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Recombinant vaccinia virus (rVV) encoding tumor-associated antigens (TAAs) and adhesion or costimulatory molecules may represent important immunogenic reagents for cancer immunotherapy. Recently, intranodal (IN) antigen administration was suggested to be more immunogenic than intradermal (ID) vaccination. However, IN rVV administration has not been attempted so far. We used a rVV encoding gp100(280-288), Melan-A/MART-1(27-35) and tyrosinase(1-9) HLA-A0201 restricted epitopes and CD80 and CD86 costimulatory molecules in stage III and IV melanoma patients in a phase 1/2 trial. Of 15 patients initiating treatment, including two cycles of IN immunization, each comprising one rVV administration and three recall injections of the corresponding peptides, accompanied by subcutaneous granulocyte macrophage-colony stimulating factor supplementation, five withdrew due to progressing disease. Of 10 remaining patients seven showed evidence of induction of cytotoxic T lymphocytes (CTLs) directed against at least one epitope under investigation, as detectable by limiting dilution analysis (LDA) of specific precursors and multimer staining. Adverse reactions were mild (National Cancer Institute (NCI) grade 1-2) and mainly represented by fever, skin rashes, and pruritus. These data indicate that IN administration of rVV encoding melanoma-associated epitopes and costimulatory molecules is safe and immunogenic.

Highly immunogenic cancers such as malignant melanoma are capable of inexorable tumor growth despite the presence of antitumor immunity. Thus, only a restricted minority of tumorigenic malignant cells may possess the phenotypic and functional characteristics needed to modulate tumor-directed immune activation. Here we provide evidence supporting this hypothesis. Tumorigenic ABCB5(+) malignant melanoma initiating cells (MMICs) possessed the capacity to preferentially inhibit IL-2-dependent T-cell activation and to support, in a B7.2-dependent manner, induction of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs). Compared with melanoma bulk cell populations, ABCB5(+) MMICs displayed lower levels of MHC class I, aberrant positivity for MHC class II, and lower expression levels of the melanoma-associated antigens MART-1, ML-IAP, NY-ESO-1, and MAGE-A. Additionally, these tumorigenic ABCB5(+) subpopulations preferentially expressed the costimulatory molecules B7.2 and PD-1, both in established melanoma xenografts and in clinical tumor specimens. In immune activation assays, MMICs inhibited mitogen-dependent human peripheral blood mononuclear cell (PBMC) proliferation and IL-2 production more efficiently than ABCB5(-) melanoma cell populations. Moreover, coculture with ABCB5(+) MMICs increased the abundance of Tregs, in a B7.2 signaling-dependent manner, along with IL-10 production by mitogen-activated PBMCs. Consistent with these findings, MMICs also preferentially inhibited IL-2 production and induced IL-10 secretion by cocultured patient-derived, syngeneic PBMCs. Our findings identify novel T-cell modulatory functions of ABCB5(+) melanoma subpopulations and suggest specific roles for these MMICs in the evasion of antitumor immunity and in cancer immunotherapeutic resistance.

Common receptor gamma chain (c-gamma) cytokines (CKs) support proliferation of CD8+ T cells in presence or absence of antigen triggering and help maintaining the immunologic memory. We addressed the effects of low (< or = 5 ng/mL)-dose interleukin (IL)-2, IL-7, or IL-15 on human naive and memory antigen-specific CD8+ T cells. Peripheral blood CD8+ lymphocytes proliferated with decreasing efficiency in response to IL-15, IL-7, and IL-2. Of note, IL-15 preferentially promoted expansion of CD45RA/CD8+ T-cell memory subset. Accordingly, cytotoxic T lymphocytes specific for cytomegalovirus-derived antigens from seropositive donors proliferated in response to IL-15 and, to lesser extent to IL-7, but poorly to IL-2. CD8+ T cells were then pretreated with CK before antigen stimulation using, as read out, specific cytotoxic activity. After the pretreatment with IL-15, but not IL-2, previously experienced viral antigens induced vigorous cytotoxic responses. Minor effects of IL-7 were also detectable. In contrast, IL-2 best supported the cytotoxic T lymphocyte generation from prevailingly naive CD8 T cells from HLA-A*0201 healthy donors, specific for L27Melan-A/MART-126-35 melanoma-associated antigen. Cells from melanoma patients were tested before and after Melan-A/MART-1-targeted antigen-specific immunotherapy. Untreated patients showed heterogeneous patterns of responsiveness to c-gamma CK. However, when naive patients whose CD8+ T cells best responded to IL-2 were vaccinated, a modified responsiveness pattern was detectable. After immunization, cells displayed a significantly higher response to IL-15 than to IL-2 pretreatment. Thus, responsiveness to c-gamma CK is critically influenced by naive or memory status of peripheral blood CD8+ T cells.

To the exception of early stages of disease, the morbidity and mortality of melanoma is considerable, with no acknowledged therapeutic options beyond surgery. Immunotherapy of melanoma has achieved some success, but further refinements are urgently needed in order to realize its potential. This paper describes a multi-centre phase I/II open labeled, controlled clinical trial investigating 2 innovative immunotherapeutic reagents. Two successive groups of 20 resected AJCC stages IIb-IV melanoma patients will be treated, first with melanoma epitopes included into Influenza virosomes (group 1), and second with a heterologous prime-boost protocol priming with a recombinant Vaccinia virus, and boosting with Influenza virosomes (group 2). Five melanoma epitopes from three different melanoma differentiation antigens were included into Influenza virosomes, that cross-stimulate CD4+ T cells and are endowed with high adjuvant capacity in the generation of CTL. The same five melanoma epitopes, two co-stimulatory molecules CD80 and CD86, and the CD40 ligand, a marker known to play a crucial role in CTL generation and memory maintenance were encoded in a recombinant Vaccinia virus. GM-CSF will be administered as a supporting cytokine. Both Influenza virosomes and octo-recombinant Vaccinia virus are innovative and original constructs assessed for the first time in human. Immunotherapy foresees 12 weekly immunizations for each group. Toxicity and adverse events will be monitored clinically. Immunological efficacy will be assessed dynamically by ex-vivo multimer analysis, Elispot, and quantitative real-time PCR for up to 3 months following completion of immunotherapy schedule. Disease free survival will be assessed by 4-monthly serial clinic visits, including physical and FDG-PET examinations, for a follow-up time of 2 years. Quality of life will be assessed with a dedicated FACT-BRM 4 questionnaire.

Because of its known heterogeneity, the analysis of antigen expression is crucial prior to the initiation of antigen-specific immunotherapy for melanoma. The melanoma differentiation antigens gp100, MART-1 and tyrosinase are involved in a common pathway of melanin synthesis. Peptides derived from these melanoma differentiation antigens are used in the immunotherapy of melanoma and antibodies recognizing these antigens are commonly applied to detect melanocytic lesions. One hundred and ninety-one paraffin-embedded melanoma metastases from 28 patients with 2-19 lesions (mean, 6.8) developing synchronously (n = 67) or asynchronously (n = 124) were analysed by immunohistochemistry for the expression of the melanoma differentiation antigens, as well as cancer/testis antigens of the melanoma antigen-A (MAGE-A) family (monoclonal antibodies 77B and 57B), anti-S100 and SM5-1. The overall reactivities were 81.6% (gp100), 79.5% (MART-1), 59.6% (tyrosinase), 59.1% (77B), 60.7% (57B), 93.2% (S100) and 91.6% (SM5-1). Twenty-seven lesions (14.1%) were positive for all tumour-associated antigens, 75 lesions (39.2%) were negative for one antigen and 87 lesions (45.5%) were negative for several tumour-associated antigens. Co-ordinated loss was found for lesions negative for gp100 and MART-1 (9.4%, P < 0.0005), gp100 and tyrosinase (11.0%, P = 0.009), MART-1 and tyrosinase (15.2%, P < 0.0005) and gp100, MART-1 and tyrosinase (8.9%, P < 0.0005), which is up to six times higher than the expected calculated loss. This co-ordinated loss of melanoma differentiation antigens in melanoma did not include cancer testis antigens and S100 or SM5-1. On average, the melanoma differentiation antigens stained 50-65% of cells within a lesion, and 10-39% of synchronous clusters were heterogeneous for melanoma differentiation antigen expression. In conclusion, broader polypeptide vaccines should be used for melanoma immunotherapy.

Production of indoleamine 2,3-dioxygenase (IDO) by tumor cells, leading to tryptophan depletion and production of immunosuppressive metabolites, may facilitate immune tolerance of cancer. IDO gene is also expressed in dendritic cells (DC) upon maturation induced by lipopolysaccarides or IFN. We investigated IDO gene expression in melanoma cell lines and clinical specimens as compared to mature DC (mDC). Furthermore, we explored effects of L-kynurenine (L-kyn) and 3-hydroxyanthranilic acid (3-HAA) on survival and antigen-dependent and independent proliferation of CD8(+) cells. We observed that IDO gene expression in cultured tumor cells and freshly excised samples is orders of magnitude lower than in mDC, providing highly efficient antigen presentation to CD8(+) T cells. Non toxic concentrations of L-kyn or 3-HAA did not significantly inhibit antigen-specific CTL responses. However, 3-HAA, but not L-kyn markedly inhibited antigen-independent proliferation of CD8(+) T cells induced by common receptor gamma-chain cytokines IL-2, -7 and -15. Our data suggest that CD8(+) T cell activation induced by antigenic stimulation, a function exquisitely fulfilled by mDC, is unaffected by tryptophan metabolites. Instead, in the absence of effective T cell receptor triggering, 3-HAA profoundly affects homeostatic proliferation of CD8(+) T cells.

Malignant tumors represent a major source of disability and account for more than one of five deaths in Western countries. Among the different cancers, melanoma harbors two distinctive features. First, its has long been recognized as an immunogenic tumor, and second, an unprecedented rise in incidence is currently observed, in face of few therapeutic options. Thus, melanoma represent an ideal target for a cancer immunotherapy program. To date, a number of immunodominant epitopes from tumor associated antigens (TAA) are used as cancer vaccines in clinical trials, in spite of an acknowledged rapid degradation in vivo and low immunogenicity. However, most of the immunotherapy trials reported so far do not achieve consistent clinical results. Hence, there is an urgent need for the development of a carrier system and strong adjuvants suitable for a TAA-based cancer immunotherapy. Liposomes and their further development as virosomes with added adjuvancy may address both these issues. We report here our experience in the tailoring of dedicated advanced liposomal vectors that were developed in the context of an upcoming immunotherapy clinical trial for melanoma.

Objective: To assess the effects of the culture of melanoma cells in 3-dimensional (3D) architectures on their immunorecognition by cytotoxic T lymphocytes (CTLs) specific for tumor-associated antigens., Summary Background Data: Growth in 3D architectures has been shown to promote the resistance of cancers to treatment with drugs, cytokines, or irradiation, thereby potentially playing an important role in tumor expansion. We investigated the effects of 3D culture on the recognition of melanoma cells by antigen-specific HLA class I-restricted CTLs., Methods: Culture of HBL melanoma cells expressing Melan-A/Mart-1 tumor-associated antigen and HLA-A0201 on poly-2-hydroxyethyl methacrylate (polyHEMA)-coated plates resulted in the generation of aggregates of 400- to 500-microm diameters containing on average 30,000 cells and characterized by slower proliferation, as compared with monolayer (2-dimensional) cultures. HLA-A0201 restricted Melan-A/Mart-127-35-specific CTL clones were used to evaluate tumor cell immunorecognition measured as specific IFN-gamma production. Comparative gene and protein expression in 2D and 3D cultures was studied by real-time PCR and flow cytometry, respectively. Overall differences in gene expression profiles between 2D and 3D cultures were evaluated by high-density oligonucleotide array hybridization., Results: HLA-A0201 restricted Melan-A/Mart-127-35 specific CTL clones produced high amounts of IFN-gamma upon short-term (4-24 hours) coincubation with HBL cells cultured in 2D but not in 3D, thus suggesting altered antigen recognition. Indeed, Melan-A/Mart-1 expression, at both gene and protein levels, was significantly decreased in 3D as compared with 2D cultures. Concomitantly, a parallel decrease of HLA class I molecule expression was also observed. Differential gene profiling studies on HBL cells showed an increased expression of genes encoding molecules involved in intercellular adhesion, such as junctional adhesion molecule 2 and cadherin-like 1 (>20- and 8-fold up-regulated, respectively) in 3D as compared with 2D cultures., Conclusions: Taken together, our data suggest that mere growth of melanoma cells in 3D architectures, in the absence of immunoselective pressure, may result in defective recognition by tumor-associated antigen-specific CTL.

Growth in three-dimensional (3D) architectures has been suggested to play an important role in tumor expansion and in the resistance of cancers to treatment with drugs or cytokines or irradiation. To obtain an insight into underlying molecular mechanisms, we addressed gene expression profiles of NA8 melanoma cells cultured in bidimensional monolayers (2D) or in 3D multicellular tumor spheroids (MCTS). MCTS containing 10-30,000 cells were generated upon overnight culture in poly-Hydroxyethylmethacrylate (polyHEMA) coated plates. Kinetics of cell proliferation in MCTS was significantly slower than in monolayer cultures. Following long-term culture (>10 days), however, MCTS showed highly compact and organised cell growth in outer layers, with necrotic cores. Oligonucleotide microarray analysis of the expression of over 20,000 genes was performed on cells cultured in standard 2D, in the presence of collagen as model of extracellular matrix (ECM), or in MCTS. Gene expression profiles of cells cultured in 2D in the presence or absence of ECM were highly similar, with >/=threefold differences limited to five genes. In contrast, culture in MCTS resulted in the significant, >/=threefold, upregulation of the expression of >100 transcripts while 73 were >/=threefold downregulated. In particular, genes encoding CXCL1, 2, and 3 (GRO-alpha, -beta, and gamma), IL-8, CCL20 (MIP-3alpha), and Angiopoietin-like 4 were significantly upregulated, whereas basic FGF and CD49d encoding genes were significantly downregulated. Oligonucleotide chip data were validated at the gene and protein level by quantitative real-time PCR, ELISA, and cell surface staining assays. Taken together, our data indicate that structural modifications of the architecture of tumor cell cultures result in a significant upregulation of the expression of a number of genes previously shown to play a role in melanoma progression and metastatic process., ((c) 2005 Wiley-Liss, Inc.)

We have comparatively evaluated the proliferative response of CTL induced in metastatic melanoma patients upon immunization against Melan-A/MART-1(27-35) tumor associated antigen (TAA) to IL-2, IL-7 or IL-15 cytokines, sharing a receptor common gamma-chain (c gamma-c cytokines). Twenty-eight CTL clones were generated from CD8+ T cells obtained from 3 patients during the contraction phase of immune response following a successful vaccine mediated expansion of specific effectors. All clones were able to kill tumor cell lines expressing HLA-A0201 and Melan-A/MART-1, and displayed phenotypic characteristics of effector/memory (CD45RA-/CCR7-) or CD45RA+/CCR7- effector cells in intermediate to late developmental stage (CD28-/CD276+/-) CTL. Proliferative responses could be elicited or enhanced by IL-2 and IL-15, but not IL-7, in the absence or in the presence of T-cell receptor (TCR) triggering, respectively. Accordingly, only IL-2 and IL-15 were able to promote the survival of the CTL clones under investigation. While all clones expressed high amounts of receptor c gamma-c (CD132), lower, but detectable, expression of IL-7 receptor alpha chain was also observed. CD8+ cells from one of the patients treated were obtained 6 months after the last vaccine boost and were cultured in the presence of Melan-A/MART-1(27-35) and each of the 3 cytokines under investigation. Consistent with data from CTL clones, expansion of Melan-A/MART-1(27-35) tetramer positive cells was only observed in the presence of IL-2 or IL-15 but not IL-7. Instead, when CD8+ cells from the same patient were sampled shortly (14 days) after an additional vaccination only IL-2 was able to promote the expansion of Melan-A/MART-1(27-35) tetramer positive cells. Taken together these data suggest a selective responsiveness of TAA-specific CTL to different c gamma-c cytokines., (Copyright 2005 Wiley-Liss, Inc)

Advanced-stage melanoma here dismal prognosis, and novel therapeutic approaches are urgently required. The possibility of taking advantage of the immune response of patients for its treatment has been an appealing concept for almost a century. Only during the last decade, however, has the molecular identification of tumor-associated antigens (TAAs) offered the possibility of vaccinating patients (e.g., active induction of TAA-specific immune responses). Active antigen-specific immunotherapy (AASIT) is currently being investigated in a number of clinical centers as a treatment option for advanced-stage melanoma. A large number of melanoma TAAs have been molecularly characterized and are being used in vaccination trials in various molecular forms and according to various immunization protocols. Here we provide a short overview on melanoma TAAs, the technologies currently in use to induce specific cytotoxic T-lymphocyte (CTL) responses in vivo, and their monitoring. We also propose a tentative AASIT agenda for the next few years, aiming at improving the capacity to induce and monitor TAA-specific immune responses and to verify their clinical effectiveness.

Objective: To evaluate MAGE tumor-associated antigen (TAA) expression in an extensive panel of normal and neoplastic tissues., Summary Background Data: TAAs of the MAGE family represent targets of active specific immunotherapy. Limited-size studies indicate that they are expressed in normal testis and tumors of different histologies. High-throughput tissue microarray (TMA) technology and MAGE TAA-specific monoclonal antibodies now allow us to comprehensively evaluate their expression in large numbers of tissues and to address clinical correlations., Methods: A TMA containing 3,520 samples from 197 different tissues and a non-small-cell lung cancer TMA including 301 specimens were stained using the MAGE TAA-specific monoclonal antibody 57B. For patients with squamous cell carcinoma of the lung, the dichotomous result (positive vs. negative) of MAGE TAA staining was used as a predictor variable along with other covariates in proportional hazard regression analysis of tumor-specific survival., Results: MAGE TAAs are expressed with frequencies ranging between 22.7% (larynx) and 50% of cases (lung) in squamous cell carcinomas from different anatomic areas and in large cell carcinomas of the lung (37.9%). The authors provide here the first description of MAGE TAA expression in basalioma (48.1%). To investigate the clinical significance of MAGE expression in a frequently positive tumor type, a non-small-cell lung cancer, TMA was then studied. In this TMA 43.2% of tumors were 57B positive. In patients with squamous cell carcinoma, MAGE TAA positivity was significantly correlated with a shorter tumor-specific survival in the proportional hazard regression analysis model., Conclusions: These data suggest novel potential therapeutic indications in different types of cancers. In lung squamous cell carcinoma, the significant association of MAGE TAA expression with poor prognosis suggests that patients with 57B-positive tumors may benefit from early, specific immunotherapy procedures.

The immunobiology of tumor-infiltrating dendritic cells (DCs) can be strongly influenced by the cytokine environment present in the malignant tissue. We have previously identified discrete melanoma lines, inducing E-cadherin expression on monocyte-derived DCs in vitro. We demonstrate here that this effect, independent of cell contact, is not inducible in the presence of tumor lysates and requires the constitutive expression of IFN stimulated gene 15 (ISG15) by malignant cells. High-density oligonucleotide arrays were used to investigate the expression pattern of 7000 genes in RNA from two melanoma cell clones competent for E-cadherin induction and two clones devoid of DC-modulating capacity. A total of 13 genes encoding soluble proteins were expressed at higher magnitude in melanomas able to induce E-cadherin expression on DCs. Combining those data with quantitative protein assays, we could narrow our investigation down to three factors: the chemokine CCL5 and the cytokines ISG15 and type I IFNs. Strikingly, >7 ng/ml of ISG15 could be detected in the corresponding melanoma-conditioned medium and induction of E-cadherin on DCs failed in the presence of antibodies neutralizing ISG15 protein. Most importantly, strong cytoplasmic expression of ISG15 was detected by immunohistochemistry in the original tumor specimen from which the melanoma cell lines under investigation were derived. These data describe a novel property of ISG15 targeting induction of E-cadherin on DCs and possibly influencing their migratory behavior.

A specific cellular immune response directed against a panel of three defined tumor-associated antigen (TAA) epitopes was induced in metastatic melanoma patients by a prime-boost strategy taking advantage of an innovative recombinant vaccinia virus as evaluated by quantitative assessment of cytotoxic T lymphocytes (CTLs) with corresponding specificity. The immunization protocol consisted of the administration of psoralen-UV-treated and replication-incompetent recombinant vaccinia virus encoding the three immunodominant HLA-A*0201-restricted epitopes Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) together with two costimulatory molecules, B7.1 and B7.2, in the context of systemic granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. Boosts were subsequently applied with corresponding synthetic nonapeptides and GM-CSF. Specific CTL induction was assessed by tetramer staining and CTL precursor (CTLp) frequency evaluation. Within 12 days of injection of the recombinant vector, cytotoxic T cell responses specific for engineered epitopes were detectable in three of three patients. During the vaccination treatment, antigen-specific CTLp frequencies exceeding 1:10,000 peripheral CD8(+) T cells could be observed. Tetramer staining also revealed significant increases in specific CD8(+) T cell numbers. We conclude that active specific antitumor vaccination can raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells with heterogeneous antigen expression. Data from this study emphasize the potency of a recombinant vaccinia virus vector encoding multiple minigenes and costimulatory molecules in the context of exogenously administered GM-CSF. Clinical effectiveness of this immunologically active protocol should therefore be explored in appropriately selected groups of patients.

In an ongoing phase I/II study, metastatic melanoma patients were treated with a replication-incompetent recombinant vaccinia virus (rVV) encoding Melan-A(27-35), gp100(280-288), and tyrosinase(1-9) HLA-A*201-restricted epitopes together with B7.1 and B7.2 co-stimulatory molecules. rVV was administered in the context of systemic GM-CSF treatment. Boosts were subsequently administered 2 weeks apart with corresponding synthetic nonapeptides and GM-CSF. Two cycles of treatment were administered 2 weeks apart from each other. Specific immune responses were evaluated by quantitative assessment of cytotoxic T-lymphocyte precursor frequency and tetramer staining. By the time the two cycles had been completed, four out of five patients showed significant (greater than threefold) increases in gp100(280-288)-specific and four out of five, in Melan-A(27-35)-specific tetramer staining of CD8+ cells. Frequencies of CTL precursors specific for gp100(280-288), tyrosinase(1-9) and Melan-A(27-35) were also significantly increased in all five, and in four and four of the five patients, respectively, in some cases within 12 days after the first injection of the recombinant vector. Thus, the innovative vector under investigation is able to raise a concurrent and specific cellular immune response against a panel of molecularly defined antigens, thereby increasing the chance of an immune hit against neoplastic cells displaying heterogeneous antigen expression.

Interferon alpha (IFN-alpha) represents an adjuvant therapy of proven effectiveness in increasing disease-free interval and survival in subgroups of melanoma patients. Since high doses of cytokine are required, the treatment is often accompanied by toxic side effects. In addition, naturally occurring insensitivity to IFN-alpha may hamper its therapeutic efficacy. Clinical, molecular or immunological markers enabling the selection of potential responders have not so far been identified. To explore the molecular basis of IFN-alpha responsiveness, we analyzed the expression pattern of about 7000 genes in IFN-alpha-sensitive and IFN-alpha-resistant cell lines using high-density oligonucleotide arrays. Melanoma cell lines were screened for their sensitivity to proliferation inhibition and HLA class I induction by IFN-alpha by standard 3H-thymidine incorporation and flow cytometry. Total cellular RNA from four sensitive and two resistant cell lines was extracted, reverse-transcribed and hybridized to high-density oligonucleotide arrays. The comparative analysis of gene expression in either set of cell lines allowed the identification of four genes (RCCl, IFI16, hox2 and h19) preferentially transcribed in sensitive cells and two (SHB and PKC-zeta) preferentially expressed in resistant cells. These data may provide a useful basis for the development of diagnostic tools to select potential IFN-alpha responders as eligible for treatment, while avoiding unnecessary toxicity to nonresponders.

Background/Aims: Members of the melanoma antigen encoding gene family are expressed in tumors of different histological types but not in normal tissue. For this reason, they are attractive targets for cancer immunotherapy.Methods: In the present study, we analyzed the expression of MAGE-1 and -3 genes in the hepatocellular carcinoma (HCC) tissue as well as frequency, phenotype and function of circulating and tumor infiltrating CD8+ cells specific for HLA-A1 and -A2 restricted epitopes of MAGE-1 and -3.Results: Our study shows for the first time the presence of MAGE/tetramer+ CD8 cells in the tumor tissue of patients with HCC. These cells are able to recognize the MAGE-1 sequence 161–169 and the MAGE-3 sequence 271–279. In a patient with a particularly high frequency of MAGE-1 sequence 161–169-specific T cells, phenotypic and functional analysis was performed showing a phenotype of recently-primed CD8 cells (CD28+CD27+CD45RA−CCR7).Conclusions: The observation of a spontaneous in vivo priming of a MAGE-specific T cell response in patients with HCC and the high frequency of MAGE antigens expression in this tumor, makes this antigen a potential candidate for a MAGE-specific immunotherapy in hepatocellular carcinoma. [Copyright &y& Elsevier]