High background in ELISpot assays is associated with elevated levels of immune activation in HIV-1-seronegative individuals in Nairobi

Abstract

Introduction: Spontaneous 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. Methods: Couples 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. Results: Among 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). Conclusions: These 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.