Protein microarray analysis as a tool for monitoring cellular autoreactivity in type 1 diabetes patients and their relatives

Background: Autoreactive T cells have a crucial role in type 1 diabetes (T1D) pathogenesis.

Objectives: The aim of our study was to monitor the in vitro production of cytokines by peripheral blood mononuclear cells (PBMCs) after stimulation with diabetogenic autoantigens.

Subjects: Ten T1D patients (tested at the time of diagnosis and 6 and 12 months later), 10 first-degree relatives of the T1D patients, and 10 controls underwent the study.

Methods: PBMCs were stimulated with glutamic acid decarboxylase 65 (GAD65) amino acids (a.a.) 247–279, 509–528, and 524–543; proinsulin a.a. 9–23; and tyrosine phosphatase (islet antigen-2)/R2 a.a. 853–872. Interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL-13, interferon (IFN)-γ, tumor necrosis factor β, transforming growth factor β1, and granulocyte colony-stimulating factor (GCSF) were analyzed by protein microarray.

Results: Differences in cytokine(s) poststimulatory and mainly in basal production were observed in all groups. The most prominent findings were in controls, the higher basal levels of IL-2, IL-4, IL-5, IL-13, and GCSF were observed when compared with relatives (p < 0.05, for all). After stimulation in controls, there was a significant decrease in IL-2, IL-13, GCSF, and IFN-γ (p < 0.05, for all). The group of relatives was the most variable in poststimulatory production. A strong correlation between cytokines production was found but groups differed in this aspect.

Conclusion: By multiplex analysis, it may be possible, for example, to define the risk immunological response pattern among relatives or to monitor the immune response in patients on immune modulation therapy.