**3.2.1 Lymphocytes**

The presence of activated T and/or natural killer (NK) cells in the tumour tissue is a positive prognostic factor in several solid cancers, including PCa (Kärjä et al., 2005; Gannon et al., 2009). CD8+ T cells are responsible for direct killing of target cells which express appropriate peptides on MHC Class I molecules, while NK cells play a role in killing tumour cells which downregulate MHC Class I molecules as an evasion mechanism from T cell recognition. Target cell killing occurs via delivering perforin and apoptosis-inducing granzyme complexes into the target cell (Thiery et al., 2011). CD4+ T cells, depending on their subtype: Th1, Th2, Th17 or T regulatory cells (Treg) produce cytokines which support pro- or anti-

Combination of Immunotherapy & Radiotherapy for the Treatment of Prostate Cancer 199

activation. They also express CCL22 which mediates Treg cell trafficking into the tumour (Miller et al., 2006). T cell clusters infiltrated by Treg cells often express high levels of PD-1 and B7-H1 markers (Ebelt et al., 2008) which indicates T cell exhaustion and functional

CD68+ monocytes and macrophages have been observed at higher frequencies in PCa compared to benign prostate tissues in a Gleason-score and disease stage-associated manner (Lindholm et al., 2010). CD68+ monocytes and less differentiated CD11b+CD33+ myeloid cells have been shown by immunohistochemistry to be present in PCa stroma (Sorrentino et al., 2011). There is no information available as to whether these cells function as tumourassociated macrophages (TAM) or myeloid-derived suppressor cells (MDSC). Monocytic MDSC have been characterised as CD11b+, CD14+, CD15+/-, CD16-, CD33+, CD66b+, CD124+, VEGFR1- and HLA-DRlow cells (Gabrilovich & Nagaraj, 2009; Marigo et al., 2008). More work is needed to establish if immunosuppressive MDSC are present in the microenvironment in PCa. Myeloid-derived dendritic cells (DC) have also been documented in PCa tissues although at relatively low frequencies and in a minimally activated state (Troy et al., 1998).

The most common immunosuppressive factors in the tumour tissue include vascular endothelial growth factor (VEGF), transforming growth factor beta (TGF-β), interleukin (IL)- 10 and adenosine which inhibit proliferation, differentiation and activation of T

VEGF supports tumour growth and metastasis by initiating endothelial cell proliferation and the formation of new blood vessels, thus providing a continuous blood supply to the tumour (Carmeliet & Jain, 2000). Increased angiogenesis has been observed in PCa tissue compared to benign prostatic hyperplasia (BPH) (Jackson et al., 1997). VEGF production by PCa cells in vitro is enhanced by addition of IL-1 and tumour necrosis factor alpha (TNF-α), both of which are found in the tumour microenvironment in PCa (Ferrer et al., 1997). VEGF is also important in immune suppression not only by inhibiting DC maturation and T cell development but also by acting as a chemoattractant for MDSC (Gabrilovich et al., 1996;

TGF-β is a pleiotropic cytokine. During PCa development it first acts as a tumour suppressor and later switches roles to become a tumour promoter and immunosuppressor in the tumour environment. TGF-β regulates immune cells by inhibiting cytotoxic T cell function, supporting the development of Treg cells and interfering with DC differentiation (Wan & Flavell, 2007; Wrzesinski et al., 2007). PCa-derived TGF-β has been shown to

 T cells into CD4+ CD25+ Foxp3+ Treg cells (Liu et al., 2007). The anti-inflammatory cytokine IL-10 secreted by tumour and stromal cells can inhibit proliferation, differentiation and activation of T lymphocytes via impaired DC (Sato et al., 2002). In the presence of IL-10, alternatively activated DC maintain an immature phenotype in the tumour microenvironment and induce tolerance rather than immune activation and

Extracellular adenosine is generated from ATP or ADP via the combined action of CD39 and CD73. These are ecto-nucleoside triphosphate diphosphohydrolases which, in the tumour tissue, are predominantly expressed on Treg cells while CD73 is also expressed on CD8+ T cells and tumour cells (CD73) (Stagg & Smyth, 2010). CD39 and CD73 are also expressed on

impairment.

**3.2.2 Myeloid cells** 

**3.3 Immune evasion in PCa** 

Ohm et al., 2003; Oyama et al., 1998).

support Treg cell development (H Huang et al., 2010).

lymphocytes and DC.

convert CD4+CD25-

inflammatory responses, respectively. CD4+, CD8+ and regulatory T cells are all present in PCa tumour tissue, with CD8+ T cells being predominant, unlike in the peripheral blood where CD4+ T cell frequencies are higher (Bronte et al., 2005). The majority of tumourinfiltrating CD8+ T cells are memory or terminally differentiated cells but their ability to upregulate activation markers is impaired (Bronte et al., 2005; Drake, 2010). In radical prostatectomy specimens of PCa, IFN-γ and perforin expression is lower than in T cells in healthy prostate tissue (Ebelt et al., 2008). Lymphocytes can mainly be observed in clusters in peritumoural areas while only a few infiltrate the tumour areas (Fig. 1).

Fig. 1. (A-C) PCa prostatectomy sections stained with haematoxylin and eosin shown at increasing magnifications; (C) a region with glandular () and stromal areas (). (D) peritumoural CD8+ T lymphocyte cluster, identified by fluorescence microscopy. (E) CD4+ cells are also present in this region.

T cells with regulatory function (CD4+CD25+Foxp3+) are present at higher frequencies in PCa than in healthy tissue. They can be found mainly in T cell clusters surrounding prostate cancer lesions or in the stroma (Ebelt et al., 2009; Miller et al., 2006; Sfanos et al., 2008). Some of these Treg cells express the glucocorticoid-induced TNF-receptor (GITR) and ICOS (a CD28-superfamily costimulatory molecule) at higher levels than in blood, indicating recent activation. They also express CCL22 which mediates Treg cell trafficking into the tumour (Miller et al., 2006). T cell clusters infiltrated by Treg cells often express high levels of PD-1 and B7-H1 markers (Ebelt et al., 2008) which indicates T cell exhaustion and functional impairment.
