Des lymphocytes T régulateurs spécifiques de MAGE-A3 sont détectés après vaccination chez des patients atteints de mélanome

Melanoma patients have been vaccinated with a MAGE-A3 peptide presented by HLA-DP4 molecules. This peptide was injected in combination with other antigens using several modalities: peptides alone, peptide-pulsed dendritic cells and protein MAGE-A3 mixed with peptides and adjuvant. Tumor regressions were observed in 14% of vaccinated patients, but only 4% of the patients had a objective clinical response. The low rate of clinical responses led us to study the presence of anti-MAGE-A3.DP4 T lymphocytes and their functional phenotype. Fourteen patients were selected. To isolate anti-vaccine T cells, we selected ex vivo the CD4+ T lymphocytes that were labeled by DP4/MAGE-A3 tetramers and the selected lymphocytes were expanded in clonal conditions. Anti-MAGE-A3.DP4 T lymphocytes were detected only after vaccination with frequencies that ranged from 2.10-6 to 2.10-3 among blood CD4+ T lymphocytes. To confirm that the tetramer+ clones recognized DP4 cells expressing MAGE-A3, we tested them for their ability to produce cytokines IFN-γ, IL-2, IL-10 or to upregulate activation marker CD25 upon stimulation with MAGE-A3-expressing cells. A total of 197 anti-MAGE-A3.DP4 clones were isolated. Interestingly, 5% of them expressed CD25 in the resting state, suggesting the presence of CD4+CD25+ regulatory T lymphocytes. A set of CD25+ clones and CD25- clones were selected and their phenotype was analysed in detail. The CD25+ clones had a high capacity to supress the proliferation of another CD4+ T cell clone after in vitro peptide stimulation. Most of them had a high FOXP3 expression in the resting state and had unmethylated sequences in the first intron of FOXP3. They produced active TGF-β but none of cytokines IFN-γ, IL-2, IL-4, IL-5 and IL-10. About 20% of CD25- clones had a signifiant but lower suppressive activity. Most of the CD25- clonal populations contained cells that expressed FOXP3 in the resting state, but lower than CD25+ clones. They had methylated sequences in the first intron of FOXP3. Our work does not provide any indication of an impact of anti-vaccine regulatory T lymphocytes on the efficacity of vaccination. But , to our knowledge, we have been the first to describe the presence of anti-vaccine regulatory T cells not only on the basis of markers such as FOXP3, but also on the basis of their suppressive activity in vitro. In addition, our work confirms that FOXP3 is not a specific marker of human regulatory T lymphocytes, because its expression is not correlated to suppressive activity and because it is expressed not only by regulatory T lymphocytes but also by resting CD4+CD25- T lymphocytes.