Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

Bovine leukemia virus (BLV) infects B cells in ruminants and causes lymphoma after an extended latency period. Previous studies have demonstrated T-cell exhaustion through the upregulation of immunoinhibitory molecules, including programmed death-ligand 1 (PD-L1) and T-cell immunoglobulin and mucin domain-3 (TIM-3), in BLV-infected cattle. However, studying T-cell exhaustion across all BLV infection stages remains challenging due to the virus's prolonged latency in cattle. Sheep provide a valuable model, as they develop lymphoma more rapidly than cattle. This study examined PD-L1 and TIM-3 expression kinetics and T-cell function in BLV-infected sheep. During persistent infection, PD-L1 expression was correlated with BLV proviral load. TIM-3 expression increased in CD4⁺, CD8⁺, and γδTCR⁺ T cells. Functional analysis revealed that TIM-3 blockade enhanced T-cell activation markers (CD25 and CD69) in cultured PBMCs from infected sheep and increased CD69⁺IFN-γ⁺ and CD69⁺TNF-α⁺ populations, particularly among CD4⁺ T cells. Combined PD-L1 and TIM-3 blockade significantly enhanced cytokine production in both CD4⁺ and CD8⁺ T cells, while PD-L1 blockade alone showed limited effects. These findings demonstrate the effect of TIM-3 blockade in restoring immune function during chronic BLV infection, effective both alone and in combination. This study validates sheep as a valuable model for investigating immune checkpoint dynamics and evaluating immunotherapies for BLV infection and other chronic diseases.