A CASP-8 mutation recognized by cytolytic T lymphocytes on a human head and neck carcinoma.

Of the antigens recognized on human tumors by autologous cytolytic T lymphocytes, all those defined thus far have been identified on melanoma or renal cell carcinoma. We report here the identification of an antigen recognized by autologous cytolytic T lymphocytes on a human squamous cell carcinoma of the oral cavity. The antigen is encoded by a mutated form of the CASP-8 gene. This gene, also named FLICE or MACH, codes for protease caspase-8, which is required for induction of apoptosis through the Fas receptor and tumor necrosis factor receptor-1. The mutation, which was found in the tumor cells but not in the normal cells of the patient, modifies the stop codon and adds an Alu repeat to the coding region, thereby lengthening the protein by 88 amino acids. The ability of the altered protein to trigger apoptosis appears to be reduced relative to the normal caspase-8. The antigenic peptide is a nonamer presented by HLA-B*3503. The five last amino acids are encoded by the extension of the reading frame caused by the mutation. This, together with previous observations of CDK4 and beta-catenin mutations, suggests that a significant fraction of the point mutations generating a tumor antigen also play a role in the tumoral transformation or progression.

Identification of MAGE-3 epitopes presented by HLA-DR molecules to CD4(+) T lymphocytes.

MAGE-type genes are expressed by many tumors of different histological types and not by normal cells, except for male germline cells, which do not express major histocompatibility complex (MHC) molecules. Therefore, the antigens encoded by MAGE-type genes are strictly tumor specific and common to many tumors. We describe here the identification of the first MAGE-encoded epitopes presented by histocompatibility leukocyte antigen (HLA) class II molecules to CD4(+) T lymphocytes. Monocyte-derived dendritic cells were loaded with a MAGE-3 recombinant protein and used to stimulate autologous CD4(+) T cells. We isolated CD4(+) T cell clones that recognized two different MAGE-3 epitopes, MAGE-3114-127 and MAGE-3121-134, both presented by the HLA-DR13 molecule, which is expressed in 20% of Caucasians. The second epitope is also encoded by MAGE-1, -2, and -6. Our procedure should be applicable to other proteins for the identification of new tumor-specific antigens presented by HLA class II molecules. The knowledge of such antigens will be useful for evaluation of the immune response of cancer patients immunized with proteins or with recombinant viruses carrying entire genes coding for tumor antigens. The use of antigenic peptides presented by class II in addition to peptides presented by class I may also improve the efficacy of therapeutic antitumor vaccination.

Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes.

Human melanoma cell line MZ2-MEL expresses several antigens recognized by autologous cytolytic T lymphocyte (CTL) clones. We reported previously the identification of a gene, named MAGE-1, that codes for one of these antigens named MZ2-E. We show here that antigen MZ2-D, which is present on the same tumor, is encoded by another member of the MAGE gene family named MAGE-3. Like MAGE-1, MAGE-3 is composed of three exons and the large open reading frame is entirely located in the third exon. Its sequence shows 73% identity with MAGE-1. Like MZ2-E, antigen MZ2-D is presented by HLA-A1. The antigenic peptide of MZ2-D is a nonapeptide that is encoded by the sequence of MAGE-3 that is homologous to the MAGE-1 sequence coding for the MZ2-E peptide. Competition experiments using single Ala-substituted peptides indicated that amino acid residues Asp in position 3 and Tyr in position 9 were essential for binding of the MAGE-1 peptide to HLA-A1. Gene MAGE-3 is expressed in many tumors of several types, such as melanoma, head and neck squamous cell carcinoma, lung carcinoma and breast carcinoma, but not in normal tissues except for testes. It is expressed in a larger proportion of melanoma samples than MAGE-1. MAGE-3 encoded antigens may therefore have a wide applicability for specific immunotherapy of melanoma patients.

A nonapeptide encoded by human gene MAGE-1 is recognized on HLA-A1 by cytolytic T lymphocytes directed against tumor antigen MZ2-E.

We have reported the identification of human gene MAGE-1, which directs the expression of an antigen recognized on a melanoma by autologous cytolytic T lymphocytes (CTL). We show here that CTL directed against this antigen, which was named MZ2-E, recognize a nonapeptide encoded by the third exon of gene MAGE-1. The CTL also recognize this peptide when it is presented by mouse cells transfected with an HLA-A1 gene, confirming the association of antigen MZ2-E with the HLA-A1 molecule. Other members of the MAGE gene family do not code for the same peptide, suggesting that only MAGE-1 produces the antigen recognized by the anti-MZ2-E CTL. Our results open the possibility of immunizing HLA-A1 patients whose tumor expresses MAGE-1 either with the antigenic peptide or with autologous antigen-presenting cells pulsed with the peptide.

A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.

Many human melanoma tumors express antigens that are recognized in vitro by cytolytic T lymphocytes (CTLs) derived from the tumor-bearing patient. A gene was identified that directed the expression of antigen MZ2-E on a human melanoma cell line. This gene shows no similarity to known sequences and belongs to a family of at least three genes. It is expressed by the original melanoma cells, other melanoma cell lines, and by some tumor cells of other histological types. No expression was observed in a panel of normal tissues. Antigen MZ2-E appears to be presented by HLA-A1; anti-MZ2-E CTLs of the original patient recognized two melanoma cell lines of other HLA-A1 patients that expressed the gene. Thus, precisely targeted immunotherapy directed against antigen MZ2-E could be provided to individuals identified by HLA typing and analysis of the RNA of a small tumor sample.

[Is it possible to correct the anergy of T lymphocytes that infiltrate tumors?]. / Est-il possible de corriger l'anergie des lymphocytes T infiltrant les tumeurs?

Human tumor metastases are often infiltrated by T lymphocytes that are specific for tumor antigens, but these metastases progress anyway. The spontaneous anti-tumor immune response seems thus to become ineffective, probably because the effector T cells become anergic. This anergy could result from inhibitory mechanisms orchestrated by the tumor cells. We have observed that recently stimulated human cytolytic T cell clones lose transiently their capacity to secrete cytokines. This anergy is correlated with the absence of colocalization of the T cell receptors (TCR) and the CD8 co-receptors. Effector functions' and TCR/CD8 colocalization are recovered by treating cells with galectin-3 ligands, suggesting that exracellular galectin-3 forms glycoprotein-galectin lattices, which decrease the TCR mobility on the surface of anergic T lymphocytes. Galectin-3 is frequently released by tumor cells. This new mechanism of anergy could thus also explain the loss of functions of the tumor-infiltrating lymphocytes, because these lymphocytes recover their effector functions and TCR/CD8 colocalization after ex vivo treatment with galectin-3 ligands. These results could lead to new therapeutical strategies.

Direct isolation, phenotyping and cloning of low-frequency antigen-specific cytotoxic T lymphocytes from peripheral blood.

Cytotoxic T lymphocytes (CTLs) play an important role in controlling viral infections and certain tumours, but characterising specific CTL responses has always been technically limited. Fluorogenic 'tetramers' of major histocompatibility complex (MHC) class I complexes have been exploited recently to quantify the massive expansion of specific CTLs in human immunodeficiency virus (HIV) infection [1]. Here, we use MHC class I complex tetramers to isolate low-frequency antigen-specific CTLs directly from human peripheral blood, allowing the simultaneous phenotypic and functional characterisation and cloning of these CTLs. We synthesised a tetramer that specifically stained human leukocyte antigen (HLA)-A2. 1-restricted CTL clones recognising the influenza matrix protein peptide 58-66, matrix 58-66 [2]. This tetramer stained between 1 in 1,500 and 1 in 58,000 peripheral blood mononuclear cells (PBMCs) from HLA-A2.1+ individuals. The surface phenotype of these cells could be analysed by fluorescence-activated cell sorting (FACS), and the cells could be directly sorted into enzyme-linked immunospot (ELISpot) plates, where they released interferon-gamma (IFN-gamma) within 1 day of antigen exposure. The same population was cloned by FACS, and the specificity of several expanded clones was confirmed. Cloning was greatly simplified and accelerated compared with standard protocols, and was highly efficient. We also used tetramer-based sorting to enrich melanoma-specific CTLs derived from a tumour-infiltrated lymph node. Direct cloning of specific CTLs from peripheral blood can provide important information about immunological memory, CTL responses against tumour antigens and CTL proliferation and function, and opens up new possibilities for generating CTLs for adoptive immunotherapy.

Molecular definition of tumor antigens recognized by T lymphocytes.

Several tumor antigens recognized by T cells have now been identified at the molecular level. Various mechanisms can account for their expression: activation of normally silent genes; point mutations; chromosome translocations; and post-translational modifications of proteins. This led to the notion that potential tumor-rejection antigens can be shared by a significant proportion of human tumors. This may have important implications in cancer immunotherapy, especially since tumors expressing a defined antigen can be identified on the basis of the expression of the relevant gene.

T cell defined tumor antigens.

T cell defined antigens have now been characterized in a large variety of tumor types, in both mice and humans. An increasing number of these antigens appear to result from tumor-specific mutations, and some of these mutations may be implicated in oncogenesis. The priority is now to demonstrate that immunization against some of these antigens is clinically valuable for antitumor therapy, and the first results of clinical pilot studies are now emerging.

A novel approach to identify antigens recognized by CD4 T cells using complement-opsonized bacteria expressing a cDNA library.

In patients with hematological malignancies receiving HLA-matched stem cell transplantation, T cells specific for minor histocompatibility antigens play a major role in graft rejection, induction of graft-versus-host disease and beneficial graft-versus-leukemia reactivity. Several human minor histocompatibility antigens recognized by T cells have been identified, but only two are presented by HLA class II molecules. In search of an efficient approach to identify antigenic peptides processed through the HLA class II pathway, we constructed a cDNA library in bacteria that were induced to express proteins. Bacteria were opsonized with complement to enforce receptor-mediated uptake by Epstein-Barr virus immortalized B cells that were subsequently used as antigen-presenting cells. This approach was validated with an HLA class II-restricted antigen encoded by gene DBY. We were able to identify bacteria expressing DBY diluted into a 300-fold excess of bacteria expressing a nonrelevant gene. Screening of a bacterial library using a DBY-specific CD4 T cell clone resulted in the isolation of several DBY cDNAs. We propose this strategy for a rapid identification of HLA class II-restricted antigenic peptides recognized by CD4 T cells.

A retrogen strategy for presentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potent antitumor T helper and CTL responses.

Induction of an effective antitumor response requires CD4+ helper T (Th) cells to recognize antigens on the same dendritic cells (DCs) that cross-present CTL antigens. Such cross-presentation is difficult to achieve by current tumor vaccine strategies. Here, we develop a novel "Retrogen" strategy for DCs to efficiently cross-present an intracellular tumor antigen, MAGE-3, to both MHC class I and MHC class II in a cognate manner. Specifically, the MAGE-3 gene was linked to a leader sequence at its NH2 terminus for secretion and to a cell-binding domain at its COOH terminus for receptor-mediated internalization. DCs transduced with the modified MAGE-3 gene produced and secreted MAGE-3 proteins, which were efficiently taken up by DCs via receptor-mediated internalization and presented as exogenous antigens to class I and class II molecules. Immunization of mice with the transduced DCs expressing the MAGE-3 fusion protein, termed "Retrogen" for its retrograde transport/internalization after secretion, efficiently induced all arms of the adaptive antitumor immune responses. Thus, this retrogen strategy of using a unifying mechanism for DCs to cross-present an intracellular tumor antigen in a cognate manner could be generally used to improve the efficacy of tumor vaccines and immunotherapies.

Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis.

Here, we report the functional expression of CD40 on human malignant melanomas (MMs). Comparison of tumor specimen from MM precursor lesions, primary tumors, and metastases revealed that CD40 surface expression is down-regulated during tumor progression. CD40 expression was confirmed in 7 human MM cell lines established from immunogenic primary tumors or metastases, whereas 11 cell lines established from advanced stages were CD40 negative. CD40 expression could be enhanced in CD40-positive MM by stimulation with IFN-gamma and tumor necrosis factor-alpha but not by interleukin (IL)-1beta or CD40 triggering. CD40 ligation on MM by CD40L-transfected murine L-cells or by a soluble CD40L fusion protein up-regulated their expression of intercellular adhesion molecule-1 and MHC class I and class II molecules and their secretion of IL-6, IL-8, tumor necrosis factor-a, and granulocyte macrophage colony-stimulating factor and also induced a rapid activation of the transcription factor nuclear factor kappaB. Furthermore, CD40 ligation of a HLA-A2+, MelanA/MART1+ MM cell line enhanced its susceptibility to specific lysis by a HLA-A2-restricted, MelanA/MART-1-specific CTL clone. Finally, CD40 ligation induced growth inhibition and apoptosis in MM. These results indicate that CD40-CD40L interactions may play an important role in augmenting antitumor immunity and inducing apoptosis in some CD40-positive immunogenic human MMs.

A MAGE-A3 peptide presented by HLA-DP4 is recognized on tumor cells by CD4+ cytolytic T lymphocytes.

Antigens encoded by MAGE-A3 and recognized by T cells are interesting targets for tumor immunotherapy because they are strictly tumor specific and shared by many tumors of various histological types. A number of MAGE-A3 antigenic peptides presented by HLA class I molecules have been used in clinical trials, and regressions of melanoma metastasis have been observed. We report here the identification of a MAGE-A3 epitope, TQHFVQENYLEY, presented to CD4+ T lymphocytes by HLA-DP4 molecules, which are expressed in approximately 76% of Caucasians. This new epitope may be useful both for therapeutic vaccination and for the evaluation of the immune response in cancer patients. Interest ingly, the CD4+ T cells lysed HLA-DP4 tumor cells expressing MAGE-A3, indicating that this epitope, in contrast to other class-II MAGE-A3 epitopes, is presented at the surface of tumor cells. The study of this disparity in the presentation of two epitopes from the same protein may lead to a better understanding of the endogenous class II presentation pathway.

Characterization of the GAGE genes that are expressed in various human cancers and in normal testis.

The GAGE-1 gene was identified previously as a gene that codes for an antigenic peptide, YRPRPRRY, which was presented on a human melanoma by HLA-Cw6 molecules and recognized by a clone of CTLs derived from the patient bearing the tumor. By screening a cDNA library from this melanoma, we identified five additional, closely related genes named GAGE-2-6. We report here that further screening of this library led to the identification of two more genes, GAGE-7B and -8. GAGE-1, -2, and -8 code for peptide YRPRPRRY. Using another antitumor CTL clone isolated from the same melanoma patient, we identified antigenic peptide, YYWPRPRRY, which is encoded by GAGE-3, -4, -5, -6, and -7B and which is presented by HLA-A29 molecules. Genomic cloning of GAGE-7B showed that it is composed of five exons. Sequence alignment showed that an additional exon, which is present only in the mRNA of GAGE-1, has been disrupted in gene GAGE-7B by the insertion of a long interspersed repeated element retroposon. These GAGE genes are located in the p11.2-p11.4 region of chromosome X. They are not expressed in normal tissues, except in testis, but a large proportion of tumors of various histological origins express at least one of these genes. Treatment of normal and tumor cultured cells with a demethylating agent, azadeoxycytidine, resulted in the transcriptional activation of GAGE genes, suggesting that their expression in tumors results from a demethylation process.

Molecular cloning and identification of murine caspase-8.

Several caspases are mediators of apoptotic cell death. We describe a novel murine member of this growing protein family. Based on homology and especially on the substrate specificity, this new procaspase is identified as the murine counterpart of human procaspase-8. The protein exhibits a rather low similarity (76%) and identity (70%) to human procaspase-8. Procaspase-8 mRNA is expressed in all adult mouse tissues examined, the highest levels being reached in kidney, liver and lung. Procaspase-8 mRNA expression is highest in seven-day old embryos, but also during later stages of development the expression was fairly high. Both human and murine procaspase-8 are very weak substrates for granzyme B as compared to procaspase-3. Murine procaspases-1, 2, 3, 6, 7, 8, 11/4 and 12 are processed by recombinant murine caspase-8, suggesting a key role in the procaspase activation cascade. In addition, murine caspase-8 induced cell death that was inhibited both by cytokine response modifier A and p35. In vitro experiments demonstrated that p35 inhibits caspase-8 directly.

Mitochondrial DNA polymerases from yeast to man: a new family of polymerases.

We report the sequence of a 4.5-kb cDNA clone isolated from a human melanoma library which bears high amino acid sequence identity to the yeast mitochondrial (mt) DNA polymerase (Mip1p). This cDNA contains a 3720-bp open reading frame encoding a predicted 140-kDa polypeptide that is 43% identical to Mip1p. The N-terminal part of the sequence contains a 13 glutamine stretch encoded by a CAG trinucleotide repeat which is not found in the other DNA polymerases gamma (Pol gamma). Multiple amino acid sequence alignments with Pol gamma from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Drosophila melanogaster, Xenopus laevis and Mus musculus show that these DNA polymerases form a family strongly conserved from yeast to man and are only loosely related to the Family A DNA polymerases.

MAGE-1 expression threshold for the lysis of melanoma cell lines by a specific cytotoxic T lymphocyte.

Human gene MAGE-1 codes for an antigen that is recognized on a melanoma by an autologous cytotoxic T lymphocyte (CTL). Because MAGE-1 is expressed on a significant proportion of tumours of various histological types and not on normal tissues, the encoded antigen may serve as a target for cancer immunotherapy. Evaluation of the expression of the gene by reverse transcription polymerase chain reaction (RT-PCR) in various tumour samples and tumour cell lines has suggested great variability in the level of expression. It was therefore important to evaluate the minimal level of expression required for lysis by CTL. We tested a number of melanoma cell lines by a quantitative RT-PCR assay to correlate their level of MAGE-1 expression and recognition by the relevant CTL clone. We found that only cell lines expressing more than 10% of the MAGE-1 messenger RNA (mRNA) level of reference cell line MZ2-MEL.3.0 (i.e. more than three mRNA molecules per cell) were lysed by the CTL or induced significant tumour necrosis factor release.

Electroporation of immature and mature dendritic cells: implications for dendritic cell-based vaccines.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial.

The long-standing quest for tumor rejection antigens.

Several tumor antigens recognized by T lymphocytes have now been identified at the molecular level. Different mechanisms could account for their expression: activation of normally silent genes, point mutations in normally expressed genes, chromosomal translocations generating combinatorial epitopes, or post-translational modifications of proteins. A major consequence of these results is the notion that potential tumor rejection antigens are often shared by a significant proportion of human tumors. This may have important implications in cancer immunotherapy, especially since tumors expressing a defined antigen can be identified clinically on the basis of the expression of the relevant gene.