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