Functional characterisation of the T cell mediated anti-tumour response in a melanoma patient : identification of a HLA-DRβ1*10011 restricted unique antigen

CD4+ T cells are central in regulating most adaptive immune responses and essential for sustaing CD8+ T cell activation and efficient tumour destruction. Therefore, a better understanding of the CD4+ T cell-mediated mechanisms involved in anti-tumour responses will be crucial to improve vaccination strategies. The objective of this thesis was to evaluate the anti-tumour CD4+ T cell response in a metastatic melanoma patient, Pt15392, disease free after 12 years from surgical removal of lymph node metastasis and who was previously shown to display a CD8+ T cell response against epitopes included in the TRP-2 and gp100 antigens. Several CD4+ T cell clones were isolated from the metastatic lymph nodes by limiting dilution assay. TCR analysis defined five groups of clones which all showed a strong TCR-dependent cytotoxic activity in response to in vitro stimulation with the autologous melanoma. Functional analysis demonstrated that all the isolated T cell clones recognised tumour cells in HLA-DR restricted fashion. In addition to exert a cytotoxic activity in response to tumor stimulation, these T cell clones released cytokines compatible with a Thl profile although they also specifically produced high amounts of IL-10. An invariant chain (Ii) - cDNA fusion library was constructed in an attempt to identify the melanoma antigen recognised by these CD4+ T cell clones. The screening resulted in the detection of a mutated form of the human protein tyrosine phosphatase receptor kappa (PTPR-k) gene, as a DRβ1*10011-restricted tumour antigen. All the T lymphocyte clones isolated in vitro were directed against a DRβ1*10011 presented peptide that contained the mutation while none of these T cells were activated by the wild type peptide. Thus, the favourable clinical outcome observed in Pt15392, may be correlated with a strong polyclonal CD4+ anti-tumour response. These data emphasise that continued monitoring of CD4+ T cell immune responses in cancer patients may allow the identification of new class II HLA-restricted antigens to be used for designing more effective cancer therapies

Functional characterisation of the T cell mediated anti tumour response in a melanoma patient Identification of a HLA DR#beta#1*10011 restricted unique antigen

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A polyclonal anti-vaccine CD4 T cell response detected with HLA-DP4 multimers in a melanoma patient vaccinated with MAGE-3.DP4-peptide-pulsed dendritic cells

During the last few years, HLA class I tetramers have been successfully used to demonstrate anti-vaccine CD8 CTL proliferation in cancer patients vaccinated with tumor antigens. Frequencies of CTL as low as 10(-6) among CD8 cells were observed even in patients showing tumor regression. Little is known about the role of tumor-antigen-specific CD4 T cells in the context of these anti-vaccine responses. Therefore, we developed a very sensitive approach using fluorescent class-II-peptide multimers to detect antigen-specific CD4 T cells in vaccinated cancer patients. We produced HLA-DP4 multimers loaded with the MAGE-3(243-258) peptide and used them to stain ex vivo PBL from melanoma patients injected with dendritic cells pulsed with several class I and class II tumor antigenic peptides, including the MAGE-3(243-258) peptide. The multimer(+) CD4 T cells were sorted and amplified in clonal conditions; specificity was assessed by their ability to secrete IFN-gamma upon contact with the MAGE-3 antigen. We detected frequencies of about 1 x 10(-6) anti-MAGE-3.DP4 cells among CD4 cells. A detailed analysis of one patient showed an anti-MAGE-3.DP4 CD4 T cell amplification of at least 3000-fold upon immunization. TCR analysis of the clones from this patient demonstrated a polyclonal response against the MAGE-3 peptide

The CD4(+) T-cell response of melanoma patients to a MAGE-A3 peptide vaccine involves potential regulatory T cells.

Melanoma patients were injected with various vaccines containing a MAGE-A3 peptide presented by HLA-DP4. Anti-MAGE-A3.DP4 T cells were not detectable in the blood before vaccination, but their frequencies after vaccination ranged from 2 x 10(-6) to 2 x 10(-3) among the CD4(+) blood T lymphocytes of the patients. The CD4(+) blood T lymphocytes that stained ex vivo with HLA-DP4 tetramers folded with the MAGE-A3 peptide were selected by flow cytometry and amplified under clonal conditions. About 5% of the CD4(+) T-cell clones that recognized the MAGE-A3.DP4 antigen had a CD25(+) phenotype in the resting state. These CD25(+) clones had a high capacity to suppress the proliferation of another T-cell clone after peptide stimulation in vitro. Most of them had high FOXP3 expression in the resting state and an unmethylated FOXP3 intron 1. They produced active transforming growth factor-beta but none of cytokines IFN-gamma, interleukin-2 (IL-2), IL-4, IL-5, and IL-10. About 20% of CD25(-) clones had a significant but lower suppressive activity. Most of the CD25(-) clonal populations contained cells that expressed FOXP3 in the resting state, but FOXP3 demethylation was not observed. We conclude that MAGE-A3.DP4 vaccination can produce CD4(+) T cells that may exert regulatory T-cell function in vivo.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Vaccination of a Melanoma Patient with Mature Dendritic Cells Pulsed with MAGE-3 Peptides Triggers the Activity of Nonvaccine Anti-Tumor Cells.

We previously characterized the CTL response of a melanoma patient who experienced tumor regression following vaccination with an ALVAC virus coding for a MAGE-A3 Ag. Whereas anti-vaccine CTL were rare in the blood and inside metastases of this patient, anti-tumor CTL recognizing other tumor Ags, mainly MAGE-C2, were 100 times more frequent in the blood and considerably enriched in metastases following vaccination. In this study we report the analysis of the CTL response of a second melanoma patient who showed a mixed tumor response after vaccination with dendritic cells pulsed with two MAGE-A3 antigenic peptides presented, respectively, by HLA-A1 and HLA-DP4. Anti-MAGE-3.A1 CD8 and anti-MAGE-3.DP4 CD4 T cells became detectable in the blood after vaccination at a frequency of approximately 10(-5) among the CD8 or CD4 T cells, respectively, and they were slightly enriched in slowly progressing metastases. Additional anti-tumor CTL were present in the blood at a frequency of 2 x 10(-4) among the CD8 T cells and, among these, an anti-MAGE-C2 CTL clone was detected only following vaccination and was enriched by >1,000-fold in metastases relative to the blood. The striking similarity of these results with our previous observations further supports the hypothesis that the induction of a few anti-vaccine T cells may prime or restimulate additional anti-tumor T cell clones that are mainly responsible for the tumor regression

Monitoring of anti-vaccine CD4 T cell frequencies in melanoma patients vaccinated with a MAGE-3 protein

Quantitative evaluation of T cell responses of patients receiving antiturnoral vaccination with a protein is difficult because of the large number of possible HLA-peptide combinations that could be targeted by the response. To evaluate the responses of patients vaccinated with protein MAGE-3, we have developed an approach that involves overnight stimulation of blood T cells with autologous dendritic cells loaded with the protein, sorting by flow cytometry of the T cells that produce IFN-gamma, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN-gamma upon. stimulation with the Ag. An important criterion is that T cell clones must recognize not only stimulator cells loaded with the protein, but also stimulator cells transduced with the MAGE-3 gene, so as to exclude the T cells that recognize contaminants generated by the protein production system. Using this approach it is possible to measure T cell frequencies as low as 10(-6). We analyzed the frequencies of anti-vaccine CD4 T cells in five metastatic melanoma patients who had been injected with a MAGE-3 protein without adjuvant and showed evidence of tumor regression. Anti-MAGE-3 CD4 T cells were detected in one of the five patients. The frequency of the anti-MAGE-3 CD4 T cells was estimated at 1/60,000 of the CD4 T cells in postvaccination blood samples, representing at least an 80-fold increase in the frequency found before immunization. The frequencies of one anti-MAGE-3 CD4 T cell clonotype were confirmed by PCR analysis on blood lymphocytes. The 13 anti-MAGE-3 clones, which corresponded to five different TCR clonotypes, recognized the same peptide presented by HLA-DR1