The Value of a Rapid Test of Human Regulatory T Cell Function Needs to be Revised

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 αCD3/28-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 αCD3/28-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 T(REG) function.