Resistance to cancer immunotherapy mediated by apoptosis of tumor-infiltrating lymphocytes.

Despite impressive clinical success, cancer immunotherapy based on immune checkpoint blockade remains ineffective in many patients due to tumoral resistance. Here we use the autochthonous TiRP melanoma model, which recapitulates the tumoral resistance signature observed in human melanomas. TiRP tumors resist immunotherapy based on checkpoint blockade, cancer vaccines or adoptive T-cell therapy.

A new transcript in the TCRB locus unveils the human ortholog of the mouse pre-Dß1 promoter.

Introduction: While most transcripts arising from the human T Cell Receptor locus reflect fully rearranged genes, several germline transcripts have been identified. We describe a new germline transcript arising from the human TCRB locus.

Methods: cDNA sequencing, promoter, and gene expression analyses were used to characterize the new transcript.

Local immunostimulation leading to rejection of accepted male skin grafts by female mice as a model for cancer immunotherapy.

Female mice of inbred strain CBA do not reject syngeneic male skin grafts even though they mount a T-cell response against the male-specific HY antigen. We show that local immunostimulation performed by injecting cytokines and Toll-like receptor ligands in close vicinity to the graft causes rejection. We feel that this approach should be tested in tumor-bearing human patients in combination with antitumor vaccination.

Antigen spreading contributes to MAGE vaccination-induced regression of melanoma metastases.

A core challenge in cancer immunotherapy is to understand the basis for efficacious vaccine responses in human patients. In previous work we identified a melanoma patient who displayed a low-level antivaccine cytolytic T-cell (CTL) response in blood with tumor regression after vaccination with melanoma antigens (MAGE). Using a genetic approach including T-cell receptor β (TCRβ) cDNA libraries, we found very few antivaccine CTLs in regressing metastases.

Contrasting frequencies of antitumor and anti-vaccine T cells in metastases of a melanoma patient vaccinated with a MAGE tumor antigen.

Melanoma patients have high frequencies of T cells directed against antigens of their tumor. The frequency of these antitumor T cells in the blood is usually well above that of the anti-vaccine T cells observed after vaccination with tumor antigens. In a patient vaccinated with a MAGE-3 antigen presented by HLA-A1, we measured the frequencies of anti-vaccine and antitumor T cells in several metastases to evaluate their respective potential contribution to tumor rejection.

High frequency of antitumor T cells in the blood of melanoma patients before and after vaccination with tumor antigens.

After vaccination of melanoma patients with MAGE antigens, we observed that even in the few patients showing tumor regression, the frequency of anti-vaccine T cells in the blood was often either undetectable or <10(-5) of CD8 T cells. This frequency being arguably too low for these cells to be sole effectors of rejection, we reexamined the contribution of T cells recognizing other tumor antigens. The presence of such antitumor T cells in melanoma patients has been widely reported.

Monoclonal anti-MAGE-3 CTL responses in melanoma patients displaying tumor regression after vaccination with a recombinant canarypox virus.

We have analyzed the T cell responses of HLA-A1 metastatic melanoma patients with detectable disease, following vaccination with a recombinant ALVAC virus, which bears short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA-A1. To evaluate the anti-MAGE CTL responses, we resorted to antigenic stimulation of blood lymphocytes under limiting dilution conditions, followed by tetramer analysis and cloning of the tetramer-positive cells. The clones were tested for their specific lytic ability and their TCR sequences were obtained.

Cytolytic T-cell responses of cancer patients vaccinated with a MAGE antigen.

'Cancer-germline' genes such as the MAGE gene family are expressed in many tumors and in male germline cells but not in normal tissues. They encode shared tumor-specific antigens, which have been used in therapeutic vaccination trials of metastatic melanoma patients. To establish whether there is a correlation between tumoral regressions and T-cell responses against the vaccine antigen, we evaluated the responses of patients vaccinated with a MAGE-3 antigenic peptide or a recombinant virus coding for the peptide.

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.

The production of a new MAGE-3 peptide presented to cytolytic T lymphocytes by HLA-B40 requires the immunoproteasome.

By stimulating human CD8(+) T lymphocytes with autologous dendritic cells infected with an adenovirus encoding MAGE-3, we obtained a cytotoxic T lymphocyte (CTL) clone that recognized a new MAGE-3 antigenic peptide, AELVHFLLL, which is presented by HLA-B40. This peptide is also encoded by MAGE-12. The CTL clone recognized MAGE-3--expressing tumor cells only when they were first treated with IFN-gamma.

DNA microarray to monitor the expression of MAGE-A genes.

Background: The MAGE-A genes encode antigens that are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. We developed a rapid method to identify the MAGE-A genes expressed in tumors.

Methods: A low-density DNA microarray was designed to discriminate between the 12 MAGE-A cDNAs amplified by PCR with only one pair of consensus primers.

A monoclonal cytolytic T-lymphocyte response observed in a melanoma patient vaccinated with a tumor-specific antigenic peptide encoded by gene MAGE-3.

Vaccination of melanoma patients with tumor-specific antigens recognized by cytolytic T lymphocytes (CTL) produces significant tumor regressions in a minority of patients. These regressions appear to occur in the absence of massive CTL responses. To detect low-level responses, we resorted to antigenic stimulation of blood lymphocyte cultures in limiting dilution conditions, followed by tetramer analysis, cloning of the tetramer-positive cells, and T-cell receptor (TCR) sequence analysis of the CTL clones that showed strict specificity for the tumor antigen.

Identification on a human sarcoma of two new genes with tumor-specific expression.

Genes MAGE, BAGE, GAGE, and LAGE-1/NY-ESO-1 code for antigens that are recognized on melanoma cells by autologous CTLs. Because the pattern of expression of these genes results in the presence of antigens on many tumors of various histological types and not on normal tissues, these antigens qualify for cancer immunotherapy. To identify new genes with tumor-specific expression, we applied a cDNA subtraction approach, ie.

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.

DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter.

A subset of male germ line-specific genes, the MAGE-type genes, are activated in many human tumors, where they produce tumor-specific antigens recognized by cytolytic T lymphocytes. Previous studies on gene MAGE-A1 indicated that transcription factors regulating its expression are present in all tumor cell lines whether or not they express the gene. The analysis of two CpG sites located in the promoter showed a strong correlation between expression and demethylation.

Quantitative evaluation of the expression of MAGE genes in tumors by limiting dilution of cDNA libraries.

The MAGE-A genes are expressed in tumor cells but not in healthy tissues, except in male germ line cells and in placenta. They encode tumor-specific antigens recognized by autologous cytolytic T lymphocytes (CTLs). On the basis of semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) assays, 6 of the 12 members of the MAGE-A family, including MAGE-A1, were previously reported to have a high level of expression in tumors, whereas 5 other members, including MAGE-A10, were expressed at a much lower level, deemed to be insufficient for CTL recognition.

Differential requirement for CD4 help in the development of an antigen-specific CD8+ T cell response depending on the route of immunization.

Previous studies in our laboratory have shown that DBA/2 mice injected i.p. with syngeneic P815 tumor cells transfected with the HLA-CW3 gene (P815-CW3) showed a dramatic expansion of activated CD8+CD62L- T cells expressing exclusively the Vbeta10 segment.

Identification of a new MAGE gene with tumor-specific expression by representational difference analysis.

Human genes expressed exclusively in tumors and male germ line cells, such as those of the MAGE, BAGE, and GAGE families, encode antigens recognized by T lymphocytes, which are potentially useful for antitumor immunotherapy. To identify new genes of this type, we generated cDNA populations enriched in sequences expressed only in testis and melanoma, using the representational difference analysis approach. A testis cDNA library enriched by subtraction with cDNA from four other normal tissues was hybridized with radiolabeled melanoma cDNA enriched by subtraction with normal skin cDNA.

Two members of the human MAGEB gene family located in Xp21.3 are expressed in tumors of various histological origins.

Genes of the MAGE family direct the expression of tumor antigens recognized on a human melanoma by autologous cytolytic T lymphocytes. Twelve closely related MAGE genes are located in the Xq28 region. These genes share 60-98% nucleotide identity in their coding region.

Identification of human testis-specific transcripts and analysis of their expression in tumor cells.

Tumor-specific antigens recognized by autologous T lymphocytes are encoded by genes, including those of the MAGE, BAGE, and GAGE gene families, that are expressed in a significant fraction of tumors of various types, but not in normal adult tissues, except for testis where they appear to be expressed in germ cells. Because male germ cells are known to express many genes that are not expressed in other normal adult tissues, we wished to determine whether most of these genes are occasionally activated in tumor cells. Representational difference analysis was used to obtain testis-specific transcripts.

Identification of genes coding for tumor antigens recognized by cytolytic T lymphocytes.

Strategies have been developed to characterize tumor antigens recognized by cytolytic T lymphocytes (CTL). We use a genetic approach based on the transfection of HLA genes and cDNA libraries in COS cells to isolate the gene producing the antigenic peptide. The tumor-specific expression of this gene can be evaluated by cDNA synthesis and quantitative PCR amplification.

Genes coding for melanoma antigens recognised by cytolytic T lymphocytes.

It is now well established that human melanoma cells express antigens that are recognised by cytolytic T lymphocytes derived from the tumour-bearing patient. The molecular definition of these antigens is progressing at an accelerated pace. The currently characterised melanoma antigens can be classified into three categories: differentiation antigens, antigens encoded by genes that are specifically expressed in tumours, and antigens encoded by mutated genes.

Induction of a cytolytic T-cell response in mice with a recombinant adenovirus coding for tumor antigen P815A.

We investigated the efficacy of a recombinant adenovirus in inducing a cytolytic T-lymphocyte (CTL) response in mice against tumor antigen P815A, which is present on mouse mastocytoma P815. The recombinant adenoviral vector (Adeno.PIA) contained the sequence coding for the antigenic nonapeptide which binds to the H-2.