The PD-1/PD-L1 Pathway Affects the Expansion and Function of Cytotoxic CD8(+) T Cells During an Acute Retroviral Infection

David, Paul; Megger, Dominik A.; Kaiser, Tamara; Werner, Tanja; Liu, Jia; Chen, Lieping; Sitek, Barbara; Dittmer, Ulf; Zelinskyy, Gennadiy

In the next step, we therefore studied proliferation and apoptosis of CD8 + T cells in mice deficient for PD-L1. We analyzed the expression of Ki67 as a marker of proliferation (34) and the expression of activated caspase 3 as marker for apoptosis (35) in virus-specific CD8 + T cells from the spleen and BM of WT, PD-1 −/− , and PD-L1 −/− mice. At day 8 after FV infection, the frequency of CD8 + tetramer + T cells expressing Ki67 was slightly enhanced, but not significantly different in both KO mouse strains compared to WT mice ( Figure 5C) . However, the number of virus-specific CD8 + T cells expressing Ki67 was significantly enhanced in PD-L1 −/− mice at day 10 and 12 after infection. In PD-1 −/− mice significantly enhanced numbers of proliferating CD8 + T cells were only observed at day 12 after infection. Thus, an enhanced proliferation of virus-specific CD8 + T cells between day 10 and 12 after FV infection likely explains the higher number of effector T cells in both KO mouse strains. The other possible mechanism, which determines the quantity of effector cells, is the programmed death of these cells The expression of activated caspase 3, a key enzyme for the induction of apoptosis, was analyzed in the spleen of day 8 FV infected B6, PD-1 −/− , and PD-L1 −/− mice ( Figure 5D) . 13% of the CD8 + tetramer + T cells expressed the activated caspase 3 enzyme in WT mice, whereas only 6% of the virus-specific CD8 + T cells from PD-1 −/− and 4% from PD-L1 −/− mice expressed caspase 3. This significant differences in CD8 + T cell number expressing the activated form of caspase 3 provides evidence that PD-1 signaling may also regulate apoptosis in virus-specific CD8 + T cells. To prove that apoptosis regulates the number of virus-specific CD8 + T cells in acutely FV-infected mice, animals were treated twice with the general caspase inhibitor Z-VAD-FMK (24) on day 6 and 7 after infection. The inhibition of caspases and subsequent reduction of apoptosis led to increased numbers of virus-specific CD8 + T cells in the spleen ( Figure 5E ) and BM (Figure 5F ) of treated mice at 8 days post infection. Thus, apoptosis plays an important role in regulating CD8 + T cell number during acute FV infection and PD-1 signaling influences levels of apoptosis.