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We compared monocyte-derived dendritic cells and transforming growth factor-[beta]1-induced Langerhans-like cells (LCs) for their capacity to cross-present exogenous NY-ESO-1 protein/antibody immune complexes to an NY-ESO-l-specific CD8+ T cell clone. In contrast to dendritic cells, LCs were not able to cross-present NY-ESO-1 to the T cell clone constitutively but did so after treatment with IFN-[gamma]. Remarkably. this IFN-[gamma]-inducible characteristic was due neither to enhanced antigen uptake nor to facilitated antigen processing in LCs. Rather, IFN-[gamma] acted at least in part by potentiating the maturation of otherwise refractory LCs, enabling in turn exogenous antigen to reach the processing machinery. This model of conditional cross-presentation establishes an original level of action for IFN-[gamma] as an effective immune modulator and supports the use of IFN-[gamma] in protein vaccination strategies targeting LCs.

NY-ESO-1 is a germ cell antigen aberrantly expressed in different tumor types that elicits strong humoral and cellular immune responses in cancer patients. Monitoring spontaneous [CD8.sup.+] T cell responses against NY-ESO-1 peptides 157-165 (s9c) and 157-167 (S11L) in a series of HLA-[A2.sup.+] cancer patients showed that these two peptides had overlapping antigenic profiles and were equally immunogenic. However, discrepancies between sgc and S11L reactivities were observed upon vaccination with both peptides to generate or boost T cell responses to NY-ESO-1 in cancer patients. We here analyze the fine specificity of these responses and describe an HLA-A2-restricted epitope, NY-ESO-1 peptide 159-167 (L9L), which is strongly recognized by [CD8.sup.+] T cells as a result of peptide vaccination of cancer patients. Responses to L9L were stimulated by S11L and appeared early in the course of vaccination, independently of S9C responses. However, L9L-specific [CD8.sup.+] T cells failed to recognize tumor cells naturally expressing NY-ESO-1 or B lymphoblastoid cells transduced with NY-ESO-1. Processing of L9L could be rescued after IFN-[gamma] treatment of tumor cells or by dendritic cells pulsed with NY-ESO-1 protein/antibody immune complexes. The present results demonstrate a dual specificity within peptide S11L, with S9C as the natural antigenic tumor epitope, and L9L as a cryptic epitope with dominant immunogenicity upon vaccination that diverts the immune response from tumor recognition. These unanticipated findings raise questions about the use of S11L in the clinic and emphasize the importance of analyzing the fine specificity of vaccine-induced T cell responses in patients as a basis for constructing effective cancer vaccines.

Dendritic cells (DCs) play a critical role in initiating antigen-specific immune responses, because they are able to capture exogenous antigens for presentation to naive T cells on both MHC class I and II molecules. As such, DCs represent important elements in the development of vaccine therapy for cancer. Although DCs are known to present antigens from phagocytosed tumor cells or preprocessed peptides, we explored whether they might also present soluble recombinant NY-ESO-1, a well characterized cancer antigen. We compared the abilities of human monocyte-derived DCs and DCs derived in vitro from CD34-positive stem cells to present NY-ESO-1 epitopes to MHC class I-restricted cytotoxic T cells. Although monocyte-derived DCs did not efficiently crosspresent free NY-ESO-1 protein, IgG-immune complexes containing NY-ESO-1 were avidly presented after uptake by Fc[gamma] receptors (Fc[gamma]RII). In contrast, CD34-derived DCs were unable to process either soluble or immune complexed NY-ESO-1, although they efficiently presented preprocessed NY-ESO-1 peptides. This difference did not necessarily correlate with endocytic capacity. Although monocyte-derived DCs exhibited greater fluid-phase uptake than CD34-derived DCs, the two populations did not differ with respect to their surprisingly limited capacity for Fc[gamma] receptor- mediated endocytosis. These results indicate that monocyte-derived DCs will be easier to load by using protein antigen in vitro than CD34-derived DCs, and that the latter population exhibits a restricted ability to crosspresent soluble exogenous antigens.

Cancer-testis antigen NY-ESO-1 is one of the most immunogenic tumor antigens defined to date. Spontaneous humoral and CD8+ T-cell responses to NY-ESO-1 are detected in 40-50% of patients with advanced NY-ESO-1-expressing tumors. A clinical trial was initiated to study the immunological effects of intradermal vaccination with 3 HLA-A2-binding NY-ESO-1 peptides in 12 patients with metastatic NY-ESO-1-expressing cancers. Seven patients were NY-ESO-1 serum antibody negative, and five patients were NY-ESO-1 serum antibody positive at the outset of the study. Primary peptide-specific CD8+ T-cell reactions and delayed-type hypersensitivity responses were generated in four of seven NY-ESO-1 antibody-negative patients. Induction of a specific CD8+ T-cell response to NY-ESO-1 in immunized antibody-negative patients was associated with disease stabilization and objective regression of single metastases. NY-ESO-1 antibody-positive patients did not develop significant changes in baseline NY-ESO-1-specific T-cell reactivity. However, stabilization of disease and regression of individual metastases were observed in three of five immunized patients. These results demonstrate that primary NY-ESO-1-specific CD8+ T-cell responses can be induced by intradermal immunization with NY-ESO-1 peptides, and that immunization with NY-ESO-1 may have the potential to alter the natural course of NY-ESO-1-expressing tumors.

NY-ESO-1 elicits frequent antibody responses in cancer patients, accompanied by strong [CD8.sup.+] T cell responses against HLA-A2-restricted epitopes. To broaden the range of cancer patients who can be assessed for immunity to NY-ESO-1, a general method was devised to detect T cell reactivity independent of prior characterization of epitopes. A recombinant adenoviral vector encoding the full cDNA sequence of NY-ESO-1 was used to transduce CD8-depleted peripheral blood lymphocytes as antigen-presenting cells. These modified antigen-presenting cells were then used to restimulate memory effector cells against NY-ESO-1 from the peripheral blood of cancer patients. Specific [CD8.sup.+] T cells thus sensitized were assayed on autologous B cell targets infected with a recombinant vaccinia virus encoding NY-ESO-1. Strong polyclonal responses were observed against NY-ESO-1 in antibody-positive patients, regardless of their HLA profile. Because the vectors do not cross-react immunologically, only responses to NY-ESO-1 were detected. The approach described here allows monitoring of [CD8.sup.+] T cell responses to NY-ESO-1 in the context of various HLA alleles and has led to the definition of NY-ESO-1 peptides presented by HLA-Cw3 and HLA-Cw6 molecules.

NY-ESO-1, a member of the cancer-testis family of antigens, is expressed in a subset of a broad range of different human tumor types. Patients with advanced NY-ESO-1-expressing tumors frequently develop humoral immunity to NY-ESO-1, and three HLA A2-restricted peptides were defined previously as targets for cytotoxic [CD8.sup.+] T cells in a melanoma patient with NY-ESO-1 antibody. The objectives of the present study were (i) to develop enzyme-linked immunospot (ELISPOT) and tetramer assays to measure [CD8.sup.+] T cell responses to NY-ESO-1, (ii) to determine the frequency of [CD8.sup.+] T cell responses to NY-ESO-1 in a series of HLA-A2 patients with NY-ESO-1 expressing tumors, (iii) to determine the relation between [CD8.sup.+] T cell and humoral immune responses to NY-ESO-1, and (iv) to compare results of NY-ESO-1 ELISPOT assays performed independently in two laboratories with T cells from the same patients. NY-ESO-1 ELISPOT and tetramer assays with excellent sensitivity, specificity, and reproducibility have been developed and found to correlate with cytotoxicity assays. [CD8.sup.+] T cell responses to HLA-A2-restricted NY-ESO-1 peptides were detected in 10 of 11 patients with NY-ESO-1 antibody, but not in patients lacking antibody or in patients with NY-ESO-1-negative tumors. The results of ELISPOT assays were concordant in the two laboratories, providing the basis for standardized monitoring of T cell responses in patients receiving NY-ESO-1 vaccines.

NY-ESO-1 is a germ cell antigen aberrantly expressed in different tumor types that elicits strong humoral and cellular immune responses in cancer patients. Monitoring spontaneous CD8[sup +] T cell responses against NY-ESO-1 peptides 157-165 (S9C) and 157-167 (S11L) in a series of HLA-A2[sup +] cancer patients showed that these two peptides had overlapping antigenic profiles and were equally immunogenic. However, discrepancies between S9C and S11L reactivities were observed upon vaccination with both peptides to generate or boost T cell responses to NY-ESO-1 in cancer patients. We here analyze the fine specificity of these responses and describe an HLA-A2-restricted epitope, NY-ESO-1 peptide 159-167 (LgL), which is strongly recognized by CD8[sup +] T cells as a result of peptide vaccination of cancer patients. Responses to L9L were stimulated by S11L and appeared early in the course of vaccination, independently of S9C responses. However, L9L-specific CD8[sup +] T cells failed to recognize tumor cells naturally expressing NY-ESO-1 or B lymphoblastoid cells transduced with NY-ESO-1. Processing of L9L could be rescued after IFN-γ, treatment of tumor cells or by dendritic cells pulsed with NY-ESO-1 protein/antibody immune complexes. The present results demonstrate a dual specificity within peptide S11L, with S9C as the natural antigenic tumor epitope, and L9L as a cryptic epitope with dominant immunogenicity upon vaccination that diverts the immune response from tumor recognition. These unanticipated findings raise questions about the use of S11L. in the clinic and emphasize the importance of analyzing the fine specificity of vaccine-induced T cell responses in patients as a basis for constructing effective cancer vaccines. [ABSTRACT FROM AUTHOR]