An optimized IFN-gamma. ELISpot assay for the sensitive and standardized monitoring of CMV protein-reactive effector cells of cell-mediated immunity

作者：Barabas Sascha; Spindler Theresa; Kiener Richard; Tonar Charlotte; Lugner Tamara; Batzilla Julia; Bendfeldt Hanna; Rascle Anne; A**ach Benedikt; Wagner Ralf; Deml Ludwig^*

来源：BMC IMMUNOLOGY, 2017, 18(1): 14.

DOI：10.1186/s12865-017-0195-y

• Cytomegalovirus
• CMV
• IE-1
• pp65
• Cell-mediated immunity
• ELISpot
• CD4(+)
• CD8(+)
• T helper (Th)
• Cytotoxic T lymphocyte (CTL)
• Natural killer (NK)
• NKT-like

Background: In healthy individuals, Cytomegalovirus (CMV) infection is efficiently controlled by CMV-specific cell-mediated immunity (CMI). Functional impairment of CMI in immunocompromized individuals however can lead to uncontrolled CMV replication and severe clinical complications. Close monitoring of CMV-specific CMI is therefore clinically relevant and might allow a reliable prognosis of CMV disease as well as assist personalized therapeutic decisions.
Methods: Objective of this work was the optimization and technical validation of an IFN-gamma ELISpot assay for a standardized, sensitive and reliable quantification of CMV-reactive effector cells. T-activated (R) immunodominant CMV IE-1 and pp65 proteins were used as stimulants. All basic assay parameters and reagents were tested and optimized to establish a user-friendly protocol and maximize the signal-to-noise ratio of the ELISpot assay.
Results: Optimized and standardized ELISpot revealed low intra-assay, inter-assay and inter-operator variability (coefficient of variation CV below 22%) and CV inter-site was lower than 40%. Good assay linearity was obtained between 6 x 10(4) and 2 x 10(5) PBMC per well upon stimulation with T-activated (R) IE-1 (R-2 = 0.97) and pp65 (R-2 = 0.99) antigens. Remarkably, stimulation of peripheral blood mononuclear cells (PBMC) with T-activated (R) IE-1 and pp65 proteins resulted in the activation of a broad range of CMV-reactive effector cells, including CD3(+) CD4(+) (Th), CD3(+) CD8(+) (CTL), CD3-CD56(+) (NK) and CD3(+) CD56(+) (NKT-like) cells. Accordingly, the optimized IFN-gamma ELISpot assay revealed very high sensitivity (97%) in a cohort of 45 healthy donors, of which 32 were CMV IgG-seropositive.
Conclusion: The combined use of T-activated (R) IE-1 and pp65 proteins for the stimulation of PBMC with the optimized IFN-gamma ELISpot assay represents a highly standardized, valuable tool to monitor the functionality of CMV-specific CMI with great sensitivity and reliability.

• 出版日期2017-3-7
• 单位河北医科大学