Cyclin D1 as a Universally-Expressed Mantle Cell Lymphoma-Associated aTumor Antigen for Immunotherapy

Abstract

Mantle cell lymphoma (MCL) accounts for 5% to 10% of all non-Hodgkin lymphomas and has the worst prognosis among all lymphomas. The hallmark of MCL is a t(11;14) translocation resulting in overexpression of cyclin D1 by tumor cells of virtually all patients. In this study we examined whether cyclin D1 could be an effective tumor-associated antigen for immunotherapy. We identified a cyclin D1 peptide (P101) for HLA-A*0201, created heteroclitic peptide (Py101) for better binding, and generated peptide-specific T-cell lines from HLA-A*0201+ blood donors and MCL patients. After 5 to 7 rounds of in vitro stimulation with peptide-pulsed autologous dendritic cells (DCs), T-cell lines were obtained, which contained about 45% CD4+ and 55% CD8+ T cells. As exemplified by the results of peptide-tetramer staining, the frequencies of peptide-specific CD8+ T cells increased during in vitro stimulation, from 5.7% of specific T cells at fourth stimulation to 19.7% at sixth stimulation. These cell lines proliferated in response to autologous DCs pulsed with cyclin D1 peptide Py101 (but not unpulsed), as measured by 3[H]-thymidine incorporation and CFSE-dilution assays. These results indicate that the T cells are indeed specific for cyclin D1 Py101 peptide. Moreover, the T cells efficiently lysed peptide-pulsed but not unpulsed T2 cell and autologous DCs, and cyclin D1+ and HLA-A*0201+ human MCL lines MINO, SP53, Jeko-1, and Granta 519, and more importantly, HLA-A*0201+ primary lymphoma cells from MCL patients. No killing was observed on HLA-A*0201− primary lymphoma cells or HLA-A*0201+ normal blood cells including B cells. These results indicate that these T cells are potent cytotoxic T cells and recognize cyclin D1 peptides naturally presented by patient lymphoma cells in the context of surface HLA-A*0201 molecules. Flow cytometry analysis was used to examine the expression of granzymes, perforin, and Fas ligand (FasL) by the T cells. Based on these results, T cells may kill their target cells via the perforin/granzyme pathways, because they express perforin and high levels of granzymes but not FasL. Two independent methods were used to examine the cytokine expression profiles of the T cells. Upon restimulation with DCs pulsed with Py101 peptide, but not with unpulsed DCs, 11.2% of the T cells expressed IFN-γ by intracellular cytokine staining. IL-4-expressing T cells were very few (0.3%). To detect cytokine secretion, an ELISPOT assay was used to enumerate IFN-γ-secreting cells. After restimulation with DCs pulsed with cyclin D1 peptides, or with cyclin D1+/HLA-A*0201+ SP53 and cyclin D1+/HLA-A*0201+ primary lymphoma cells from patients, large numbers of IFN-γ-secreting cells were detected. Taken together, our work identifies cyclin D1 as a potentially important antigen for immunotherapy in MCL.