2.1 Effector T cells and cancer immunotherapy

The main purpose of immunotherapy is to enhance the body's immune response and one of the most common ways is to enhance T cell function, especially CD8+ T cells, so that they can more effectively recognize and eliminate cancer cells. There are many studies targeting effector T cells to enhance immune efficacy. CD8+ T cell phenotype is markedly increased after receptor activator of nuclear factor-κB deletion, which enhances the efficacy of breast cancer immunotherapy.¹⁹ Metformin was found to improve the survival and the effector function of CD8+ T cells under hypoxic conditions as well as reduce hypoxia-induced apoptosis, while inhibiting PD1 and LAG3, thus improving the efficacy of cancer immunotherapy.²⁰ The stress response states (STRs), which is characterized as expressing heat shock genes, were significantly upregulated in immune checkpoint blockade treated unresponsive tumors, and T cells could link STR to immunotherapy resistance, suggesting that T cells may take a role in immunotherapeutic efficiency.²¹ Studies have demonstrated that the utilization of EP4 antagonist, YY001, enhances the proliferation and anticancer function of T cells while inhibiting the differentiation and maturation of MDSCs, thereby reversing the level of infiltration of MDSCs and T cells in the tumor microenvironment, and can be used for enhancing the prostate cancer immunotherapy efficiency.²² In conclusion, in many cases, effector T cells are a reflection of cancer immunotherapy efficiency. The number, function, and activation of effector T cells, represented by CD8+T, can often be used as an observable indicator for the efficacy of cancer immunotherapy.

2.2 Other T cells and cancer immunotherapy

In addition to effector T cells, other T cells are also critical for cancer immunotherapy efficacy. So far it has been discovered that tissue-resident memory T cells are prognostic markers for a variety of cancers, including breast and lung cancers, and potential biomarkers for predicting the efficacy of immunotherapies such as immune checkpoint blockade and cancer vaccines.²³ In addition, the tissue-resident memory T cells are regulated by TGF-β and they interact with tumor cells, DC cells, and other T cells in the TIME to ensure a positive T cell effector state.²⁴ In addition, it has been demonstrated that neoantigen-specific T cells have high affinity and perform an essential role in adoptive cell therapy (ACT).^{25, 26} Recent studies have revealed that T cells associated with recognizing cancer-independent antigens (“bystander T cells”) also perform important functions in cancer immunotherapy and they differ in specificity, activation state, and effector function.²⁷ Meanwhile, the exhausted T cells and immunosuppressive macrophages exhibited enhanced interactions in urothelial carcinoma of the bladder and performed significant functions in antitumor immunotherapy.²⁸ Deletion of sorting nexin 9 (SNX9) in CD8+ T cells reduces the exhaustion of CD8+ T cells and enhances both T memory cell differentiation and interferon-γ (IFNγ) secretion of adoptively transferred T cells, thereby enabling effective CAR-T cells immunotherapy.²⁹ In addition, the differentiation and modulation of T(H) subpopulations as well as γδ T cells in the TIME is critical for cancer immunotherapy efficacy.^{30, 31} Cellular senescence of tumor infiltrating lymphocytes (TIL) has recently been identified as an important state of T cell dysfunction in malignancy. Cancer cells and Treg cells are capable to induce senescence of responsive T cells through MAPK signaling, thus suppressing antitumor immunity and immunotherapy.³² In a word, besides effector T cells, tissue-resident memory T cells, neoantigen-specific T cells, “bystander T cells”, exhausted T cells, T memory cells, Th subpopulations, γδ T cells, senescence of TILs, and Treg cells also are significant index of cancer immunotherapy efficiency and worthy to be further studied.

2.3 Other immune cells and cancer immunotherapy

It is generally accepted that the eventual aim of cancer therapy is to induce a prolonged memory T cell response. So, the degree and function of T cell infiltration exerts an essential role in the response to tumor therapy. However, Chen et al.³³ predicted the efficacy of anti-PD1/PD-L1 immunotherapy and survival rates in gastric cancer patients by multidimensional tumor-infiltrating immune cells (TIICs) labeling. Also, Jiang et al.³⁴ constructed a TIICs model for immune cell-targeted therapy of gastric cancer and predicted the immunotherapy effect, which revealing that other immune cells in the TIME, such as B cells, macrophage cells, DC, and myeloid cells, also positively or negatively modulate the efficacy of antitumor immunity.

Here are some specific examples. B cells have been found to trigger antitumor immune responses either by direct tumor killing or by interacting with T cells and follicular DCs in tertiary lymphoid structures (TLSs). However, B cells can also acquire a suppressive function under certain conditions, becoming transformed into regulatory B cells that inhibit the tumor immune response.³⁵ Depletion of tumor-infiltrating B cells (TIBs) to enhance antitumor immunity is a novel and promising immunotherapeutic approach. Remodeling the TIME by decreasing the proportion of TIBs, enhancing the infiltration of CD8+/CD4+ T cells, and inhibiting the proliferation of Treg cells, as well as increasing the secretion of IL-2 and IFN-γ and decreasing the secretion of IL-10, IL-4, and TGF-β, could improve cancer immunotherapy efficiency.³⁶ Bufalin, as an antitumor immunomodulator, activates antitumor immune response by promoting the polarization of tumor-infiltrating macrophages (TIMs) toward the M1 phenotype, and meanwhile facilitates the production of immunostimulatory cytokines by regulating the NF-κB signaling pathway, thus effectively improving the therapeutic efficacy of HCC immunotherapy.³⁷ Poly(I:C) and resiquimod combination therapy provides a synergistic activation of macrophage-mediated antitumor immune response.³⁸ DC vaccines induces specific CD8+/CD4+ T cell responses, and the DC-cell-based cancer immunotherapy possesses promising future perspectives.³⁹ In addition, tumor-infiltrating plasmacytoid DCs (pDCs) are known to have critical roles in the triggering and maintenance of antitumor immunity. The activated pDCs (IRF7+) were identified as a key regulator of adaptive antitumor immunity in CRC.⁴⁰ In addition, TIL cells can also be used as markers to predict immunotherapy responsiveness and survival outcomes in melanoma,^{26, 41, 42} breast cancer,^43-45 osteosarcoma,⁴⁶ and other cancers.^{47, 48} All in all, current researches indicated that large amounts of immune cells possess the potential to affect cancer immunotherapy efficiency. And some of these immune cells such as different subset of B cells and T cells might exert extremely different effect (to be better or worser indicator for cancer immunotherapy efficiency), and these different isoforms can transform into each other under certain conditions.

Based on the important role of immune cells in tumor immunotherapy, we will next explore the function and mechanism of cancer immunomodulation in terms of epigenetics modification, metabolism modulation, and cell-to-cell communication in immune cells to provide new possible directions for tumor immunotherapy.

3.1 Epigenetic modifications in tumors

Cancer is known to be a disease triggered and driven by genetic abnormalities, but epigenetic pathways also play an important role in tumorigenesis. And many features of cancer, including differentiation blockade, self-renewal, cell death evasion, and invasiveness, are deeply affected by epigenomic changes.⁴⁹ Epigenetic modifications have attracted increasing attention as anticancer strategies in recent years, which is largely based on their direct impact on cancer cells.

The epigenetic modification such as DNA and RNA methylation modifications have been shown to play an important role in tumorigenesis and diagnosis. For example, the researchers analyzed DNA methylation profiles in colon cancer and discovered that the levels of 5-hmdC and 5-mdC modifications were low, 5-fdC modifications were the lowest, and 5-cadC modifications were high, suggesting a relationship between aberrant patterns of DNA epigenetic modifications and cancer development.⁵⁰ Liang et al.⁵¹ conducted DNA methylation analysis using high-throughput DNA bisulfite sequencing of early-stage lung cancer tissue samples and found that DNA methylation patterns were differentially expressed in tumors and benign lesions in circulating tumor DNA (ctDNA). In addition, deficiency of ALKBH5-mediated m6A modification in osteosarcoma causes an increase expression of histone deubiquitinase USP22 and ubiquitin ligase RNF40, which inhibits histone H2AK199 monoubiquitination, induces the expression of genes related to DNA repair, and promotes the progression of osteosarcoma.⁵² The m6A RNA methylation in LINC01559 was found to inhibit the progression of colorectal cancer (CRC) by modulating the miR-106b-5p/PTEN axis.⁵³ The m6A, m5C, m7G, and m1A-related genes have also been demonstrated to have a correlation with immunotherapy response in cervical cancer,⁵⁴ HCC,⁵⁵ and glioma.⁵⁶ However, the mechanisms and exact functions of these modifications in cancer still need further investigation.

In addition, histone modifications are another common epigenetic mark that involves the maintenance of stability of chromatin, as well as dynamic cellular processes like transcription and DNA repair.⁵⁷ A recent research demonstrated that the lactonization of H3 histone modification sites H3K9la and H3K56la promoted the progression of HCC.⁵⁸ In addition, H3K14, H4K5, and H4K12 histone acetylation promote non-small cell lung carcinoma (NSCLC) cell growth.⁵⁹ Different modifications of histones can be mutually modulated. For example, the deacetylation of AKAP12 at K531 by HDAC6 can increase its level of ubiquitination, promotes the proteasome-dependent degradation of AKAP12 and facilitates colon cancer metastasis.⁶⁰ H3K27 acetylation-activated COL6A1 inhibits STAT1 by interacting with SOCS5 in a ubiquitinated and proteasomal degradation manner thereby promoting osteosarcoma progression.⁶¹ Inhibition of histone deacetylase protein 6 (HDAC6) decreases ERK phosphorylation levels and inhibits cancer proliferation by upregulating microtubule protein acetylation.⁶² Histone methyltransferase DOT1L affects prostate cancer progression by increasing the methylation at H3K79 of MYC and decreasing expression of E3 ubiquitin ligases HECTD4 and MYCBP2.⁶³ The methyltransferase SMYD3 mediates H3K4 trimethylation at the cold shock structural domain-containing protein E1 (CSDE1) and contributes to STAT1 dephosphorylation through stabilization of the T cell protein tyrosine phosphatase, which alters the antigenicity of tumor cells.⁶⁴ The methyltransferases SMYD3 promotes RNF113A K20me3 methylation modification while impairing the interaction with phosphatase PP4, which downregulates its phosphorylation level, promotes and maintains RNF113A E3 ligase activity, and inhibits chemo-sensitivity in small cell lung cancer.⁶⁵ In a word, histone modifications act as a part of epigenetic area, they could participate in cancer progress alone or together with several different histone modifications form. Also, some epigenetic modifications among cancer seem to be involved in immune cells or immunotherapy. As the epigenetic modifications and immunotherapy are believed to be hot pot in recent cancer researches. So, it is urgent to figure out the specific mechanism of epigenetic modifications in immune modulation.

3.2 Epigenetic modifications of immune cells in the TIME

In recent years, the contribution of the immune system in restraining the development and progression of cancer has been largely acknowledged. New evidence is emerging that tumors usually hijack diverse epigenetic mechanisms to evade immune system constraints, and the immune cells engaged in antitumor response also may be influenced by altered epigenomes.⁶⁶

3.2.3 Other chromosome modifications of immune cells in TIME

Polycomb family

The Polycomb family (PcG) is a group of highly conserved chromatin modifiers, mainly comprising Polycomb repressive complex 2 (PRC2), a methyltransferase for histone H3K27 methylation, and PRC1, which has E3 ubiquitin ligase activity.⁸⁰ Members of PRC1 include the CBX family and the PHC family, while members of PRC2 include the EZH family, SUZ12, and EED. This family mainly works by modifying chromatin structure in order to maintain the silenced state of genes.

The PcG has been discovered to participate in modulating immune cell fate. CBX7 can activate CD4+ T cell polarization and apoptosis by regulating FasL expression.⁸¹ CBX4 deficiency leads to reduced accumulation of inhibitory histone modifications such as H2AK119ub1 and H3K27me3 of the PDCD1 promoter in T cells, and thus inhibiting antitumor immunity.⁸² In addition, the role of PRC2 member in immune cells also been researched. Inhibition of EZH1 can epigenetically remodel iPSCs to efficiently generate mature T cells for immunotherapy.⁸³ Inhibition of EZH2 reduces antitumor immune efficiency by reprogramming CD8+ T cells and reducing their survival and effector functions.⁸⁴ In bladder cancer, inhibition of EZH2 induces NK cell-mediated immune responses leading to tumor cell differentiation and death.⁸⁵ Inhibition of EZH2 stimulated the production of chemokines such as CCL2 and CXCL9/10, leading to NK cell and T cell infiltration in pancreatic ductal adenocarcinoma.⁸⁶ EZH2 maintains silencing on the FOXP3 gene through H3K27me3 labeling, which prevents overactivation of T cells and helps maintain immune tolerance.⁸⁷ Similarly, EZH2 has been identified to inhibit NK cell proliferation and promote the progress of cancer cells through modulating the H3K27 methylation and downregulate NKG2D ligands.⁸⁸ Also, EZH2 is involved in the regulation of epigenetic reprogramming in Treg cells, which resulted in a proinflammatory function and maintains the survival and function of effector CD4+/CD8 T cells through remodeling TIME.⁸⁹ Currently, most studies on the effects of PcG members on immune cells have focused on PRC2, especially EZH2, while studies on PRC1 members such as the PHC family are almost absent.

CTCF

In addition, some transcription factors bind to DNA to ensure precise regulation of gene expression and maintenance of structural stability of chromatin. They are critical for biological processes such as cell development and differentiation. CTCF, a DNA-binding protein involved in the formation of chromatin circuits, which has two main functions, one is to act as a chromatin terminator, preventing chromatin regions from interacting, and the other is to act as a medium of chromatin interconnection, facilitating chromatin regions to interact.^90-92 It has been shown that Tcf1 recruits CTCF and promotes chromatin interactions to regulate the genomic structure and homeostatic proliferation of CD8+ T cells.⁹³ CTCF regulates CD8+ T cell heterogeneity and promotes terminal differentiation of CD8+ T cells by altering the transcription factor landscape and transcriptome interactions.⁹⁴ CTCF is required to establish NF-κB-dependent chromatin interactions in DCs to promote inflammatory response.⁹⁵ However, another research declaims that CTCF is dispensable for immune cell trans-differentiation although it affects the topologically associating domains but rather facilitates rapid responses to external stimuli.⁹⁶ All in all, CTCF is involved in the regulation of gene expression in immune cells by modulating chromatin structure to ensure the differentiation and function of immune cells. Investigating the function of CTCF can help reveal the epigenetic regulatory mechanisms of immune cells and provide new targets and strategies for the cancer immunotherapy treatment. In conclusion, epigenetic modifications can modulate tumor immunity by regulating the function of multiple immune cell subtypes and can also reshape the immune response by regulating the expression of immune-related genes (Figure 2). Consequently, specific reprogramming through epigenetic modification of immune cells holds great promise for remodeling aspects of tumor microenvironment. Epigenetic modifications are largely dependent on various enzymes, and specific functions of epigenetic regulation are investigated by knocking down these enzymes. A number of drugs targeting these enzymes have also been developed, such as targeting DNA methyltransferases (e.g., 5-azacytidine, decitabine, and guadecitabine), EZH2 inhibitors (e.g., Tazemetostat and GSK126), HDAC inhibitors (e.g., vorinostat, romidepsin, and panobinostat) making research more convenient and laying the foundation for clinical translation of epigenetic modifications.

4.1 Metabolic reprogramming in tumors

With a more sophisticated understanding of tumor biology and the complexity of tumor metabolism, it is now believed that the metabolic reprogramming is one of the hallmarks of cancer.⁹⁷ There is no doubt that cancer cells have different metabolic states compared to normal cells, and cancer cells can show great metabolic diversity and plasticity in different tumors.⁹⁸ Studies have shown that metabolic phenotypes vary at different stages of cancer development. For example, tumors need more nutrients to grow in the early stages, while during metastasis stage, new metabolic phenotypic dependencies often emerge due to the treatment resistance in tumors.⁹⁹ The metabolic resources within local tumor tissues are limited, creating a tumor microenvironment of nutrient depletion and metabolic waste accumulation. In order to maintain growth under various conditions of nutrient distribution in the body, cancer cells utilize different metabolic adaptations. And the metabolites of cancer cells not only can be used as substrates for a source of energy, but also can adjust the expression of genes and proteins in normal cells so as to influence their behaviors.¹⁰⁰ Luo et al.¹⁰¹ analyzed the metabolic phenotype of HCC cells and found that the expression of CD36 was positively correlated with the degree of glycolysis. Overexpression of CD36 induced an increase in mammalian target of rapamycin (mTOR)-mediated glycolytic flux and lactate generation through activation of the Src/PI3K/AKT signaling pathway, thereby promoting the progression of HCC.¹⁰¹ In non-small cell lung cancer, LINE-1 is activated by L1 antisense promoter (L1- asp) or reverse transcription to form L1 gene chimeric transcript, which participates in the metabolic process of tumors and the regulation of mitochondrial function, facilitating lung cancer progression.¹⁰² In addition, a strong link exists between tumor metabolism and immune cell metabolism, as metabolic changes in the TIME can affect immune cell function and response.

4.2 Metabolic modulation of immune cells

4.2.7 Relationship between ions metabolic pathways and immune cells

In recent years, multiple ion metabolisms have been found to be involved in modulating cancer immunity. For example, magnesium ions could induce conformational changes of LFA-1 by binding to β1-MIDAS and α1-MIDAS on the membrane of CD8+ T cells, thus altering their cytotoxicity and promoting tumor cell killing functions.¹⁴³ OTUD1 promotes the deubiquitination of IREB2, which inhibits the degradation of IREB2 protein and activates the expression of TFRC, thereby increasing the concentration of iron ions in tumor cells and enhancing the sensitivity to ferroptosis as well as the infiltration of CD8+ T cells, improving antitumor immune response.¹⁴⁴ In addition, high potassium ions concentrations in tumors disrupt glutamine uptake and reprogram TAM metabolism from oxidative phosphorylation to glycolysis, thereby inhibiting the antitumor capacity of TAM.¹⁴⁵ Studies have revealed that Mn²⁺ can effectively activate the cGAS–STING pathway, which significantly promotes the function of DC and enhances the survival, proliferation, and function of cytotoxic T cells and memory T cells and NK cells, thus promoting the tumor immunosurveillance effect.¹⁴⁶ The researchers found that zinc ions released from ZnS@BSA nanoclusters under acidic TIM significantly enhanced cGAS/STING signaling and promoted the function of CD8+ T cells and DCs, which improved the immunotherapy effect of HCC¹⁴⁷ (Figure 3).

Immunogenic cell death (ICD) is a novel tumor cell death manner that could promote the antitumor immune responses. Recent studies have indicated that some ions may modulate tumor immunity by triggering the ICD. For example, researchers used nanomaterials to construct a cocarrier with Cu²⁺ and DSF, which generate the cytotoxic metabolite DTC-Copper complex for tumor therapy. One of the active metabolites, CuET, can effectively induce ICD to modulate the TIME.¹⁴⁸ In addition, other ions such as calcium was also reported to induce ICD in cancer.¹⁴⁹ Therefore, ion metabolism targeting immune cells is also significant part in cancer immune therapy, and their roles and mechanisms for tumor immunotherapy are worth exploring. With the development of nano-based therapeutic biomolecule immunotherapies, targeted ion metabolism will have a broader future.

Cancer cells produce metabolites that can have profound effects on immune cells in the TIME. Targeting metabolic pathways in tumors facilitates alteration of metabolic competition between tumors and immune cells that increases the immunogenicity of tumors. The metabolic products produced by tumor cells are various and have different effects on immune cells. Immune cells can employ a variety of programs to respond to different incentives in the microenvironment, and they need to adapt to the cellular metabolism reprogramming in order to match the biochemical demands for each different functional state. For example, different states of T cell activation need metabolic programs appropriate to their functional requirements. Transitions among states are associated with active cellular metabolic reprogramming. And this requires the coordinated adaptation by a network of signaling and transcription factors for each transition.¹⁵⁰ The shift from a resting to an activated state of immune cells involves the distribution of nutrients into different pathways, and therefore studying how metabolic pathways are modulated to directly change the immune cell function is critical to the immunotherapy of tumors.

5.1 Exosome

Exosomes, as an emerging part of tumor–host interactions, which are key mediators of signal transfer between secretory cells and target cells, are now increasingly considered as message carriers and key molecules in TIME. Exosomes derived from immune cells have immunomodulatory properties and promising therapeutic properties that providing an important role in cancer therapy. For example, M2 macrophage-derived exosomes miR21 interacts with CD8+ T cells through inhibiting the expression of PEG3, which results in a decrease amount of CD8+T cells and inhibits immune response in glioma.¹⁵¹ In addition, M1 macrophage-derived exosomes can facilitate T-cell production of IFN-γ and thus promoting immune response.¹⁵² DC-derived exosomes directly interact with specific cytotoxic T lymphocytes (CTL) and promote the activation of CD8+ and CD4+ T cells to inhibit tumor growth.¹⁵³ DC-derived exosomes express IL-15Rα, leading to amplification of NKs and secretion of IFN-γ, which induces tumor regression.¹⁵⁴ B cell-derived exosomes with CD73, CD19, CD39, C3, FasL proteins, and integrins on their surface can regulate T cells function and thus influence tumor progression through some specific factors.¹⁵⁵ MHCII(+) FasL(+) exosomes secreted by B cells that induce the apoptosis of CD4+ T cell.¹⁵⁶ Treg-derived exosomes containing miR-150-5p and miR-142-3p can be delivered to DCs to produce more IL-10 and less IL-6 thereby suppressing immune cell activity.¹⁵⁷ TAM-secreted exosomes containing miR-21-5p and miR-29-3p promote the progression of epithelial ovarian cancer by suppressing STAT3 expression and inducing an imbalance of Treg/T helper cell (Th)17.¹⁵⁸ In addition, mast cell-derived exosomes contained heat shock proteins have been reported to induce DC maturation.¹⁵⁹ NK cells secrete exosomes contained miR-186 that stimulate other immune cell (such as T cells and monocytes) functions and also attenuate the immunosuppressive effects of tumor cells by reducing PD1 expression.¹⁶⁰ As mentioned above, a variety of immune cells possess the function of secreting exosomes, which contain complex biological signals that interact with other immune cells as well as tumor cells, thereby killing tumor cells through a variety of mechanisms. Exosomes carry biomolecules with the capacity to regulate the immune response, such as cytokines, miRNAs, and other regulatory substances. These molecules, delivered through exosomes, can influence the immune status of recipient cells and thus regulate the activity and function of immune cells. Exosomes transmit information between different types of immune cells, prompting them to collaborate with each other to perform specific immune tasks. This intercellular communication helps to create synergies and increase the efficiency of the immune response.

5.2 TIME

The TIME, which offers conditions for the tumor cells to grow, is a deeply immunosuppressive environment. In this section, we will focus on immune cell types that are directly engaged in regulating tumor-killing activity, such as T cells, NK cells, and MDSC. Among them, MDSC are considered to be the immune cells that make the greatest contribution to the development of the TIME.¹⁶¹ The MDSC usually including tumor-associated macrophages (TAM) and tumor-associated neutrophils. MDSC can not only act as a source of TAM in tumors, it can also influence macrophage polarization, activation and function.¹⁶² In the tumor environment, MDSC produce IL10 to regulate IL12 production in macrophages, thus playing an important role in macrophages’ polarization toward the M2 phenotype.¹⁶³ In addition, MDSC have also been reported to affect MHC II expression in macrophages possibly.¹⁶⁴ The interactions that occur between MDSC and macrophages aggravate the suppression of immune cells in TIME through changing the production of cytokines and the expression of some key molecules. However, there is very few literatures to introduce the interaction between neutrophil and MDSC, and these field remains to be investigated.

As reported, researchers cocultured MDSCs with NK cells and found that the MDSCs can inhibit the tumor cytotoxic activity of NK cells by regulating TGFβ and resulted in immune tolerance.¹⁶⁵ Previous studies have suggested that the ability of MDSC to mediate NK cell unresponsiveness may be related to the MDSCs-mediated downregulation of CD247 expression on the surface of NK cells.¹⁶⁶ Furthermore, MDSCs can also excrete diverse soluble elements, such as nitric oxide-inducible nitrogen oxygenase and ROS in order to enhance immunosuppression and suppress NK cell activation.¹⁶⁷ In addition, MDSCs also generate IDO to reduce the expression of DNAM1, NKG2D, and NCR, thereby blocking NK cell activation.¹⁶⁸

Recently, it was demonstrated that MDSCs could secrete itaconate, which could be absorbed by CD8+ T cells and inhibit the properties of CD8+ T cells proliferation and also inhibit the production and activity of cytokine, thus promoting the growth of melanoma.¹⁶⁹ Another study demonstrated that dysregulation of notch signaling in immature T cells facilitated the formation of CD11b+Gr-1+ MDSCs, while deficiency of anti-Gr-1-mediated MDSCs reduces the malignant T-cell proliferation and expansion in acute lymphoblastic leukemia (ALL).¹⁷⁰ In oral squamous cell carcinoma, MDSCs induced the generation of Treg cells by inhibiting TGF-β and promoted the differentiation of Th17 cells by secreting PGE2, IL-1β, IL-6, and IL-23.¹⁷¹ In addition, MDSC facilitate natural Treg cell expansion and promote the development of induced Treg cells by producing IFN-γ, TGFβ, and IL-10.¹⁷² MDSC suppresses T cell activation by generating ROS and RNS and depleting arginine and cysteine, which are both required for the activation and proliferation of T cells.¹⁷³ In addition, mutual crosstalk between T follicular helper cells and B cells in TLSs plays an important function in antitumor immunotherapy.¹⁷⁴

MDSC can increase immunosuppressive function through interactions with DC. The impaired cross-presentation of DC in cancer may be one of the barriers to successful cancer immunotherapy. In melanoma, MDSC reduces antigen uptake and prevents DC maturation, while simultaneously obstructing the capacity of DCs to activate T cells.¹⁷⁵ It has been reported that MDSC prevents cross-presentation of DC while not interfering the direct presentation of DC to antigen, which process was linked to lipid peroxidation and requires no direct cell-to-cell contact.¹⁷⁶ The MDSCs have been shown to inhibit not only DC function, but also their maturation and progression. A recent study examined the expression of CD11b and CD33 (MDSC markers) as well as CD303 and IDO1 (DC markers) in patients with OSCC and analyzed their relationship with clinicopathological parameters, and the results showed a positive correlation between these biomarkers. In addition, MDSC and DC were highly expressed in OSCC patients, suggesting that MDSCs and DCs infiltration was significantly correlated with the progression of OSCC.¹⁷⁷

In addition, apart from immune cells, TIME contains fibroblasts, endothelial cells, epithelial cells, and so on; these nonhematopoietic cells have the ability to generate immunomodulatory factors and also participate in the interactions between the tumor and the immune system.¹⁷⁸ Some components of TIME can produce cytokines, chemokines, and so on, which modulate the function of immune cells through a variety of signaling mechanisms. For instance, cancer-associated fibroblasts modulates the tumor immune response by secreting cytokines, chemokines, and so on, which regulate macrophage polarization^{179, 180} and influence the interaction between immune cells such as DC cells and T cells.^{181, 182} In addition, TIME also have affected the activity of immune cells and thus impacted their ability to attack cancer cells. Comprehension of the interactions between immune cells in TIME is crucial for the clinical application of tumor immunotherapy (Figure 4).

The communications between immune cells have important implications in cancer immunotherapy. In recent years, some bispecific antibodies have brought different immune cells closer together (e.g., DCs and T cells) and increased mutual communication, which is considered a novel cancer immunotherapy option.^{183, 184} This novel therapy using bispecific antibodies to increase DC-T cell interactions promotes both T cell activation and function as well as cDC1 maturation.¹⁸⁵ On the one hand, communication between immune cells helps modulate and coordinate the immune response. Different types of immune cells can interact with each other through cytokines, chemokines, and other signaling molecules to ensure the timely, intensity, and direction of the immune response. On the other hand, lack of communication or miscommunication between immune cells might result in the inability of immune cells to recognize and attack tumor cells in a prompt manner. Thus, tumor cells could take advantage of the communication between immune cells to establish immune tolerance and thus evade immune attack. In conclusion, understanding and regulating the communication between different immune cells can provide important guidance for the development of more effective immunotherapies.

7.1 Inhibitors in the field of cancer immunotherapy

Different immune cell compositions in TIME can influence the immune checkpoint inhibitor (ICI) response to tumor therapy.^{202, 203} Immune checkpoint receptors such as CTL-associated protein 4 (CTLA4), PD1, T-cell immunoreceptor with Ig and ITIM structural domains (TIGIT), and lymphocyte activation gene 3 (LAG3) could modulate the activation, differentiation, and function of T cells. For example, PD1 directly modulates TCR signaling to attenuate T cell activity. Additionally, deficiency of CTLA4 in Tregs induces aberrant T cell activation and causes autoimmunity. And deletion of Treg could counteract the expansion of Treg cells induced by CTLA4 blockade, thereby improving the efficacy of anti-CTLA4 therapy.²⁰⁴ Anti-TIGIT targeting Tregs possesses strong potential for cancer immunotherapy,²⁰⁵ and targeting Tregs may also contribute to cancer immunotherapy efficacy when used as monotherapy or in combination with ICB antibodies.²⁰⁶ Recent studies have shown that sialic acid-binding immunoglobulin-like lectin (Siglec) receptors on TIICs have an interplay with glycan-containing sialic acid in the tumor microenvironment, which could be a novel immune checkpoint and a promising potential target for cancer immunotherapy. The Siglec receptor is widely expressed on different immune cells including T cells, DCs, NK cells, neutrophils, and macrophages. And the interaction between Siglec receptors and sialic acid glycan ligands on these immune cells helps to establish an immune suppressive microenvironment and suppress antitumor immunity in the cancer microenvironment.²⁰⁷ More importantly, studies have reported that Siglec-15 can be targeted by the blocking antibody NC318, and it has been shown in early clinical trials that the antibody is responsive to cancer immunotherapy in some patients.²⁰⁸ This suggests that various immune checkpoints can influence tumor immunotherapy by influencing the function and activation of immune cells and modulating the activity and specificity of the immune system.

ICI therapy is one of the most widely utilized cancer immunotherapies at present. Currently, drugs against immune checkpoints focus on targets such as PD1, PDL1, and CTLA4. Among them, PD1 inhibitor nivolumab (trade name Opdivo) used to treat a variety of cancers, including melanoma, NSCLC, renal cell carcinoma, and head and neck cancer.^209-212 Pembrolizumab (Keytruda): used to treat melanoma, NSCLC, head and neck cancer, Hodgkin's lymphoma, and so on.^213-215 And PDL1 inhibitor, atezolizumab (Tecentriq), is mainly used for the treatment of uroepithelial cancer, HCC, and NSCLC.^216-218 Avelumab (trade name Bavencio) is mainly used for the treatment of Merkel cell carcinoma and uroepithelial carcinoma.^{219, 220} Durvalumab (trade name Imfinzi) is mainly used for the treatment of NSCLC and bladder cancer.²²¹ Moreover, CTLA4 Inhibitors Ipilimumab (trade name Yervoy) initially used to treat advanced melanoma, now also used for other cancer types.²²² Notably, the range of indications for these drugs will continue to expand based on new clinical trial results and regulatory approvals. In addition, certain drugs may be more effective in specific patient populations.

While ICIs have achieved remarkable success in cancer treatment, they may also affect noncancer cells, leading to a range of immune-related side effects such as autoimmune reactions, inflammatory responses, and so on.^{223, 224} Some patients may acquire resistance to ICIs, and further research is needed to address the challenges associated with side effect management, resistance, treatment failure, and cost and accessibility. The combination of ICIs with other treatments, such as radiotherapy, chemotherapy, targeted therapy, or other immunotherapies, is being studied extensively. Such combination therapies may improve treatment efficacy and overcome the limitations of monotherapy.

7.2 Latest clinical trials of immune cell-based cancer immunotherapy

There are two main types of immune cell-based cancer immunotherapies currently on the market or approved, namely DC therapy and CAR-T cell therapy. In 2010, the DC therapy Sipuleucel-T (trade name: Provenge) was approved for the treatment of metastatic desmoplasia-resistant prostate cancer that is asymptomatic or only mildly symptomatic.^{225, 226} In 2017, the CAR-T therapy Tisagenlecleucel (trade name: Kymriah) was approved for the treatment of ALL,^{227, 228} and in the same year, the Axicabtagene ciloleucel (trade name: Yescarta) was approved for the treatment of diffuse large B-cell lymphoma.^{229, 230} In addition, Brexucabtagene Autoleucel (Tecartus) is approved for the treatment of relapsed or refractory B-cell precursor ALL in adults.²³¹ Lisocabtagene Maraleucel (Breyanzi) is approved for the treatment of relapsed or refractory large B-cell lymphoma.²³² Additionally, there are also many immune cell therapies in clinical trials.

Clinical trials allow doctors to assess the accuracy and reliability of new diagnostic and therapeutic techniques to provide more appropriate treatment options for patients. Today, immune cell therapies are attracting a lot of attention in cancer immunotherapy, and scientists are continuing to improve therapies and trial methods. Below are some examples of clinical trial results for cancer immunotherapy based on immune cell therapies that have been published in the recent 2 year, providing new discoveries and understanding for fighting cancer (Table 1).

Researchers conducted a large-scale randomized controlled phase 3 clinical trial (NCT03607539) to explore the relationship between TIL and the efficacy of immunotherapy in 397 advanced NSCLC patients, where the primary endpoint was progression-free survival (PFS) while OS and objective remission rates (ORR) were secondary endpoints. The results showed that combination therapy of chemotherapy plus sintilimab (anti-PD1) was associated with statistically significant improvements in PFS (HR = 0.12, 95% CI: 0.06–0.25, p < 0.001) and OS (HR = 0.27, 95% CI: 0.13–0.55, p < 0.001) only for subtype II (PDL1+ and TIL+) compared with chemotherapy, which further illustrates the critical role of immune cells in cancer immunotherapy.²³³ Recently researchers conducted a phase 3 multicenter open-label clinical trial (NCT02278887) in which 168 patients with advanced melanoma were randomly assigned to receive either TIL or ipilimumab, with the results revealing a median PFS of 7.2 months (95% CI, 4.2–13.1) and median overall survival was 25.8 months (95% CI, 18.2 to not reached) in the TIL group while PFS of 3.1 months (95% CI, 3.0–4.3) and 18.9 months (95% CI, 13.8–32.6) in ipilimumab group.²³⁴ Researchers reported a phase I clinical trial (NCT02652455) of nivolumab in combination with TIL ACT for the treatment of melanoma which proved to be a safe and feasible treatment.²³⁵ A phase I clinical trial (NCT02421640) of 156 nasopharyngeal carcinoma patients using TILs following concurrent chemoradiotherapy was determined to be safe and objective.²³⁶ This demonstrates the potential survival benefit of TILs in patients with low levels of circulating CD8+ T cells and PDL1 expression.

Recent results from a Phase I clinical trial (NCT04555551) of CAR-T cell therapy (MCARH109) targeting GPRC5D confirmed that GPRC5D is an important immunotherapeutic target in multiple myeloma.²³⁷ Researchers conducted a phase 1 clinical trial (NCT04093596) in 43 patients with multiple myeloma evaluating allogeneic anti-BCMA CAR-T cell therapy (ALLO-715). Overall, 24 patients (55.8%) responded, with 17 (70.8%) in remission, 11 (45.8%) in partial remission or better, and six (25%) in complete remission, demonstrating the viability and safety of allogeneic CAR-T cell therapy.²³⁸ In addition, a phase II clinical trial (NCT02640209) of autologous anti-CD19 humanized binding domain T cells (huCART-19) added to Bruton's tyrosine kinase (BTK) inhibitor ibrutinib for 20 patients with B-cell chronic lymphatic leukemia and the results indicated that patients in the huCART-19 group had higher rates of remission with favorable safety and feasibility outcomes.²³⁹ Investigators conducted a phase II clinical trial (NCT04148430) of the IL-1 receptor antagonist (IL-1Ra) anakinra in patients with relapsed/refractory large B-cell lymphoma who were treated with anti-CD19 CAR-T cells. The results showed that anakinra treatment reduced immune effector cell-associated neurotoxicity syndrome (ICANS) while not damaging the efficacy of anti-CD19 CAR-T therapy.²⁴⁰

In addition, there are many ongoing clinical trials of immune cell-based cancer immunotherapies, including NKT cell therapy, NK cell therapy, CAR-M, LAK cell therapy, TCR-T cell therapy, DC-CIK cell therapy, and so on. For details on the progress of these clinical trials, please refer to https://clinicaltrials.gov/. Among them, CAR-M and NK cell therapies are already in the late clinical stages, and the results of their clinical trials are extremely promising.²⁴¹

7.3 Potentially available biomarkers for evaluation of cancer immunotherapy

There is no doubt that the advent of immunotherapy has provided a new and more effective therapy for the prevention and treatment of tumors. However, it is urgent to evaluate and predict the efficacy of immunotherapy and thus improving the effectiveness of immunotherapy, since immunotherapy cannot be suitable for all patients. The Cancer ImmunoMonitoring and Analysis Center and the Cancer ImmunoData Commons have been established and are able to systematically identify biomarkers and correlate them with clinical outcomes, which is essential for enhancing immunotherapeutic efficiency for cancer patients.²⁴²

It has been believed that CTLA4, TMB, and SMACA4 mutations are considered as potential predictive biomarkers of ICB efficacy in EBV virus-associated gastric cancer.²⁴³ A recent study showed that the expression of PDL1, B7-H3 and VISTA as well as some tumor necrosis factor receptor superfamily such as OX40L, CD27, 4-1BB, CD40, and CD95/Fas were correlated with the head and neck squamous cell carcinoma immunotherapy efficiency, and could serve as potential biomarkers to predict the immunotherapy response.²⁴⁴ Using meta-analysis, Zhou et al.²⁴⁵ discovered that high expression of PDL1 in tumor cells instead of tumor tissues was associated with better prognosis, and PDL1 could be used as a prognostic biomarker of immunotherapy efficacy for HCC.

However, tumor tissue biopsies are invasive and cannot obtain the full spatial heterogeneity of the tumor, as well as being difficult to follow up on treatment efficacy. In recent years, it has been found that cancer immunotherapy efficacy can be reflected by comprehensive analysis of systemic immune events through peripheral blood immune cell subpopulations. Thus, circular immune cell-based biomarkers can assess and provide guidance for cancer immunotherapy.²⁴⁶ Liquid biopsies can be used to assess the progression of tumors and the efficacy of clinical therapies during cancer immunotherapy. Among them, ctDNA could be used as a tumor load biomarker to assess the efficacy of immunotherapies.²⁴⁷ In addition, PDL1 in circulating tumor cells and CD4/CD8+ T cell populations may serve as prognostic biomarkers for immunotherapy in metastatic genitourinary cancer patients.²⁴⁸ GNG4 is expressed typically in exhausted CD4+ T cells, and its high expression is correlated with a higher immune cell infiltration level and a better immunotherapy response, which could be recognized as a biomarker for assessing the immunotherapy efficacy in bladder cancer.²⁴⁹ A recent study has indicated that the tumor mutational load in blood is expected to be a biomarker for the prediction of the efficacy of immunotherapy in NSCLC.²⁵⁰ Researchers found that patients with high expression of LAG-3 in the surface of CD8+ T cells in peripheral blood were less responsive to immunotherapy through analysis of peripheral blood cells from melanoma patients. At the same time, they suggested that LAG-3 protein could serve as a potentially vital biomarker for predicting the efficacy of ICB.²⁵¹ Study demonstrates that circulating extracellular vesicles expressing CD81 may serve as a possible biomarker of ICI response in patients with low levels of PDL1 expression in advanced NSCLC.²⁵² Serum biomarkers, the CEA and Ca-125, potentially serve as biomarkers for predicting the immunotherapy efficacy in NSCLC treated with PDL1 inhibitors.²⁵³ Circulating soluble programmed death ligand 1 (sPDL1) is a prospective dynamic biomarker that negatively regulates the function of T cells, evaluates the efficacy of immunotherapy in pMMR CRCs, and can serve as a predictive prognostic marker in a diverse range of cancers.²⁵⁴

In recent years, with the development of bioinformatics, some novel potential markers to predict cancer immunotherapy efficacy have been identified such as PTTG,²⁵⁵ EXTL3,²⁵⁶ CTNNB1,²⁵⁷ CXCL5,²⁵⁸ APC,²⁵⁹ and TASL.²⁶⁰ Researchers identified 11 immune-related gene prognostic indicators that have the capacity to predict immune cell infiltration in the tumor microenvironment, the efficacy of immunotherapy and can more accurately predict the survival of HCC patients.²⁶¹ In addition, the researchers compiled a list of 52 candidate genes that could possibly predict the efficacy of ICB therapy, based on data from a cohort of 350 patients with NSCLC. These candidate genes are characterized by compound mutations that might be better predictors than TMB and could serve as new biomarkers for assessing the NSCLC immunotherapy efficacy.²⁶² Researchers developed a computational model of resilient T cells (Tres) using single-cell transcriptomics data to recognize immunosuppressive signatures (e.g., TGFβ1 and PGE2). It is a reliable predictor of clinical response to immunotherapy in a wide range of tumors including melanoma, TNBC, and lung cancer. The Tres revealed that FIBP is the major negative biomarker of tumor resilient T cells and can be used to assess the immunotherapy response in solid tumors.²⁶³ An immune infiltration score (IIS) was calculated based on immune cell infiltration in the cancer microenvironment. And patients with higher IIS had significantly higher PDL1 expression and shorter PFS time, which can be used as a novel and reliable biomarker to assess the prognosis of TNBC patients.²⁶⁴ A recent study found that using spatial quantification of CD8 and PDL1 markers to quantify immune scoring is a novel in vitro diagnostic methods, which is capable to predict the efficacy of anti-PD1/PDL1 immunotherapy in NSCLC, and is a potential powerful tool for evaluating the outcomes of cancer immunotherapy.²⁶⁵ The researchers tried a new method to automatically select tissues and calculate the frequency of cell to cell spatial interactions happening in the PD1/PDL1 pathway, which could be used to reveal spatial interactions for immune escape and provide potential prognostic information for oropharyngeal squamous cell carcinoma.²⁶⁶

In addition to the classical immune checkpoints and immune cells, other new areas such as cancer stemness, ferroptosis, pyroptosis, lncRNAs, and radiogenomic traits have also been considered as potential markers for cancer immunotherapy efficiency. Researchers have found that cancer stemness is a key reason for ICI resistance by comprehensively analyzing single-cell and bulk RNA sequencing data, and that cancer stemness gene expression profiles can be used as biomarkers of cancer immunotherapy efficacy.²⁶⁷ In addition, the ferroptosis score is strongly associated with the efficacy of immunotherapy in colon cancer. Where patients with high ferroptosis score (characterized by high TMB and MSI-high subtype) showed poor prognosis using immunotherapy. Among them, ALOX5 is a key ferroptosis gene identified as a predictive marker for immunotherapy efficacy.²⁶⁸ Yet another study found that cancer patients with high ferroptosis scores displayed high CD8+ T cell and TIL infiltration as well as enrichment of immune-related signaling pathways, which could be a good prognostic factor and an assessment of the immunotherapy effect.²⁶⁹ In addition, the researchers also elucidated that tumor-infiltrating B-lymphocyte lncRNA signature can be used as an index of immune cell infiltration in TIME and lncRNA is expected to be used as a potential biomarker for the prediction of immunotherapy response in bladder cancer.²⁷⁰ Also, some pyroptosis-related genes and immune-related lncRNAs have been recognized as potential prognostic markers of cancer immunotherapy.^{271, 272} In addition, radiogenomic biomarkers to predict therapeutic response to PD1/PDL1 immunotherapy in NSCLC have also been discovered.²⁷³

In summary, cancer immunotherapy is a rapidly evolving field, and a growing number of potentially usable biomarkers for predicting and evaluating cancer immunotherapy have emerged. Taken together, these biomarkers allow researchers and clinicians to better understand which patients are most likely to benefit from immunotherapy, thereby personalizing treatment plans and improving outcomes. Currently, there are two major limitations in evaluating potential biomarkers for cancer immunotherapy: first, although many promising markers have been identified, such as PD-L1 expression, tumor mutational load (TMB), and immune cell infiltration, the predictive power of these markers is not always consistent and exhibits variability in different types of cancer. Second, the process of biomarker discovery and validation is complex and time consuming, and there is a lack of uniform standards and methods to assess and validate the clinical application value of new markers, which limits their widespread use in personalized immunotherapy. As more studies are conducted, it is expected that additional biomarkers will be identified and existing assessment methods will be improved (Figure 6).

7.4 Mouse tumor models for immunotherapy

Immunotherapy has yielded benefits for some patients with tumors. However, cure rates for most patients remain low, and many patients do not even respond to these treatments. Preclinical in vivo models play a crucial role in the clinical efficacy assessment of tumor immunotherapy. Mouse tumor models are one of the key tools for studying tumor pathogenesis. In recent years, with the advances of tumor immunotherapy, mouse models have been further developed.²⁷⁴ Currently, the main mouse models for tumor immune efficacy evaluation include syngeneic model, genetically engineered mouse models, humanized mouse models with immune system reconstitution.