PD-L1/BTLA Checkpoint Axis Exploited for Bacterial Immune Escape by Restraining CD8⁺ T Cell-Initiated Adaptive Immunity in Zebrafish

Programmed death-ligand 1/programmed cell death 1 (PD-L1/PD-1) is one of the most important immune checkpoints in humans and other mammalian species. However, the occurrence of the PD-L1/PD-1 checkpoint in evolutionarily ancient vertebrates remains elusive because of the absence of a PD-1 homolog before its appearance in tetrapods. In this article, we identified, to our knowledge, a novel PD-L1/B and T lymphocyte attenuator (BTLA) checkpoint in zebrafish by using an Edwardsiella tarda-induced bacterial infection model. Results showed that zebrafish (Danio rerio) PD-L1 (DrPD-L1) and BTLA (DrBTLA) were differentially upregulated on MHC class II+ macrophages (M phi s) and CD8(+) T cells in response to E. tarda infection. DrPD-L1 has a strong ability to interact with DrBTLA, as shown by the high affinity (K-D 5 5.68 nM) between DrPD-L1/DrBTLA proteins. Functionally, the breakdown of DrPD-L1/DrBTLA interaction significantly increased the cytotoxicity of CD8(+)BTLA(+) T cells to E. tarda-infected PD-L1(+) Mf cells and reduced the immune escape of E. tarda from the target Mf cells, thereby enhancing the antibacterial immunity of zebrafish against E. tarda infection. Similarly, the engagement of DrPD-L1 by soluble DrBTLA protein diminished the tolerization of CD8(+) T cells to E. tarda infection. By contrast, DrBTLA engagement by a soluble DrPD-L1 protein drives aberrant CD8(+) T cell responses. These results were finally corroborated in a DrPD-L1-deficient (PD-L12/2) zebrafish model. This study highlighted a primordial PD-L1/BTLA coinhibitory axis that regulates CD8(+) T cell activation in teleost fish and may act as an alternative to the PD-L1/PD-1 axis in mammals. It also revealed a previously unrecognized strategy for E. tarda immune evasion by inducing CD8(+) T cell tolerance to target Mf cells through eliciting the PD-L1/BTLA checkpoint pathway.