Effect of radiotherapy on T cell and PD-1 / PD-L1 blocking therapy in tumor microenvironment

ABSTRACT

Cancer is a worldwide problem that threatens human health. Radiotherapy plays an important role in a variety of cancer treatment methods. The administration of radiotherapy can alter the differentiation pathways and functions of T cells, which in turn improves the immune response of T cells. Radiotherapy can also induce up-regulation of PD-L1 expression, which means that it has great potential for enhancing the therapeutic effect of anti-PD-1/PD-L1 inhibitors and reducing the risk of drug resistance toward them. At present, the combination of radiotherapy and anti-PD-1/PD-L1 inhibitors has shown significant therapeutic effects in clinical tumor research. This review focuses on the mechanism of radiotherapy on T cells reported in recent years, as well as related research progress in the application of PD-1/PD-L1 blockers. It will provide a theoretical basis for the rational clinical application of radiotherapy combined with PD-1/PD-L1 inhibitors.

KEYWORDS:

• Radiotherapy
• t cell
• cd4-CD8
• pd-1/PD-L1
• immune resistance

Introduction

According to the latest global cancer statistics from the International Agency for Research on Cancer, in 2018, 18.1 million newly diagnosed cancer cases were recorded worldwide, and the number of deaths reached 9.6 million.^1,2 Radiotherapy is one of the most commonly used methods of cancer treatment.^3,4 About 60% of newly diagnosed cancer patients, as well as many existing or recurrent cancer patients, choose RT as part of their treatment plan.⁵ RT affects the differentiation pathways and functions of T cells in the tumor microenvironment. It then affects the anti-tumor immune response.⁶ However, the application of RT is also accompanied by corresponding side-effects. In recent years, immunotherapy has become the focus of cancer treatment due to its limited side effects,⁷ and its application has evolved from being an end-line treatment for some “hot tumors” to being the first-line. It has even entered the field of neoadjuvant therapy, where it has achieved amazing results.⁸ Recently, it has been found that radiotherapy also plays an important role in immunotherapy.^9,10 It can not only enhance the anti-tumor immune response, but can also prevent tumors cells from becoming resistant to the drug. These results are a key reason for the impact of radiotherapy on T cells in the tumor microenvironment. This article summarizes the recent scientific findings on this issue.

1. T-cell functional classification and transformation relationship in tumor microenvironment

Tumor microenvironment (TME) is the internal environment in which tumor cells are produced and survived, has complex regulatory mechanisms. In 1880, Paget¹¹ first proposed the renown “seed and soil” theory, which proposed the notion of TME as a kind of soil that provides the basis for the occurrence, development, invasion, and metastasis of tumors.

Recently, more and more researches have focused on the subsets of T cell. As an important component of TME, these subsets of T cell interact and restrict each other to form a complex network structure, and thereby maintaining a dynamic balance. In TME, antigen-presenting cells (APCs) are mostly needed for the process using T cells to killing tumors. Their surface includes a high variety of costimulatory factors, which can induce T cell activation by binding to corresponding antibodies on the surface of T cells. This specific process is shown in Figure 1.

Figure 1. Interacti of T cells and tumor cells in tumor microenvironment

1.1 Classification of T cells

The intensity of the immune response in TME is regulated by various T cell types.¹² Functionally, T cells in TME, it can be classified into cytotoxic T cells, regulatory T cells, auxiliary T cells and memory T cells.

1.1.1 Cytotoxic T cells

Cytotoxic T cell (CTL): This type of T cell can identify the infected cells by recognizing the pathogenic antigens, and subsequently kill them. CTLs are the main force behind cellular immunity, and their main surface marker is CD8. It can mediate the selective apoptosis of tumor cells by releasing perforin, granzyme, Fas or tumor necrosis factor (TNF).¹³ It has been confirmed that the number, location, and quality of CD8 + T cells positively correlate with the prognosis of many malignant tumors.¹⁴

1.1.2 Regulatory T cells

Regulatory T cells (Treg): These cells are responsible for up- and downregulation of the immune response of the body. They are an essential force that maintains immune tolerance and prevents autoimmune reactions.^15,16 Tregs have many phenotypes, among which are the CD4⁺ T cell, which can inhibit the immune response against autologous tumor cells. This inhibition is considered to be the main reason for the failure of immunotherapy.^17,18

1.1.3 Helper T cells

Helper T cell (TH): These cells play an intermediate role in the immune response. They can activate other types of immune cells through amplification of their own numbers, causing them to directly participate in the immune response. The main surface marker is CD4. Studies have found that the expression of TH17 is associated with poor prognosis in patients with resectable colorectal cancer (CRC),¹⁹ while the presence of TH1 is correlated with longer disease-free survival.

1.1.4 Memory T cells

Memory T cells (TM): These cells play an immune role when they are re-exposured to known antigens. However, no specific surface markers have been found. A large number of research data have shown that the long-term anti-tumor function of the body largely depends on the cell differentiation state during TM metastasis.²⁰ T memory stem cells (TSCM) and central memory (TCM) T cells, which have lower differentiation, longer life span²¹ and higher recombination ability.^22,23 They are potentially effective in mediating anti-tumor immunity.

1.2 Transformation between CD4-CD8 lineages

Mature T cells mainly differentiate into two lineages, CD4⁺ and CD8⁺ T cells, which are different in MHC limitation and function.^24,25 CD4⁺ T cells are usually restricted by MHC-II, while CD8⁺ T cells require MHC-I to exert cytotoxicity. Both CD4⁺ and CD8⁺ T cells can participate in cellular immunity, and there is a transformational relationship between them.

T cells are produced by hematopoietic progenitor cells and they mature in the thymus. They transform from CD4⁻ CD8^–double negative (DN) stage²⁶ into CD4⁺ CD8⁺-double positive (DP) thymocytes, producing CD4⁺CD8lo intermediate cells. When they receive TCR-mediated differentiation signals, negative or positive selection will occur. Only positive cells could receive TCR signals and further differentiate into CD4⁺- or CD8⁺- single positive (SP) T cells. However, the time, intensity, and duration of TCR signals influence the lineage stereotypes. For example, CD4 differentiation requires a longer stimulation time of TCR signals.²⁷

In addition to the stimulation of TCR signals, the finalization of the differentiation into CD4 or CD8 also requires the participation of many transformation factors. The differentiation into CD4 requires zinc finger proteins Th-POK and GATA3,^28,29 as well as the trans-acting T cell factor 1 (TCF-1) and lymphoid enhancer factor 1 (LEF-1)³⁰ while the differentiation into CD8 is inseparable from the regulation of RUNX factor.³¹ It has been found that Th-POK-RUNX is a key adjustment axis in the determining whether T cells differentiate into the CD4 or CD8 lineage, it plays a regulatory role through a negative feedback mechanism.^32,33 When the presence of key transforming factors is absent or decreased in the course of the T cells maturation, the intermediate cells of CD4⁺CD8lo that should have developed into CD4⁺ SP, may instead develop into CD8⁺ SP, and vice versa. Therefore, we may be able to enhance the immune response of T cells in TME by re-engineer the differentiation of T cells into either CD4 or CD8 lineage.

1.3 Stem-like and terminally differentiated CD8⁺ T cells

In recent years, the team of Professor Haydn Kissick has found that tumor-infiltrating CD8⁺ T cells can be divided into two categories: stem-like CD8⁺ T cells and terminally differentiated CD8⁺ T cells.³⁴ Between them, the stem-like CD8⁺ T cells express low checkpoint molecules, high co-stimulatory molecules CD28, and transcription factor TCF-1, whereas the terminally differentiate type highly expresses immune checkpoint molecules TIM3, PD-1, CTLA4, and TIGIT. Stem-like and terminally differentiated CD8⁺ T cells accumulate in areas where antigen-presenting cells are abundant. Their expression levels are closely related to the efficacy of patients’ anti-tumor therapy, so they can be used as biomarkers for predicting treatment efficacy.³⁵

Differentiated lineage of CD8⁺ T cells has been found in patients with chronic lymphocytic choriomeningitis virus (LCMV) infection.³⁶ The general direction is that TCF-1⁺ stem-like CD8⁺ T cells first differentiate into CD101-Tim-3⁺ transient cells, and then transformed into CD101⁺ Tim-3⁺ terminally differentiated cells. The differentiation process of CD8⁺ T cells in tumors is similar.^37,38,38,39 It can be seen that TCF-1 is a key transcription factor for stem-like CD8⁺ T cells,^39,40,42 which is essential for T cell proliferation and activation.^39,41 TCF-1/TCF-7⁺ CD8⁺ T cells have been detected in human tumors, and their increased expression is associated with positive responses to immune checkpoint blockade therapy.^37,42–44 Professor Zhang Zemin’s research team⁴⁵ developed the STARTRAC method and found that there was a significant TCR overlap between the stem-like type and the terminally differentiated type of CD8⁺ T cells, which may determine whether tumor-infiltrating effector memory CD8⁺ T cells differentiate into effector T cells or “exhausted “ CD8⁺ T cells. This discovery also explained the source of the terminally differentiate type T cells in TME. Other studies have also found that the response of T cells in tumors depends on the ability of CD8 stem-like cells to produce terminally differentiated cells.^{34,39,46–49} In conclusion, reversing the status of CD8 + T cells may change the efficacy of tumor immunotherapy.

2. Mechanism and effect of radiotherapy on T cells in TME

Radiotherapy plays an important role in the treatment of many tumors, such as neoadjuvant radiochemotherapy⁵⁰ and neoadjuvant radiotherapy⁵¹ in patients with stage II and III rectal cancer, as well as concurrent postoperative radiotherapy and chemotherapy.^52,53 RT can play an anti-tumor role by increasing the release of cytokines, recruiting antigen-presenting cells and their surface epitopes⁵⁴and enhancing the expression of antigens or receptors on the membrane of tumors or immune cells.^55–57 RT can not only control local lesions, but also produce distant effects,⁵⁸ which may be related to biological characteristics of tumor cells and factors such as TME remodeling caused by RT.⁵⁹ Previous studies have shown that RT can regulate the innate and adaptive immune systems,^60,61 which mean that they have potential immunomodulatory effects. Therefore, it is evident that RT can affect T cells in TME. The following sections will list the various mechanisms of how RT regulates T cells in TME:

1. RT can enhance the binding of T cells onto antigens. RT can directly kill or induce tumor cell apoptosis by inducing DNA double-strand breaks,⁶² up-regulating and releasing tumor-associated antigens (TAAs),^63–65 including adenosine triphosphate (ATP) and HMGB1 protein^66,67 released by stress cells, and increasing the expression level of damage-related model molecules (DAMPs)^68–70 to induce DC maturation. Studies have also confirmed that RT can induce the diversity of TCR sequences⁷¹ and increase the expression of MHC-I,⁷² which enhances the directional selection of CD8⁺ T cells. Furthermore, RT can also increase the effectiveness of antigen presentation, thereby kickstarting the initial differentiation of T cells, and finally activating the memory of adaptive immunity of the body.

2. RT induces the production and release of cytokines. This is done by activating the cGAS-STING signaling pathway and increasing the sensitivity of tumor cells to RT.^73–78 The activation of this pathway increases the release of interferon regulatory factors (IRFs) and NF-κB, and the high expression of the latter can induce the release of type I interferons (INF-α and INF-β),⁷⁹ which is used in responding to the activation of pattern recognition receptors. The expression of NF-κB is also correlated with the release of type II interferon (IFN-γ). IFN-γ is mainly produced by T cells when recognizing homologous antigens. RT can up-regulate the expression of IFN-γ⁸⁰ thereby activating anti-tumor immune response. Other studies have shown that RT promotes the secretion of CXCL16 in tumor cells. This cytokine can be combined with TH1 cells and CXCR6 on activated CD8 ⁺ T cells⁸⁰ to promote the infiltration of local immune cells⁸¹ and induce the anti-tumor response of the body.^73,82,83

3. RT can regulate the ratio of CD4/CD8 T cells in TME by increasing the number of Tregs cells in TME.⁸⁴ Treg cells can convert ATP into adenosine, and they participate in immunosuppression by increasing the production of CTLA4, TGF β, IL-10, CD39, and CD73.^85,86 According to various reports, RT can increase the number of Foxp3⁺ CD4⁺ Tregs in tumors, while the ratio of CD8⁺ T/Tregs decreases,⁸⁷ exerting a negative anti-tumor immunomodulatory effect.^88,89.

4. RT changes the physical properties of TME, which in turn improves the recruitment of T cells. For example, the intercellular adhesion of molecule-1 (ICAM-1) on the cell surface is enhanced through the regulation of the extracellular matrix (ECM) interstitial fluid pressure,⁹⁰ and tissue stiffness,⁹¹ as well as the oxygen content and pH value of tumor nourishment vessels. The expression of ICAM-1 in endothelial cells promotes the translocation of leukocytes to the endothelial cells, and induces the occurrence of inflammatory TME,⁹² so as to increase the infiltration of T cells into tumor tissues.

5. RT increases the expression of PD-L1 in immune checkpoint. This is achieved through activating the cGAS-STING pathway, which influences the production of interferon through IRFs and NF-κB,⁷⁹ and promotes the expression of PD-L1 on the surface of tumor cell membranes,⁹³ thus affecting the response of immune checkpoint inhibitors.^94,95 RT has the potential to trigger the anti-tumor immune responses by improving the efficacy of immune checkpoint blockade therapy (ICBs).^96,97

6. RT may alter the differentiation between CD4-CD8 categories. In the study of Jacek et al., GATA-3 expression was significantly increased after stereotactic ablation and radiotherapy, which was the main regulator of humoral immune responses.⁹⁸ GATA3, was the first transcription factor identified in CD4 differentiation. In the absence of GATA3 expression, MHC-II restricted thymocytes can be “redirected” to differentiate into CD8⁺ T cells.⁹⁹ At present, there is no research on the relationship between radiotherapy and the Th-POK-RUNX axis.

3. Limitations of the application of PD-1/PD-L1 inhibitors

In recent years, PD-1/PD-L1 immune checkpoint inhibitors have become the focus of tumor treatment,^100,101 and their expression level has been found to be related to the pathological type, clinical pathological stage, drug resistance,¹⁰² and the prognosis of some tumors.^103–105 The expression level of PD-1/PD-L1 is significantly different in different types of tumors or different subtypes of the same tumor.⁷⁸ While there are some tumor patients with positive PD-L1 who do not respond to the treatment, there are also some patients with negative PD-L1 who can benefit from it.^106,107 Therefore, the efficacy of ICBs may be related to the MSI classification and drug resistance of tumors.

3.1 The effect of MSI classification on efficacy

The results of clinical trials of anti-PD-1/PD-L1 drugs suggest that, compared with MSS tumors, patients with MSI tumors can achieve better clinical remission and longer survival time.^108,109 These advantages may be attributed to the expression of multiple immune molecules in TME of patients with MSI type. At present, solid tumors with high incidence of MSI-H are known to include endometrial cancer (20% ~ 30%), gastric cancer (15% ~ 20%) and colorectal cancer (12 ~ 15%, of which stage IV colorectal cancer is 4% ~ 5%).¹¹⁰ It has been pointed out that MSI-H is the key factor for the benefits of ICBs, which has nothing to do with specific type of cancer.⁵⁹ It can be seen that MSI classification has an important impact on the application of ICBs, while MSS tumors account for a large proportion. The quest to reverse the sensitivity of MSS to ICBs, so that more cancer patients can benefit from it, will be a long-term and difficult one in anti-tumor treatment.

3.2 The effect of drug resistance on efficacy

The difference in efficacy of PD-1/PD-L1 blockers may be attributed to tumors that become drug-resistant.¹¹¹ The emergence of drug resistance is not only related to the heterogeneity of tumor,¹¹² but also caused by the changes in the composition and function of T cells in TME.¹¹³ At present, the mechanism of resistance to PD-1/PD-L1 is generally divided into two types: primary resistance and acquired resistance.

3.2.1 Primary resistance

Primary drug resistance means that the patient’s display drug resistance upon his/her first use of ICBs, also known as “congenital drug resistance”⁸⁰ The primary resistance toward PD-1/PD-L1 blocking therapy is mainly caused by the lack of PD-L1 expression and the production of inhibitory cytokines by some immune cells in TME. These cytokines inhibit the activation and function of T cells by influencing the activation of corresponding pathways and inducing adaptive immune response. The specific mechanisms include:

3.2.1.1 Activation of MAPK pathway

The MAPK pathway plays a vital role in cell proliferation, differentiation, migration, apoptosis, and survival.¹¹⁴ It can promote the production of immunomodulatory cytokines,¹¹⁵ and promote the activation and infiltration of CD8⁺ T cells.

3.2.1.2 Loss of PTEN expression

The absence of PTEN increases the expression of immunosuppressive cytokines,¹¹⁶ and decreases the cytotoxicity CD8⁺ T cells and the killing effect of NK cells¹¹⁷ in TME. These lead to an increase of resistance in tumor cells and inhibition of their apoptosis, which in turn leads to poor prognosis.¹¹⁸

3.2.1.3 Enhancement of PI3K/Akt signaling pathway

The abnormal activation of the PI3K/AKT pathway can be observed in many human tumors.^119,120 The activation of this pathway leads to an increase in the expression of PD-L1, mediates the immune escape of tumor cells, and promotes the occurrence and progress of tumors.^80,121

3.2.1.4 Wnt-β-catenin signaling pathway is continuously activated

The typical WNT-β-catenin signal transduction pathway is activated due to the binding of WNT family proteins to cell surface receptors, which leads to the nuclear translocation and transcriptional activation of β-catenin,¹²² reduction in the production of immunosuppressive cytokines, and inhibition of T cell recruitments,^80,123 eventually allowing for the occurrence of immune tolerance.¹²⁴

3.2.1.5 Loss of IFN-γ signaling pathway

IFN-γ can up-regulate the expression of MHC and PD-L1, thereby increasing the positive response to PD-1 therapy.¹²⁵ Therefore, the absence of IFN-γ and its receptor may cause the occurrence of or maintain drug resistance in tumor cells,^126,127 resulting in no response to ICBs.^128,129

3.2.1.6 Epigenetic modification changes

Epigenetic modification of cells, including DNA methylation, histone methylation/acetylation and microRNA regulation, can regulate not only the processing and presentation of antigens, but also the expression of PD-1¹³⁰/PD-L1.¹³¹ Therefore, altering or regulating the process of epigenetic modification of cells may exacerbate the drug resistance of tumors.

3.2.1.7 Overexpression of CDK4/CDK6

The activation and overexpression of the cell division cycle (CDK) genes and defects in the CDK functional inhibitors (CDKIs) are found in most tumor cells.¹³² Among them, CDK4/CDK6 are related to the tumorigenesis.^133–135 When combined with Cyclin D, they can cause cells to transition from the G1 phase to the S phase in the cell cycle,¹²² which in turn drives tumor resistance.¹³⁶

3.2.2 Acquired resistance

Acquired drug resistance means that patients initially respond to ICBs treatment, but as the disease progress or recur during treatment (rather than after discontinuation), reapplying immune checkpoint blockers becomes ineffective. The mechanisms of acquired drug resistance mostly involve some changes the activity of T cells,¹³⁷ which are triggered by a mutation of β2 M gene in MHC-I molecules, the up-regulation of other immune checkpoints, and the activation of JAK/STAT/IFN-γ pathway.

3.2.2.1 β2 M gene mutation

Immune editing can cause mutations in the β2 M gene of MHC-I, resulting in the loss of HLA and reduce diversity,^43,125 altering the presentation of antigens,^59,138,139 thereby inhibiting the activation and function of T cells.¹⁴⁰ Therefore, the mutation of β2 M gene is the key to the development of resistance toward PD-1/PD-L1 therapy.^{125,128,141,142}

3.2.2.2 Up-regulation of other immune checkpoints

A study has found that CLTA-4, TIM-3, LAG-3, B and T lymphocyte attenuators (BTLA), NKG2A, VISTA, and TIGIT are involved in the regulation of PD-1/PD-L1 in TME.^143–145 If PD-1/PD-L1 checkpoint is blocked, the expression of other molecular immune monitoring points of tumor cells will be up-regulated,¹³⁰ enabling those cells to evade the anti-tumor immune response of the body.

3.2.2.3 Activation of JAK/STAT/IFN-γ pathway

JAK1 and JAK2 are the key signals of IFN.^80,125 The activation of IFN-γ signaling pathway can be found in drug-resistant tumor cells, and it prompts high transcription of genes such as JAK1 and STAT1, as well as the expression of Apelin receptor, which encodes interferon signal regulators. They can increase the sensitivity of tumor cells to IFN-γ¹²² The JAK2 gene is on the same chromosome as PD-L1 and PD-L2, which can be further enhanced by co-amplification.⁸⁰ Therefore, mutations in JAK1/2 play an important role in anti-PD-1/PD-L1 therapy.¹⁴⁶

4. Application of radiotherapy combined with PD-1/PD-L1 blocker

Previous studies have found that the response rate of PD-1/PD-L1 blockade therapy is extremely low,^45,147 indicating that there are limitations to using this treatment alone. At present, RT combined with an anti-PD-1/PD-L1 treatment is more effective than only using radiotherapy ¹⁴⁸ or anti-PD-1/PD-L1 antibody treatment alone.^149,150Many clinical trials have confirmed that RT, when combined with anti-PD-1/PD-L1 therapy, has a synergistic sensitization effect.^151,152 Therefore, combination therapy strategies can provide more drug targets and improve efficacy.

4.1 The mechanisms of synergy between RT and PD-1/PD-L1 therapies

As described above in detail, RT can induce DNA double-strand breaks in tumor cells, up-regulate the expression of TAAs⁷⁰ and DAMPs,⁷⁵ and then promote the maturation of DCs. Mature DCs can recruit and activate naive T cells in TME. The effect of RT on the differentiation of CD4-CD8 lineages can also increase the infiltration of CD8 T cells in TME,⁹⁹ which leads to the increase of the PD-1 expression. In addition to its positive immunomodulatory effect, RT can also induce an immunosuppressive microenvironment.¹⁵³ Recent preclinical studies have shown that RT can up-regulate the expression of PD-L1 through the use of three mechanisms. However, they are ultimately achieved through STAT/IRF pathway.¹⁵⁴ The details are as follows: 1) DNA damage response activated the ATM/ATR/Chk1 kinase and cGAS/STING pathway.^155,156 2) Increased secretion of IFN,¹⁵⁷ especially IFN – γ¹⁵⁸ can continuously stimulate the JAK-STAT-IRF pathway, thereby promoting the production of PD-L1 for a long time.^129,153 3) After irradiation, the secretion of epidermal growth factor (EGF) and IL-6 increased, which enhanced the EGFR signal and activated the IL-6/JAK/STAT3 pathway. The combination of the PD-1 of T cells with the PD-L1 of tumor cells will make T cells lose their ability to kill tumor cells, and lead to immune escape and resistance. Therefore, if PD-1/PD-L1 blockers are administered in the early stage of RT, the activity of “exhausted” CD8⁺ T cells can be restored and an effective anti-tumor immune response can be produced.¹⁵⁹ The detailed process is shown in Figure 2.

Figure 2. The proposel mechanisms of synergy between RT and PD-1/PD-L1 therapies

4.2 Influencing factors of combination therapy

Evidently, if one hopes to achieve the synergistic effect of combination therapy,^{55,93,160,161} they first need to formulate a reasonable treatment plan and analyze possible influential factors. The combined application can regulate and amplify the remote effect of RT,⁵⁴ increase the mutation load,¹⁶² and improve the immune function and anti-tumor capabilities.^163,164 With further research, we have learned that the therapeutic effect and risk of toxicity of radiotherapy combined with anti-PD-1/PD-L1 depend on the sequence of its application,¹⁶⁵ as well as the grade and dose of radiation therapy.¹⁶⁶

Dove-di et al.¹⁶⁷ compared three different combined treatment regimens using the mouse model. The results showed that in order to achieve long-term tumor control, anti-PD-L1 therapy should be administered simultaneously with radiotherapy. The experiments of Chen et al.¹⁶⁸ also showed that the simultaneous application of radiotherapy and PD-1/PD-L1 monoclonal antibody treatment regimen was significantly better than sequential therapy and radiotherapy alone, in terms of survival period and 1-year progression-free survival rate. Recently, the PFS of patients with locally advanced unresectable stage III non-small-cell lung cancer (NSCLC), who were treated with pembrolizumab and concurrent chemoradiotherapy, was 69.7% after 12 months, and had a high toxicity tolerance.¹⁶⁹ However, the PEMBRO-RT trial showed that the administration of anti-PD-1/PD-L1, if administered within one week following radiotherapy, significantly prolonged OS and PFS in PD-L1 negative NSCLC patients, thus supporting the feasibility of sequential therapy.¹⁵² The initial publication of the KEYNOTE-001 and PACIFIC trials demonstrated the feasibility and effectiveness of sequential radiotherapy combined with PD-1/PD-L1 inhibitors.^149,170 Additionally, the clinical results of stereotactic radiotherapy and anti-PD-1 therapy for the treatment of melanoma brain metastases showed no significant difference in OS and local control.¹⁷¹ Komori T et al. discovered that the toxicity risk demonstrated no impact from the different treatment sequences.¹⁷² At present, there is no worldwide consensus on the optimal dosage and mode of combination therapy. For optimal dose fractions, one report found that a single high-dose irradiation (20 Gy in a single fraction) can lead to reduced immunogenicity.⁷⁴ However, in another clinical trial, where patients were divided into three radiotherapy regimens (15–24 Gy in 1 fraction, 18–24 Gy in 3 fractions, or 25 Gy in 5 fractions), the results showed that there was no difference in OS and PFS.¹⁶⁸ Therefore, in order to improve the efficiency of the treatment and achieve long-term control of the disease, more clinical trials are needed to verify the sequence, dose, and grading of the combined treatment.

4.3 Screening of benefit groups in combination therapy

In recent years, many preclinical and clinical studies have shown that,^79,173–175 in advanced or metastatic melanoma, colorectal cancer, Hodgkin’s lymphoma, breast cancer, non-small cell lung cancer, renal cell carcinoma and esophageal cancer, radiotherapy combined with PD-1/PD-L1 inhibitors can improve the chances of long-term survival and prevent tumor recurrence.^176–179 So, figuring out how to select the beneficiaries of this treatment more accurately and effectively has become an urgent task.

A variety of biomarkers have been observed, including the expression level of CTL,^35,180 PD-1/PD-L1 in TME,¹⁸¹ the apparent diffusion coefficient,^182,183 mismatch repair (MMR), microsatellite instability,^184,185 and some neutrophil-lymphocyte ratio,¹⁸⁶which can predict the efficacy of RT combined with PD-1/PD-L1 inhibitors to some extent. Although no precise predictive biomarkers have been found, previous studies have found that patients with a history of RT have higher PFS, OS, and response rates than those who do not receive anti-PD-1/PD-L1 antibodies.^187,188 The OS difference was only found in the PD-L1 negative subgroup when combined therapy was used.¹⁵² These results suggest that radiotherapy may be an important factor in reversing PD-L1 expression. Therefore, the accuracy of predictions can be improved by combining multiple prediction indicators.

5. Summary

In recent years, with the continuous development of treatment methods, health-care technologies, and patients’ growing demands for a better quality of life, the treatment plan of tumor patients has gradually changed from standardization to precision and individualization. Radiotherapy can not only reshape T cells in TME, but also provide a basis for immune checkpoint-blocked therapy. It also shows great potential in reversing the immune resistance of tumor cells. One study found that the combination of radiotherapy and immunotherapy can benefit patients with giant lung metastatic melanoma.¹⁸⁹ It is imperative that, when combining radiotherapy with anti-PD-1/PD-L1 antibody treatment, medical professionals pay attention to its adverse reactions, such as the increase in the incidence of radiation pneumonia.¹⁴⁹ At present, more clinical studies are needed to confirm whether (1) radiotherapy can facilitate the conversion between T cell subsets and their specific transformation mechanism; (2) the combined treatment of radiotherapy with PD-1/PD-L1 inhibitors can be used as a first-line treatment program for more tumor patients; (3) the combined application can reverse the immune tolerance of MSS patients and the mechanism of action. It is believed that in the field of tumor therapy, the combined treatment of radiotherapy and immunotherapy will have a broader application prospects in the near future.

Author contributions

Liu Yanlong and Chen Chen made substantial contributions to the conception and design of this study. Chen Chen wrote the first draft of the manuscript. All authors made substantial contributions to the acquisition or analysis of data used in this article. Cui Binbin revised the manuscript for the purpose of important intellectual content.

Ethical approval and ethical standards

The study was performed in accordance with the Declaration of Helsinki. This study does not contain any studies with human participants or animals performed by any of the authors.

Informed consent

Informed consent is not required for this study.

Disclosure of potential conlficts of interest

The authors declare no potential conflict of interest. All authors approve of this manuscript.