Correlation of different cellular assays to analyze T cell-related cytokine profiles in vitamin D-3-supplemented patients with multiple sclerosis

Different laboratory approaches have been exploited to analyze an effect of vitamin D-3 supplements on T cell cytokine profiles in multiple sclerosis, with poorly reproducible results. We assessed the correlation between intra-cellular flowcytometry analysis of CD4 T cell-enriched CD3(+)CD8(-) lymphocytes after PMA/ionomycin stimulation directly ex-vivo or after 72 h pre-stimulation with anti-CD3, and cytokine levels excreted in culture supernatants. Pre-stimulation with anti-CD3 resulted in higher proportions of cells positive for IFN-gamma, IL-17A, IL-4, IL-10 and GM-CSF (all P <0.001), but not TNF-alpha. Positive correlation between approaches was highly variable, but most eminent for IFN-gamma and IL-4 (R = 0.608-0.612 and R = 0.677-0.777, resp., all P <0.001). No effect of 16-weeks vitamin D-3 supplements on any outcome was found except for a decreased TNF-alpha concentration in culture supernatants. Choice of immune-assay is, apparently, a relevant confounder for the reproducibility of individual studies.