Generation of Virus-Specific CD8+ T Cell Lines

EBV, CMV, or influenza A (IAV)-specific CD8⁺ T cell lines were generated from chronically-infected individuals following in vitro expansion of PBMC stimulated with gamma-irradiated peptide-pulsed autologous cells (1 μM peptide, 3,000 Rads) at a 2:1 ratio in RF10 [composed of RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 2 mM MEM non-essential amino acid solution (Life Technologies), 100 mM HEPES (Life Techologies), 2 mM L-glutamine (Life Technologies), penicillin/streptomycin (Life Technologies), 50 mM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, MO), 10% heat-inactivated FCS (Sigma-Aldrich)] supplemented with 20 U/mL IL-2 (PeproTech, Rocky Hill, NJ) for 13 days at 37°C, 5% CO₂ as previously described (4 (link), 11 (link)). Peptides for CMV: HLA-A^*02:01-restricted pp65-derived NLVPMVATV (A2_NLV) epitope, EBV: HLA-B^*07:02-restricted EBNA-3A-derived RPPIFIRRL (B7_RPP) epitope and IAV: HLA-A^*02:01-restricted matrix protein-derived GILGFVFTL (A2_GIL) epitope. Virus-specific CD8⁺ T cell clones from chronically-infected individuals were generated following single-cell sorting based on tetramer staining using the HLA-B^*57:01-restricted TSTLQEQIGW (B57_TW10) epitope derived from HIV-1 Gag protein for A16 and 457 (20 (link)) or EBV: B7_RPP epitope for HD9G6 (21 (link)), as previously described (2 (link), 22 (link), 23 (link)).

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Rowntree L.C., van den Heuvel H., Sun J., D'Orsogna L.J., Nguyen T.H., Claas F.H., Rossjohn J., Kotsimbos T.C., Purcell A.W, & Mifsud N.A. (2020). Preferential HLA-B27 Allorecognition Displayed by Multiple Cross-Reactive Antiviral CD8+ T Cell Receptors. Frontiers in Immunology, 11, 248.

Publication 2020

RPMI 1640 MEM non-essential amino acid solution L-glutamine penicillin/streptomycin 2-mercaptoethanol heat-inactivated FCS IL-2

Cd8 t cell Cell lines Cells Clones Epitope Essential amino acid Gag protein Gilgfvftl Glutamine Hepes Hiv 1 Hla b 07 Hla b 57 Il 20 Influenza Irradiated gamma Mercaptoethanol Penicillin Peptides Pp65 Protein Rads Streptomycin Tetramer Virus

Corresponding Organization : Australian Regenerative Medicine Institute

Other organizations : Leiden University Medical Center, Fiona Stanley Hospital, University of Western Australia, Peter Doherty Institute, University of Melbourne, Australian Research Council, Cardiff University

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Variable analysis

independent variables

Stimulation of PBMC with gamma-irradiated peptide-pulsed autologous cells at a 2:1 ratio

dependent variables

Generation of EBV-, CMV-, or influenza A (IAV)-specific CD8+ T cell lines

control variables

Composition of RF10 media (RPMI 1640, non-essential amino acids, HEPES, L-glutamine, penicillin/streptomycin, 2-mercaptoethanol, 10% heat-inactivated FCS)
Incubation conditions (37°C, 5% CO2)
Concentration of IL-2 (20 U/mL)

positive controls

Tetramer staining using HLA-B*57:01-restricted TSTLQEQIGW (B57_TW10) epitope derived from HIV-1 Gag protein for A16 and 457 cell clones
Tetramer staining using EBV: B7_RPP epitope for HD9G6 cell clone

negative controls

Not explicitly mentioned

Annotations

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