Cd4 pe

Cryopreserved PBMC samples were labelled using CD4-PE, CD8-APC-Cy7, CD45RA-APC (all Becton Dickinson) and CD62L-ECD (Beckman Coulter) on ice, following overnight rest to restore CD62L expression (Figure A in S1 File). CD4+ and CD8+ gated lymphocytes were sorted (Influx; Becton Dickinson) on the basis of CD45RA and CD62L expression [20 (link)]. Sort purities were regularly >98%. We confirmed naïve and memory populations by stimulating naïve or memory CD8+ T cells with cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza derived peptides (CEF peptides; Mabtech) and performing an IFN-γ ELISpot assay as described above. CEF peptides responses were only detected in stimulated memory phenotype T cells, but not naïve populations (Figure B in S1 File).

CD4-PE, CD25-FITC, CD127-APC, FITC-lineage-cocktail (CD3, CD14, CD16, CD19, CD20, CD56), HLA-DR-PerCP, CD123-PE, CD11c-APC, CD3-PE-Cy5, CD4-FITC, CD8-PE, mouse isotype controls, and lysis solution were purchased from Becton Dickinson (Franklin Lakes, NJ, USA).

Mononuclear cells were isolated from mice spleens by Ficoll-Hypaque density gradient centrifugation and the count and viability of the lymphocytes were determined using a Guava EasyCyte Flow Cytometer and Viacount Kit (both Merck, USA). For this, 2 × 10⁶ cells/ml were placed in RPMI containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml of streptomycin, and with/without 10 μg/ml of recombinant gB. After incubation for 20 h at 37°C, the cells were used for phenotypic analysis of lymphocytes. Then, the cells were washed three times with PBS and viability staining was performed with FVS780 dye (Becton Dickinson, USA) according to the manufacturer’s protocol. Subsequently, the cells were washed and placed in vials containing PBS supplemented with 2% FBS and 0.09% sodium azide (staining buffer). Cell surface marker staining was then performed on the combination with the following conjugated antibodies: CD3-FITC, CD4-PE, CD8-BV650, and CD69-BV421 (all from Becton Dickinson). After incubation in the dark for 30 min on ice, the cells were washed three times with staining buffer. Subsequently, at least 10.000 CD3⁺ events were analyzed by flow cytometric acquisition using the Guava EasyCyte 16ST Flow Cytometer (Merck). Data were analyzed using InCyte software (Merck).

Total viable CD3+ CD4+ T cells, and CD3+ CD8+ T cells (replication samples only) were positively selected for from the PBMC population using flow cytometry (DAPI: Cat ID D9542, Sigma Aldrich, St Louis, MO, USA; CD8-FITC: Cat ID 55536, CD3-APC Cat ID 340440, CD4-PE Cat ID 347327, Beckton Dickinson, San Jose, CA, USA). T cell purities were typically above 98% for CD3+CD4+ T cells and above 95% for CD3+CD8+ following cell sorting. DNA was extracted using the Flexigene DNA extraction kit (Qiagen). Where CD4+ T cell numbers were sufficient, an aliquot of cells was used for RNA extraction using a standard Trizol extraction adapted from Chomczynski, et al.46 (link), adjusting reagent volume to suit cell numbers. All RNA samples were DNase treated using the Ambion DNA Free kit (Life Technologies, Austin, TX USA).