Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes.

Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100(mel)47-52/40-42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100(mel)47-52/40-42 generation is enhanced in the presence of the ß5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8(+) T cell response. Importantly, we demonstrate that different gp100(mel)-derived spliced epitopes are generated and presented to CD8(+) T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100(mel)-derived spliced epitopes trigger activation of CD8(+) T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes.

Rheumatoid arthritis synovial fibroblasts produce a soluble form of the interleukin-7 receptor in response to pro-inflammatory cytokines.

We previously demonstrated that baseline synovial overexpression of the interleukin-7 receptor α-chain (IL-7R) is associated with poor response to tumour necrosis factor (TNF) blockade in rheumatoid arthritis (RA). We found that IL-7R gene expression is induced in fibroblast-like synovial cells (FLS) by the addition of TNF-α, IL-1ß and combinations of TNF-α+ IL-1ß or TNF-α+ IL-17, thereby suggesting that these cytokines play a role in the resistance to TNF blockade in RA. Because FLS and CD4 T cells also produce a soluble form of IL-7R (sIL-7R), resulting from an alternative splicing of the full-length transcript, we wondered whether expression of sIL-7R is similarly regulated by pro-inflammatory cytokines. We also investigated whether sIL-7R is detectable in the serum of RA patients and associated with response to TNF blockade. RA FLS were cultured in the presence of pro-inflammatory cytokines and sIL-7R concentrations were measured in culture supernatants. Similarly, sIL-7R titres were measured in sera obtained from healthy individuals, early untreated RA patients with active disease and disease-modifying anti-rheumatic drug (DMARD)-resistant RA patients prior to initiation of TNF-blockade. Baseline serum sIL-7R titres were correlated with validated clinical measurements of disease activity. We found that exposure of RA FLS to pro-inflammatory cytokines (TNF-α, IL-1ß and combinations of TNF-α and IL-1ß or TNF-α and IL-17) induces sIL-7R secretion. Activated CD4 T cells also produce sIL-7R. sIL-7R serum levels are higher in RA patients as compared to controls. In DMARD-resistant patients, high sIL-7R serum concentrations are strongly associated with poor response to TNF-blockade. In conclusion, sIL-7R is induced by pro-inflammatory cytokines in RA FLS. sIL-7R could qualify as a new biomarker of response to therapy in RA.

A peptide derived from melanocytic protein gp100 and presented by HLA-B35 is recognized by autologous cytolytic T lymphocytes on melanoma cells.

A panel of autologous cytolytic T lymphocyte (CTL) clones have been isolated from blood lymphocytes of a melanoma patient after in vitro stimulation with autologous tumor cells. We previously reported the molecular definition of three distinct antigens recognized by some of these CTL clones. We describe here, the identification of a fourth antigenic peptide expressed by this melanoma line and recognized by a CTL clone restricted by HLA-B*3503. The antigenic peptide, which is nine-amino acid long, has the sequence LPHSSSHWL and is derived from melanocyte differentiation antigen gp100. As HLA-B35 is one of the most frequent HLA-B alleles, being present in 20% of the Caucasian individuals, this peptide may be a good target for peptide-based immunotherapy of melanoma.

An alternative open reading frame of the human macrophage colony-stimulating factor gene is independently translated and codes for an antigenic peptide of 14 amino acids recognized by tumor-infiltrating CD8 T lymphocytes.

We show that cytotoxic T lymphocytes (CTLs) infiltrating a kidney tumor recognize a peptide encoded by an alternative open reading frame (ORF) of the macrophage colony-stimulating factor (M-CSF) gene. Remarkably, this alternative ORF, which is translated in many tumors concurrently with the major ORF, is also translated in some tissues that do not produce M-CSF, such as liver and kidney. Such a dissociation of the translation of two overlapping ORFs from the same gene is unexpected. The antigenic peptide encoded by the alternative ORF is presented by human histocompatibility leukocyte antigen (HLA)-B*3501 and has a length of 14 residues. Peptide elution indicated that tumor cells naturally present this 14 mer, which is the longest peptide known to be recognized by CTLs. Binding studies of peptide analogues suggest that it binds by its two extremities and bulges out of the HLA groove to compensate for its length.

Differential processing of class-I-restricted epitopes by the standard proteasome and the immunoproteasome.

Upon exposure to IFN-gamma, the standard proteasome is replaced by the immunoproteasome, which contains LMP2, LMP7 and MECL1, and is considered more efficient at producing antigenic peptides presented to CD8(+) T cells. This view has been challenged this year by reports showing that some epitopes, mainly of self origin, are not processed by the immunoproteasome and that mature dendritic cells constitutively express immunoproteasomes and therefore cannot efficiently present such epitopes.

Cytolytic T lymphocytes raised against a human bladder carcinoma recognize an antigen encoded by gene MAGE-A12.

Human bladder carcinoma line LB831-BLC expresses several distinct Ags that are recognized by different autologous CTL. Here, we show that one of these Ags is presented by HLA-Cw7 and encoded by gene MAGE-A12. This is the first time that CTL directed against a MAGE-encoded Ag have been derived from the lymphocytes of a patient with cancer other than melanoma. This new Ag was found to be nonapeptide VRIGHLYIL, corresponding to position 170-178 of the MAGE-A12 protein. Gene MAGE-A12 is silent in normal tissues except in male germline cells, which do not express HLA molecules. It is expressed in 26-62% of melanomas, infiltrating bladder carcinomas, lung carcinomas, esophageal carcinomas, and head and neck carcinomas. Because HLA-Cw7 is present in 43% of Caucasians, this new Ag is shared by many tumors and should be a useful target for cancer immunotherapy.

Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells.

By stimulating human lymphocytes with an autologous renal carcinoma, we obtained CTL recognizing an antigen derived from a novel, ubiquitous protein. The CTL failed to lyse autologous EBV-transformed B cells, even though the latter express the protein. This is due to the presence in these cells of immunoproteasomes, which, unlike standard proteasomes, cannot produce the antigenic peptide. We show that dendritic cells also carry immunoproteasomes and fail to present this antigen. This may explain why the relevant CTL escape thymic deletion and are not regularly activated in the periphery. Lack of cleavage by the immunoproteasome was also observed for melanoma differentiation antigen Melan-A26-35/HLA-A2, currently used for antitumoral vaccination. For immunization with such antigens, proteins should be less suitable than peptides, which do not require proteasome digestion in dendritic cells.

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 new antigen recognized by cytolytic T lymphocytes on a human kidney tumor results from reverse strand transcription.

By stimulating blood lymphocytes from a renal cell carcinoma patient in vitro with the autologous tumor cells, we obtained cytolytic T lymphocyte (CTL) clones that killed several autologous and allogeneic histocompatibility leukocyte antigen (HLA)-B7 renal carcinoma cell lines. We identified the target antigen of these CTLs by screening COS cells transfected with the HLA-B7 cDNA and with a cDNA library prepared with RNA from the tumor cells. The antigenic peptide recognized by the CTLs has the sequence LPRWPPPQL and is encoded by a new gene, which we named RU2. This gene is transcribed in both directions. The antigenic peptide is not encoded by the sense transcript, RU2S, which is expressed ubiquitously. It is encoded by an antisense transcript, RU2AS, which starts from a cryptic promoter located on the reverse strand of the first intron and ends up on the reverse strand of the RU2S promoter, which contains a polyadenylation signal. This mechanism of antigen expression is unprecedented and further illustrates the notion that many peptides recognized by T cells cannot be predicted from the primary structure of the major product of the encoding gene. Antisense transcript RU2AS is expressed in a high proportion of tumors of various histological types. It is absent in most normal tissues, but is expressed in testis and kidney, and, at lower levels, in urinary bladder and liver. Short-term cultures of normal epithelial cells from the renal proximal tubule expressed significant levels of RU2AS message and were recognized by the CTLs. Therefore, this antigen is not tumor specific, but corresponds to a self-antigen with restricted tissue distribution.

Identification of a second major tumor-specific antigen recognized by CTLs on mouse mastocytoma P815.

Murine mastocytoma P815 induces CTL responses against at least four distinct Ags (AB, C, D, and E). Recent studies have shown that the main component of the CTL response against the P815 tumor is targeted against Ags P815AB and P815E. The gene P1A has been well characterized. It encodes the P815AB Ag in the form of a nonameric peptide containing two epitopes, P815A and P815B, which are recognized by different CTLs. Here, we report the identification of the P815E Ag. Using a cDNA library derived from tumor P815, we identified the gene coding for P815E. We also characterized the antigenic peptide that anti-P815E CTLs recognize on the MHC class I molecule H-2Kd. The P815E Ag results from a mutation within an ubiquitously expressed gene encoding methionine sulfoxide reductase, an enzyme that is believed to be important in the protection of proteins against the by-products of aerobic metabolism. Surprisingly, immunizing mice i.p. with syngeneic tumor cells (L1210) that were constructed to express B7-1 and P815E did not induce resistance against live P815, even though a strong anti-P815E CTL response was observed with splenocytes from immunized animals.

The shared tumor-specific antigen encoded by mouse gene P1A is a target not only for cytolytic T lymphocytes but also for tumor rejection.

A number of human tumor antigens have been characterized recently using cytolytic T lymphocytes (CTL) as screening tools. Some of them are encoded by MAGE-type genes, which are silent in normal tissues except in male germ cells, but are activated in a variety of tumors. These tumor-specific shared antigens appear to be promising targets for cancer immunotherapy. However, the choice of these antigens as targets has been questioned because of the lack of direct evidence that in vivo responses against such antigens can lead to tumor rejection. The antigen encoded by the mouse gene P1A represents the only available animal model system for MAGE-type tumor antigens. We show here that mice immunized by injection of L1210 leukemia cells expressing P1A and B7-1 (L1210.P1A.B7-1) are efficiently protected against a challenge with a lethal dose of mastocytoma P815 tumor cells, which express P1A. Mice immunized with L1210 cells expressing B7-1 but not P1A were not protected. Furthermore, we observed that P1A-transgenic mice, which are tolerant to P1A, were not protected after immunization with L1210.P1A.B7-1. These results demonstrate that the immune response to P1A is the major component of the tumor rejection response observed in normal mice, and support the use of tumor-specific shared antigens as targets for the immunotherapy of human cancer.

An antigen recognized by autologous CTLs on a human bladder carcinoma.

By stimulating blood lymphocytes with autologous bladder carcinoma cells that had been transfected with B7-1, we obtained a panel of CTL clones which lyse specifically the bladder tumor cells in an MHC class I-restricted fashion. Based on inhibition with anti-HLA Abs and the recognition of allogeneic tumor cells, we could distribute our clones in three groups that recognized three distinct Ags. We characterized one of these Ags by screening a cDNA library prepared with the RNA from this bladder tumor line. This new tumor Ag is a peptide presented by HLA-B4403 molecules. It is produced by a point mutation in a gene that is recorded in databases under the name KIAA0205, is ubiquitously expressed, and whose function is unknown. We also found this mutation in the tumor sample that was originally resected from this patient, but the mutation was not found in the 100 or more independent tumors of various histologic types that were tested. This report is the first to describe the isolation of CTL clones directed against human bladder cancer and the molecular characterization of a bladder tumor Ag.

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.

The expression of mouse gene P1A in testis does not prevent safe induction of cytolytic T cells against a P1A-encoded tumor antigen.

Tumor antigen P815AB is recognized by cytolytic T lymphocytes (CTL) on mouse mastocytoma P815. This antigen is encoded by P1A, a gene activated in several tumors but silent in normal tissues except for testis and placenta. Notwithstanding the expression of P1A in testis, we found that male mice mounted P815AB-specific CTL responses as efficiently as females. The responding males remained fertile and no autoimmune lesions were observed in their testes. By immunohistochemistry with a rabbit antiserum directed against the P1A protein, we identified spermatogonia as the testicular cells expressing P1A. The absence of MHC class-I molecules on spermatogonia could be one of the mechanisms of protection against testicular autoimmunity, as the antigenic peptide should not be displayed at the cell surface. Human genes MAGE, BAGE and GAGE, which also code for tumor antigens recognized by autologous CTL, are not expressed in normal tissues other than testis. The results obtained in mice with antigen P815AB suggest that immunization of human males with such antigens will not generate autoimmune side-effects. Although P1A is strongly expressed in placenta, we also found that gestation did not prevent generation of CTL responses against antigen P815AB, and that such CTL responses did not affect gestation outcome. We identified labyrinthine trophoblasts as the placental cells expressing P1A. Again, the absence of MHC class-I molecules on these cells provides a plausible explanation for placental protection, although other mechanisms may also play a role.

Tumor antigens recognized by T lymphocytes.

In the last five years, knowledge of human tumor antigens recognized by autologous cytolytic T lymphocytes (CTL) has increased considerably. So far, genetic and biochemical approaches have led to the molecular identification of three classes of antigens. Most of these antigens consist of peptides that are presented to T cells by HLA molecules. The first class comprises antigens encoded by genes such as MAGE, BAGE, and GAGE, which are expressed in various tumors of different histological origins, but not in normal tissues other than testis. The second class represents differentiation antigens encoded by genes that are only expressed in melanoma and normal melanocytes like tyrosinase, Melan-A/MART-1, gp100 and gp75. The third class includes antigens produced by unique point mutations in genes that are ubiquitously expressed. In most cases, the antigenic peptide is encoded by the mutated region of the gene. A number of these antigens provide promising targets for new protocols of specific cancer immunotherapy.

Renal-cell carcinoma-specific lysis by cytotoxic T-lymphocyte clones isolated from peripheral blood lymphocytes and tumor-infiltrating lymphocytes.

Melanoma and renal-cell carcinoma (RCC) are generally considered to be relatively immunogenic tumor types in humans. In the case of melanoma, many major histocompatibility complex (MHC) class I-restricted tumor-specific cytotoxic T lymphocytes (CTL) have been isolated from either tumor-infiltrating lymphocytes (TIL) or autologous peripheral blood lymphocytes (PBL). In contrast, such CTL have only incidentally been described in the case of RCC. It has often been reported that TIL lines isolated from RCC display non-MHC-restricted and non-specific activity. Here, we report the isolation and characterization of tumor-specific CTL from PBL of one RCC patient and from TIL of another RCC patient. CTL clones 263/17 and 263/45, isolated from the PBL of patient LE-9211, were restricted by HLA-B7. CTL clone 5E, isolated from the TIL of patient LE-8915, was restricted by HLA-B37. The autologous RCC cell lines were efficiently lysed by the CTL clones, whereas normal epithelial cells of the proximal tubuli matched for the restriction element and K562 were not. From a panel of allogeneic RCC cell lines, CTL 5E recognized MZ-1940-RCC. Reactivity to allogeneic RCC sharing HLA-B7 was also observed with CTL 263/17 and 263/45, both of which could lyse the HLA-B7-positive cell line MZ-1851-RCC. Our data provide evidence that common tumor antigens are recognized by CTL on RCC.

A new gene coding for an antigen recognized by autologous cytolytic T lymphocytes on a human renal carcinoma.

Previous reports have described antigens that are recognized on human melanoma cells by autologous cytolytic T lymphocytes (CTL). The genes coding for a number of these antigens have been identified. Here we report the cloning of a gene that codes for an antigen recognized by autologous CTL on a human renal carcinoma cell line. This antigen is presented by HLA-B7 and is encoded by a new gene that we have named RAGE1. No expression of RAGE1 was found in normal tissues other than retina. RAGE1 expression was found in only one of 57 renal cell carcinoma samples, and also in some sarcomas, infiltrating bladder carcinomas, and melanomas. This represents the first identification of an antigen recognized by autologous CTL on a renal tumor.