BackgroundPools of overlapping synthetic peptides are routinely used for ex vivo monitoring of antigen-specific T-cell responses. However, it is rather unlikely that these peptides match those resulting from naturally processed antigens. T-activated proteins have been described as immunogenic and more natural stimulants, since they have to pass through antigen processing and comprise activation of all clinically relevant effector cell populations.MethodsWe performed comparative analysis of numbers and cytokine expression pattern of CD4 and CD8 T cells after stimulation with recombinant, urea-formulated T-activated EBV-BZLF1, -EBNA3A, and HCMV-IE1, and -pp65 proteins or corresponding overlapping peptide pools. Freshly isolated and cryopreserved PBMC of 30 EBV- and 19 HCMV-seropositive and seven EBV- and HCMV-seronegative subjects were stimulated ex vivo and analysed for IFN-gamma, TNF and IL-2 production by flow cytometry-based intracellular cytokine staining.ResultsT-activated proteins showed a high specificity of 100% (EBV-BZLF1, HCMV-IE1, and -pp65) and 86% (EBV-EBNA3A), and a high T-cell stimulatory capacity of 73-95% and 67-95% using freshly isolated and cryopreserved PBMC, respectively. The overall CD4 T-cell response rates in both cohorts were comparable after stimulation with either T-activated protein or peptide pools with the exception of lower numbers of CD8 T cells detected after stimulation with T-activated EBV-EBNA3A- (p=0.038) and HCMV-pp65- (p=0.0006). Overall, the number of detectable antigen-specific T cells varied strongly between individuals. Cytokine expression patterns in response to T-activated protein and peptide pool-based stimulation were similar for CD4, but significantly different for CD8 T-cell responses.ConclusionEBV and HCMV-derived T-activated proteins represent innovative, highly specific recall antigens suitable for use in immunological endpoint assays to evaluate success or failure in immunotherapy clinical trials (e.g. to assess the risk of EBV and/or HCMV reactivation after allogenic hematopoietic stem cell transplantation). T-activated proteins could be of particular importance, if an impaired antigen processing (e.g. in a post-transplant setting) must be taken into account.