Characterization of zebrafish Ctla-4.

A Alignment of the Ctla-4 homologs from different species generated with ClustalX and Jalview. The conserved and partially conserved amino acid residues in each species are colored in hues graded from orange to red, respectively. Key features, including conserved cysteine residues, functional motifs, such as B7-binding motif, tyrosine phosphorylation site, and potential tyrosine phosphorylation site, were indicated separately. The signal peptide, IgV-like domain, transmembrane (TM) domain, and cytoplasmic domain were marked above the sequence. B The tertiary structure of the zebrafish Ctla-4 ectodomain, as predicted by AlphaFold2, was compared with that of humans. The two pairs of disulfide bonds (Cys20-Cys91/Cys46-Cys65 in zebrafish and Cys21-Cys92/Cys48-Cys66 in humans) used to connect the two-layer β-sandwich, and the separate Cys residue (Cys119 in zebrafish and Cys120 in humans) involved in the dimerization of the proteins are indicated. Cysteine residues are represented in purple ball-and-stick models, and the identified or potential B7 binding sites are highlighted in blue. C Dimer of Ctla-4 was identified by Western blot under reducing (+β-ME) or non-reducing (−β-ME) conditions. The ctrl represents a control sample derived from cells transfected with an empty plasmid. The monomers and dimers were indicated by single and double arrows, respectively. D The subcellular localization of Ctla-4 protein was assessed in HEK293T cells transfected with pEGFPN1-Ctla-4 for 48 hours, imaged using a two-photon laser-scanning microscope (Original magnification, 630×). Nuclei were stained with DAPI (blue), and cell membranes were stained with DiI (red). E UMAP plots showing the relative distribution of common T cell markers (cd4-1, cd8a and ctla-4) based on a splenic single-cell RNA sequencing (scRNA-seq) dataset we recently established [37]. F Immunofluorescence staining of lymphocytes isolated from zebrafish blood, spleen, and kidney. Cells were stained with mouse anti-Ctla-4, together with rabbit anti-Cd4-1 or rabbit anti-Cd8α. DAPI stain shows the location of the nuclei. Images were obtained using a two-photon laser-scanning microscope (Original magnification, 630×).

Examination on the IBD-like phenotype in ctla-4−/− zebrafish.

A Generation of a homozygous ctla-4-deficient (ctla-4−/−) zebrafish line through CRISPR/Cas9-based knockout of ctla-4 gene on chromosome 9. A 14-bp deletion mutation in exon 2 results in a premature stop at codon 82, which is predicted to produce a defective Ctla-4 protein containing 81 amino acids. B Genotyping of the deficiency of ctla-4 gene by Sanger sequencing. C Knockout efficiency of Ctla-4 selectively examined in spleen and gut tissues of ctla-4−/− zebrafish by Western blot analysis. Gapdh serves as a loading control. D Normal gross appearance of adult wild-type (WT) and ctla-4−/− zebrafish. E Body weight statistics of WT and ctla-4−/− zebrafish (n = 30). F The change of intestine length in WT and ctla-4−/− zebrafish. G The change of splenic size in WT and ctla-4−/− zebrafish. H Representative H&E staining analysis of histopathological changes and quantitation of histology scores in the anterior, mid and posterior intestines from WT and ctla-4−/− zebrafish. Red arrows denote mucosal inflammatory cell infiltration, and black arrow indicates transmural inflammatory cell infiltration. I AB-PAS staining was used to analyze the mucin components and the number of goblet cells in anterior intestine from WT and ctla-4−/− zebrafish (n = 5). J Quantitation analysis of goblet cells of each villus in the foregut of WT and ctla-4−/− zebrafish (n = 8). K Observation of cell junctions between intestinal epithelial cells in posterior intestines from WT and ctla-4−/− zebrafish under TEM (Hitachi Model H-7650). White triangles indicate tight junctions, black triangle indicates adhesion junctions, and red triangles indicate desmosomes. Data are presented as mean ± standard deviation (SD). Statistical significance was assessed through an unpaired Student’s t test (*p < 0.05; **p <0.01; ***p < 0.001; ****p < 0.0001).

RNA-sequencing analysis of the molecular implications associated with the IBD-like phenotype in ctla-4−/− zebrafish.

A Heatmap of different expressed genes between the intestines from wild-type (WT) and ctla-4−/− zebrafish. B Volcano plot showing the up-/down-regulated genes in the intestines of ctla-4−/− zebrafish compared with those of WT zebrafish. Red represents up-regulated genes, while blue denotes down-regulated genes. C GO analysis showing top 10 terms in biological processes of DEGs. D GO analysis showing top 10 terms in biological processes of all up-regulated genes. E Heatmap showing row-scaled expression of the representative differently expressed inflammation and chemotaxis-related genes. F The mRNA expression levels of important genes associated with inflammation and chemokines confirmed by real-time qPCR. G Protein-protein interaction network was constructed using the DEGs. The nodes represent the proteins (genes); the edges represent the interaction of proteins (genes). H The MPO activity in the intestines (up) and peripheral blood (down). I Heatmap showing row-scaled expression of the IBD biomarker genes and IBD-related genes. J The mRNA expression levels of representative IBD biomarker genes and IBD-related genes were analyzed by real-time qPCR. Data are presented as mean ± standard deviation (SD). Statistical significance was assessed through an unpaired Student’s t test (**p < 0.01; ***p < 0.001; ****p < 0.0001).

Single-cell RNA sequencing analysis of the major cell types associated with the IBD-like phenotype in ctla-4−/− zebrafish.

A Classification of cell types from zebrafish intestines by tSNE embedding. B Dot plot showing the expression levels of lineage marker genes and percentage of cells per cluster that express the gene of interest. C Expression maps of T cell associated markers within the cell populations of the zebrafish intestines. D Heatmap showing the mean expression levels of genes associated with tight and adhesion junctions in enterocytes across samples from wild-type (WT) and ctla-4−/− zebrafish. E Heatmap showing the mean expression levels of inflammation-related genes involved in cytokine-cytokine receptor interactions in neutrophils from WT and ctla-4−/− zebrafish samples. F KEGG enrichment analysis showing the top 15 terms of up-regulated genes in neutrophils from the ctla-4−/− sample versus the WT sample. G KEGG enrichment analysis showing the top 15 terms of up-regulated genes in macrophages from the ctla-4−/− sample versus the WT sample.

Single-cell RNA sequencing analysis of the subset immune-cells associated with the IBD-like phenotype in ctla-4−/− zebrafish.

A Classification of subset cells from the T/NK/ILC-like group by tSNE embedding. B Dot plot showing the mean expression levels of subset marker genes and percentage of cells per cluster that express the gene of interest. C Marker gene expression in individual cluster identifying this cluster as ILC3-like cells. D Changes in the composition of subset cells between samples from wild-type (WT) and ctla-4−/− zebrafish. A significantly increased Th2 subset (referred to as Th2 cells 2) in the ctla-4−/− sample was highlighted with a black dashed circle. E Histogram showing the different ratios of subset cells between the WT and ctla-4−/− samples. F Histogram presenting the different numbers of subset cells between the WT and ctla-4−/− samples. G Mean expression levels of the cytokine il13 within different subset cells between the WT and ctla-4−/− samples. H Dot plot illustrating the mean expression of il13 in T/NK/ILC-like cells from WT and ctla-4−/− zebrafish. I KEGG enrichment analysis showing the top 15 terms of the Th2 cells 2 genes from ctla-4−/− zebrafish. J KEGG enrichment analysis showing the top 15 terms of up-regulated genes in NKT-like cells. K Scatter plot showing the DEGs of ILC3-like cells in WT and ctla-4−/− zebrafish. The il17a/f1 and il17a/f3 was shown in the scatter plot.

Alteration in microbial composition in the intestines of ctla-4−/− zebrafish.

A Venn diagram showing the number of ASVs in zebrafish intestinal microbiota. B Alpha-diversity of microbes was calculated through Shannon index and Simpson index. C Beta-diversity analyzed based on PCoA was shown by using Bray Curtis distance. D The relative abundance of intestinal microbiota at the class level. E-G The relative abundance of Alphaproteobacteria (E), Bacilli (F) and verrucomicrobiae (G) in the intestines from the wild-type (WT) and ctla-4−/− zebrafish. *p < 0.05. H The relative abundance of intestinal microbiota at the family level. I Heatmap showing row-scaled expression of the differential abundances of bacterial communities at family level in the WT and ctla-4−/− zebrafish (p < 0.05). J Cladogram representation of LEfSe analysis showing the differentially abundant bacterial taxa between the intestines from WT (red) and ctla-4−/− (green) zebrafish (p < 0.05).

Examination on the inhibitory function of Ctla-4 in T cell activation.

A Assessment of the proliferative activity of T cells from wild-type (WT) and ctla-4−/− zebrafish by a mixed lymphocyte reaction combined with PHA-stimulation. The CFSE dilution, which served as an indicator of lymphocyte proliferation, was measured through flow cytometry. B Assessment of the proliferative activity of lymphocytes from ctla-4−/− zebrafish by the administration of sCtla-4-Ig. C Assessment of the proliferative activity of lymphocytes from WT zebrafish by supplementing anti-Ctla-4 antibody. D Assessment of the proliferative activity of lymphocytes from ctla-4−/− zebrafish by the administration of sCd28-4-Ig. E Assessment of the proliferative activity of lymphocytes from ctla-4−/− zebrafish by the administration of recombinant sCd80/86 protein. F, G Interactions between Cd80/86 and Cd28 (F), and Cd80/86 and Ctla-4 (G) as predicted by AlphaFold2. On the left are structural models depicting Cd80/86 in complex with Cd28 or Ctla-4. The center panels display per-residue model confidence scores (pLDDT) for each structure, using a color gradient from 0 to 100, where higher scores indicate increased confidence. The right panels show the predicated aligned error (PAE) scores for each model. The well-defined interfaces between Cd28 or Ctla-4 and Cd80/86 are highlighted with red dashed squares. H The interaction between Cd80/86 and Cd28 (left), and Cd80/86 and Ctla-4 (right) were verified by Co-IP. I Binding affinities of the Cd80/86 protein for the Cd28 and Ctla-4 proteins were measured by the microscale thermophoresis (MST) assay. The KD values are provided. Data are presented as mean ± standard deviation (SD), which were calculated from three independent experiments. Statistical significance was assessed through an unpaired Student’s t test (**p < 0.01; ***p < 0.001; ns denotes no statistical significance).

In vivo inhibition of intestinal inflammation by sCtla-4-Ig.

A Percent initial weight of zebrafish after injection of the sCtla-4-Ig or the IgG isotype control. Each group consisted of six zebrafish (n = 6). Data show means with SEM analyzed by two-way ANOVA with Sidak’s correction for multiple comparisons. B Representative H&E staining analysis of histopathological changes and quantitation of histology scores in the posterior intestine from ctla-4−/− zebrafish treated with sCtla-4-Ig or IgG isotype control. Scale bar: 50 μm. C The mRNA expression levels of inflammation-related genes in ctla-4−/− zebrafish treated with sCtla-4-Ig or IgG isotype control. D The mRNA expression levels of IBD biomarker genes and IBD-related genes in ctla-4−/− zebrafish treated with sCtla-4-Ig or IgG isotype control. The p value was generated by unpaired two-tailed Student’s t-test. **p < 0.01; ***p < 0.001; ****p < 0.0001.

The organization, sequence and phylogenetic analysis of zebrafish ctla-4 and cd28 genes.

A Comparison of the intron/exon organizations of ctla-4 gene in zebrafish and humans. Exons and introns are shown with black boxes and lines, and their size are indicated by the numbers found above and below the sequences respectively. B The nucleotide and amino acid sequences of ctla-4 gene and Ctla-4 protein. The underline indicates the signal peptide, the circles represent the conserved cysteine residues. C Phylogenetic analysis of the relationship of Ctla-4 and Cd28 between zebrafish and other species. An unrooted phylogenetic tree was constructed through the neighbor-joining method, based on amino acid sequence alignments generated by ClustalX. Bootstrap confidence values, derived from 500 replicates, are indicated as percentages at each node. D Alignment of the Cd28 homologs from different species generated with ClustalX and Jalview. The conserved and partially conserved amino acid residues in each species are colored in hues graded from orange to red, respectively. The conserved functional motifs, such as B7-binding motif, tyrosine phosphorylation site, and potential tyrosine phosphorylation site, were indicated separately. The signal peptide, IgV-like domain, transmembrane (TM) domain and cytoplasmic domain were marked at the top of the sequence.

Preparation of mouse anti-Ctla-4 antibody.

A SDS-PAGE detection of the recombinant Ctla-4 protein with extracellular domain (ECD). Lane 1, 2 and 3 represent the protein markers, blank, and target protein, respectively. B Western blot analysis of the mouse anti-EGFP and anti-Ctla-4 antibodies that bind to the recombinant Ctla-4-EGFP fusion proteins expressed in HEK293T cells. C Western blot analysis of native Ctla-4 protein in zebrafish intestinal tissues using mouse anti-Ctla-4 antibody.

Histopathological analysis of intestines.

A Periodic Acid-Schiff (PAS) staining was used to analyze the mucin components in anterior intestine from wild-type (WT) and ctla-4−/− zebrafish (n = 5). B The ratio of intestinal villi length to intestinal ring radius was measured in the anterior, mid, and posterior intestines of WT and ctla-4−/− zebrafish (n = 6). Statistical significance was assessed through an unpaired Student’s t test (*p < 0.05; **p <0.01).

Examination on the functional genes and pathways associated with the IBD-like phenotype in ctla-4−/− zebrafish.

A Top 5 KEGG enrichment bar plot of up-regulated genes in ctla-4−/− zebrafish intestines versus wild-type (WT) zebrafish intestines. B Top 10 KEGG enrichment bar plot of down-regulated genes in ctla-4−/− zebrafish intestines versus WT zebrafish intestines. C, D Changes in the expression of genes associated with lymphocyte chemotaxis, positive regulation of ERK1/ERK2 cascades, Calcium and MAPK signaling pathways in the ctla-4−/− zebrafish intestines analyzed by using a collection of pre-defined gene sets retrieved from GO (C) and KEGG (D) database. The p value, false discovery rates (FDR) and normalized enrichment score (NES) are shown above each pathway graph.

Quality control analysis of single-cell RNA sequencing data.

A Identification of the effective cell number of the sample. The blue line represents the effective cells corresponding to barcodes, while the gray line denotes the background noise. B The basic cellular metrics before and after filtering, including the total number of detected genes (nFeature_RNA), the total number of UMIs (nCount_RNA), and the percentage of reads mapped to mitochondrial genes (Percent.mito). C The scatter plot comparing the cellular metrics before and after filtering, showing the relationship between nCount_RNA and nFeature_RNA. The Pearson correlation coefficients are indicated above the graph.

Examination on the involvement of apoptotic process in epithelial cells and expression of inflammation-related genes in neutrophils and B cells in the intestines of ctla-4−/− zebrafish.

A Expression map of the epithelial markers within the cell populations of the zebrafish intestines. B Expression map of the neutrophil markers within the cell populations of the zebrafish intestines. C KEGG enrichment bar plot of all differentially expressed genes (DEGs) from epithelial cells. D Quantification of TUNEL-positive cells per 1 × 104 μm2 in WT and ctla-4−/− posterior intestines (n = 5). E Heatmap of inflammation-related genes in B cells from wild-type and ctla-4−/− intestines. Statistical significance was assessed through an unpaired Student’s t test (***p < 0.001).

Examination on the activation of T cell subsets in the intestines of ctla-4−/− zebrafish.

A Marker gene expression in individual cluster identifies the cluster as NKT-like cells. B RT-qPCR confirms the mRNA expression levels of Th2 cell marker genes in the intestines of wild-type (WT) and ctla-4−/− zebrafish. C RT-qPCR validates the mRNA expression levels of NKT-like cell marker genes in the intestines of WT and ctla-4−/− zebrafish. D Heatmap illustrates up-regulated genes involved in cytokine-cytokine receptor interaction in NKT-like cells from WT and ctla-4−/− samples. E KEGG enrichment analysis reveals the top 15 terms of up-regulated genes in Cd8+ T cells in ctla-4−/− samples versus WT samples. F Heatmap displays up-regulated genes involved in cytokine-cytokine receptor interaction in Cd8+ T cells from WT and ctla-4−/− samples. Statistical significance was assessed through an unpaired Student’s t test (*p < 0.05; **p <0.01; ***p < 0.001; ****p < 0.0001).

Preparation of recombinant proteins and examination of their molecular interactions.

A-C SDS-PAGE detection of the purified recombinant soluble Ctla-4-Ig (sCtla-4) (A) and sCd28-Ig (B) proteins and the Cd80/86 extracellular domain (ECD) (C) with Coomassie brilliant blue staining. D-E The predicted molecular interactions between Cd80/86 and Cd28 (D), as well as Cd80/86 and Ctla-4 (E), as modeled by AlphaFold2. The structures are represented in a cartoon style, with Cd80/86, Cd28, and Ctla-4 colored cyan, green, and magenta, respectively. A total of 25 models were predicted for each complex and aligned with Cd80/86. F Flow cytometry analysis of the interactions between Cd80/86 and Cd28 (top), and Cd80/86 and Ctla-4 (bottom). Cd80/86 was expressed on HEK293T cells and incubated with varying concentrations of fluorescently labeled sCd28-Ig or sCtla-4-Ig. Fluorescence intensity was detected by flow cytometry to determine molecular interactions. Data are presented as mean ± SD, derived from three independent experiments. Statistical significance was evaluated using an unpaired Student’s t test (**p < 0.01; ***p < 0.001).