Immunological techniques for monitoring of tumour antigen specific CD4+ T cell responses

We have developed immunological techniques for monitoring of tumour antigen specific CD4+ T cell responses. We set an in vitro system able to detect fona fide in vivo primed CD4+ T cells. Purified CD4+ T cells are first stimulated in vitro once in the presence peptides, previously identified as containing naturally processed epitopes on tumour-associated antigens. After two weeks, proliferation and cytokine secretion (IFN-?, IL-5, IL-4, IL-2, TNF-?, IL-10, TGF-?, IL-17, granzyme and perforin) are tested. To estimate the frequency of antigen specific CD4+ T cell we used intracellular staining for IFN-? and/or IL-13, following indications from restimulation assays. We investigated the spontaneous CD4+ T cell immunity to MAGE-A3 and CEA in normal donors and in patients with advanced melanoma and pancreas carcinoma, respectively. We found that MAGE-A3 specific CD4+ T cells, which showed an unpolarized or Th2 skewed phenotype, were present in the blood of a high percentage of melanoma patients. We also found that pancreatic cancer patients compared to normal donors have impaired quantitative (i.e., reduced number of responders) and qualitative (i.e., Th2 skew) anti-CEA CD4+ T cell response. Importantly, anti-viral CD4+ T immunity was preserved. These results suggest that local more than systemic immunoregulatory mechanisms are active in pancreatic cancer. We have optimized the MAGE-A3 peptides to load onto HLA-DR*1101 tetramers by generating minimal peptide analogues containing a single anchor residue and we succeeded to produce functional HLA-DR*1101 tetramers.

We plan to continue the monitoring of vaccine and tumour specific T cell responses in vaccinated melanoma patients and the spontaneous anti-tumour CD4+ T cell immunity in patients with MAGE-A3, CEA and HPV expressing neoplastic lesions by the use of the strategies developed inclusive of the HLA-DR*1101 tetramers.

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