Pepmix hcmva pp65

The pp65 peptide NLVPMVATV (HLA-A 0201 CMVpp65) at >90% purity was synthesized using automated solid phase peptide synthesis in the TVR (Beckman Research Institute of City of Hope). MP1 GIGFVFTL peptide (HLA-A 0201 influenza) was synthesized at the City of Hope DNA/RNA Peptide Synthesis Facility, (Duarte, CA). pepMix HCMVA (pp65) (pp65pepmix) was purchased from JPT peptide Technologies (GmbH, Berlin Germany). All peptides were used according to the manufacturer’s instructions.

Intracellular staining of pSAMHD1 was performed according to manufacturer’s instructions. Briefly, cells were fixed and permeabilized with methanol (Sigma Aldrich). After washing, cells were stained with anti-pSAMHD1-PE and then analyzed by flow cytometry. For intracellular staining of IFNγ, cells were treated for 4 h at 37°C with PepMix HCMVA pp65 (lower matrix protein 65) and IE1 (immediate-early-1 protein) (JPT Peptide Technologies, Berlin, Germany) to induce specific CD8 response against CMV or with Hsp70 peptide (Abcam, Cambridge, UK) to stimulate cytolytic activity of NK cells, in the presence of brefeldin A and co-stimulator CD28/CD49D (BD Biosciences). Cells were then stained with antibodies against CD3 and CD8 conjugated to PE-Cy7 and APC-H7, respectively, or with CD3, CD56 and CD16 conjugated to APC, FITC and PercP, respectively. After fixation and permeabilization with IntraPrep Permeabilization Reagent (Beckman Coulter), cells were stained with an antibody against IFNγ-PE and then analyzed by flow cytometry.
Flow cytometry data acquisition was performed using BD LSR Fortessa X-20 flow cytometer (BD Biosciences) with BD FACSDiva software. Data analyses were carried out with FlowJo v10 software (TreeStar, Ashland, OR).