title: Establishment of an alternative intracellular cytokine staining assay for HIV/AIDS clinical studies.
abstract: To evaluate immune responses in large-scale clinical studies, it is crucial to use assays that are not only sensitive and specific, but also standardized and easily transferable. To this end, an intracellular cytokine staining (ICS) assay for quantifying HIV-specific CD8+ T cells has been established and its performance characteristics determined. PBMCs were stimulated using recombinant vaccinia expressing HIV-Env, -Gag, -Pol and -Nef proteins, or a protein from Escherichia coli (Eco); CD8+ T cell responses were assessed by intracellular IFN-gamma staining and flow cytometric (FC) analysis. In 15 HIV seronegative and 11 HIV seropositive individuals, a minimal background IFN-gamma staining was found after Eco stimulation with a median [inter-quartile range (IQR)] of 0.005% (0.000%, 0.030%) irrespective of HIV infection status. Recent smallpox vaccination was associated with a small but significant increase in background staining [0.02% (0.02%, 0.04%) versus 0.0% (0.0%, 0.01%) (p=0.039)] in HIV seronegative individuals. This assay detected moderate (>0.10%) HIV-specific responses in 64% (7/11) of HIV seropositive individuals. The results suggest that this ICS assay format is sensitive and specific, and is amenable to standardization for screening for HIV-specific CD8+ T cells in clinical trials.
authors.affiliation: Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York, NY 14642, USA.
authors.forename: Huiyuan
authors.initials: H
authors.lastname: Chen
authors.forename: Richard
authors.initials: R
authors.lastname: Reichman
authors.forename: Michael
authors.initials: M
authors.lastname: Keefer
authors.forename: Michael P
authors.initials: MP
authors.lastname: McDermott
authors.forename: Xia
authors.initials: X
authors.lastname: Jin
mesh_terms: D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism / Q000821:virology; D002986:Clinical Trials as Topic; D016207:Cytokines / Q000032:analysis; D005434:Flow Cytometry; D015658:HIV Infections / Q000378:metabolism*; D006679:HIV Seropositivity; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis*; D013194:Staining and Labeling / Q000379:methods*; D014616:Vaccinia virus / Q000235:genetics
chemical_list: D016207:Cytokines; D007371:Interferon-gamma