Ex Vivo Staining of Metastatic Lymph Nodes by Class I Major Histocompatibility Complex Tetramers Reveals High Numbers of Antigen-experienced Tumor-specific Cytolytic T Lymphocytes

Abstract

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

Decreased number of mast cells infiltrating into needle biopsy specimens leads to a better prognosis of prostate cancer

“…Most attention focused on the role of infiltration lymphoid cells such as tumour-infiltrating lymphocytes, which represent the host immune reaction against the tumour (Romero et al, 1998;Molldrem et al, 2000;Zhang et al, 2003). There have been a number of reports regarding mast cell infiltration around a variety of human and experimentally induced tumours (Ueda et al, 1988;Dimitriadou and Koutsilieris, 1997;Takanami et al, 2000;Ribatti et al, 2004;Tuna et al, 2006).…”

Long‐lasting cross‐presentation of tumor antigen in human DC

“…The present observations may have important consequences on the modes of antigen delivery for immunotherapy. Both MAA selected for the present in vitro study (MelanA/MART-1 and gp100) are the targets of CTL that can be identified in blood and tumor infiltrates of melanoma patients [47]. Nevertheless, the in vivo anti-tumoral efficiency of these T cells has not been demonstrated [48].…”

Inhibition of spontaneous development of liver tumors by inoculation with dendritic cells loaded with hepatocellular carcinoma cells in C3H/HeNCRJ mice

“…13,14 T cells that recognize TAAs are present in the blood of cancer patients. [15][16][17] However, development of antitumor immunity is inhibited by peripheral tolerance and immunosuppression due to cytokines secreted by tumors. 17,18 Furthermore, cancer cells use various methods to escape recognition and killing by T cells.…”