Abstract
The inhibitory receptor programmed death-1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1) are involved in immune evasion mechanisms for several pathogens causing chronic infections. Blockade of the PD-1/PD-L1 pathway restores anti-virus immune responses, with concomitant reduction in viral load. In a previous report, we showed that, in bovine leukemia virus (BLV) infection, the expression of bovine PD-1 is closely associated with disease progression. However, the functions of bovine PD-L1 are still unknown. To investigate the role of PD-L1 in BLV infection, we identified the bovine PD-L1 gene, and examined PD-L1 expression in BLV-infected cattle in comparison with uninfected cattle. The deduced amino acid sequence of bovine PD-L1 shows high homology to the human and mouse PD-L1. The proportion of PD-L1 positive cells, especially among B cells, was upregulated in cattle with the late stage of the disease compared to cattle at the aleukemic infection stage or uninfected cattle. The proportion of PD-L1 positive cells correlated positively with prediction markers for the progression of the disease such as leukocyte number, virus load and virus titer whilst on the contrary, it inversely correlated with the degree of interferon-gamma expression. Blockade of the PD-1/PD-L1 pathway in vitro by PD-L1-specific antibody upregulated the production of interleukin-2 and interferon-gamma, and correspondingly, downregulated the BLV provirus load and the proportion of BLV-gp51 expressing cells. These data suggest that PD-L1 induces immunoinhibition in disease progressed cattle during chronic BLV infection. Therefore, PD-L1 would be a potential target for developing immunotherapies against BLV infection.
Highlights
The immune response to bovine leukemia virus (BLV) in cattle is an important factor to determine the outcome of BLV infection
These findings indicated that high expression of programmed death-1 (PD-1) and programmed death-ligand 1 (PD-L1) in retrovirus infection leads to T cell dysfunction, suggesting that the reinvigoration of immune dysfunction has a potential for application in clinical immunotherapy against these chronic infections
Total RNA was extracted from cultivated peripheral blood mononuclear cells (PBMC) using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions, and residual DNA was removed from the RNA samples by treatment with Deoxyribonuclease I (Invitorogen). cDNA was synthesized from the RNA samples using Moloney murine leukemia virus reverse transcriptase (Takara, Shiga, Japan) according to the manufacturer’s instructions. 3’- and 5’- rapid amplification of cDNA ends (RACE) were performed using the 3’-/5’- RACE System for Rapid Amplification of cDNA Ends (Invitrogen) with PD-L1 gene-specific primers, 5’-ACG TGT CAG GCT GAG GGT TAC CCT GAA GC-3’ and 5’GTC ACA TTT TTC TAC ATC-3’
Summary
The immune response to bovine leukemia virus (BLV) in cattle is an important factor to determine the outcome of BLV infection. Recent in vitro and in vivo studies have shown the importance of the PD-1/PD-L1 pathway in retroviral infections, such as human immunodeficiency virus (HIV), HTLV-1 and simian immunodeficiency virus (SIV). Blocking of the PD-1/PD-L1 pathway by antibodies specific to PD-1 or PD-L1 has been shown to restore T cell function during HIV and HTLV infection in vitro [6,8,9]. In the SIV model for potential immunotherapy, the viral load was significantly reduced by the inoculation of anti PD-1 antibody in vivo [10,11] These findings indicated that high expression of PD-1 and PD-L1 in retrovirus infection leads to T cell dysfunction, suggesting that the reinvigoration of immune dysfunction has a potential for application in clinical immunotherapy against these chronic infections
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