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Monitoring of Anti-Vaccine CD4 T Cell Frequencies in Melanoma Patients Vaccinated with a MAGE-3 Protein¹

Yi Zhang^*, Zhaojun Sun^*, Hugues Nicolay^*, Ralf G. Meyer, Nicolina Renkvist^*, Vincent Stroobant^*, Jurgen Corthals, Javier Carrasco^*, Alexander M. M. Eggermont, Marie Marchand^*, Kris Thielemans, Thomas Wölfel, Thierry Boon^* and Pierre van der Bruggen^2,*

^* Ludwig Institute for Cancer Research, Brussels Branch and Cellular Genetics Unit, Université de Louvain, Brussels, Belgium; Third Department of Internal Medicine, Johannes Gutenberg University, Mainz, Germany; Laboratory of Molecular and Cellular Therapy, Medical School of the Vrije Universiteit Brussel, Brussels, Belgium; and Erasmus Medical Center-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands

Quantitative evaluation of T cell responses of patients receiving antitumoral 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-, cloning of these cells, and evaluation of the number of T cell clones that secrete IFN- 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.

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