title: The effect of apoptotic cells on virus-specific immune responses detected using IFN-gamma ELISPOT.
abstract: Interferon (IFN)-gamma ELISPOT can be used to monitor the magnitude of virus-specific cellular immune responses in vaccine trials. Often, IFN-gamma ELISPOT is performed with cryopreserved peripheral blood mononuclear cells (PBMC). However, it has not been well defined yet to what extent diminished cell viability of PBMC following cryopreservation affects IFN-gamma responses in ELISPOT. Therefore, we assessed the influence of apoptotic cells on the number of spot-forming cells (SFC) in IFN-gamma ELISPOT using a gradient of UV-irradiated apoptotic PBMC and viral antigens derived from varicella zoster virus (VZV) and cytomegalovirus (CMV). No SFC were observed when UV-irradiated apoptotic cells were stimulated with VZV or CMV antigens. Moreover, presence of apoptotic cells among viable T cells hampered the detection of SFC following stimulation with VZV or CMV cell lysates, but not with CMVpp65 peptide pool. Statistical analysis showed that mainly late apoptotic cells, staining both Annexin V and 7-amino-actinomycin D (7-AAD), were associated with a decreased number of SFC. In conclusion, it is recommended to use highly viable thawed PBMC for the detection of virus-specific cellular immune responses by IFN-gamma ELISPOT, since the detection of CMV- and VZV-specific T cell responses stimulated by cell lysates was significantly impeded by the presence of apoptotic cells.
authors.affiliation: Laboratory of Experimental Hematology, Vaccine & Infectious Disease Institute (Vaxinfectio), University of Antwerp, Antwerp University Hospital, Wilrijkstraat 10, B-2650 Edegem, Belgium.
authors.forename: Kevin
authors.initials: K
authors.lastname: Lenders
authors.forename: Benson
authors.initials: B
authors.lastname: Ogunjimi
authors.forename: Philippe
authors.initials: P
authors.lastname: Beutels
authors.forename: Niel
authors.initials: N
authors.lastname: Hens
authors.forename: Pierre
authors.initials: P
authors.lastname: Van Damme
authors.forename: Zwi N
authors.initials: ZN
authors.lastname: Berneman
authors.forename: Viggo F I
authors.initials: VF
authors.lastname: Van Tendeloo
authors.forename: Evelien L J M
authors.initials: EL
authors.lastname: Smits
mesh_terms: D000328:Adult; D000956:Antigens, Viral / Q000276:immunology*; D017209:Apoptosis / Q000276:immunology* / Q000528:radiation effects; D015925:Cryopreservation; D003587:Cytomegalovirus / Q000276:immunology*; D005260:Female; D014645:Herpesvirus 3, Human / Q000276:immunology*; D006801:Humans; D007163:Immunosorbent Techniques; D007371:Interferon-gamma / Q000096:biosynthesis / Q000276:immunology*; D008297:Male; D013601:T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D014466:Ultraviolet Rays / Q000009:adverse effects
chemical_list: D000956:Antigens, Viral; D007371:Interferon-gamma