Combined blockade of PSGL-1 and PD-1 ameliorates bovine peripheral blood lymphocyte function during cytopathic BVDV infection via the PI3K/AKT/mTOR and ERK signaling pathways.

Cytopathic (CP) bovine viral diarrhea virus (BVDV) infection is a major cause of lymphocyte dysfunction and immunosuppression in cattle; however, the involvement of the immune checkpoint P-selectin glycoprotein ligand-1 (PSGL-1) in this process remains poorly defined. In this study, bovine peripheral blood lymphocytes (PBL) - rather than total peripheral blood mononuclear cells (PBMCs) - were infected with CP BVDV in vitro. PBL were used because they consist primarily of T cells, B cells, and NK cells without the interference of monocytes/macrophages, allowing a more specific assessment of lymphocyte-intrinsic responses to BVDV infection and antibody blockade. The dynamic expression of PSGL-1, programmed death-1 (PD-1), and their ligands was analyzed by quantitative real-time PCR and Western blotting. Functional antibody blockade was employed to evaluate the effects of individual and combined PSGL-1 and PD-1 inhibition on lymphocyte proliferation, apoptosis, cytokine secretion, and viral replication. We found that CP BVDV infection significantly upregulated PSGL-1 and PD-1 expression in PBL. While blockade of either PSGL-1 or PD-1 partially restored lymphocyte function, combined blockade produced a pronounced synergistic effect, characterized by enhanced proliferation, reduced apoptosis, increased interferon-γ (IFN-γ) and interleukin-2 (IL-2) secretion, and suppression of viral replication. Mechanistically, this synergistic restoration was associated with reactivation of the PI3K/AKT/mTOR and ERK signaling pathways. Collectively, these findings identify PSGL-1 as a critical immune checkpoint involved in BVDV-induced lymphocyte exhaustion and demonstrate that combined PSGL-1/PD-1 blockade effectively restores antiviral lymphocyte function. This study provides new insight into host immune regulation during BVDV infection and highlights a potential combinatorial immunotherapeutic strategy for viral immunosuppression.