Fig. 1

Characterization of zebrafish Cytotoxic T lymphocyte antigen-4 (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 hr, 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 (Hu et al., 2023). (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×).