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<h4>Background</h4>In contrast to many animal model studies, immunotherapeutic trials in humans suffering from cancer invariably result in a broad range of outcomes, from long-lasting remissions to no discernable effect.<h4>Methods and findings</h4>In order to study the T cell responses in patients undergoing a melanoma-associated peptide vaccine trial, we have developed a high-throughput method using arrays of peptide-major histocompatibility complexes (pMHC) together with antibodies against secreted factors. T cells were specifically immobilized and activated by binding to particular pMHCs. The antibodies, spotted together with the pMHC, specifically capture cytokines secreted by the T cells. This technique allows rapid, simultaneous isolation and multiparametric functional characterization of antigen-specific T cells present in clinical samples. Analysis of CD8+ lymphocytes from ten melanoma patients after peptide vaccination revealed a diverse set of patient- and antigen-specific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNgamma and TNFalpha did so.<h4>Conclusion</h4>Such multiparametric analysis of T cell antigen specificity and function provides a valuable tool with which to dissect the molecular underpinnings of immune responsiveness and how this information correlates with clinical outcome.