Regulatory role of BTLA and HVEM checkpoint inhibitors in T cell activation in a perciform fish Larimichthys crocea.

The BTLA and HVEM are two well-characterized immune checkpoint inhibitors in humans and other mammalian species. However, the occurrence and functionality of these two molecules in non-mammalian species remain poorly understood. In the present study, we identified the BTLA and HVEM homologs from large yellow croaker (Larimichthys crocea), an economically important marine species of the perciform fish family. The Larimichthys crocea BTLA and HVEM (Lc BTLA and Lc HVEM) share conserved structural features to their mammalian counterparts, and they were expressed in various tissues and cells examined at different transcriptional levels, with particular abundance in immune-relevant tissues and splenic leukocytes. Immunofluorescence staining and flow cytometry analysis showed that Lc HVEM and Lc BTLA proteins were distributed on MHC-II+ APCs and CD4-2+ T cells, and a strong interaction between Lc BTLA and Lc HVEM was detected in splenic leukocytes in the mixed lymphocyte reaction (MLR). By blockade assays using anti- Lc BTLA and anti- Lc HVEM Abs as well as recombinant soluble Lc BTLA and Lc HVEM proteins in different combinations, it was found that Lc BTLA- Lc HVEM interactions play an important inhibitory role in the activation of alloreactive T cells using MLR as a model, and APC-initiated antigen-specific CD4-2+ T cells in response to A. hydrophila (A. h) stimulation. These observations highlight the extensive functional roles of Lc BTLA and Lc HVEM immune-checkpoint inhibitors in allogeneic T cell reactions, and CD4-2+ T cell-mediated adaptive immune responses in Larimichthys crocea. Thus, the BTLA-HVEM checkpoint may represent an ancient coinhibitory pathway, which was originated in fish and was conserved from fish to mammals throughout the vertebrate evolution. • Identification of BTLA-HVEM checkpoint inhibitors in perciform fish Larimichthys crocea. • Regulatory role of BTLA-HVEM checkpoint in the activation of alloreactive T and antigen-specific CD4+ T cells. • BTLA-HVEM checkpoint is an ancient coinhibitory mechanism originated as early as in teleost fish. [ABSTRACT FROM AUTHOR]