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

Proteome Analysis of Transferred CD8 + T Cells Isolated From WT and PD-L1 −/− Mice We used the previously described transfer model for analyzing the influence of PD-L1 on the population of virus-specific CD8 + T cells. For a detailed characterization of the molecular mechanisms regulated by PD-L1, a global analysis of the CD8 + T cell proteome was performed by label-free quantification using liquid chromatography and tandem mass spectrometry (LC-MS/MS). FV-specific CD8 + T cells were isolated from TCR-Tg-CD45.1 mice and transferred into WT or PD-L1 −/− mice. One day after transfer the recipients were infected with FV. Expanded donor CD8 + CD45.1 + T cells were sorted from splenocytes at day 12 after FV infection. Cells were lysed and subjected to proteome analysis. 2673 proteins were quantitated and compared between CD8 + CD45.1 + T cells isolated from WT vs. PD-L1 KO mice (Supplementary Table 1. Excel data) . From all these proteins, only 40 molecules significantly changed expression levels in the absence of PD-L1 (Figures 5A,B; Supplementary Table 2 ). The concentration of 37 proteins was reduced and only three proteins [General transcription factor IIE subunit 1 (Gtf2e1), Pyridoxal kinase (Pdxk), and U3 small nucleolar RNA-associated protein 14 homolog A (Utp14a)] were enhanced in virus-specific CD8 + T cells isolated from PD-L1 KO mice compared to WT mice. From these differently expressed proteins 8 are involved in regulation of the cell cycle [indicated by functional analysis using DAVID (26, 27) ] and the expression of caspase 3 was reduced in cells isolated from PD-L1 −/− mice, mice were infected with FV and splenocytes were isolated at day 8 after infection. Flow cytometry was used to detect the expression of activated caspase 3 in the cytoplasm of the virus-specific Tetramer + CD8 + T cells. C57BL/6 mice were infected with FV and at day 6 and 7 after infection they were treated with pan-caspase inhibitor Z-WAD-FMK. Flow cytometry was used to detect numbers of virus-specific Tetramer + CD8 + T cells in spleen (E) and bone marrow (F) of 8 days infected mice. Each dot represents an individual mouse and the mean numbers and SD are indicated. Differences were analyzed by Benjamini-Hochberg corrected one-way ANOVA (A,B) , one-way ANOVA with a Tukey post-test (C,D), or an unpaired t-test (E,F). Statistically significant differences between the groups are indicated (*p < 0.05, ***p < 0.0005).