Computational protocol: Asymptomatic Primary Infection with Epstein-Barr Virus: Observations on Young Adult Cases

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Protocol publication

[…] Tetramers were used to identify and analyze the surface marker phenotype of EBV-specific CD8+ T cells. In addition to CD3 and CD8, cell surface CD45RA and CCR7 were used to identify the naive (CD45RA+ CCR7+), central memory (CD45RA− CCR7+), effector memory (CD45RA− CCR7−), and revertant memory (CD45RA+ CCR7−) T cell subsets. Staining for cell surface HLA-DR and CD38 and for intracellular Bcl2 was used to identify activated (HLA− DR+ CD38+ Bcl2low) T cells. Tetramer staining identified CD8+ T cells specific for the following epitopes derived from individual EBV lytic or latent cycle proteins (see below): YVLDHLIVV (lytic, BRLF1), GLCTLVAML (lytic, BMLF1), LLIEGIFFI (lytic, BaRF1), or CLGGLLTMV (latent, LMP2), which are restricted through HLA-A*0201, or RPRATWIQEL (lytic, BaRF1), TPSVSSSISSL (lytic, BFRF3), or RPPIFIRRL (latent, EBNA3A) (), which are restricted through HLA-B*0702. Epitopes are designated by their initial three residues (underlined).Tetramer and antibody staining of cryopreserved PBMCs was performed as follows. Cells were thawed and stained with LIVE/DEAD fixable Aqua Dead cell stain (Molecular Probes, Thermo Fisher Scientific) for 15 min at room temperature, washed, and stained with tetramer-phycoerythrin (PE) for 15 min at 37°C. Following two further washes, surface staining with the following was performed in Brilliant stain buffer (BD Biosciences) for 30 min at 4°C: anti-CD14-Pacific Green (clone SJ25-C1) and anti-CD19-Pacific Green (clone TüK4) (both from Molecular Probes, Thermo Fisher Scientific); anti-CD3-BV786 (clone SK7), anti-CD8-allophycocyanin (APC)-H7 (clone SK1), anti-CD45RA-BV605 (clone HI100), and anti-CD45RO-BV711 (clone UCHL1) (all from BD Biosciences); anti-CD38-peridinin chlorophyll protein (PerCP)-Cy5.5 (clone HIT2) and anti-HLA-DR-APC (clone L243) (both from BioLegend); and anti-CCR7-fluorescein isothiocyanate (clone 150503; R&D Systems). Following fixation and permeabilization using a Cytofix/Cytoperm kit (BD Biosciences), intracellular staining with anti-Ki67-BV421 (clone B56) and anti-Bcl-2-PE-CF594 (clone 563601) (both from BD Biosciences) was performed in Brilliant stain and Perm/Wash buffer (both from BD Biosciences) for 30 min at 4°C.Flow cytometry data were acquired with an LSR Fortessa X20 analyzer (BD Biosciences) with standard filter sets. Data were analyzed using Kaluza (v1.3) software (Beckman Coulter), and figures were created using FlowJo software (TreeStar). CD3+ T cells were gated on within the single, viable, CD14− CD19− lymphocyte population, before CD8+ T cells and CD8+ tetramer-positive cells were selected for further analysis of surface and/or intracellular marker expression. […]

Pipeline specifications

Software tools Kaluza, FlowJo
Application Flow cytometry
Organisms Homo sapiens, Human poliovirus 1 Mahoney