There is a network of cell surface molecules and cytokines involved in humoral and cellular immune responses (441). Cellular immune responses have an important role in stimulating a response against tumour growth (442). Recently, researchers have emphasised the importance of active suppressor mechanisms arising from the tumour and from the immune system itself that can inhibit anti-tumour immune reactions in vivo (157), (181), (443). A wide array of regulatory mechanisms of the immune system is exploited by tumours to avoid or suppress immune responses (444), (445). These include a network of soluble factors such as cytokines and specialised T cell subsets that repress cellular immunity known as Tregs.
The past 10 years have witnessed an increased interest in the field of active mechanisms of immune regulation, and much of this renewed interest has been centred on Tregs (446), (447). Tregs keep the immune system in check, limiting the potential for auto reactivity and autoimmune diseases (448). Most of the early studies of human cancer defined Tregs using a platform of CD4 and CD25 co-expressions. Some studies have characterised Tregs as being unnecessary for the activation of cells that express CD69, lacking in CD62L, exhibiting a blastoid phenotype or expressing Ki67 and corroborating the CD4+ CD25+ Treg phenotype and CTLA-4 (18), (250), (449), (450), (451). However, the core features of Tregs now include the expression of FOXP3, the constitutive expression of CTLA4 and GITR, an inability to proliferate in response to
113 TCR-driven signals in vitro, a dependence on IL-2 for expansion, and the ability to suppress the proliferation of other T cells in vitro and in vivo (202), (446), (452), (453), (454). Despite their differences, the most physiologically relevant Treg population is CD4+ CD25+ Tregs, which are naturally present in the immune system as 10% of CD4+ T cells and which specifically express the transcription factor FOXP3 (449).
Numerous studies in recent years have found increased frequencies of CD4+ CD25+ T cells, with some or all of the features of Tregs in the peripheral blood of patients with a wide array of cancers, including head and neck cancer, lung and ovarian cancer, gastrointestinal cancers, pancreatic and breast cancers and skin cancer (250), (254), (450), (455), (456), (457), (458). In addition, in several studies the tumour associated Tregs that were identified via phenotypic criteria were further isolated and shown to be highly suppressive in in vitro functional assays (459). The two most frequent observations collectively provide strong evidence that Tregs do improve immunity against a wide variety of human tumours. Firstly, the frequency of Tregs is increased in the peripheral blood of cancer patients and enriched in frequency among TILs or within tumour draining lymph nodes. Secondly, an accumulation of Tregs in tumour- associated tissue predicts poor prognosis or survival (459). How and why CD4+ CD25+ FOXP3+ Tregs increase in cancer patients may be due firstly, to the possibility that proliferating and dying tumour cells provide a large amount of self-antigens; secondly, the inflammatory milieu in tumours might also recruit Tregs; thirdly, the fact that it has been proposed that tumour cells and tumour infiltrating macrophages produce CCL22; and finally, the fact that it is likely the FOXP3+ Tregs are induced from non-Tregs
114 because of high concentrations of TGF-β secreted by tumour cells and DCs present in tumours (18), (197), (460), (461), (462), (463).
There are clearly soluble factors present in the tumour microenvironment of cancer
patients that encourage Tregs recruitment and differentiation. Therefore, it would be
useful to characterise these soluble factors and see if it would be possible to predict who is likely to have immunosuppression leading to tumour escape. In general, most studies have failed to identify a soluble suppressor cytokine (278), (279), (280), (281), (282), (464), (465). Therefore, besides characterising the Tregs population in ovarian cancer patients in the circulating blood, ascites and tumour tissues, a comprehensive study is included to characterise the cytokines present in the ovarian cancer peritoneal tumour microenvironment that is the ascites supernatant. Previous studies have been conducted to understand immune tolerance in ovarian cancer using tumour cells and immune cells obtained from patients‟ tumour tissues, peripheral blood and ascites. Cells isolated from tumour tissues, peripheral blood and ascites are termed TILs, PBMCs and TALs, respectively.
In this project, results are included from the flow cytometric analysis of lymphocytic and monocytic populations in blood, ascites or tumour tissue derived lymphocytes. The results from this approach could contribute to findings of prognostic significance of immunological markers which have not been systematically studied up to now. Therefore, this study, which consists of data produced using the bioinformatics approach with further validation using qRT-PCR and IHC simultaneously, is the first to be conducted in ovarian cancer research.
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3.1.1 Chapter 3: Aims and hypotheses
In this chapter, both the clinical data and experimental data are analysed. The association between selected cell surface marker protein expression and other phenotypic markers such as FOXP3 were looked at with patients‟ histological diagnosis, surgical debulking status, disease free survival and prognosis.
The hypotheses is that immune responses (as characterized by studying phenotypic markers and cytokines) with either regulatory or inhibitory function may be associated with clinical diagnois of ovarian cnacer and prognosis from cancer. There is evidence in the literature to suggest immune regulation in patients with ovarian cancer and these hypotheses is likely to be significant and may be used to aid diagnosis and predict prognosis in these patients.
The aims in this chapter are:
To identify the specific immune signatures associated with clinical parameters such as patients‟ histological diagnosis, surgical debulking status, diagnosis, disease free survival and prognosis. We have used a panel of markers identified from the literature to do this.
To characterise the distribution of Tregs in peripheral blood, ascites and tumour tissue of pateints with ovarian tumours.
To identify the patterns of cytokine expression associated with clinical diagnosis in patients‟ ascitic supernatant from patients with both benign and malignant ovarian disease.
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