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Quantitative, absolute count-based T cell analysis of CD69 upregulation as a new methodology for in vitro diagnosis of delayed-type nickel hypersensitivity.

BACKGROUND
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 proliferation and cytokine production. The aim of our study was to develop a novel, whole blood-based, quantitative, absolute count activation index (AI) analysis of CD69 upregulation on different subsets of T cells in nickel hypersensitive patients and compare it with the previously reported approaches.


METHODS
Ten patients with nickel allergy and nine healthy controls were included. 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 h. The absolute cell count of CD69+ cells was determined with microbeads. The production of the cytokines IL-2, IL-5, IL-13, and IFN-γ was determined after nickel sulfate stimulation of PBMNCs for 48 h.


RESULTS
We showed that the most sensitive methodology is the absolute AI, which was calculated as the ratio between the absolute count of CD69-positive T cells stimulated with nickel and the absolute count of CD69-positive T cells in non-stimulated blood. This novel quantitative approach was more discriminative than the previously reported approaches in which T cell CD69 percentage AI and cytokine production are measured.


CONCLUSIONS
Our results demonstrated that measuring the absolute CD69 AI is an accurate new approach to quantify 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.