**4.3 Irradiated tumour cells become sensitive to immune cell attack**

In tumour cells, not killed by IR, surface molecules such as MHC, the death receptor Fas and heat-shock proteins become upregulated (Shiao & Coussens, 2010; Garnett et al., 2004; Lugade et al., 2008). IR-induced upregulation of MHC Class I molecules, on both tumour cells and APC, improves antigen presentation and may enhance tumour cell recognition by activated CD8+ cells that infiltrate the tumour at an enhanced rate following radiation (Reits et al., 2006; Lugade et al., 2005).

Adhesion molecules, such as intracellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1 and platelet endothelial cell adhesion molecule (PECAM)-1, along with integrins, selectins and cadherins are also upregulated in the tumour tissue by IR (Baluna et al., 2006; Lugade et al., 2008). ICAM-1 is known to be upregulated by inflammatory cytokines such as TNFα, IL-1α/β and IL-6 thus resulting in lymphocyte and macrophage accumulation in inflamed tissues. As previously discussed, these cytokines are upregulated in the tumour tissue as a response to IR. ICAM-1 has an important role in enhancing T cell killing via cell-cell adhesion to lymphocyte function-associated antigen (LFA)-1 and by directly co-stimulating activated T cells (Garnett et al., 2004; Baluna et al., 2006). PCa cells express ICAM-1 and VCAM-1 and in tissue areas of high lymphocyte and neutrophil accumulation the expression of ICAM-1 is significantly elevated (Fujita et al., 2008; Rokhlin & Cohen, 1995). These data suggest that ICAM-1 upregulation by IR may facilitate immune responses by recruiting lymphocytes and macrophages to the tumour site. Increased adhesion between tumour cells with upregulated ICAM-1 and activated CD8+ T cells expressing LFA-1+ may result in more powerful cytotoxic T cell activity.

#### **4.4 Direct effect of IR on immune cells**

Immune cells are highly susceptible to IR-induced damage and readily undergo apoptosis. Therapeutic doses of RT often result in lymphopenia. One of the potential immunologically positive effects of direct IR on tumour-infiltrating immune cells is the depletion of Treg cells. However, there is some controversy regarding this question. Cao et al. observed that the proportion of Foxp3+ cells within purified CD4+CD25+ T cell population decreased significantly (48.2% to 23.3%) following irradiation with 1.8 Gy in vitro and was abolished (1.2%) by 30 Gy. The suppressive function of these Treg cells was also impaired by IR

Combination of Immunotherapy & Radiotherapy for the Treatment of Prostate Cancer 203

The use of IL-2 as a monotherapy in cancer has been extensively researched but due to issues with toxicity its clinical use has been limited. In the murine renal adenocarcinoma model, IR was found to augment the response of pulmonary metastases to IL-2 therapy (Younes et al., 1995). Following IR to one lung, plus systemic IL-2 treatment, a reduction in tumour size was observed in both lungs. The effect is dose-dependent and immunohistological studies show significant infiltration of T cells and macrophages at the tumour site. IL-2 is capable of rescuing T cells from IR-induced apoptosis and restores T cell proliferation after RT (Tabi et al., 2010). Its use in combination with RT may minimise the immunosuppressive effects of RT and enhance tumour cell killing via T cells (Mor & Cohen, 1996). In PCa, the combination of IL-2 and radiation in a mouse bone metastases model demonstrated a ~50% inhibition of tumour growth (Hillman et al., 2003). There was a greater degree of tumour destruction in IL-2-treated irradiated tumours than in irradiated tumours alone and the histology revealed increased fibrosis and elevated numbers of

Antitumour effects were also observed in a model utilising IL-12 and RT. IL-12 is secreted by mature DC and macrophages and required for IFNγ and TNFα production from T cells and mediates a Th1-type immune response. Adenovirus-derived IL-12 plus RT significantly increased local antitumour and systemic antimetastatic effects in a preclinical model of metastatic PCa when compared to either treatment alone (Fujita et al., 2008). This antimetastatic activity is due to the antitumoural activities of natural killer (NK) cells. These results were also observed in the 4T1 mammary carcinoma. The combination of RT and an adenoviral vector encoding IL-12 and the co-stimulatory molecule CD80 resulted in a significant reduction in tumour growth (Lohr et al., 2000). The antitumour effect observed in the combination therapy group was far superior if the IL-12 and CD80-expressing

Further cytokine studies have evaluated the combined effect of IL-3 and RT. IL-3 differentiates haematopoietic stem cells into myeloid progenitor cells and stimulates the proliferation of myeloid-derived cells such as DC and monocytes. In mouse models of fibrosarcoma and PCa, IL-3 was found to increase the tumour response to radiation. Combining adenoviral-IL-3 and radiation in the TRAMP-C1 mouse prostate model caused significant delays in tumour growth. Further reports indicated that adenoviral-IL-3 plus radiation enhanced IFNγ-producing CD4+ and CD8+ T cell responses in the spleen (Oh et al., 2004). This shifted the immune response to a Th1–type response from a suppressive Th2-

The combination of the pro-inflammatory cytokine TNFα and RT in mouse mammary carcinoma delayed tumour growth at a greater extent than either treatment alone (Nishiguchi et al., 1990). Similar synergistic effects have also been observed in mouse melanoma, lung adenocarinoma and brain tumours. The combined effects were attributed to increased recruitment and enhanced activation of lymphocytes and neutrophils (Gridley et

Adoptive-cell-transfer (ACT) therapy is the passive transfer of tumour-specific T cells that have been expanded ex vivo. Local tumour irradiation can enhance the therapeutic efficacy of ACT therapy (Teitz-Tennenbaum et al., 2009). Combination of RT with ACT of carcinoembryonic antigen (CEA)-specific CD8+ T cells in a mouse colon carcinoma

al., 1996; Gridley et al., 2002; Gridley et al., 2000; Jin et al., 2005; Li et al., 1998).

**5. RT and immunotherapy combination modalities** 

adenovirus was administered after the final fraction of radiation.

**5.1 Pre-clinical models** 

infiltrating inflammatory immune cells.

type response (Tsai et al., 2006).

probably due to loss of membrane-bound TGF-β (Cao et al., 2009). Tregs were especially sensitive to low-dose radiation compared to effector T cells (Cao et al., 2011). Another study, in TRAMP mice, had the opposite findings: Treg cell numbers increased in immune organs after local or whole body irradiation without changes to their functional activity (Kachikwu et al., 2010), indicating relative resistance to 0-20 Gy radiation. It remains to be seen what happens to Tregs in situ during RT of PCa.

We showed recently that lymphopenia following prostate and pelvic RT causes preferential death of naïve or unstimulated T-cells (Tabi et al., 2010). Elevated frequencies of Treg cells were observed in the circulation following 44 Gy radiation in 20 fractions to the pelvic nodes and 55 Gy to the prostate (Fig.2). T cell proliferative function was also impaired (Tabi et al., 2010) but it was restored in vitro with exogenous IL-2 without increasing Treg frequencies (Fig. 2). This indicates that IL-2 maybe used to support T cell function after patients completed standard RT.

Fig. 2. IL-2 response of Treg cells from the peripheral blood of PCa patients undergoing standard RT. Frequencies of CD4+CD25+Foxp3+ T cells (see gate in insert) were measured before (RT0), immediately after radical radiation in 20 fractions (RT20) and 4 weeks after the last fraction (pRT4). Means and SD of triplicates are shown from a representative patient. Frequencies of Tregs were elevated at RT20, but returned to pre-radiation (RT0) level at pRT4. Unlike at RT0 or RT20, exogenous IL-2, added to the cells in vitro, did not increase Treg frequencies at pRT4.

Most importantly, we identified novel TAA-specific T-cell responses post-RT (Tabi et al., 2010), which were not present before RT. Similar findings were observed by others (Nesslinger et al., 2007; Schaue et al., 2008), indicating the ability of radiation to shift the balance between tumour-specific regulatory and effector immune mechanisms.
