title: Processing of blood samples influences PBMC viability and outcome of cell-mediated immune responses in antiretroviral therapy-naïve HIV-1-infected patients.
abstract: Intracellular cytokine staining (ICS) assay is increasingly used in vaccine clinical trials to measure antigen-specific T-cell mediated immune (CMI) responses in cryopreserved peripheral blood mononuclear cells (PBMCs) and whole blood. However, recent observations indicate that several parameters involved in blood processing can impact PBMC viability and CMI responses, especially in antiretroviral therapy (ART)-naïve HIV-1-infected individuals. In this phase I study (NCT01610427), we collected blood samples from 22 ART-naïve HIV-1-infected adults. PBMCs were isolated and processed for ICS assay. The individual and combined effects of the following parameters were investigated: time between blood collection and PBMC processing (time-to-process: 2, 7 or 24 h); time between PBMC thawing and initiation of in vitro stimulation with HIV-1 antigens (resting-time: 0, 2, 6 and 18 h); and duration of antigen-stimulation in PBMC cultures (stimulation-time: 6h or overnight). The cell recovery after thawing, cell viability after ICS and magnitude of HIV-specific CD8(+) T-cell responses were considered to determine the optimal combination of process conditions. The impact of time-to-process (2 or 4 h) on HIV-specific CD8(+) T-cell responses was also assessed in a whole blood ICS assay. A higher quality of cells in terms of recovery and viability (up to 81% and >80% respectively) was obtained with shorter time-to-process (less than 7 h) and resting-time (less than 2 h) intervals. Longer (overnight) rather than shorter (6 h) stimulation-time intervals increased the frequency of CD8(+)-specific T-cell responses using ICS in PBMCs without change of the functionality. The CD8(+) specific T-cell responses detected using fresh whole blood showed a good correlation with the responses detected using frozen PBMCs. Our results support the need of standardized procedures for the evaluation of CMI responses, especially in HIV-1-infected, ART-naïve patients.
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: PATRICIA.BOURGUIGNON@GSK.COM.
authors.forename: Patricia
authors.initials: P
authors.lastname: Bourguignon
authors.affiliation: Center for Vaccinology, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: Frederic.Clement@UGent.be.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Clément
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: FREDERIC.X.RENAUD@GSK.COM.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Renaud
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: VIVIEN.X.LE-BRAS@GSK.COM.
authors.forename: Vivien
authors.initials: V
authors.lastname: Le Bras
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: MARGUERITE.KOUTSOUKOS@GSK.COM.
authors.forename: Marguerite
authors.initials: M
authors.lastname: Koutsoukos
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: WIVINE.BURNY@GSK.COM.
authors.forename: Wivine
authors.initials: W
authors.lastname: Burny
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: PHILIPPE.MORIS@GSK.COM.
authors.forename: Philippe
authors.initials: P
authors.lastname: Moris
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: CLARISSE.M.LORIN@GSK.COM.
authors.forename: Clarisse
authors.initials: C
authors.lastname: Lorin
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: ALIX.COLLARD@GSK.COM.
authors.forename: Alix
authors.initials: A
authors.lastname: Collard
authors.affiliation: Center for Vaccinology, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: Geert.LerouxRoels@ugent.be.
authors.forename: Geert
authors.initials: G
authors.lastname: Leroux-Roels
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: FRANCOIS.P.ROMAN@GSK.COM.
authors.forename: François
authors.initials: F
authors.lastname: Roman
authors.affiliation: GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: MICHEL.JANSSENS@GSK.COM.
authors.forename: Michel
authors.initials: M
authors.lastname: Janssens
authors.affiliation: ARC (AIDS Reference Center), Department of Internal Medicine, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: Linos.Vandekerckhove@UGent.be.
authors.forename: Linos
authors.initials: L
authors.lastname: Vandekerckhove
mesh_terms: D016915:AIDS Vaccines / Q000276:immunology* / Q000627:therapeutic use; D000293:Adolescent; D000328:Adult; D044966:Anti-Retroviral Agents / Q000627:therapeutic use*; D000937:Antigen-Antibody Reactions; D001800:Blood Specimen Collection / Q000592:standards*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002470:Cell Survival; D015925:Cryopreservation; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015658:HIV Infections / Q000276:immunology / Q000628:therapy*; D006679:HIV Seropositivity / Q000276:immunology; D015497:HIV-1 / Q000276:immunology; D006403:Hematologic Tests / Q000379:methods; D006801:Humans; D007111:Immunity, Cellular; D066298:In Vitro Techniques; D008297:Male; D008875:Middle Aged; D013194:Staining and Labeling / Q000379:methods; D013997:Time Factors; D019562:Viral Load; D055815:Young Adult
chemical_list: D016915:AIDS Vaccines; D044966:Anti-Retroviral Agents
keywords: Antiretroviral therapy-naïve; Blood processing; CD8(+) T-cell response; HIV-1; Intracellular cytokine staining; Peripheral blood mononuclear cells