Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells.

The goal of tumor immunotherapy is to elicit immune responses against autologous tumors. It would be highly desirable that such responses include multiple T cell clones against multiple tumor antigens. This could be obtained using the antigen presenting capacity of dendritic cells (DCs) and cross-priming. That is, one could load the DC with tumor lines of any human histocompatibility leukocyte antigen (HLA) type to elicit T cell responses against the autologous tumor. In this study, we show that human DCs derived from monocytes and loaded with killed melanoma cells prime naive CD45RA(+)CD27(+)CD8(+) T cells against the four shared melanoma antigens: MAGE-3, gp100, tyrosinase, and MART-1. HLA-A201(+) naive T cells primed by DCs loaded with HLA-A201(-) melanoma cells are able to kill several HLA-A201(+) melanoma targets. Cytotoxic T lymphocyte priming towards melanoma antigens is also obtained with cells from metastatic melanoma patients. This demonstration of cross-priming against shared tumor antigens builds the basis for using allogeneic tumor cell lines to deliver tumor antigens to DCs for vaccination protocols.

In vivo expression of natural killer cell inhibitory receptors by human melanoma-specific cytolytic T lymphocytes.

Natural killer (NK) receptor signaling can lead to reduced cytotoxicity by NK cells and cytolytic T lymphocytes (CTLs) in vitro. Whether T cells are inhibited in vivo remains unknown, since peptide antigen-specific CD8(+) T cells have so far not been found to express NK receptors in vivo. Here we demonstrate that melanoma patients may bear tumor-specific CTLs expressing NK receptors. The lysis of melanoma cells by patient-derived CTLs was inhibited by the NK receptor CD94/NKG2A. Thus, tumor-specific CTL activity may be decreased through NK receptor triggering in vivo.

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.

Characterization 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.

APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.

Expression of gene rrg is associated with reversion of NIH 3T3 transformed by LTR-c-H-ras.

A partial complementary DNA was isolated for a gene (rrg) that is normally expressed in mouse NIH 3T3 cells, but is down-regulated after cellular transformation by long terminal repeat (LTR)-activated c-H-ras (LTR-c-H-ras). This gene was reexpressed in a nontumorigenic persistent revertant cell line created by prolonged treatment of the transformed cells with mouse interferon alpha/beta. Persistent revertants stably transfected with rrg complementary DNA antisense expression vectors appeared transformed, had decreased amounts of rrg messenger RNA, and were tumorigenic in nude mice. Stable transfection with sense constructs did not alter the normal morphology, message level, or nontumorigenicity of the persistent revertant cell line.

Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape.

Malignant melanoma accounts for most of the increasing mortality from skin cancer. Melanoma cells were found to express Fas (also called Apo-1 or CD95) ligand (FasL). In metastatic lesions, Fas-expressing T cell infiltrates were proximal to FasL+ tumor cells. In vitro, apoptosis of Fas-sensitive target cells occurred upon incubation with melanoma tumor cells; and in vivo, injection of FasL+ mouse melanoma cells in mice led to rapid tumor formation. In contrast, tumorigenesis was delayed in Fas-deficient lpr mutant mice in which immune effector cells cannot be killed by FasL. Thus, FasL may contribute to the immune privilege of tumors.

Potential target antigens for immunotherapy in human pancreatic cancer.

BACKGROUND: To be effective and selective, immunotherapy ideally targets specifically tumor cells and spares normal tissues. Identification of tumor specific antigens is a prerequisite to establish an effective immunotherapy. Still very little is known about the expression of tumor-related antigens in pancreatic neoplasms. Cancer Testis antigens (CT) are antigens shared by a variety of malignant tumors, but not by normal tissues with the exception of germ cells in testis. Restricted expression in neoplastic tissues and inherent immunogenic features make CT antigens ideal for use in immunotherapy. We analyzed the expression of a selected panel of nine CT antigens that have been proven to elicit an efficient immunogenic response in other malignancies. In addition we analyzed the expression of HERV-K-MEL, an immunogenic antigen of viral origin. METHODS: Pancreatic adenocarcinoma tumor samples (n=130) were obtained intraoperatively, control tissues (n=23) were collected from cadaveric donor and from patients with chronic pancreatitis. Tumor-associated antigen expression of MAGE-A1, MAGE-A3, MAGE-A4, MAGE-A10, LAGE-1, NY-ESO-1, SCP-1, SSX-2, SSX-4 and HERV-K-MEL was assessed by PCR. Sequencing of PCR products were performed to assess the expression of SSX-4 in neoplastic and normal pancreatic tissues. RESULTS: Three of 10 tested antigens were expressed in over 10% of malignant pancreatic tissue samples. SSX-4 was found positive in 30% of cases, SCP-1 in 19% and HERV-K-MEL in 23% of cases. No expression of CT antigens was found in non-malignant pancreatic tissue with the exception of SSX-4 and and SSX-2. CONCLUSIONS: Fifty two percentage of the analyzed tissues expressed at least one CT antigen. The concomitant expression of SSX-4 in both malignant and non-malignant pancreatic tissue is a new finding which may raise concerns for immunotherapy. However, HERV-K-MEL is expressed with a relatively high prevalence and may be a candidate for specific immunotherapy in a large subgroup of pancreatic cancer patients. This study advocates the analysis of patients with regard to their immunogenic profile before the onset of antigen-specific immunotherapy.

Sentinel lymph node involvement and a high Breslow index are independent factors of risk for early relapse of melanoma.

AIM: The clinical relevance of sentinel lymph node (SLN) analysis was evaluated prospectively and compared with other known risk factors of relapse in early stage melanoma. METHODS: Surgery was guided by lymphoscintigraphy, blue dye and gamma probe detection. SLN were analysed by haematoxylin eosin (HE) histochemistry and multimarker immunohistochemistry (IHC). Disease free survival (DFS) was evaluated with Kaplan-Meier plots according to different parameters and Cox analyses of variance. RESULTS: From 210 patients a total of 381 SLN were excised. Lymphoscintigraphy identified all excised SLN with only 2 false positive lymphatic lakes. Fifty patients (24%) had tumour positive SLN. With a mean follow-up of 31.3 months, 29 tumour recurrences were observed, 19 (38%) in 50 SLN positive and 10 (6%) in 160 SLN negative patients. Strong predictive factors for early relapse (p < 0.0005) were SLN positivity and a high Breslow index. CONCLUSION: SLN tumour positivity is an independent factor of high risk for early relapse with a higher power of discrimination than the Breslow index.

Transcriptional inhibition of Ha-ras in interferon-induced revertants of ras-transformed mouse cells.

Elevation in Ha-ras expression, due to transcriptional activation or gene amplification, is associated with oncogenic transformation of NIH 3T3 cells. We have previously shown that murine interferon (IFN)-alpha/beta induced phenotypic reversion of NIH 3T3 cells transformed by long terminal repeat (LTR)-activated c-Ha-ras. The revertants produced decreased amounts of ras-encoded Mr 21,000 protein. We have now determined the molecular level at which LTR-ras regulation occurred. Nuclear run-on experiments revealed a selective inhibition of ras transcription in IFN-treated revertants. There was no apparent additional posttranscriptional control by IFN as judged by the unchanged half-life of ras transcripts. Inhibition of ras RNA synthesis was seen only in conjunction with long-term IFN treatment and was limited to the revertants, a population of cells that maintained sensitivity to IFN during the prolonged exposure. The reduction in ras activity appeared responsible in part for the loss of tumorigenicity in treated cells and was stable for several weeks after treatment had been discontinued.

Increased sensitivity of nontumorigenic fibroblasts expressing ras or myc oncogenes to malignant transformation induced by 5-aza-2'-deoxycytidine.

The hypomethylating chemotherapeutic drug 5-aza-2'-deoxycytidine (5AzadC) has been shown to induce cell differentiation in some systems, while promoting neoplastic transformation in others. Using both in vitro and in vivo models, we have explored the relationship between oncogene expression and the susceptibility of cells to malignant transformation by 5AzadC. The study involved several nontumorigenic subclones of NIH3T3 fibroblasts, including cells transfected with deregulated c-myc, as well as phenotypic revertants expressing v-Ki-ras or long terminal repeat-activated c-Ha-ras. Transient 5AzadC treatment of the oncogene-bearing cell lines was associated with a rapid and efficient neoplastic transformation. In some cases, over 50% of the cell population exhibited loss of contact inhibition of growth within 1 week of treatment. The transformants were capable of forming s.c. tumors and experimental lung metastases in recipient nude mice. In contrast, 5AzadC failed to induce malignant properties in control 3T3 cultures transfected with the bacterial neor gene; rather, treatment of these cells was associated with differentiation into adipocytes and myotubes. The differential response to 5AzadC was also observed in vivo, in mice first inoculated s.c. with the premalignant cells and then treated with 5AzadC 24 h later. In agreement with the in vitro model, tumor development in mice correlated with the presence of cells with activated ras or myc oncogenes. Cytidine analogs that do not inhibit DNA methylation (i.e., 6-azacytidine and 1-beta-D-arabinofuranosyl cytosine) had no effect on cell phenotype. The results indicate that exposure of cells to 5AzadC can lead to tumor progression both in vitro and in vivo and suggest that preexisting alterations in oncogene expression may facilitate the evolution of cancerous growth induced by hypomethylating agents.

Analysis of MAGE-3-specific cytolytic T lymphocytes in human leukocyte antigen-A2 melanoma patients.

The MAGE-3 gene is a member of a multigene family that is selectively expressed by subsets of different human tumor types, including malignant melanoma, but not by normal tissues except for testis and placenta. A cytolytic T lymphocyte (CTL)-defined MAGE-3 antigen, corresponding to the MAGE-3 peptide 271-279 associated with the human leukocyte antigen (HLA)-A2 molecule, has been recently identified using T lymphocytes from a normal individual stimulated in vitro with peptide-pulsed autologous antigen-presenting cells. Because MAGE-3 is expressed in 76% of metastatic melanomas, the HLA-A2-restricted MAGE-3 antigen should be expressed by approximately 37% of Caucasians bearing a metastatic melanoma tumor, thus representing an attractive candidate for the elicitation of specific CTL immune responses in vivo. In this study, we determined the proportion of HLA-A2+ melanoma patients displaying detectable MAGE-3 peptide 271-279-specific CTL precursors in peripheral blood. Peptide-specific CTL populations were obtained from at least 4 of 11 HLA-A2+ patients. Peptide-specific CTL lines derived from these populations readily lysed HLA-A2-positive target cells that were pulsed with MAGE-3 peptide 271-279 at nanomolar concentrations yet were unable to recognize (as assessed by cytolysis and cytokine production) MAGE-3-expressing autologous or allogeneic HLA-A2-positive melanoma lines. Similarly, the CTL lines failed to recognize MAGE-3-negative HLA-A2-positive tumor lines after transfection with the MAGE-3 gene, although they were able to recognize COS-7 cells transfected with MAGE-3. In contrast, HLA-A1-positive melanoma lines transfected with MAGE-3 were efficiently recognized by CTL lines directed against the MAGE-3 peptide 168-176, a known HLA-A1-restricted CTL epitope. These results suggest that the expression level of the MAGE-3 peptide 271-279, unlike that of MAGE-3 peptide 168-176, in melanomas may be too low to allow efficient recognition by specific CTLs. Thus, it appears that despite the presence of CTL precursors against MAGE-3 peptide 271-279 in some HLA-A2+ melanoma patients, the usefulness of this peptide for specific immunotherapy of melanoma may be limited.

Development of resistance to the growth inhibitory effects of transforming growth factor beta 1 during the spontaneous transformation of rat liver epithelial cells.

The temporary maintenance of a rat liver epithelial cell population at confluence before passaging followed by periods of rapid proliferation resulted in the generation of spontaneous transformants after about 108 population doublings. The appearance of morphologically aberrant transformants correlated directly with an increased resistance of the population to the growth inhibitory effects of transforming growth factor beta 1 (TGF-beta 1). Clonal cell lines derived from the transformants were resistant to TGF-beta 1 dependent inhibition of DNA synthesis. These cell lines were also highly tumorigenic and aneuploid, with characteristic gross chromosomal abnormalities, and they expressed a number of phenotypic markers common to rat liver epithelial cells transformed by oncogenes or chemicals. In contrast, apparently normal looking cell lines cloned from the same population were nontumorigenic and near diploid, with few chromosomal abnormalities, and they were as sensitive to TGF-beta 1 as early passage normal rat liver epithelial cells. Morphologically normal late passage rat liver epithelial cells were sensitive to transformation by the DNA hypomethylating agent 5-aza-2-deoxycytidine, in contrast to earlier passage cells, and this transformation was accompanied by the development of resistance to the growth inhibitory effects of TGF-beta 1. These findings suggest that acquisition of resistance to the effects of growth inhibitors such as TGF-beta 1 is an important and possibly essential stage in the spontaneous transformation of rat liver epithelial cells.

Analysis of the cytolytic T lymphocyte response of melanoma patients to the naturally HLA-A*0201-associated tyrosinase peptide 368-376.

The human tyrosinase gene codes for two distinct antigens that are recognized by HLA-A*0201-restricted CTLs. For one of them, tyrosinase peptide 368-376, the sequence identified by mass spectrometry in melanoma cell eluates differs from the gene-encoded sequence as a result of posttranslational modification of amino acid residue 370 (asparagine to aspartic acid). Here, we used fluorescent tetrameric complexes ("tetramers") of HLA-A*0201 and tyrosinase peptide 368-376 (YMDGTMSQV) to characterize the CD8+ T-cell response to this antigen in lymphoid cell populations from HLA-A2 melanoma patients. Taking advantage of the presence of significant numbers of tetramer-positive CD8+ T cells in tumor-infiltrated lymph node cells from a melanoma patient, we derived polyclonal and monoclonal tyrosinase peptide 368-376-specific CTLs by tetramer-guided flow cytometric sorting. These CTLs efficiently and specifically lysed HLA-A*0201- and tyrosinase-positive melanoma cells. As assessed with tyrosinase peptide variants, the fine antigen specificity of the CTLs was quite diverse at the clonal level. Flow cytometric analysis of PBMCs stained with tetramers showed that tyrosinase peptide 368-376-specific CD8+ T cells were hardly detectable in peripheral blood of melanoma patients. However, significant numbers of such cells were detected after short-term stimulation of CD8+ lymphocytes with tyrosinase peptide 368-376 in 6 of 10 HLA-A2 melanoma patients. Taken together, these findings emphasize the significant contribution of the natural tyrosinase peptide 368-376 to the antigenic specificities recognized by the tumor-reactive CTLs that may develop in HLA-A2 melanoma patients.

An antigen-targeted approach to adoptive transfer therapy of cancer.

Previous attempts to treat human malignancies by adoptive transfer of tumor-specific CTLs have been limited by the difficulty of isolating T cells of defined antigen specificity. The recent development of MHC class I/antigenic peptide tetrameric complexes that allow direct identification of antigen-specific T cells has opened new possibilities for the isolation and in vitro expansion of tumor-specific T cells. In the present study, we have derived polyclonal monospecific cell lines from circulating Melan-A-specific CTL precursors of HLA-A*0201+ melanoma patients by combining stimulation with recently identified peptide analogues of the immunodominant epitope from the melanoma-associated antigen Melan-A with staining with fluorescent HLA-A*0201/Melan-A peptide tetramers. In vitro expansion of antigen-specific CD8+ T cells was monitored by flow cytometry with the fluorescent tetramers and anti-CD8 monoclonal antibody. This analysis revealed that Melan-A 26-35 peptide analogues were much more efficient than the parental peptides in stimulating a rapid in vitro expansion of antigen-specific CD8+ T cells. These cells were then isolated by tetramer-guided cell sorting and subsequently expanded in vitro by mitogen stimulation. The resulting polyclonal but monospecific CTLs fully cross-recognized the parental peptides and were able to efficiently lyse Melan-A-expressing tumor cells. Altogether, these results pave the way to a molecularly defined approach to antigen-specific adoptive transfer therapy of cancer.

Naturally occurring human lymphocyte antigen-A2 restricted CD8+ T-cell response to the cancer testis antigen NY-ESO-1 in melanoma patients.

Cancer testis (CT) antigens are particularly interesting candidates for cancer vaccines. However, T-cell reactivity to CT antigens has been detected only occasionally in cancer patients, even after vaccination. A new group of CT antigens has been recently identified using the SEREX technique based on immunoscreening of tumor cDNA expression libraries with autologous sera. We have used fluorescent HLA-A2/peptide tetramers containing an optimized antigenic peptide to directly identify HLA-A2-restricted CD8+ T cells specific for the SEREX-defined CT antigen NY-ESO-1 in melanoma patients. High frequencies of NY-ESO-1-specific CD8+ T cells were readily detected in peptide-stimulated peripheral blood mononuclear cells as well as in lymphocytes infiltrating melanoma lesions from patients with measurable antibody responses to NY-ESO-1. NY-ESO-1-specific CD8+ T cells were also detectable in peptide-stimulated peripheral blood mononuclear cells from some seronegative patients. Whereas the frequencies of NY-ESO-1-specific CD8+ T cells in circulating lymphocytes were usually below the limit of detection by tetramer staining, the presence of NY-ESO-1 CD8+ T cells displaying a memory phenotype was clearly detectable ex vivo in blood from a seropositive patient over an extended period of time. These results indicate that sustained CD8+ T-cell responses to CT antigens can naturally occur both locally and systemically in melanoma patients.

Frequent cytolytic T-cell responses to peptide MAGE-A10(254-262) in melanoma.

MAGE genes encode tumor-specific shared antigens that are among the most interesting candidates for cancer vaccines. Despite extensive studies, however, CD8+ T-cell responses to MAGE-derived epitopes have been detected only occasionally in cancer patients, even after vaccination. In contrast with these findings, we report here that HLA-A2 melanoma patients respond frequently to the recently identified peptide MAGE-A10(254-262). Indeed, as assessed by staining with fluorescent HLA-A2/peptide MAGE-A10(254-262) tetramers, CD8+ T cells directed against this peptide were readily detectable in a large proportion of HLA-A2+ melanoma patients. These results provide new insight into the immunogenicity of MAGE antigens and underline the potential usefulness of MAGE-A10 peptide-based cancer vaccines.

Heterogeneous T-cell response to MAGE-A10(254-262): high avidity-specific cytolytic T lymphocytes show superior antitumor activity.

MAGE-encoded antigens, which are expressed by tumors of many histological types but not in normal tissues, are suitable candidates for vaccine-based immunotherapy of cancers. Thus far, however, T-cell responses to MAGE antigens have been detected only occasionally in cancer patients. In contrast, by using HLA/peptide fluorescent tetramers, we have observed recently that CD8(+) T cells specific for peptide MAGE-A10(254-262) can be detected frequently in peptide-stimulated peripheral blood mononuclear cells from HLA-A2-expressing melanoma patients and healthy donors. On the basis of these results, antitumoral vaccination trials using peptide MAGE-A10(254-262) have been implemented recently. In the present study, we have characterized MAGE-A10(254-262)-specific CD8(+) T cells in polyclonal cultures and at the clonal level. The results indicate that the repertoire of MAGE-A10(254-262)-specific CD8(+) T cells is diverse both in terms of clonal composition, efficiency of peptide recognition, and tumor-specific lytic activity. Importantly, only CD8(+) T cells able to recognize the antigenic peptide with high efficiency are able to lyse MAGE-A10-expressing tumor cells. Under defined experimental conditions, the tetramer staining intensity exhibited by MAGE-A10(254-262)-specific CD8(+) T cells correlates with efficiency of peptide recognition so that "high" and "low" avidity cells can be separated by FACS. Altogether, the data reported here provide evidence for functional diversity of MAGE-A10(254-262)-specific T cells and will be instrumental for the monitoring of peptide MAGE-A10(254-262)-based clinical trials.

Compact solid-state CMOS single-photon detector array for in vivo NIR fluorescence lifetime oncology measurements.

In near infrared fluorescence-guided surgical oncology, it is challenging to distinguish healthy from cancerous tissue. One promising research avenue consists in the analysis of the exogenous fluorophores' lifetime, which are however in the (sub-)nanosecond range. We have integrated a single-photon pixel array, based on standard CMOS SPADs (single-photon avalanche diodes), in a compact, time-gated measurement system, named FluoCam. In vivo measurements were carried out with indocyanine green (ICG)-modified derivatives targeting the αvß 3 integrin, initially on a genetically engineered mouse model of melanoma injected with ICG conjugated with tetrameric cyclic pentapeptide (ICG-E[c(RGD f K)4]), then on mice carrying tumour xenografts of U87-MG (a human primary glioblastoma cell line) injected with monomeric ICG-c(RGD f K). Measurements on tumor, muscle and tail locations allowed us to demonstrate the feasibility of in vivo lifetime measurements with the FluoCam, to determine the characteristic lifetimes (around 500 ps) and subtle lifetime differences between bound and unbound ICG-modified fluorophores (10% level), as well as to estimate the available photon fluxes under realistic conditions.

Vaccination of stage III/IV melanoma patients with long NY-ESO-1 peptide and CpG-B elicits robust CD8+ and CD4+ T-cell responses with multiple specificities including a novel DR7-restricted epitope.

Long synthetic peptides and CpG-containing oligodeoxynucleotides are promising components for cancer vaccines. In this phase I trial, 19 patients received a mean of 8 (range 1-12) monthly vaccines s.c. composed of the long synthetic NY-ESO-179-108 peptide and CpG-B (PF-3512676), emulsified in Montanide ISA-51. In 18/18 evaluable patients, vaccination induced antigen-specific CD8+ and CD4+ T-cell and antibody responses, starting early after initiation of immunotherapy and lasting at least one year. The T-cells responded antigen-specifically, with strong secretion of IFNγ and TNFα, irrespective of patients' HLAs. The most immunogenic regions of the vaccine peptide were NY-ESO-189-102 for CD8+ and NY-ESO-183-99 for CD4+ T-cells. We discovered a novel and highly immunogenic epitope (HLA-DR7/NY-ESO-187-99); 7/7 HLA-DR7+ patients generated strong CD4+ T-cell responses, as detected directly ex vivo with fluorescent multimers. Thus, vaccination with the long synthetic NY-ESO-179-108 peptide combined with the strong immune adjuvant CpG-B induced integrated, robust and functional CD8+ and CD4+ T-cell responses in melanoma patients, supporting the further development of this immunotherapeutic approach.

CD8+ T-cell response to NY-ESO-1: relative antigenicity and in vitro immunogenicity of natural and analogue sequences.

We have shown previously that HLA-A*0201 melanoma patients can frequently develop a CTL response to the cancer testis antigen NY-ESO-1. In the present study, we have analyzed in detail the relative antigenicity and in vitro immunogenicity of natural and modified NY-ESO-1 peptide sequences. The results of this analysis revealed that, although suboptimal for binding to the HLA-A*0201 molecule, peptide NY-ESO-1 157-165 is, among natural sequences, very efficiently recognized by specific CTL clones derived from three melanoma patients. In contrast, peptides NY-ESO-1 157-167 and NY-ESO-1 155-163, which bind very strongly to HLA-A*0201, are recognized less efficiently. In agreement with previous data, substitution of peptide NY-ESO-1 157-165 COOH-terminal C with various other amino acids resulted in a significantly increased binding to HLA-A*0201 molecules as well as in an increased CTL recognition, although variable at the clonal level. Among natural peptides, NY-ESO-1 157-165 and NY-ESO-1 157-167 exhibited good in vitro immunogenicity, whereas peptide NY-ESO-1 155-163 was poorly immunogenic. The fine specificity of interaction between peptide NY-ESO-1 C165A, HLA-A*0201, and T-cell receptor was analyzed at the molecular level using a series of variant peptides containing single alanine substitutions. The findings reported here have significant implications for the formulation of NY-ESO-1-based vaccines as well as for the monitoring of either natural or vaccine-induced NY-ESO-1-specific CTL responses in cancer patients.