Effects Of PD-1 Blocking On The Development And Function Of Effector And Memory CD8~+T Lymphocytes In BVDV Acute Infected Mice

Bovine viral diarrhea virus(BVDV)is an important infectious agent that infects ruminants and pigs,causing damage to the respiratory,digestive and reproductive systems,inducing peripheral blood lymphopenia and immunosuppression,and posing a serious threat to the health of cattle.Programmed cell death protein 1(PD-1)is a central inhibitory receptor that regulates CD8 T cell depletion during chronic viral infections and cancer.PD-1 is also transiently expressed by activated CD8~+T cells during acute viral infections,but the role of PD-1 in regulating T cell effector differentiation and function is not clear.It has been shown that PD-1pathway is blocked with anti-PD-L1 or anti-PD-1 antibodies during the early stages of acute lymphocytic choroid plexus meningitis virus infection,that PD-1 plays an inhibitory role in the transition from naive to effector CD8 T cells,and that lack of PD-1 can also lead to subsequent memory cell failure and loss of function.In studies of influenza virus X31 infection,early loss of PD-1 leads to its overactivation in CD8~+T cells,and mice with constitutive cellular deficiency of PD-1 or PD-L1 during acute infection develop impaired CD8~+T cell memory cell-intrinsic PD-1signaling that inhibits the expansion of effector cells,leading to impaired memory formation.In a mouse model of acute BVDV infection,PD-1 monoclonal antibody blockade restored the proliferation of peripheral blood CD4~+T and CD8~+T lymphocytes and restored their organismal antiviral function,but its effects on effector CD8~+T lymphocyte and memory CD8~+T cell differentiation and proliferation remain unclear.Therefore,this study applied the BVDV acute infection mouse model to investigate the effects of PD-1 monoclonal antibody blockade on CD8~+T lymphocyte activation,differentiation and memory CD8~+T lymphocyte formation in BVDV-infected mice.Firstly,mice were injected intraperitoneally with CP and NCP type BVDV and blocked with PD-1 monoclonal antibody at an interval of 24 h.The activation and differentiation of CD8~+T lymphocytes and their replication during the viral infection period were detected by fluorescence quantitative PCR and flow cytometry.The results showed that compared with the virus-infected group,the blood and spleen CD8~+T lymphocytes of mice showed significant activation and differentiation after PD-1 blockade,in which the peripheral blood CD8~+CD25 activation increased in the CP+PD-1 group in a highly significant upward trend,and the differentiation important index KI-67 was highly expressed in the blood CD8~+T cells of the CP+PD-1 group in a highly significant upward trend,and in the spleen The NCP+PD-1 group showed a highly significant increase in CD8~+T cells in both blood and spleen.Viral load in spleen and peripheral blood CD8~+T cells peaked at the 7th d after BVDV infection and was completely cleared within15 d.Secondly,the effect of PD-1 monoclonal antibody blockade on the differentiation and function of effector CD8~+T lymphocytes in the BVDV-infected mouse model was examined.The results showed that the differentiation of short-lived effector cells(SLECs)in peripheral blood CD8~+T lymphocytes was highly significantly increased in both CP+PD-1 and NCP+PD-1groups by flow cytometry at day 7 after BVDV infection,and a significant decrease in the differentiation of memory precursor cells(MPECs)was observed in the CP+PD-1 group,while the NCP+PD-1 group showed the opposite trend of significantly increased differentiation of MPECs in CP-type BVDV infection.T-bet and Eomes are important transcription factors that regulate the differentiation and function of type I effector T cells such as CD4 Th1 cells and CD8~+CTLs.The results showed that the expression of transcription factor T-bet m RNA in peripheral blood CD8~+T lymphocytes was significantly upregulated at day 7 and 10 of infection in the CP+PD-1 group,while the expression in the NCP+PD-1 group was highly significantly increased at d 4,7 and 10;the expression of transcription factor Eomes m RNA in the CP+PD-1group was not significantly changed,in contrast to the NCP+The expression of IL-2 and IFN-γin CD8~+T lymphocytes was significantly increased in CP+PD-1 and NCP+PD-1 groups.Finally,the effects of PD-1 monoclonal antibody blockade on memory cell differentiation,and changes in short-term effector cells(SLECs),cytokine expression and viral load after secondary viral infection were examined by flow cytometry,fluorescence quantitative PCR and ELISA in a BVDV-infected mouse model.The results showed that the differentiation of CD8~+T central type memory cells(TCM)and effector memory cells(TEM)was significantly decreased in the CP+PD-1 group at 60 d post-infection,and there were no significant changes in CD8~+TCM and TEM in the NCP+PD-1 group.The expression of SLEC in peripheral blood CD8~+T lymphocytes was significantly reduced in the CP+PD-1 and NCP+PD-1 groups 48 h after secondary BVDV infection.IL-2 was significantly reduced in the supernatant of peripheral blood CD8~+T cell cultures in the CP+PD-1 group,and INF-γwas extremely significantly reduced in the supernatant of peripheral blood CD8~+T cell cultures in the NCP+PD-1 group.In summary,PD-1 monoclonal antibody blockade promotes the activation,differentiation and antiviral effects of early CD8~+T cells in CP and NCP type BVDV acutely infected mice,but PD-1 deficiency causes severe effects on the formation of memory CD8~+T cells and antiviral function in CP type BVDV infected mice,suggesting that PD-1 signaling may be an important signaling molecule for memory cell formation.This study provides a scientific basis for exploring antiviral therapeutic strategies and mechanism of action studies based on the immunodetection site PD-1.