No abstract

Using fluorescent HLA-A*0201 tetramers containing the immunodominant Melan-A/MART-1 (Melan-A) tumor-associated antigen (Ag), we previously observed that metastatic lymph nodes of melanoma patients contain high numbers of Ag-experienced Melan-A–specific cytolytic T lymphocytes (CTLs). In this paper, we enumerated and characterized ex vivo Melan-A–specific cells in peripheral blood samples from both melanoma patients and healthy individuals. High frequencies (≥1 in 2,500 CD8+ T cells) of Melan-A–specific cells were found in 10 out of 13 patients, and, surprisingly, in 6 out of 10 healthy individuals. Virtually all Melan-A–specific cells from 6 out of 6 healthy individuals and from 7 out of 10 patients displayed a naive CD45RAhi/RO− phenotype, whereas variable proportions of Ag-experienced CD45RAlo/RO+ Melan-A–specific cells were observed in the remaining 3 patients. In contrast, ex vivo influenza matrix–specific CTLs from all individuals exhibited a CD45RAlo/RO+ memory phenotype as expected. Ag specificity of tetramer-sorted A2/Melan-A+ cells from healthy individuals was confirmed after mitogen-driven expansion. Likewise, functional limiting dilution analysis and interferon γ ELISPOT assays independently confirmed that most of the Melan-A–specific cells were not Ag experienced. Thus, it appears that high frequencies of naive Melan-A–specific CD8+ T cells can be found in a large proportion of HLA-A*0201+ individuals. Furthermore, as demonstrated for one patient followed over time, dramatic phenotype changes of circulating Melan-A–specific cells can occur in vivo.

SummaryCharacterization of cytolytic T lymphocyte (CTL) responses to tumor antigens has been impeded by a lack of direct assays of CTL activity. We have synthesized reagents ("tetramers") that specifically stain CTLs recognizing melanoma antigens. Tetramer staining of tumor-infiltrated lymph nodes ex vivo revealed high frequencies of tumor-specific CTLs which were antigen-experienced by surface phenotype. In vitro culture of lymph node cells with cytokines resulted in very large expansions of tumor-specific CTLs that were dependent on the presence of tumor cells in the lymph nodes. Tetramer-guided sorting by flow cytometer allowed isolation of melanoma-specific CTLs and confirmation of their specificity and their ability to lyse autologous tumor cells. Our results demonstrate the value of these novel reagents for monitoring tumor-specific CTL responses and for generating CTLs for adoptive immunotherapy. These data also indicate that strong CTL responses to melanoma often occur in vivo, and that the reactive CTLs have substantial proliferative and tumoricidal potential. It is now well established that human tumor cells, notably cutaneous melanoma, may express multiple CTL-defined antigens that are shared among tumors, providing the prospect for generic vaccines applicable to large subsets of cancer patients (1, 2). Hence, numerous clinical trials are underway aimed at inducing vigorous CTL responses against defined tumor antigens. However, further progress in understanding natural or vaccine-induced CTL responses to tumor antigens has been prevented by the lack of direct assays of CTL activity. Previous assays for detection of antigen-specific CTLs have depended on their ability to proliferate extensively and acquire lytic activity, or to release relatively large amounts of cytokine (3), such that accurate quantification of these cells has not been possible. The necessity of stimulating CTLs with antigen in order to detect them has also prevented characterization of their phenotype in vivo. Recently, tetrameric arrays of soluble class I MHC-peptide complexes ("tetramers") have been used to identify antigen-specific CTLs (4), and techniques for detecting and isolating low-frequency CTLs using these reagents have been developed and validated (5). We report here the first analyses of patient samples using tetramers incorporating melanoma-derived antigenic peptides. These reagents were used to directly enumerate and phenotype melanoma-specific CTLs ex vivo from both metastatic lesions and peripheral blood, using multiparameter flow cytometry. Materials and MethodsTissues and Cells. Melanoma patients subjected to therapeutic surgical LN resection were selected for this study on the basis of P. Romero and P.R. Dunbar contributed equally to this work. T on