Fig. 7

Examination on the inhibitory function of Cytotoxic T lymphocyte antigen-4 (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 carboxyfluorescein succinimidyl ester (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 predicted 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).