Aline Van Pel

Structure of the gene of tum− transplantation antigen P91A: The mutated exon encodes a peptide recognized with Ld by cytolytic T cells

Mutagen treatment of mouse P815 tumor cells produces immunogenic mutants that express new transplantation antigens (tum- antigens) recognized by cytolytic T cells. We found that the gene conferring expression of tum- antigen P91A contains 12 exons, encoding a 60 kd protein lacking a typical N-terminal signal sequence. The sequence shows no significant similarity with sequences in current data bases. A mutation that causes expression of the antigen is located in exon 4; it is the only apparent difference between the normal and the antigenic alleles. A short synthetic peptide corresponding to a region of exon 4 located around this mutation makes P815 cells sensitive to lysis by anti-P91A cytolytic T cells. The mutation creates a strong aggretope enabling the peptide to bind the H-2 Ld molecule. Several secondary tumor cell variants that no longer express tum- antigen P91A were found to carry deletions in the gene.

Polyclonal CTL Responses Observed in Melanoma Patients Vaccinated with Dendritic Cells Pulsed with a MAGE-3.A1 Peptide

Vaccination with mature, monocyte-derived dendritic cells (DC) pulsed with the MAGE-3168–176 peptide, which is presented by HLA-A1, has been reported to induce tumor regressions and CTL in some advanced stage IV melanoma patients. We present here a precise evaluation of the level of some of these anti-MAGE-3.A1 CTL responses and an analysis of their clonal diversity. Blood lymphocytes were stimulated with the MAGE-3.A1 peptide under limiting dilution conditions and assayed with an A1/MAGE-3 tetramer. This was followed by the cloning of the tetramer-positive cells and by TCR sequence analysis of the CTL clones that lysed targets expressing MAGE-3.A1. We also used direct ex vivo tetramer staining of CD8 cells, sorting, and cloning of the positive cells. In three patients who showed regression of some of their metastases after vaccination, CTL responses were observed with frequencies ranging from 7 × 10−6 to 9 × 10−4 of CD8+ blood T lymphocytes, representing an increase of 20- to 400-fold of the frequencies found before immunization. A fourth patient showed neither tumor regression nor an anti-MAGE-3.A1 CTL response. In each of the responses, several CTL clones were amplified. This polyclonality contrasts with the monoclonality of the CTL responses observed in patients vaccinated with MAGE-3.A1 peptide or with an ALVAC recombinant virus coding for this antigenic peptide.

A new tumor-specific antigen encoded by MAGE-C2 and presented to cytolytic T lymphocytes by HLA-B44

A panel of cytolytic T lymphocyte (CTL) clones was isolated from metastases and blood samples of a melanoma patient vaccinated with MAGE-3.A1-pulsed autologous dendritic cells. We report here the identification of a new antigen encoded by the MAGE-C2 cancer-germline gene. This antigen is recognized by some of these CTL on HLA-B*4403. The sequence of the peptide is SESIKKKVL. It is processed in various melanoma cell lines expressing MAGE-C2 and HLA-B*4403. Because of the expression pattern of gene MAGE-C2, this new antigen is strictly tumor-specific and could therefore be used for peptide-based antitumoral vaccination.

Stimulation of Cytolytic T Lymphocytes By Azaguanine-resistant Mouse-tumor Cells in Selective Hat Medium

Primed syngeneic or unprimed allogeneic mouse spleen cells were stimulated with azaguanine-resistant P815 tumor cells that were killed by the addition of aminopterin to the stimulation medium. The recovery of lymphocytes and their cytolytic activity and specificity were similar to those obtained after stimulation with irradiated cells. This method conveniently replaces the inactivation of stimulatory cells by irradiation or mitomycin treatment. Moreover, it has the advantage of inactivating not only the stimulatory cells but also the tumor cells that often contaminate the spleens of tumor-bearing animals, provided these animals have been inoculated with azaguanine-resistant tumor cell mutants.

Genes Coding for TUM- Transplantation Antigens. A Model for TSTA?

It has been known for a long time that some experimental tumors express antigens that constitute targets for immune rejection by the syngencic host. The existence of these tumor-specific transplantation antigens (TSTA) was first demonstrated with chemically induced mouse sarcomas: each independent tumor was found to express a different antigen (Prchn 1957). Later, these findings were extended to ultraviolet-induced tumors (Kripke 1981). The generality of the existence of transplantation antigens specific for each tumor was however put under serious question when spontaneous mouse tumors were found to be completely incapable of eliciting an immune rejection response (Hewitt 1976). But further, experiments demonstrated that even these tumors express weak TSTA that arc potential targets for immune rejection (Van Pel 1983).