Neoantigen-specific T cells function as the primary effectors in neoantigen-based immunotherapies (72–74). Advanced DNA and RNA sequencing technologies, coupled with sophisticated bioinformatics algorithms, have deepened our understanding of these cells (75, 76). Single-cell RNA sequencing (scRNA-seq) and immune repertoire profiling have revolutionized the characterization of T cell features and the ability to predict therapeutic outcomes (77, 78). The integration of paired single-cell T cell receptor sequencing (scTCR-seq) and scRNA-seq enables tracking TCR clonotype frequencies before and after vaccination, while concurrently providing insights into the cellular states and functions of T cells with specific TCR clonotypes (76, 77, 79–82). These findings have led to the identification of neoantigen-specific T cell gene signatures, facilitating the prediction of antitumor responses and the development of more effective immunotherapies.

Over 100 clinical trials of personalized neoantigen vaccines have been registered in ClinicalTrials.gov, with encouraging results in melanoma, lung cancer, glioblastoma, and pancreatic cancer (6, 26, 30, 83–85). Following administration, these vaccines are processed by APCs, triggering T cell-mediated anti-tumor immune responses (86, 87). The resulting neoantigen-reactive T cell clones demonstrate diverse characteristics, including antigen recognition specificity, TCR diversity, functional heterogeneity, and HLA restriction (73, 88, 89). Recent research have revealed the transcriptome profiles of these neoantigen-reactive T cells (90), providing insights that validate vaccine efficacy and inform the development of optimized neoantigen-specific T cell therapies.

Several phase I clinical trials demonstrate that neoantigens activate CD8+ T cells fostering long-term immune memory (26, 29, 30, 85, 88). A phase Ib trial of a personalized neoantigen vaccine for glioblastoma showed that vaccine-induced T cells infiltrate the brain tumor microenvironment and elicit a robust immune response. CD8+ T cells exhibited cytotoxicity markers (granzymes, perforin) without CCR7 expression, while some CD4+ T cells displayed regulatory properties through IL2RA and FOXP3 expression (26). Similar results in pancreatic ductal adenocarcinoma revealed expanded CD8+ T cells expressing cytotoxicity genes (PRF1, GZMB) and IFN-γ (30). Remarkably, numerous studies have identified CXCL13 as a key marker in neoantigen-recognizing T cells, correlating with immune checkpoint blockade (ICB) efficacy (25, 90–93). Lung cancer research has revealed tissue-resident memory T cell programs, including CD103, CD69, HOBIT, LINC02446, and CXCL13 expression (90). CXCL13, CD103, and GZMA can serve as markers for neoantigen-specific CD8+ T cells and show promise as potential predictors of neoantigen-driven therapy efficacy (91, 92). Researchers have developed a neoantigen-reactive T cell signature based on clonotype frequency, CD39 protein, and CXCL13 mRNA expression for identifying reactive TCRs in non-small cell lung cancer (NSCLC) (93). Current research indicates that neoantigen-specific CD8+ T cells express characteristic genes including cytotoxicity genes (PRF1, GZMB), exhaustion genes (PDCD1, LAYN, ENTPD1, TIGIT, LAG3, HAVCR2), and specific markers (CD103, CD95, CXCL13). However, a complete gene profile of neoantigen-specific T cells remains to be established.

Recent advances in paired scRNA-seq and TCR-seq have revealed clonotypic T cell signatures, enabling deeper insights into T cell responses to antigen exposure. A landmark study by Steven A. Rosenberg’s team in 2022 provided a comprehensive transcriptome profiling of neoantigen-specific T cells (94). Through analyzing transcriptomic profiles from CD8+ and CD4+ neoantigen-reactive TILs in 10 metastatic cancer patients, the study revealed distinct molecular patterns. Both CD4+ and CD8+ TILs with neoantigen-specific TCRs (NeoTCRs) expressed chemokine-related genes (CXCL13, CXCR6) and inhibitory/exhaustion markers (TIGIT, PDCD1, CD39, LAG3, TOX). CD8+ NeoTCR TILs specifically showed cytotoxicity genes (GZMA, GZMB, GZMK, IFN-γ, PRF1) and the tissue-residency marker ITGAE (CD103). Most notably, the study identified over 200 NeoTCR signature genes specific to neoantigen-specific CD8+ and CD4+ T cells, opening new possibilities for developing patient-specific neoantigen-targeting TCR immunotherapies.

Notably, neoantigen-reactive CD8+ T cells from peripheral blood lymphocytes (PBLs) exhibit distinct features (95). These rare cells, comprising less than 0.005% of PBLs, express memory and quiescence markers (SELL, LTB, KLF2, LGALS3) while showing reduced expression of dysfunction-associated genes (TOX, CXCL13, PRF1, GZMH, GNLY). The cells show reduced expression of dysfunctional genes (TOX, CXCL13, PRF1, GZMH, GNLY) while maintaining higher levels of immune function-related genes (COTL1, PASK, ALOX5AP, HLA-DRB1, HLA-DPA). The study also identified specific surface markers (CD8, CD45RO, HLA-DR, CD39, CD103) for isolating these cells (95). Supporting these findings, other research groups have identified a minor population of neoantigen-specific CD8+ T cells in peripheral blood, characterized by high expression of CD39, PDCD1, and Ki67 (96). While circulating neoantigen-reactive CD8+ T cells share several markers with their tumor-derived counterparts, including CD39, CD103, IFN-γ, and PDCD1, recent research has uncovered important distinctions. Notably, Circulating cells express higher levels of IFN-γ and MHC class II-associated genes but lower levels of exhaustion-related genes (ENTPD1, PDCD1, LAYN) compared to their tumor-resident counterparts (97).

Despite less extensive study, neoantigen-specific CD4+T cells also play a crucial role in the anti-tumor response, as demonstrated by multiple recent studies (25, 98, 99). Research using B16F10 murine melanoma models has shown that neoantigen-specific CD4+ T cells induce strong, long-lasting antitumor immune responses. The study revealed that CD4+ T cells with high or moderate TCR avidity showed similar in vivo proliferation when exposed to cross-presented tumor antigens, resulting in comparable therapeutic effects (98). This finding is consistent with previous study in neoantigen-specific CD8+ T cells, where high structural avidity correlates with enhanced tumor localization and elimination. These findings establish TCR avidity as a critical determinant of both CD4+ and CD8+ T cell responses in cancer immunotherapy (100). Recent phenotypic profiling have revealed diverse characteristics of neoantigen-reactive CD4+ T cells. In melanoma studies, these cells display CXCL13 expression and can be categorized into distinct subsets, including those expressing memory and T follicular helper markers, cytotoxicity markers (GZMA/K and PRF1), inhibitory receptors (PD1 and TIM3), and IFN-γ (25, 101, 102). Consistent with this, studies of stage IIIB/C or IVM1a/b melanoma patients demonstrated long-term persistence of neoantigen-specific T cell responses, with CD4+ T cells exhibiting both memory and exhaustion phenotypes (27, 101).

These studies have illuminated the genetic characteristics of neoantigen-specific CD4+ and CD8+ T cells, revealing shared markers including exhaustion-associated genes, cytotoxicity markers, and IFN-γ expression. As summarized in Table 1 , the comprehensive gene profile of neoantigen-reactive T cells highlights their potential as both predictive biomarkers for vaccine efficacy and therapeutic targets for enhancing antitumor responses.

B cells contribute to neoantigen-specific immune responses through dual mechanisms: generating antibodies against tumor-specific antigens and serving as APCs (103). These antibodies can mediate antibody-dependent cellular cytotoxicity (ADCC) and opsonization, resulting in the destruction of tumor cells (12, 104–106). In the meanwhile, B cells function as APCs, presenting neoantigens to T cells and facilitating their activation. The role of B cells in neoantigen vaccine responses represents an emerging area of research, holding significant potential for enhancing vaccine efficacy (11, 107).

Within the tumor microenvironment, various B cell populations orchestrate anti-tumor immunity, including follicular B cells, germinal center B cells, and plasma cells. Among them, follicular B cells of the B2 lineage with bimodal IgD expression predominate among tumor-infiltrating B cells (11, 105, 108–111). Different B cell subsets exhibit distinct roles in tumor neoantigen-driven immune responses (11). Specifically, follicular B cells and marginal zone B cells have been implicated in the production of antibodies against tumor neoantigens (103, 112). Furthermore, germinal center (GC) B cells posse the ability to identify neoantigens and actively enhance anti-tumor immunity by orchestrating the activation of autologous CD8+ T cells (113). Essentially, the recognition of neoantigens by B cells triggers tumor-specific responses involving both B cells and T follicular helper (TFH) cells, which subsequently amplify CD8+ T cell anti-tumor immunity via the cytokine IL-21. This mechanism highlights the pivotal role of B cell recognition of neoantigens in eliciting effective anti-tumor responses by CD8+ T cells (113). Further evidence of B cell functionality come from a murine glioblastoma model, where B_Vax derived from GBM patients induced the generation of 4-1BBL+ B cells, successfully triggering autologous CD8+ T cell activation through CD40/CD40L interaction and IFN-γ stimulation (114). Moreover, B cells can differentiate into memory B cells following neoantigen exposure, providing long-term immunity through their prolonged lifespan and ability to rapidly generate antibodies upon subsequent antigen encounters (115, 116). The clinical significance of these mechanisms is supported by recent findings showing that renal cell carcinoma patients with tumor cells bearing IgG antibodies demonstrate higher response rates to immune checkpoint inhibitors and experience prolonged progression-free survival (PFS) (117).

Tertiary lymphoid structures (TLS) act as sites for the maturation of B cells in situ, resulting in the generation of immunoglobulins (Ig)-producing plasma cells (118, 119). These Ig-producing plasma cells are often found in association with IgG antibodies bound to apoptotic tumor cells, correlating with a positive response to immunotherapy (117, 119, 120). Scientists hypothesize that this correlation stems from the direct anti-tumor effects of these cells, ultimately enhancing treatment efficacy (118). Recent advances in understanding B cell-specific checkpoint molecules have revealed their role in engaging adaptive immunity to promote anti-tumor responses and control tumor growth. Particularly significant is the discovery by Vijay K. Kuchroo’s team that B cell subsets expressing T cell immunoglobulin and mucin domain 1 (TIM-1, encoded by Havcr1) specifically proliferate in draining lymph nodes and enhance anti-tumor CD8+ and CD4+ T cell responses (121). A breakthrough study by Gao and colleagues has provided comprehensive insights into the pan-cancer B cell landscape, establishing a clear connection between TLS maturation, GC B cells, and favorable immunotherapy outcomes. Their work has identified two distinct B cell differentiation pathways in human cancers, with memory B cells (Bm) correlating with improved treatment responses and survival, while atypical memory (AtM) cells are associated with treatment resistance and an exhaustion phenotype developing independently of germinal centers (122). These collective findings highlight B cells as crucial players in neoantigen-driven antitumor immune responses, offering promising opportunities for cancer prevention and treatment. Continued advancement in our understanding of B cell biology and their interactions with other immune cells opens new avenues for developing effective cancer immunotherapies.

DCs, as highly specialized professional APCs, play a fundamental role in orchestrating cancer immunity (123). These cells are not only integral components of the TME but also possess the distinctive ability to cross-present tumor-associated antigens to T cells, thereby initiating specific antitumor immune responses (124, 125). A remarkable feature of DCs is their capacity to activate between 100 and 3000 T cells individually, demonstrating an efficiency 100-1000 fold higher than that of macrophages and B cells (126, 127).

The DC population exhibit significant heterogeneity and can be classified into different subsets based on their anatomical location, phenotypic characteristics, and functional properties. The principal subsets of DCs encompass conventional DCs (cDCs), plasmacytoid DCs (pDCs), and monocyte-derived DCs (moDCs) (128, 129). cDCs are further delineated into two main subsets: cDC1 and cDC2. Each subset serves distinct functions: cDC1s specialized in cross-presenting antigens to CD8+ T cells, while cDC2s present MHC II-associated antigens to CD4+ T cells, promoting Th1, Th2, and Th17 polarization (129–131). pDCs, predominantly present in the blood and lymphoid organs, are characterized by their production of type I interferons, which are essential for antiviral immune responses. Additionally, pDCs contribute to the activation of other immune cells, including NK cells, and B cells (132). In contrast, moDCs arise through differentiation from monocytes and predominantly reside in inflamed tissues, particularly within the TME. These cells share functional similarities with cDCs in their ability to capture antigens and present them to CD8+ T cells. Of particular significance is their crucial role in neoantigen-driven therapeutic strategies, where they effectively present tumor-specific neoantigens to T cells, thereby initiating and sustaining immune responses against cancer cells (125, 133–135).

Recent research has extensively mapped tumor-infiltrating DC characteristics across various cancer types, such as melanoma, hepatocellular carcinoma, head and neck squamous cell carcinoma, non-small cell lung cancer, cutaneous squamous cell carcinoma, ovarian cancer, breast cancer, and colorectal cancer (136, 137). Of particular interest is the identification of LAMP3+ DC clusters in multiple tumor types, which may originate from cDC1 and cDC2 and migrate to lymph nodes (136, 138, 139). Additionally, scientists have identified a conserved DC cluster enriched in immunoregulatory molecules (CD274, Pdcd1lg2, and CD200) and maturation genes(CD40, CCR7, and IL12b) in non-small-cell lung cancers (140). This DC cluster represents a molecular state acquired by both cDC1s and cDC2s upon sensing or uptake of cell-associated antigens, encompassing the induction of molecular programs specialized in antigen presentation and associated with the induction of regulatory, immunogenic, and migratory gene programs. Recent advances have led to the reclassification of certain cDC2s as ‘DC3s’, which show distinctive capabilities in priming naive CD8+ T cells into tissue-homing CD103+ T cells. DC3s have also been further characterized, and their development has been shown to be dependent on GM-CSF (141, 142).

The development of neoantigen-based DC vaccines has shown remarkable progress, with extensive evidence supporting their efficacy across various cancer types. These highly personalized vaccines utilize DCs derived from autologous peripheral blood monocytes. Multiple clinical studies have demonstrated promising results (143–145). Yang, Li et al. successfully developed a personalized neoantigen peptide-pulsed autologous DC vaccine for lung cancer treatment, demonstrating both safety and efficacy in eliciting neoantigen-specific T-cell responses (134). Similar success has been observed in breast cancer, where neoantigen-pulsed DCs effectively induced cytotoxic T lymphocyte responses when co-cultured with autologous peripheral lymphocytes (146, 147). Studies have explored the combination of personalized neoantigen-loaded DC vaccines with T-cell therapy for hepatocellular carcinoma, while further research demonstrated promising results by combining anti-PD1 with Neo-MoDC vaccines, achieving complete tumor regression in metastatic gastric cancer patients for over 25 months (135, 148).

However, despite these promising results, autologous DC-based vaccines face significant challenges, including limited reproducibility and manufacturing constraints. To overcome these limitations, researchers have developed alternative approaches using allogeneic dendritic cells. A notable breakthrough emerged with the development of an innovative allogeneic plasmacytoid dendritic cell (pDC) line platform. This system was specifically engineered to enhance the priming and expansion of neoantigen-specific T cells for cancer vaccines, offering advantages in both scalability and consistency of production (149). The effectiveness of this novel approach was demonstrated in a phase Ib clinical trial involving nine patients with metastatic stage IV melanoma, where the vaccine successfully primed antitumor CD8+ responses without triggering allogeneic reactions. Particularly encouraging was the observation that two patients showed significant increases in circulating anti-tumor-specific T lymphocytes, with a beneficial transition from naïve to memory phenotype (150). These comprehensive findings support the potential of pDC line-based vaccines, particularly when used in conjunction with immune checkpoint inhibitors, representing a significant advancement in cancer immunotherapy strategies.

Natural killer (NK) cells represent essential components of the innate immune system, demonstrating remarkable efficacy in cancer immunotherapy, particularly in treating advanced-stage leukemia (151, 152). Their therapeutic value stems from their unique ability to recognize stressed cells independently of neoantigen presentation, enabling them to target tumor cells that have lost MHC class I expression, which represents a common mechanism of tumor immune evasion (153, 154). With the growing recognition of tumor neoantigens as potential immunotherapy targets, research has increasingly focused on understanding NK cell characteristics in neoantigen-driven immune responses. Current investigations are exploring the interactions between NK cells and tumor neoantigens, as well as strategies to enhance NK cell activity for improved cancer treatment outcomes (155, 156). This chapter aims to understand the mechanisms by which NK cells recognize and eliminate neoantigens-presenting tumor cells, ultimately working toward developing strategies to fully harness the potential of NK cells in cancer immunotherapy.

NK cell function is primarily mediated through their recognition of HLA molecules via specific receptors, notably killer cell immunoglobulin-like receptors (KIRs) and natural killer group 2 A (NKG2A). Studies have shown that the interaction affinity between HLA and KIR is peptide-dependent and can influence NK cell effector function (157, 158). Therefore, the altered peptide repertoire displayed by MHC molecules in cancer cells may significantly influence NK cell activity. Supporting this concept, a study has demonstrated that NK cells’ cytotoxic effects on tumor cells are modulated by the binding affinity of KIR peptide-MHC interactions (159). A significant advancement in this field emerged with the development of a cancer vaccine targeting MICA and MICB (MICA/B). This innovative approach induces a coordinated attack by both T cell and NK cell populations, maintaining effectiveness against MHC class I-deficient tumors through the combined action of NK cells and CD4+ T cells (160).

Natural killer T (NKT) cells represent a distinctive cell subset of the innate immune system, characterized by CD1d restriction and lipid antigen reactivity, serving as immunoregulatory T lymphocytes (161, 162). While sharing many phenotypic and functional similarities with conventional T cells in anti-tumor immunity, NKT cells are unique in their ability to recognize lipid-based antigens presented on CD1d molecules rather than peptide-MHC complexes (162–164). The therapeutic potential of this pathway was demonstrated by a single-domain antibody targeting CD1d, which successfully induced robust NKT cell activation and enhanced anti-tumor responses, as validated in multiple myeloma and acute myeloid leukemia models (165). Further supporting their therapeutic relevance, strong evidence indicates that tumor cells generate specific lipid species that function as activating antigens for NKT cells via CD1d presentation, playing a crucial role in cancer immune surveillance (166).

These tumor-associated lipid antigens are unique to tumor cells and can be recognized as foreign by the immune system, particularly through NKT cell-mediated responses.These lipid antigens play a role in the participation of NKT cells in cancer immune surveillance (167). Up to now, various tumor-associated lipid antigens have been identified, such as glycolipids (Globo H, Globo A, Lewis Y, and GM2), phospholipids (phosphatidylinositol and phosphatidylglycerol), glycosphingolipids (GM3, GD2, and GD3), and phosphatidylethanolamines (including PE and PG). Several of these antigens have emerged as promising candidates for cancer vaccine development, owing to their distinctive immunogenic properties (167–169). This recognition mechanism offers potential avenues for immunotherapy and cancer treatment. While scientists are working to harness these antigens to enhance anti-tumor immune responses and overcome immune evasion mechanisms, significant research is still needed to fully understand their role in tumor immunity and optimize therapeutic approaches.

In conclusion, NK cells have shown great potential in cancer immunotherapy due to their ability to recognize and eliminate tumor cells. However, crucial questions remains to be learned about NK cell characteristics and their interactions with neoantigen. Further research is needed to understand the factors that regulate NK cell activation and function within the tumor microenvironment. As our understanding of these processes deepens, it will likely lead to the development of more effective and targeted immunotherapies for cancer treatment.

Tumor-Associated Macrophages (TAMs), a heterogeneous population of myeloid cells infiltrating neoplastic tissues, play a critical role in tumor development and progression. TAMs accumulate within tumor tissues and predominantly exhibit tumor-promoting activity, promoting angiogenesis and immune evasion (170–173). With advancing understanding of tumor neoantigens, research has increasingly focused on characterizing macrophages in neoantigen-driven immune responses.

Macrophages employ multiple mechanisms to recognize tumor cells, including direct recognition through receptors such as CD11c, CD14, and CD40, and indirect recognition via DC presentation. This recognition enables macrophages to interact with various immune cell types, including T cells, B cells, and NK cells, coordinating the immune response against tumors (174–176). Recent experimental studies have validated the antigen-presenting function of MHC II-expressing tumor-associated macrophages (TAMs) to tumor-infiltrating CD4 T cells (177, 178). In a bladder carcinoma mouse model, TAM-mediated antigen presentation to effector CD4 T cells was demonstrated to be crucial for tumor rejection. These immunologically active TAMs exhibited a distinctive immunophenotype characterized by CD11b+, MHCII, F4/80, CD11C, CD80, CD86, CD64, and PD-L1 expression. The antitumor response was found to be dependent on IFN-γ production by CD4 T cells (177).

Macrophages have established themselves as crucial players in tumor neoantigen-driven immune responses. However, significant challenges persist in understanding their complete characteristics within the context of neoantigen-based cancer immunotherapy. Key obstacles include the inherent heterogeneity and plasticity of macrophages within the tumor microenvironment, which complicate effective targeting and manipulation strategies. Moreover, the precise mechanisms governing their recruitment and activation in response to neoantigens require further elucidation.

The collective evidence demonstrates that tumor neoantigens initiate a complex, multifaceted immune response, orchestrated through the coordinated actions of T cells, DCs, B cells, NK cells, NKT cells, and macrophages ( Figure 1 ). While these diverse immune cell populations utilize distinct recognition patterns and mechanistic pathways to mount a comprehensive systemic immune response, several critical challenges remain unresolved. These include gaining a deeper understanding of macrophage heterogeneity and plasticity within the tumor microenvironment, and fully characterizing the mechanisms controlling their recruitment and activation in response to neoantigens.