Investigating the role of the PD-1 pathway in human ovarian cancer

Abstract

Tumours have been shown to have a number of mechanisms to suppress immunosurveillance allowing them to survive and grow. One of these mechanisms involves expression of molecules which inhibit T cells such as the molecules involved in the PD-1 pathway. PD-1 is expressed on activated T cells and one of its ligands, PD-L1 has been shown to be expressed on a wide range of cell types including antigen presenting cells (APCs), endothelial cells as well as tumour cells. This pathway is important in maintaining T cell tolerance and homeostasis and as a result we hypothesised that this pathway may be an important target for immunotherapy in ovarian cancer. Ascites and blood from patients with ovarian cancer were collected over a two year period and analysis into the phenotype of immune cells and the cytokine content of the ascites was carried out. The major finding during this analysis was that APCs from ascites and blood of patients with malignant cancer showed a significantly higher level of PD-L1 than on APCs from patients with benign/borderline cancer. Furthermore the monocytes from ascites could suppress T cell proliferation in a co-culture which could be partially reversed when blocking PD-1. The cytokine content of the tumour revealed high levels of immunosuppressive (TGF-β and IL-10 correlated with malignancy) and pro-angiogenic cytokines which may contribute to the suppression of T cell proliferation observed when stimulated T cells were cultured in the ascites supernatant. These cytokines may also be responsible for the upregulation of PD-L1 observed on normal blood monocytes following culture in the ascites supernatant. Real time PCR analysis of tumour mass mRNA revealed higher levels of PD-L1 in borderline and malignant mRNA compared to normal ovary and flow cytometry showed that PD-L1 is expressed on ovarian cancer cell lines of serous histology. The second part of this project concentrated on the cloning of a PD-1 KDEL construct. PD-1 KDEL was to be used as a novel mechanism to down regulate PD-1 ligands on ovarian cancer cell lines. The construct was not successful in knocking down expression of PD-L1 on cancer cell lines and reasons were put forward for this lack of functionality. Each reason was tested experimentally and a hypothesis is put forward as to the reason of its lack of function. Alternatives to PD-1 KDEL were also tested.