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

Cells were lysed by sonication (6 10-s pulses on ice) in sample buffer (50 mM NH4HCO3; 0.1% Rapigest). Protein isolation, digestion, amino acid analysis, and liquid chromatographytandem mass spectrometry (LC-MS/MS) analysis were performed by following previously described procedures (25) . Progenesis QI for proteomics (v. 2.0.5387.52102; Nonlinear Dynamics, Newcastle upon Tyne, UK) and Proteome Discoverer (v. 1.4; Thermo Scientific, Bremen, Germany) software packages were used for protein quantification and identification, respectively. For protein identification, LC-MS/MS runs were searched against the UniProt-SwissProt database (Release 2014_10; v. 2.5; 546,790 sequences) with taxonomy restriction to mus musculus. Further search parameters matched previously reported ones (25) . Non-unique peptides associated to more than one protein accession were not used for quantification. Differentially expressed proteins (p < 0.05) were functionally annotated using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, ver. 6.8) (26, 27) .