Abstract
Cyclin D1 is over-expressed in various human tumors and therefore can be a potential oncogenic target antigen. However, only a limited number of T cell epitopes has been characterized. We aimed at identifying human cyclin D1-derived peptides that include both CD4 and CD8 T cell epitopes and to test if such multi-epitope peptides could yield improved cytotoxic CD8 T cell responses as well as cytotoxic CD4 T cells. Five HLA-DR.B1-binding peptides containing multiple overlapping CD4 epitopes and HLA-A0201-restricted CD8 T cell epitopes were predicted by computer algorithms. Immunogenicity of the synthetic peptides was assessed by stimulating T cells from healthy donors in vitro and the epitope recognition was measured by IFN-γ ELISPOT and 51Chromium release assays. A HLA-DR.B1 peptide, designed “DR-1”, in which a HLA-A0201-binding epitopes (D1-1) was imbedded, induced CD3 T cell responses against both DR-1 and D1-1 peptides in IFN-γ ELISPOT assay. This suggested processing of the shorter D1-1 epitope from the DR-1 sequence. However, only DR-1-stimulated CD4 or CD3 T cells possessed cytotoxicity against peptide-pulsed autologous DCs and a cancer cell line, that expresses a high level of cyclin D1. Monoclonal antibody to HLA-DR abrogated the epitope-specific responses of both CD3 and CD4 T cells, demonstrating class II-mediated killing. Our studies suggest a possible role of CD4 T cells in anti-tumor immunity as cytotoxic effectors against HLA-DR expressing cancers and provide a rationale for designing peptide vaccines that include CD4 epitopes.
Highlights
Cyclin D1 is a key regulatory protein during the progression of cell cycle through G1 phase
Physical linking of T helper and cytotoxic T lymphocytes (CTLs) epitopes increases the magnitude and duration of the CTL response, suggesting that the presentation of both T helper and CTL epitopes on a single antigen presenting cell is more efficient than when two epitopes are presented on different antigen presenting cells (APCs) [18]
Our approach included the following steps: A) Prediction of potential epitopes by computer algorithms. This would serve to limit the number of possible sequence choices; B) Stimulation of T cells with selected peptides to confirm the immunogenicity of the epitopes; C) Testing of the response of the peptide-stimulated T cells against tumor cells that highly express cyclin D1, to confirm the processing and the presentation of the epitopes
Summary
Cyclin D1 is a key regulatory protein during the progression of cell cycle through G1 phase. The active kinase triggers phosphorylation of the retinoblastoma protein (RB), relieving its transcriptional repressive activities and its capacity to regulate components of the DNA replication and G2/M progression [1,2]. It is normally expressed at low levels in some organs and tissues, but is over-expressed in a number of human cancers, including mantle cell lymphoma (MCL), breast cancer, esophageal cancer and non-small cell lung cancer (NSCLC) [3,4,5,6]. Studies from esophageal cancer and murine model of MCL have suggested that this mutant protein is oncogenic [7,8,9]
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