CD4(+)CD25(+) regulatory T cells (Tregs) are considered to play a key role as 
suppressors of immune mediated reactions. The analysis of Treg function in 
patients with autoimmune, allergic or oncogenic diseases has emerged over the 
past years. In the present study we describe a CFSE based protocol to measure 
Treg mediated suppression of CD4(+) T cells. Measuring Treg suppressive capacity 
towards proliferation of anti-CD3 Ab stimulated CD4(+)CD25(-) T cells in 
coculture experiments by means of a CFSE based and a classical [(3)H]thymidine 
incorporation assay gave similar results, provided that CD4(+)CD25(+) T cells 
were anergic. However, when CD4(+)CD25(+) T cells proliferated upon mitogenic 
stimulation, data obtained by the CFSE assay allowed the detection of a 
significant Treg suppression whereas this was clearly underestimated using the 
[(3)H]thymidine assay. In addition, an indirect CFSE based method was developed 
to analyze antigen specific responses of total CD4(+) T cells and Treg depleted 
CD4(+) T cells (i.e. CD4(+)CD25(-) T cells). Our results indicate that, in 
healthy individuals, CD4(+) T cell responses against the multiple sclerosis (MS) 
auto-antigens, myelin basic protein (MBP) and myelin oligodendrocyte 
glycoprotein (MOG), were increased in Treg depleted CD4(+) T cells as compared 
to total CD4(+) T cells. Our initial data suggest that Tregs in MS patients show 
an impaired suppression of myelin reactive T cells when compared to healthy 
controls. Moreover, this experimental setup permits the measurement of cytokine 
production of the antigen proliferated CFSE(low) T cells by additional flow 
cytometric analyses. In conclusion, the described CFSE based Treg suppression 
assay is a valuable tool to study suppressor T cells in (auto)immune disorders.