T cell subsets. Dot size indicates the fraction of expressing cells, colored based on normalized expression levels. d UMAP plot showing T cell activation-related gene expression in different T cell subsets. e UMAP plot showing Th2-related gene expression across T cell subsets. f Heatmap showing CD4+ T cell subset transcription factor expression across T cell subsets. g UMAP plot showing CD8+ T cell and CD4+ T cell key transcription factor expression across T cell subsets and immune cell lineages.We divided T cells from the bamboo shark into six subclusters (Fig. 5b; Supplementary Fig. 10a, b and Supplementary Data 9). We analyzed the expression of TCR genes and found high tcrb expression and low tcrd expression (Supplementary Fig. 10c, d, and Supplementary Data 7). We then annotated each subset based on DEG expression (Fig. 5c). T-0 cells were characterized by tissue-resident memory T cell-related genes such as itgae-like and btnl2. The naïve T cell marker genes lef1, ccr7, and tcf7 were highly expressed by T-1 cells. T-2 cells primarily expressed transcription factors (junb-like32 and aiolos-like33), and CD4+ T cells differentiation-related genes (il12rb234 and sh2d1a-like35), exhibiting characteristics of the helper T cell subset. Interestingly, T-3 cells highly expressed many genes associated with B cells, including blnk and igj-like. The genes flt3 and tmem119b, which were DEGs of bamboo shark B cells (see above), were also expressed in this cluster. Cluster T-4 was characterized as proliferating T cells based on the high expression levels of cell proliferation-related genes (e.g., pcna, myc-like, and eif1ad). T-5 cells specifically expressed T cell activation-related genes such as myb, egr2b and tagapb (Fig. 5c).Many studies have investigated the presence of canonical CD4 and T cell lineage diversification36,37,38. Our study aimed to elucidate the diversification of the T cell lineage in the white-spotted bamboo sharks using single-cell data. We found that T-2 with helper T cell characteristics is CD8a negative (Fig. 5d). We identified the sequence of CD4 gene in white-spotted bamboo shark (Supplementary Fig. 11) based on a previous study21. However, the expression of the CD4 gene was not subset specific (Supplementary Fig. 12a), which may be related to the structural differences (stemming from a highly diverged gene locus and protein sequence) of CD4 in cartilaginous and bony fish. Like other species, MHCI genes were expressed by most immune cell types in the bamboo shark, while MHCII genes were specifically expressed in antigen-presenting cells (Supplementary Fig. 12b).Antigens presented by the MHC are recognized by different T cell subsets, and through the cascade signaling of lck and zap70, ultimately lead to T cell activation and differentiation39. Lck was primarily expressed in the T-2 subset and zap70 was ubiquitously expressed in all T cell sub-clusters, indicating different mechanisms of T cell activation in the bamboo shark (Fig. 5d). In addition, some Th1-related genes were expressed in the T-2 subset, including ifng-like, cxcr3-like, and cxcl10-like (Fig. 5e). However, we failed to detect the expression of other Th2 effectors, including il12 and tnfa in T-2. Furthermore, ccl11-like and ccl20-like, involved in the IL-17 signaling pathway40, were also expressed in the T-2 subset (Supplementary Fig. 12c).Transcription factors are crucial in determining cell fate. We analyzed the expression of transcription factors of bamboo shark T cells (Fig. 5f). Some of the signature transcription factors of CD4+ T cells were expressed (Fig. 5f and Supplementary Fig. 12d). Both the CD4+ T cell-related transcription factor gata3 and the CD8+ T cell-related transcription factor runx3 were ubiquitously expressed in all T cell subsets (Fig. 5g). The transcription factor ZBTB7B is essential for the development of the CD4 lineage, and its lack results in the commitment to the CD8 lineage41. We confirmed that the zbtb7b-like gene in the bamboo shark is intact at its conserved domain; however, the gene was not specifically expressed by T cells (Fig. 5g). In conclusion, we have characterized the subsets of T cells in a cartilaginous fish and further elucidated the function of the T-2 subset in a bamboo shark. Our results indicate that T-2 is a Th-like subpopulation in sharks. Moreover, it may have cell fate determinants that differ from those identified in bony vertebrates.A T cell subtype co-expressed T and B cell markers in cartilaginous fishesUnexpectedly, the differentially expressed genes of the bamboo shark T cell subcluster T-3 included some B cell-related genes (Fig. 5c). Genome studies have reported a close linkage between immunoglobulin and T-cell receptor genes12. To demonstrate that our result did not occur due to cell admixture, we used co-expression analysis to verify that these genes were expressed by T cells. Many cells in T-3 showed co-expression of blnk, flt3, and tmem119b with the T cell markers cd247 (Fig. 6a). We verified the expression of tcf7, cd247, blnk, and flt3 by fluorescence in situ hybridization (FISH), and detected the cells co-expressing tcf7 and blnk, cd247 and flt3 (Fig. 6b). Furthermore, we also analyzed the cell populations that co-expressing tcf7, cd247 and IgNAR (Supplementary Fig. 13a). In addition, many immunoglobulin genes were expressed by bamboo shark T cells, especially in the T-3 and T-4 clusters (Fig. 6c). We also found a substantial overlap between bamboo shark T and B cell DEGs (Fig. 6d). The correlation of the gene expression pattern of T and B cells from the bamboo shark was higher than that of zebrafish (Fig. 6e).Fig. 6: Co-expression of T cell and B cell marker genes in T-3 subsets.a UMAP plots showing co-expression of cd247 and blnk, flt3, tmem119b, respectively, in T cell subsets of bamboo shark. The co-expression color threshold for all genes is based on the same standard of 0.5. b fluorescence in situ hybridization (FISH) showing co-expression of B cell markers (blnk and flt3) and T cell markers (cd247 and tcf7). c Violin plots showing immunoglobulin gene expression in T cell subsets. d Venn diagram showing the intersection of all DEGs in T cells and B cells of bamboo shark (n = 3 biological replicates), Scale bar = 10 μm. e Scatter plots show the correlation of gene expression in T cells and B cells in bamboo shark and zebrafish, respectively. The correlation coefficient R is calculated by Pearson correlation test (two-sided).To further expand on our results, we analyzed the scRNA-seq dataset of the nurse shark (Ginglymostoma cirratum)42 with the same quality control flow performed in this study. T and B cell markers co-expressed in a specific subset of T cells in nurse sharks (Supplementary Fig. 13b). Meanwhile, there is no subcluster co-expressing T and B cell markers in Chinese tongue sole or zebrafish (Supplementary Fig. 13c, d). This observation suggests that the T-3 subset (co-expressing T-cell and B-cell markers) appears to be specific to cartilaginous fishes. Furthermore, we added additional cross-species analysis. We extracted zebrafish and Chinese tongue sole T cells for clustering and identified several subgroups. Cross-species analysis revealed that there are no T cell subpopulations in zebrafish and Chinese tongue soles with similar transcriptional characteristics to T-3 in sharks (Supplementary Fig. 14). These results support the claim that the T-3 subset is exclusive to cartilaginous fishes. This cell type may represent a unique branch of T cell evolution, conferring specific immune advantages to cartilaginous fishes. However, whether the T-3 is a unique group that distinguishes cartilaginous fish from all bony fishes still needs to be determined. Further expansion of the species range for in-depth comparative analysis is needed.
