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 previous experiments we observed that the deficiency of the PD-1 ligand had a more profound influence on the expansion and function of virus-specific CD8 + T cells than the deficiency of the PD-1 receptor. Therefore, in the following experiments we decided to focus on the role of PD-L1 on the functionality of cytotoxic CD8 + T cells. In these cells the effects of PD-L1 knockout on the production of different cytotoxic molecules was characterized in greater detail. It is known that polyfunctional CD8 + T cells simultaneously producing different cytokines correlates with the long-term control of HIV in elite controllers (31) . However, the role of polyfunctionality regarding the production of different granzymes by T cells has not been characterized. The granzymes produced by antiviral CTLs have a different spectrum of target proteins and mediate a diverse range of cytotoxic, proinflammatory, and antiviral functions (32) . To characterize the simultaneous production of different granzymes, the frequency of CD8 + CD43 + T cells expressing granzyme A (GzmA), GzmB, and granzyme K (GzmK) were determined in the spleen of WT and PD-L1 −/− mice at day 8 ( Figure 3A) , day 10 ( Figure 3B) , and day 12 ( Figure 3C ) after FV infection. Significantly more CTLs from PD-L1 −/− than WT mice produced GzmB or GzmK at day 8 after FV infection ( Figure 3A) . The frequencies of CD8 + CD43 + T cells, which were GzmA + GzmB + or GzmB + GzmK + double-positive, were also significantly higher in the PD-L1 −/− mouse strain. Two days later at day 10 after infection the frequencies of effector cells producing all three granzymes were also significantly higher in mice without PD-L1. At day 12 after infection significant differences were observed only in the frequencies of total effector cells producing single GzmA and GzmB. The population of FV-specific Tetramer + CD8 + T cells was also analyzed for the production of different granzymes molecules. At day 8 after infection significantly more virus-specific CD8 + T cells from the spleens of PD-L1 −/− than WT mice produced GzmK or all three Gzms simultaneously ( Figure 3D) . Similar results were obtained on day 10 after infection ( Figure 3E) . Interesting, at the late time point day 12 after FV infection significant differences were only found for the production of single Gzms (GzmA, GzmB) ( Figure 3F) . The analysis of the PD-L1 effects on the FIGURE 2 | Cytotoxic CD8 + T cell responses in mice without PD-1 signaling. C57BL/6, PD-1 −/− and PD-L1 −/− mice were infected with FV and splenocytes and bone marrow were isolated at different time points after infection. Flow cytometry was used to detect intracellular granzyme B in effector CD8 + T cells (CD43 + ) and in CD8 + T cells specific for the FV gagL epitope (Tetramer + ). A kinetic analysis was performed at different time points after FV infection. Shown are the numbers of effector CD8 + T cells expressing B (GzmB) from spleen (A) and bone marrow (B) and the numbers of virus-specific CD8 + tetramer + CD8 + T cells expressing granzyme B in spleen (C) and bone marrow (D). Each dot represents the mean number plus SEM per one million nucleated cells for a group of 5-9 mice. Data were pooled from three independent experiments with similar results. One-way ANOVA with a Tukey post-test was used for the comparison of both KO mice strains with WT animals at every analyzed time point. Statistically significant differences between the groups (blue for comparison with PD-1