Abstract
IntroductionSpontaneous interferon‐γ (IFNγ) released detected by enzyme‐linked immunospot (ELISpot) assays may be a biological phenomenon. Markers of immune activation levels were assessed as correlates of high background among individuals in Kenya.MethodsCouples concordantly seronegative for HIV‐1 were enrolled. IFN‐γ ELISpot assays were conducted and negative control wells were categorized as having either high or low background (≥50 and <50 SFU/106 peripheral blood mononuclear cells [PBMC], respectively). PBMC were stained for CD4, CD8, and immune activation markers (CD38 and HLA‐DR) and analyzed using flow cytometry. Proportions of activated T‐cells were compared between those with low and high background by Mann–Whitney U test. Correlates of background SFU and immune activation were assessed using regression models.ResultsAmong 58 individuals, 14 (24%) had high background. Frequencies of CD4+CD38+HLA‐DR+ and CD8+CD38+HLA‐DR+ cells were higher in individuals with high background compared to those with low background (P = 0.02). Higher background SFU was associated with history of sexually transmitted infections (P = 0.03), and illness in the past 3 months (P = 0.005), in addition to increased levels of activated CD4+ and CD8+ cells (P range = 0.008–0.03). Female gender and male circumcision decreased levels of CD4+ and CD8+ immune activation (P range = 0.002–0.03). Additionally, higher background SFU and activated CD4+ and CD8+ cells were individually associated with positive ELISpot responses to HIV‐1 peptide pools (P range = 0.01–0.03).ConclusionsThese findings suggest that increased basal immune responses may be a biological mechanism contributing to higher background ELISpot SFU. Systematic exclusion of data from individuals with increased background in IFN‐γ release assays may bias results in population‐based studies.
Highlights
The IFN-γ enzyme-linked immunospot (ELISpot) assay is widely used to quantify viral, tumor, allo- and auto-antigenic cellular immune responses in clinical and vaccine trials
In this study we found of high spontaneous IFN-γ release in ELISpot assays conducted on PBMC from ~25% of individuals and these individuals were more likely to have elevated levels of immune activation in both CD4+ and CD8+ T cells
Naïve T cells differentiate into memory T cells after being activated {Hazenberg, 2000 #1530}, and the activated effector cells are responsible for producing cytokines in vivo, most typically only during the acute immune response before the cells die or differentiate into resting memory cells
Summary
The IFN-γ enzyme-linked immunospot (ELISpot) assay is widely used to quantify viral, tumor, allo- and auto-antigenic cellular immune responses in clinical and vaccine trials. This assay is quick, cost-effective and one of the most sensitive methods to detect antigen-specific T cell responses {Schmittel, 1997 #1657;Schmittel, 2001 #235}. It is necessary to minimize background SFU in negative control wells and to maximize antigen-induced spots in experimental wells to optimize the signal-to-noise ratio. Methodological issues are commonly cited as possible reasons for spot production in negative control wells {Streeck, 2009 #1677}. The general consensus is that high SFU in unstimulated wells are due to assay-specific problems and should be excluded from analyses, ELISpot assays are not standardized and exclusion criteria based on high background varies depending on protocols {Streeck,
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