Development of therapeutic cancer vaccines based on cancer immunity cycle

Abstract

Therapeutic cancer vaccines have experienced a resurgence over the past ten years. Cancer vaccines are typically designed to enhance specific stages of the cancer-immunity cycle, primarily by activating the immune system to promote tumor regression and overcome immune resistance. In this review, we summarize the significant recent advancements in cancer immunotherapy based on the cancer-immunity cycle, including the effector cell function, infiltration, initiation, and exhaustion. We summarize the identification of tumor antigens and their delivery through cancer vaccines. We discuss how specific stages of the cancer-immunity cycle have been leveraged to augment anti-tumor immune responses and improve vaccine efficacy. Additionally, the impact of aging and myelosuppression, two prevalent forms of immunological stress, on the effectiveness of therapeutic cancer vaccines is deliberated. Finally, we summarize the current status of various therapeutic cancer vaccines at different clinical trial phases.

Keywords

therapeutic cancer vaccines / cancer-immunity cycle / cancer neoantigen / combination therapy / aging / myelosuppression

1 Introduction

The great success of vaccines for their prevention of many infectious diseases has considerably inspired the study of vaccines for cancer treatment. However, from infection prevention to cancer treatment, vaccines are facing at least two major dramatic context changes: (1) the recipients are usually infection-free and healthy for the former while cancer-bearing and disordered for the latter; (2) the antigen targets are way more complex in most cancers than in infections. These have been the intensively focused in the past one or two decades.

The cancer-immunity cycle is a fundamental concept in immuno-oncology, describing the process by which the immune system recognizes, targets, and eliminates cancer cells. Cancer vaccines are usually designed to enhance specific steps of the cancer-immunity cycle, primarily by stimulating the immune system to better recognize and attack cancer cells. Therefore, understanding this cycle is crucial for designing effective cancer vaccines, which aim to boost the immune system’s ability to fight tumors. In addition, cancer cells often develop mechanisms to evade immune detection and destruction. They may suppress immune activity in the tumor microenvironment by expressing inhibitory molecules like PD-L1 or creating an immunosuppressive environment that dampens T cell activity. Understanding these immune evasion tactics is critical for designing vaccines that can overcome such barriers.

A tumor antigen is a molecule, typically a protein or peptide that is expressed by cancer cells and recognized by the immune system, particularly by T cells or antibodies. Tumor antigens can be presented on the surface of cancer cells in association with major histocompatibility complex (MHC) molecules, making them targets for immune recognition and potential therapeutic intervention. Tumor antigens are central to the development of cancer vaccines and other immunotherapies, as they provide a way for the immune system to distinguish between normal cells and cancer cells. As research advances, the identification of novel tumor antigens and the development of personalized therapies hold great promise for improving cancer treatment outcomes and providing more targeted, effective options for patients.

In this review, we first summarize the recent major advances on the cancer-immunity cycle, tumor antigens identification, particularly neoantigens, and their delivery in vaccines. We then discuss how the specific steps of the cancer-immunity cycle were utilized to enhance anti-tumor immune response, and to enhance vaccine efficacy. We also discuss how aging and myelosuppression, two common immunological stresses may affect the efficacy of therapeutic cancer vaccines. At last, the current status of all therapeutic cancer vaccines in different clinical stages is summarized.

2 Cancer-immunity cycle, basis for therapeutic immunotherapy

2.1 Effector cells against cancers

In the cancer-immunity cycle, immunosurveillance by immune effector cells is the key step in removing cancer cells. Numerous cell types, innate and adaptive immune cells, are involved in the effect process.

2.1.1 CD8⁺ T cells

CD8⁺ T cells are essential for immune surveillance and the elimination of malignant cells. Traditionally, cytotoxic CD8⁺ T cells, known for generating cytotoxic granules and effector cytokines to directly destroy target cells, have been considered the primary effector T cell population. Recent research suggests that effector CD8⁺ T cells have different Tc subsets, each of which has specific effector roles and potential therapeutic uses.

2.1.1.1 Tc1 cells

Tc1 cells are cytotoxic CD8⁺ T lymphocytes that help eliminate tumor cells by producing granzyme B, perforin, IFN-γ, and TNF-α. Their induction is augmented by interleukin (IL)-12, usually released by antigen-presenting cells (APCs) that have been stimulated by maturation signals derived from pathogens [1]. Key transcription factors including T-bet and STAT4 are essential for Tc1 cell polarization [2,3]. Functionally, Tc1 cells deliver apoptotic factors like granzymes, perforin, and Fas ligand (FASL) to target cells, leading to cancer cell death. Additionally, recent research indicates that Tc1 cells employ alternative cytotoxic mechanisms, including ferroptosis and pyroptosis [4]. These cells are commonly found among tumor-infiltrating lymphocytes in various cancers, such as breast cancer [5], chronic lymphocytic leukemia [6], and lung cancer [7], which are linked with positive prognoses [8–10].

2.1.1.2 Tc2 cells

Tc2 cells are distinguished by their production of type 2 cytokines, such as IL-4, IL-5, and IL-13, while exhibiting reduced secretion of IFN-γ [11,12]. The differentiation of Tc2 cells is stimulated by IL-4, leading to the activation of STAT6 and GATA3, promoting the expression of genes necessary for the Tc2 [13]. In antitumor settings, the transfer of Tc2 effector cells into a B16 melanoma model induced tumor regression and improved mouse survival [14]. However, several studies indicate that Tc2 cells are less effective at inducing robust antitumor immunity compared to Tc1 cells [15,16]. Tc2 cells have been identified within CD8⁺ tumor-infiltrating lymphocytes (TILs), sometimes in higher percentages than Tc1 cells in certain cervical cancer patients. Cancer cells can directly influence T cells, steering them toward a Tc2 phenotype, which increases IL-4 production and decreases IFN-γ production, thereby facilitating tumor immune escape [17].

2.1.1.3 Tc9 cells

Tc9 cells, which produce IL-9, are transcriptionally regulated by STAT6 and IRF4. In mouse tumor models, Tc9 cells demonstrate significantly higher therapeutic effectiveness compared to Tc1 cells. According to the research by Lu et al., Tc9 cells secrete distinct cytokines and exhibit lower cytotoxic activity in vitro; however, they unexpectedly generate stronger antitumor responses against advanced tumors in melanoma model [18]. Compared to Tc1 cells, Tc9 cells exhibit longer in vivo persistence and a less worn-out phenotype in vitro. Moreover, Tc9 cells have the capability to transform into Tc1 like effector cells within the body [19]. The therapeutic impact of Tc9 cells is critically dependent on IL-9 production [18]. Mechanistically, IL-9 produced by Tc9 cells activates STAT3, enhancing fatty acid oxidation and mitochondrial activity, which results in reduced lipid peroxidation and increased resistance to tumor- or reactive oxygen species (ROS)-induced ferroptosis within the tumor microenvironment [20].

2.1.1.4 Tc17 cells

Tc17 cells constitute a subset of CD8⁺ T cells distinguished by the ability to produce IL-17 and the expression of STAT3 and RORγt [21]. The function of Tc17 cells in tumor continues to be a topic of ongoing debate. Research has indicated that patients with head and neck cancer or gastric cancer who exhibit high levels of Tc17 cells in TILs or in circulation tend to have a notably reduced overall survival in contrast to individuals who have lower levels of Tc17 cells [22,23]. Mechanistically, IL-17 produced by these cells can directly interact with tumor cells within the TME, promoting the generation of angiogenic and inhibitory factors [24–26]. For example, tumor-infiltrating Tc17 cells stimulate the secretion of CXCL12 by tumor cells, which subsequently facilitates the migration of myeloid-derived suppressor cells (MDSCs) through CXCR4-dependent mechanisms [22]. Nevertheless, in some contexts, such as esophageal cancer, Tc17 cells may serve as a positive prognostic indicator [27]. Adoptive transfer of Tc17 cells specific to tumors has demonstrated significant antitumor activity in specific mouse cancer models, attributed to the enhanced survival capacity of Tc17 cells and their higher expression of stemness markers compared to Tc1 cells [28–31]. Additionally, IFN-γ-producing Tc17 (Tc17-1) cells have exhibited anti-glioma activity when combined with vaccination strategies [32]. It has also been observed that Tc17 cells, when generated in vitro, can readily transform into Tc1 cells once within the tumor environment, thereby acting as an in vivo reservoir for Tc1 cells [33].

2.1.1.5 Tc22 cells

Tc22 cells are distinguished by their capacity to generate IL-22. The aryl hydrocarbon receptor transcription factor and IL-6 are required for Tc22 to become polarized. When generated in vitro, Tc22 cells exhibit potent cytotoxic activity and are capable of controlling tumor growth upon adoptive transfer into hosts with tumors, operating more efficiently than Tc1 cells [34]. Studies have revealed that Tc22 cells infiltrate tumors in human ovarian cancer patients, making up around 30% of expanded CD8⁺ TILs and this infiltration correlates with improved recurrence-free survival [34]. Moreover, CoA-driven mitochondrial metabolism is known to enhance the anti-tumor capabilities of Tc22 cells [35]. On the other hand, in transplant-associated squamous cell carcinoma, an elevation in Tc22 cells has been linked to a quicker tumor growth, suggesting that they may act as a negative indicator for prognosis [36].

2.1.1.6 Follicular cytotoxic T cells (Tfc)

CXCR5 expression is a defining feature of Tfc cells. Cytokines including IL-6, IL-21, IL-23, and transforming growth factor (TGF)-β, transcription factors like BCL-6, TCF-1, and Runx3 drive their differentiation [37,38]. In non-small cell lung cancer, Tfc cells show notably higher levels of degranulation and increased secretion of proinflammatory cytokines like IFN-γ, IL-2, and TNF, along with the multifunctional cytokine IL-10. Nonetheless, the expression of granzyme B and perforin is considerably lower [39]. Furthermore, the disease-free survival rates of pancreatic cancer patients after tumor resection have been positively linked to the presence of both circulating and tumor-infiltrating Tfc cells. Similar positive correlations have been observed in gastric cancer, muscle-invasive bladder cancer, and colorectal cancer patients [40–43].

2.1.2 CD4⁺ T cells

The many roles of CD4⁺ T lymphocytes within malignancies have attracted a great deal of study. When APCs and environmental cues activate CD4⁺ T lymphocytes, they upregulate transcription factors that direct their differentiation into discrete subpopulations with discrete roles.

2.1.2.1 Th1 cells

Naïve CD4⁺ T cells differentiate into Th1 cells under the induction of IL-12 and IFN-γ, with the expression of key transcription factors such as T-bet, members of the STAT family, IRF1, and RUNX3 [44–47]. Th1 cells contribute to the antitumor response through multiple mechanisms. Cytotoxic T lymphocytes (CTLs) activation requires the help of Th1 cells via secretion of IFN-γ and CD40L signaling. Additionally, Th1 cells stimulate macrophages to produce IL-1β and IL-6. IFN-γ derived from Th1 cells can directly make macrophages cytotoxic to cancer cells and also stimulate macrophages to release angiostatic chemokines [48]. Furthermore, Th1 cell-derived IFN-γ enhances the expression of MHC on dendritic cells (DCs), improving antigen presentation, inhibiting the suppressive tumor microenvironment, and facilitating the development of early vasculature within the tumor [49,50]. IFN-γ is also linked to a positive prognosis in immune checkpoint blockade (ICB) therapies for several cancers [51–53].

2.1.2.2 Th2 cells

Th2 cells develop under the influence of IL-4 and STAT6, which promote GATA3 expression while inhibiting the expression of STAT4 and T-bet. Th2 cells release cytokines including IL-4, IL-5, and IL-13, thereby contributing to the antitumor response via altering the TME and enlisting more effector cells [54]. Th2 can enhance antitumor immunity in organs with low tolerance, such as the lungs and brain, by controlling excessive tissue damage caused by Th1-mediated cytotoxicity [55]. It has been demonstrated that adoptively transferring Th2 cells specific to tumor antigens efficiently eradicates subcutaneous myeloma by causing type 2 inflammation at the tumor location, characterized by a significant infiltration of M2-type macrophages that produce arginase [56]. Blocking the IL-5 pathway can promote tumor growth, whereas overexpression of IL-5 or IL-4 enhances eosinophil and macrophage infiltration, thus boosting tumor clearance [54,57]. However, Th2 cells also influence the differentiation of CD8⁺ T cells, leading to the generation of Tc2 cells, which exhibit diminished antitumor activity compared to Tc1 cells. Studies have indicated that Th2 cells are associated with poorer prognoses in breast cancer, cervical tumors, pancreatic cancer, neuroblastoma, lung adenocarcinoma, and oropharyngeal squamous cell carcinoma [58–63].

2.1.2.3 Th9 cells

When IL-4 and TGF-β1 are present, naïve CD4⁺ T cells can transform into Th9 cells, which produce IL-9. The impact of Th9 and IL-9 signaling on tumor suppression and promotion remains contentious. Th9-derived IL-9 triggers the phosphorylation of STAT3 at serine 727, activating the JAK/STAT pathway, which regulates the proliferation of cancer cells and prevents their apoptosis in lung, leukemia, thyroid, and breast cancers [64–68]. Conversely, there is evidence suggesting that Th9 cells are effective effector T cells in cancer immunotherapy. Introducing Th9 cells into animals that have lung cancer or melanoma, for instance, or giving these mice recombinant IL-9 protein, has been shown to significantly impede tumor growth [64]. Th9 cells enhance the host’s anti-tumor response by facilitating the recruitment of DCs to tumor sites, thereby boosting CD8⁺ CTL activity [69]. Furthermore, Th9 cells also support anti-tumor immunity by secreting IL-21 and IL-24 [70,71].

2.1.2.4 Th17 cells

Th17 cells are a specific type of CD4⁺ T cells distinguished by high IL-17 production. It is widely accepted that TGF-β, IL-1β, and IL-6 are necessary for Th17 cell development. Studies have reported that Th17 cells possess both tumor-promoting and tumor-suppressive activities. Th17 generates GM-CSF, IL-17, IL-21, and IL-22 pro-inflammatory cytokines, which can lead to carcinogenesis, facilitate angiogenesis, and attract MDSCs to the microenvironment within the tumor [72–74]. For instance, Th17 cells infiltrating lung cancer tissue can trigger an epithelial-mesenchymal transition (EMT) in lung cancer, facilitating their migration and metastasis via the secretion of IL-17 [75]. Additionally, Th17 cells may convert into a regulatory T (Treg) cell phenotype within the tumor microenvironment [76]. However, adoptive transfer experiments have provided evidence that Th17 cells exert anticancer immunity in mice [77]. Th17 cells secrete cytokines such IFN-γ, TNF-α, IL-17F, IL-21, and IL-22, which restrict tumor development, accelerate cancer cell death, and hinder angiogenesis. They also secrete chemokines to attract DCs, macrophages, NK cells, granulocytes, and CD8⁺ T cells, and use IFN-γ to eliminate tumors [78,79]. Moreover, Th17 cells have the ability to change into Th1 cells, leading to the production of TNF-α and IFN-γ, which helps in tumor killing and reduces the function of Treg cells [80].

2.1.2.5 Th22 cells

Th22 cells are distinguished by their inability to produce IFN-γ, IL-4, or IL-17, and exclusively secreting IL-22. Studies have shown that they are linked to the progression of various human cancers [81–84]. IL-22 has been found to counteract the apoptotic and cell cycle arrest effects induced by chemotherapy and molecularly targeted drugs in non-small cell lung cancer (NSCLC) cell lines, thereby promoting tumor cell proliferation and tumor growth. Additionally, IL-22 activates the JAK-STAT signaling pathway in both experimental models and living organisms [85].

2.1.2.6 Tfh cells

Tfh cells are characterized by Bcl-6 and STAT3 expression, as well as IL-4 and IL-21 secretion. Their impact on tumor progression varies widely. They can either fuel malignancies through their inherent ability to assist B cells or participate in the immune response against solid tumors. For example, Tfh cells have the potential to be used as diagnostic and therapeutic targets because of their important contribution to the course of B cell malignancies [86]. However, in most solid tumors of non-lymphocytic origin, Tfh cells are associated with enhanced immune responses and improved clinical outcomes. By secreting CXCL13, Tfh cells aid in the attraction of B and CD8⁺ T cells, and by secreting IL-21, they facilitate the development of B cells into antibody-producing plasma cells, thereby fostering an immune-active tumor microenvironment [87]. Tfh cells are essential for tertiary lymphoid structures (TLSs) formation and secrete cytokines like IL-21, which affect both B cells and NK cells, influencing their effector functions [88]. Treatment with anti-PD-1 immunotherapy enhances Tfh cell function, though this enhancement can also lead to the development of immune-related adverse events (irAEs) [86].

2.1.2.7 Treg cells

Elevated TGF-β has been shown to upregulate FOXP3 expression and encourage Treg cell polarization. IL-10 and TGF-β are then secreted by these Treg cells, contributing to immune tolerance and suppression by inhibiting IFN-γ and IL-2, while enhancing the expression of CTLA-4 and CD25 [89,90]. Tregs produce an immunosuppressive milieu by secreting TGF-β and IL-10 [91,92]. Moreover, the tumor microenvironment can convert effector CD4⁺ T cells into Tregs or facilitate naïve CD4⁺ T cells differentiation into induced Tregs [93,94]. Additionally, Treg cells have the ability to create cytotoxic materials such as perforins and granzymes, which can eliminate antitumor effector cells, thereby dampening immune responses [95,96]. Clinically, elevated levels of Tregs are often correlated with poorer prognoses for cancer patients [97–100]. Thus, targeting Tregs is an efficient way to evoke an anti-tumor immune response. Strategies such as depleting/inhibiting Tregs, blocking their recruitment, and attenuating their immunosuppressive function aim to enhance tumor elimination [101,102]. Moreover, the perturbation of tumor-infiltrating Tregs through the IFNγ–IFITM3–STAT1 feedback loop is essential for anti-tumor immunity [103].

Recent studies have suggested the intriguing possibility that Tregs might be convertible into anti-tumor effector cells. Research by Shimon Sakaguchi’s team demonstrated that a subset of Tregs characterized by low FOXP3 expression (FoxP3lo) isolated from colorectal cancer patients can be influenced by the Th1 cytokine IL-12 and can release pro-inflammatory mediators [98]. Patients with higher infiltration of FoxP3lo cells exhibited better clinical outcomes relative to those with lower infiltration, suggesting that these cells might possess anti-tumor effector capabilities [104].

2.1.3 γδ Τ cells

γδ T cells were initially discovered by Tonegawa in 1984 [105]. γδ T cells express γδ T cell receptors (TCRs), in contrast to typical αβ T cells, which constitute most CD3⁺ T cells and mainly detect peptide antigens presented by MHC molecules. This enables them to identify a wider range of antigens without relying on MHC. The antigens that γδ T cells may identify include stress-induced antigens, phosphoantigens, self-antigens, and other non-peptide compounds [106–108].

γδ T cells possess potent anti-tumor properties and can eliminate tumor cells via both direct and indirect mechanisms [109,110]. Stress-induced molecules, such as MHC class I polypeptide-related sequence A/B (MICA/B), are expressed by transformed tumor cells and can interact with γδ T cells through receptors such as NKG2D and NCR2 (NKp44) [111]. This interaction triggers the cytotoxic activity of antitumor γδ T cells. γδ T cells can migrate to tumor sites and secrete granzymes and perforins to eliminate cancer cells. They can also induce direct cytotoxicity through Fc receptor-mediated antibody-dependent cellular cytotoxicity (ADCC), FASL, and TRAIL ligand interactions [112]. Indirectly, activated antitumor γδ T cells secrete IFN-γ and TNF, which enhance antitumor immunity by activating other γδ T cells, upregulating MHC molecules on tumor cells, and promoting the maturation of DCs. Furthermore, γδ T cells have the ability to induce B cells to produce IgE, which possesses antitumor properties. Studies have demonstrated that γδ T cells effectively target a wide variety of tumor cells from both solid and hematological malignancies [113,114], and their presence within tumors is linked to positive prognoses in many types of cancer [115].

Conversely, IL-17-producing γδ T cells are regarded as promoting tumor growth. IL-17 can stimulate angiogenesis by directly communicating with endothelial cells [116] and by inducing angiogenic factors production [117]. IL-17 also promotes the polarization of M2 macrophages [118]. The production of IL-17 by γδ T cells leads to a G-CSF-dependent increase and directional shift in neutrophils, which gain the ability to suppress CTLs [119]. Additionally, IL-17 regulates the PI3K/AKT signaling pathway, which promotes tumor cell motility and invasion [120]. Apart from IL-17, other compounds released by γδ T cells may also play a role in the development of tumors. For example, CD39⁺γδ T cells may decrease immunological responses via the adenosine route and attract MDSCs in colorectal cancer [121].

2.1.4 B cells

B cells are crucial for generating antibodies and playing pivotal roles in humoral immunity. Research has indicated that B cells display both anti-tumor and pro-tumor activities within tumor tissues. Tumor-associated antigens induce the antibody response inside the tumor microenvironment [122]. These antibodies can trigger potent innate effector mechanisms, including ADCC, antibody-dependent cellular cytotoxicity (ADCP), and complement-dependent cytotoxicity (CDC), which help eliminate tumor cells by the complement system, macrophages, and natural killer (NK) cells [123–125]. Tumor binding IgG antibodies can be recognized by DCs, enabling them to capture tumor antigens and then induce cytotoxic T cell responses [126]. B cells can also directly cause apoptosis in malignant cells via the FASL/FAS [127] and TRAIL/Apo-2L pathways [128].

Furthermore, B cells can acquire cytotoxic capabilities and produce granzyme B following IL-21 activation in chronic lymphocytic leukemia [129]. Similar to DCs and macrophages, B cells are capable of acting as professional APCs to activate CD4⁺ T cells, promoting the differentiation of Th1 or Treg cells [130].

B cells can destroy tumor cells, but they can also encourage tumor development. Circulating immune complexes (CICs) include antibodies bound to tumor antigens [131]. CICs can trigger chronic inflammation by activating myeloid cells through the engagement of FcRs, thereby enhancing tumor growth [132]. Regulatory B cells (Breg) defined as CD5⁺CD24^hiCD27⁺CD38^hi, also maintain tumor-promoting potential with their immunoinhibitory abilities [133]. Bregs can inhibit the proliferation of CD4⁺ T cells and promote Foxp3 expression in Tregs. Bregs are correlated with poor prognosis in tumor patients [134].

2.1.5 NK cells

NK cells are a type of lymphocyte with cytotoxic properties, innately programmed to detect and eliminate stressed cells through spontaneous killing mechanisms. The development of NK cells is influenced by early ID2 expression, while the late-stage maturation and cytotoxic capabilities are driven by the later expression of T-bet and EOMES [135,136]. Much of the evidence supporting the anticancer activities of NK cells comes from mouse models utilizing NK cell-depleting antibodies or strains lacking NK cells [137]. In human cancer studies, particularly those involving acute leukemia patients, the transfer of NK cells has demonstrated a protective effect [138–140].

NK cells destroy cancer cells directly by releasing perforin and granzyme or through interactions between Fas and FasL, TRAILR and TRAIL [141,142]. They also secrete cytokines, such as IFN-γ and TNF-α, which can directly suppress tumor cell growth or promote apoptosis. Research conducted using a mouse B16 melanoma model indicates that NK cell activation via the NKp46 receptor leads to increased IFN-γ production, which upregulates fibronectin-1 on tumor cells, thus preventing metastasis [143]. Additionally, NKp80, an activating receptor of NK cells, interacts with AICL, stimulating monocytes to produce proinflammatory cytokines [144]. Moreover, NK cells support T cell priming by aiding in the maturation of DCs, eliminating immature and potentially tolerogenic DCs, and facilitating DC phagocytose antigen. Studies in mice have demonstrated that NK cells, when activated by tumors expressing low levels of MHC-I, can stimulate DCs to generate IL-12, which results in protective CD8⁺ T cell responses [145]. Furthermore, NK cells significantly contribute to the accumulation of cDC1s within tumors, a crucial step for initiating cytotoxic T cell responses, via the secretion of CCL5, XCL1, and/or Flt3L [146].

2.1.6 Neutrophils

Neutrophils, the predominant myeloid cell type in blood, are increasingly recognized for their regulatory roles in cancer. Their influence on tumors is diverse, both inhibiting tumor growth and accelerating its progression. Neutrophil-generated reactive oxygen species (ROS) exacerbate DNA damage during carcinogen exposure, thus fostering tumorigenesis directly [147]. Neutrophils enhance tumor cell growth or antagonize senescence by releasing prostaglandin E2 [148], IL-1 receptor antagonist (IL-1RA) [149], and elastase [150]. Additionally, neutrophils induce zinc-finger protein SNAI1 expression and facilitate epithelial-to-mesenchymal transition in cancer cells under hypoxic conditions, with SNAI1 further promoting neutrophil recruitment [151]. Neutrophils also contribute to angiogenesis by activating dormant pancreatic vasculature through matrix metalloproteinase 9 (MMP9) and BV8 [152]. Another critical aspect of neutrophil function involves modulating other immune cells via mediators such as ROS, inducible nitric oxide synthase, and arginase 1 [153]. Moreover, neutrophils express ligands for immune checkpoint inhibitory receptors that lead to T cell exhaustion [154]. Notably, dcTRAIL-R1⁺ reprogrammed neutrophils tend to accumulate within the glycolytic and hypoxic niche at the tumor core, where they exhibit pro-angiogenic properties supporting tumor growth [155].

In terms of anti-tumor activities, neutrophils counteract uterine carcinogenesis by prompting the detachment of neoplastic cells [156]. When stimulated by hepatocyte growth factor (HGF), anticancer MET⁺ neutrophils eliminate melanoma cells through the release of nitric oxide [157]. Additionally, neutrophils can destroy cancer cells by Fc-mediated ingestion of their plasma membrane, a process known as trogoptosis [158]. Neutrophils contribute to an IFNγ-enriched microenvironment that enhances the antitumor functions of macrophages via unconventional αβ T cells in dermal sarcoma [159].

2.2 Exhaustion of effector T cells

T cell exhaustion acts as a regulatory mechanism to control the intensity and functionality of T cells that are exposed to persistent antigen stimulation. T cell exhaustion is primarily caused by chronic TCR engagement, along with the influence of cytokines such as IL-6, IL-10, IL-35, and IL-27. Other contributing factors include alterations in the microenvironment like a scarcity of nutrients and hypoxic conditions. Characteristics of exhausted T cells include diminished effector functions, decreased cytokine secretion, reduced ability to proliferate, lowered cytotoxic potential, continuous high-level expression CTLA4, PD-1, LAG3, TIM-3, and TIGIT, metabolic shifts, and modifications in both transcriptional and epigenetic patterns [160].

2.2.1 CD8⁺ T cell exhaustion

CD8⁺ T cell exhaustion was initially coined when T cells are persistently stimulated by antigens over an extended period in chronic viral infections mouse models. Despite displaying markers indicative of activation, these CD8⁺ exhausted T cells (Tex) are incapable of eliminating virus-infected cells [161,162]. Subsequently, CD8⁺ T cells exhibiting similar functional impairments were observed in various human chronic viral infections, such as hepatitis C virus (HCV) and the human immunodeficiency virus (HIV) [163–166]. The concept of exhaustion is also used to describe tumor-infiltrating CD8⁺ T cells, which have impaired functionality [167–169].

During chronic infections, CD8⁺ T cells evolve into precursor exhausted cells (Tex pre), marked by the presence of TCF1, EOMES, and TOX. With ongoing exposure to the antigen, these cells develop into a progenitor exhausted subset (Tex prog), which represents an intermediate exhausted state and retains the ability to self-renew. These intermediate cells eventually lose the expression of TCF1 and mature into terminally exhausted cells (Tex term), characterized by weak cytokine secreting and elevated levels of PD-1 [160]. In murine B16 melanoma, a specific group of TCF1⁺PD-1⁺ stem-like cells have been identified within tumors, which has been identified as vital for controlling tumors via immunotherapy [170]. Various prior studies have noted the presence of exhausted cell populations within the TME, which loses its proliferative capability and responsiveness to ICB therapies [171,172]. T cell exhaustion is affected by both intrinsic and extrinsic factors combined.

2.3 Intrinsic factors

Several transcription factors, such as TOX, EOMES, NR4A1, T-BET, BLIMP1, BATF, and NFAT, play a role in regulating the expression of PD-1 and are associated with T cell exhaustion [173,174]. Epigenetic elements, such as DNA methylation and histone modifications, can also influence the expression of PD-1 and contribute to T cell exhaustion [175,176]. Moreover, intrinsic metabolic pathways in dysfunctional T cells appear to be altered, impacting the state of exhaustion [177–179].

2.4 Transcriptional factors

NR4A1: NR4A1 is observed to be substantially expressed in defective T cells in animal models. Its overexpression hinders the differentiation of effector T cell, while its deletion reverses the tolerance of T cell, boosts the proliferation of T cell, and enhances antitumor ability. In Nr4a1-deficient mice, PD-1 and TIM-3 expression in the T cell are notably reduced [174].

TOX: This factor is pivotal in CD8⁺ T cell exhaustion. In the mouse model of hepatocellular carcinoma (HCC), TOX is increased in CD8⁺ exhausted T cells, compromising the antitumor activity by reducing PD-1 degradation and promoting its recycling back to the cell surface. Reducing TOX levels of CD8⁺ T cells amplify their antitumor effects, demonstrating synergy with anti-PD-1 therapy [180].

EOMES: Expression of Eomes is heightened in tumor-infiltrating PD-1⁺Tim-3⁺ exhausted CD8⁺ T cells. Total absence of Eomes in CD8⁺ T cells impairs the antitumor CTLs development. On the other hand, Eomes gene deletion in T cells reduces the formation of exhausted T cells, leading to improved tumor control [181].

NFAT: NFAT governs the process of T cell exhaustion by weakening TCR signaling, elevating inhibitory surface receptors expression, and interfering with the capacity of CD8⁺ T cells to inhibit tumor growth in vivo [182].

2.5 Epigenetic factors

DNA methylation enzymes like DNMT1, DNMT3B, and DNMT3A exhibit significantly elevated expression levels in exhausted T cells [183]. Inhibiting DNMT3A enhances the differentiation of T cells into memory cells. When TCR stimulation is paired with treatments involving IL-6 or IL-12, there is an increase in both H3K4me3 and H3K27 acetylation, contributing to higher PD-1 expression [184]. miR-155 strengthens the antitumor functionality of T cells by preventing their senescence and functional exhaustion, achieved through the epigenetic silencing of factors that drive terminal differentiation [185].

2.6 Metabolism factors

As T cells progress from a naïve state to becoming effectors, they transition metabolically from oxidative phosphorylation to aerobic glycolysis. Conversely, after the clearance of antigens, during the contraction phase and memory formation, they revert back to relying on oxidative phosphorylation [186]. In tumor, persistent antigen encounter and the nutrient-poor hypoxic environment subvert the normal metabolic regulation of Tex cells [187–189]. This disruption manifests as diminished glucose uptake, decreased mitochondrial mass and function, and reduced expression of insulin receptors (IRs) on Tex cells [188]. Disruptions in Tex cell metabolism have been associated with lowered expression of PGC1α, a critical regulator of cellular bioenergetics. Overexpressing PGC1α has been shown to partially restore metabolic capabilities and enhance the effector capabilities of CD8⁺ T cells [190].

2.7 Extrinsic factors

The components of TME, such as tumor cells, stromal cells, immunosuppressive cells, and cytokines, significantly influence T cell activity by restraining their differentiation and promoting dysfunction [191].

2.8 MDSCs

MDSCs contribute significantly to T cell exhaustion in TME by releasing high levels of ROS, arginase (ARG)-1, indoleamine 2, 3-dioxygenase (IDO), and inducible nitric oxide synthase (iNOS). ROS generated by MDSCs in cancer can induce the hypo-responsiveness of CD8⁺ T cells by leading to oxidative stress [192]. The expression of ARG-1 has been reported to impair the function of T cells by decreasing CD3 zeta chain biosynthesis [193]. In breast cancer, IDO expression by MDSCs mediates immunosuppressive effects, significantly inhibiting CD8⁺ T cell proliferation and IFN-γ production [194]. NO inhibits the function of CD8⁺ T cells by preventing STAT5 and JAK3 activation, as well as promoting death in T cells [195].

2.9 Tumor-associated macrophages (TAMs)

TAMs are generally defined by their M2-like macrophage phenotype, which is activated by Th2 cytokines. TAMs produce high quantities of immunosuppressive factors, contributing to immune suppression and facilitating tumor growth [196]. Additionally, TAMs can inhibit the cytotoxicity of CD8⁺ T cells by depleting L-arginine and tryptophan directly. Furthermore, they produce significant amounts of IDO to further suppress CD8⁺ T cell activity [197]. In patients with NSCLC, TAM-derived IL-10 enhances tumor progression, leading to a correlation between elevated IL-10 levels and a worse prognosis for these patients [198].

2.10 Cancer-associated fibroblasts (CAFs)

CAFs, the most common stromal cells, release immunomodulatory substances inside the TME and are becoming recognized as antitumor immunity inhibitors. These cells attract myeloid cells to the tumor site by secreting chemokines, subsequently polarizing them toward an M2 phenotype [199]. CAFs can process and cross-present antigens, leading to the targeted elimination of CD8⁺ T cells through interactions involving PD-L2 and FASL. Expression of inhibitory ligands, such as PD-L2 and FASL, has been detected in CAFs isolated from human tumors. Blocking these ligands restores T cell cytotoxic function both in experimental settings and within living organisms [200].

2.11 Treg cells

Treg cells, a suppressive fraction of CD4⁺ T cells, increase in the peripheral blood and tumor microenvironment of cancer patients, permitting immune escape [201]. Tregs produce immunosuppressive cytokines such as TGF-β, IL-10, and VEGF. These factors facilitate the transformation of conventional T cells into Treg cells, and simultaneously, suppress the activity of effector T cells and antigen-presenting cells [93,94,202]. Treg cells have the ability to create cytotoxic materials such as perforins and granzymes to kill T cells, NK cells, and antigen-presenting cells [95,96]. Furthermore, tumor-infiltrating Treg cells produce inhibitory cytokines such as IL-10 and IL-35, which contribute to the exhaustion of CD8⁺ T cells in tumor through mechanisms dependent on BLIMP-1 [203]. Treg cells constitutively express CTLA-4, which leads to a reduction in the expression of CD80/CD86 on APCs via trans-endocytosis, thereby limiting the co-stimulatory signals available to responding T cells. Treg cells also impair DCs functionality through interactions involving LAG-3 and MHC class II molecules [202]. Additionally, the high expression of CD25 on Treg cells leads to the consumption of significant amounts of local IL-2, impairing T cell functionality [204]. Treg cells produce the ectoenzymes CD39 and CD73, which are known to create adenosine and suppress the function of effector T cells by binding the adenosine receptor [205,206]. FOXP3⁺ Treg cells can promote effector T cell senescence through metabolic competition. FOXP3⁺ Treg cells demonstrate increased glycolysis, accelerating glucose depletion and diminishing glucose availability in the TME, which induce senescence in both CD4⁺ and CD8⁺ T cells [202,207].

2.11.1 CD4⁺ T cell exhaustion

CD4⁺ T cells, like CD8⁺ T cells, can be exhausted in numerous tumor types due to the presence of inhibitory receptors such as PD-1, CTLA4, TIM-3, and LAG-3 [208,209]. In a melanoma model, transferring CD4⁺ T cells into RAG1 knockout recipients led to tumor regression. However, some mice experienced recurrence. CD4⁺ T cells isolated from these recurring tumors exhibited reduced cytokine production, elevated levels of co-inhibitory receptors, and failed to induce tumor extinction when transferred into another hosts, indicating the onset of exhaustion that limits therapeutic responses. Combination therapy using PD-L1 and LAG3 blocking was shown to reduce the expression of checkpoint, enhance effector functions of CD4⁺ T cells, and achieve sustained tumor control [210,211]. Clinical studies have also revealed that higher levels of co-inhibitory markers are expressed by CD4⁺ T cells found in tumor compared to those found in circulation or adjacent tissues [212–217].

In a murine melanoma model, the expression of TCF1 and SLAMF6 in CD4⁺ T cells is lower compared to those in the spleen, suggesting a more advanced state of terminal exhaustion. Treatment with PD-L1 blockage increased the expression of TCF1 and decreased the expression of TIM3 and LAG3, respectively, indicating the preservation of a progenitor exhausted subset [218]. These findings are supported by experiments conducted on human samples from ovarian, head and neck, and cervical cancers [219]. Tumor-infiltrating CD4⁺ T cells expressed CD39, as well as increased PD-1 levels and decreased cytokine output. Following anti-PD-1 treatment, these CD4⁺ T cells demonstrated increased cytokine production. Additionally, anti-PD-1 enhanced DC maturation and promoted the proliferation of CD8⁺ T cells [219].

2.12 Infiltration and maintenance of immune cells in cancers

Tumors exhibit various immunological phenotypes: immune-inflamed, immune-excluded, and immune-desert. These terms refer to tumors with significant immune infiltration, those where immune cells are limited to the tumor stroma rather than the parenchyma, and tumors devoid of immune infiltration, respectively [220]. For the effective destruction of tumor cells, effector T cells need to actively migrate and engage with cancer cells [221]. Chemokines are crucial for directing the movement of both stimulatory and inhibitory immune cells.

2.12.1 T cell infiltration

CD8⁺ effector cells enhance the expression of the chemokine CXC receptor 3 (CXCR3), which is indispensable for the migration of adoptive-transferred CD8⁺ T cells through the tumor’s blood vessels [222]. Studies in mice have shown that T cell infiltration depending on CXCR3 occurs in many tumor models, such as melanoma, lymphoma, breast cancer, and renal cell carcinoma [222–226].

Likewise, heightened levels of CXCL9 and CXCL10 have been linked to greater activated T cells infiltration in several human cancers, such as ovarian, melanoma, and colon cancer [227–230]. Treating prostate cancer samples in with immune stimulant in vitro can induce the expression of chemokine, like CXCL9, CXCL10, and CCL5, leading to T cell infiltration in a preclinical model [231]. Reactive nitrogen produced within tumors can lead to the nitration of the CCL2 chemokine, obstructing T cell infiltration and trapping them in the surrounding stroma [232].

The vascular system facilitates immune cell migration to tumors. When influenced by VEGF and TGF derived from tumor cells, vascular endothelial cells have lower expression of adhesion molecules [233]. This phenomenon results in T cells being unable to adhere to the tumor vasculature, thereby hindering their access to the tumor site.

2.12.2 Trm maintenance

Solid tumors often harbor CD8⁺ tissue-resident memory T (Trm) cells in tumor tissue, with the characteristics of expressing CD103, CD69, and CD49a [234]. CD69 inhibits S1PR1 signaling, thereby preventing Trm cells from leaving the tissue [235]. CD103 and CD49a act as “anchors,” retaining Trm cells within the tissues [236]. A significant presence of CD103⁺CD8⁺ T cells is observed in both the epithelium and stroma of tumors, correlating with extended tumor-free and overall survival in patients with breast cancer [237], lung cancer [238,239], endometrial adenocarcinoma [240], ovarian cancer [241], cervical cancer [242], and urothelial carcinoma of the bladder [243]. Blocking CD103 with antibodies or genetically eliminating CD103 leads to a decrease in tumor-infiltrating T cells and faster tumor progression in mice [244–246]. Additionally, Notch regulates the maximum expression of CD103 in tumor-associated Trm cells [247]. Moreover, Runx3 is a critical regulator of Trm maintenance within tumors. CD8⁺ T cells deficient in Runx3 were unable to accumulate in melanoma tumors after adoptive T cell therapy, leading to increased tumor growth and higher mortality rates [248].

2.12.3 Treg infiltration

The CCR4–CCL22/17 axis has been repeatedly demonstrated to facilitate the migration of Tregs to the TME across different cancer models. In ovarian carcinoma, both tumor cells and TAMs produce CCL22, which acts as a ligand for CCR4 and is responsible for directing Treg cells to the tumor site [249]. Similarly, CCL22 plays a key role in recruiting Tregs in breast cancer [250]. Furthermore, heightened expression of CCL22 and CCL17, another ligand for CCR4, is linked to increased Treg infiltration in esophageal squamous cell carcinoma and gastric cancer [251,252].

2.12.4 TAMs and MDSC infiltration

The CCR2/CCL2 axis is crucial for mediating the recruitment of TAMs and MDSCs into solid tumors. In Ccr2 knockout mice, tumors exhibit minimal infiltration by MDSCs, leading to decreased tumor growth and metastasis in esophageal carcinogenesis [253–255]. Tumor cells and the associated stromal components are substantial producers of CCL2, which is the ligand for CCR2 [256]. Increased CCL2 expression in tumors has been shown to attract large numbers of monocytes or monocyte-derived cells in several mouse models [257–261], as well as in some tumor patients [262–265]. Moreover, the CCR5 axis also contributes to MDSC infiltration in tumors. In a melanoma transgenic mouse model, MDSCs induced immunosuppression by CCR5 expressing within the tumor microenvironment. Blocking the CCR5 axis, enhanced the survival of tumor-bearing mice and correlated with reduced migration and immunosuppressive activity of MDSCs within tumor lesions [266]. In a breast cancer mouse model, overexpression of CCL5 induces tumor recurrence by attracting macrophages expressing CCR5 [267].

2.13 Initiation of antitumor T cell response in cancer-immunity cycle

To trigger an effective antitumor response, the presentation of tumor antigens must occur successfully at two distinct stages: initially, cancer antigens need to be released and then captured by DCs for cross-presentation, which primes CD8⁺ T cells. On the other hand, these antigens must be directly presented by the tumor cells themselves, allowing for recognition by the primed CD8⁺ T cells and subsequent tumor cell elimination.

2.13.1 Release of cancer cell antigens

2.13.1.1 Immunogenic cell death

Damage-associated molecular patterns (DAMPs) and other endogenous immunoadjuvants, such as tumor antigens, are examples of endogenous immunoadjuvants that are exposed during immunogenic cell death (ICD). By increasing the adjuvanticity of dying cancer cells, this exposure encourages antigen-presenting cells to be recruited and activated [268]. ICD that is driven by radiation therapy and conventional chemotherapies can stimulate anticancer immune responses [269–271]. DAMPs include calreticulin (CALR), high-mobility group box 1 (HMGB1), the cytoplasmic protein annexin A1 (ANXA1) and ATP [268,272–274]. Different PRRs can identify DAMPs, which causes the DC to cross-present tumor antigens to CTLs that are highly stimulated by their immune system [275,276].

Normally found in the ER lumen, CALR migrates to the surface of cancer cells in response to ER stress and ICD. There, it interacts with CD91 on DCs to act as a crucial “eat-me” signal [277]. Interestingly, anthracycline-treated tumor cells’ ability to produce CALR is considerably reduced, which lowers DC-mediated anti-tumor immunity [278,279]. HMGB1 activates TLR4 and plays a crucial role in DCs cross-presentation, avoiding the lysosomal degradation required for cross-presentation [280,281]. In preclinical models, the absence of HMGB1 in cancer cells and TLR4 neutralization by antibodies in hosts impair the immune responses triggered by anthracyclines, cyclophosphamide, or oxaliplatin [280]. ANXA1 serves as a unique homing factor that guides DCs toward cancer cells undergoing ICD. Cancer cells deficient in ANXA1 show reduced sensitivity to chemotherapy in vivo [282]. Exocytosis of ATP-containing vesicles via pannexin channels is one of the processes by which autophagy-dependent ATP is released [283–285]. Extracellular ATP binds to the purinergic receptor P2Y2, aiding the attraction of myeloid cells to areas of ongoing ICD [286,287]. Furthermore, ATP released by dying tumor cells interacts with P2X7 purinergic receptors on DCs, triggering the secretion of IL-1β and IL-18, which help prime CD8⁺ T cells [288,289].

Roles of ICD in enhancing antigen presentation by APCs are shown in Fig.1.

2.13.1.2 Other forms of tumor cell death

Necroptosis, a kind of caspase-independent cell death marked by the phosphorylation of MLKL, RIPK1, and RIPK3, along with the induction of necrosome formation, activates DCs and promotes adaptive immune responses against tumor cells [290]. The signaling cascade initiated by RIPK1 and the subsequent activation of NF-κB within dying cells, accompanied by the DAMPs’ release, facilitates the priming of CD8⁺ T cells [291]. Studies have shown that vaccination using necroptotic cells can effectively boost anti-tumor immunity [292]. Intratumoral administration of engineered necroptotic cells expressing constitutively active RIPK3 into B16 tumors has been observed to trigger a RIP1/RIP3/NF-κB pathway-dependent anti-tumor response mediated by CD8⁺ T cells and cDC1, without requiring cell lysis [293].

Pyroptosis, distinguished by cell swelling and pro-inflammatory cytokines release such as IL-1β and IL-18, involves the assembly of the inflammasome and the ultimate lytic cell death facilitated by the pore-forming protein gasdermin [294]. This process is closely linked with antitumor immunity. Expression of gasdermin E (GSDME) enhances the phagocytosis of tumor cells by TAMs and increases the quantity and functionality of tumor-infiltrating NK cells and CD8⁺ T lymphocytes. Research conducted by Feng Shao’s team indicated that approximately 15% of tumor cells undergoing pyroptosis is sufficient to eliminate an entire tumor in animal models [295]. Pyroptotic cell death in tumors can be induced through a bioorthogonal system utilizing NP-GSDMA3 and Phe-BF3, leading to the increase of cytotoxic T cells and NK cells while simultaneously reducing the numbers of monocytes, neutrophils, and myeloid-derived suppressor cells [295].

2.13.2 Cancer antigen presentation and T cell priming

DCs are a diverse collection of myeloid-derived APCs with specific functions that work together to orchestrate the immune system’s response to cancer.

2.13.2.1 DC heterogeneity

cDC1 subsets have been the subject of much research, primarily because of their capacity to identify dying and dead cells, present antigens via MHC-I to drive CD8⁺ T cell responses, and promote tumor rejection [296]. cDC1 is characterized by its dependence on major transcription factors such as IRF8, ID2, and BATF3, as well as the selective expression of CLEC9A and XCR1 [297,298]. Studies have shown that BATF3-deficient mice experience impaired cDC1 development and maturation, resulting in an inability to reject malignancies. Research has demonstrated that BATF3-deficient mice have impaired cDC1 maturation and development, which makes them unable to reject immunogenic tumors [299].

Through the presentation of soluble antigens via MHC class II, cDC2 directly stimulates CD4⁺ helper T cell responses, which is a critical component of host barrier defense and immunization against external infections and allergens [300]. This subset is characterized by high IRF4 expression and is also influenced by other key transcriptional regulators such as RELB, ZEB2, KLF4, and NOTCH2 [301]. The involvement of cDC2s in cancer immunology is still being elucidated. Nonetheless, it has been shown that the ratio of cDC2s to Tregs is connected to the infiltration of CD4⁺ T cells into the TME. An increased percentage of cDC2s in the TME is associated with a greater infiltration of CD4⁺ T cells [302].

pDCs are specialized in anti-viral defense and serve as a critical source of interferon-I, along with interferon-III, during viral infections. They are thought to play a comparable role in enhancing anti-tumor immune responses by producing IFN-I, which aids in the maturation of cDC1 and stimulates the local activities of CD8⁺ CTLs and NK cells [303,304]. Nevertheless, within the context of malignancies, pDCs can also contribute to immune tolerance and suppression. Across various types of cancer, pDC hypofunction has been noted, characterized by a diminished response to TLR stimulation and reduced induction of IFN-I responses. The tolerogenic actions of pDC in these scenarios are often linked to immunosuppressive factors present in the TME [305–310].

MoDCs are typically found in inflammatory contexts and exhibit characteristics and functionalities similar to those of monocytes and cDC2s. These MoDCs are known to be IRF4-dependent and capable of cross-priming CD8⁺ T cells [311]. Research indicates that the recruitment of MoDCs into the TME is facilitated through the CCR6/CCL20 axis. Recently, the presence of MoDCs has been linked to improved responses to checkpoint blockade therapy, both when used alone and in combination with other treatments [312].

Tumor-infiltrating DC3 cells are characterized by the concurrent presence of maturation and activation markers, along with an immunoregulatory profile. This profile encompasses markers indicative of DC migration, DC maturation, and immune regulation [313]. Functionally, DC3 cells have the capacity to activate naïve T cells and may be essential for TRM cell programming [314,315]. Upon stimulation through TLR3, TLR4, and TLR7/8, activated DC3 cells enhance their expression of CCR7 and secrete cytokines like IL-12, as well as chemokines such as CXCL9 and CXCL10, which aid in T cell homing [314]. Additionally, both CD8⁺ and CD4⁺ T cells express CD103 when stimulated by DC3 cells, perhaps via a process controlled by TGF-β [302,313].

2.13.2.2 T cell priming

Within the TME, locally generated growth factors and chemokines that support DC recruitment, differentiation, and extension have an impact on the localization and accumulation of DCs [316,317]. For instance, chemokines produced by NK cells function as chemo-attractants to help draw cDC1 into the TME [146]. Tumor-derived CCL4 has also been implicated in the recruitment of cDC1 [318]. After absorbing antigens, DCs go to secondary lymphoid organs, where they engage in interactions with naïve CD8⁺ and CD4⁺ T lymphocytes, ultimately causing their activation. After maturing, migratory DCs upregulate CCR7, which allows them to be attracted to T cell-rich areas inside tdLNs by means of a gradient of CCL21 chemokines released by the lymphatic endothelium [319,320].

DCs interact directly with T cells in the TME to improve antitumor immunity in addition to their functions in antigen presentation and T cells priming within the tdLNs. Tumor-infiltrating CD103⁺ DCs produce CXCR3-ligand chemokines, which are essential for attracting effector T cells to the TME in some models and tumor types [222,321].

3 De novo generation of anti-tumor T cell response in lymphoid tissues

3.1 What antigen to target?

Cancer vaccines stimulate the body’s cellular immunity to eliminate tumor cells. They can utilize various methods to present tumor antigens and initiate the process of cell-immunity cycle [322]. The choice of antigen largely determines the efficacy of vaccine. The ideal antigen should be specifically expressed on the tumor cells and has great immunogenicity. Here we outline the four primary candidates for targeting antigens.

3.1.1 Self-antigen

In the study of immunology, self-antigens refer to molecules produced by our body’s normal cells, which should be recognized by the immune system as part of the “self. ” However, when it comes to cancer, the situation becomes complex. For example, overexpressed antigens, which are antigens expressed at excessively high levels in tumor cells, can be identified as possible non-self targets by the immune system; differentiation antigens are molecules specifically expressed during certain stages of cell differentiation or in particular cell types; and cancer-testis antigens are a particularly interesting class of antigens because they are not only found in some tumor cells but also in normal testicular tissue, yet they are generally not expressed in other normal tissues. The distinct properties of these antigens make them important targets for the development of cancer vaccines and immunotherapies.

A prime example of a therapy targeting overexpressed antigens is the groundbreaking autologous dendritic cell vaccine known as sipuleucel-T. This treatment has been clinically proven to enhance the lifespan of individuals suffering from metastatic, castration-resistant prostate cancer by an additional period ranging from two to four months. The mechanism of action for this therapeutic strategy zeroes in on prostate acid phosphatase (PAP), a tumor-associated antigen (TAA) that is prominently displayed on the surface of prostate cancer cells [323,324].

The most frequently targeted differentiation antigens are produced from proteins like gp100, tyrosinase, and TRP1 that are involved in the melanin production or biogenesis of melanosomes [325,326]. The same as overexpression antigens, the differentiation antigens are expressed on normal cells and tumor cells. Targeting these antigens presents a complex dual challenge in oncological therapeutics. On the one hand, potential toxicity to low-expressing cells needs to be considered. On the other hand, due to the existence of immune tolerance, these antigens may take advantage of immune escape and affect vaccine efficacy [327].

Cancer-testis antigens, also known as cancer-germline antigens, such as MAGE [328], NY-ESO1 [329], GAGE, and BAGE [330], demonstrate a unique expression profile. They are primarily confined to the germ cells residing in immune-privileged environments, notably the testes. However, these antigens are strikingly overexpressed in numerous types of tumor cells, which renders them promising candidates for targeted immunotherapeutic interventions, given their limited expression in normal tissues alongside their pronounced presence in malignancies.

3.1.2 Non-self antigen

The non-self-antigens, mostly from virus, is a kind of TSAs, which are distinctively found in tumor cells. As the first human virus identified to be associated with human cancers [331], Epstein-Barr virus encodes multiple antigens such as EBNA1, EBER, and the latent membrane proteins LMP1 and LMP2, which may be expressed in various tumors including natural killer-T cell lymphoma and nasopharyngeal carcinoma [332]. Statistics indicate that the E6 and E7 proteins (HPV E6 and E7) are present in approximately 60% of oropharyngeal cancers and in virtually all cases of cervical cancer [333]. Over the past decade, human papillomavirus (HPV) vaccine delivers strong protection against cervical cancer. Several such vaccines against E6/E7 or other epitopes are under investigation [334,335]. For example, immunization with an mRNA vaccine expressing E6 and E7 proteins significantly induced robust T cell-mediated immune responses in subcutaneous and orthotopic tumor implantation mouse models, along with significant suppression of tumor growth and significant infiltration of immune cells into tumor tissues [334].

3.1.3 Neoantigens

Neoantigens have garnered increased attention as a result of advances in modern high-throughput gene sequencing technology and a growing comprehension of neoantigen generation [336]. They have advantage of tumor-specific and are absent from normal cells. Neoantigens can be derived into genomic variants, transcriptomic variants, and proteomic variants (Fig.2).

For the genomic variants, there are single-nucleotide variants (SNVs), which are similar to self-antigens and seldom shared between patients, but they have the advantage of simple prediction and relatively elevated burden. However, genomic variants from alternative sources, insertions and deletion (INDEL) and fusion genes are relatively low burden, more immunogenic because they are more dissimilar from self-antigens [337–340]. The last source group is chromosomal rearrangements, which are the most complex type of structural variants with strong immunogenicity. Structural variations (SVs) typically encompass genetic alterations larger than 50 base pairs in size, including various types of changes including insertions, deletions, inversions, translocations, and duplications or amplifications. These also cover a broader range of chromosomal events such as additions and deletions, plus other complex chromosomal rearrangements [322].

Transcriptomic variants include polyadenylation (pA) and RNA editing, RNA splicing, and allegedly non-coding regions. The former are easily predicted, while the latter two have fewer tools available. A significant number of predicted targets have been identified through RNA splicing. Studies that detect alternative splicing events and tumor variants by analyzing RNA and whole-exome sequencing data from 8705 patients demonstrate that tumors exhibit up to 30% more alternative splicing events compared to normal samples [341].

Proteomic variants have post-translational modifications (PTMs), proteasome processing, and T cell epitopes associated with impaired peptide processing (TEIPP). Aberrant PTMs shared between patients, which may involve processes such as phosphorylation, glycosylation, and O-linked β-N-acetylglucosamine (O-GlcNAc), can result in the generation of neoantigenic peptides. These peptides are often displayed by MHC molecules on the surface of tumor cells [342]. As an illustration, a neoantigen derived from the post-translationally modified protein MUC1 was specifically presented by MHC class I and uniquely recognized by a TCR that has specificity for a particular glycoform of this antigen [343].

Neoantigen identification and prediction is a critical process in personalized cancer immunotherapy, aiming to discover tumor-specific antigens that can be targeted by the immune system. This process involves several interrelated steps, each requiring sophisticated computational and experimental techniques.

3.1.3.1 Computational prediction of neoantigens

Step 1: Identification of somatic mutations

The immunogenomic strategy for neoantigen identification hinges on a meticulous comparative analysis of the genomic landscapes between tumor cells and their corresponding normal cells. This approach involves the systematic detection of somatic mutations that are present in the cancerous tissue but absent from healthy counterparts. In 2012, two distinct research teams concurrently and pioneeringly employed next-generation sequencing (NGS) technology to pinpoint immunogenic neoantigens within murine tumor models. Matsushita et al. identified mutant spectrin-β2 as a potential antigen of the d42m1 sarcoma using exome sequencing including cDNA capture and Illumina deep sequencing (that is cDNA CapSeq). Castle et al. further documented the prophylactic impact of neoantigen-based vaccines in the context of the B16 melanoma model, showcasing the potential of personalized immunotherapeutic strategies [344,345].

Step 2: Prediction of mutated peptides

By combining WES, which focuses on sequencing the protein-coding regions of the genome where most known disease-causing mutations reside, with transcriptome profiling, which captures the active transcriptional output of cells, researchers can achieve a thorough understanding of the functional consequences of genetic changes in cancer. This synergistic method allows for a more accurate determination of which mutations are likely to play a role in the growth and advancement of tumors by affecting gene expression patterns and protein function [345]. Various integrated bioinformatic tools, such as pTuneos [346], pVAC-seq [347], TIminer [348], ProGeo-Neo [349], and INTEGRATE-Neo [350], leverage inputs from whole exome sequencing (WES) and RNA sequencing (RNA-seq) data sets. These tools utilize stringent filtering processes, based on predetermined threshold criteria, to systematically eliminate potential false positives. The objective is to refine the identification of somatic mutations that are both expressed at the transcript level and have the potential to alter the proteome, thus serving as neoantigens or drivers of disease pathology.

There are numerous prediction tools based on machine learning, such as ConvMHC [351], NetMHCpan [352], MHCflurry [353], PLAtEAU [354], NetMHCIIpan [355], and NetCTLpan [356]; however, these tools are highly reliant on the availability of robust data sets for both training and testing purposes. A statistical analysis of T cell reactivity from 13 studies focusing on neopeptide-HLA interactions revealed that only 53 out of 1943 peptides were reported to elicit a T cell response [357]. To enhance the validation rate of these sequence-based predicted antigen peptides as true neoepitopes, it is imperative to adhere to the fundamental principle governing how antigen peptides become epitopes: they must undergo processing and presentation by human leukocyte antigen (HLA) molecules and subsequently be recognized by T cells.

Step 3: MHC binding affinity prediction

Immune Epitope Database (IEDB) established in 2004 has been publicly accessible for 20 years, provides experimental data presented in figures, text, and tables from scientific literature. Nevertheless, these entries have been accumulated through a variety of experimental techniques that lack uniformity, particularly when it comes to data derived from cancer models [358]. EDGE, a computational models of antigen presentation, uses deep learning on a large data set (N = 74 patients) of HLA peptides and genomics from various human tumors, enhancing the positive predictive value by as much as nine times compared to previous methods [359]. Multiple in silico algorithms have been developed to improve the likelihood of identifying clinically relevant neoepitopes, such as MuPeXI algorithm [360] and EpitopeHunter algorithm [361]. For the personalized vaccine, one of the most crucial steps is to identify the patient’s HLA genotypes. Tools such as HISAT Genotype, ATHLATES, and HLA-HD are versatile and can be employed for the typing of both Class I and Class II HLA alleles. Other tools such as NetCTL/NetCTLpan, McPAS-TCR, VDJdb, and pMTnet can be utilized to predict the binding affinity of TCRs.

3.1.3.2 Experimental validation of neoantigens

Step 4: Immunogenicity assessment

To assess the immunogenicity of candidate neoantigens, experimental testing of their MHC binding and T cell responses is required. The direct assay for the validation of MHC binding peptides is the mass spectrometry (MS) approach. Despite its utility, the MS approach faces limitations, including sensitivity issues and the potential to overlook significant epitopes [362]. To date, no single high-throughput technique has been able to comprehensively and definitively identify potential neoepitopes. The most reliable and precise method remains the detection of naturally processed peptides through CD8⁺ T cell-mediated cytotoxicity, as even a single MHC-peptide complex can effectively trigger both recognition and lysis by T cells [363]. As an example, one might employ T cells derived from HLA transgenic mice that have been immunized with the reference neoepitope.

Step 5: In vivo validation

Recent clinical trials involving neoantigen vaccines have demonstrated their ability to induce both CD4⁺ and CD8⁺ T cell responses. Sahin et al. manufactured the RNA-based poly-neoantigen vaccine unique for each patient with melanoma, responses of CD4 and CD8 T cells were detected against 60% of the predicted neo-epitopes by ELISpot, approximately one-fifth of the immunogenic mutations elicited detectable responses in the blood without the need for in vitro stimulation [364]. Similarly, a peptide neoantigen vaccine has shown the ability to induce T cell responses and promote T cell infiltration into the tumor microenvironment (TME) in patients with high-risk melanoma. Specifically, 60% of neoantigens in this vaccine triggered CD4⁺ T cell responses, whereas 16% induced CD8⁺ T cell responses. [365].

3.1.3.3 Integration of steps: a seamless process

It is worth noting that the computational prediction and experimental validation steps are not strictly sequential but rather iterative and complementary. Initial predictions guide subsequent validations, while experimental results refine computational models. There is a continuous feedback loop between computational and experimental data to improve neoantigen discovery.

3.1.3.4 Challenges and future directions for neoantigen identification and use

• Limitations exist in current prediction algorithms, highlighting the need for more accurate predictors.

• The heterogeneity of tumors and the dynamic nature of the tumor microenvironment pose significant challenges.

• Ethical considerations and obtaining patient consent are crucial aspects of personalized medicine.

An important point to mention is that the neoantigen landscape of patients are not invariable, therapeutic strategies should aim at inducing broad neoantigen-specific T cell responses to thwart tumor resistance. Verdegaal et al. elucidated the dynamic interplay between human melanoma and T cells. Their findings revealed that T cell-recognized neoantigens can be selectively excluded from the tumor cell population, either due to decreased expression of the associated genes or loss of the mutant alleles. Importantly, this loss of neoantigen expression was associated with the appearance of neoantigen-specific T cell responses in tumor-infiltrating lymphocytes [366]. Tumor mutation burden (TMB) indicates the number of mutations per megabase (Mut/Mb) in the sequenced DNA of a particular cancer. Tumor mutation burden is related to the number of neoantigens, with high TMB often indicating more potential neoantigens. TMB is frequently used as a biomarker for immunogenicity and as a criterion for immune checkpoint inhibitor (ICI) therapy.

3.2 How to deliver antigens?

The design of tumor vaccines not only relies on identifying effective antigens, but also needs to consider how to efficiently deliver these antigens into the body to trigger a specific immune response. The choice of delivery system is critical to improving the effectiveness of the vaccine. Among the many delivery platforms, polypeptide vaccines, microbial vectors, and nucleic acid vaccines (such as DNA or mRNA vaccines) each have their own unique advantages and limitations.

3.2.1 Polypeptide

Polypeptide vaccines generally refer to vaccines that use peptides or proteins as their active components, are one of the most direct forms of vaccines that mimic natural antigens. These can be further categorized based on how the antigens are produced and presented, such as peptide-based vaccines, recombinant protein vaccines and some of the subunit vaccines.

Peptide vaccines typically consist of purified short peptides that can be part of a tumor-associated antigen and can be presented to T cells and activate an immune response against a specific tumor cell. Despite their ability to activate CTLs, short peptides, such as those serving as CD8⁺ T cell epitopes, have a limited half-life. Research supports the notion that extended CTL peptides can increase the duration of antigen presentation and elevate the quality of the antigenic signal [367]. Peptide-based vaccines are primarily produced using chemical synthesis methods [368]. Synthetic peptide vaccines offer several benefits, including cell-free production, automated synthesis, and demonstrated clinical efficacy. However, they also pose challenges, such as the lack of clinical-grade manufacturability for many sequences, high variability in the physicochemical properties of longer peptides, and irrelevant immune responses to artificial epitopes generated by peptide degradation in the extracellular environment.

A significant limitation of peptide vaccines is their inefficient transport to lymph nodes following administration. In a study conducted by Mehta et al., the pharmacokinetics of peptide antigens conjugated to albumin were monitored. The results demonstrated that the use of carrier proteins significantly facilitated the transport of antigens from the injection site to the draining lymph nodes. This approach not only effectively mitigated antigen degradation but also minimized antigen presentation in uninflamed, distal lymphoid organs. Consequently, this enhancement in antigen delivery led to an overall improvement in T cell responses elicited by peptide vaccines [369].

With advancements in biotechnology, particularly in genetic engineering, recombinant protein vaccines have become an important branch of vaccine research, showing great potential in cancer therapy.

Recombinant protein vaccines involve the use of genetic engineering to clone key antigen genes from pathogens into suitable host cells, enabling the efficient production and purification of antigens for vaccine preparation. Common host cells include E. coli, yeast, insect cells, and mammalian cells [370]. Zare et al. developed a cGRP78-based vaccine using an E. coli expression system, which demonstrated a significant inhibitory effect against B16F10 melanoma cells [371].

Recombinant protein vaccines, when applied as tumor vaccines, can leverage genetic engineering methods to produce high-quality antigens on a large scale, facilitating industrial production. This approach makes it easier to control product quality and ensure consistency between batches. Additionally, different antigen proteins can be combined to form multivalent vaccines.

Despite the numerous advantages of recombinant protein vaccines, there are still several challenges that must be addressed. First, enhancing immunogenicity is a critical area of research. Often, antigens alone are insufficient to elicit a strong immune response, necessitating the use of appropriate adjuvants to boost efficacy. Secondly, optimizing expression systems to improve both the yield and quality of antigen proteins is also a key issue. Moreover, identifying suitable protective antigens remains a challenge for certain pathogens.

To tackle these challenges, scientists are continuously exploring new solutions. For example, using virus-like particles (VLPs) as carriers for delivering recombinant proteins can maintain the spatial conformation of antigens while mimicking the structure of natural viruses, thereby enhancing the immune response by facilitating cellular uptake and augment the interaction between antigens and immune cells [372]. Furthermore, advancements in nanotechnology offer new delivery platforms for recombinant protein vaccines, with the potential to further improve their stability and targeting capabilities. An innovative chimeric protein vaccine platform called KISIMA has self-adjuvanting properties, which integrates a ZEBRA-derived cell-penetrating peptide (CPP), a multiantigenic domain (Mad) with epitopes recognized by multiple MHC alleles, and a self-adjuvanting TLR2/4 agonist. This recombinant vaccine construct elicits robust CD8 and CD4 antigen-specific immune responses, leading to enhanced immunological memory and vaccine efficacy in preclinical mouse tumor models, while demonstrating safety and efficacy in nonhuman primates [373].

3.2.2 Microbial vectors

Viral vectors have the advantage of being recognized by the immune system naturally, inducing potent innate and adaptive responses. Traditionally, the viral vectors used to deliver tumor antigens include adenovirus vector [374], poxvirus vector [375], herpes simplex virus vector [376], adeno-associated virus vector [377], etc. Oncolytic viruses (OV) are a special class of viral vectors, which can dissolve tumors and promote the release and recognition of related antigens. At present, OV has been studied for a variety of tumor treatment through natural or engineered [378]. VSV-GP is an engineered viral vector based on vesicular stomatitis virus (VSV), pseudotyped with the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) [379]. This combination significantly reduces the neurotoxicity of the original virus while retaining its potent lytic activity and broad tumor tropism. When used as a vaccine platform, VSV-GP, by carrying specific target antigens, induces robust and durable CTL responses and antibody production in a homologous prime-boost strategy, thereby enhancing the immune memory against tumor antigens [373].

To effectively overcome tumor tolerance and the immunosuppressive microenvironment, vaccine therapies often require multiple boosting doses to induce a sustained and robust immune response. However, one limitation of viral vector-based vaccines is the generation of neutralizing antibodies against the vector. Pre-existing immunity can hinder subsequent responses, but this challenge has been addressed through the use of heterologous prime-boost strategies, where different viral vector platforms are employed for priming and boosting.

In both preclinical and clinical studies, a prime-boost approach combining a recombinant vaccinia virus vector for priming and a recombinant avipox virus, such as fowlpox (rF-), for boosting has shown success. Multiple boosting doses using recombinant avipox viruses have also proven feasible [380]. The TRICOM vaccine platform serves as a potent example of this strategy, utilizing a recombinant vaccinia virus (rV-) for the initial priming dose and a recombinant avipox virus (rF-) for subsequent booster vaccinations [381].

Another potential candidate carrier for anti-cancer treatment is bacteria, which are active in anaerobic environment and has tumor targeting properties. In a study of cancer vaccine combined with intravesical Bacillus-Calmette-Guérin (BCG) therapy, local BCG immune stimulation directed vaccine-specific T cells to the tumor site, taking advantage of the activation of Bacillus in the hypoxic conditions including tumor environment [382].

3.2.3 Nucleic acids

Compared with the above vaccines, nucleic acid vaccines may have more advantages in preparation time, which are worth considering for personalized vaccines. Nucleic acid vaccines deliver tumor antigens into the body in the form of nucleic acids, including DNA, RNA, circular RNA, etc. They can deliver multiple antigens simultaneously within the same construct.

DNA vaccines based on plasmids are straightforward to design, boasting relatively low production costs, excellent stability (they remain stable at temperatures between +2 °C to +8 °C), and good solubility. Moreover, they can be rapidly modified to adapt to new requirements [383]. A DNA vaccine platform known as ImmunoBody™ has been developed, in which CTL and helper T cell epitopes replace the complementarity-determining regions (CDRs) within the framework of a human IgG1 antibody. Upon immunization, these T cell epitopes are efficiently processed and presented, leading to robust helper and CTL responses at high frequencies [384]. Although no definitive DNA tumor vaccine has been fully approved for marketing, several candidates are actively undergoing clinical trials, such as INO-3107 for HPV-6 and/or HPV-11-associated recurrent respiratory papillomatosis [385].

However, numerous limitations persist in the clinical application of DNA vaccines. For DNA to effectively deliver antigens, it must overcome both extracellular and intracellular barriers to reach the cell nucleus, presenting a significant challenge. Physical methods, such as gene gun, electroporation and sonoporation, are commonly used for administration but pose difficulties in clinical implementation and scalability. With the development of nanotechnology, nanoparticles synthesized from new materials are aiming to deliver DNA based on convenient injection methods. Zhao et al. developed a DNA-engineered nanoparticle based on Ag2S quantum dot- and nanoliposome for tumor-targeted delivery. This system achieves an elevated T/Li ratio (up to approximately 5.1) in near-infrared-II (NIR-II) fluorescence imaging of mouse hepatic tumors [386].

mRNA vaccines deliver mRNA coding for TAAs directly into host cells, where it is translated into the corresponding proteins. Unlike DNA vaccines, mRNA does not require nuclear entry, simplifying the process and reducing the risk of integration into the host genome. The first clinical trials using RNA were conducted by Weide et al., who administered naked mRNA coding for TAAs intradermally [387,388]. Their trials demonstrated that the vaccinations induced CD8⁺ and CD4⁺ immune responses in patients, as analyzed by IFN-γ enzyme-linked immunospot (ELISpot) and chromium-release assays [388].

The emergency use authorization of two mRNA COVID-19 vaccines has brought mRNA vaccines back into the spotlight. However, mRNA is inherently unstable and requires meticulous handling and storage. Mature eukaryotic mRNAs include a 5′-cap, a coding region, UTR, and a 3′ terminal poly A tail [389]. Furthermore, the modification of mRNA and the design of its regulatory and coding sequences such as enhancing GC content and introducing pseudouridine are critical factors that determine its stability and translation efficiency [390–392]. Various materials for mRNA delivery have been developed, including protein derivatives, polymers, lipids and lipid-like materials [393]. Notably, lipid nanoparticles (LNPs) have been extensively studied and have successfully advanced into clinical use [394,395].

Autogene cevumeran (BNT122) is a personalized cancer therapy based on mRNA vaccine technology. When combined with the anti-PD-L1 antibody atezolizumab, personalized RNA neoantigen vaccines against pancreatic cancer have elicited neoantigen-specific T cell responses in 8 out of 16 patients (50%) [396]. A recent study published in Cell reported a novel mRNA cancer vaccine, RNA-LPAs. Using mRNA as a molecular bridge with cationic liposomes results in the formation of self-assembling lamellar aggregates that mimic infectious emboli. Systemically administered RNA-LPAs activate RIG-I in stromal cells, triggering a robust cytokine and chemokine response and enhancing the mobilization of dendritic cells and lymphocytes. In human trials, this approach has shown significant efficacy against glioblastoma [397].

Circular RNAs (circRNAs) are typically generated through reverse splicing, and their covalently closed loop structure protects them from exonuclease degradation, providing greater stability compared to linear mRNAs. Novel vaccines based on circRNAs are emerging as a promising field. Wang et al. utilized the permuted intron-exon (PIE) strategy [398] to generate circRNAs containing functional IRES elements and antigen-coding sequences. The results indicated that the antigen expression levels from this vaccine were higher and more durable than those achieved with mRNA vaccines, ultimately demonstrating superior antitumor immune effects in HCC model [399].

3.3 Enhancing vaccine efficacy by adjuvants

Adjuvants are essential components in the development of effective tumor vaccines, designed to optimize the activation of APCs and subsequent stimulation of T cell immunity. The success of tumor vaccines is heavily dependent on the ability of adjuvants to enhance the functionality of APCs, particularly DCs, and to promote a robust T cell response against cancer cells. These adjuvants operate via several mechanisms, including engagement with Toll-like receptors (TLRs), induction of inflammation, and direct activation of T cells.

For instance, TLR agonists, such as CpG oligodeoxynucleotides (CpG ODNs), have been extensively tested in clinical trials for various cancers, including melanoma, breast, and glioblastoma [400]. CpG ODNs, which are recognized by TLR9 on DCs, enhance DC maturation and antigen presentation, thereby boosting the immune response against tumors. Similarly, imiquimod (R837) [401] and resiquimod (R848) [402], which are agonists of TLR7 and TLR8 respectively, have shown promise in clinical applications. Imiquimod is currently approved for treating genital warts and basal cell carcinoma, demonstrating its efficacy in stimulating a Th1-type immune response characterized by the production of IL-12, IFN-γ, and TNF-α.

Inflammatory modulators, such as aluminum salts (Alum), are utilized to promote inflammation, which aids in recruiting and activating immune cells [403]. While Alum predominantly induces Th2-type responses, which are less favorable for cancer vaccines, formulations like AS04, combining aluminum salts with MPL (a detoxified form of lipopolysaccharide), have proven effective in evoking a Th1-type response and producing INF-γ [404]. This combination has been shown to enhance the activation of cytotoxic T lymphocytes (CTLs), a crucial aspect of tumor immunotherapy.

Another notable example is the use of cytokine-based adjuvants. GM-CSF, when used as a vaccine adjuvant, has been tested in clinical trials for anti-tumor immunotherapy in lung, skin, prostate and breast cancers [405]. These trials have shown that GM-CSF supports the expansion and activation of T cells, promoting their migration to lymph nodes and tumor locations. Additionally, combinations of cytokines, such as IL-2 and TLR7 agonists, have been explored to boost CD8⁺ T cell responses and improve anti-tumor effects.

Innovative approaches involving nanomaterials have also emerged as promising adjuvant platforms. For example, multifunctional adjuvant platforms based on two-dimensional nanomaterials, like black phosphorus nanosheets (BPs), have been designed to provide both efficient antigen codelivery and potent immune activation. Studies have demonstrated that these nanomaterials can interact with APCs, triggering pro-inflammatory responses and contributing to the generation of antigen-specific antitumor immune responses. The integration of these nanomaterials with other adjuvant strategies, such as the inclusion of STING agonists, has further enhanced their efficacy in preclinical models [406].

In conclusion, the use of adjuvants tailored to the specific needs of tumor vaccines has become increasingly sophisticated, incorporating a range of mechanisms designed to maximize the immune response against cancer. These adjuvants not only aid in the activation of APCs but also support the priming and proliferation of T cells, ultimately leading to more effective tumor control.

4 Combination with cancer-immunity cycle

The purpose of therapeutic cancer vaccines is to suppress tumor growth and induce regression of established tumors by administration of sufficient quantities of high-quality tumor antigens along with adjuvants to DCs, or by vaccinating with autologous DCs loaded with tumor antigens to activate tumor-specific adaptive immune responses [407]. However, as single-agent therapies, the therapeutic efficacy of cancer vaccines is limited by species of tumor antigens and adjuvants, platform design, administration route, and effective production, especially tumor-induced immunosuppressive microenvironment, which lead to immune cells dysfunction and exhaustion [408]. Considering that substantial factors affect the antitumor efficacy of cancer vaccines, various studies have investigated combination therapy approaches. Next, we introduce that how cancer vaccines can be combined with other immunotherapies to achieve a successful and durable antitumor immune response. We focus on five key steps in the cancer-immunity cycle: the release of cancer cell antigens, the presentation of these antigens, the priming and activation of APCs and T cell, the infiltration of T cell into the tumor and surrounding stroma, and the recognition and killing of cancer cell by T cell [220].

4.1 Release of cancer cell antigen

Tumor antigen release can be enhanced using oncolytic viruses, radiotherapy, and chemotherapy. Combining these methods with cancer vaccines can significantly improve antitumor efficacy. By killing host cancer cells, localized oncolytic virus treatment can induce the release of tumor antigens along with DAMPs and PAMPs. This creates a pro-immune cytokine environment that enhances the function of APCs, leading to the priming of tumor-specific T cell responses [409]. Treating mice bearing B16-GP33 tumors with the oncolytic virus rVSV-LCMVG combined with gp33 peptide-expressing mRNA vaccines significantly inhibited tumor growth [410]. In addition, alphavirus M1 (OVM) can strongly promote the antitumor efficacy of a DC-based vaccine-sipuleucel-T against numerous mouse tumor models [411].

Chemotherapy can also trigger immunogenic cell death, thereby activating the adaptive immune response and alleviating tumor-mediated immunosuppression to achieve rapid tumor debulking [412]. A series of studies have shown that combining chemotherapy with therapeutic cancer vaccines has several potential advantages. IFNα-DC-based vaccine plus lenalidomide was more effective than single agent treatment, leading to marked eradication of established lymphoma tumor and decreased tumor regrowth in mouse model [413]. In 2022, a phase Ib clinical trial (NCT03380871) evaluated the neoantigen cancer vaccine NEO-PV-01 in combination with pemetrexed, carboplatin, and pembrolizumab for advanced non-squamous non-small cell lung cancer. After vaccination, specific CD4⁺ T cells and CD8⁺ T cells responses against non-vaccinating neoantigens were detected, including KRAS, G12C, and G12V mutations. Furthermore, infiltration of CD4⁺ T cells into tumor site was increased [414].

Radiotherapy induces abundant DNA damage, which leads to mutations, tumor cell death, and neoantigen formation. One study showed that radiotherapy combined with a DC vaccine triggers the specific immune responses in patients with esophageal cancer. After vaccination, the levels of Th1 cytokines and CD8⁺ T cells were significantly elevated compared to both baseline levels and the control group [415]. In another study, a combination therapy of radiotherapy and FAPα-based cancer vaccine suppressed tumor growth in 4T1 tumor mouse model. The promising results are associated with the inhibition of extracellular matrix production, tumor vascularization, and lymphangiogenesis, as well as enhanced activation of CD8⁺ T cells and reprogramming of immunosuppressive cells [416].

Nano delivery systems can be used to enhance the release of tumor antigens. In addition to utilizing the mechanism of tumor cells themselves to promote antigen release, there are also exogenous methods that can achieve the same effect. Some chemotherapy drugs, physical methods, and biomolecules have been shown to induce tumor cell lysis and release antigens, but using them alone cannot achieve the expected effect. Combining them with nano delivery technology can significantly improve their efficacy in promoting tumor antigen release. The delivery system based on nanoparticles can improve the accumulation of chemotherapy drugs in tumor tissues by enhancing permeability and retention effects [417]. Nanoparticles modified with PEG can increase the circulation time of drugs in the blood, while nanoparticles bound to targeted ligands can improve tumor specific delivery and tumor antigen release [418,419]. For physical therapy, nanoparticles can serve as sensitizers to enhance the release of tumor antigens and also increase the specificity of physical therapy. The use of nanoparticles to deliver biomolecules can to some extent alleviate the body’s immune response to biomolecules and improve their ability to lyse tumor cells.

4.2 Presentation of cancer cell antigen

Immune responses exert selective pressures on tumor cells, leading to the survival and proliferation of variants with diminished immunogenicity. Given that the variant tumor cell experiencing the high mitotic rate and genetic instability, it is common that the genes of encoding tumor antigen mutate or delete. Besides the loss of tumor specific antigens, numerous tumor cells downregulate the expression of class I MHC or suppress the process of antigen presentation, which allows tumor cells to evade CTL-mediated immune responses. To activate tumor specific CTL immune responses, providing sufficient numbers of cancer cell antigen to APCs and stimulating presentation of cancer cell antigen are very important strategy. Therapeutic cancer vaccines are usually designed to achieve this purpose. Furthermore, APC-mediated cancer cell antigen processing and presentation can be improved by agonists of pattern recognition receptors, which is always used as cancer vaccine adjuvant [420]. For example, a phase I clinical study investigated the antitumor efficacy of recombinant NY-ESO-1 protein combined with the TLR9 agonist CpG as an adjuvant. After combination therapy, a specific CD8⁺ T cell response was induced in nine patients, five of whom did not express NY-ESO-1 in their tumors [421]. TLR3-agonist Poly-ICLC is also widely used as cancer vaccine adjuvant, a phase I/Ib study confirmed that personalized neoantigen vaccines admixed with poly-ICLC can change the immune milieu of glioblastoma through promoting migration of neoantigen-specific T cells. And neoantigen specific CD4⁺ T cells and CD8⁺ T cells had memory phenotype [422]. Apart from TLR agonist, activating CD40 pathway in APCs can promote presentation of cancer cell antigen, PDAC tumor bearing mice received FGK45 (CD40 agonist) therapy after DC vaccination which loaded with either pancreatic cancer or mesothelioma lysate. The results showed that the combination therapy induced significant changes in the tumor transcriptome and alleviated the expression of inhibitory markers on CD8⁺ T cells [423].

4.3 Priming and activation of APCs and T cell

Besides antigen-induced signals, the differentiation of naïve T cells into effector T cells requires additional signals provided by co-stimulatory molecules on APCs. In cancer immunotherapy, agents that stimulate co-stimulatory molecules are used to enhance the activation of APCs, thereby improving the priming of effector T cells [412]. For example, targeting ICOS, ICOSL, 4-1BB, OX40, CD40, and CD40L with agonist antibodies can promote the priming and activation of APCs and T cells. Nevertheless, the outcome of T cell activation is influenced by the expression of co-inhibitory receptors CTLA-4 and PD-1, which are upregulated on T cells during stimulation to prevent overstimulation. Tumors often avoid detection and attack by T cells through the engagement of inhibitory molecules. These molecules typically serve to prevent autoimmune reactions and modulate immune responses to pathogens. Thus, immune checkpoint therapy (ICT) is designed to block inhibitory signals or facilitate stimulatory signals of T cells activation to promote T cells anti-tumor immune responses.

Several studies have indicated that the therapeutic efficacy of cancer vaccines can be augmented by combining them with immune checkpoint therapy. For instance, in a phase II clinical trial, the human papillomavirus 16 (HPV-16) vaccine ISA101 combined with the anti-PD-1 antibody nivolumab, achieved an overall response rate of 33%. This compares favorably with response rates of 16% to 22% observed with PD-1 inhibition alone in similar patient groups (NCT02426892) [424]. Similar results were observed in a phase 1b Trial (NCT02897765) involving personalized neoantigen therapy combined with nivolumab. The combination was shown to be safe, and neoantigen-specific CD4⁺ and CD8⁺ T cell responses were detected in all patients following vaccination [425]. In addition, a variety of studies are investigating combination therapy approaches between mRNA cancer vaccines and immune checkpoint inhibitors. Melanoma FixVac (BNT111), encoding four tumor-associated antigens, NY-ESO-1, tyrosinase, TPTE, and MAGE-A3, is undergoing clinical testing in adults. In a phase I trial (NCT02410733), the data indicated that the combination therapy induced a robust CD4⁺ and CD8⁺ T cell responses [426]. mRNA-4157 is an mRNA-based personalized neoantigen vaccine. When combined with pembrolizumab, it has been shown to enhance survival in patients with cutaneous melanoma [427].

4.4 Infiltration of T cells into tumor and stroma

Once activated antigen specific CTLs in tumor-draining lymph nodes migrate into tumors, where they can trigger apoptosis of the tumor cell by delivering cytotoxic proteins (perforins and granzymes) to target cells or through membrane protein Fas ligand and Fas interaction. Sufficient population of TILs is beneficial to the prognosis of tumor patients and may sever as a predictive biomarker of response to a variety of immunotherapy [428]. CTLs trafficking into tumor and stroma involves vascular angiogenesis and expression of chemokines and cognate chemokine receptors.

For instance, the tumor microenvironment produces specific chemokines to recruit immune cells, such as CXCL9, CXCL10, CCL2, CCL5, and so on. And immune cells express different combinations of chemokine receptors to bind to blood vessels and migrate into tumor site. One study showed that chemokine CCL11-fused E6/E7 nucleic acid vaccine improved the humoral and cellular immune responses and induced potent antitumor efficacy in TC-1 mouse models. Further analyses demonstrated that CCL11-E6/E7 treatment not only stimulated infiltration of T cells, dendritic cells, macrophages and neutrophils, but also raised the amount of stem-like CD8⁺ T cells [429].

In addition, the vasculature is vital for immune cells to infiltrate target tissue. One of hallmark of cancer is aberrant angiogenesis, and it is associated with immunosuppression and immune evasion. Therefore, normalizing of abnormal tumor vasculature is beneficial to the infiltration of CTLs. VEGF is a crucial factor for promoting vascular permeability and angiogenesis. A series of studies have demonstrated that anti-VEGF antibody or tyrosine kinase inhibitors (TKIs) targeting VEGF can normalize tumor vessels, increase T cell infiltration, and decrease tumor hypoxia [430]. Thereby, VEGF blocking combined with cancer vaccines is potential strategy for facilitating antitumor efficacy. A randomized phase 2 study (NCT02432846) has evaluated DC vaccine ilixadencel plus tyrosine kinase inhibitor sunitinib in patients with synchronous metastatic renal cell carcinoma (mRCC). The patients that received treatment with ilixadencel followed by sunitinib after nephrectomy exhibited a trend of improved objective response rate (42.2%) and overall survival (54%), compared with sunitinib alone after nephrectomy [431].

4.5 Recognition and killing of cancer cells by T cells

The primary mechanism of immune system against cancer is that tumor cells are killed by CD8⁺ CTLs. To achieve efficient killing, T cells can be isolated from patient’s blood or solid tumor, genetically modified by loading specific tumor antigen receptors (or not be modified) and expanded to large numbers through cytokines (IL2, IL7, IL15) in vitro, and then reinforced back into the patient to directly eradicate tumor. The approach is called adoptive cellular transfer (ACT) immunotherapy. To achieve increased therapeutic efficacy, especially in solid tumors, several preclinical studies about ACT immunotherapy combination with therapeutic cancer vaccines are on the rise.

A personalized DC vaccine loaded with oxidized autologous whole-tumor lysate (OCDC) plus bevacizumab has elicited T cell responses and mutated neoepitopes in patients with recurrent ovarian cancer [432]. In the follow-on phase 1 clinical trial (NCT01312376/UPCC26810), peripheral blood T cells were collected from 17 patients vaccinated with OCDC during NCT01132014 trial, and were expanded their numbers with IL2 plus anti-CD3/CDC28 beads in vitro, T cells then were reinfused back into patients subsequently followed by personalized OCDC vaccine and bevacizumab. The combination treatment was safe, and epitope spreading with novel neopeptide reactivities was observed in ovarian cancer patients [433].

CAR-T cell therapy has shown significant antitumor efficacy in patients with various hematologic malignancies. However, its success in treating solid tumors has been limited due to the heterogeneity and loss of tumor antigens. Several studies have shown that cancer vaccines can enhance the tumor-killing effects of CAR-T cells and lead to antigen spreading (AS), which induces an endogenous T cell immune response against additional tumor antigens beyond the original therapeutic targets [434]. For instance, one combination therapy approach that enhanced CLDN6-CAR-T function against solid tumor by directly delivering CAR antigen into lymphoid compartments through a nanoparticulate RNA vaccine (CARVac), induced profound expansion and activated phenotype of CAR-T cells in mouse model [435]. In the subsequently ongoing phase 1/2 BNT211-01 clinical trial (NCT04503278), CLDN6-CAR-T therapy combination with CAR-amplifying vaccine exhibited 67% disease control rate, with 14 out of 21 patients achieving control, and seven patients experiencing stable disease [417]. In another study, an amph-ligand-based vaccine, which is comprised of a hydrophobic phospholipid tail link to an mEGFRvIII-derived peptide epitope via a poly spacer, co-injected with vaccine adjuvant cyclic di-GMP after mEGFRvIII-CAR-T cells administration. The amph ligand that binds to albumin migrated to draining lymph nodes, and then transferred to the surface of APCs. Additionally, DCs were activated by co-administered cyclic di-GMP. This activation led to the initiation of mEGFRvIII-CAR-T cell priming by ligand-decorated DCs within the native lymph node microenvironment. The use of an amph-ligand vaccine booster promoted the effector function and expansion of CAR-T cells. Further analysis revealed that treatment with the vaccine-boosted CAR-T cells enhanced the production of IFN-γ, a key factor in antigen spreading [418].

TCR-T immunotherapy has also demonstrated promising prospect for treatment of various cancers. However, the benefit to patients has been limited, especially in the case of solid tumors. Several studies showed that combination with cancer vaccine may be essential to enhance TCR-T therapy clinical success. In a phase 1 trial, the antitumor efficacy of combining autologous TCR-T cells, which specifically recognize NY-ESO-1 in conjunction with the HLA-A02:01 or A02:06 complex, with a pullulan nanogel:LPA vaccine containing NY-ESO-1 peptides was evaluated in three patients. The results showed that two patients developed cytokine release syndrome and one patient achieved durable persistence of TCR-T cells leading to long-lasting (more than 2 years) tumor shrinkage [419]. Furthermore, combining amphiphile (AMP) vaccine including gp100 peptides and CpG adjuvant with Pmel-1 TCR T cells specific for gp10025-33 enhanced antitumor efficacy against B16F10 tumor in mouse model. Compared with TCR-T cells treatment alone or control, tumor growth was significantly delayed and survival was prolonged. In addition, the AMP-based vaccine combined with human TCR-T cells specific to NY-ESO-1, mutated KRAS, or HPV16 E7 was demonstrated to boost the expansion, activation, and tumor-killing capabilities of TCR-T cells in vitro [436].

5 Challenges from body’s homeostasis stress and imbalance

5.1 Aging and therapeutic cancer vaccines

Aging is associated with cancer induction and progression. The incidence of most cancers rises sharply with age [437]. Those aged 70 years and over account for about half of all cancer-related deaths, while another 40% occur in people aged between 50 and 70 years [438]. Aging is characterized as a steady functional deterioration over time, and it has many characteristics with tumors, such as increased cellular senescence, telomere attrition, genomic instability, and heightened inflammation. Tumor formation is strongly correlated with age, particularly immunosenescence. Meanwhile, a number of components inside the TME can cause immune cells to undergo senescence and have a significant impact on how they function [439]. Studying how the aging TME affects cancer development and the effectiveness of therapeutic vaccines is essential.

5.1.1 Degeneration of thymus and lymph node

One of the hallmarks of immunosenescence is thymic involution, which causes the imbalance of immune cell proportions. The thymic epithelial space (TES), which supports the development of thymocytes, decreases with advancing age, benefiting the perivascular space (PVS) that contains adipocytes [440,441]. This process of thymic involution results in a reduction of naïve T cells, triggers a compensatory clonal expansion of memory T cells in peripheral tissues, and limits the migration of naïve T cells to the periphery [440,441]. Similar to thymic involution, lymph node degeneration impairs the homeostasis of naïve T cells and B cells, manifesting as disturbances in the structure and organization of nodal zones, an increased degree of fibrosis and lipomatosis, and a loss of lymphocytes and high endothelial venules (HEVs) [442,443]. With aging and degeneration of thymus and lymph node, diversity of TCR repertoire decreases, impairing effectiveness of therapeutic cancer vaccines. A higher diversity repertoire is essential for an effective immune response, as it provides the flexible TCR reserve to meet the challenge of clearing pathogens and their various antigenic variants, especially tumor cells that carry many neoantigens [444]. Better survivability in a number of malignancy histotypes has been linked to high TCR diversity in the tumor-infiltrating T cells [445].

To recover an effective immune response, it is crucial to enhance thymic output and increase T cell repertoire diversity to reverse aging. To achieve this goal, clinical trials have employed hormones, cytokine, and growth factors therapy with promising results, such as IL-7, growth hormone, dehydroepiandrosterone, and so on [446,447]. In aged mice, a study has found IL-7 and GM-CSF fusion cytokine can reverse aging-related lymphoid organ atrophy, as adjuvant for a tumor vaccine can increase TCR productive frequency and generate durable long-term antitumor immunity specifically [448]. These findings could help in the design of therapeutic cancer vaccines.

5.1.2 Priming and activation

Lymph nodes are key locations for cancer antigen presentation, T cell priming, and activation. However, lymph node degeneration not only impairs T cell homeostasis, but also negatively impacts the migration and recruitment of immune cells, intercellular interactions, and response of T and B cells [443]. For example, a reduced amount of germinal centers (GCs), follicular dendritic cells, and T follicular helper cells affects co-stimulatory, survival, and differentiation signals to B cells within the GCs, leading to the age-related fall in vaccination effectiveness [449].

Dendritic cells (DCs) are professional antigen-presenting cells that drive T cell responses. DCs’ abilities are weakened with age, contributing to decreased vaccination responsiveness in older adults [450]. DCs from older individuals show a notably diminished ability to phagocytose antigens through macropinocytosis and endocytosis and impaired migration capacity [451]. In an in vitro activation experiment, youthful CD8⁺ T cells showed lower activation when mixed with DCs from older individuals. These T cells exhibit low proliferation and production of IL-12 and IFN-γ [452]. These alterations may contribute to the decreased vaccine responses among the elderly population. Furthermore, vaccines consisted of tumor antigens and DC hyperactivators (LPS and an oxidized phospholipid known as PGPC) increased the production of cytotoxic CD4⁺ T cells, enhancing immune response against tumors in aged mice [453].

Cytotoxic T cells serve as critical effector cells in tumor immunity; consequently, investigating the effects of aging on T cell priming and activation is essential. Hallmarks of T cell aging, in addition to thymic involution, naïve-memory imbalance, and TCR repertoire reduction mentioned above, also include cellular intrinsic aging characteristics such as genetic and epigenetic alterations, loss of proteostasis, mitochondrial dysfunction, and the accumulation of senescent T cells [454]. We shall initially address how aging-related cellular intrinsic characteristics influence the activation of naïve T cells, and subsequently delve into the impact of senescent T cells on tumor immune escape and the efficacy of cancer therapies within aging TME. Naïve T cell aging showing partial differentiation into memory and effector T cells, such as miRNA expression pattern changes and expression of CD25 and CD95, may have detrimental consequences of their function [455]. Using an in vitro experiment, a study found that CD8⁺ T cells from older individuals show reduced T cell priming effectiveness [456]. Another study has shown that decreased miR-181a expression and lower TCR sensitivity to stimulation are caused by the aging-related decrease in transcription factor YY1 expression [457].

Targeting to reverse cellular senescence and induce efficient T cell priming and activation, is an ideal approach for enhancing therapeutic cancer vaccine response. Senescent T cells fail to proliferate in response to TCR stimulation because of the loss of several critical components of the TCR signaling pathway. The proliferative impairment of senescent T cells was restored by blocking the AMPK-TAB1-dependent activation of p38 [458]. Moreover, the knockout of sestrins, which bind to and coordinate the simultaneous activation of Erk, Jnk, and p38 MAPK, enhances responsiveness to vaccination in older mice by promoting the expansion of CD62L⁻CD44⁺ T effector cells [459].

5.1.3 Immune cell trafficking and infiltration

Fibroblasts are the most common stromal cell type in tissues. In aged tissue microenvironments, there is an accumulation of senescent fibroblasts, along with a shift toward more immunosuppressive cells, including MDSCs and Tregs, compared to younger tissue microenvironments [460]. Inhibiting the ROS-producing enzyme nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4), which is expressed by cancer-associated fibroblasts, can promote the infiltration of CD8⁺ T cells and enhance the tumor’s responsiveness to immune checkpoint blockade therapy [461]. This study can also provide insights for the design of tumor vaccines.

The extracellular matrix (ECM) functions as a structural scaffold that facilitates the specific trafficking of cells across the body, which would remodel and matrix stiffen in the local microenvironment as we age [460]. A study demonstrated that the loss of ECM remodelling protein hyaluronan and proteoglycan link protein 1 (HAPLN1) in aged fibroblasts leads to a more aligned ECM, promoting the metastasis of melanoma cells. Decreased HAPLN1 levels inhibit the migration and infiltration of effector CD8⁺ T cells into the tumor, while promoting the infiltration of Treg cells [462].

5.1.4 Cancer cell killing within aging environment

The cytotoxic activity of effector cells against tumors is the most critical component of the immune response elicited by therapeutic vaccines. Undoubtedly, this process is subject to dual inhibition by both the aging microenvironment and the tumor microenvironment. Cancer cells orchestrate an immunosuppressive inflammatory TME to facilitate immune escape [463]. During aging, this inflammatory and immunosuppressive microenvironment is exacerbated, manifesting as a senescence-associated secretory phenotype (SASP) unfavorable to immune responses and an increase in the proportion of various immunosuppressive cells, such as MDSCs, Tregs, M2 macrophages, and N2 neutrophils [460]. Additionally, cellular senescence of effector cells induced by aging TME directly impair their response of vaccine. We will now discuss these changes and how they influence tumor progression and the efficacy of vaccine treatments.

“Inflammaging,” characterized by systemic low-grade chronic inflammation, is a defining hallmark of aging [441,464]. Inflammaging can arise from multiple factors, including the accumulation of proinflammatory tissue damage, impaired clearance of pathogens, and the persistence of senescent host cells [464]. Senescent cells significantly enhance the secretion of proinflammatory cytokines, chemokines, growth factors, and proteases; this secretory profile is referred to as the SASP [465]. Concurrently, chronic inflammation facilitates immune cell senescence, which results in weakened immunity and an incapacity to remove inflammatory agents and senescent cells. The downward spiral of inflammation and cellular senescence is therefore maintained [466]. Furthermore, certain cytokines involved in chronic inflammation can help to boost the amount of immunosuppressive MDSCs [467] and Treg cells [468]. Blocking inflammation may delay aging and enhance the effectiveness of tumor vaccines [469,470]. Inhibition of mTOR signaling, which is involved in many inflammatory processes, has demonstrated the ability to prolong lifespan and put off the invention of age-related illnesses. Using the mTOR inhibitor RAD001 improved immunosenescence in older adults, as it boosted the response to the influenza vaccine and decreased the proportion of CD4 and CD8 T cells expressing the PD-1 receptor [471].

With the rise of proinflammatory cytokines essential for the differentiation of MDSCs, such as TNF-α, IL-6, and IL-1β, the number of MDSCs increases with age [472]. MDSCs secrete arginase 1 (ARG1), TGF-β, and reactive oxygen species (ROS) to suppress adaptive immune responses [473]. An example used the melanoma model and demonstrated that MDSC are accumulated in skin tumors and metastatic LNs, along with increased levels of inflammatory factors such as IL-1β, GM-CSF, and IFN-γ. In vitro coculture experiments demonstrate that tumor-derived MDSCs suppress T cell proliferation [474]. Oral treatment of Lentinula edodes mycelia extract improves anticancer vaccination effectiveness in elderly mice by suppressing IL-6 and TNF-α levels and lowering MDSCs [475].

Tregs are another critical cell population implicated in tumor immune escape. However, alterations in Treg cell numbers and function in age-related immune dysfunction show inconsistent patterns [460]. The impact of aging on Treg functions is context-dependent and varies according to the specific function investigated [476]. For example, more Tregs were found in the spleen and lymph nodes of older animals, depletion of which by anti-CD25 mAb could induce tumor rejection in aged mice and restore the activity of cytotoxic T cells [477]. However, another study demonstrated that depleting Tregs did not delay tumor growth or enhance tumor-specific immunity, due to the significant increase in MDSC numbers following Treg reduction in old, but not in youthful mice [478]. Disturbing both Tregs and MDSCs was more immunologically and clinically effective in aged mice, similar to the effect of Treg depletion alone in young mice [478]. A clinical trial data set indicates that low Treg ratios both before and after vaccination in elderly patients (median age 61.5 years) were associated with a good prognosis [479]. In TC1 tumor-bearing mice, depletion of Treg cells can enhance antitumor activity of vaccine [480].

With advancing age, neutrophils and macrophages typically undergo immunosuppressive N2 [481] and M2 [482] states, respectively. Aged mice contained significantly more splenic IL-10 secreting M2-macrophages than young mice. Macrophages from aged mice produced high levels of IL-4 and could inhibit T cell secretion of IFN-γ. IL-2/CD40-stimulated macrophages restored IFN-γ production by T cells derived from elderly mice [483]. A study [484] employed a Listeria-based hepatocellular carcinoma vaccine that altered the phenotype of tumor-associated macrophages from an M2 to an M1 polarization state. This shift was accompanied by a transformation of the cytokine profile within the TME to one that is more antagonistic to tumor growth. The vaccine was used in conjunction with anti-PD-1 therapy to recover T cell responsiveness.

Patients with various forms of tumors have a higher number of senescent CD8⁺ T cells in tumor infiltrating lymphocytes, metastatic satellite lymph nodes, and peripheral blood [439]. In addition to accumulating with age, senescent T cells can be induced by the TME [485]. Glucose deprivation in the TME can induces telomere erosion, DNA damage, and cell cycle arrest of T cells through MAPK p38 regulatory network [439,458]. The TME-induced reduction of CD28 and TCR signaling can stimulate the MAPK p38 pathway, which in turn prevents autophagy and causes mitochondrial dysfunction in addition to raising ROS levels in senescent T cells [439,459,486]. Senescent T cells display aberrant phenotypes, characterized by the downregulation of costimulatory molecules CD27 and CD28, the upregulation of terminal-differentiation markers, such as CD57 and KLRG1, and the upregulation of immune checkpoint-related molecules, such as Tim-3, TIGIT, and CTLA-4, resulted in the loss of their capacity for antitumor immunity [487]. T cells’ cytotoxic activity declines as they age, as does the generation of cytotoxic molecules, such as granzyme B and perforin [487,488]. Blocking p38 MAPK signaling [454] and utilizing nutraceuticals [489] can delay or reverse T cell senescence. Moreover, activating TLR8 signaling through downregulating cAMP in cancer cells can inhibit the development of senescent T cells and reverse their suppressive effects [485]. These approaches can provide insights for therapeutic tumor vaccines. In addition, vaccines targeting antigens of senescent cell to eliminate senescent T cells may represent a feasible approach to promote tumor clearing. Indeed, a study reported the creation of a vaccine utilizing a CD153 epitope, which increases anti-CD153 antibody levels and prevents the accumulation of CD153⁺ senescent T cells in mice [490].

In summary, the aging TME and cellular senescence collectively influence the activation, proliferation, migration, infiltration, and cytotoxic function of vaccine-induced effector cells. Naïve-memory imbalance and reduced TCR diversity result in the disproportionate expansion of T cells, posing a challenge in generating a diverse repertoire of vaccine-responsive tumor-specific clones, particularly in creating neoantigen-specific T cells. The aging TME is infiltrated by more immunosuppressive cells, which not only directly inhibit the cytotoxic function of effector cells but also further promote cellular senescence and dysfunction. Additionally, the aging TME disrupts cDC1-CD8⁺ T cell cross-talk and impairs the priming and expansion of CD8⁺ T cells. Therefore, facilitating effective T cell responses and cytotoxicity in the aging TME is vital for the innovation of tumor vaccines.

5.2 Myelosuppression and therapeutic cancer vaccines

5.2.1 Symptoms of myelosuppression

Myelosuppression, also known as bone marrow suppression, is one of the manifestations of homeostatic imbalance that cancer patients often face. The main causes of myelosuppression are radiation therapy and chemotherapy. In addition, some cancers can directly affect bone marrow function, including leukemia, lymphoma, myeloma, and neuroblastoma [491–493]. Myelosuppression is defined as a decrease in bone marrow activity, which leads to a drop in the amount of red blood cells, white blood cells, and platelets [494,495]. Patients with myelosuppression may experience a range of serious complications, such as anemia, bleeding disorders, immunodeficiency, and potentially life-threatening infections [494,495]. These adverse effects can directly impact the effectiveness of treatments, including vaccines, and may even lead to treatment interruption.

5.2.2 Mechanism of myelosuppression

Chemotherapy medications are designed to aggressively expand cells. Besides tumor cells that exhibit malignant proliferation, bone marrow also contains rapidly renewing cells. Chemotherapy inhibits the proliferation and function of these cells through restraining cell replication, disrupting the cell cycle, affecting the bone marrow microenvironment, or altering hematopoietic regulatory factors [496]. Therefore, almost all chemotherapeutic medications are thought to have myelosuppressive effects; the main distinction between them is how severe they are.

Cancer induced inflammation not only promotes tumor progression, but also can play a major role in myelosuppression [491,497]. Moreover, gangliosides derived from neuroblastoma suppress the functional activity of T and NK cells, contributing to tumor induced bone marrow suppression and causing multiple alterations in hematopoiesis [493]. Mechanically, ganglioside exposure decreased TLR dependent p38 signaling of DCs and impaired DCs stimulatory potential of CD8⁺ T cells [498]. Both inflammation and the deficiency or dysfunction of APCs and T cells can disrupt immune homeostasis and impair tumor suppression, thus affecting the efficacy of immunotherapies. Below, we will discuss methods that may reverse bone marrow suppression, as well as combination vaccine therapies that hold promise for reducing or even eliminating chemotherapy-induced bone marrow suppression.

5.2.3 Viable vaccine therapies

Many cytokines and small molecule drugs have been used to alleviate bone marrow suppression with some success, providing insights for the creation of tumor vaccines. A clinical trial has shown that prophylactic use of granulocyte colony-stimulating factor (G-CSF) significantly reduces the incidence of febrile neutropenia (FN) following systemic chemotherapy [499]. Another study demonstrated that treatment with recombinant human growth hormone mitigates the effects of lethal irradiation and increases the frequency of hematopoietic stem/progenitor cells and CD4⁺ and CD8⁺ T cell subsets in mice and nonhuman primates [500]. Moreover, the combination of neutral polysaccharide with cyclophosphamide (one of the chemotherapy drugs) ameliorated the immunosuppression and myelosuppression caused by cyclophosphamide in H22 tumor-bearing mice, as demonstrated by increased proliferation of T lymphocytes, higher serum hemolysin antibody content, and enhanced NK cell activity [501].

The above studies suggest that improving vaccine design can facilitate the combined use of vaccines with chemotherapy or radiation therapy, thereby maintaining therapeutic efficacy while reducing the side effects of bone marrow suppression. Elderly individuals with esophageal cancer were treated in a clinical study using intensity-modulated radiation treatment in combination with adoptive cytokine induced killer cell and DCs therapy. This combination therapy could decrease bone marrow suppression and extend survival duration and boost T and NK cell counts [502].

6 Clinical development of cancer therapeutic vaccines

As of August 2024, our database focuses on 281 ongoing therapeutic cancer vaccine clinical trials. We categorize cancer vaccines into 20 groups by tumor type, including lung, liver, bladder and breast cancers, among others, and then track the number of clinical trials from early phase I through phase IV. Most oncology vaccine clinical trials are in early phases (phase I/II), whereas fewer have advanced to later phases (phase III/IV). The primary focus areas are solid tumor vaccines (33 trials), breast cancer (37 trials), pancreatic cancer (29 trials), lung cancer (27 trials), and brain tumors (33 trials) (Fig.3).

Neoantigens are tumor-specific and usually not expressed in normal tissues, divided into shared antigens which are common to multiple cancer types and personalized antigens based on specific mutations of individual tumors. The identification of neoantigens is a key step in personalized tumor vaccines. Self-antigens typically refer to antigens that are expressed at higher levels in tumor cells. There is a wide variety of self-antigens, each with varying degrees of expression and immunogenicity in different contexts. Neoantigens account for 41.4% of the trials, self-antigens for 45.7%, and non-self-antigens for 13.1%. HPV (human papillomavirus) is the most targeted antigen, with a significant number of trials focusing on HPV16/18, E6/E7, and L1. Other antigens, such as CMV (cytomegalovirus) and allogeneic tumor cells, have fewer trials associated with them (Fig.4).The table below lists the specific self-antigens that are the focus of ongoing clinical trials for cancer vaccines (Tab.1).

Adjuvants, also refered to as immunomodulatory substances, are key components in therapeutic cancer vaccine formulations used to boost immune system function and modulate the type of immune response. They work through various mechanisms, including activating dendritic cells to improve efficiently antigen uptake, processing, and presentation. Adjuvants can also promote cytokine production, which can stimulate immunocyte proliferation and differentiation. Additionally, some adjuvants can activate inflammasomes, further promoting inflammatory responses. Adjuvant classifications cover a range from traditional options such as Freund’s adjuvants, oil emulsion adjuvants, and aluminum salt adjuvants, to newer types including liposomes, herbal adjuvants, small molecule peptide adjuvants, cytokines adjuvants, and nano-adjuvants. Adjuvants targeting CD4 are the most commonly used adjuvants in therapeutic tumor vaccines, accounting for 29.8% of trials. Next is adjuvants targeting APC, which was used in 24.6% of trials. Other adjuvants, such as adjuvants targeting TLR3, aluminum, GM-CSF, gp96, poly-ICLC, β-glucan, IL-2/12 (interleukin-2/12), IFA (incomplete Freund’s adjuvant), OBI-821, QS-21, BCG (Bacillus-Calmette-Guérin), and GPI-0100, each have a usage frequency of 19.3%. This suggests that these adjuvants are used with similar frequency in clinical trials (Fig.5).

Cancer vaccine carriers are biomaterials or systems used to deliver tumor antigens to immunocytes and activate a specific anti-tumor immune response. These carriers can be viral, bacterial, liposomal, polymer nanoparticles, or other biocompatible materials. The carriers for cancer vaccines under development primarily consist of APCs (72 trials), followed by viral vectors (36 trials) and lipid-based particles (24 trials) (Fig.6).

7 Conclusions

The basis of therapeutic cancer vaccines is a comprehensive understanding of the anti-tumor immune response and immune escape mechanism. Based on cancer-immunity cycle, we reviewed the function, circulation, initiation, and exhaustion of the effector cells that play a critical role in antitumor immunity. In addition to the well-recognized roles of CTLs and NK cells in tumor suppression, the contributions of CD4⁺ T cells, non-conventional γδ T cells, and B cells in antitumor responses are also summarized. From the perspective of vaccine design, antigens, adjuvants, and delivery systems are three indispensable aspects. A comprehensive review has been undertaken regarding these three aspects, especially neoantigen identification and prediction. Given that numerous factors influence the antitumor effectiveness of cancer vaccines, multiple studies have explored combination therapy strategies. We reviewed how cancer vaccines can be combined with other immunotherapies to achieve a successful and durable antitumor immune response based on key steps in the cancer-immunity cycle. The homeostatic balance of cancer patients, particularly those disrupted by age and bone marrow suppression, affects the effectiveness of therapeutic vaccinations. These dysregulations may result in an inflammatory environment, impair the structure and function of immune organs, and induce defects in immune and associated cells. This disturbance creates a detrimental loop that leads to tumor formation, which poses significant obstacles for vaccine therapies and is an important matter that has to be addressed. As of August 2024, this review emphasizes ongoing research into therapeutic cancer vaccines, covering 281 clinical trials. The current state of therapeutic cancer vaccines is analyzed and summarized according to tumor type, clinical stage, antigen specificity, adjuvant use, and delivery mechanisms. This data set encapsulates the prevailing trends and developmental dynamics within the therapeutic cancer vaccine field.

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