Effects of combined cytotoxic T-lymphocyte antigen 4 and programed death 1 ligand-receptor blockade on interferon-gamma production in bovine leukemia virus-infected cattle.

Background and aim: In chronic viral infections, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and programmed death ligand 1 (PD-L1) significantly suppress immune responses. The CTLA-4 receptor abundance in regulatory T cells showed a positive association with viral load and a negative association with interferon-gamma (IFN-γ) production in bovine leukemia virus (BLV)-infected cattle. Blocking this receptor boosted IFN-γ production, recovering immune response against this illness. In human cancer patients, not everyone responded positively to non-immunotherapy using CTLA-4 receptor antibodies. The present study analyzed the synergistic effects of CTLA-4 and PD-L1 receptor blockade on IFN-γ production in BLV+ cattle in vitro.

Materials and methods: The genes for bovine CTLA-4 and PD-L1 were artificially produced. The amino acid sequences of the extracellular receptor domains were sourced from the National Center for Biotechnology Information PubMed database. The western blotting and liquid chromatography with tandem mass spectrometry (LC-MS/MS) techniques were employed for the characterization of recombinant CTLA-4 (rCTLA-4) and recombinant PD-L1 (rPD-L1) proteins. The immunoinhibitory effects of recombinant proteins in Staphylococcus enterotoxin B (SEB)-stimulated cattle peripheral blood mononuclear cells (PBMCs) were investigated. Enzyme-linked immunosorbent assay (ELISA) was used to analyze monoclonal antibodies against rCTLA-4 and rPD-L1. Antibodies generated from peripheral blood mononuclear cells of healthy and BLV-seropositive cows were employed to evaluate their blocking capabilities.

Results: The resulting recombinant proteins specifically reacted with commercial homogeneous monoclonal antibodies (mAbs) using ELISA and anti-His-tag mAbs using western blotting. Analysis of the proteins using LC-MS/MS revealed correspondence with the sequences of rCTLA-4 and rPD-L1 located in the Mascot database. rCTLA-4 and rPD-L1 proteins inhibited IFN-γ production in bovine PBMCs of activated SEB. When PBMCs from cows were cultured with activated SEB containing rCTLA-4 and rPD-L1, the mAbs increased IFN-γ production in PBMCs. The combined cultivation of mAbs and PBMCs from BLV+ cattle enhanced IFN-γ production in the cells.

Conclusion: These findings suggest that the combined blockade of bovine CTLA-4 and PD-L1 receptors can be used as a therapy for bovine leukemia. However, it was shown that a single PBMC sample from a BLV-positive donor did not amplify the synergistic effect. Therefore, it is necessary to perform further studies on a larger population and assessing a wider range of cytokines.