Abstract
BackgroundRecent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient’s body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. Approaches based on antigen presentation to CTLs by dendritic cells (DCs) are currently being intensively studied, because DCs are more efficient in tumor antigen presentation to T cells through their initiation of strong specific antitumor immune responses than other types of antigen-presenting cells. Today, it has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. This enables direct and specific cell-mediated immune responses against cells carrying certain antigens. The aim of the present study was to develop an optimized protocol for generating CTL populations specific for epitopes of tumor-associated antigen HER2/neu, and to assess their cytotoxic effects against the HER2/neu-expressing MCF-7 tumor cell line.MethodsThe developed protocol included sequential stages of obtaining mature DCs from PBMCs from HLA A*02-positive healthy donors, magnet-assisted transfection of mature DCs with the pMax plasmid encoding immunogenic peptides HER2 p369–377 (E75 peptide) and HER2 p689–697 (E88 peptide), coculture of antigen-activated DCs with autologous lymphocytes, magnetic-activated sorting of CTLs specific to HER2 epitopes, and stimulation of isolated CTLs with cytokines (IL-2, IL-7, and IL-15).ResultsThe resulting CTL populations were characterized by high contents of CD8+ cells (71.5% in cultures of E88-specific T cells and 90.2% in cultures of E75-specific T cells) and displayed strong cytotoxic effects against the MCF-7 cell line (percentages of damaged tumor cells in samples under investigation were 60.2 and 65.7% for E88- and E75-specific T cells, respectively; level of spontaneous death of target cells was 17.9%).ConclusionsThe developed protocol improves the efficiency of obtaining HER2/neu-specific CTLs and can be further used to obtain cell-based vaccines for eradicating targeted tumor cells to prevent tumor recurrence after the major tumor burden has been eliminated and preventing metastasis in patients with HER2-overexpressing tumors.
Highlights
Recent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient’s body
Evaluation of the phenotype and functional activity of the resulting dendritic cells (DCs) Phenotyping of adherent mononuclear cells (MNCs), immature DCs (iDCs), and mature DCs (mDCs) using specific antibodies against surface markers described in the literature as DC markers [23,24,25] was performed to evaluate the effectiveness of the protocol for producing DCs from the adherent fraction of MNCs
Significant differences in expression of the following markers were demonstrated for the populations of iDCs and mDCs: downregulation of CD14 expression (10.25 and 1.75%, respectively) and upregulation of CD83 expression (24.25 and 37.95%, respectively)
Summary
Recent fundamental and clinical studies have confirmed the effectiveness of utilizing the potential of the immune system to remove tumor cells disseminated in a patient’s body. Cytotoxic T lymphocytes (CTLs) are considered the main effectors in cell-mediated antitumor immunity. It has become possible to isolate CTLs specific for certain antigenic determinants from heterogeneous populations of mononuclear cells. The minimal residual disease remaining after resection of the major tumor burden underlies the existing problems of tumor recurrence and metastasis, which increase the mortality and morbidity rates among cancer patients In this connection, there is obviously a need for the development of new technologies that can improve the recognition and elimination of single cancer cells remaining in a patient’s body after radiation therapy, chemotherapy, or surgical resection. Cytotoxic effects of antigen-specific CTLs against cells of different tumor types have been demonstrated in a number of studies. Bernhard et al demonstrated that CTLs specific for E75 peptide of the HER2/neu tumor antigen could eliminate breast cancer cells in patients after adoptive transfer of HER2-specific CTL populations [3]
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