T cell immunity is traditionally assessed through functional recall assays, which detect the consequences of the T cells' antigen encounter, or via fluorescently labeled multimers that selectively bind peptide-specific T cell receptors. Using either approach, if the wrong antigen or peptide of a complex antigenic system, such as a virus, is used for immune monitoring, either false negative data will be obtained, or the magnitude of the antigen-specific T cell compartment will go largely underestimated. In this work, we show how selection of the “right” antigen or antigenic peptides is critical for successful T cell immune monitoring against human cytomegalovirus (HCMV). Specifically, we demonstrate that individual HCMV antigens, along with previously reported epitopes, frequently failed to detect CD8+ T cell immunity in test subjects. Through systematic assessment of T cell reactivity against individual nonamer peptides derived from the HCMVpp65 protein, our data clearly establish that (i) systematic testing against all potential epitopes encoded by the genome of the antigen of interest is required to reliably detect CD8+ T cell immunity, and (ii) genome-wide, large scale systematic testing of peptides has become feasible through high-throughput ELISPOT-based “brute force” epitope mapping.

Monitoring antigen-specific T cell immunity relies on functional tests that require T cells and antigen presenting cells to be uncompromised. Drawing of blood, its storage and shipment from the clinical site to the test laboratory, and the subsequent isolation, cryopreservation and thawing of peripheral blood mononuclear cells (PBMCs) before the actual test is performed can introduce numerous variables that may jeopardize the results. Therefore, no T cell test is valid without assessing the functional fitness of the PBMC being utilized. This can only be accomplished through the inclusion of positive controls that actually evaluate the performance of the antigen-specific T cell and antigen presenting cell (APC) compartments. For Caucasians, CEF peptides have been commonly used to this extent. Moreover, CEF peptides only measure CD8 cell functionality. We introduce here universal CD8+ T cell positive controls without any racial bias, as well as positive controls for the CD4+ T cell and APC compartments. In summary, we offer new tools and strategies for the assessment of PBMC functional fitness required for reliable T cell immune monitoring.

Monitoring antigen-specific T cell immunity relies on functional tests that require T cells and antigen presenting cells to be uncompromised. Drawing of blood, its storage and shipment from the clinical site to the test laboratory, and the subsequent isolation, cryopreservation and thawing of peripheral blood mononuclear cells (PBMC) before the actual test is performed can introduce numerous variables that may jeopardize the results. Therefore, no T cell test is valid without assessing the functional fitness of the PBMC being utilized. This can only be accomplished through inclusion of positive controls that actually evaluate the performance of the antigen-specific T cell and antigen presenting cell (APC) compartments. CEF peptides have been commonly used to this extent. Here we show that CEF peptides fail as a positive control in nearly half of test subjects. Moreover, CEF peptides only measure CD8+ T cell functionality. More reliable alternatives for the assessment of CD8+ T cells are introduced here, as well as positive controls for the CD4+ T cell and APC compartments. In sum, we offer new tools and strategies for the assessment of PBMC functional fitness required for reliable T cell immune monitoring.