PMID- 25224748 OWN - NLM STAT- MEDLINE DCOM- 20150312 LR - 20141208 IS - 1872-7905 (Electronic) IS - 0022-1759 (Linking) VI - 414 DP - 2014 Dec 1 TI - Processing of blood samples influences PBMC viability and outcome of cell-mediated immune responses in antiretroviral therapy-naive HIV-1-infected patients. PG - 1-10 LID - S0022-1759(14)00258-0 [pii] LID - 10.1016/j.jim.2014.09.001 [doi] AB - 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)-naive HIV-1-infected individuals. In this phase I study (NCT01610427), we collected blood samples from 22 ART-naive 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-naive patients. CI - Copyright (c) 2014. Published by Elsevier B.V. FAU - Bourguignon, Patricia AU - Bourguignon P AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: PATRICIA.BOURGUIGNON@GSK.COM. FAU - Clement, Frederic AU - Clement F AD - Center for Vaccinology, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: Frederic.Clement@UGent.be. FAU - Renaud, Frederic AU - Renaud F AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: FREDERIC.X.RENAUD@GSK.COM. FAU - Le Bras, Vivien AU - Le Bras V AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: VIVIEN.X.LE-BRAS@GSK.COM. FAU - Koutsoukos, Marguerite AU - Koutsoukos M AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: MARGUERITE.KOUTSOUKOS@GSK.COM. FAU - Burny, Wivine AU - Burny W AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: WIVINE.BURNY@GSK.COM. FAU - Moris, Philippe AU - Moris P AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: PHILIPPE.MORIS@GSK.COM. FAU - Lorin, Clarisse AU - Lorin C AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: CLARISSE.M.LORIN@GSK.COM. FAU - Collard, Alix AU - Collard A AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: ALIX.COLLARD@GSK.COM. FAU - Leroux-Roels, Geert AU - Leroux-Roels G AD - Center for Vaccinology, Ghent University and Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. Electronic address: Geert.LerouxRoels@ugent.be. FAU - Roman, Francois AU - Roman F AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: FRANCOIS.P.ROMAN@GSK.COM. FAU - Janssens, Michel AU - Janssens M AD - GlaxoSmithKline Vaccines, Rue de l'institut 89, Rixensart 1330, Belgium. Electronic address: MICHEL.JANSSENS@GSK.COM. FAU - Vandekerckhove, Linos AU - Vandekerckhove L AD - 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. LA - eng SI - ClinicalTrials.gov/NCT01610427 PT - Clinical Trial, Phase I PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20140916 PL - Netherlands TA - J Immunol Methods JT - Journal of immunological methods JID - 1305440 RN - 0 (AIDS Vaccines) RN - 0 (Anti-Retroviral Agents) SB - IM MH - AIDS Vaccines/*immunology/therapeutic use MH - Adolescent MH - Adult MH - Anti-Retroviral Agents/*therapeutic use MH - Antigen-Antibody Reactions MH - Blood Specimen Collection/*standards MH - CD4-Positive T-Lymphocytes/immunology MH - CD8-Positive T-Lymphocytes/*immunology MH - Cell Survival MH - Cryopreservation MH - Female MH - Flow Cytometry/methods MH - HIV Infections/immunology/*therapy MH - HIV Seropositivity/immunology MH - HIV-1/immunology MH - Hematologic Tests/methods MH - Humans MH - Immunity, Cellular MH - In Vitro Techniques MH - Male MH - Middle Aged MH - Staining and Labeling/methods MH - Time Factors MH - Viral Load MH - Young Adult OTO - NOTNLM OT - Antiretroviral therapy-naive OT - Blood processing OT - CD8(+) T-cell response OT - HIV-1 OT - Intracellular cytokine staining OT - Peripheral blood mononuclear cells EDAT- 2014/09/17 06:00 MHDA- 2015/03/13 06:00 CRDT- 2014/09/17 06:00 PHST- 2014/05/09 00:00 [received] PHST- 2014/09/01 00:00 [revised] PHST- 2014/09/05 00:00 [accepted] PHST- 2014/09/17 06:00 [entrez] PHST- 2014/09/17 06:00 [pubmed] PHST- 2015/03/13 06:00 [medline] AID - S0022-1759(14)00258-0 [pii] AID - 10.1016/j.jim.2014.09.001 [doi] PST - ppublish SO - J Immunol Methods. 2014 Dec 1;414:1-10. doi: 10.1016/j.jim.2014.09.001. Epub 2014 Sep 16.