title: Overnight resting of PBMC changes functional signatures of antigen specific T- cell responses: impact for immune monitoring within clinical trials.
abstract: Polyfunctional CD4 or CD8 T cells are proposed to represent a correlate of immune control for persistent viruses as well as for vaccine mediated protection against infection. A well-suited methodology to study complex functional phenotypes of antiviral T cells is the combined staining of intracellular cytokines and phenotypic marker expression using polychromatic flow cytometry. In this study we analyzed the effect of an overnight resting period at 37 °C on the quantity and functionality of HIV-1, EBV, CMV, HBV and HCV specific CD4 and CD8 T-cell responses in a cohort of 21 individuals. We quantified total antigen specific T cells by multimer staining and used 10-color intracellular cytokine staining (ICS) to determine IFNγ, TNFα, IL2 and MIP1β production. After an overnight resting significantly higher numbers of functionally active T cells were detectable by ICS for all tested antigen specificities, whereas the total number of antigen specific T cells determined by multimer staining remained unchanged. Overnight resting shifted the quality of T-cell responses towards polyfunctionality and increased antigen sensitivity of T cells. Our data suggest that the observed effect is mediated by T cells rather than by antigen presenting cells. We conclude that overnight resting of PBMC prior to ex vivo analysis of antiviral T-cell responses represents an efficient method to increase sensitivity of ICS-based methods and has a prominent impact on the functional phenotype of T cells.
authors.affiliation: Institute of Virology, Technische Universität München/Helmholtz Zentrum München, Munich, Germany ; Cooperation Group 'Immune Monitoring', Helmholtz Zentrum München, Munich, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Kutscher
authors.forename: Claudia J
authors.initials: CJ
authors.lastname: Dembek
authors.forename: Simone
authors.initials: S
authors.lastname: Deckert
authors.forename: Carolina
authors.initials: C
authors.lastname: Russo
authors.forename: Nina
authors.initials: N
authors.lastname: Körber
authors.forename: Johannes R
authors.initials: JR
authors.lastname: Bogner
authors.forename: Fabian
authors.initials: F
authors.lastname: Geisler
authors.forename: Andreas
authors.initials: A
authors.lastname: Umgelter
authors.forename: Michael
authors.initials: M
authors.lastname: Neuenhahn
authors.forename: Julia
authors.initials: J
authors.lastname: Albrecht
authors.forename: Antonio
authors.initials: A
authors.lastname: Cosma
authors.forename: Ulrike
authors.initials: U
authors.lastname: Protzer
authors.forename: Tanja
authors.initials: T
authors.lastname: Bauer
mesh_terms: D000328:Adult; D000368:Aged; D000938:Antigen-Presenting Cells / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology* / Q000821:virology; D002470:Cell Survival; D018925:Chemokines / Q000378:metabolism; D002986:Clinical Trials as Topic; D015925:Cryopreservation; D000939:Epitopes / Q000276:immunology*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology; D005260:Female; D006801:Humans; D018836:Inflammation Mediators / Q000378:metabolism; D042541:Intracellular Space / Q000378:metabolism; D018655:Lymphocyte Count; D008297:Male; D008875:Middle Aged; D015166:Monitoring, Immunologic; D013194:Staining and Labeling; D013997:Time Factors
chemical_list: D000941:Antigens; D018925:Chemokines; D000939:Epitopes; D018984:Epitopes, T-Lymphocyte; D018836:Inflammation Mediators