Neoadjuvant immunotherapy in squamous cell carcinoma of the head and neck: current evidence and future perspectives.

Introduction

Head and Neck Squamous Cell Carcinoma (HNSCC), encompassing tumors located at anatomical sites such as the oral cavity, oropharynx, larynx, and hypopharynx, ranks as the sixth most common malignant tumor worldwide. According to GLOBOCAN 2022, there are over 900,000 new cases and 450,000 deaths annually from this disease [ 1 ]. The current standard treatment modality for locally advanced head and neck squamous cell carcinoma (LA-HNSCC) primarily involves radical surgery combined with postoperative adjuvant concurrent chemoradiotherapy. Although this strategy enhances local control rates, the median disease-free survival (DFS) for patients remains at only 18–24 months, with a 5-year OS rate of less than 50% [ 2 ]. Additionally, approximately 30%−50% of patients experience acute toxic reactions of grade 3 or higher (such as radiation-induced mucositis and dysphagia), while over 50% of patients suffer from chronic xerostomia or neck fibrosis, significantly reducing their quality of life [ 3 – 5 ]. More critically, distant metastases occur in approximately 20% of LA-HNSCC patients after traditional treatment, and long-term smokers face a cumulative risk of developing a second primary cancer of up to 20%−30% within 10 years [ 6 ]. Postoperative chemoradiotherapy can eliminate residual tumor cells, it may induce CD8 + T cell exhaustion and regulatory T cells (Tregs) amplification, leading to a suppressive “immune desert” microenvironment, significantly reducing the response rate to subsequent immunotherapy [ 7 , 8 ].

Neoadjuvant therapy has emerged as a promising strategy to overcome these challenges caused by adjuvant therapy. However, traditional neoadjuvant chemotherapy regimens have not been demonstrated superiority in HNSCC. A randomized controlled trial conducted by the Licitra team showed there were no differences between oral squamous cell carcinoma patients received neoadjuvant therapy with cisplatin or fluorouracil [ 9 ]. The randomized phase III NCT01542931 trial further confirmed that there were no significant differences in median OS and DFS between the TPF induction chemotherapy group and the direct surgery group [ 10 ]. Besides, Chaukar et al. indicated that neoadjuvant chemotherapy commonly encountered chemotherapy-related toxicities without any improvement in survival benefits [ 11 ]. These findings suggest an urgent need for novel neoadjuvant therapy approaches.

The breakthrough of immune checkpoint inhibitors (ICIs) has revolutionized the treatment paradigm for HNSCC. Based on the survival advantages demonstrated by ICIs in the treatment of advanced HNSCC, neoadjuvant immunotherapy (NIT) has been highly anticipated. NIT is conceptualized around two key scientific hypotheses: Firstly, in situ antigen priming, where tumor-associated antigens (TAAs) and neoantigens released from the tumor in situ before surgery are more likely to activate dendritic cells (DCs) and initiate a systemic T-cell response [ 12 ]. Secondly, the immune memory protective effect, where studies have shown that neoadjuvant PD-1 inhibitor therapy can induce the expansion of antigen-specific memory T-cell (Tmem) pools, with effects persisting after surgery and reducing the rate of distant metastasis by three times [ 13 – 15 ]. Currently, several pivotal clinical trials, such as NCT02296684 [ 16 ], CheckMate-358 ( NCT02488759 ) [ 17 ], CIAO ( NCT03144778 ) and IMCISION ( NCT03003637 ) [ 18 , 19 ], have validated that NIT can improve the treatment in certain sub-population of patiens with HNSCC. Furthermore, multicenter studies have indicated that NIT does not significantly increase the risks of surgical delays, perioperative complications, or accelerated tumor progression. However, key issues such as the heterogeneity of NIT’s efficacy in HNSCC, unclear mechanisms of durable response, and the lack of predictive markers still need to be addressed. This article will start from the molecular mechanisms of immunotherapy, summarize potential strategies to enhance the durable response of NIT, and propose future research directions for improving the effectiveness of NIT for HNSCC patients.

Treatment challenges in locally advanced head and neck squamous cell carcinoma

Locally advanced head and neck squamous cell carcinoma (LA-HNSCC) often presents with complex pathological features and significant clinical heterogeneity, typically requiring a multimodal approach integrating surgery, radiotherapy, chemotherapy, and targeted/immunotherapy [ 20 , 21 ]. However, current treatment options remain controversial. The overall efficacy of targeted therapy combined with chemoradiotherapy has not met expectations, and significant toxic side effects, such as myelosuppression and gastrointestinal toxicity, force nearly half of the patients to frequently adjust dosages or discontinue treatment, affecting long-term survival [ 22 ]. While ICIs have demonstrated breakthrough efficacy in advanced HNSCC, their application in the locally advanced stage is still limited by issues such as immune microenvironment suppression and inadequate response rates [ 23 ]. Regarding efficacy improvement, multiple clinical studies have initially validated the core advantages of NIT. A systematic review including ten clinical studies showed that the pathological complete response (pCR) rate in the NIT group was significantly higher than that in the traditional chemotherapy group (32% vs. 12%, OR = 3.27; p < 0.001), without increasing the risk of postoperative complications or delayed healing [ 24 ]. In terms of safety optimization, NIT exhibits unique potential. For instance, a prospective study targeting HPV-negative patients found that chemotherapy combined with PD-1 inhibitors could induce a deep pathological response in 58% of cases, making it possible to adaptively reduce subsequent chemoradiotherapy dosages. This treatment significantly reduced the incidence of acute toxicities such as grade 3 or higher mucositis and dysphagia (21% vs. 42%) [ 25 ]. Despite these advances, the clinical translation of NIT still faces multiple challenges. Phase III randomized controlled trials are urgently needed to clarify long-term survival benefits. The mechanisms to improve the durability of responses are poorly understood, and there is a lack of a biomarker system to guide treatment selection. Strategies such as combination regimens need to be explored to enhance durable immune responses, and artificial intelligence should be utilized to integrate multi-dimensional data from genomics and the tumor microenvironment (TME) to build individualized screening models. Overall, NIT provides a new perspective for breaking through the treatment bottlenecks of LA-HNSCC.

Advances in neoadjuvant therapy for head and neck squamous cell carcinoma

The purpose of neoadjuvant therapy is to reduce tumor volume, increase the possibility of surgical resection, enhance the chance of organ preservation, and treat potential micrometastases. Although traditional neoadjuvant therapies, such as radiotherapy and chemotherapy combined with targeted therapy, can achieve partial reduction in tumor load and improve the R0 resection rate, their benefits for long-term survival of patients are limited, with less improvement in the 5-year OS rate, and high incidence of significant toxic reactions [ 26 ]. Although a high-dose TPF regimen (Docetaxel 75 mg/m²) increased the pRR (15.5% vs. 10.7%), it also led to an increased incidence of grade 3–4 neutropenia up to 63% [ 27 ], which highlights the dual challenges of efficacy and toxicity associated with traditional neoadjuvant therapies. A pivotal moment occurred in 2016 when the U.S. FDA first approved PD-1 inhibitors Pembrolizumab and Nivolumab for the treatment of HNSCC, based on the groundbreaking results of two key studies: the KEYNOTE-012 trial [ 28 ] and the CheckMate-141 trial [ 29 ]. Since then, a series of studies have accelerated the evolution of the treatment strategy (Fig. 1 ). The KEYNOTE-040 study found that Pembrolizumab could prolong patients’ OS (median OS 8.4 vs. 6.9 months) with fewer treatment-related adverse events (13% vs. 36%) compared to standard of care (SOC) therapy [ 30 ]; The KEYNOTE-048 study established Pembrolizumab ± chemotherapy as a first-line treatment for patients with PD-L1 CPS ≥ 1, significantly extending OS (median OS: 14.9 vs. 10.7 months, HR = 0.74) and reducing the incidence of grade 3 or higher toxic reactions (63% vs. 85%) [ 31 ]. Apart from PD-1/PD-L1, another immune checkpoint widely used as a therapeutic target is CTLA-4. Currently, no immunotherapy based on anti-CTLA-4 has been approved for the treatment of HNSCC, but related clinical trials are underway. The EAGLE study targeting PD-1/CTLA-4 dual targets (Durvalumab ± Tremelimumab vs. SOC) did not reach the OS/PFS endpoint (median OS: 7.6/6.5 vs. 8.0 months), but it provided clues for the study of immune resistance mechanisms [ 32 ]; Additionally, the NCT04080804 trial innovatively combined PD-1 inhibitor (Nivolumab) with LAG-3 inhibitor (Relatlimab)/CTLA-4 inhibitor (Ipilimumab). This trial introduced anti-LAG-3 as a therapeutic target, aiming to reshape the microenvironment through immune synergy and enhance adaptive immunity before surgical resection, thereby improving the antitumor activity of anti-PD-1 antibodies [ 33 ]. The findings of the above studies have laid the foundation for NIT in HNSCC and inspired researchers to explore shifting the immunotherapy window to the preoperative neoadjuvant stage.

Safety of neoadjuvant immunotherapy

The safety evaluation of NIT has formed a systematic evidence-based chain. Early concerns focused on the risks associated with early tumor progression and the impact of immune-related adverse events (irAEs) on perioperative care. A retrospective study by Phillips et al. found no statistically significant difference in the 30-day surgical complication rate between HNSCC patients who received NIT (PD-1/PD-L1 inhibitors) and those who did not (15.7% vs. 18.4%). Phase II clinical trial NCT02296684 and related meta-analyses reported that the incidence of grade 3/4 adverse events in HNSCC patients receiving monotherapy with NIT was only 0%−8.4% [ 34 , 35 ], However, immunotherapy may increase the risk of wound healing complications (such as infection, fistulas, etc.) in patients undergoing flap reconstruction [ 36 ]. Multiple studies have shown minimal impact of NIT on surgical delays, intraoperative complication risks, postoperative outcomes, or tumor progression. Phillips et al.‘s retrospective analysis of 463 HNSCC surgical patients receiving NIT revealed no statistically significant difference in perioperative outcomes (30-day complication rates) compared to the control group. Regarding concerns about surgical delays, a meta-analysis by Masarwy et al. of 10 studies involving 344 patients showed that only 2 out of 344 patients (0.6%) receiving immune neoadjuvant therapy had their surgery delayed due to irAEs, and both recovered their surgical window with short-term hormone therapy [ 35 ]. Currently, the FDA’s adverse reaction database indicates that severe irAEs such as pneumonia (1.8% incidence) and colitis (1.2%) associated with NIT for HNSCC can achieve clinical remission in 95% of cases after standardized management, providing strong support for perioperative safety. These data have led to widespread recognition of the safety of NIT.

Exploration of neoadjuvant immunotherapy strategies

Neoadjuvant immunotherapy monotherapy

Neoadjuvant therapy with PD-1 inhibitors has demonstrated considerable potential for pathological response in HNSCC. NCT02296684 was the first to verify the safety and efficacy of neoadjuvant Pembrolizumab in patients with LA-HNSCC. This trial evaluated neoadjuvant treatment with Pembrolizumab for 2 cycles in LA-HNSCC, with the primary endpoints being pTR-2 in all patients and the 1-year recurrence rate in high-risk pathological patients. Additionally, the correlation between baseline PD-L1 and T-cell infiltration with pTR, as well as tumor clonal dynamics, were assessed. Among the 36 patients treated, 8 (22%) achieved pTR-2, while another 8 (22%) achieved pTR-1. The 1-year recurrence rate for the 18 high-risk pathological patients was 16.7% (95% CI, 3.6%−41.4%), which was significantly lower than historical data (35%) [ 34 ]. The NCT02641093 trial, with an expanded sample size ( n = 92), evaluated the efficacy of neoadjuvant treatment with Pembrolizumab for 1 cycle in patients with resectable LA-HNSCC. The results showed that the 1-year DFS rates for high-risk (positive margins or extracapsular lymph node invasion) and intermediate-risk (negative margins, no extracapsular spread) patients were 67% and 93%, respectively. However, patients with PD-L1 CPS < 1 did not benefit (DFS HR = 1.12) [ 37 ]. Compared to the traditional RTOG9501 trial results [ 38 ], the 1-year DFS rate improved by 29% in the high-risk group and by 28% in the intermediate-risk group. It’s worth noting that in NCT02296684 , neoadjuvant Pembrolizumab for 2 cycles resulted in a 22% higher pRR (44% vs. 22%) compared to the 1-cycle regimen in NCT02641093 [ 34 , 37 ]. The NCT03021993 trial evaluated 12 patients with resectable LA-OCSCC who received two cycles of Nivolumab. The major pathological response rate (MPR) was 24%, with 1 patient achieving a complete pathological response (pCR). Single-cell sequencing revealed that the proportion of CD8 + T cells among tumor infiltrating lymphocytes (TILs) in MPR patients increased by 3.7 times compared to baseline ( P = 0.02), and there was a positive correlation between T cell receptor (TCR) clonal expansion efficiency and pathological response ( r = 0.68) [ 39 ]. ICI used in neoadjuvant therapy has shown short-term superiority over traditional neoadjuvant treatment regimens, with a lower incidence of severe adverse reactions. These results provide experimental evidence for further research on combining immunotherapy drugs with other agents in neoadjuvant therapy. Although monotherapy with ICIs has demonstrated a survival advantage in recurrent/metastatic (R/M) HNSCC, with the 2-year OS rate for patients with high PD-L1 expression increasing by nearly two times compared to traditional chemotherapy, the objective response rate remains relatively low [ 40 ]. This has further driven research towards combination strategies.

Neoadjuvant dual immunotherapy

Dual immunotherapy combinations, such as CTLA-4/PD-1 dual blockade, enhance efficacy through synergistic mechanisms. Numerous Phase II trials (Table 1 ) have shown that this approach can extend the median progression-free survival by several months. Additionally, by controlling the dosage, the incidence of Grade 3–4 toxic side effects is significantly reduced, establishing a scientific foundation for combined efficacy enhancement and toxicity reduction [ 41 , 42 ].The IMCISION trial in Netherlands enrolled 40 patients with locally advanced HNSCC, comparing neoadjuvant therapy with Nivolumab monotherapy to combination immunotherapy with Nivolumab and Ipilimumab. The postoperative MPR rates were 17% and 35%, respectively, and a significant correlation was found between pathological response and CD8 + T-cell expansion. However, the toxic effects were not negligible, with a 38% incidence of grade 3–4 irAEs, leading to surgical delays of ≥ 2 weeks in 7% of patients [ 43 ]. Interestingly, in another phase II NCT03708224 trial, low-dose Ipilimumab (1 mg/kg) combined with Nivolumab maintained an MPR rate of 28% while reducing severe irAEs to 9%, suggesting the possibility of improved safety through dose optimization. The NCT04080804 study compared the efficacy of neoadjuvant Nivolumab monotherapy to neoadjuvant Nivolumab combined with Relatlimab (anti-LAG⁃3)/Ipilimumab in patients with resectable LA-HNSCC. The results showed that the combined immunotherapy group had higher pPR (15.4% and 20%) and MPR (7.7% and 10%) rates compared to the Nivolumab monotherapy group, with no serious adverse events or surgical delays related to the study drugs [ 33 ]. The CIAO trial compared neoadjuvant Durvalumab monotherapy to combination immunotherapy with Durvalumab and Tremelimumab. Postoperatively, the combined immunotherapy group showed a significant improvement in MPR compared to the monotherapy group, with a 14% incidence of grade 3–4 irAEs [ 18 ]. Similarly, Kim et al. conducted a similar comparison in the NCT03737968 trial, but the latest results have not been reported yet [ 44 ]. Besides the aforementioned immunotherapy drugs, several other drug-related clinical trials are currently underway (Table 1 ). Available clinical trial data indicate that compared to immunotherapy monotherapy groups, combination immunotherapy groups can significantly improve pCR without significantly increasing the risk of severe adverse events (≥ grade 3). Based on the positive correlation between the depth of pathological response and long-term relapse-free survival (RFS) in cancer patients, the potential survival benefits of these combination therapy models are noteworthy. To further validate their clinical value, it is essential to conduct phase III randomized controlled trials based on rigorous screening of biomarker-expressing populations.

Neoadjuvant immunotherapy–targeted therapies combinations

The neoadjuvant immunotherapy-targeted combination strategy (NITT) is considered a promising approach due to its roles in reactivating the immune system, disrupting the immunosuppressive microenvironment, and delaying drug resistance. The Phase I NCT04722523 clinical trial evaluated the feasibility of combining Cemiplimab and Cetuximab for resectable HNSCC. The results indicated that 90% of patients achieved clinicopathological downstaging after combination therapy, with MPR and pCR rates reaching 60% and 40%, respectively. Among the patients, 75% with mandibular invasion avoided bone resection, 70% were spared flap repair, 40% did not require postoperative radiotherapy, and there was no recurrence in any patient after a median follow-up of 16 months [ 59 ]. Another Phase I clinical study, NCT04393506 , investigated the use of Camrelizumab combined with Apatinib before surgery for oral squamous cell carcinoma ( n = 20). In the CPS >10 subgroup, the MPR rate was 100%, and the 18-month survival rate was remarkably high at 95% [ 47 ]. Based on the Phase I safety verification, the Phase II EACH study employed Pembrolizumab plus Cetuximab as neoadjuvant therapy for hypopharyngeal cancer ( n = 25). The MPR rate increased to 44%, and there was a significant reduction in intratumoral Tregs (−71%, P = 0.004) [ 60 ]. The results obtained in the Phase I/II clinical studies demonstrate good safety and tolerable serious adverse event rates for the neoadjuvant combination of targeted and immune therapies, along with promising tumor response rates. However, it’s important to note that the primary objective of Phase I/II clinical trials is to assess the initial safety and efficacy of treatment regimens. Therefore, conducting large-scale Phase III clinical trials remains essential.

Neoadjuvant immunotherapy–radiotherapy combinations

Radiotherapy can not only directly kill tumor cells but also enhance the effectiveness of immunotherapy by inducing local and systemic antitumor immune responses. Neoadjuvant immunotherapy combined with radiotherapy (NIRT) has shown good potential in studies of other cancer types, such as non-small cell lung cancer (NSCLC), as well as HNSCC [ 61 , 62 ]. In the phase Ib NCT03247712 clinical study, stereotactic body radiotherapy (SBRT, 24 Gy/3f) combined with two cycles of nivolumab was used to treat unresectable HNSCC. The results showed that 90% of patients achieved clinicopathological downstaging, with a 28.6% incidence of grade 3/4 treatment-related adverse events. The overall response rate (ORR) reached 72%, with 42% of patients converting to resectable status. Among HPV-positive patients, the MPR rate was as high as 100%, and the pCR rate was 90% [ 55 ]. The phase I/Ib NCT03635164 clinical trial evaluated the safety and efficacy of SBRT combined with a single dose of durvalumab as neoadjuvant treatment for HPV-negative locally advanced HNSCC (LA-HNSCC). SBRT was administered using a dose-escalation approach (starting at 12 Gy/2 fractions and escalating to 24 Gy/3 fractions) to compare the effects of different radiotherapy doses. The results indicated that 75% of patients achieved MPR or pCR, with an OS rate of 80.1%, a PFS rate of 75.8%, and a grade 3/4 irAE rate of 28.6%. There were no surgical delays or serious complications, and the treatment was well-tolerated. It is noteworthy that the 24 Gy dose demonstrated better efficacy, and higher doses may affect the required adjuvant radiotherapy dose after surgery, potentially leading to the omission of postoperative radiotherapy. However, this needs to be validated in phase II trials [ 56 ]. Current research suggests that the combination of radiotherapy and ICIs can be safely used as neoadjuvant treatment for LA-HNSCC, exhibiting a high rate of pathologically significant response. It is important to note that higher doses of radiotherapy yield better results, providing the possibility for subsequent omission of postoperative radiotherapy. The combination of radiotherapy and immunotherapy in neoadjuvant treatment highlights the synergistic effects of immunotherapy and novel local intervention modalities, which deserve further exploration. In future studies, consideration can be given to replacing radiotherapy with alternative local treatment modalities such as minimally invasive thermal ablation techniques (e.g., radiofrequency or microwave thermal coagulation therapy) to further reduce treatment invasiveness while maintaining efficacy. However, radiotherapy was able to deplete key immune cell pools, particularly within cervical lymph nodes, which serve as the primary source of lymphocytes, and within the primary tumor, thereby forming an immunosuppressive environment that favors tumor cell immune evasion [ 63 ].

Neoadjuvant immunotherapy–chemotherapy combinations

Neoadjuvant immunotherapy combined with chemotherapy (NICT) increases the pCR rate (pCR) in some types of cancers [ 64 – 66 ]. Multiple studies indicated NICT was emerged as the preferred neoadjuvant treatment strategy for HNSCC. For instance, results of the phase I/II trial showed a significantly improved pCR rate to 38.1% with three cycles of Carboplatin, Paclitaxel, and Pembrolizumab combination treatment compared to other neoadjuvant treatment regimens [ 67 ]. Similarly, the NCT05522985 trial, utilizing a neoadjuvant treatment regimen of Toripalimab combined with Nab-paclitaxel and Cisplatin, achieved a high MPR rate of 81.58% (immunotherapy group vs. chemotherapy group = 81.58% vs. 53.55%, p = 0.01) and a pCR rate of 55.3% (immunotherapy group vs. chemotherapy group = 55.3% vs. 36.7%) [ 53 ]. Furthermore, various other trials have reported impressive pCR and MPR rates with neoadjuvant chemoimmunotherapy (NICT). The NCT03342911 trial showed a 45% pCR and 73% MPR with a significant trend towards survival benefit, with a 3-year OS of 83% and recurrence-free survival (RFS) of 74%. The NCT03426657 trial reported a 52% pCR in different arms [ 49 ], while the ChiCTR2100043743, ChiCTR1900025303, and ChiCTR2200055719 trials demonstrated pCR rates of 16.7%, 37%, and 36.4%, respectively, with corresponding MPR rates [ 50 – 52 ]. The NCT04826679 trial also exhibited a noTable 55.6% pCR and 63% MPR. The combination of immunotherapy and chemotherapy has shown superior efficacy in terms of pCR and MPR compared to other neoadjuvant regimens (Table 1 ) and has also demonstrated improvements in OS and DFS [ 54 ]. Studies suggest that this significant efficacy may be attributed to the ability of preoperative induction chemotherapy to reduce tumor burden and promote the release of tumor antigens, as well as the synergistic effect when combined with ICIs to induce a systemic antitumor immune response [ 68 ]. Despite the abundance of research on neoadjuvant chemoimmunotherapy, most existing studies are limited to phase II clinical trials (Table 1 ). The efficacy and safety of this approach are still lacking large-scale phase III study data. Future prospective randomized controlled trials are needed to further validate the clinical translational value of this strategy.

How to improve the efficacy of neoadjuvant immunotherapy?

Targeting immune checkpoint is one of the most important strategy of immunotherapy. The PD-1/PD-L1 pathway maintains immune homeostasis by negatively regulating T-cell activity under physiological conditions [ 69 ], while tumor cells tumor cells hijack this pathway to suppress the immune response. Hence, immune checkpoint blockers (ICBs) disturbed PD-1/PD-L1 interaction to reverse T-cell suppression and reactivate antitumor immune responses [ 70 ]. The phase II NCT03698019 trial indicated the addition of neoadjuvant Pembrolizumab to surgery with adjuvant Pembrolizumab improved event-free survival (EFS) among patients with resectable stage IIIB-IVC melanoma, confirming the advantage of the neoadjuvant treatment in re-priming the immune response compared to the adjuvant treatment [ 71 , 72 ]. Furthermore, KEYNOTE-689 ( NCT03765918 ), a phase III open-label trial, confirmed that the addition of perioperative pembrolizumab to standard care with surgery and adjuvant therapy improved event-free survival among patients with locally advanced HNSCC [ 73 ]. However, there is still 37.5% participants faced recurrence, progression or death. A study found that TIM3 + T cells and LAG3 + T cells were enriched in non-response HNSCC cohort after NIT, indicating novel combination strategy might improve the efficacy of NIT. This section will discuss the way that improves the efficacy of NIT, including enhancement of antigen presentation, priming and expansion of T-cell, reverse of immunosuppression environment, and disruption of stromal barrier and vascular abnormalities [ 74 – 77 ].

Enhanced antigen presentation

Exposing tumor antigens

Tumor cells evade immune recognition through antigen-shielding mechanisms (e.g., glycosylation) or by activating the coagulation system to form protective fibrin “cocoons” [ 78 , 79 ]. Therefore, exposing tumor antigenicity is crucial for enhancing antitumor immunity. Dead tumor cells (DTCs) can induce immunogenic cell death (ICD) in other tumor cells. ICD modulates the interaction between DTCs and immune cells, with DTCs releasing damage-associated molecular patterns (DAMPs) such as adenosine triphosphate (ATP) and high-mobility group box 1 (HMGB1). These DAMPs serve as danger signals to initiate tumor-specific immune responses, enhance tumor immunogenicity, recruit and activate DCs, and ultimately guide cytotoxic T lymphocytes (CTLs) to attack other tumor cells. This suggests that ICD induction represents a potential strategy for NIT [ 80 ]. In HNSCC, ICIs trigger ICD effects by killing tumor cells, releasing TAAs, DAMPs, and viral-derived antigens (VDAs; e.g., HPV E6/E7) [ 81 ]. This not only enhances tumor immunogenicity and adjuvant exposure but also drives the transformation of the TME from “cold” to “hot,” persistently amplifying DC- and T cell-mediated antitumor immune responses [ 80 ]. Furthermore, the STING pathway plays a pivotal role in tumor immunity. The STING-dependent cytosolic DNA-sensing mechanism significantly enhances DC antigen presentation by promoting type I interferon (IFN-I) production, effectively activating T cells to potentiate antitumor immunity [ 82 – 84 ]. In HPV-positive HNSCC, viral HPV E6/E7 DNA activates IFN-I via the cGAS-STING pathway, robustly inducing DC maturation (characterized by CD80/86 upregulation) and improving cross-presentation efficiency, thereby markedly expanding virus antigen-specific T cells [ 82 , 85 , 86 ].

From an immune recognition perspective, neoantigen diversity constitutes the key driver of T cell clonal expansion. Although tumor mutational burden (TMB) and PD-L1 expression serve as predictive biomarkers for ICI response (with high expression generally correlating with better outcomes), their mechanistic interplay requires further elucidation [ 87 ]. In HNSCC, smoking-associated HPV-negative subtypes frequently exhibit high TMB, generating richer neoantigen repertoires. Under neoadjuvant therapy, these neoantigens activate diverse TCR clones, enhancing cross-recognition potential [ 88 , 89 ]. Notably, post-neoadjuvant treatment peripheral blood analyses revealed a 2- to 5-fold increase in clonally expanded T cells, which correlated with improved 1-year recurrence-free survival (HR = 0.34) [ 90 , 91 ]. Moreover, antigen exposure and immune responses in HNSCC demonstrate molecular subtype-dependent heterogeneity. In the phase II CheckMate-358 trial, HPV-positive patients—leveraging inherent immunogenicity from viral antigens—exhibited significantly higher intratumoral E7-specific T cell expansion rates after neoadjuvant ICI therapy compared to HPV-negative patients. This supports combinatorial strategies of E6/E7-targeted vaccines with ICIs to enhance synergistic efficacy [ 92 ].

Remodeling dendritic cell function and antigen presentation

CD103 + cross-presenting DCs play a pivotal role by orchestrating the cascade of antigen presentation and T cell priming during the antitumor immune response. PD-1/PD-L1 blockade therapy remodels DC function through a dual mechanism: (1) it alleviates PD-L1-mediated suppression of DCs, restoring the expression of key antigen-presenting molecules such as MHC-I and co-stimulatory molecules (CD80/CD86) [ 93 ]; (2) it enhances the migration and infiltration of CD103 + DCs into tumor beds and their capacity to transport tumor antigens to lymph nodes via chemokine receptor (CCR5/CCR7)-mediated signaling pathways [ 94 , 95 ]. Activated CD103 + DCs deliver tumor antigens to CD8 + T cells through cross-presentation, driving the selection and expansion of high-affinity TCR clones [ 96 , 97 ]. Single-cell RNA-seq study has confirmed a significant positive correlation between intratumoral functional DC density and TCR clonal diversity [ 98 ], indicating the decisive role of mature DC antigen presentation in T cell-mediated antitumor responses.

The innovative application of TLR agonists provides novel strategies for targetedly enhancing DC function. TLR9 agonists significantly promote DC maturation and activation as well as Th1-type immune responses through the TLR/MyD88 pathway, particularly when combined with PD-1 blockade, which effectively improves the antigen-processing efficiency of DCs [ 99 ]. This synergistic effect not only optimizes DC function but also induces the expansion of GZMB + CTLs to enhance antitumor cytotoxicity [ 100 ]. Combinatorial use of different TLR agonists can also markedly augment DC immunogenicity and antigen-presentation efficiency while reinforcing Th1-type immune polarization through increased IL-12 secretion [ 101 ]. Distinct TLR combinations (e.g., TLR7/8, TLR2/4) have been demonstrated to exhibit synergistic effects via mechanisms such as enhancing NK cell antitumor activity or overcoming TGF-β1-mediated immunosuppression [ 102 , 103 ]. Furthermore, preclinical studies show that TLR agonists combined with other immunomodulators (e.g., CD40L) effectively suppress tumor progression by altering cellular composition within the TME (e.g., reducing CD11c + DCs while increasing CD8 + T cell infiltration) and promoting the differentiation of exhausted T cells into memory precursor cells [ 104 ]. These findings robustly demonstrate the broad potential of TLR agonist combinations with PD-1 inhibitors and other immunotherapies in inducing durable antitumor immune responses (Fig. 2 ).

Priming and expansion of T-cell

Activation of T cells

T-cell activation within lymph nodes plays a decisive role in cancer immunotherapy. Recently, novel neoadjuvant combination strategies based on dual blockade of the PD-1 and CTLA-4 signaling pathways have emerged as a research focus, with their mechanisms closely linked to the functional remodeling of T cells in lymph nodes. PD-1/PD-L1 checkpoint blockade has been demonstrated to enhance antitumor T-cell immune responses, while CTLA-4 regulates immune responses by suppressing the effector functions of T cells [ 105 ]. Studies have revealed that CTLA-4 checkpoint blockade significantly augments antitumor immune responses in tumor-draining lymph nodes (TDLNs), where CTLA-4 + tumor-specific T cells are densely colocalized with CTLA-4 + conventional dendritic cells (cDCs). Targeted blockade of CTLA-4 effectively unleashes the antitumor potential of these T cells [ 106 ]. Notably, CTLA-4 inhibitors exert their effects not only by blocking T-cell inhibitory signals but also through unique mechanisms in the TME that have been progressively untangled. Mechanistic studies indicate that anti-CTLA-4 antibodies selectively deplete Tregs via Fcγ receptor-mediated macrophage activation. This depletion effect, dependent on local microenvironmental features, significantly attenuates Treg-mediated suppression of effector T cells [ 107 ]. When combined with PD-1 blockade, this dual immunotherapy creates a spatial synergy: PD-1-mediated suppression during T-cell priming is alleviated in TDLNs, while Treg-driven immunosuppression is eliminated within the tumor, thereby systemically enhancing antitumor immune responses [ 108 ]. Mechanistic insights into this combination strategy provide critical implications for clinical trial design. Precise optimization of drug sequencing and administration routes, based on the spatiotemporal characteristics of distinct immune checkpoints, may represent a key breakthrough for improving therapeutic outcomes in solid tumors. Ongoing refinements of PD-1/CTLA-4 dual blockade therapies are propelling cancer immunotherapy into a new era of precision modulation.

Enhanced TCR clonal expansion and diversity

The clonal expansion and diversity of TCR represent a core mechanism by which the immune system recognizes and eliminates foreign antigens. The diversity of the TCR repertoire, generated through V(D)J gene rearrangement, enables the organism to recognize a broad spectrum of pathogens and tumor antigens. Meanwhile, dynamic changes in TCR clonality—such as dominant expansion or reduced diversity—have emerged as a critical biological window for evaluating responses to cancer immunotherapy. High-throughput TCR sequencing (TCR-seq) studies have untangled characteristic patterns of immune responses. Compared to healthy individuals, cancer patients exhibit significant heterogeneity in TCR repertoires across tumor tissues, adjacent normal tissues, and peripheral blood. These spatial distribution differences provide crucial insights into the dynamics of antitumor immunity [ 109 ]. Another study has revealed that neoadjuvant therapy leads to a 2- to 5-fold increase in the proportion of clonally expanded T cells in the peripheral blood of HNSCC patients, which correlated with improved 1-year recurrence-free survival [ 110 ]. Additionally, studies have demonstrated that a significantly higher frequency of TCR repertoire was detected in responders of HNSCC patients after cetuximab and nivolumab treatment [ 111 , 112 ]. Single-cell multi-omics studies further elucidate the systemic-local immune response relationship. The presence of circulating T cells and TILs sharing identical TCRαβ clonotypes indicates that certain peripheral T cell clones can home to the TME and exert effector functions. Transcriptomic profiling of these circulating T cells reveals that they do not enter a terminally exhausted state but retain activation potential, suggesting their utility as biomarkers for dynamically monitoring treatment responses [ 113 ]. These findings indicated that TCR profiling may propel cancer immunotherapy into a new era of precision assessment. Pretreatment peripheral blood exhibiting high clonality and high Shannon diversity in TCR repertoires was demonstrated to correlate with superior treatment outcomes across multiple cancer types [ 114 ]. In classical Hodgkin lymphoma (cHL), responders to PD-1 blockade exhibit a post-treatment surge in CD4 + T-cell diversity, with complete responders showing the most pronounced elevation [ 115 ]; Metastatic melanoma cohort analyses reveal that peripheral CD8 + T-cell clonal expansion post-ICI directly correlates with sustained clinical responses, suggesting clonality as a predictor of long-term efficacy [ 116 ]; For multiple myeloma, daratumumab—by targeting CD38 + immunoregulatory cells—induces synchronized TCR clonal expansion and CD8 + T-cell proliferation, mechanistically linking clonal dynamics to therapeutic benefit [ 93 , 94 ]. The CheckMate 358 trial further substantiates these findings in HPV-associated cancers: neoadjuvant pembrolizumab triggered HPV-E7-specific T-cell expansion in 78% of patients, significantly prolonging EFS and validating the clinical relevance of antigen-driven clonal amplification [ 117 ]. These reports indicated that integrated anlysis of single-cell RNA-seq and TCR-seq is a promising approach for predicting immune response and thus optimizes treatment plans (Fig. 3 ).

Remodeling the tumor microenvironment

Restoring CD8 + T cell infiltration and function

Remodeling the TME represents a pivotal strategy for enhancing the efficacy of cancer immunotherapy. Converting “cold tumors” into “hot tumors” can improve the immune system’s ability to recognize and attack tumors. The depth of T cell infiltration and their functional remodeling capacity within the TME are critical determinants of immunotherapy response efficacy. Studies indicate that the distribution patterns and functional activity of T cells in the TME are regulated at multiple levels [ 118 ]. Under persistent antigen stimulation, tumor-infiltrating CD8 + T cells exhibit a characteristic exhausted state, marked by the progressive upregulation of immune checkpoints such as TIM-3 and LAG-3, accompanied by a gradual loss of effector functions. This functional exhaustion can be partially reversed through ICI intervention. Anti-PD-1 monoclonal antibodies restore T cell priming by blocking the PD-1/PD-L1 signaling axis, thereby promoting CD8 + T cell proliferation and functional recovery, as well as enhancing the secretion of effector molecules such as IFN-γ and GZMB [ 119 ]. Notably, sustained PD-1 activation suppresses the PI3K-AKT-mTOR pathway, whereas PD-1 blockade restores its activity, significantly improving mitochondrial metabolism and glycolysis levels in exhausted T cells, thereby providing the energy required for functional reinvigoration [ 120 , 121 ]. Additionally, PD-1 blockade supports long-term T cell survival and functional maintenance by promoting fatty acid oxidation [ 122 ].

Co-expression of multiple checkpoints reveals heterogeneity in T cell exhaustion. The PD-1 + TIM-3 + double-positive CD8 + T cells in the TME exhibit a markedly suppressed functional state, characterized not only by reduced proliferative capacity but also by impaired cytokine production. In multiple cancer types, these double-positive cells are recognized as a hallmark of functional exhaustion. For instance, in renal cell carcinoma, the co-expression of PD-1 and TIM-3 on CD8 + T cells correlates with more aggressive tumor phenotypes and poorer clinical outcomes [ 123 ]. Similarly, in gastric cancer, PD-1 and TIM-3 co-expression is strongly associated with significant functional impairment of CD8 + T cells, evidenced by diminished IFN-γ production [ 124 ]. In a murine model of acute myeloid leukemia (AML), the co-expression of PD-1 and TIM-3 was also linked to CD8 + T cell exhaustion, rendering these cells incapable of effectively producing IFN-γ, TNF-α, or IL-2 upon encountering AML cells expressing PD-1 and TIM-3 ligands [ 125 ]. This exhausted state not only compromises cellular proliferation but also attenuates the efficacy of antitumor immune responses. Furthermore, in ovarian cancer, while PD-1 + TIM-3 + CD8 + T cells retain partial cytokine-producing capability, their cytotoxic activity is significantly impaired, which may contribute to unfavorable patient prognosis [ 126 ]. These findings collectively demonstrate that PD-1 and TIM-3 co-expression serves as a critical indicator of CD8 + T cell exhaustion in the TME. Targeting these pathways may represent a key strategy to enhance durable immune responses and improve the efficacy of cancer immunotherapy.

Breakthrough advances in single-cell omics and spatial transcriptomics have enabled researchers to delineate the dynamic behavioral characteristics of T cells at single-cell resolution. Studies revealed that CD8 + T cells exhibit a progressive infiltration trajectory from the tumor periphery toward the core following anti-PD-1 therapy [ 127 ]. This phenomenon is closely associated with increased expression of IFN-γ and the chemoattractant CXCL10 within tumor sites in response to anti-PD-1 treatment [ 128 ]. Furthermore, anti-PD-1 therapy reduces the population of TAMs, thereby diminishing their inhibitory effects on CD8 + T cells and further promoting CD8 + T cell infiltration [ 129 ]. Additional investigations identified therapy-induced T cell clusters forming “immune activation hotspots.” These regions are enriched with T cells expressing high levels of GZMB and IFN-γ, accompanied by elevated immune activity and cytokine release. The emergence of “immune activation hotspots” not only reflects the redistribution of T cells within tumors but also suggests that anti-PD-1-induced alterations in the TME may provide more favorable conditions for T cell survival and functional efficacy [ 130 , 131 ]. Consequently, the formation of these hotspots represents a pivotal event in antitumor immune responses, elucidating both the spatiotemporal dynamics of immune cells in the TME and novel strategies to enhance immunotherapeutic outcomes.

Further single-cell multi-omics studies have untangled the functional stratification of exhausted T cell subsets. A precursor exhausted T cell population (Tpex) exhibiting high expression of stemness-associated genes such as TCF-1 and CXCR5 demonstrates self-renewal capacity. These Tpex cells display a partially exhausted phenotype within the TME while retaining stem-like plasticity and cytotoxic potential [ 132 ]. Research indicates that Tpex cells can partially reactivate effector functions upon PD-1 pathway blockade [ 133 ], whereas terminally exhausted T cells exhibit poorer responsiveness [ 134 ]. Moreover, investigations into the developmental trajectory of exhausted T cells have delineated four distinct differentiation stages, including two TCF1 + precursor subsets: one tissue-resident and quiescent, and another more readily detectable in peripheral blood. With successive cell divisions, these precursor populations progressively lose TCF1 expression and transition into a third intermediate exhausted subset, which shows partial functional restoration upon PD-L1 blockade before ultimately differentiating into a fourth terminally exhausted subset [ 135 ]. These studies, through transcriptomic profiling, have elucidated the regulatory mechanisms underlying subset transitions, providing novel opportunities for improving cancer immunotherapy.

Recent studies have untangled the critical immunosurveillance role of tissue-resident memory T cells (TRM) in the tumor immune microenvironment, with their enrichment in the tumor bed showing a significant positive correlation with improved patient prognosis. Cohort analysis of patients with oropharyngeal squamous cell carcinoma undergoing neoadjuvant therapy revealed that the prolonged persistence (>6 months) of CD69 + CD103 + TRM cells in primary lesions was strongly associated with enhanced 5-year recurrence-free survival (HR = 0.24) [ 136 , 137 ]. The uniqueness of TRM cells lies in their ability to persist long-term in tissues and exert localized immunosurveillance functions. In hepatocellular carcinoma (HCC), the dynamic expansion of HBV-specific CD8 + TRM subsets is pivotal for antitumor immune responses. IL-15 plays a central role in sustaining TRM cell survival and function; these cells maintain self-renewal capacity via the IL-15-PI3K/mTOR pathway and release GZMB to enhance cytotoxic activity [ 138 ]. In lung cancer, M1-polarized TAMs recruit TRM cells by secreting chemoattractants such as CXCL9 and sustain their survival through the provision of essential fatty acids, thereby amplifying antitumor efficacy [ 139 ]. In summary, the contribution of TRM cells to tumor immunity is undeniable. Their presence not only correlates with superior clinical outcomes but may also offer translational avenues for improving durable immune responses.

Depletion of immunosuppressive cells

In the process of TME remodeling, targeted depletion of immunosuppressive Tregs has emerged as a critical breakthrough to improve immunotherapy responses. Studies have revealed that Tregs establish an immunosuppressive barrier by secreting TGF-β/IL-10 and upregulating PD-1/CTLA-4 double-positive TILs. Selective depletion of Tregs enhances the expression of activation markers CD69/GZMB in effector T cells (Teff) and significantly prolongs median progression-free survival [ 140 ]. However, systemic Treg depletion may lead to severe autoimmune diseases, highlighting the necessity of developing strategies to specifically target intratumoral Tregs [ 141 ]. A promising approach involves the selective clearance of CCR8 + intratumoral Tregs via FcγRIII + macrophage-mediated antibody-dependent cellular cytotoxicity (ADCC). Anti-CTLA-4 antibodies (e.g., ipilimumab) exploit this mechanism to preferentially deplete intratumoral Tregs [ 107 ]. Furthermore, combining anti-CTLA-4 therapy with other modalities, such as radiotherapy, enhances intratumoral Treg clearance [ 142 ]. This synergistic effect was demonstrated in the NCT03247712 trial, achieving a pCR rate of 67% [ 55 ]. Metabolic reprogramming of Tregs also represents a potential therapeutic target. Tregs utilize CD36-mediated fatty acid oxidation (FAO) to convert lactate into acetyl-CoA, sustaining their survival and function within the TME. CD36 is selectively upregulated in tumor-infiltrating Tregs and serves as a central metabolic regulator, fine-tuning mitochondrial adaptability through the peroxisome proliferator-activated receptor-β (PPAR-β) signaling pathway, enabling Tregs to thrive in lactate-rich TMEs [ 143 ]. Elevated CD36 expression correlates with the upregulation of fatty acid metabolism genes, a metabolic shift critical for Treg development. Targeting CD36 effectively downregulates Foxp3, a lineage-defining transcription factor for Tregs that modulates fatty acid metabolism and induces CD36 transcription [ 144 ]. This regulatory mechanism underscores the importance of CD36 in Treg metabolic adaptation within the TME and offers a potential therapeutic strategy to augment antitumor immunity via metabolic intervention. Additionally, Treg depletion may promote the formation of high endothelial venules (HEVs), vascular structures that facilitate lymphocyte recruitment and infiltration, thereby further enhancing immune responses [ 145 ]. In summary, direct elimination or functional modulation of Tregs can effectively convert “cold tumors” into “hot tumors,” improving immunotherapy efficacy in HNSCC. This strategy provides novel directions and possibilities for enhancing immunotherapy responsiveness.

In the study of immune reprogramming within the TME, disrupting the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) represents a critical pathway for converting “cold tumors” into “hot tumors.” In patients with HNSCC, the proportion of CD11b + CD33 + HLA-DR − MDSCs in peripheral blood is significantly elevated, consistent with observations in multiple cancer types [ 146 , 147 ]. MDSCs mediate immunosuppression through dual metabolic axes: the tryptophan-IDO1-kynurenine pathway and the arginine-Arg-1-urea cycle. The key enzyme in tryptophan metabolism, indoleamine 2,3-dioxygenase (IDO), can be targeted by its inhibitors to enhance immunotherapy efficacy by blocking tryptophan catabolism. Studies demonstrate that IDO inhibitors reprogram inflammatory processes, promote tumor clearance, and restore immune surveillance [ 148 ]. Furthermore, combining IDO inhibitors with ICBs effectively reverses Arg-1-driven T-cell dysfunction, thereby augmenting antitumor immunity [ 149 ]. Notably, MDSCs induce functional exhaustion of natural killer (NK) cells via the TGF-β/ROS signaling axis, a mechanism validated across multiple studies [ 150 ]. For instance, membrane-bound TGF-β1 from MDSCs suppresses NK-cell cytotoxicity and IFN-γ production, leading to significant functional impairment [ 151 ]. To enhance NK-cell infiltration and activity in tumors, MDSC migration may be blocked. CCR2 inhibitors (e.g., PF-04136309) effectively reduce MDSC trafficking, thereby improving NK-cell infiltration and antitumor function in the TME [ 152 ]. The CCR2/MCP-1 signaling pathway plays a pivotal role in MDSC recruitment; its inhibition markedly decreases MDSC accumulation in tumors and potentiates antitumor immunity [ 152 ]. Additionally, CCR2 pathway blockade not only limits MDSC migration but also enhances NK-cell antitumor activity by suppressing the IL-6-STAT3 pathway [ 153 ]. These findings collectively suggest that targeting MDSC migration and function can significantly improve NK-cell-mediated antitumor responses in HNSCC, offering novel directions for future immunotherapeutic strategies(FIGURE 4 ).

Disruption of stromal barrier and vascular abnormalities

Targeting the core signaling pathways of cancer-associated fibroblast (CAF)-mediated immunosuppressive barriers in the TME has emerged as a novel research direction to improve immune therapy responses in HNSCC. In HNSCC, FAP + CAFs establish a physical barrier through CXCL12 secretion, significantly inhibiting CD8 + T cell infiltration into the tumor core and diminishing the clinical response rate to ICBs [ 154 ]. The mechanism is directly associated with the hyperactivation of the CXCL12/CXCR4 signaling axis. As a well-documented pro-fibrotic and immunosuppressive pathway, this axis promotes immune evasion in solid tumors such as breast cancer by inducing stromal remodeling and T lymphocyte exclusion. Targeted blockade of the CXCL12/CXCR4 axis not only reduces tumor fibrosis but also significantly enhances intratumoral T-cell infiltration and improves responsiveness to immunotherapy, providing experimental evidence for combination treatment strategies in HNSCC [ 155 ]. Notably, the combined application of ICIs and CXCR4 inhibitors in the NIT setting demonstrates synergistic effects. This approach disrupts CAF-mediated CXCL12-dependent stromal barriers to facilitate T-cell transmigration while simultaneously normalizing tumor vasculature to enhance the trafficking efficiency of immune effector cells, thereby systemically activating antitumor immune responses [ 156 , 157 ]. Further studies reveal that the functional heterogeneity of CAFs extends to multidimensional regulation of TME homeostasis. For instance, the TGF-β signaling pathway can activate fibroblast transition into pro-tumorigenic CAF phenotypes, driving sustained CXCL12 secretion and forming a self-reinforcing immunosuppressive loop. Combined interventions targeting both the TGF-β pathway and the CXCL12/CXCR4 axis may more comprehensively remodel the immune microenvironment. Targeting these pathways, particularly the CXCL12/CXCR4 axis, offers a comprehensive strategy to reshape the TME and enhance the efficacy of cancer therapies. CXCL12/CXCR4 axis blockade-based combination strategies, through dual modulation of CAF-mediated physical barrier disruption and tumor vessel normalization, hold promise for overcoming the bottleneck of immune cell stromal migration. This approach ultimately enables deep intratumoral T-cell infiltration and functional activation, providing a critical scientific foundation for optimizing NIT regimens in HNSCC and other stroma-rich tumors.

In cancer therapies, the combination of VEGF inhibitors and PD-1 inhibitors demonstrates significant therapeutic potential. VEGF inhibitors drive tumor vascular normalization by suppressing pathological angiogenesis and restoring microvascular perfusion efficiency. This process not only optimizes intratumoral drug delivery but also significantly enhances the infiltration of effector immune cells (e.g., CD8 + T cells) into tumor parenchyma through upregulation of vascular endothelial adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1) [ 158 ]. Further studies indicate that VEGF inhibitors reverse the dysfunctional state of tumor endothelial cells and augment their responsiveness to inflammatory signals (e.g., IFN-γ/TNF-α), thereby improving leukocyte transendothelial migration efficiency and remodeling the tumor immune-inflammatory microenvironment [ 158 ]. When combined with PD-1 inhibitors, this strategy further enhances intratumoral lymphocyte homing and cytotoxic function by inducing the formation of HEVs, resulting in multidimensional immune activation [ 159 ]. Preclinical models have confirmed that dual VEGF/PD-1 blockade significantly increases the density of activated CD8 + T cells within tumors, promotes the polarization of TAMs toward the M1 antitumor phenotype, and suppresses the immunosuppressive activity of Tregs, thereby amplifying antitumor immune responses [ 160 ]. Preclinical studies and preliminary clinical trials (e.g., NCT04040361 , NCT04736823 ) have demonstrated that the VEGF/PD-1 co-inhibition strategy exhibits favorable safety profiles and objective response rates in neoadjuvant settings for solid tumors such as HNSCC. The dual mechanism of action provides an innovative approach for suppressing tumor metastasis and improving patient prognosis [ 161 ] (Fig. 5 ).

Current limitations in mechanistic studies and future research directions

Current studies have revealed that although circulating TILs are generally considered a favorable prognostic marker for cancer immunotherapy, the phenomenon that some patients with high TILs still fail to respond to ICBs warrants extensive investigation. Some reports suggest that the presence of TILs does not always correlate directly with the success of immunotherapy, which may be attributed to the complexity and heterogeneity of the TME [ 162 ]. Several hypotheses have been proposed: Immunosuppressive factors within the TME may impair TIL functionality. For instance, even in cases with abundant TILs, the presence of Tregs or other immunosuppressive cell populations may hinder their antitumor efficacy [ 163 ]. Additionally, tumor cells may evade immune surveillance by expressing immunosuppressive molecules such as PD-L1, thereby attenuating the antitumor activity of TILs [ 164 ]. The functional state and quality of TILs may be critical. Studies have demonstrated that dysfunctional or exhausted TILs, despite their numerical abundance, may fail to mediate effective antitumor immune responses [ 165 ]. Therefore, assessing the functional status and activity of TILs may provide a more reliable predictor of immunotherapy outcomes than mere quantification. Finally, the genomic characteristics of tumors, such as TMB and neoantigen diversity, may also influence TIL efficacy. Tumors with high mutational burden typically generate more neoantigens, potentially enhancing immune recognition and attack. However, excessive intratumoral heterogeneity may contribute to immune evasion, thereby diminishing the effectiveness of TILs [ 166 ]. In summary, although high TIL levels are generally regarded as a favorable prognostic marker in current immunotherapy, their efficacy is influenced by multiple factors, including the immunosuppressive state of the TME, the functional status of TILs, and the genomic features of the tumor. Thus, in-depth mechanistic studies on these factors are essential for improving prognostic monitoring in cancer immunotherapy and enhancing the success rate of NIT.

Current research on the interactions between the microbiome and cancer immunotherapy has expanded to encompass the modulation of ICIs. Interindividual heterogeneity in the microbiome may contribute to differential responses to ICI therapy [ 167 ]. In certain cases, the presence of specific commensal bacteria has been demonstrated to modulate the efficacy of ICIs, suggesting that microbiome manipulation could represent a viable strategy to enhance treatment response rates [ 168 ]. Of particular interest is the emerging role of the oral microbiota in HNSCC [ 169 ], especially regarding its potential impact on immunotherapy. Studies indicate that the oral microbiota may influence HNSCC pathogenesis and progression through multiple mechanisms, potentially affecting immunotherapy outcomes [ 170 ]. Dysbiosis of the oral microbiota is closely associated with HNSCC development. Research has identified significant enrichment of certain bacterial species (e.g., Fusobacterium and Peptostreptococcus) in HNSCC tumor tissues, while other bacterial populations are markedly reduced. These HNSCC-associated bacteria may promote tumorigenesis and progression by modulating host gene methylation and immune responses [ 171 ]. The oral microbiota may participate in carcinogenesis by influencing the TME and facilitating malignant transformation [ 172 ]. Interactions between the oral microbiota and the host immune system may impact immunotherapy efficacy. Notably, the oral microbiota can influence cancer immunotherapy outcomes—particularly in treatments involving PD-1 and CTLA-4 ICIs—by regulating host immune responses. Certain microbial species may exert positive or negative modulatory effects by either enhancing or interfering with immunotherapy effectiveness [ 167 ]. Furthermore, the oral microbiota may serve as biomarkers for immunotherapy response. Studies demonstrate that the composition of oral (salivary) microbiota correlates with NIT response in oral squamous cell carcinoma (OSCC) patients. Specifically, low abundance of particular bacterial taxa in saliva is associated with poor immunotherapy responsiveness, suggesting their potential utility as biomarkers for distinguishing responders from non-responders [ 173 ]. Similarly, research indicates that the immune microenvironment of HNSCC patients is closely linked to prognosis, with specific immune infiltration patterns potentially influencing immunotherapy sensitivity. Analysis of immune infiltration-related genes may predict patient responses to immunotherapy and provide novel prognostic biomarkers for HNSCC [ 174 ]. In summary, the oral microbiota plays multifaceted roles in HNSCC, potentially influencing tumorigenesis and immunotherapy outcomes through modulation of host immune responses and gene expression. Future studies should further elucidate the precise mechanisms by which oral microbiota affects HNSCC immunotherapy, thereby informing novel strategies for NIT.

Epigenetic interventions are playing an increasingly profound role in cancer therapies, particularly in augmenting the efficacy of immunotherapy. For instance, knock out experiments have demonstrated that EZH2 deficiency induces spontaneous autoimmunity, and modulation of EZH2 expression enhances antitumor immune responses during anti-CTLA-4 therapy. This provides a robust theoretical foundation for combining EZH2 inhibitors with anti-CTLA-4 treatment [ 175 ]. Furthermore, studies indicate that epigenetic drugs can reverse the immunosuppressive state of cancer cells, thereby improving the effectiveness of immunotherapy. Specifically, these drugs enhance tumor immunogenicity and promote immune cell activation, leading to improved outcomes with ICBs [ 176 ]. For example, DNA methylation inhibitors and histone deacetylase (HDAC) inhibitors have been shown to potentiate immunotherapy in certain cancers [ 177 ]. In the treatment of HNSCC, NIT combined with epigenetic modulation may emerge as an effective strategy. Preoperative administration of these therapies can reduce tumor burden, mitigate the risk of distant metastasis, and potentially improve OS [ 178 ]. However, identifying optimal patient selection and determining the most effective therapeutic combinations remain key research priorities [ 179 ]. In conclusion, the integration of epigenetic interventions with NIT offers new promise for HNSCC treatment. Future studies should further elucidate the underlying mechanisms and clinical applications of this combinatorial approach to enhance patient outcomes and quality of life.

The development of personalized vaccines has demonstrated significant potential in cancer immunotherapy, particularly neoantigen-based mRNA vaccines. Neoantigens are protein fragments generated by tumor-specific mutations that evade central tolerance and elicit robust immune responses. Recognized as “non-self” antigens by the immune system, they induce specific T-cell-mediated immunity [ 180 , 181 ]. This specificity renders neoantigens ideal targets for personalized cancer vaccines. In recent years, mRNA vaccines have emerged as a promising platform for cancer immunotherapy due to their high-efficiency antigen expression and rapid development cycle. By encoding neoantigens into mRNA and utilizing delivery systems such as lipid nanoparticles (LNPs), these vaccines effectively activate antigen-presenting cells (APCs), thereby stimulating potent T-cell responses [ 182 ]. Clinical trials have demonstrated the therapeutic potential of mRNA vaccines against various solid tumors, particularly when combined with ICIs, which significantly enhance antitumor immunity [ 183 ]. For instance, studies have shown that adjuvant therapy with mRNA-4157 in combination with pembrolizumab prolongs recurrence-free survival (RFS) in resected high-risk melanoma patients compared to pembrolizumab monotherapy, with a manageable safety profile [ 184 ]. Similarly, BNT113 ( NCT04534205 ), a neoantigen-based mRNA vaccine developed by BioNTech, demonstrated significantly improved objective response rates (ORR) in HPV16-positive, PD-L1-expressing R/M HNSCC patients when combined with neoadjuvant pembrolizumab in a phase II trial, compared to pembrolizumab alone. This combination strategy not only expands the antigen-specific T-cell repertoire but may also delay tumor recurrence and improve patient survival. These findings provide robust support for the clinical application of personalized cancer vaccines and offer promising directions for future NIT.

Conclusions and prospects

Neoadjuvant pembrolizumab has been confirmed to improve event-free survival among patients with locally advanced HNSCC, which laid the foundation for clinical use of NIT in HNSCC. More high-level evidence for combination stratege is urgently needed to enhance therapeutic efficacy as well as minimize adverse reactions. Additionally, evaluating the efficacy of NIT presents another major challenge. Clinical observations reveal frequent discrepancies between pathological and radiological responses following NIT, raising concerns about the accuracy of traditional response evaluation criteria in solid tumors (RECIST) criteria and complicating precise response evaluation. Future research must prioritize the personalization and precision of NIT to address these challenges,. On one hand, predictive models based on tumor genomic variation profiles, immune microenvironment characteristics, and host-microbiome interactions should be established to enable precise patient stratification. On the other hand, novel combination strategies should be developed to enhance immune response, including targeted modulation of the immune microenvironment, regulation of the oral microbiota-immune axis to improve PD-1 inhibitor sensitivity, and sequential therapies incorporating personalized neoantigen vaccines. These approaches will facilitate the transition of NIT from empirical broad-spectrum responses to precision-enhanced efficacy.

Supplementary Information

Supplementary Material 1.

Acknowledgements

We thank FIGDRAW for providing the useful online darwing tool.

Authors’ contributions

Bocheng Zhang, Haolei Tan, Bo Zhou, Xiao Pang, Shixuan Liang collected the related paper and drafted the manuscript. Hailiin Zhang, Ying Long, Yu Ouyang participated in the design of the review and review the manuscript. Bocheng Zhang, Jingyu Kuang, Zhang Zhang and Ying Long revised the manuscript and figures. All authors read and approved the final version of manuscript.

Funding

The present study was supported in part by National Natural Science Foundation of China (Grant No. 82273466), the Hunan Provincial Science and Technology Department (grant nos. 2023ZJ1120, Yu Ouyang), the Natural Science Foundation of Hunan Province (grant nos. 2024JJ9264, Haolei Tan, 2024JJ2039, Ying Long, 2024JJ8117, Hailin Zhang and 2023JJ60468, Bo Zhou), the Health Commission of Hunan Province (grant nos. 20230355, Ying Long), Hunan Cancer Hospital Climb Plan (No. ZX2021002, Hailin Zhang), and High-Level Talent Support Program of Hunan Cancer Hospital (grant nos. 20250730-1011, Zhang Zhang, and 20250731-1015, Ying Long).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Bocheng Zhang, Haolei Tan and Jingyu Kuang these authors contributed equally to this work.

Contributor Information

Yu Ouyang, Email: ouyangyu@hnca.org.cn.

Zhang Zhang, Email: zz_sunny@126.com.

Ying Long, Email: longying@hnca.org.cn.

Hailin Zhang, Email: Zhahali@163.com.

References

1. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74:229–63. 10.3322/caac.21834.

2. Gulati S, Desai J, Palackdharry SM, Morris JC, Zhu Z, Jandarov R, et al. Phase 1 dose-finding study of Metformin in combination with concurrent cisplatin and radiotherapy in patients with locally advanced head and neck squamous cell cancer. Cancer. 2019;126:354–62. 10.1002/cncr.32539.

3. Henke M, Alfonsi M, Foa P, Giralt J, Bardet E, Cerezo L, et al. Palifermin decreases severe oral mucositis of patients undergoing postoperative radiochemotherapy for head and neck cancer: a randomized, placebo-controlled trial. J Clin Oncol. 2011;29:2815–20. 10.1200/JCO.2010.32.4103.

4. Hutcheson KA, Lewin JS. Functional outcomes after chemoradiotherapy of laryngeal and pharyngeal cancers. Curr Oncol Rep. 2012;14:158–65. 10.1007/s11912-012-0216-1.

5. Lambert L, Fortin B, Soulières D, Guertin L, Coulombe G, Charpentier D, et al. Organ preservation with concurrent chemoradiation for advanced laryngeal cancer: are we succeeding? Int J Radiat Oncol. 2010;76:398–402. 10.1016/j.ijrobp.2009.01.058.

6. Hui ABY, Bruce JP, Alajez NM, Shi W, Yue S, Perez-Ordonez B, et al. Significance of dysregulated Metadherin and microRNA-375 in head and neck cancer. Clin Cancer Res. 2011;17:7539–50. 10.1158/1078-0432.CCR-11-2102.

7. Whiteside TL. Head and neck carcinoma immunotherapy: facts and hopes. Clin Cancer Res. 2018;24:6–13. 10.1158/1078-0432.CCR-17-1261.

8. Cavalieri S, Rivoltini L, Bergamini C, Locati LD, Licitra L, Bossi P. Immuno-oncology in head and neck squamous cell cancers: news from clinical trials, emerging predictive factors and unmet needs. Cancer Treat Rev. 2018;65:78–86. 10.1016/j.ctrv.2018.03.003.

9. Licitra L, Grandi C, Guzzo M, Mariani L, Lo Vullo S, Valvo F, et al. Primary chemotherapy in resectable oral cavity squamous cell cancer: a randomized controlled trial. J Clin Oncol. 2003;21:327–33. 10.1200/JCO.2003.06.146.

10. Zhong L, Zhang C, Ren G, Guo W, William WN, Sun J, et al. Randomized phase III trial of induction chemotherapy with docetaxel, cisplatin, and fluorouracil followed by surgery versus up-front surgery in locally advanced resectable oral squamous cell carcinoma. J Clin Oncol. 2013;31:744–51. 10.1200/JCO.2012.43.8820.

11. Chaukar D, Prabash K, Rane P, Patil VM, Thiagarajan S, Ghosh-Laskar S, et al. Prospective phase II open-label randomized controlled trial to compare mandibular preservation in upfront surgery with neoadjuvant chemotherapy followed by surgery in operable oral cavity cancer. J Clin Oncol. 2022;40:272–81. 10.1200/JCO.21.00179.

12. Vonderheide RH. The immune revolution: a case for priming, not checkpoint. Cancer Cell. 2018;33:563–9. 10.1016/j.ccell.2018.03.008.

13. Ribas A, Wolchok JD. Cancer immunotherapy using checkpoint blockade. Science. 2018;359:1350–5. 10.1126/science.aar4060.

14. Topalian SL, Weiner GJ, Pardoll DM. Cancer immunotherapy comes of age. J Clin Oncol. 2011;29:4828–36. 10.1200/JCO.2011.38.0899.

15. Lauret Marie Joseph E, Kirilovsky A, Lecoester B, El Sissy C, Boullerot L, Rangan L, et al. Chemoradiation triggers antitumor Th1 and tissue resident memory-polarized immune responses to improve immune checkpoint inhibitors therapy. J Immunother Cancer. 2021;9:e002256. 10.1136/jitc-2020-002256.

16. Uppaluri R, Campbell KM, Egloff AM, et al. Neoadjuvant and adjuvant pembrolizumab in resectable locally advanced, human papillomavirus-unrelated head and neck cancer: a multicenter, phase II trial. Clin Cancer Res. 2020;26(19):5140–52. 10.1158/1078-0432.CCR-20-1695.

17. Ferris RL, Spanos WC, Leidner R, Gonçalves A, Martens UM, Kyi C, et al. Neoadjuvant nivolumab for patients with resectable HPV-positive and HPV-negative squamous cell carcinomas of the head and neck in the CheckMate 358 trial. J Immunother Cancer. 2021;9:e002568. 10.1136/jitc-2021-002568.

18. Ferrarotto R, Bell D, Rubin ML, Hutcheson KA, Johnson JM, Goepfert RP, et al. Impact of neoadjuvant durvalumab with or without tremelimumab on CD8 + tumor lymphocyte density, safety, and efficacy in patients with oropharynx cancer: CIAO trial results. Clin Cancer Res. 2020;26:3211–9. 10.1158/1078-0432.CCR-19-3977.

19. Zuur L, Vos JL, Elbers JB, Krijgsman O, Qiao X, van der Leun A, et al. LBA40 Neoadjuvant nivolumab and nivolumab plus ipilimumab induce (near-) complete responses in patients with head and neck squamous cell carcinoma: The IMCISION trial. Ann Oncol. 2020;31:S1169-9. 10.1016/j.annonc.2020.08.2270.

20. Zheng S, Feng Y, Li C, Zhang J, Xie K. Induction therapy for locally advanced head and neck squamous cell carcinoma. Oncol Ther. 2023;11:185–98. 10.1007/s40487-023-00226-7.

21. Wang H, Zheng Z, Zhang Y, Bian C, Bao J, Xin Y, et al. Locally advanced head and neck squamous cell carcinoma treatment efficacy and safety: a systematic review and network meta-analysis. Front Pharmacol. 2023;14:1269863. 10.3389/fphar.2023.1269863.

22. Mollnar S, Pondorfer P, Kasparek A-K, Reinisch S, Moik F, Stotz M, et al. Decrease in treatment intensity predicts worse outcome in patients with locally advanced head and neck squamous cell carcinoma undergoing radiochemotherapy. Clin Transl Oncol. 2020;23:543–53. 10.1007/s12094-020-02447-y.

23. Nindra U, Hurwitz J, Forstner D, Chin V, Gallagher R, Liu J. A systematic review of neoadjuvant and definitive immunotherapy in locally advanced head and neck squamous cell carcinoma. Cancer Med. 2023;12:11234–47. 10.1002/cam4.5815.

24. Zhu X, Qiu J, Zhang Y, Lin C, Wang X, Shi X, et al. Neoadjuvant chemoimmunotherapy for locally advanced squamous cell carcinoma of the head and neck: systematic review and meta-analysis. Pharmacol Res. 2025;212:107598. 10.1016/j.phrs.2025.107598.

25. Rosenberg AJ, Juloori A, Jelinek MJ, Agrawal N, Cursio JF, Cipriani N, et al. Neoadjuvant nivolumab plus chemotherapy followed by response-stratified chemoradiation therapy in HPV-negative head and neck cancer: the DEPEND phase 2 nonrandomized clinical trial. JAMA Oncol. 2025;e250081. 10.1001/jamaoncol.2025.0081.

26. Colevas AD. Systemic therapy for metastatic or recurrent squamous cell carcinoma of the head and neck. J Natl Compr Canc Netw. 2015;13:e37–48. 10.6004/jnccn.2015.0080.

27. Noronha V, Patil V, Chaturvedi P, Mathrudev V, Menon N, Bhattacharjee A, et al. Phase 3 RCT comparing docetaxel-platinum with docetaxel-platinum-5FU as neoadjuvant chemotherapy in borderline resectable oral cancer. Eur J Cancer. 2024;200:113560. 10.1016/j.ejca.2024.113560.

28. Haddad RI, Seiwert TY, Chow LQM, Gupta S, Weiss J, Gluck I, et al. Influence of tumor mutational burden, inflammatory gene expression profile, and PD-L1 expression on response to pembrolizumab in head and neck squamous cell carcinoma. J Immunother Cancer. 2022;10:e003026. 10.1136/jitc-2021-003026.

29. Harrington KJ, Ferris RL, Blumenschein G, Colevas AD, Fayette J, Licitra L, et al. Nivolumab versus standard, single-agent therapy of investigator’s choice in recurrent or metastatic squamous cell carcinoma of the head and neck (CheckMate 141): health-related quality-of-life results from a randomised, phase 3 trial. Lancet Oncol. 2017;18:1104–15. 10.1016/S1470-2045(17)30421-7.

30. Cohen EEW, Soulières D, Le Tourneau C, Dinis J, Licitra L, Ahn M-J, et al. Pembrolizumab versus methotrexate, docetaxel, or cetuximab for recurrent or metastatic head-and-neck squamous cell carcinoma (KEYNOTE-040): a randomised, open-label, phase 3 study. Lancet. 2019;393:156–67. 10.1016/S0140-6736(18)31999-8.

31. Burtness B, Harrington KJ, Greil R, Soulières D, Tahara M, de Castro G, et al. Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048): a randomised, open-label, phase 3 study. Lancet. 2019;394:1915–28. 10.1016/S0140-6736(19)32591-7.

32. Ferris RL, Haddad R, Even C, Tahara M, Dvorkin M, Ciuleanu TE, et al. Durvalumab with or without tremelimumab in patients with recurrent or metastatic head and neck squamous cell carcinoma: EAGLE, a randomized, open-label phase III study. Ann Oncol. 2020;31:942–50. 10.1016/j.annonc.2020.04.001.

33. Ferris RL, Gooding WE, Chiosea SI, et al. Neoadjuvant nivolumab alone or in combination with relatlimab or ipilimumab in resectable head and neck squamous cell carcinoma (HNSCC) [J]. J Clin Oncol. 2023;41(16suppl):6018. 10.1200/JCO.2023.41.16_suppl.6018.

34. Uppaluri R, Campbell KM, Egloff AM, Zolkind P, Skidmore ZL, Nussenbaum B, et al. Neoadjuvant and adjuvant pembrolizumab in resectable locally advanced, human papillomavirus–unrelated head and neck cancer: a multicenter, phase II trial. Clin Cancer Res. 2020;26:5140–52. 10.1158/1078-0432.CCR-20-1695.

35. Masarwy R, Kampel L, Horowitz G, Gutfeld O, Muhanna N. Neoadjuvant PD-1/PD-L1 inhibitors for resectable head and neck cancer: a systematic review and meta-analysis. JAMA Otolaryngol Head Neck Surg. 2021;147:871. 10.1001/jamaoto.2021.2191.

36. Mays AC, Yarlagadda B, Achim V, Jackson R, Pipkorn P, Huang AT, et al. Examining the relationship of immunotherapy and wound complications following flap reconstruction in patients with head and neck cancer. Head Neck. 2021;43:1509–20. 10.1002/hed.26601.

37. Wise-Draper TM, Takiar V, Mierzwa ML, Casper K, Palackdharry S, Worden FP, et al. Association of pathological response to neoadjuvant pembrolizumab with tumor PD-L1 expression and high disease-free survival (DFS) in patients with resectable, local-regionally advanced, head and neck squamous cell carcinoma (HNSCC). J Clin Oncol. 2021;39:6006–6006. 10.1200/JCO.2021.39.15_suppl.6006.

38. Bernier J, Domenge C, Ozsahin M, Matuszewska K, Lefèbvre JL, Greiner RH, et al. European organization for research and treatment of cancer trial 22931. Postoperative irradiation with or without concomitant chemotherapy for locally advanced head and neck cancer. N Engl J Med. 2004;350(19):1945–52. 10.1056/NEJMoa032641.

39. Knochelmann HM, Horton JD, Liu S, Armeson K, Kaczmar JM, Wyatt MM, et al. Neoadjuvant presurgical PD-1 inhibition in oral cavity squamous cell carcinoma. Cell Rep Med. 2021;2:100426. 10.1016/j.xcrm.2021.100426.

40. Le X, Ferrarotto R, Wise-Draper T, Gillison M. Evolving role of immunotherapy in recurrent metastatic head and neck cancer. J Natl Compr Canc Netw. 2020;18:899–906. 10.6004/jnccn.2020.7590.

41. Cramer JD, Burtness B, Le QT, Ferris RL. The changing therapeutic landscape of head and neck cancer. Nat Rev Clin Oncol. 2019;16:669–83. 10.1038/s41571-019-0227-z.

42. Vasudevan HN, Yom SS. Combining systemic therapy with radiation: head and neck cancer treatments in an era of targeted agents and immunotherapy. J Natl Compr Canc Netw. 2020;18:907–13. 10.6004/jnccn.2020.7594.

43. Vos JL, Elbers JBW, Krijgsman O, Traets JJH, Qiao X, Van Der Leun AM, et al. Neoadjuvant immunotherapy with nivolumab and ipilimumab induces major pathological responses in patients with head and neck squamous cell carcinoma. Nat Commun. 2021;12:7348. 10.1038/s41467-021-26472-9.

44. Cha J, Kim DH, Kim G, et al. Single-cell analysis reveals cellular and molecular factors counteracting HPV-positive oropharyngeal cancer immunotherapy outcomes. J Immunother Cancer. 2024;12(6):e008667. 10.1136/jitc-2023-008667.

45. Ferrarotto R, Amit M, Nagarajan P, Rubin ML, Yuan Y, Bell D, et al. Pilot phase II trial of neoadjuvant immunotherapy in locoregionally Advanced, resectable cutaneous squamous cell carcinoma of the head and neck. Clin Cancer Res. 2021;27:4557–65. 10.1158/1078-0432.CCR-21-0585.

46. Luoma AM, Suo S, Wang Y, Gunasti L, Porter CBM, Nabilsi N, et al. Tissue-resident memory and Circulating T cells are early responders to pre-surgical cancer immunotherapy. Cell. 2022;185:2918–e293529. 10.1016/j.cell.2022.06.018.

47. Ju W-T, Xia R-H, Zhu D-W, Dou S-J, Zhu G-P, Dong M-J, et al. A pilot study of neoadjuvant combination of anti-PD-1 camrelizumab and VEGFR2 inhibitor apatinib for locally advanced resectable oral squamous cell carcinoma. Nat Commun. 2022;13:5378. 10.1038/s41467-022-33080-8.

48. Rahim MK, Okholm TLH, Jones KB, McCarthy EE, Liu CC, Yee JL, et al. Dynamic CD8 + T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes. Cell. 2023;186:1127–e114318. 10.1016/j.cell.2023.02.021.

49. Hecht M, Gostian AO, Eckstein M, Rutzner S, von der Grün J, Illmer T, et al. Safety and efficacy of single cycle induction treatment with cisplatin/docetaxel/ durvalumab/tremelimumab in locally advanced HNSCC: first results of CheckRad-CD8. J Immunother Cancer. 2020;8:e001378. 10.1136/jitc-2020-001378.

50. Huang X, Liu Q, Zhong G, Peng Y, Liu Y, Liang L, et al. Neoadjuvant Toripalimab combined with gemcitabine and cisplatin in resectable locally advanced head and neck squamous cell carcinoma (NeoTGP01): an open label, single-arm, phase Ib clinical trial. J Exp Clin Cancer Res. 2022;41:300. 10.1186/s13046-022-02510-2.

51. Zhao X, Zhou Y, Peng G, Wen L, Hong X, Hu Y, et al. Final analysis of a phase II trial of neoadjuvant chemoimmunotherapy for locoregionally advanced head and neck squamous cell carcinoma. Oral Oncol. 2024;156:106918. 10.1016/j.oraloncology.2024.106918.

52. Wang K, Gui L, Lu H, He X, Li D, Liu C, et al. Efficacy and safety of pembrolizumab with preoperative neoadjuvant chemotherapy in patients with resectable locally advanced head and neck squamous cell carcinomas. Front Immunol. 2023;14:1189752. 10.3389/fimmu.2023.1189752.

53. Wang H, Wang X, Zhou X, Li Y, Wang P, Zhang X, et al. Neoadjuvant PD-1 inhibitor combined with Nab-paclitaxel and cisplatin in resectable locally advanced head and neck squamous cell carcinoma ( NCT05522985 ): A randomized, controlled, open label, phase II clinical trial. Ann Oncol. 2023;34:S571–2. 10.1016/j.annonc.2023.09.2040.

54. Liu C, Li M, Liu X, Shi T, Wang Y, Sui C, et al. Evaluating the efficacy and safety of different neoadjuvant immunotherapy combinations in locally advanced HNSCC: a systematic review and meta-analysis. Front Immunol. 2024;15:1467306. 10.3389/fimmu.2024.1467306.

55. Leidner R, Crittenden M, Young K, Xiao H, Wu Y, Couey MA, et al. Neoadjuvant immunoradiotherapy results in high rate of complete pathological response and clinical to pathological downstaging in locally advanced head and neck squamous cell carcinoma. J Immunother Cancer. 2021;9:e002485. 10.1136/jitc-2021-002485.

56. Darragh LB, Knitz MM, Hu J, Clambey ET, Backus J, Dumit A, et al. A phase I/Ib trial and biological correlate analysis of neoadjuvant SBRT with single-dose durvalumab in HPV-unrelated locally advanced HNSCC. Nat Cancer. 2022;3:1300–17. 10.1038/s43018-022-00450-6.

57. Jm J, Ia V, La H, V AA. Nivolumab and ipilimumab in combination with radiotherapy in patients with high-risk locally advanced squamous cell carcinoma of the head and neck. J Immunother Cancer. 2023;11:e007141. 10.1136/jitc-2023-007141.

58. Redman JM, Friedman J, Robbins Y, Sievers C, Yang X, Lassoued W, et al. Enhanced neoepitope-specific immunity following neoadjuvant PD-L1 and TGF-β blockade in HPV-unrelated head and neck cancer. J Clin Invest. 2022;132:e161400. 10.1172/JCI161400.

59. Wong W, Cracchiolo JR, Riaz N, et al. Neoadjuvant Cemiplimab with platinum-doublet chemotherapy and cetuximab to de‐escalate surgery and omit adjuvant radiation in locoregionally advanced head & neck squamous cell carcinoma (HNSCC) [J]. J Clin Oncol. 2023;41(16suppl):6019. 10.1200/JCO. 2023.41.16_ suppl.6019. n.d.

60. Siu LL, Even C, Le Tourneau C, et al. Phase II study of neoadjuvant pembrolizumab plus cetuximab in resectable hypopharyngeal squamous cell carcinoma (EACH trial): Biomarker correlates of response. Annals of Oncology. 2022;33(7):S1428. 10.1016/annonc/annonc1103. n.d.

61. Shi Y, Ma X, He D, Dong B, Qiao T. Neoadjuvant SBRT combined with immunotherapy in NSCLC: from mechanisms to therapy. Front Immunol. 2023;14:1213222. 10.3389/fimmu.2023.1213222.

62. Zhang H, Ye L, Yu X, Jin K, Wu W. Neoadjuvant therapy alters the immune microenvironment in pancreatic cancer. Front Immunol. 2022;13:956984. 10.3389/fimmu.2022.956984.

63. Moreira D, Sampath S, Won H, White SV, Su Y-L, Alcantara M, et al. Myeloid cell-targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell-mediated immunity. J Clin Invest. 2021;131:e137001. 10.1172/JCI137001.

64. Economopoulou P, Kotsantis I, Psyrri A. The promise of immunotherapy in head and neck squamous cell carcinoma: combinatorial immunotherapy approaches. ESMO Open. 2016;1:e000122. 10.1136/esmoopen-2016-000122.

65. Qin H, Liu F, Zhang Y, Liang Y, Mi Y, Yu F, et al. Comparison of neoadjuvant immunotherapy versus routine neoadjuvant therapy for patients with locally advanced esophageal cancer: a systematic review and meta-analysis. Front Immunol. 2023;14:1108213. 10.3389/fimmu.2023.1108213.

66. Liu Y, Bao Y, Yang X, Sun S, Yuan M, Ma Z, et al. Efficacy and safety of neoadjuvant immunotherapy combined with chemoradiotherapy or chemotherapy in esophageal cancer: a systematic review and meta-analysis. Front Immunol. 2023;14:1117448. 10.3389/fimmu.2023.1117448.

67. Wise-Draper T, Gibson MK, Ferrarotto R, et al. Phase Ib/II neoadjuvant pembrolizumab with chemotherapy in local-regionally advanced head and neck squamous cell carcinoma: immunotherapy assessment of surgical specimens (IMPASSE). J Clin Oncol. 2022;40(16suppl):6007. 10.1200/JCO.2022.40.16_suppl.6007.

68. Hughes PE, Caenepeel S, Wu LC. Targeted therapy and checkpoint immunotherapy combinations for the treatment of cancer. Trends Immunol. 2016;37:462–76. 10.1016/j.it.2016.04.010.

69. Errico A. PD-1-PD-L1 axis: efficient checkpoint blockade against cancer. Nat Rev Clin Oncol. 2014;12:63–63. 10.1038/nrclinonc.2014.221.

70. Ferris RL, Blumenschein G, Fayette J, Guigay J, Colevas AD, Licitra L, et al. Nivolumab for recurrent squamous-cell carcinoma of the head and neck. N Engl J Med. 2016;375:1856–67. 10.1056/NEJMoa1602252.

71. Zhao M, Schoenfeld JD, Egloff AM, Hanna GJ, Haddad RI, Adkins DR, et al. T cell dynamics with neoadjuvant immunotherapy in head and neck cancer. Nat Rev Clin Oncol. 2024;22:83–94. 10.1038/s41571-024-00969-w.

72. Patel SP, Othus M, Chen Y, Wright GP, Yost KJ, Hyngstrom JR, et al. Neoadjuvant-adjuvant or adjuvant-only pembrolizumab in advanced melanoma. N Engl J Med. 2023;388:813–23. 10.1056/NEJMoa2211437.

73. Uppaluri R, Haddad RI, Tao Y, Le Tourneau C, Lee NY, Westra W, et al. Neoadjuvant and adjuvant pembrolizumab in locally advanced head and neck cancer. N Engl J Med. 2025;393:37–50. 10.1056/NEJMoa2415434.

74. He F, Wu Z, Liu C, Zhu Y, Zhou Y, Tian E, et al. Targeting BCL9/BCL9L enhances antigen presentation by promoting conventional type 1 dendritic cell (cDC1) activation and tumor infiltration. Signal Transduct Target Ther. 2024;9:139. 10.1038/s41392-024-01838-9.

75. Ascic E, Åkerström F, Sreekumar Nair M, Rosa A, Kurochkin I, Zimmermannova O, et al. In vivo dendritic cell reprogramming for cancer immunotherapy. Science. 2024;386:eadn9083. 10.1126/science.adn9083.

76. Ma HS, Poudel B, Torres ER, Sidhom J-W, Robinson TM, Christmas B, et al. A CD40 agonist and PD-1 antagonist antibody reprogram the microenvironment of nonimmunogenic tumors to allow T-cell-mediated anticancer activity. Cancer Immunol Res. 2019;7:428–42. 10.1158/2326-6066.CIR-18-0061.

77. Horn LA, Fousek K, Hamilton DH, Hodge JW, Zebala JA, Maeda DY, et al. Vaccine increases the diversity and activation of intratumoral T cells in the context of combination immunotherapy. Cancers (Basel). 2021;13:968. 10.3390/cancers13050968.

78. Höchst B, Diehl L. Antigen shedding into the circulation contributes to tumor immune escape. Oncoimmunology. 2012;1:1620–2. 10.4161/onci.21514.

79. Meyaard L, Smyth MJ. Liberating tumor immunity. Curr Opin Immunol. 2012;24:204–6. 10.1016/j.coi.2012.03.001.

80. X DT. Inducing Immunogenic cell death in immuno-oncological therapies. Chin J Cancer Res. 2022;34:1–10. 10.21147/j.issn.1000-9604.2022.01.01.

81. Krysko DV, Garg AD, Kaczmarek A, Krysko O, Agostinis P, Vandenabeele P. Immunogenic cell death and DAMPs in cancer therapy. Nat Rev Cancer. 2012;12:860–75. 10.1038/nrc3380.

82. Woo S-R, Fuertes MB, Corrales L, Spranger S, Furdyna MJ, Leung MYK, et al. STING-dependent cytosolic DNA sensing mediates innate immune recognition of Immunogenic tumors. Immunity. 2014;41:830–42. 10.1016/j.immuni.2014.10.017.

83. Deng L, Liang H, Xu M, Yang X, Burnette B, Arina A, et al. STING-dependent cytosolic DNA sensing promotes radiation-induced type I interferon-dependent antitumor immunity in immunogenic tumors. Immunity. 2014;41:843–52. 10.1016/j.immuni.2014.10.019.

84. Ho SSW, Zhang WYL, Tan NYJ, Khatoo M, Suter MA, Tripathi S, et al. The DNA Structure-Specific endonuclease MUS81 mediates DNA sensor STING-Dependent host rejection of prostate cancer cells. Immunity. 2016;44:1177–89. 10.1016/j.immuni.2016.04.010.

85. Donnelly CR, Jiang C, Andriessen AS, Wang K, Wang Z, Ding H, et al. STING controls nociception via type i interferon signalling in sensory neurons. Nature. 2021;591:275–80. 10.1038/s41586-020-03151-1.

86. Carroll EC, Jin L, Mori A, Muñoz-Wolf N, Oleszycka E, Moran HBT, et al. The vaccine adjuvant chitosan promotes cellular immunity via DNA sensor cGAS-STING-dependent induction of type I interferons. Immunity. 2016;44:597–608. 10.1016/j.immuni.2016.02.004.

87. Rizvi H, Sanchez-Vega F, La K, Chatila W, Jonsson P, Halpenny D, et al. Molecular determinants of response to anti-programmed cell death (PD)-1 and anti-programmed death-ligand 1 (PD-L1) blockade in patients with non-small-cell lung cancer profiled with targeted next-generation sequencing. J Clin Oncol. 2018;36:633–41. 10.1200/JCO.2017.75.3384.

88. Samstein RM, Lee C-H, Shoushtari AN, Hellmann MD, Shen R, Janjigian YY, et al. Tumor mutational load predicts survival after immunotherapy across multiple cancer types. Nat Genet. 2019;51:202–6. 10.1038/s41588-018-0312-8.

89. Cao H, Lan T, Kuang S, Wang L, Li J, Li Q, et al. FAT1 as a tumor mutation burden specific gene affects the immunotherapy effect in head and neck squamous cell cancer. Drug Resist Updat. 2024;76:101095. 10.1016/j.drup.2024.101095.

90. Duhen R, Ballesteros-Merino C, Frye AK, Tran E, Rajamanickam V, Chang S-C, et al. Neoadjuvant anti-OX40 (MEDI6469) therapy in patients with head and neck squamous cell carcinoma activates and expands antigen-specific tumor-infiltrating T cells. Nat Commun. 2021;12:1047. 10.1038/s41467-021-21383-1.

91. Kansy BA, Shayan G, Jie H-B, Gibson SP, Lei YL, Brandau S, et al. T cell receptor richness in peripheral blood increases after cetuximab therapy and correlates with therapeutic response. Oncoimmunology. 2018;7:e1494112. 10.1080/2162402X.2018.1494112.

92. Bhatia S, Topalian SL, Sharfman W, Meyer T, Steven N, Lao CD, et al. Nivolumab with or without ipilimumab in patients with recurrent or metastatic Merkel cell carcinoma: a nonrandomized, open-label, international, multicenter phase I/II study. J Clin Oncol. 2025. 10.1200/JCO-24-02138.

93. Peng Q, Qiu X, Zhang Z, Zhang S, Zhang Y, Liang Y, et al. PD-L1 on dendritic cells attenuates T cell activation and regulates response to immune checkpoint blockade. Nat Commun. 2020;11:4835. 10.1038/s41467-020-18570-x.

94. Mayoux M, Roller A, Pulko V, Sammicheli S, Chen S, Sum E, et al. Dendritic cells dictate responses to PD-L1 blockade cancer immunotherapy. Sci Transl Med. 2020;12:eaav7431. 10.1126/scitranslmed.aav7431.

95. Roberts EW, Broz ML, Binnewies M, Headley MB, Nelson AE, Wolf DM, et al. Critical role for CD103(+)/CD141(+) dendritic cells bearing CCR7 for tumor antigen trafficking and priming of T cell immunity in melanoma. Cancer Cell. 2016;30:324–36. 10.1016/j.ccell.2016.06.003.

96. Sugiura D, Maruhashi T, Okazaki I-M, Shimizu K, Maeda TK, Takemoto T, et al. Restriction of PD-1 function by cis-PD-L1/CD80 interactions is required for optimal T cell responses. Science. 2019;364:558–66. 10.1126/science.aav7062.

97. Maier B, Leader AM, Chen ST, Tung N, Chang C, LeBerichel J, et al. A conserved dendritic-cell regulatory program limits antitumour immunity. Nature. 2020;580:257–62. 10.1038/s41586-020-2134-y.

98. He J, Shen J, Luo W, et al. Research progress on application of single-cell TCR/BCR sequencing technology to the tumor immune microenvironment, autoimmune diseases, and infectious diseases. Front Immunol. 2022;13:969808. 10.3389/fimmu.2022.969808.

99. Li S, Yao JC, Li JT, Schmidt AP, Link DC. TLR7/8 agonist treatment induces an increase in bone marrow resident dendritic cells and hematopoietic progenitor expansion and mobilization. Exp Hematol. 2021;96:35–43.e7. 10.1016/j.exphem.2021.02.001.

100. Mullins SR, Vasilakos JP, Deschler K, et al. Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies. J Immunother Cancer. 2019;7(1):244. 10.1186/s40425-019-0724-8.

101. Das S, Rani M, Rabidas V, Pandey K, Sahoo GC, Das P. TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin-miltefosine combination therapy in visceral leishmaniasis. Br J Pharmacol. 2014;171:1260–74. 10.1111/bph.12530.

102. Kim H, Khanna V, Kucaba TA, Zhang W, Sehgal D, Ferguson DM, et al. TLR7/8 agonist-loaded nanoparticles augment NK cell-mediated antibody-based cancer immunotherapy. Mol Pharm. 2020;17:2109–24. 10.1021/acs.molpharmaceut.0c00271.

103. Koido S, Homma S, Okamoto M, Namiki Y, Takakura K, Uchiyama K, et al. The combination of TLR2 and TLR4 agonists promotes the immunogenicity of dendritic cell/cancer cell fusions. Oncoimmunology. 2013;2:e24660. 10.4161/onci.24660.

104. Stone GW, Barzee S, Snarsky V, Santucci C, Tran B, Langer R, et al. Nanoparticle-delivered multimeric soluble CD40L DNA combined with toll-like receptor agonists as a treatment for melanoma. PLoS ONE. 2009;4:e7334. 10.1371/journal.pone.0007334.

105. Buchbinder E, Hodi FS. Cytotoxic t lymphocyte antigen-4 and immune checkpoint blockade. J Clin Invest. 2015;125:3377–83. 10.1172/JCI80012.

106. Dammeijer F, van Gulijk M, Mulder EE, Lukkes M, Klaase L, van den Bosch T, et al. The PD-1/PD-L1-Checkpoint restrains T cell immunity in Tumor-Draining lymph nodes. Cancer Cell. 2020;38:685–e7008. 10.1016/j.ccell.2020.09.001.

107. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med. 2013;210:1695–710. 10.1084/jem.20130579.

108. Duraiswamy J, Freeman GJ, Coukos G. Dual blockade of PD-1 and CTLA-4 combined with tumor vaccine effectively restores T-cell rejection function in tumors–response. Cancer Res. 2014;74:633–4. 10.1158/0008-5472.CAN-13-2752.

109. Li N, Yuan J, Tian W, Meng L, Liu Y. T-cell receptor repertoire analysis for the diagnosis and treatment of solid tumor: a methodology and clinical applications. Cancer Commun (Lond). 2020;40(10):473–83. 10.1002/cac2.12074.

110. Zhigalova EA, Izosimova AI, Yuzhakova DV, Volchkova LN, Shagina IA, Turchaninova MA, et al. RNA-seq-based TCR profiling reveals persistently increased intratumoral clonality in responders to anti-PD-1 therapy. Front Oncol. 2020;10:385. 10.3389/fonc.2020.00385.

111. Ge H, Ferris RL, Wang JH. Cetuximab responses in patients with HNSCC correlate to clonal expansion feature of peripheral and Tumor-Infiltrating T cells with top T-Cell receptor clonotypes. Clin Cancer Res. 2022;29:647–58. 10.1158/1078-0432.CCR-22-2355.

112. Wang X, Muzaffar J, Kirtane K, Song F, Johnson M, Schell MJ, et al. T cell repertoire in peripheral blood as a potential biomarker for predicting response to concurrent cetuximab and nivolumab in head and neck squamous cell carcinoma. J Immunother Cancer. 2022;10:e004512. 10.1136/jitc-2022-004512.

113. Lucca LE, Axisa P-P, Lu B, Harnett B, Jessel S, Zhang L, et al. Circulating clonally expanded T cells reflect functions of tumor-infiltrating T cells. J Exp Med. 2021;218:e20200921. 10.1084/jem.20200921.

114. Joshi K, Milighetti M, Chain BM. Application of T cell receptor (TCR) repertoire analysis for the advancement of cancer immunotherapy. Curr Opin Immunol. 2022;74:1–8. 10.1016/j.coi.2021.07.006.

115. Cader FZ, Hu X, Goh WL, Wienand K, Ouyang J, Mandato E, et al. A peripheral immune signature of responsiveness to PD-1 blockade in patients with classical hodgkin lymphoma. Nat Med. 2020;26:1468–79. 10.1038/s41591-020-1006-1.

116. Fairfax BP, Taylor CA, Watson RA, Nassiri I, Danielli S, Fang H, et al. Peripheral CD8 + T cell characteristics associated with durable responses to immune checkpoint blockade in patients with metastatic melanoma. Nat Med. 2020;26:193–9. 10.1038/s41591-019-0734-6.

117. Naumann RW, Hollebecque A, Meyer T, Devlin M-J, Oaknin A, Kerger J, et al. Safety and efficacy of nivolumab monotherapy in recurrent or metastatic cervical, vaginal, or vulvar carcinoma: results from the phase I/II checkmate 358 trial. J Clin Oncol. 2019;37:2825–34. 10.1200/JCO.19.00739.

118. Lanitis E, Dangaj D, Irving M, Coukos G. Mechanisms regulating T-cell infiltration and activity in solid tumors. Ann Oncol. 2017;28:xii18–32. 10.1093/annonc/mdx238.

119. McLane LM, Abdel-Hakeem MS, Wherry EJ. CD8 T cell exhaustion during chronic viral infection and cancer. Annu Rev Immunol. 2019;37:457–95. 10.1146/annurev-immunol-041015-055318.

120. Zhao R, Song Y, Wang Y, et al. PD-1/PD-L1 blockade rescue exhausted CD8+ T cells in gastrointestinal stromal tumours via the PI3K/Akt/mTOR signalling pathway. Cell Prolif. 2019;52(3):e12571. 10.1111/cpr.12571.

121. Lastwika KJ, Wilson W, Li QK, Norris J, Xu H, Ghazarian SR, et al. Control of PD-L1 expression by oncogenic activation of the AKT-mTOR pathway in non-small cell lung cancer. Cancer Res. 2016;76:227–38. 10.1158/0008-5472.CAN-14-3362.

122. Patsoukis N, Bardhan K, Chatterjee P, Sari D, Liu B, Bell LN, et al. PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation. Nat Commun. 2015;6:6692. 10.1038/ncomms7692.

123. Granier C, Dariane C, Combe P, Verkarre V, Urien S, Badoual C, et al. Tim-3 expression on tumor-infiltrating PD-1 + CD8 + T cells correlates with poor clinical outcome in renal cell carcinoma. Cancer Res. 2017;77:1075–82. 10.1158/0008-5472.CAN-16-0274.

124. Takano S, Saito H, Ikeguchi M. An increased number of PD-1+ and Tim-3+ CD8+ T cells is involved in immune evasion in gastric cancer. Surg Today. 2016;46(11):1341–47. 10.1007/s00595-016-1305-9.

125. Zhou Q, Munger ME, Veenstra RG, Weigel BJ, Hirashima M, Munn DH, et al. Coexpression of Tim-3 and PD-1 identifies a CD8 + T-cell exhaustion phenotype in mice with disseminated acute myelogenous leukemia. Blood. 2011;117:4501–10. 10.1182/blood-2010-10-310425.

126. Sawada M, Goto K, Morimoto-Okazawa A, Haruna M, Yamamoto K, Yamamoto Y, et al. PD-1 + Tim3 + tumor-infiltrating CD8 T cells sustain the potential for IFN-γ production, but lose cytotoxic activity in ovarian cancer. Int Immunol. 2020;32:397–405. 10.1093/intimm/dxaa010.

127. John M, Helal M, Duell J, Mattavelli G, Stanojkovska E, Afrin N, et al. Spatial transcriptomics reveals profound subclonal heterogeneity and T-cell dysfunction in extramedullary myeloma. Blood. 2024;144:2121–35. 10.1182/blood.2024024590.

128. Peng W, Liu C, Xu C, et al. PD-1 blockade enhances T-cell migration to tumors by elevating IFN-γ inducible chemokines. Cancer Res. 2012;72(20):5209–18. 10.1158/0008-5472.CAN-12-1187.

129. K YK, A Y-K TTK. Lenvatinib plus anti-PD-1 antibody combination treatment activates CD8 + T cells through reduction of tumor-associated macrophage and activation of the interferon pathway. PLoS ONE. 2019;14:e0212513. 10.1371/journal.pone.0212513.

130. Krishna C, DiNatale RG, Kuo F, Srivastava RM, Vuong L, Chowell D, et al. Single-cell sequencing links multiregional immune landscapes and tissue-resident T cells in CcRCC to tumor topology and therapy efficacy. Cancer Cell. 2021;39:662–e6776. 10.1016/j.ccell.2021.03.007.

131. Zhang H, AbdulJabbar K, Moore DA, et al. Spatial positioning of immune hotspots reflects the interplay between B and T cells in lung squamous cell carcinoma. Cancer Res. 2023;83(9):1410–25. 10.1158/0008-5472.CAN-22-2589.

132. Brummelman J, Mazza EMC, Alvisi G, Colombo FS, Grilli A, Mikulak J, et al. High-dimensional single cell analysis identifies stem-like cytotoxic CD8 + T cells infiltrating human tumors. J Exp Med. 2018;215:2520–35. 10.1084/jem.20180684.

133. Giles JR, Ngiow SF, Manne S, Baxter AE, Khan O, Wang P, et al. Shared and distinct biological circuits in effector, memory and exhausted CD8 + T cells revealed by temporal single-cell transcriptomics and epigenetics. Nat Immunol. 2022;23:1600–13. 10.1038/s41590-022-01338-4.

134. Bc M, Dr S, R AA, Yv KB, Mw V. Subsets of exhausted CD8 + T cells differentially mediate tumor control and respond to checkpoint Blockade. Nat Immunol. 2019;20:326–36. 10.1038/s41590-019-0312-6.

135. Beltra J-C, Manne S, Abdel-Hakeem MS, Kurachi M, Giles JR, Chen Z, et al. Developmental relationships of four exhausted CD8 + T cell subsets reveals underlying transcriptional and epigenetic landscape control mechanisms. Immunity. 2020;52:825–e8418. 10.1016/j.immuni.2020.04.014.

136. Hewavisenti R, Ferguson A, Wang K, Jones D, Gebhardt T, Edwards J, et al. CD103 + tumor-resident CD8 + T cell numbers underlie improved patient survival in oropharyngeal squamous cell carcinoma. J Immunother Cancer. 2020;8:e000452. 10.1136/jitc-2019-000452.

137. von Witzleben A, Ellis M, Thomas GJ, Hoffmann TK, Jackson R, Laban S, et al. Tumor-infiltrating CD103 + tissue-resident memory T cells and CD103-CD8 + T cells in HNSCC are linked to outcome in primary but not metastatic disease. Clin Cancer Res. 2023;30:224–34. 10.1158/1078-0432.CCR-23-0445.

138. Cheng Y, Gunasegaran B, Singh HD, Dutertre C-A, Loh CY, Lim JQ, et al. Non-terminally exhausted tumor-resident memory HBV-specific T cell responses correlate with relapse-free survival in hepatocellular carcinoma. Immunity. 2021;54:1825–e18407. 10.1016/j.immuni.2021.06.013.

139. Garrido-Martin EM, Mellows TWP, Clarke J, Ganesan A-P, Wood O, Cazaly A, et al. M1hot tumor-associated macrophages boost tissue-resident memory T cells infiltration and survival in human lung cancer. J Immunother Cancer. 2020;8:e000778. 10.1136/jitc-2020-000778.

140. Knitz MW, Bickett TE, Darragh LB, Oweida AJ, Bhatia S, Van Court B, et al. Targeting resistance to radiation-immunotherapy in cold HNSCCs by modulating the Treg-dendritic cell axis. J Immunother Cancer. 2021;9:e001955. 10.1136/jitc-2020-001955.

141. Wang H, Franco F, Ho P-C. Metabolic regulation of Tregs in cancer: opportunities for immunotherapy. Trends Cancer. 2017;3:583–92. 10.1016/j.trecan.2017.06.005.

142. Liu Y, Zheng P. Preserving the CTLA-4 checkpoint for safer and more effective cancer immunotherapy. Trends Pharmacol Sci. 2020;41:4–12. 10.1016/j.tips.2019.11.003.

143. Wang H, Franco F, Tsui Y-C, Xie X, Trefny MP, Zappasodi R, et al. CD36-mediated metabolic adaptation supports regulatory T cell survival and function in tumors. Nat Immunol. 2020;21:298–308. 10.1038/s41590-019-0589-5.

144. Dhar S, Sarkar T, Bose S, Pati S, Chakraborty D, Roy D, et al. FOXP3 transcriptionally activates fatty acid scavenger receptor CD36 in tumour-induced Treg cells. Immunology. 2024;174:296–309. 10.1111/imm.13887.

145. Hindley JP, Jones E, Smart K, Bridgeman H, Lauder SN, Ondondo B, et al. T-cell trafficking facilitated by high endothelial venules is required for tumor control after regulatory T-cell depletion. Cancer Res. 2012;72:5473–82. 10.1158/0008-5472.CAN-12-1912.

146. Li J, Srivastava RM, Ettyreddy A, Ferris RL. Cetuximab ameliorates suppressive phenotypes of myeloid antigen presenting cells in head and neck cancer patients. J Immunother Cancer. 2015;3:54. 10.1186/s40425-015-0097-6.

147. Tian T, Gu X, Zhang B, et al. Increased circulating CD14(+)HLA-DR-/low myeloid-derived suppressor cells are associated with poor prognosis in patients with small-cell lung cancer. Cancer Biomark. 2015;15(4):425–32. 10.3233/CBM-150473.

148. Prendergast GC, Mondal A, Dey S, Laury-Kleintop LD, Muller AJ. Inflammatory reprogramming with IDO1 inhibitors: turning immunologically unresponsive cold tumors hot. Trends Cancer. 2018;4:38–58. 10.1016/j.trecan.2017.11.005.

149. Fletcher M, Ramirez ME, Sierra RA, Raber P, Thevenot P, Al-Khami AA, et al. L-arginine depletion blunts antitumor T-cell responses by inducing myeloid-derived suppressor cells. Cancer Res. 2015;75:275–83. 10.1158/0008-5472.CAN-14-1491.

150. Tumino N, Di Pace AL, Besi F, Quatrini L, Vacca P, Moretta L. Interaction between MDSC and NK cells in solid and hematological malignancies: impact on HSCT. Front Immunol. 2021;12:638841. 10.3389/fimmu.2021.638841.

151. L H, H Y, G Q, Z M, C X. Cancer-expanded myeloid-derived suppressor cells induce anergy of NK cells through membrane-bound TGF-beta 1. J Immunol. 2009;182:240–9. 10.4049/jimmunol.182.1.240.

152. L PM, Ca WTA, Jc L, Mr C. The CCR2/MCP-1 chemokine pathway and lung adenocarcinoma. Cancers. 2020;12:3723. 10.3390/cancers12123723.

153. Q T. CCR5 Blockade suppresses melanoma development through Inhibition of IL-6-Stat3 pathway via upregulation of SOCS3. Inflammation. 2015;38:2049–56. 10.1007/s10753-015-0186-1.

154. Fearon DT. The carcinoma-associated fibroblast expressing fibroblast activation protein and escape from immune surveillance. Cancer Immunol Res. 2014;2:187–93. 10.1158/2326-6066.CIR-14-0002.

155. Chen IX, Chauhan VP, Posada J, Ng MR, Wu MW, Adstamongkonkul P, et al. Blocking CXCR4 alleviates desmoplasia, increases T-lymphocyte infiltration, and improves immunotherapy in metastatic breast cancer. Proc Natl Acad Sci U S A. 2019;116:4558–66. 10.1073/pnas.1815515116.

156. Sledge GW. Targeting CXCR4-induced desmoplasia to improve checkpoint inhibition in breast cancer. Proc Natl Acad Sci U S A. 2019;116:4769–71. 10.1073/pnas.1900368116.

157. Wu T, Wang W, Shi G, Hao M, Wang Y, Yao M, et al. Targeting HIC1/TGF-β axis-shaped prostate cancer microenvironment restrains its progression. Cell Death Dis. 2022;13:624. 10.1038/s41419-022-05086-z.

158. Nowak-Sliwinska P, van Beijnum JR, Griffioen CJ, Huinen ZR, Sopesens NG, Schulz R, et al. Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy. Angiogenesis. 2022;26:279–93. 10.1007/s10456-022-09863-4.

159. Allen E, Jabouille A, Rivera LB, Lodewijckx I, Missiaen R, Steri V, et al. Combined antiangiogenic and anti-PD-L1 therapy stimulates tumor immunity through HEV formation. Sci Transl Med. 2017;9:eaak9679. 10.1126/scitranslmed.aak9679.

160. Shigeta K, Datta M, Hato T, Kitahara S, Chen IX, Matsui A, et al. Dual programmed death receptor-1 and vascular endothelial growth factor receptor-2 blockade promotes vascular normalization and enhances antitumor immune responses in hepatocellular carcinoma. Hepatology. 2019;71(4):1247–61. 10.1002/hep.30889.

161. Vathiotis IA, Johnson JM, Argiris A. Enhancing programmed cell death protein 1 axis inhibition in head and neck squamous cell carcinoma: combination immunotherapy. Cancer Treat Rev. 2021;97:102192. 10.1016/j.ctrv.2021.102192.

162. Stanton SE, Disis ML. Clinical significance of tumor-infiltrating lymphocytes in breast cancer. J Immunother Cancer. 2016;4:59. 10.1186/s40425-016-0165-6.

163. Pawelec G. Unexpected benefits of aging for favorable responses to PD-1 blockade in melanoma? Clin Cancer Res. 2018;24:5193–4. 10.1158/1078-0432.CCR-18-1475.

164. Chumsri S, Sokol ES, Soyano-Muller AE, Parrondo RD, Reynolds GA, Nassar A, et al. Durable complete response with immune checkpoint inhibitor in breast cancer with high tumor mutational burden and APOBEC signature. J Natl Compr Canc Netw. 2020;18:517–21. 10.6004/jnccn.2020.7543.

165. Callahan MK, Wolchok JD. Recruit or reboot? How does anti-PD-1 therapy change tumor-infiltrating lymphocytes? Cancer Cell. 2019;36:215–7. 10.1016/j.ccell.2019.08.009.

166. Trinh A, Polyak K. Tumor neoantigens: when too much of a good thing is bad. Cancer Cell. 2019;36:466–7. 10.1016/j.ccell.2019.10.009.

167. Matson V, Chervin CS, Gajewski TF. Cancer and the microbiome-influence of the commensal microbiota on cancer, immune responses, and immunotherapy. Gastroenterology. 2021;160:600–13. 10.1053/j.gastro.2020.11.041.

168. Yi M, Yu S, Qin S, Liu Q, Xu H, Zhao W, et al. Gut microbiome modulates efficacy of immune checkpoint inhibitors. J Hematol Oncol. 2018;11:47. 10.1186/s13045-018-0592-6.

169. Metsäniitty M, Hasnat S, Salo T, Salem A. Oral microbiota-a new frontier in the pathogenesis and management of head and neck cancers. Cancers. 2021;14:46. 10.3390/cancers14010046.

170. Burcher KM, Burcher JT, Inscore L, Bloomer CH, Furdui CM, Porosnicu M. A review of the role of oral microbiome in the development, detection, and management of head and neck squamous cell cancers. Cancers. 2022;14:4116. 10.3390/cancers14174116.

171. Chen Z, Wong PY, Ng CWK, Lan L, Fung S, Li JW, et al. The intersection between oral microbiota, host gene methylation and patient outcomes in head and neck squamous cell carcinoma. Cancers. 2020;12:3425. 10.3390/cancers12113425.

172. Su L, Yang R, Sheng Y, Ullah S, Zhao Y, Shunjiayi H, et al. Insights into the oral microbiota in human systemic cancers. Front Microbiol. 2024;15:1369834. 10.3389/fmicb.2024.1369834.

173. Wang XX, Liu YT, Ren JG, Liu HM, Fu Q, Yang Y, et al. Salivary microbiome relates to neoadjuvant immunotherapy response in OSCC. J Dent Res. 2024;103:988–98. 10.1177/00220345241262759.

174. Ding Y, Chu L, Cao Q, Lei H, Li X, Zhuang Q. A meta-validated immune infiltration-related gene model predicts prognosis and immunotherapy sensitivity in HNSCC. BMC Cancer. 2023;23:45. 10.1186/s12885-023-10532-y.

175. Goswami S, Apostolou I, Zhang J, Skepner J, Anandhan S, Zhang X, et al. Modulation of EZH2 expression in T cells improves efficacy of anti-CTLA-4 therapy. J Clin Invest. 2018;128:3813–8. 10.1172/JCI99760.

176. Carter CA, Oronsky BT, Roswarski J, Oronsky AL, Oronsky N, Scicinski J, et al. No patient left behind: the promise of immune priming with epigenetic agents. Oncoimmunology. 2017;6:e1315486. 10.1080/2162402X.2017.1315486.

177. Jones PA, Ohtani H, Chakravarthy A, De Carvalho DD. Epigenetic therapy in immune-oncology. Nat Rev Cancer. 2019;19:151–61. 10.1038/s41568-019-0109-9.

178. Cao L-M, Zhong N-N, Chen Y, Li Z-Z, Wang G-R, Xiao Y, et al. Less is more: exploring neoadjuvant immunotherapy as a de-escalation strategy in head and neck squamous cell carcinoma treatment. Cancer Lett. 2024;598:217095. 10.1016/j.canlet.2024.217095.

179. Smussi D, Mattavelli D, Paderno A, Gurizzan C, Lorini L, Romani C, et al. Revisiting the concept of neoadjuvant and induction therapy in head and neck cancer with the advent of immunotherapy. Cancer Treat Rev. 2023;121:102644. 10.1016/j.ctrv.2023.102644.

180. Jiang T, Shi T, Zhang H, Hu J, Song Y, Wei J, et al. Tumor neoantigens: from basic research to clinical applications. J Hematol Oncol. 2019;12:93. 10.1186/s13045-019-0787-5.

181. Keskin DB, Anandappa AJ, Sun J, Tirosh I, Mathewson ND, Li S, et al. Neoantigen vaccine generates intratumoral T cell responses in phase Ib glioblastoma trial. Nature. 2018;565:234–9. 10.1038/s41586-018-0792-9.

182. Miao L, Zhang Y, Huang L. mRNA vaccine for cancer immunotherapy. Mol Cancer. 2021;20:41. 10.1186/s12943-021-01335-5.

183. Fan T, Xu C, Wu J, Cai Y, Cao W, Shen H, et al. Lipopolyplex-formulated mRNA cancer vaccine elicits strong neoantigen-specific T cell responses and antitumor activity. Sci Adv. 2024;10:eadn9961. 10.1126/sciadv.adn9961.

184. Weber JS, Carlino MS, Khattak A, Meniawy T, Ansstas G, Taylor MH, et al. Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): a randomised, phase 2b study. Lancet. 2024;403:632–44. 10.1016/S0140-6736(23)02268-7.

Associated Data

Supplementary Materials

Supplementary Material 1.

Data Availability Statement

No datasets were generated or analysed during the current study.