ABSTRACT: The combination of radiation therapy and immunotherapy holds particular promise as a strategy for cancer therapeutics. Evidence suggests that immunotherapy is most beneficial alone when employed early in the disease process or in combination with standard therapies (eg, radiation) later in the disease process. Indeed, radiation may act synergistically with immunotherapy to enhance immune responses, inhibit immunosuppression, and/or alter the phenotype of tumor cells, thus rendering them more susceptible to immune-mediated killing. As monotherapies, both immunotherapy and radiation may be insufficient to eliminate tumor masses. However, following immunization with a cancer vaccine, the destruction of even a small percentage of tumor cells by radiation could result in crosspriming and presentation of tumor antigens to the immune system, thereby potentiating antitumor responses. Learning how to exploit radiation-induced changes to tumor-cell antigens, and how to induce effective immune responses to these cumulatively immunogenic stimuli, is an exciting frontier in cancer therapy research. This review examines mechanisms by which many forms of radiation therapy can induce or augment antitumor immune responses as well as preclinical systems demonstrating that immunotherapy can be effectively combined with radiation therapy. Finally, we review current clinical trials where standard-of-care radiation therapy is being combined with immunotherapy.
Radiation is often considered immunosuppressive, an activity that is most likely a result of the complex interplay of hormesis and the abscopal effect. The abscopal effect, also called the “distant bystander” effect, is a paradoxical effect of radiation on cellular systems whereby local radiation may have an antitumor effect on tumors distant from the site of radiation. Indeed radiation’s ability to enhance distinct immune responses by inducing a “danger” signal that excites and activates the immune system has recently come under investigation. In the context of tumors, radiation has been hypothesized to cause tumor disruption and a type of “danger” signal that could be successfully exploited to improve the effectiveness of immunotherapy.
Radiation therapy is conventionally used for local tumor control. Although local control of the primary tumor can usually prevent development of subsequent systemic metastases, tumor radiation fails to control preexisting systemic disease, which may be present only as micrometastatic (and therefore undetectable) deposits. Combining radiation therapy with immunotherapy allows one to exploit two broad areas: (1) radiation-induced tumor-cell death as a potential source of tumor antigens for immunotherapy, and (2) postirradiation tumor-cell modulation that allows more efficient immune-cell access and increased sensitivity to T-cell killing. These tumor-specific T cells could arise endogenously or be induced from active vaccination strategies.
Many clinical trials exploring the use of radiation and vaccines in the treatment of cancer are currently underway. As knowledge of the synergistic effects of radiation and immunotherapy increases, the translational use of this strategy for a variety of carcinomas will become more feasible.
Combining Radiation Therapy and Immunotherapy
Local irradiation of tumor is the standard of care for many cancer types. Traditionally, it is employed to destroy tumor cells or to alter tumor/stroma architecture with either curative or palliative intent. However, it is often the case that not all tumor cells in a given mass receive a lethal dose of radiation due to dose constraints mandated by the need to limit damage to normal tissue. Nevertheless, even sublethal doses of radiation can generate potent immmune responses by altering tumor cells in a variety of ways.
Antigen Release From Dying Tumor Cells Can Activate Immune Responses
On their own, tumor cells do not generate potent antitumor immune responses due to their inefficient expression of molecules important for antigen processing and presentation. Tumor cells may not express the antigen transporter gene product TAP-2 and class I major histocompatibility complex (MHC) molecules, and they lack T-cell costimulatory molecules such as B7-1 (CD80). Irradiation can induce recognition and phagocytosis signals for dendritic cells (DCs), such as membrane-bound calreticulin, as well as release “danger” signals for DC activation, such as various heat shock proteins (HSP) and high-mobility group protein B1 (HMGB1).
Antigens released by dying tumor cells can activate the immune system to induce immunogenic cancer cell death, thus contributing to the eradication of residual tumor cells (Figure 1).[1,4,5] In order to induce this immune response, dying tumor cells need to provide two signals for DCs. First, a specific phagocytosis/recognition signal is presented by the translocation of cytoplasmic calreticulin to the cell membrane, which allows DCs to engulf dying tumor cells. Second, a specific “danger” signal is released by the dying cell that activates DCs and stimulates antigen processing and presentation to T cells.
It was recently demonstrated that irradiated, dying tumor cells release the nuclear nonhistone protein HMGB1, which binds to Toll-like receptor 4 (TLR4), thereby providing a “danger” signal to DCs for TLR4-dependent antigen processing (Figure 3). In addition, several groups have demonstrated that one class of endogenous “danger” signals is provided by stress proteins, or HSPs, which are released from dying tumor cells and actively taken up by DCs for cross-presentation via HSP receptors (CD91 for gp96, calreticulin, HSP70, and HSP90; CD14 for HSP70).[8-11] In other experiments, Sozzani et al demonstrated that hepatocellular carcinoma cells switched chemokine-receptor expression from CCR1 to CCR7 following irradiation. This chemokine switching acted as a homing receptor for activated DCs and facilitated the DC migration to draining lymph nodes.
The immunologic consequences of radiation therapy–induced tumor-cell death are thus twofold: providing a source of tumor antigens for presentation by circulating DCs and providing “danger” signals for DC activation (Figure 1). Radiation-treated tumor cells would thus serve as an in situ autologous tumor vaccine, inducing a strong tumor-specific immune response that could eradicate residual tumor cells in primary tumors and distant micrometastases (Figure 1).
Irradiation Modulates Tumor-Cell Phenotype and Increases Immune Recognition
Neoplastic cells may evade the adaptive immune system by altering expression of specific molecules such as tumor-associated antigens (TAAs) or MHC molecules. Studies investigating the mechanism by which local tumor irradiation enhances therapeutic response to immunotherapy have established that nonlethal doses of radiation may alter the phenotype of target tissue by upregulating some gene products and making tumor cells more susceptible to T cell–mediated immune attack (Figures 2 and 3).
MHC class I is responsible for direct presentation of tumor antigen peptides to cytotoxic T lymphocytes (CTLs) via peptide-MHC complexes. ICAM-1 and other cell adhesion molecules enhance T cells’ ability to kill target cells by improving cell-to-cell adhesion [14,15]. Studies have demonstrated that nonlethal doses of radiation induce a two-phase, dose-dependent increase in MHC class I presentation in human tumor cells.. MHC class I molecules present endogenous peptides to CTLs. Many of these peptides are generated by the proteasome from newly synthesized but rapidly degraded proteins (RDPs). Within 4 hours after exposure, the protein degradation triggered by radiation damage leads to an increased peptide pool (Figures 2 and 3).
During the latter phase of ionizing radiation (> 4 hours after exposure) the mammalian target of rapamycin (mTOR) pathway is activated, resulting in translation of proteins and increased generation of peptides from the RDPs of these new proteins. At each of these stages, unique proteins are expressed and upregulated in response to ionizing radiation, resulting in novel peptide presentation (Figure 2). These novel peptides could cause activation of resting T cells specific for these epitopes, leading to an antitumor immune response.
Radiation has also been shown to alter the cell-surface expression of a variety of immunomodulatory molecules. Garnett et al examined 23 human carcinoma cell lines (12 colon, 7 lung, and 4 prostate) for their response to nonlytic doses of radiation (10 or 20 Gy). They examined changes in surface expression of Fas and other molecules involved in T cell–mediated immune attack, such as the adhesion molecule ICAM-1, TAAs such as carcinoembryonic antigen (CEA) and mucin-1 (MUC-1), and MHC class I. Radiation upregulated at least one of these surface molecules in 21 of 23 (91%) cell lines studied. Furthermore, all five irradiated CEA-positive/A2-positive colon tumor-cell lines demonstrated significantly enhanced killing by CEA-specific HLA-A2-restricted CD8-positive CTLs compared to nonirradiated cell lines.
Microarray analysis of gene-expression changes revealed that many additional genes had been modulated by irradiation. These upregulated gene products may further enhance the tumor cells’ susceptibility to T cell–mediated immune attack or serve as additional targets for immunotherapy. Taken together, these results suggest that nonlethal doses of radiation render human tumor cells more amenable to immune system recognition and attack.
Radiation Effects on the Immune System
Radiotherapy that involves high doses to multiple lymph node chains can decrease nonspecific immune system responses, and these responses may remain suppressed for several months following irradiation. In addition, there may be decreases in T-cell subsets following radiation therapy, largely in the naive T-cell populations. However, for radiation therapy that does not involve high doses to multiple lymph node chains within the standard treatment ports, the potential benefits of combining radiation with immunotherapy remain very appealing.
In examining the potential mechanisms of the combination of radiation therapy with immunotherapy, it has recently been reported in murine systems that lymphodepletion from 5 Gy total-body irradiation followed by adoptive transfer of tumor-specific T cells resulted in significantly improved antitumor activity. There, Gattinoni et al found that the irradiation removed endogenous cell populations that acted as sinks for the needed interleukin (IL)-7 and IL-15 cytokine supporting the tumor-reactive T cells.
Another factor being examined is the removal of regulatory T cells (Tregs) by irradiation[4,21] and the role of TLR4 from radiation-injured gut on the activation of immune cells.[22,23] These mechanisms are also being seen in patients. Improved understanding of the intertwined mechanisms involved in the augmentation of antitumor immunity by the addition of immunotherapy and radiation can further optimize this combination.