Radiation therapy and the innate immune response: Clinical implications for immunotherapy approaches

Radiation therapy is an essential component of cancer care, contributing up to 40% of curative cancer treatment regimens. It creates DNA double‐strand breaks causing cell death in highly replicating tumour cells. However, tumours can develop acquired resistance to therapy. The efficiency of radiation treatment has been increased by means of combining it with other approaches such as chemotherapy, molecule‐targeted therapies and, in recent years, immunotherapy (IT). Cancer‐cell apoptosis after radiation treatment causes an immunological reaction that contributes to eradicating the tumour via antigen presentation and subsequent T‐cell activation. By contrast, radiotherapy also contributes to the formation of an immunosuppressive environment that hinders the efficacy of the therapy. Innate immune cells from myeloid and lymphoid origin show a very active role in both acquired resistance and antitumourigenic mechanisms. Therefore, many efforts are being made in order to reach a better understanding of the innate immunity reactions after radiation therapy (RT) and the design of new combinatorial IT strategies focused in these particular populations.

Radiation therapy is an essential component of cancer care, contributing up to 40% of curative cancer treatment regimens. It creates DNA double-strand breaks causing cell death in highly replicating tumour cells. However, tumours can develop acquired resistance to therapy. The efficiency of radiation treatment has been increased by means of combining it with other approaches such as chemotherapy, molecule-targeted therapies and, in recent years, immunotherapy (IT).
Cancer-cell apoptosis after radiation treatment causes an immunological reaction that contributes to eradicating the tumour via antigen presentation and subsequent T-cell activation. By contrast, radiotherapy also contributes to the formation of an immunosuppressive environment that hinders the efficacy of the therapy. Innate immune cells from myeloid and lymphoid origin show a very active role in both acquired resistance and antitumourigenic mechanisms. Therefore, many efforts are being made in order to reach a better understanding of the innate immunity reactions after radiation therapy (RT) and the design of new combinatorial IT strategies focused in these particular populations.

K E Y W O R D S
damage-associated molecular patterns, dendritic cells, immunotherapy, innate and adaptive immunity, myeloid-derived suppressor cells, natural killer cells, radiation therapy, tumourassociated macrophages

| INTRODUCTION
Radiation therapy is an essential component of cancer care, contributing up to 40% of curative cancer treatment regimens. It creates DNA double-strand breaks causing cell death in highly replicating tumour cells. However, tumours can develop acquired resistance to therapy.
The efficiency of radiation treatment has been increased by means of combining it with other approaches such as chemotherapy, moleculetargeted therapies and, in recent years, immunotherapy (IT).
Cancer-cell apoptosis after radiation treatment causes an immunological reaction that contributes to eradicating the tumour via antigen presentation and subsequent T-cell activation. By contrast, radiotherapy also contributes to the formation of an immunosuppressive environment that hinders the efficacy of the therapy. Innate immune cells from myeloid and lymphoid origin show a very active role in both acquired resistance and antitumourigenic mechanisms.
Therefore, many efforts are being made in order to reach a better understanding of the innate immunity reactions after radiation therapy (RT) and the design of new combinatorial IT strategies focused in these particular populations. Valentí Gómez, Rami Mustapha and Kenrick Ng contributed equally to this work.
RT-alone or in combination with surgery and/or chemotherapyis 1 of the main treatments for cancer. Over 50% of patients will receive some form of RT (external beam, brachytherapy or systemic RT) both in the curative and palliative settings. 1,2 RT relies on the ability of ionising radiation to create double-strand breaks in highly proliferating tumour cells thus provoking their death by mechanisms such as apoptosis, radiation-induced senescence, mitotic catastrophe, autophagy or necrosis. [2][3][4] However, tumours can acquire resistance despite the development of novel combination therapies involving RT and molecular-targeted therapies. 4,5 Abscopal effect, a phenomenon where local RT is associated with cancer regression at the metastatic site, has been linked to the patient immune status at the time of therapy. 6 This has changed the vision from cancer-cell oriented RT (and its subsequent RT-acquired resistance) to the consideration of tumour microenvironment (TME) as a key element in both the pro-and antitumourigenic activities after RT. 7 Cancer-cell apoptosis due to RT triggers a series of molecular events known as damage-associated molecular patterns (DAMPs). 8 Examples of DAMPs include: (i) translocation of calreticulin; (ii) extracellular release of ATP; (iii) extracellular release of high-mobility group box 1; and (iv) production of cytokines such as type I interferon (IFN-I). 9 These signals trigger a series of immunological reactions that affect both innate and adaptive immunity ( Figure 1). Innate immunity refers to nonspecific defence mechanisms that act immediately after the antigen's appearance. It is activated by the chemical properties of the antigen and include different immune cells (dendritic, mast and natural killer [NK] cells, monocytes and macrophages, granulocytes and the complement system). It also includes anatomical and physical barriers such as skin, internal mucosa, pH or temperature. It is present at birth and generally inherited and has the ability to fight against any foreign invading presence. Its potency has generally been considered lower and limited due to the lack of memory mechanisms, despite certain evidence showing a capacity of adaptation, named trained immunity or innate immune memory. 10,11 By contrast, adaptive immunity is based in the antigen-specific response. It is more complex than the innate as the antigen first must be processed and recognised hence being a slower but much powerful response. Adaptive immunity is mediated by lymphocytes (T and B cells) and is also characterised by immunological memory that allows a long-lasting response. The randomisation of immunoglobulin (Ig) superfamily genes and the selection of multiple cell types during active responses confers adaptive immunity a great plasticity and adaptability. 12 DAMPs elicit immunological reactions such as recruitment of antigen presenting cells (APCs) [13][14][15] and subsequent T-cell activation and establishment of immunological memory. By contrast, IFN-γ release upregulates programmed death ligand-1 (PD-L1) expression in cytotoxic CD8+ T-cells, therefore silencing the adaptive immune response. 16 In addition, CD8+ T cells increase regulatory T-cell (Treg) recruitment via CCR4. 16 Therefore, combination of radio and IT such as PD-L1 or CTLA4 blockade can result in an effective T cell-mediated tumour clearance. 17 Extensive literature has already discussed the effects on RT and adaptive immunity [18][19][20] and is out of the scope of this review. The review will focus on the implications of the innate myeloid and lymphoid lineages in both anti-and protumourigenic processes induced by RT and the potential benefit of a combinatorial RT and IT approach. The understanding of the synergy between RT and F I G U R E 1 Effect of radiation therapy (RT) over the innate immune system. RT causes tumour cell death and damage-associated molecular pattern (DAMP) release. These signals (grey circles: interferon [IFN]-I, IFN-γ, transforming growth factor-β [TGF-β], tumour necrosis factor-α [TNF-α], colony stimulating factor-1 [CSF1], inducible nitric oxide synthase [iNOS], CXCL6 among many others) trigger both antitumourigenic (blue boxes) and protumourigenic (red boxes) effects in the different components of the innate immune system: dendritic cells, macrophages, myeloidderived suppressor cells (MDSC) and natural killer (NK) cells the immune system will also be illustrated by a brief overview of the published and ongoing clinical trials in this area.

| MYELOID LINEAGE
Myeloid cells constitute a highly diverse population evolved as an innate mechanism against pathogen infection. They also participate in the elimination of dying cells and tissue remodelling after wound healing. In cancer, the contributing myeloid types are mainly dendritic cells (DCs), monocyte and macrophages, and myeloid-derived suppressor cells (MDSCs). 21

| DCs
DCs are specialised APCs derived mainly from a common myeloid progenitor (CMP) although there is a minor subset of DCs from lymphoid origin. They play a crucial role in T-cell activation after RT-induced damage in cancer cells. 8,21,22 DAMPs are recognised by specific receptors in sentinel DCs, 23,24 which undergo maturation and in turn stimulate cytotoxic CD8+ T cells by antigen presentation and release of activating cytokines. 25 Based on these principles, DCs are capable of enhancing RT treatments. [26][27][28][29][30] In patients, the combinatorial effect of RT and DC-based IT have started to be exploited in the form of therapeutic cellular vaccines, 31 which will be discussed later in this review.
Interestingly, number and intensity of radiation doses are important in order to activate DCs. In a murine mammary carcinoma model, repeated low-irradiation doses will create cytosolic DNA in tumour cells, thus activating the cGAS-STING pathway and the release of DCactivating IFN-γ and subsequent T-cell activation. However, a higher single dose will increase the expression of the DNA-exonuclease Trex1. Trex1 action will reduce the amount of cytosolic DNA and minimise the immunogenic effect of RT. 32 The antitumourigenic action of DCs depends on 3 simultaneous signals: antigen presentation, costimulation and secretion of proinflammatory cytokines. If full DC maturation does not occur, antigen presentation can lead to T cell anergy and immune tolerance. 33 In contrast, mature DCs (i.e. after RT) express TRAIL, a protein belonging to the tumour necrosis factor (TNF) superfamily. DC-secreted TRAIL is involved in the induction of apoptosis in cytotoxic Th1 T cells and promotes the proliferation of immunosuppressive Tregs, hence promoting suppression of antitumour immunity. 34

| MDSCs
MDSCs are a heterogeneous population of immature myeloid cells that exhibit immunosuppressive properties, therefore contributing to tumour progression and the establishment of a premetastatic niche. 21,35 Two main MDSC populations have been characterised: monocytic MDSCs and polymorphonuclear MDSCs (also known as granulocytic MDSCs). 36 MDSCs exert their immunosuppressive function through different mechanisms: (i) T-cell inhibition; (ii) promotion and activation of regulatory Tregs; (iii) inhibition of NK and NK T cells activation. The main secreted factors involved in MDSC-mediated immune suppression include arginase 1, nitric oxide, interleukin (IL)-10, transforming growth factor-β (TGFβ) and COX2 among others. 21,37 The STING-type I IFN pathway triggered after RT as part of the DAMP-mediated signalling plays an important role in MDSCs recruitment, therefore counteracting the activation of dendritic cells previously described. This phenomenon is partially regulated via CCR2, thus combining anti-CCR2 treatments with RT will enhance the immune STING-dependent response while minimising MDSC-derived immunosuppression. 38 Colony stimulating factor-1 (CSF1)-CSF1 receptor is a second mechanism described to contribute to MDSC recruitment with potential clinical implications. 39 However, the effect of RT on MDSC activation appears to be tumour-type and RT-regimen dependent. It has been shown that ablative hypofractionated RT (AHFRT) decreases MDSC recruitment when compared with conventional fractionated RT. 40,41 AHFRT reduces the appearance of intratumoural hypoxia and, consequently, HIF1α expression, which drives VEGF and PD-L1 expression, 2 known MDSC activators. 42 Reduction in MDSC levels within the tumour microenvironment might be the reason behind the better outcome of AHFRT therapies in some cancer types.
Therefore, MDSCs are also considered a promising target for IT treatments. A summary of ongoing preclinical approaches and clinical trials can be found in Yin et al. 37

| Monocytes, macrophages and tumourassociated macrophages
While it can be stated that RT increases tumour immunogenicity or immunosuppression by respectively recruiting DCs and MDSCs, the picture becomes much more complicated when assessing the role of macrophages after RT. Macrophages and monocytic precursors constitute the major myeloid population infiltrating the tumour microenvironment and display great heterogeneity and plasticity both phenotypically and functionally. Bone-marrow derived precursors are the main source for macrophage recruitment but tissue-resident macrophages derived from erythro-myeloid precursors can also be found within the tumour microenvironment. 43,44 Tumoricidal M1-like or proinflammatory macrophages (also known as classically activated macrophages) represent 1 edge of the spectra while on the other end of the continuum (alternatively activated) M2-like or anti-inflammatory macrophages contribute to tumour progression. Tumours have the ability to bias the original inflammatory macrophages towards the M2-like phenotype upon the secretion of a broad cytokine and chemokine array (i.e CCL2, IL-4, IL-13, CSF1, TGFβ or IL-10). 45 Re-educated tumour-associated macrophages (TAMs) show different phenotypes (and capacity to change from 1 to another) and contribute to tumour progression by enhancing immunosuppression, angiogenesis, invasion and metastasis. [46][47][48][49][50][51][52][53] Therefore, TAM accumulation generally correlates with poor prognosis in various types of cancer. [54][55][56][57][58] However, in colorectal cancer, the presence of TAMs correlated with a better patient outcome 59 and remains controversial in lung cancer where there is coexistence of both populations. 60 Inflammation and wound healing (or removal of apoptotic cells) are the 2 main processes occurring after RT that modulate the physiology of TAM in the affected tissues. Irradiated cells secrete CCL-2 and CSF1 that are responsible for the recruitment and skewing of macrophages towards the protumourigenic phenotype. 39,61 The tumourigenic polarisation of TAMs is also enhanced by the secretion of TGFβ and the accumulation of adenosine within the irradiated tumour microenvironment. 62,63 In addition to cytokine secretion, RT creates a hypoxic environment within the damaged tissue. Hypoxia allows for the stabilisation of the transcription factor HIF1α, which has been shown to contribute to the skewing of TAMs. 64 In addition, irradiated cells secrete TNFα, which has antitumour effects at high concentrations but is able to support survival, angiogenesis and metastases at lower levels. Blockage of the TNF-TNF receptor axis abrogates the radio-protective effect of macrophages. 65 This increased knowledge about the mechanisms underlying TAM involvement in tumour radio-resistance and relapse have allowed developing IT strategies in order to combine RT with TAM-targeted therapies (for depletion or re-education). 44,45,66,67 By contrast, different RT strategies might result in alternative scenarios where recruited TAMs can contribute to immunostimulation and antitumour activity. A local low-dose of ionising radiation causes differentiation of inducible nitric oxide synthase (iNOS)+ M1-like macrophages leading to the recruitment of tumour-specific T cells and tumour regression in human pancreatic carcinomas and insulinomas. 68,69 Furthermore, this proinflammatory macrophage skewing modulates endothelial cells activation and angiogenesis, thus collaborating with IT treatments. 70 This process is shown to be mediated by the DNA-damage repair related kinase ATM in HCT116 xenografts. 71 Surprisingly, a fractionated low dose cumulative regime (2Gy/fraction/day) polarised human monocyte-derived macrophages towards the proinflammatory phenotype without being able to revert their proinvasive and proangiogenic features. 72 In summary, macrophage responses to RT will range from antitumourigenic to promoting tumour progression depending on tumour type and environment, IR and dose and fractionation and additional treatments (chemo and/or IT). The whole landscape is extremely complicated and needs to be completely understood to take full advantage of macrophage-targeted therapy.

| LYMPHOID LINEAGE
Innate lymphoid cells (ILCs) derive from a common lymphoid progenitor and are defined by the absence of antigen specific B or T cell receptor because of the lack of recombination activating gene. In addition, ILCs do not express myeloid markers. They are associated with inflammation, tissue remodelling and homeostasis and, in a similar manner to their myeloid partners, ILCs can display both pro-and antitumourigenic activities. 73 ILCs are divided into 3 main groups, ILC1s, ILC2s and ILC3s, according to the expression of transcription factors and cytokine production. 74 In this section, we will focus on the role of the better studied NK cells, a specific subpopulation of ILC1s.  76 Investigation of ex vivo work found that NK cell sensitivity to ionising radiation varied between individuals 77 and between NK cell subsets with the more cytotoxic subsets showing increased resistance. In fact, low-dose fractionated RT in ex vivo experiments seems to increase NK activity and cell cytotoxic potential. 78 Nevertheless, NK cells are considered more sensitive to ionising radiation that T lymphocytes and their activation response is that of a typical response to radiation characterised by increased ATP production. Nowadays, RT is intensity modulated and utilises image-guided treatment targeting to minimise the effect on surrounding healthy tissue. Hence, the deleterious effect on the immune cells observed in early studies or from in vitro data is minimal and will only impact tumour-infiltrating lymphocytes. 79 In fact, it has long been accepted that RT stimulates NK cell function and, in return,  86 The effect of these soluble ligands seems to be regulatory as their binding to NKG2D leads to internalisation of the receptor and a desensitisation of the cells. 84 Soluble NKG2DL has been detected in multiple cancer patients and correlated with poor prognosis. 87,88 The major stress-inducible heat shock protein 70 (Hsp70) is a cytoplasmic chaperone that is overexpressed in multiple cancer types and associated with higher aggressiveness and resistance to standard chemo-RT by reducing therapy-induced stress. It plays a role in correct protein folding of nascent and misfolded proteins, transport across membranes and prevents protein aggregation. Hsp70 has been shown to be overexpressed following RT and its presence on the membrane of tumour cells renders them more susceptible to lysis by NK and not T cells. 89 A recent retrospective study in a squamous cell carcinoma of the head and neck patient cohort correlated high levels of Hsp70 and low levels on tumour infiltrating NK cells with unfavourable outcome following radio-chemotherapy. 90 Moreover, soluble Hsp70 has been shown to be very effective in stimulating NK cell function in the presence of inflammatory cytokines that it is now being tested in phase II clinical trials in combination with radiochemotherapy with promising results. 91,92 One of the most described effects of ionizing radiation on cancer cells is the upregulation of MHC1 and this in turn enhances the antitumoural T cell specific response 93 driven by an upregulation of IFN-γ in the TME. 94 In an ideal situation, the T cell response should be sufficient to eliminate the tumour. However, tumour-infiltrating lym-

| CLINICAL IMPLICATIONS AND ONGOING CLINICAL TRIALS
The combination of RT with immunomodulatory biological agents is a rapidly growing field. The trials differ in design, dose fractionation, sequencing and endpoints, but exhibit a conceptual theme of harnessing the abscopal effect, mainly in the context of advanced disease. Due to the extensive number of trials across different solid tumour types, we are unable to cover all ongoing trials in this review but aim to provide a representative clinical trial for each class of mechanism of action in Tables 1 and 2. The combination of immune checkpoint inhibitors (anti-PD1, anti-PD-L1, anti-CTLA4 and OX40 agonists) and RT have been covered comprehensively in other reviews. 102,103 They will not be discussed in this section, which will instead focus on modulation of the innate immune system including but not limited to: (i) autologous dendritic cell vaccination; Clinical trials of radiation therapy and stimulants of the innate immune response.  85 In addition, RT changes the vesicle-secreted patterns in the irradiated area, which may explain some of the effects observed in distant sites. 110 Our recent work shows that PD-L1 is secreted in exosomes, thus contributing to the generation of an immunosuppressive environment in distant sites of the tumour. 111 How the immune environment and more specifically, the innate compartment, contributes to these processes remains largely unknown and requires further investigation.

| Nomenclature of targets and ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY.

T A B L E 2
Ongoing clinical trials of radiation therapy and stimulants of the innate immune response. Status of clinical trials obtained from www.clinicaltrials.gov as of March 2020).