Quantitative, Absolute Count-Based T-Cell Analysis of CD69 Upregulation as a New Methodology for In Vitro Diagnosis...

T cells play a major role in delayed-type hypersensitivity reactions. Their reactivity can be assessed by measuring the upregulation of the activation marker CD69, followed by assessment of proliferation and cytokine production. The aim of our study was to develop a novel, whole blood-based, quantitative, absolute count activation index (AI) for analysis of CD69 upregulation in various subsets of T cells in nickel-hypersensitive patients and compare it with previously reported approaches.

The study population comprised 10 patients with nickel allergy and 9 healthy controls. CD69 expression of CD3+, CD3+CD4+, and CD3+CD8+ T cells in heparinized blood was determined with flow cytometry after incubation with nickel sulfate for 48 hours. The absolute count of CD69+ cells was determined using microbeads. Production of the cytokines IL-2, IL-5, IL-13, and IFN-γ was determined after stimulation of peripheral blood mononuclear cells with nickel sulfate for 48 hours.

We showed absolute AI to be the most sensitive approach. The index was calculated as the ratio of the absolute count of nickel-stimulated CD69-positive T cells to the absolute count of CD69-positive T cells in nonstimulated blood. This novel quantitative approach was more discriminative than previously reported approaches in which the T-cell CD69 percentage AI and cytokine production are measured.

Our results demonstrated that measuring the absolute CD69 AI is a novel and accurate approach for quantification of antigen-specific T cells in the blood of patients with hypersensitivity reactions to nickel. This approach may be useful for better in vitro assessment of patients with delayed-type hypersensitivity reactions.