Study On The Effect Of BTLA And PD-1 Monoclonal Antibody Blockade On The Proliferation Of CD8~+T Lymphocytes In Peripheral Blood Of NCP-type BVDV-infected Cattle

Bovine viral diarrhea(BVD)is a contact infection of cattle and other ruminants and pigs caused by Bovine viral diarrhea virus(BVDV).The main clinical manifestations of BVDV infected cattle are respiratory,reproductive and digestive symptoms and persistent infection.The main clinical manifestations of BVDV infected cattle are respiratory,reproductive and digestive symptoms and persistent infection,which seriously endanger the cattle industry.BTLA is an immune checkpoint molecule in the CD28 family with structural and functional similarity to PD-1,which is mainly expressed on the surface of mature T and B lymphocytes and plays a role in suppressing lymphocyte proliferation and immune function.),human immunodeficiency virus(HIV),and melanoma synergistically promote the proliferation and function of T lymphocytes(TLs).However,there are no studies on its role in BVDV infection.Pre-laboratory studies have confirmed that PD-1 in BVDV infection inhibits the proliferation and activation of CD4~+T cells and CD8+T lymphocytes and induces apoptosis through downregulation of PI3K/AKT/mTOR,caspase 9/caspase 3 and ERK pathways.However,PD-1 blockade in NCP-type BVDV infection did not completely restore the proliferation of CD8~+TLs and the expression of p-ERK.Thus,there may be other immune checkpoint molecules that synergistically regulate the proliferation and antiviral function of CD8~+TLs with PD-1.The aim of this study was to clarify the immunomodulatory role of BTLA in the reduction of CD8~+TLs due to NCP-type BVDV infection and whether there is a synergistic effect with PD-1.First,to clarify the correlation between BTLA and PD-1 and the proliferation and apoptosis of CD8~+TLs in NCP-type BVDV infection.Healthy bovine peripheral blood single nucleated cells(PBMC)were isolated by gradient density centrifugation,inoculated with NCP-type BVDV(NY-1 strain),and CD8~+TLs and CD14~+PBM were sorted by immunomagnetic beads.cell purity was analyzed by flow cytometry,and CD8~+TLs BTLA,PD-1 and CD14~+were detected by q RT-PCR and Western Blot.PBM HVEM(BTLA ligand),and PD-L1 expression levels.The proliferation and apoptosis of CD8~+TLs were detected by CCK-8 method and flow cytometry.The results showed that the mRNA expression of BTLA,HVEM,PD-1 and PD-L1 gradually increased with the prolongation of NCP-type BVDV infection,and the mRNA expression of BTLA and HVEM peaked at 72 h(BTLA,p<0.001;HVEM,p<0.05),PD-1 and PD-L1 mRNA expression peaked at 96 h(PD-1,p<0.0001;PD-L1,p<0.001),and protein expression levels of all four molecules were significantly upregulated at 72 h(BTLA,p<0.001;HVEM,p<0.01;PD-1,p<0.0001;PD-L1,p<0.01),accompanied by a significant decrease in the proliferation of CD8~+TLs(p<0.0001)and a significant increase in apoptosis(p<0.01).Second,to clarify the effects of BTLA and PD-1 monoclonal antibody blockade on the proliferation,apoptosis and antiviral function of CD8~+TLs in NCP-type BVDV infection.We selected NCP-type BVDV-infected healthy bovine PBMC,applied commercial monoclonal antibodies to block BTLA and PD-1,sorted CD8~+TLs by immunomagnetic beads,detected CD8~+TLs proliferation by CCK-8,detected CD8~+TLs apoptosis and CD8~+granzyme B~+TLs ratio by flow cytometry,detected viral replication level by q RT-PCR,and detected cytokines by ELISA.levels.The results showed that blocking BTLA significantly restored the proliferation of CD8~+TLs(72 h,p<0.01;96 h,p<0.05;120 h,p<0.01),reduced the apoptosis of CD8~+TLs(72 h,p<0.05),and increased the secretion of IL-2(72 h,p<0.05)compared with the attack group control.Synergistic blockade of BTLA+PD-1 significantly restored the proliferation of CD8~+TLs compared with blockade of BTLA or PD-1 alone(96 h,p<0.05).Finally,to clarify the molecular mechanism by which BTLA and PD-1 blockade restores the proliferation of CD8~+TLs in NCP-type BVDV infection.BTLA and PD-1 were blocked by monoclonal antibodies,CD8~+TLs were sorted by immunomagnetic beads,and Western bolt detected the expression of key signaling molecules associated with proliferation downstream of BTLA and PD-1.The results showed that blocking BTLA or synergistic blockade of BTLA+PD-1 significantly upregulated the expression of p-PI3K(p<0.01),p-AKT(p<0.05),p-mTOR(p<0.05)and p-ERK(p<0.001)in CD8~+TLs compared with the viral infection group.In conclusion,BTLA is involved in regulating the proliferation,activation and antiviral function of bovine peripheral blood CD8~+TLs in NCP-type BVDV infection.synergistic blockade of BTLA and PD-1 significantly restored the proliferation of CD8~+TLs,and BTLA or synergistic with PD-1 could inhibit the proliferation of CD8~+TLs by suppressing the downstream PI3K/AKT/mTOR and ERK pathways.This study provides a basis for exploring the molecular mechanisms of lymphopenia and immunosuppression caused by BVDV infection.