[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.

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.

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.

Identification of a MAGE-1 peptide recognized by cytolytic T lymphocytes on HLA-B*5701 tumors.

"Cancer germline" genes such as those of the MAGE family are expressed in many tumors and in male germline cells but are silent in normal tissues. They encode shared tumor-specific antigens that have been used in therapeutic vaccination trials of cancer patients. We report the identification of a new MAGE-1-encoded peptide that is recognized by a cytolytic T-lymphocyte (CTL) clone on human leukocyte antigen (HLA)-B*5701. The sequence of the peptide, corresponding to position 102-112 of the MAGE-1 protein sequence, is ITKKVADLVGF. When tumor cells expressing MAGE-1 were transfected with HLA-B*5701, they were lyzed by the CTL clone, indicating that the peptide is processed in tumor cells and can, therefore, be used as a target for anti-tumoral vaccination.

New MAGE-4 antigenic peptide recognized by cytolytic T lymphocytes on HLA-A1 tumor cells.

'Cancer-germline' genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in other normal tissues. They encode shared tumor-specific antigens, which have been used in small therapeutic vaccination trials of cancer patients. Gene MAGE-4, which is expressed in more than 50% of carcinomas of esophagus, head and neck, lung, and bladder, has two known alleles. Using PCR amplifications and digestions of the amplified product, we found that one third of the MAGE-4-positive samples expressed MAGE-4a. We folded HLA-A1 tetramers with peptide MAGE-4a169-177 EVDPASNTY, which is homologous to MAGE-1- and MAGE-3-encoded peptides recognized on HLA-A1 by cytolytic T lymphocytes. Blood lymphocytes from an individual without cancer were directly labelled with these A1/MAGE-4 tetramers. The very rare cells that were stained were sorted by flow cytometry and cloned. We isolated a cytolytic T-lymphocyte clone that lyzed specifically cells pulsed with this MAGE-4 peptide and HLA-A1 tumor cells expressing MAGE-4a, demonstrating that this antigenic peptide is processed efficiently in tumor cells. This peptide might therefore be useful for therapeutic antitumoral vaccination.

A MAGE-A4 peptide presented by HLA-B37 is recognized on human tumors by cytolytic T lymphocytes.

'Cancer-germline' genes such as those of the MAGE family are expressed in many tumors and in male germline cells, but are silent in normal tissues. They encode shared tumor-specific antigens, which have been used in therapeutic vaccination trials of cancer patients. MAGE-A4 is expressed in more than 50% of carcinomas of esophagus, head and neck, lung, and bladder. We report here the identification of a new MAGE-A4 encoded peptide, which is recognized by a cytolytic T lymphocyte (CTL) clone on HLA-B*3701. The sequence of the peptide is SESLKMIF. It corresponds to the MAGE-A4156-163 protein sequence. When tumor cells expressing MAGE-A4 were transfected with HLA-B*3701, they were recognized by the CTL clone, demonstrating that the peptide ought to be processed in tumor cells and could therefore serve as a target for therapeutic antitumoral vaccination.

A MAGE-3 peptide presented by HLA-B44 is also recognized by cytolytic T lymphocytes on HLA-B18.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their tumoral specificity and because they are shared by many tumors. Antigenic peptide MEVDPIGHLY, which is encoded by MAGE-3 and is known to be presented by human leukocyte antigen (HLA)-B44, is currently being used in therapeutic vaccination trials. We report here that a cytolytic T lymphocyte (CTL) clone, which is restricted by HLA-B*1801, recognizes the same peptide and, importantly, lyzes HLA-B18 tumor cells expressing MAGE-3. These results imply that the use of peptide MEVDPIGHLY can now be extended to HLA-B18 patients. We also provide evidence that, under limiting amounts of protein MAGE-3, HLA B*1801 and B*4403 compete for binding to the peptide.

A MAGE-1 peptide recognized on HLA-DR15 by CD4(+) T cells.

Antigens encoded by MAGE genes and recognized by T cells are of interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Several MAGE-1 peptide that are recognized by CD8(+) cytolytic T lymphocytes have been used in therapeutic vaccination trials. To obtain anti-tumor immune response, vaccines combining peptides recognized by CD8(+) and peptides recognized by CD4(+) T cells might be optimal. We focused therefore on the identification of MAGE peptides recognized by CD4(+) T cells. We report here the identification of MAGE-1 epitope EYVIKVSARVRF, which is presented to CD4(+) T lymphocytes by HLA-DR15. This HLA allele is present in 29 % of Asians and 17 % of Caucasians.

A MAGE-3 peptide recognized on HLA-B35 and HLA-A1 by cytolytic T lymphocytes.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Antigenic peptide EVDPIGHLY encoded by MAGE-3 and known to be presented by HLA-A*0101 is currently being used in therapeutic vaccination trials. We report here that a cytolytic T-lymphocyte (CTL) clone, which is restricted by HLA-B*3501, recognizes the same peptide and, importantly, lyses HLA-B*3501 tumor cells expressing MAGE-3. These results infer that the current clinical use of peptide EVDPIGHLY can now be extended to HLA-B*3501 patients.

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.

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.

A MAGE-A1 peptide presented to cytolytic T lymphocytes by both HLA-B35 and HLA-A1 molecules.

Antigens encoded by melanoma antigen (MAGE) genes are of particular interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. Antigenic peptide EADPTGHSY encoded by MAGE-A1 and known to be presented by HLA-A1 is currently being used in therapeutic vaccination trials. We report here that a cytotoxic T-lymphocyte (CTL) clone, which is restricted by HLA-B35, recognizes the same peptide and, importantly, lyses HLA-B35 tumor cells expressing MAGE-A1. This peptide can be presented to CTL by both HLA-B*3501 and HLA-B*3503 molecules, which are expressed by approximately 19% of Caucasians. These results infer that the current clinical use of peptide EADPTGHSY can now be extended to HLA-B35 patients.

A MAGE-A1 peptide is recognized on HLA-B7 human tumors by cytolytic T lymphocytes.

Antigens encoded by MAGE genes are of particular interest for cancer immunotherapy because of their strict tumoral specificity and because they are shared by many tumors. MAGE antigenic peptides are currently used in therapeutic vaccination trials. The identification of additional antigenic peptides is likely to be important for the future of these clinical trials in order to increase the number of patients eligible for these vaccinations and to analyze in detail the T-cell response of vaccinated patients. We describe here the isolation of a cytolytic T lymphocyte (CTL) clone which recognizes a new MAGE-A1 peptide, RVRFFFPSL (MAGE-A1(289-297)), which is presented by HLA-B7. This CTL clone lysed HLA-B7 tumor cells expressing MAGE-A1. HLA-B7 is expressed by approximately 20% of Caucasians

A human CTL recognizes a caspase-8-derived peptide on autologous HLA-B*3503 molecules and two unrelated peptides on allogeneic HLA-B*3501 molecules.

A CTL clone that recognizes autologous tumor cells was previously isolated from the blood of a head-and-neck cancer patient. The Ag was identified as peptide FPSDSWCYF presented by autologous HLA-B*3503 molecules. This peptide was encoded by a mutated CASP-8 gene, which is implicated in the triggering of apoptosis. Here, we show that this CTL clone, which expresses a single TCR, also recognizes two unrelated peptides on allogeneic HLA-B*3501 molecules. One peptide, HIPDVITY, is encoded by squalene synthase, and the other one, QFADVIVLF, is encoded by 2-hydroxyphytanoyl-CoA lyase. Both genes are expressed ubiquitously. These antigenic peptides are processed and presented by HLA-B*3501 cells. The two HLA-B35 alleles are closely related. Our results might reinforce the notion that the recognition of allogeneic HLA molecules depends on the presence in their groove of a limited number of peptides processed from ubiquitous proteins.

A tyrosinase peptide presented by HLA-B35 is recognized on a human melanoma by autologous cytotoxic T lymphocytes.

We previously described different cytotoxic T lymphocyte (CTL) clones isolated from the blood lymphocytes of a melanoma patient after in vitro stimulation with autologous tumor cells. These CTL clones recognized at least 2 distinct antigens on the melanoma cells. Here, we show that one of them consists of a peptide derived from tyrosinase and presented by HLA-B35. The peptide is 9 amino acids long and has the sequence LPSSADVEF. It can be presented by the 2 major B35 allelic subtypes, B*3501 and B*3503. As HLA-B35 is one of the most frequent HLA-B specificities, being present in about 20% of Caucasian individuals, it may be a useful target for peptide-based immunotherapy of melanoma.

A MAGE-A4 peptide presented by HLA-A2 is recognized by cytolytic T lymphocytes.

The MAGE-encoded antigens that are recognized by cytolytic T lymphocytes (CTL) are shared by many tumors and are strictly tumor specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes. We have used a method to identify CTL epitopes, which selects naturally processed peptides. CD8(+) T cells, obtained from individuals without cancer, were stimulated with autologous dendritic cells infected with a recombinant adenovirus containing the MAGE-A4 coding sequence. Responder cell microcultures that specifically lysed autologous EBV-transformed B cells infected with vaccinia-MAGE-A4 were cloned using autologous stimulator cells infected with a Yersinia enterocolitica carrying the MAGE-A4 sequence. An anti-MAGE-A4 CTL clone was obtained and the epitope was found to be decapeptide GVYDGREHTV (amino acids 230-239) presented by HLA-A2 molecules. The CTL clone lysed HLA-A2 tumor cells expressing MAGE-A4. This is the first reported antigenic peptide encoded by MAGE-A4. It may be valuable for cancer immunotherapy because MAGE-A4 is expressed in 51% of lung carcinomas and 63% of esophageal carcinomas, whereas about 50% of Caucasians and Asians express HLA-A2.

Identification of five MAGE-A1 epitopes recognized by cytolytic T lymphocytes obtained by in vitro stimulation with dendritic cells transduced with MAGE-A1.

MAGE genes are expressed by many human tumors of different histological types but not by normal cells, except for male germline cells. The Ags encoded by MAGE genes and recognized by T cells are therefore strictly tumor-specific. Clinical trials involving therapeutic vaccination of cancer patients with MAGE antigenic peptides or proteins are in progress. To increase the range of patients eligible for therapy with peptides, it is important to identify additional MAGE epitopes recognized by CTL. Candidate peptides known to bind to a given HLA have been used to stimulate T lymphocytes in vitro. In some instances, CTL clones directed against these synthetic peptides have been obtained, but these clones often failed to recognize tumor cells expressing the relevant gene. Therefore, we designed a method to identify CTL epitopes that selects naturally processed peptides. Monocyte-derived dendritic cells infected with a recombinant canarypoxvirus (ALVAC) containing the entire MAGE-A1 gene were used to stimulate CD8+ T lymphocytes from the blood of individuals without cancer. Responder cell microcultures that specifically lysed autologous cells expressing MAGE-A1 were cloned using autologous stimulator cells either transduced with a retrovirus coding for MAGE-A1 or infected with recombinant Yersinia-MAGE-A1 bacteria. The CTL clones were tested for their ability to lyse autologous cells loaded with each of a set of overlapping MAGE-A1 peptides. This strategy led to the identification of five new MAGE-A1 epitopes recognized by CTL clones on HLA-A3, -A28, -B53, -Cw2, and -Cw3 molecules. All of these CTL clones recognized target cells expressing gene MAGE-A1.

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.

Effect of epitope flanking residues on the presentation of N-terminal cytotoxic T lymphocyte epitopes.

We here demonstrate that placing two distinct influenza virus nucleoprotein epitopes at the N terminus of a cytosolic protein selectively blocks their presentation to specific cytotoxic T lymphocytes. The block is a cytosolic phenomenon, which can be overcome by distancing the epitope from the protein N terminus by two or more amino acids. Shortening the protein's C terminus fails to relieve the antigen presentation block. These results demonstrate that events at the N terminus of the target protein, rather than at its C terminus, are responsible for the lack of presentation of N-terminal epitopes. We also show that lack of presentation of N terminal epitopes is associated with a modification of the target protein which affects its electrophoretic mobility and isoelectric focusing point. This modification can be prevented by mutating the epitope's N-terminal flanking sequence, which results in an efficient presentation of the N-terminal epitope. Lack of presentation of the N-terminal epitopes results in a reduced ability of influenza-primed mice to clear acute infection with vaccinia virus encoding an N-terminal nucleoprotein epitope. Our results demonstrate that presentation of epitopes localized at the N terminus of cytosolic proteins can be modulated by events occurring at early stages of antigen processing.

Cytolytic T lymphocytes recognize an antigen encoded by MAGE-A10 on a human melanoma.

From melanoma patient LB1751, cytolytic T lymphocytes (CTL) were generated that lysed specifically autologous tumor cells. To establish whether these CTL recognized one of the Ags that had previously been defined, a CTL clone was stimulated with cells expressing various MAGE genes. It produced TNF upon stimulation with target cells expressing MAGE-A10. The Ag was found to be nonapeptide GLYDGMEHL (codons 254-262), which is presented by HLA-A2.1. This is the first report on the generation of anti-MAGE CTL by autologous mixed lymphocyte-tumor cell culture (MLTC) from a melanoma patient other than patient MZ2, from whom the first MAGE gene was identified. MAGE genes are expressed in many tumors but not by normal tissues except male germline cells and placenta, which do not express HLA molecules. Therefore, the identification of an antigenic peptide derived from MAGE-A10 adds to the repertoire of tumor-specific shared Ags available for anti-tumoral vaccination trials.