**2. Immune response of glioma**

#### **2.1 T cell infiltration in tumours and prognosis**

Many tumours are potentially immunogenic and exhibit tumour-specific immune responses in vivo (Curiel 2008; Curiel, et al. 2004). Tumour-specific antigens are released from the tumour cells and then captured by antigen presenting dendritic cells (Huang, et al. 2010). Dendritic cell migration brings tumour antigen to the lymphoid organ where the antigen presentation stimulates immature T cells to become either "cytotoxic" CD8(+) T-cells (CTL), "helper" CD4(+) T-cells or memory T-cells (Fig. 1). Lymphocytes and some innate immune cells (macrophages, natural killer cells) migrate to the tumour in order to kill and eliminate tumour cells. Patients with high infiltration of lymphocytes in their tumours have usually found to have a better prognosis of survival.

Fig. 1. Tumour immune response

2004; Gulley, et al. 2005; Sharp, et al. 2007). There is also an ongoing pre-clinical search for methods to enhance the therapeutic response of malignant glioma by combining immunotherapy with single fraction or hypo-fractionated radiation therapy (Demaria, et al. 2005a; Graf, et al. 2002; Lumniczky, et al. 2002; Newcomb, et al. 2006; B. R. R. Persson, et al. 2002; B. R. R. Persson, et al. 2003; B. R. R. Persson, et al. 2010; B. R. R. Persson, et al. 2008).

This chapter will summarize the aspects of the interaction of ionizing radiation with the immune system and its immunomodulatory effects and its implications for glioma therapy (Friedman 2002). Preclinical studies of the combinational approaches of radiation and immune therapies, which results in high fractions complete remissions of glioma in animal models, is reviewed. Various clinical studies towards combination of radiation- and

Many tumours are potentially immunogenic and exhibit tumour-specific immune responses in vivo (Curiel 2008; Curiel, et al. 2004). Tumour-specific antigens are released from the tumour cells and then captured by antigen presenting dendritic cells (Huang, et al. 2010). Dendritic cell migration brings tumour antigen to the lymphoid organ where the antigen presentation stimulates immature T cells to become either "cytotoxic" CD8(+) T-cells (CTL), "helper" CD4(+) T-cells or memory T-cells (Fig. 1). Lymphocytes and some innate immune cells (macrophages, natural killer cells) migrate to the tumour in order to kill and eliminate tumour cells. Patients with high infiltration of lymphocytes in their tumours have usually

The clinical trials using this approach, however, are still very sparse.

immune-therapy for treatment of glioma are summarized in a final section.

**2. Immune response of glioma** 

**2.1 T cell infiltration in tumours and prognosis** 

found to have a better prognosis of survival.

Fig. 1. Tumour immune response

Tumour infiltrating lymphocytes (TILs) of various subtypes represent the host-to-tumour reaction. Anti-tumour immune response is mediated by infiltrating CD8(+) T cells which have been shown to lyses tumour cells directly via recognition of the major histocompatibility complex class I (MHC-I) present on most tumour cells. But some tumours, which have low or none expression of MHC-I, are not affected by the CTL. Tumour infiltrating CD4() helper T cells seems to play a role in regulating and amplifying tumours response by priming tumour-specific cytotoxic CD8(+) T cells, as well as macrophages involved in clearance of dead tumour cells (Toes, et al. 1999; Vesalainen, et al. 1994).

In Fig. 1 is shown how tumour antigens are captured by antigen presenting cells such as dendritic cells, which migrate to regional lymph nodes. There they present the antigen to Tcells which differentiate into CD8(+) cytotoxic T-cells, CD4(+) helper T-cells, and memory Tcells. The cytotoxic CD8(+) T-cells (CTL) are transferred to the tumour in order to kill the tumour cells. The CD4(+) release IL2 which help the CD8(+) T-Cells to proliferate. But the CD4(+) can also form CD4(+)CD25(+) regulatory T-cells which excrete IL10 to suppress the activity of the CD8(+) cytotoxic T-cells.

The number of tumour infiltrating lymphocytes can be used as prognostic factor for several types of cancer (Cho, et al. 2003; Rauser, et al. 2010; Schumacher, et al. 2001; Zingg, et al. 2010). But in malignant glioma the use of tumour infiltrating lymphocytes as a prognostic factor seems to be more complex. The overall reports on tumour-infiltrating CD8(+), CD4(+) T-cells and major histocompatibility complex class I (MHC-I) expression in malignant glioma do not yield consistent correlation with clinical outcome (Dunn, et al. 2007). There seems to be factors present in patients with glioma that suppress the action of tumour infiltrated lymphocytes, and it has been demonstrated that glioma cells can actively paralyze T cell migration by the expression of Tenascin-C (Huang, et al. 2010).

Regulatory CD4(+)CD25(+)FoxP3(+) T cells (Treg) have been shown to play a major role in suppression of the immune response to malignant glioma. In human CNS tumor samples both CD4(+) and Treg infiltration have found to be significantly increased throughout the time of metastatic tumor progression. Thus immunotherapeutic strategies for treating metastatic CNS tumors must fight against Treg (Sugihara, et al. 2009). In an experimental GL261 intracranial tumor model, it was shown that depletion of CD25(+) regulatory T-cells (Treg) using anti-CD25 antibodies enhance the efficacy of DC immunotherapy (Maes, et al. 2009).

Infiltration of myeloid suppressor cells (MSC) is another factor inhibiting the function of the CD8(+) T cells, which results in tumour progression (Graf, et al. 2005). Other studies indicate that glioma seems to secrete factors such as TGF and prostaglandins (PGE2) that depress the cell-mediated immunity by down regulating the function of infiltrated CD8(+) T-cells and monocytes (Dix, et al. 1999; Farmer, et al. 1989). This might be one of the reasons why anti-tumour response of the immune system is decreased in patients with primary glioma (Brooks, et al. 1972).

#### **2.2 Radio-immune-modulating effects by local irradiation**

Recent studies have shown that local single fraction radiotherapy stimulates the immune response by enhancing the antigen presentation of MHC class I (Liao, et al. 2004). The mechanism underlying these effects is probably at the level of the proteasome in the cytoplasm of the tumour cell, which are essential for production of antigenic peptides for

Radiation Immune Modulation Therapy of Glioma 367

these preclinical experiments, which were performed already 2001, showed that a single fraction of RT combined with immunotherapy resulted in a significantly increased survival time of rats with intra-cranially implanted N29 or N32 glioblastoma. Further there were significant numbers of complete remissions of the most infiltrative N29 tumour implanted in Fischer-344 rats (B.R.R. Persson, et al. 2010). Other researchers have also reported substantial tumour regression by single fraction radiation therapy combined with various regimes of immune therapy (Bradley 1999; Chakraborty, et al. 2003; Demaria, et al. 2005a; Friedman

**3.1 The Lund experience of combined single fraction RT and Immunization with IFN-**

Fischer-344 rats were maintained by continuous, single-line brother to sister mating in the laboratory at Lund. During the experiments rats of both sexes, females weighing around 190 g and males 370 g respectively, were housed in a climate controlled cabinet. Otherwise they were kept in Macralon cages provided with food pellets and water *ad libitum*. All experimental animal procedures were approved by the Animal Ethical Committee in

All cells were maintained in culture flasks (Nunc, Denmark) and harvested by treatment with trypsin/EDTA. The culture medium was antibiotic-free RPMI-1640 medium supplemented with 5-10% foetal calf serum, L-glutamine (2 mM), HEPES (10 mM), pyruvate (0.5 mM) and NaHCO3 (11 mM). The cell-cultures were regularly checked for contaminating microbes by staining with the fluorescent dye Hoechst 32 258 and examined with fluorescent microscopy. If *Mycoplasma* infection was indicated the cultures were discharged or treated with *Mycoplasma Removal Agent* (Hoechst, Germany) twice with 7 days interval,

The tumour cells (N29 or N32) used for immunization were interferon-gamma (IFN-) gene modified to enhance secretion of IFN. The cells were cultured for one week, washed twice, and suspended in serum free medium (IMDM-0) to a cell density of 2104 cells/ml. Just before immunization the cells were transferred from the culture flasks to 15 ml centrifuge test tubes (Nanclon) and stored on melting ice to prevent the cells to grow during the procedure. Irradiation of the cells was performed during 20 minutes at room temperature to an absorbed dose of 70 Gy by using a 137Cs gamma-ray source (*Gammacell 2000;* Mølsgaard

Inoculation was performed by injecting 5 000 tumour cells in 5 l nutrient solution into the head of Fischer 344 rats, using a stereotactic technique with a Hamilton syringe. To avoid extra-cranial tumour growth, the injection site was cleaned with 70% ethanol after injection and the borehole was sealed with wax. The animals were arranged into 6 groups, which included: controls, RT with either 5 or 15 Gy, immunization with IFN- gene modified

Animals were given a single radiation treatment using a 60Co radiotherapy unit (Siemens Gammatron S) with a source-skin distance (SSD) of 50 cm and the maximum absorbed dose rate 0.65-0.70 Gy/min. The radiation field size was collimated to cover the brain. The adsorbed dose of either 5 or 15 Gy was measured both by an dose-meter diode and TLD

Medical, Risø, Denmark) (Siesjö, et al. 1996; Sjögren, et al. 1996; Visse, et al. 1999).

tumour cell, and RT with either 5 or 15 Gy combined with immunization (Table 1).

**3.1.2 Inoculation and treatment of intracerebrally tumours** 

2002; Garnett, et al. 2004; Graf, et al. 2002; Lumniczky, et al. 2002).

Malmö/Lund (Lunds tingsrätt, Box 75, 22100 Lund Sweden).

**secreting tumour cells** 

**3.1.1 Animals and tumour cell lines** 

and repeatedly confirmed free of infection.

loading onto MHC class I molecules. The proteasome in tumour cells is a sensitive target for radiation, resulting in decreased processing of endogenous self antigens. The processing of tumour antigens is, however, increased by radiation, which enhance the accumulation of antigen/MHC class I complexes on the cell surface (Pajonk &Mcbride 2001 ).

Radiation therapy also causes an increase in production of the cytokine IFN in the target region which up-regulates low levels of MHC class I, creating a tumour microenvironment conducive for CD8(+) T cell infiltration and their recognition of tumour cells (Lugade, et al. 2008).

It has been demonstrated that antigen presentation by MHC class I is increased for many days by single fraction radiation therapy. The most pronounced effect was recorded at 7 days after irradiation with an absorbed dose of 8 Gy. This might be one of the reasons why the efficacy of tumour immunotherapy is most effective in combination with single fraction radiation therapy (Reits, et al. 2006). Maximum loading of the tumour micro-environment with cancer antigen occurred 2 days after radiation therapy and coincided with the optimal time for CD8(+) T cell transfer (Bin Zhang, et al. 2007).
