Processing of blood samples influences PBMC viability and outcome of cell-mediated immune responses in antiretroviral therapy-naïve HIV-1-infected patients.

Autor: Bourguignon P; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: PATRICIA.BOURGUIGNON@GSK.COM., Clément F; Center for Vaccinology, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: Frederic.Clement@UGent.be., Renaud F; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: FREDERIC.X.RENAUD@GSK.COM., Le Bras V; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: VIVIEN.X.LE-BRAS@GSK.COM., Koutsoukos M; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: MARGUERITE.KOUTSOUKOS@GSK.COM., Burny W; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: WIVINE.BURNY@GSK.COM., Moris P; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: PHILIPPE.MORIS@GSK.COM., Lorin C; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: CLARISSE.M.LORIN@GSK.COM., Collard A; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: ALIX.COLLARD@GSK.COM., Leroux-Roels G; Center for Vaccinology, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: Geert.LerouxRoels@ugent.be., Roman F; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: FRANCOIS.P.ROMAN@GSK.COM., Janssens M; GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: MICHEL.JANSSENS@GSK.COM., Vandekerckhove L; 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.
Jazyk: angličtina
Zdroj: Journal of immunological methods [J Immunol Methods] 2014 Dec 01; Vol. 414, pp. 1-10. Date of Electronic Publication: 2014 Sep 16.
DOI: 10.1016/j.jim.2014.09.001
Abstrakt: 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.
(Copyright © 2014. Published by Elsevier B.V.)
Databáze: MEDLINE
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