Macrophages play critical roles in tissue homeostasis by regulating tissue development, mediating inflammatory responses and clearing pathogens and cell debris (DeNardo and Ruffell, 2019; Zheng et al., 2021). They are inducible in function, with the classically activated M1 type exerting pro-inflammatory and antitumor activities while the M2 type functioning basically the opposite (Mills et al., 2016). Local ROS concentration has an obvious influence on the polarization of macrophages, and based on current evidences ROS may induce pro-inflammatory macrophages more dominantly than doing the opposite. They can activate nuclear factor κB (NFκB) and p38 mitogen-activated protein kinase (MAPK) signaling pathways and promote the expression of M1-associated pro-inflammatory cytokines and chemokines (Rendra, et al., 2019). This mechanism is widely accepted in innate immunity and has also been reported to augment the antitumor immunity. For example, iron overload, which rapidly induced ROS production, polarized macrophages to pro-inflammatory phenotype, enhanced the activity of p300/CBP acetyltransferase and improved p53 acetylation (Zhou et al., 2018). However, other studies indicated the M2-promoting function of ROS. Typical studies involving diverse M2-promiting mechanisms have been summarized in Table 1.

Therefore, ROS can induce the differentiation of macrophages to both M1 and M2 types, raising uncertainty for the direction of ROS modulation (Rendra et al., 2019; Zhou et al., 2020). What further complicates the situation is that factors inducing M1 polarization may not provide benefits for cancer suppression. For example, it was shown that black raspberries, which served as an antioxidant, reduced the incidence of esophageal cancer by suppressing oxidative stress and NFκB/MAPK signaling (Shi, et al., 2017). Given the presence of pathways that lead to contrary results, it can be envisioned that ROS may simultaneously exert opposite influences on macrophages, and the ultimate impact may depend on ROS concentration (including the relative concentration of different species), location and their interaction with therapeutic agents. It is noteworthy that besides M1/M2 polarization, ROS influence macrophages in many other aspects. Roux et al. showed that ROS mediated the immunosuppression effect of macrophages by up-regulating the expression of programmed death ligand-1 (PD-L1) (Roux et al., 2019). When treated with ROS inducers such as paclitaxel, PD-L1 expression was up-regulated on the surface of tumor-associated (TAMs) in a mouse model of triple negative breast cancer, via the activation of NFκB signaling. Note that both M1 and M2 signatures positively correlated with the expression of PD-L1.

Using monoclonal antibodies to block the immune checkpoint-mediated immune escape has been one of the most promising strategy for tumor control (Havel et al., 2019). The immune microenvironment exerts a great influence on the treatment efficacy, while ROS serve as critical mediators. A large number of studies have shown that ROS generation would lead to augmented expression of the programmed death-ligand 1 (PD-L1) on cancer cells. A straight forward mechanism is that ROS elevation leads to the upregulation or stabilization of multiple transcription factors such as NFκB and HIF-1α, while NFκB initiates PD-L1 expression by binding to the PD-L1 promoter (Guo et al., 2019), and HIF-1α directly binds to a transcriptionally active hypoxia-response element in the PD-L1 proximal promoter (Noman et al., 2014). Note that hypoxia-induced HIF-1α activation can either elevate ROS level via NOX or reduce ROS by inhibiting the tricarboxylic acid cycle (Chen et al., 2018). There are also studies reporting enhanced ROS generation with reduced PD-L1 expression or vice versa, as summarized in a review article (Bailly, 2020). Nevertheless, in most occasions the PD-L1 expression is positively correlated with ROS level, as demonstrated by using a large variety of ROS-modulating small molecules and human oncoviruses (Montani, et al., 2018). Meanwhile, the role macrophage plays in the connection between ROS and PD-L1 expression is worth noting. ROS is a critical mediator of macrophage polarization, while PD-L1 high expression has been found to be correlated with M2-polarization of macrophages (Zhu, et al., 2020). Therefore, skewing the M1/M2 balance of macrophages may be a potential route by which ROS modulate PD-L1 expression.

ROS also have an impact on programmed death-1 (PD-1) blocking therapy at least due to the ligand/receptor relationship of PD-L1/PD-1. Chamoto et al. reported that tumor-reactive T cells boosted by PD-L1 blockade possessed activated mitochondria with augmented ROS production, and improving ROS generation using ROS precursors or mitochondrial uncouplers synergized the antitumor activity of PD-1 blockade by expansion of effector/memory cytotoxic T cells in draining lymph nodes (Chamoto et al., 2017). A more recent study reported similar results, that in a “bilateral tumor model”, ROS increment in CD8⁺ T cells was observed only in tumors that were responsive to PD-1 blockade therapy (Kumar et al., 2020). Therefore, ROS level in tumoral and lymphatic cells might be a potential indicator of the responsiveness to PD-1/PD-L1 blockade therapy, especially considering that the expression of immune checkpoints has been accepted as a tumor-intrinsic sign of the vulnerability of tumors to ICB therapy (Zappasodi et al., 2018).

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is a vital regulator of T cell function. Studies directly exploring the effect of ROS on CTLA-4 therapy are rare to the best of our knowledge, but ROS have an obvious influence on the development of T regulatory cells (Tregs), on which CTLA-4 is constitutively expressed (Walker 2013). The effects are still multi-faced. For example, macrophage-generated ROS were functional for the induction of Tregs (Kraaij et al., 2010), while neutrophil cytosolic factor 1-deficient mice with a lower level of ROS also carried Tregs more reactive than those from wild mice (Kim et al., 2014). Obviously, the expansion of CTLA-4 blocking therapy warrants further studies on the effects of ROS.

Activation of T cells, as the pivotal step in cellular immunity, relies on the binding with main histocompatibility complex, the stimulation by co-stimulatory molecules on antigen-presenting cells, and a suitable biochemical environment that allows these processes to happen. ROS have a great influence on T cell activation. An example is that the local number and phenotype of macrophages, which often function as antigen presenting cells, can be re-/programmed by ROS as described above. Another aspect is the ROS-tuned expression of immune checkpoint molecules on immune cells and cancer cells. More directly, ROS can create an oxidative environment to inactivate T cells. It was reported that the redox level on cell surface physically determines the reactivity of T cells (Sahaf et al., 2003). Researchers found that mice were more susceptible to develop severe arthritis if ROS production was diminished, and then revealed that lower ROS level would increase the number of reduced thiol groups on T cell membrane surface and make T cells more prone to be activated (Gelderman et al., 2006). This is a necessary mechanism to prevent the over-activation of T cells in inflammatory sites, while for cancer treatment this is commonly undesirable (Moro-Garcia et al., 2018; Yin et al., 2021). Artificially increasing cell surface thiol by adding antioxidants (e.g., glutathione, GSH) or reducing ROS generation has been employed to increase the sensitivity of T cells to stimulatory signals (Kesarwani, et al., 2013).

Efficient expansion of tumor-specific T cells upon activation is necessary in cell therapy, and the failure to do so has been a major limitation for adoptive cell therapy to achieve broader application. It was shown that the persistence of effector CD8⁺ T cells and CD62L^hi central memory T cells were obviously longer if the cytosolic GSH and surface thiol were higher (Kesarwani et al., 2015), while GSH depletion prevented T cell proliferation despite the stimulation using antigens (Mak et al., 2017). Pretreatment with antioxidant N-acetyl cysteine (NAC) during ex vivo T cell expansion process significantly improved the persistence of adoptively transferred cells and led to more effective tumor control in a mouse model of melanoma (Scheffel et al., 2016). The underlying mechanism was revealed to be reduction in DNA damage by reducing ROS and the resultant reduced activation-induced cell death (an immunosuppressive process known to be induced by repeated stimulation of T cell receptor) of T cells in the presence of NAC (Scheffel et al., 2016). All these evidence suggests the necessity of adding antioxidants to the culture media of therapeutic T cell survival and expansion. Note that both GSH and NAC contain thiol groups as potent reducing moiety to scavenge electrons from highly reactive molecules, e.g., to consume ROS.

Cancer develops with mutations, resulting in the emergence of abundant neoepitopes (sequence-altered nucleic acids and proteins) that are foreign to host’s immune system. Immune responses induced by a specific neoepitope may fail to damage tumor cells that do not contain this neoepitope, while immunogenic cell death (ICD), which is featured by the release of tumor-associated antigens and danger-associated molecular patterns, will provide a full spectrum of neoepitopes to eliminate immune escape caused by tumor heterogeneity. ROS generally have an inducing effect to ICD occurrence. The induction of endoplasmic reticulum (ER) stress, surface exposure of calreticulin, and secretion of adenosine triphosphate (ATP), high-mobility group box 1 (HMGB1) and heat shock protein 70 (HSP70) are requisites for ICD (Bugaut et al., 2013; Van Loenhout et al., 2020), while many of these processes can be triggered by ROS. An example is that bleomycin (an anticancer drug relying on its ability to generate ROS) induced ER stress and autophagy, which then led to calreticulin exposure and release of HMGB1 and ATP to trigger ICD (Bugaut et al., 2013). Actually, many chemotherapeutic small molecules known to kill cancer cells via ROS generation are undergoing clinical trials as ICD inducers beyond chemo drugs, such as doxorubicin, bortezomib, and epirubicin (Vanmeerbeek et al., 2020). Other kinds of agents are also under exploration. For example, a fluorinated mitochondria-disrupting helical polypeptides, which could destabilize mitochondrial outer membrane, was developed to over-produce intracellular ROS (iROS), induce ICD and enhance PD-L1 blockade therapy (Jeong et al., 2021).

Particularly, a number of treatment modalities have intrinsic capability to induce ICD by producing ROS or other critical stimulators. 1) Photodynamic therapy (PDT), which kills cancer cells by generating abundant ROS with the assistance of photosensitizers and light irradiation, can induce ICD and antitumor immunity (Panzarini et al., 2013). Cellular internalization of photosensitizers causes high iROS level and ER stress especially when photosensitizers localizes near the ER. Using a ER-associated photosensitizer, hypericin, Garg et al. found that PDT generated obvious ER stress, and caused cancer cells to secrete ATP, display damage-associated molecular patterns on cell surface and undergo immunogenic apoptosis (Garg et al., 2012; Garg et al., 2012). The display of calreticulin was crucial by providing the motifs needed for the engulfment of PDT-treated cells by dendritic cells (Garg et al., 2012). Using other photosensitizers other than hypericin failed to induce the exposure of calreticulin on cell surface (Garg et al., 2012), suggesting the necessity of choosing suitable photosensitizers or choosing suitable drug carriers to afford enhanced affinity to ER. 2) Sonodynamic therapy is similar with PDT but employs ultrasound as the energy source (Li et al., 2021), and has been reported to elicit ICD. For instance, a nanocomposite loaded with chlorin e6 (as a sonosensitizer) induced ICD via receptor-interacting protein kinase 3–dependent cell necroptosis (Um et al., 2020). 3) Radiotherapy produces ROS via radiolysis and induce ICD, although the break of double-strand DNA was previously considered as the primary mechanism of tumor suppression in radiotherapy. Actually, ICD-mediated antitumor immunity has been recognized as the origin of abscopal effect in radiotherapy.

There are also studies reporting obvious inhibition of ICD-induced immune response by elevated ROS. Kazama et al. reported that ROS would neutralize the stimulatory capacity of dying cells (Kazama et al., 2008). The results showed that caspase activation against mitochondria promoted immune tolerance of apoptotic cells by generating ROS to oxidize the HMGB1 (Kazama et al., 2008). HMGB1 potently act on dendritic cells to stimulate immunity (Dumitriu et al., 2005), so its inactivation promotes immune tolerance. Using a ROS scavenger to consume extracellular ROS (eROS) enhanced the stimulatory effect of dying cells by avoiding the oxidation of HMGB1 (Deng et al., 2020). Therefore, as depicted in Figure 1, there might be a need to induce ER stress via iROS and simultaneously eliminate eROS to avoid the oxidization of the exposed calreticulin and the released stimulatory molecules.