Objective Autoreactive CD4 T cells specific for nuclear peptide antigens play an important role in tolerance breakdown during the course of systemic lupus erythematosus (SLE). However, reliable detection of these cells is limited due to their low frequency in peripheral blood. The authors assess autoreactive CD4 T cells in a representative SLE collective (n=38) by flow cytometry and study the influence of regulatory T cells (Treg) on their antigenic challenge. Methods CD4 T-cell responses were determined according to intracellular CD154 expression induced after 6-h short-term in-vitro stimulation with the SLE-associated autoantigen SmD1(83-119). To clarify the influence of Treg on the activation of autoreactive CD4 T cells, CD25 Treg were depleted by magnetic activated cell sorting before antigen-specific stimulation in selected experiments. Results In the presence of Treg, autoreactive CD4 T-cell responses to SmD1(83-119) were hardly observable. However, Treg removal significantly increased the frequency of detectable SmD1(83-119)-specific CD4 T cells in SLE patients but not in healthy individuals. Consequently, by depleting Treg the percentage of SmD1(83-119)-reactive SLE patients increased from 18.2% to 63.6%. This unmasked autoreactivity of CD4 T cells correlated with the disease activity as determined by the SLE disease activity index (p=0.005*, r=0.779). Conclusions These data highlight the pivotal role of the balance between autoreactive CD4 T cells and CD25 Treg in the dynamic course of human SLE. Analysing CD154 expression in combination with a depletion of CD25 Treg, as shown here, may be of further use in approaching autoantigen-specific CD4 T cells in SLE and other autoimmune diseases.