Introduction

Toll-like receptors (TLRs) are a large family of proteins and a class of pattern recognition receptors, which are not only expressed in immune cells, but also in various tumor cells. As the key components, TLRs are evolutionarily conserved innate immune molecules that play an important part in the innate immune system and promote adaptive immune responses as a bridge between innate immunity and adaptive immunity. Different TLRs are expressed differently in different target cells and play different functions by activating different immune cascades [1]. There are ten kinds of TLRs in the human (TLR1-TLR10) and 13 kinds of TLRs in mice. TLR1, TLR2, TLR4, TLR5, TLR6, TLR10, and TLR11 are receptors located on the surface of the cell membrane that recognize extracellular components of pathogens, and TLR3, TLR7, TLR8, TLR9, and TLR13 are located on endosomes where they recognize foreign nucleic acids [2, 3]. As a member of the TLR family with less known functions, TLR13 can participate in the immune and inflammatory reactions for recognizing the conserved sequence of 23S rRNA in bacteria and induce immune response in mice [4, 5]. TLRs can recognize pathogen-related and structurally conserved molecules like single-stranded (ss) or double-stranded (ds) RNAs or DNAs, lipoproteins and lipopolysaccharides derived from microbes, and then activate immune cell responses to all externally attacking microbiota [2]. They participate in tumor immune surveillance and play different roles in tumor growth [6]. Activation of TLR4/9-COX2 signaling was involved in the metastasis of hepatocellular carcinoma. Inhibition of TLR4/9-COX2 signaling abrogated the neutrophils to form extracellular traps (NETs)-aroused metastatic potential [7].

The activation of TLRs downstream signal mainly depends on two types of transcription factors: NF-κB and interference regulatory factors (IRFs), which mainly induce the production of pro-inflammatory cytokines. In addition, it can induce the production of type I interferon (IFN) [2]. Activation of TLR7 and TLR8 triggers induction of a Th1-type innate immune response. The emergence of new structural and small molecule information generated in the last decade has contributed enormously to our understanding of this highly sophisticated process of innate immunity signaling with the recent developments in the small molecule activation of TLR7 and TLR8 [8].

Meanwhile, many studies have revealed that the expression levels of TLR7 and TLR8 are altered in some autoimmune diseases, such as arthritis, cancers [9,10,11,12,13], or in antiviral regimes, including coronavirus and human immunodeficiency virus(HIV) prevention [14]. TLR agonists have potential therapeutic prospects and are one of the research hotspots in the field of immunotherapy. Thus, novel drugs of TLR7 and TLR8 agonists with different scaffolds have been developed. These agonists can induce certain cytokines and chemokines that can be used as good adjuvants for vaccines in the treatment of some diseases. Furthermore, TLR7/8 agonists as potential therapeutics for tumor-targeted immunotherapy have been developed [8, 15, 16].

In this review, we summarize the frontiers of TLR 7 and 8 related research, especially, in the field of new drug development of TLR agonists for cancer immunotherapy.

TLR family and its expression patterns

A typical TLR is a single-spanning receptor consisting of three domains: an extracellular domain (ECD) for the recognition of pathogen-associated molecular patterns (PAMPs), a transmembrane domain (TMD), and an intracellular Toll-interleukin 1 receptor (TIR) domain for initiating downstream signaling [17]. Each of these receptors has a unique antigen-recognizing domain [18]. TLR3, TLR7, TLR8, and TLR9 act as sensors of nucleic acids. Specifically, TLR3 recognizes viral dsRNA, and TLR9 senses unmethylated cytosine phosphate guanosine (CpG) containing DNA, whereas TLR7 and TLR8 function as viral ssRNA sensors [19]. TLR4 identifies bacterial lipopolysaccharide (LPS) found in gram-negative bacteria, whereas TLR5 recognizes flagellin, and TLR9 subfamily members (TLR7, TLR8, and TLR9) recognize microbial DNA and RNA [18]. TLR10 is the latest discovered human TLR, and its ligands are still unknown (Fig. 1). However, TLR10 is the only known member of the TLR family that can elicit an anti-inflammatory effect [3]. The intracellular TLRs (TLR3, TLR7, TLR8, and TLR9 in human) can detect viral and bacterial nucleic acids, playing an important role in host immune response [20,21,22,23,24,25] and potentially in the treatment of cancer [26].

Fig. 1
figure 1

TLRs and their ligands. TLR1–7 and TLR9 have been characterized to recognize microbial components. TLR3 is essential for the recognition of microbial lipopeptides. TLR1 and TL6 associate with TLR2, and discriminate subtle differences between triacyl- and diacyl lipopeptides, respectively. TLR4 recognizes LPS. TLR9 is the CpG DNA receptor, whereas TLR3 is implicated in the recognition of viral dsRNA. TLR5 is a receptor for flagellin. TLR10 is the latest human TLR to be discovered and its function and ligands are still unclear

Full size image

In contrast, extracellular TLRs, such as TLR1, TLR2, TLR4, TLR5, and TLR6, can be located at the plasma membrane where they recognize macromolecules exposed on the surface of pathogens [20,21,22,23,24,25]. Emerging evidence has suggested that dysfunction of TLRs has been correlated with inflammation associated with tumorigenesis (carcinogenesis), such as esophageal cancer [20] and other gastrointestinal tract cancers [27, 28]. Specifically, expression of TLR3, TLR4, TLR5, and TLR9 has been suggested as a potential mediator of the progression from reflux disorders to esophageal adenocarcinoma [29] Meanwhile, the increased expression levels of TLR3, TLR4, and TLR9 have been observed in esophageal squamous cell carcinoma (SCC) associated with lymphatic metastasis, with increased expression of TLR7 and TLR9 associating with advanced disease [30]. TLRs’ expression may be used as a valuable diagnostic or prognostic factor for esophageal cancer [20] .

TLR7, TLR8, and TLR9 display similarities in structure and endosomal localization, and natural agonists composed of nucleic acids, such as ssRNA or DNA with CpG motifs, activate the innate immune cells through these TLRs. The modulation of TLR7 and TLR8 responses is independent of CpG motifs or the nature of the oligodeoxynucleotides (ODNs) backbone structure, and the crosstalk between ODNs, IRMs, and TLR7 and TLR8 may be used for different clinical implications, including tumor therapy [31].

Although TLR7 and TLR8 show a high degree of sequence homology and similar structure, their biological responses to small molecule binding are very different. A recent study on molecular dynamics simulations reveals the selectivity mechanism of structurally similar agonists to TLR7 and TLR8, each with their own specific adapter proteins [32, 33]. While TLR7 is mainly expressed in antigen-presenting cells (APCs), such as plasmacytoid dendritic cells (pDCs) and B-cells, TLR8 is primarily expressed in myeloid cells, such as monocytes, macrophages, and myeloid dendritic cells [33]. Furthermore, TLR8 is considered biologically inactive in mice because they are unresponsive to imidazoquinolines (TLR7/8 agonists) when TLR7 expression has been knocked down. Hence, murine TLR8 cannot be triggered by TLR7/8 agonists [62]. Analysis results of normalized gene expression and corresponding clinical data of patients with skin cutaneous melanoma demonstrated that TLR7 and 8 expressions correlated with the expression of immune biomarkers and positively predicted the clinical outcome of patients with melanoma [63]. Meanwhile, in a preclinical melanoma mouse model, the TLR7 agonist IMQ improves T and NK cell function during BRAF-targeted therapy [64]. The topical application of TLR7 agonist IMQ in combination with other drugs, such as ipilimumab, for patients with malignant melanoma has been reported with successful results [65, 66].

Non-small cell lung cancer (NSCLC)

The gene expression analysis on a total of 33 advanced NSCLC patients treated with immune checkpoint inhibitors (ICI) evaluating the expression levels of 365 immune-related genes showed that high TLR7 expression levels were significantly associated with a lack of response to immunotherapy and the multivariate analysis confirmed TLR7 RNA expression as an independent predictor for both poorPFS and overall survival (OS) in advanced NSCLC patients treated with immunotherapy [67]. Furthermore, TLR7 expressed by malignant cells promotes tumor progression and metastasis through the recruitment of myeloid-derived suppressor cells in NSCLC [68]. A preclinical study showed that TLR7 agonists inhibit the growth and metastasis of lung cancer cells through immune activated mesenchymal stem cells [69]. Another study showed that R848-based stimulation of APCs in the tumor microenvironment resulted in the mobilization of an antitumor CD8+ immune response for treating metastatic NSCLC [70]. In addition, the TLR7/8 agonist R848 optimizes host and tumor immunity to improve therapeutic efficacy in murine lung cancer [71]. Although many studies have demonstrated that TLR7 agonists can enhance anti-tumor immune responses, these agonists also stimulate TLR7-expressing tumor cells. High TLR7 expression in the primary tumor confers poor clinical outcome and resistance to chemotherapy in lung cancer patients. This pro-tumorigenic effect of TLR7 has been validated in murine models of lung carcinoma [72].

Stomach adenocarcinoma.

A study in 30 patients with gastric cancer showed that the mRNA and protein expression levels of TLR7 were significantly downregulated in gastric cancer tissues. IMQ significantly increased TLR7 protein expression levels in SGC-7901 cells and promoted the secretion of proinflammatory cytokines such as TNF and IL-6 [73].

Overexpression of TLR7 in patients with advanced stomach adenocarcinoma (STAD) indicates a higher degree and poorer prognosis. In addition, TLR7 expression was positively correlated with immune cell infiltration and immune checkpoint expression. Therefore, TLR7 can be used as a novel diagnostic biomarker, progression and prognostic indicator, and immunotherapeutic target for stomach adenocarcinoma [74]. Conjugation of TLR7 agonist to gastric cancer antigen MG7-Ag exerts antitumor effects [75] and have synergistic antitumor effects with 5-fluorouracil via T cell activation and MDSCs inhibition [57, 76].

Other types of cancer

A meta-analysis of the prognostic role of TLRs in cancer showed that higher expression levels of TLR7 in tumor tissues could predict poorer survival, suggesting the expression level of TLR7 in cancerous tissue may have a prognostic value in patients with various cancers [181,182]. So far, there has been no breakthrough in the research of innate immune drugs regarding TLRs, and the selectivity and effectiveness are not clear yet. Emerging immunotherapies were proposed to overcome the primary and secondary resistance to existing immune checkpoint inhibitors, activate effector cells, and target immunosuppressive mechanisms in tumor microenvironment [141, 183,184,185]. More research needs to be explored regarding the mechanisms of the innate immune therapeutic agents. Autophagy pathway to inflammasome activation may influence the outcome of pro-tumor or anti-tumor responses depending on the cancer types, microenvironment, and the disease stage. Targeting macrophage approaches for either autophagy or inflammasome may be potential as anti-cancer strategies [186, 187].

Immunotherapy targeting inhibitory molecules like anti-CTLA-4 and anti-PD-1/PD-L1 were developed to overcome the immunosuppressive effects. These agents have demonstrated remarkable, durable responses in a small subset of patients [84]. TLRs agonists in combination of these inhibitory molecules and focus on the microenvironment and metabolic characteristics of tumor cells as well as the regulatory immune cells should be further explored [188]. Although no drug targeting TLR7/8 has been approved for cancer immunotherapy clinical application worldwide, but great efforts of global research and development have been made by focusing on TLR7/8-targeted agents for cancer immunotherapy and other types of diseases, such as patients with chronic hepatitis B. TLR agonists is considered a promising therapeutic strategy in the immunotherapy of solid tumors [189].

Clinical applications with a special subclass of patients may need to be categorized with either TLR agonist alone or agonist in combination with ICBs to specifically target that special population for cancer target immunotherapy, before it could be applied to a broad range of clinical applications. With the optimal antitumor immunity for robust enhancement of the effector T-cell response induced by tumor antigenic peptides and control or elimination of Treg cell-suppressive function, the combination of immune check point inhibitors with TLR agonists, in particular, the TLR8 agonist, may greatly improve the therapeutic potential of cancer immunotherapy. Furthermore, more novel targeting agents also need to be explored. We believe that with the continuous progress of research, the new TLR7/8 targeted drugs will improve the treatment efficiency and survival rate of patients with malignant tumors and benefit more patients.