Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

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Abstract
T‐helper (Th) cells play an important role in orchestrating the effector function of CTL in anti‐tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen‐presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA‐bound peptides and identification of the antigen‐derived peptides by mass spectrometric comparison to the non‐transfected cells. In vitro‐stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA‐DR4‐restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.