CD4(+)CD25(+)FoxP3(+) human regulatory T-CELLS (T-REG) are promising candidates for reshaping undesired immunity/inflammation by adoptive cell transfer, yet their application is strongly dependent on robust assays testing their functionality. Several studies along with first clinical data indicate T-REG to be auspicious to use for future cell therapies, e.g., to induce tolerance after solid organ transplantation. To this end, T-REG suppressive capacity has to be thoroughly evaluated prior to any therapeutic application. A 7 h-protocol for the assessment of T-REG function by suppression of the early activation markers CD154 and CD69 on CD4(+)CD25(-) responder T-CELLS (T-RESP) upon polyclonal stimulation via alpha CD3/28-coated activating microbeads has previously been published. Even though this assay has since been applied by various groups, the protocol comes with a critical pitfall, which is yet not corrected by the journal of its original publication. Our results demonstrate that the observed decrease in activation marker frequency on T-RESP is due to competition for alpha CD3/28-coated microbeads as opposed to a T-REG-attributable effect and therefore the protocol cannot further be used as a diagnostic test to assess suppressive TREG function
