Marked Differences in Human Melanoma Antigen-Specific T Cell Responsiveness after Vaccination Using a Functional Microarray

DOI: 10.1371/journal.pmed.0020265 Marked Differences in Human Melanoma Antigen-Specific T Cell Responsiveness after Vaccination Using a Functional Microarray Daniel S. Chen 0 1 2 3 4 5 6 7 Yoav Soen 0 1 2 3 4 5 6 7 Tor B. Stuge 0 1 2 3 4 5 6 7 Peter P. Lee 0 1 2 3 4 5 6 7 Jeffrey S. Weber 0 1 2 3 4 5 6 7 Patrick O. Brown 0 1 2 3 4 5 6 7 Mark M. Davis mdavis@cmgm 0 1 2 3 4 5 6 7 0 University, Stanford, California, United States of America, 3 Department of Biochemistry, Stanford University, Stanford, California, United States of America, 4 Department of 1 1 Department of Internal Medicine/Division of Oncology, Stanford University, Stanford, California, United States of America, 2 Howard Hughes Medical Institute , Stanford 2 Open access , freely available online 3 Citation: Chen DS , Soen Y, Stuge TB, Lee PP, Weber JS, et al. (2005) Marked differences in human melanoma antigen-specific T cell responsiveness after vaccination using a functional microarray. PLoS Med 2(10): e265 4 Academic Editor: Jonathan Rees, University of Edinburgh , United Kingdom 5 Author Contributions: DSC and YS conceived of the experiments and designed the study. DSC, YS, and TBS performed the experiments. DSC, YS, and TBS analyzed the data. JSW enrolled patients. DSC , YS, TBS, JSW, POB , and MMD contributed to writing the paper. POB and MMD suggested ideas and experiments 6 America, 6 Department of Microbiology and Immunology, Stanford University , Stanford, California , United States of America 7 Medicine, Stanford University, Stanford, California, United States of America, 5 Norris Cancer Center, University of Southern California , Los Angeles, California , United States of 8 www.plosmedicine.org A B S T R A C T - Methods and Findings 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 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 antigenspecific profiles of cytokine secretion, indicating surprising differences in their responsiveness. Four out of four patients who showed moderate or greater secretion of both interferon-c (IFNc) and tumor necrosis factor-a (TNFa) in response to a gp100 antigen remained free of melanoma recurrence, whereas only two of six patients who showed discordant secretion of IFNc and 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. Antigen-specific cellular immune responses are mediated by abT cell receptor (TCR)-bearing T cells that recognize specific peptides bound to major histocompatibility complex (MHC) molecules on the surfaces of other cells. These T cells form a major part of the adaptive immune response. CD8 T cells mediate direct lysis of infected or aberrant cells, whereas CD4 T helper cells modulate antibody (B cell) responses and those of other cells. T cells may become activated following antigen recognition and respond by secreting soluble factors, which include mediators of target cell lysis, pleiotropic effector factors, growth factors, and inflammatory and regulatory cytokines (Table 1). This is a highly regulated and complex process. In many cases, antigen recognition by primed CD8 T cells leads to the lysis of cellular targets and the release of inflammatory cytokines. Alternatively, this response may be partially or completely anergic. For many years, investigators have sought to direct T cell responses against tumors by vaccination [1]. These efforts have been greatly aided by the discovery of many peptide antigens that are displayed on MHC molecules on the surface of tumor cells and that have been shown to elicit T cell responses both in vitro and in vivo [2,3]. This discovery has given rise to a variety of strategies, including protein and peptide vaccination [4], adoptive cellular therapy [5], cytokine therapy (i.e., interleukin [IL]-2, granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon [IFN] a) [68], and immune response modifiers such as anti-CTLA4 [9,10]. Despite intense efforts, the success of most of these protocols has been mixed. Although in many cases, specific T cell responses can be generated in patients (or expanded ex vivo and reintroduced intravenously), they are not usually effective against the tumor. A large part of the problem may be that most of these tumor-associated antigens are normal self peptides, and responses may be naturally suppressed. In this context, it is important to monitor the precise functional status of T cells that are elicited by a particular immunization protocol, and to determine what conditions result in T cells that are the most effective in bringing about clinically significant results. For this purpose, the ability to track antigen-specific T cells with peptide-MHC (pMHC) tetramers [11] has been an important tool in the identification and characterization of lymphocytes capable of recognizing specific tumor antigens. This technique, together with other assays (e.g., intracellular cytokine staining, CD107, ELISpot, killing assay) have been used to try to address T cell function [1215]. However, these assays are labor intensive, require large quantities of clinical peripheral blood mononuclear cell (PBMC) specimens for a comprehensive analysis, have poor spatial resolution and/or low sensitivity for secreted responses, and do not address the growing need to track multiple T cell specificities for different functional events. To overcome these limitations, we previously reported on an array-based approach to capture and quantitate antigen-specific T cells based on their adherence to pMHC complexes [16]. Here, we report a further development of this technology, in which we combined the high-throughput capture and activation of antigen-specific T cells described previously with the simultaneous analysis of the secretion of a wide variety of factors with single-cell resolution. Using this technique, we assess antigen-specific T cells from different vaccine recipients and analyze different functional profiles following antigen recognition in an attempt to explore the variability of clinical outcomes that (...truncated)