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

The effect of the PD-1 receptor on the proliferation of effector CD8 + T cells is one of the known mechanisms regulating the numbers of CTLs in infected organ (38) . Apoptosis is another mechanism that determines the quantity of activated effector cells. The phenomenon of activation induced cell death (AICD) which is involved in the pathogenesis of autoimmune disorders also determines the magnitude of Influenza virusspecific CD8 + T cells (46) . Apoptosis plays a key role in dampening immune responses and for establishing long-lasting T cell memory during the contraction phase of antiviral immune responses (47) . In previous studies it was shown that PD-L1 deficiency was associated with diminished expression of the pro-apoptotic molecule BCL-2-interacting mediator of cell death (Bim) in activated antigen-specific CD8 + T cells (48) . Moreover, a similar reduction of Bim in PD-1 + CD8 + T cells was observed in melanoma patients successfully treated with anti-PD-1 antibodies (49) . However, the role of apoptosis during the expansion phase of virus-specific CTLs is not well-characterized. Activated caspase 3 is the important executor molecule managing the apoptosis process (50) . This enzyme cleaves different intracellular substrate molecules and starts the cascade of irreversible changes ultimately resulting in programmed cell death. Previous studies showed that the presence of caspase 3 is a phenotypic marker of activated effector T cells and not always associated with apoptosis of these cells (51) . Our findings, based on proteome analysis and flow cytometry, suggest that the level of activated caspase 3 in effector CD8 + T cells was regulated by the PD-1 receptor. Effector cells that expanded without PD-1/PD-L1 regulation expressed reduced levels of activated caspase 3 in the cytoplasm. Cells with high levels of caspase 3 expression were predisposed to apoptotic death (35) . The treatment of FV-infected mice with the low molecular pancaspase inhibitor Z/VAD-FMK resulted in enhanced numbers of virus-specific CD8 + T cells during the early phase FV infection. Thus, apoptosis was involved in regulating numbers of expanded effector T cells. The regulation of T cell apoptosis through PD-1/PD-L1 molecules is most likely an important mechanism determining the magnitude of the CTL responses in infected organs. The quantity of effector CD8 + T cells is one important aspect of the PD-1 regulation, whereas the functional maturation toward cytotoxic T cells is another. In the current study, we analyzed the role of PD-1 signaling for the ability of CD8 + CTLs to simultaneously produce the different cytotoxic effector molecules granzyme A, B, and K. Interestingly, effector CD8 + T cells that produced two or three granzymes simultaneously were impaired by the PD-1 checkpoint mechanism. Deficiency for PD-L1 clearly enhanced numbers and presence of polyfunctional cytotoxic cells in FV-infected organs. Thus, PD-1 signaling not only regulates the quantity of antiviral T cells through the inhibition of proliferation and the induction of apoptosis, but is also critical for inhibiting the differentiation of these cells into polyfunctional cytotoxic T cells. These observations from KO mice allow us to better understand the role of PD-1 during the early phase of antiviral immune responses and may provide new strategies for the development of successful immunmodulatotry treatments of viral infections or tumors and for the development of efficient prophylactic