The presence of sufficient neoantigens recognizable by the immune system is the first step in initiating antitumor immunity. Enhancing effective antigen release is a key strategy for improving the efficacy of anti-PD-1/PD-L1 inhibitors. Epigenetic drugs represent a classic approach to restoring tumor immunogenicity. However, early clinical trials revealed limited clinical activity when combining histone deacetylase (HDAC) inhibitors with PD-1/PD-L1 inhibitors^[39]. In addition, novel epigenetic drugs, such as bromodomain and extraterminal (BET) inhibitors, have shown potential for enhancing the efficacy of anti-PD-1/PD-L1 therapies^[40]. Oncolytic virus therapy is a novel antitumor strategy that enhances immunogenic cell death (ICD) in tumors. Mechanistic studies and early clinical trials have confirmed its role in sensitizing tumors to immunotherapy^[41]. Recent research has focused on engineering oncolytic viruses that express multiple molecules, such as OX40L, to further enhance the effects of PD-1/PD-L1 inhibitors^[42]. Antibody-drug conjugates (ADCs) carrying ICD-inducing payloads have also been shown to promote ICD, providing a rationale for combination strategies with immunotherapy. Several clinical studies have preliminarily demonstrated synergistic effects between ADCs and PD-1/PD-L1 inhibitors. Next-generation conjugates, such as bispecific ADCs, stimulator of interferon genes (STING) agonist-loaded ADCs, and conditionally active ADCs, hold promise for enhancing the efficacy of combination therapy while reducing treatment-related adverse events^[43,44]. Additional methods to promote antigen release are currently being explored in preclinical studies. Antigen release often depends on ICD processes, such as apoptosis, necrosis, and regulated cell death (RCD), including ferroptosis, cuproptosis, pyroptosis, and programmed necrosis. However, most ICD occurs through apoptosis, which is generally immunotolerant and unable to activate antitumor immunity^[45]. As a result, inducing RCD is emerging as a promising strategy for immunotherapy. Ferroptosis is a major focus in this field. In several mouse tumor models, ferroptosis inducers, such as GPX4 inhibitors and translocator protein (TSPO) inhibitors, have shown strong synergy with anti-PD-1/PD-L1 therapy^[46,47]. Ferroptosis not only directly suppresses tumor cell proliferation, but also activates dendritic cells (DCs) by releasing damage-associated molecular patterns (DAMPs)^[48]. Furthermore, induction of ferroptosis by targeting upstream or downstream molecules can also enhance immunotherapy efficacy, as demonstrated by TMOD3 knockdown or heterogeneous nuclear ribonucleoprotein L (HnRNP L) knockdown^[49,50]. However, ferroptosis-induced immunogenicity is context-dependent^[51]. Maximizing its therapeutic efficacy requires improving tumor selectivity while reducing the suppressive effects of oxidized lipids on immune cells^[52,53]. Nanoparticles and similar materials represent a potential solution^[54,55]. Cuproptosis, proposed in 2022, also shows promise for enhancing anti-PD-1/PD-L1 efficacy, but its clinical application still requires further preclinical research^[56].

Tumor antigens are captured by antigen-presenting cells (APCs) and presented to T cells through major histocompatibility complex (MHC) molecules. Reduced numbers of DCs in draining lymph nodes and impaired T cell activation hinder antitumor immunity and limit the efficacy of PD-1/PD-L1 inhibitors. Clinical trials have evaluated antigen vaccines, CD40 agonists, and Toll-like receptor (TLR) agonists to improve responses to anti-PD-1/PD-L1 therapy, but the results have been mixed. A phase I trial showed that the TLR9 agonist vidutolimod plus pembrolizumab induced durable tumor regression in 25% of patients with metastatic melanoma resistant to PD-1 inhibitors^[57], whereas a separate phase I study of a TLR7 agonist showed limited benefit either as monotherapy or in combination with a PD-1 inhibitor^[58]. Preclinical studies are now exploring TLR agonists incorporated into nanomedicines or co-delivery systems to better reverse the suppressive tumor microenvironment (TME) and improve anti-PD-1/PD-L1 activity^[59]. Neoantigen vaccines activate APCs by directly delivering antigens, and studies have shown that vaccine-induced memory T cells can provide long-term protection^[60]. Personalized neoantigen vaccines combined with PD-1/PD-L1 blockade have demonstrated preliminary antitumor activity in early-phase trials^[61]. Current preclinical studies aim to further optimize these combinations by refining neoantigen design, adjusting dosing schedules, and integrating multi-drug strategies. The functional state of DCs also shapes antigen presentation. Sánchez-Paulete et al. identified BATF3-dependent DCs as essential for cross-presenting tumor antigens and mediating anti-PD-1 responses^[62]. The interaction between the STAT3 and STAT5 transcriptional pathways determines DC phenotypes in the TME, and STAT3 degraders represent a potential immunotherapeutic approach^[63]. Another study found that TP63 and STAT1 mutually inhibit each other and regulate the IFN-γ signaling pathway through co-occupation and co-regulation of promoters and enhancers, and TP63 silencing can enhance PD-1 blockade efficacy^[64].

Loss of co-stimulatory factors such as 4-1BB/4-1BBL and CD28/CD80 also impairs T cell activation and contributes to immunotherapy resistance. Combinations of 4-1BB agonists with PD-1/PD-L1 inhibitors, as well as bispecific antibodies, have entered clinical trials. Although the results have not been entirely satisfactory, high response rates have been observed in certain cancers (e.g., melanoma)^[65]. CD28, another primary co-stimulatory receptor, provides the second activation signal by binding CD80/CD86. Despite early safety concerns, CD28 remains a viable therapeutic target. Majocchi et al. recently reported early results for NI-3201, a bispecific antibody that mediates PD-L1-dependent CD28 co-stimulation, and clinical studies are being planned^[66]. However, another study has identified a distinct function of CD28 in tumor cells - promoting immune evasion. Knockdown of tumor-cell CD28 may overcome anti-PD-1 resistance by affecting PD-L1^[67]. Because CD28 exerts different functions in tumor cells and T cells, therapeutic targeting requires strict cell specificity. RNA sequencing studies have also identified novel targets, such as mitochondrial antiviral signaling protein (MAVS) and FTSJ3, which modulate antitumor immunity through IFN signaling and may sensitize tumors to PD-1/PD-L1 blockade^[68,69]. These findings offer new insights into combination strategies, though clinical translation remains ongoing.

Abnormal metabolism contributes to an immunosuppressive TME. Enhancing the therapeutic efficacy of PD-1/PD-L1 blockade through metabolic reprogramming is an emerging and promising approach. In a preclinical study of colorectal cancer, H₂S promoted Treg activation and inhibited CD8⁺ T cell migration via ENO1 and ELK4 post-sulfation. Conversely, reducing H₂S can enhance the antitumor activity of ICIs^[106]. Boelaars et al. reported that sialic acid deficiency increases CD4⁺ and CD8⁺ T cells while reducing CD4⁺ Tregs, thereby sensitizing pancreatic cancer cells to immunotherapy^[107]. Elevated blood ammonia levels in colorectal cancer patients were associated with non-response to anti-PD-L1 therapy^[108]. Another study indicated that supplying L-arginine to immune cells while limiting its uptake by tumor cells may be an optimal strategy^[109]. Dynamic labeling of L-arginine using nanomaterials can improve its delivery and boost ICI efficacy^[110]. Other research also showed that inhibiting glutamine metabolism in tumor cells enhances immune cell function and synergizes with PD-L1 blockade^[111]. Hypoxia in the TME is another factor contributing to anti-PD-1/PD-L1 resistance. Alleviating hypoxia through direct or indirect oxygen delivery has significantly improved antitumor efficacy. Researchers developed alginate microcapsules encapsulating cyanobacteria that generate sustained oxygen via photosynthesis. In a breast cancer mouse model, this approach demonstrated strong synergy with anti-PD-1 therapy^[112]. A recent study proposed that targeting lipid rafts and mitochondrial respiration with albumin-bound statins can reduce hypoxia and improve PD-1 blockade efficacy in NSCLC^[113].

Activated T cells recognize tumor cells through T cell receptor (TCR)-MHC-peptide interactions and eliminate them with the support of co-stimulatory molecules. Studies have shown that increasing MHC levels through PCSK9 inhibition, α-ketoglutarate supplementation, or G3BP1 inhibition can enhance T cell recognition of tumor cells^[114-116]. Recent research revealed that valosin-containing protein (VCP) stabilizes GPD1L, causing downstream glycerol-3-phosphate (G3P) accumulation and subsequent TCR impairment. Dual inhibition of VCP and PD-1 enhanced antitumor activity in a mouse model of HCC^[117]. The integration of PD-1/PD-L1 blockade and adoptive cell therapy (ACT) has also been actively investigated. Physical and immunologic barriers suppress ACT efficacy within solid malignancies, and the addition of PD-1/PD-L1 inhibitors could help overcome these challenges^[118]. Giuffrida et al. reported that interleukin (IL)-7/IL-15 pretreatment improves chimeric antigen receptor T cell (CAR-T) responses to anti-PD-1/PD-L1 therapy^[119]. Advances in the ex vivo expansion and delivery of specific effector T cells may further strengthen this combination therapy. Simultaneous blockade of PD-1 and other immune checkpoints has shown favorable safety profiles and antitumor activity in multiple phase III trials. However, limited efficacy and safety concerns in certain cancers persist, highlighting the need for further mechanistic studies^[120]. A preclinical study suggested that a TIGIT and 4-1BB bispecific antibody (ABL112), when combined with pembrolizumab, inhibits tumor growth more effectively than pembrolizumab plus an anti-TIGIT antibody^[121]. Hu et al. found that immunoradiotherapy with dual LAG-3 and TIGIT blockade effectively overcomes anti-PD-1 resistance^[122]. Targeting multiple immune checkpoints remains a promising strategy to strengthen PD-1/PD-L1 inhibitors, and advances in multi-target inhibitors and delivery technologies may further improve clinical responses.

Beyond the microenvironment, host factors such as age, sex, microbiota, and diet also influence the therapeutic outcomes of PD-1/PD-L1 blockade. With increasing age, lymphoid tissues undergo structural and cellular alterations that lead to diminished immune surveillance and a peripheral pro-inflammatory immune environment. This decline in immune adaptability is not only associated with increased cancer risk but also impacts the efficacy of immunotherapy^[123]. Although immune dysfunction worsens with age across most tumor types, the roles of specific immune cell subsets are cancer type-specific. Furthermore, aging also reshapes the immune microenvironment through alterations in the ECM, blood vessels, adipocytes, and other components.

A meta-analysis showed that males may benefit more from immunotherapy, but another meta-analysis in NSCLC reported the opposite finding. Sex-related differences arise from complex interactions among genetic, hormonal, behavioral, and environmental factors. Among these, sex hormones - particularly estrogen and androgen - are key regulators of antitumor immunity and responses to ICIs. Estrogen and its receptors (ERα and ERβ) influence the formation of the TME through both tumor cell-intrinsic and -extrinsic mechanisms. Studies in NSCLC have shown that tumor cells produce estrogen through aromatase expression and activate ERα via an autocrine mechanism, thereby promoting CD274/PD-L1 overexpression^[124]. Another preclinical study indicated that estrogen promotes the recruitment of TAMs and induces their polarization toward the M2 phenotype^[125]. Adurthi et al. found that ERα signaling stimulates Treg infiltration by directly binding to the FOXP3 promoter and upregulating the expression of immunosuppressive molecules^[126]. Furthermore, in melanoma, lung cancer, and ovarian cancer models, estrogen was shown to promote the immunosuppressive activity of MDSCs and their infiltration into tumor sites by activating the JAK2-STAT3 or TNFα pathways^[127,128]. Preclinical models have demonstrated that blocking estrogen signaling with selective ER-degrading agents effectively restores CD8⁺ T cell function and enhances the efficacy of ICIs^[125,129]. Notably, estrogen-mediated regulation of T cell function exhibits complexity that is dependent on receptor subtype. Binding to ERα restricts the proliferation and intratumoral infiltration of cytotoxic T cells by inducing mitochondrial dysfunction, shortening telomere length, and suppressing hTERT expression^[130,131]. However, activation of ERβ in CD8⁺ T cells enhances TCR signaling by modulating tumor-derived phosphotyrosine switches, thereby increasing anti-PD-1 activity^[132]. The effects of androgens in the TME primarily involve driving CD8⁺ T cell exhaustion and suppressing innate immune cell function. The androgen receptor (AR) directly impairs CD8⁺ T cell function by binding to androgen response elements (AREs) within open chromatin regions of the IFN-γ and GZMB genes, thereby suppressing their expression^[133]. Studies in bladder cancer models have also found that AR promotes the early differentiation of CD8⁺ T cells into a “progenitor-exhausted” phenotype by upregulating the transcription factors TCF7/TCF1^[134]. Furthermore, Liu et al. found that anti-androgen therapy or AR knockdown can reduce PD-L1 expression in NK cells by downregulating circ_0001005^[135]. Based on these mechanisms, blocking androgen signaling via androgen deprivation therapy (ADT) or AR antagonists has been shown to exert synergistic effects with anti-PD-1/PD-L1 therapy in several preclinical models^[133,136]. In addition to estrogen and androgen, progesterone and human chorionic gonadotropin (hCG) have also been found to influence T cell epigenetic and metabolic reprogramming by upregulating progesterone-induced inhibitory factors and promoting histone methylation^[9]. Recent studies have further revealed sex-specific differences in the function of certain enzymes within T cells. In female mice, the absence of diacylglycerol acyltransferase 1 (DGAT1) in T cells enhances antitumor immunity by improving mitochondrial function and expanding precursor-depleted T cells. However, in male mice, DGAT1 deficiency leads to fatty acid peroxidation, endoplasmic reticulum stress, and T cell death via AR signaling, thereby impairing antitumor immunity^[137]. The mechanisms underlying sex-related differences in tumor immunity and immunotherapy responses are complex. Current studies suggest that modulating sex hormones to enhance sensitivity to anti-PD-1/PD-L1 therapy is a promising direction, but further mechanistic studies are needed.

Long-distance regulation by gut microbiota and local remodeling via intratumoral microbiota together influence the TME. Gut microbiota not only contribute to local tumorigenesis but also influence tumor immunity through long-distance regulation of the TME^[138]. Studies in mice have revealed differential effects of the gut microbiota on patient responses to anti-PD-1/PD-L1 treatment^[139]. Fidelle et al. found that antibiotic-induced dysbiosis increases intestinal Enterocloster abundance and causes a loss of MAdCAM-1 expression, thereby triggering immune-suppressive Treg17 cell infiltration into the TME^[140]. Another study showed that specific gut microbiota can downregulate PD-L2 and its ligand RGMb, and that blocking this pathway may overcome anti-PD-1 resistance^[141]. Ex-Th17 cells induced by Enterobacteriaceae were found to produce high levels of IFN-γ and TNF in the TME, thereby promoting antitumor immunity and achieving anti-PD-1-mediated tumor control^[142]. Furthermore, soluble metabolites from gut bacteria, such as short-chain fatty acids, inosine, and bile acids, can reach the TME via systemic circulation and influence tumor immunity^[138]. Additional clinical evidence suggests that the use of broad-spectrum antibiotics disrupts the gut microbiome, thereby impairing immunotherapy efficacy^[143]. Conversely, increased gut microbiota diversity (e.g., Bifidobacterium spp.) has been associated with higher response rates to ICIs^[139]. Modulation of the gut microbiota via fecal microbiota transplantation has also been shown to enhance the efficacy of PD-1 inhibitors in early clinical trials^[144]. Recently, a synthetic microbiota composed of 15 gut bacterial strains was successfully engineered and demonstrated the potential to reverse anti-PD-1 antibody resistance, regardless of individual gut microbiota backgrounds^[145]. Unlike gut microbiota, microorganisms colonizing tumor sites directly participate in TME remodeling. Although the biomass of intratumoral bacteria is low (typically 1-10 bacteria per 100,000 tumor cells)^[146], these microorganisms can influence the TME through direct contact and local metabolism. H. pylori and S. Typhimurium promote immune evasion by inducing PD-L1 expression in host cells^[147,148]. Hezaveh et al. found that indole metabolites secreted by Lactobacillus promote the polarization of TAMs toward an immunosuppressive phenotype by activating the AhR pathway^[149]. Conversely, some bacteria are considered beneficial for immunotherapy, with baseline enrichment of Clostridium at tumor sites found to be associated with response to ICIs^[150]. Intratumoral injection of Burkholderia cepacia, Priestia megaterium, or Corynebacterium kroppenstedtii in melanoma-bearing mice was found to synergistically inhibit tumor growth when combined with anti-PD-1 therapy^[151]. With the deepening understanding of the microbiome, clinical intervention strategies are shifting toward precision medicine. Canale et al. found that engineered tumor-colonizing Escherichia coli can convert ammonia into L-arginine, thereby improving immunotherapy efficacy^[152].

In addition to the factors discussed above, diet, exercise, and chronic stress may also influence tumor immunity and responses to anti-PD-1/PD-L1 therapy. Preclinical studies have shown that a ketogenic diet alters the immunosuppressive TME through multiple mechanisms, increasing sensitivity to ICIs^[153]. Regular exercise has been shown to enhance NK cell cytotoxicity and reshape the T cell repertoire, supporting antitumor immunity^[154]. Growing interest in host-related factors underscores the potential of modulating host physiology to improve immunotherapy efficacy.

With increasing research attention focused on the neuro-immune system, its role in ICI resistance has gradually been recognized in recent years. Abnormal nerve fiber growth or irregular neurotransmitter secretion can lead to immune escape. Targeting neurotransmitters and their receptors has shown potential for overcoming resistance. Plasma neurotransmitter analyses in patients who were sensitive or resistant to anti-PD-1 antibodies revealed that norepinephrine influences CXCL9 and adenosine (ADO) secretion in tumor cells, thereby inhibiting the chemotaxis and function of CD8⁺ T cells^[155]. Qiao et al. reported that norepinephrine inhibits TCR signaling by activating β-adrenergic receptors (β-ARs) on CD8⁺ T cells while promoting metabolic dysfunction and exhaustion^[156]. A meta-analysis showed that β-blocker use may correlate with improved ICI efficacy^[157]. The safety and antitumor activity of combining β-AR antagonists with PD-1/PD-L1 inhibitors are being investigated in several clinical trials. In contrast to the β-AR pathway, α2-adrenergic receptor (α2-AR) agonists have shown the ability to induce MDSC apoptosis and enhance CD8⁺ T cell infiltration, thereby exhibiting effective antitumor activity even in ICI-resistant tumor models^[158]. Furthermore, activation of the α7 nicotinic acetylcholine receptor has been shown to suppress TNF-α release in macrophages and modulate T cell function^[159]. Schneider et al. revealed that platelet-derived serotonin upregulates PD-L1 on cancer cells, thereby driving CD8⁺ T cell exhaustion^[160]. Combining serotonin-targeting drugs with PD-1/PD-L1 inhibitors has achieved long-term tumor inhibition. Further studies have also revealed the role of sensory neurons in tumor immunity. In preclinical melanoma models, calcitonin gene-related peptide (CGRP), released by nociceptors, increased CD8⁺ T cell exhaustion^[161]. Although the mechanism by which CGRP regulates T cells remains incompletely understood, it may involve Gαs signaling mediated by the RAMP1–CALCRL receptor complex^[162]. In summary, neurotransmitter signaling pathways play critical roles in resistance to PD-1/PD-L1 inhibitors. Further studies in this field may reveal novel therapeutic targets and combination strategies for overcoming immunotherapy resistance.