TCR signaling cascade starts with antigen recognition, leading to the recruitment of the Src family kinase, Lck, to the TCR complex. Lck phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMs) on CD3 subunits, a process that subsequently recruits and activates the protein tyrosine kinase zeta-chain–associated protein kinase of 70 kDa (ZAP-70) (7). Once activated, ZAP-70 phosphorylates the key adaptor proteins LAT and SLP-76 (7), which are essential for assembling signaling complexes. Phosphorylated LAT acts as a docking site for several proteins, including PLCγ, GRB2, and Gads (7), which initiates downstream signaling. The activation of PLCγ is crucial for the calcium signaling pathway. This signaling cascade enhances T-cell activity and triggers cellular responses.

While recognition of peptide–MHC complexes by TCRs provides the initial activation signal, this alone is insufficient for full T-cell activation. A crucial second signal is delivered through co-stimulatory pathways, most prominently the interaction of CD28 on T cells with CD80 (B7.1) or CD86 (B7.2) on APCs (8). This engagement promotes T-cell proliferation, cytokine secretion, survival, and effector functions. These co-stimulatory signals activate downstream pathways such as PI3K–AKT and ERK/MAPK, reinforcing T-cell activation ( Figure 1 ).

The design of CAR-T cells is a direct clinical application of these TCR signaling principles. CARs incorporate ITAM-containing CD3ζ domains to initiate activation and costimulatory domains (e.g., CD28, 4-1BB) to enhance T cell persistence and function. This synthetic biology approach exemplifies how targeted manipulation of T cell signaling pathways can generate potent antitumor immunity. Table 1 presents the mechanisms of T cell activation.

The corresponding immunotherapy strategies directly target these signals: dendritic cell vaccines aim to optimize antigen presentation and co-stimulation. Cytokine therapy provides strong cytokine signals to drive T cell expansion and memory differentiation. Both are clinical transformation examples targeting the fundamental mechanism of the APC-T cell interaction.

Targeted optimization can be achieved by modifying the antigen presentation process, thereby addressing the issue of limited T cell persistence. MAPK inhibitors effectively suppress the proliferation of cancer cells and enhance the transcription of MHC-I genes, promoting the activation of CD8+ T cells and increasing their infiltration into solid tumors (26). Additionally, researchers have engineered dynamic lipid bilayers that co-present TCR and co-stimulatory signals in a physiologically ordered manner, sustaining T-cell expansion and cytokine (e.g., IL-2) support while avoiding the abrupt over-activation linked to single high-dose stimulation (27).

Despite their therapeutic potential, MAPK pathway inhibitors (such as BRAF/MEK inhibitors) are associated with hepatotoxicity (28), and in clinical practice adaptive or acquired resistance frequently emerges. Meanwhile, artificial cell systems or carriers based on lipid bilayers continue to face engineering and translational challenges in terms of large-scale manufacturing and in vivo delivery (29). Therefore, at the current stage, these strategies are more appropriately considered as adjuncts or components of combination therapies rather than standalone treatments, unless substantive breakthroughs are achieved in safety and delivery technologies.

Optimizing T cell activation signals, enhancing their differentiation status, and regulating their resistance to exhaustion are key research directions in the future. Dual costimulation (4-1BB/ICOS) amplifies the persistence of CD8+ T cells through the interaction of NF-κB/NFATc1 (30), significantly enhancing the efficacy of adoptive cell therapy and helping to prolong the disease-free survival of patients and reduce the risk of recurrence (25, 31).

The regulation of T-cell differentiation and an increase in the proportion of memory T-cells can enhance T-cell persistence. Tcf1 + PD-1 + T cells exhibit characteristics similar to those of stem cells (25, 31). By self-renewal, they continuously produce Tcf1− cells to participate in the killing of tumor cells, thereby supporting a long-term anti-tumor response. The regulation of the NR4A family receptors (NR4A1, NR4A2, NR4A3) can effectively enhance T cell activation and persistence by regulating the early activation state and promoting the differentiation of memory T cells (32). Moreover, epigenetic regulation via the inhibition of BCL-6 and BLIMP-1, key regulators of T follicular helper cells, can prolong the survival of CAR T cells (33). CD4 + CAR T cells not only show strong cytotoxic activity but also show less sensitivity to activation-induced cell death and express fewer inhibitory immune checkpoint receptors than CD8 + CAR T cells, resulting in greater persistence and antitumor activity (34, 35). This finding may enhance therapeutic effects by regulating the differentiation of CD4 + T cells. Furthermore, regulation of T cell polarization, particularly toward Th2 or Th9 cells, can significantly enhance CAR T cell efficacy. Th9-polarized CAR-T cells show superior efficacy in preclinical models (36). Cord blood-derived hematopoietic stem cells (HSCs) allow large-scale expansion of precursor T cells prior to terminal differentiation (33)preserving proliferative capacity and functionality during manufacturing, Together, these strategies highlight the potential of differentiation and polarization control to improve the durability of T cells therapy.

Although these strategies expand the design space for CAR-T optimization and show promise in enhancing persistence and functionality, dual costimulation may increase the risks of cytokine release and neurotoxicity (37); lineage skewing (such as Th2/Tfh bias) or unstable Th9 phenotypes limit reproducibility (38); transcriptional or epigenetic modulation introduces risks of off-target effects and autoimmunity; and large-scale production of hematopoietic stem cell–derived products remains constrained by manufacturing and regulatory hurdles. At present, control of differentiation represents the most translationally promising avenue. Future research should focus more on rational costimulatory design and the enrichment of stem cell–like T cells, in order to balance persistence with effector potency and ultimately achieve safer, more predictable, and durable clinical outcomes.

Aging leads to enhanced immunosuppression in the tissue microenvironment and impairs T cell function. CAR-T cells in this environment are more likely to exhibit senescent phenotypes, thereby reducing their anti-tumor efficacy.

Optimizing the culture conditions for CAR-T cells can help mitigate this issue. IL-7 and IL-15 can direct T cells toward a memory stem cell-like phenotype, improve their expansion and viability, delay terminal differentiation, and enhance antitumor efficacy (39–41). Prolonged or high-dose IL-2 administration leads to T cell over-differentiation, whereas short-term or low-dose administration can generate early memory T cells (42, 43). Therefore, it can be seen that properly regulating the combination and concentration of cytokines is the core strategy for enhancing the function of CAR-T cells. Aging causes a decline in thymic function and T-cell generation. Supplementation with young thymic epithelial cells, gene therapy (such as the FOXN1 gene), and cytokines such as IL-7 can help restore thymic function (44).

This strategy is most immediately translatable, but dosing precision is critical. High-dose IL-2 toxicity and IL-7/IL-15 is activated abnormally in the immune system or works in synergy with other pro-inflammatory cytokines, it may be involved in the worrying pathological process of cytokine storm. Standardizing cytokine regimens across donors for GMP manufacturing is a priority.

Telomere transfer technology slows T-cell aging by extending telomere length, thereby enhancing immune function. Telomere transfer from APC-derived vesicles to T cells, triggered by ionomycin-mediated calcium signaling, delays senescence and enhances immune activity (45–47). This strategy may play a key role in the development of therapies to enhance immune function and extend the lifespan of T cells. Artificial telomere elongation has been linked to risks of tumorigenesis and genomic instability (48). Ionomycin, although widely used as a calcium ionophore, exhibits broad cytotoxicity and poor dose controllability. In addition, the specificity and reproducibility of telomere transfer techniques remain under debate, and regulatory hurdles for clinical translation add further complexity.

T-cell reprogramming and gene editing techniques hold promise for delaying T-cell aging and enhancing antitumor immune functions. Redifferentiating T-induced pluripotent stem cells (T-IPSCs) into naïve, cytotoxic, or dedifferentiated T cells can help delay T cell depletion and aging (49–51). CLASH, a novel CRISPR system used to create CAR T cells with enhanced memory and stem cell properties by targeting genes such as PRDM1 to extend their longevity (52). I find these strategies compelling, yet their long-term efficacy and safety remain open questions. Similarly, blocking inhibitory receptors such as ILT4 shows preclinical potential to rejuvenate T cells and reprogram tumor metabolism (53), but translating these results to patients will likely face biological and regulatory hurdles.

Regulation of signaling pathways and use of anti-aging drugs can help mitigate T cell aging. Modulating the Wnt/β-catenin and mTOR pathways has been shown to reverse T cell aging, particularly in memory T cells. And in vivo administration of small-molecule Wnt agonists may help achieve sustained effects (54). Drugs such as rapamycin and quercetin can restore CAR T-cell function and extend their persistence and antitumor activity by inhibiting mTOR signaling or clearing senescent cells (55). This is attractive due to oral drug accessibility, but systemic Wnt activation risks oncogenesis (56), and rapamycin causes immune suppression and metabolic toxicity (57). We advocate for short-term, perimanufacturing exposure rather than long-term systemic therapy, coupled with biomarker monitoring.

Physical removal of senescent T cells can restore function and promote the expansion of memory and effector subsets. Engineered peptides can disrupt the binding of FOXO 4 and p53 to induce apoptosis in senescent cells (58). UPAR-specific CAR T cells efficiently eliminate senescent cells by targeting the surface receptor uPAR and prolonging survival in mouse models (59). These approaches highlight the potential of selectively clearing senescent T cells to enhance immunotherapy. However, concerns remain regarding off-target effects, durability of responses, and safety of translating senolytic strategies into humans, underscoring the need for careful evaluation before clinical application.

Autologous stem cell transplantation (ASCT) can restore naïve, memory, and effector T-cell function, with notable success in autoimmune diseases and hematological malignancies (60–63). Early clinical trials (NCT00133367) shows that umbilical cord blood–derived HSCs, and young stem cells more broadly, may help regenerate a youthful immune system and mitigate T-cell aging (64–66). However, such strategies face major limitations, including conditioning regimen toxicity, graft availability, risk of graft-versus-host disease, and uncertain long-term rejuvenation effects. Careful patient selection and integration with safer rejuvenation approaches will be critical for translation.

Restoring and maintaining the thymic environment can reverse the effects of thymic involution, thereby promoting the production of new T-cells. Bioengineered thymic organoids incorporating cytokines such as IL-21 have been shown to enhance T-cell output in aged mice (67). Whether bioengineered thymic organoids, when combined with other cytokines or immunomodulators, can support T cell maturation and adaptability remains to be investigated. The intrathymic injection of allogeneic hematopoietic cells successfully restores functional T cell development after thymic reconstitution in a mouse model of severe combined immunodeficiency (68). However, thymic organoids still face challenges in restoring T cell function, including establishing immune tolerance, replicating complex thymic stroma, supporting thymic epithelial cells and optimizing T cell maturation (69, 70). While promising for immune rejuvenation in the elderly or immunocompromised, their clinical utility will ultimately depend on overcoming these barriers and demonstrating durable, safe T-cell reconstitution.

Effector T cells rely on glycolysis for rapid cytokine production and cytotoxicity. In the tumor microenvironment (TME), glucose competition with tumor cells impairs T cell energy supply. For instance, increased GLUT1 expression promotes glucose uptake, thereby enhancing T cell function (71). PQDN enhances the antitumor capacity of CD8+ T cells by activating the mitochondrial electron transport chain and promoting glucose uptake and glycolysis (72). While glycolytic enhancement can restore immediate function, excessive reliance accelerates exhaustion, promotes lactate accumulation, and risks metabolic toxicity. This approach is better positioned as a short-term, manufacturing-phase intervention rather than systemic therapy.

Although glycolysis is crucial for effector T cell function, excessively high levels can hinder the long-term survival and differentiation of memory T cells (73). Restricting glycolysis using the HK2 inhibitor 2-deoxyglucose (2-DG) promoted the formation of memory CD8+ T cells and enhanced their antitumor capacity (73, 74). Metformin shifts metabolism toward fatty acid oxidation (FAO) through AMPK activation, enhancing long-term survival and antitumor capacity (75). Proper regulation of the balance between glycolysis and other metabolic pathways is crucial for optimizing CD8+ T cell function. Yet systemic drugs like 2-DG and metformin carry risks of hypoglycemia, lactic acidosis, and variable efficacy in patients with metabolic comorbidities. Their use should focus on ex vivo programming rather than chronic in vivo exposure.

In TME and autoimmune settings, glycolytic suppression drives CD8⁺ T cell dysfunction (76). The combination of anti-PD-1 therapy, and glycolysis-promoting drugs can partially restore CD8+ T-cell function (77). Anti-PD-1 enhances glycolysis by inhibiting pyruvate entry into fatty acid oxidation, promoting oxidative phosphorylation (OXPHOS) and energy production, and further increasing glycolysis via the PI3K-mTOR pathway through the AGK kinase. Additionally, regulating mitochondrial function or neutralizing reactive oxygen species (ROS) can significantly improve CD8+ T-cell metabolism and enhance antitumor capacity (78). This dual strategy is compelling but risky—checkpoint blockade already predisposes to immune-related adverse events, and additional metabolic activation could exacerbate toxicity or benefit tumor metabolism. Precise timing, dosing, and biomarker-guided monitoring are critical.

Quiescent T cells depend on OXPHOS, whereas activated T cells integrate glycolysis with OXPHOS (79). Upon T-cell activation, mitochondrial fragmentation reduces OXPHOS (80). AMPK activators (e.g., metformin) or mTOR inhibitors (e.g., rapamycin) stimulate FAO, promoting the generation of memory CD8+ T cells (79), thereby extending their longevity and enhancing immunological memory. Studies have shown that PGC1α and PPAR agonists (e.g., bezafibrate and fenofibrate) can enhance FAO, boosting the antitumor function of memory T cells (81–84). In adoptive cell therapy, metabolic interventions through in vitro reprogramming can enhance the long-term survival and anti-cancer capacity of T cells. Targeting FAO and OXPHOS holds promise for durable antitumor immunity, yet systemic administration of metabolic modulators faces significant tolerability constraints. Rapalogs and fibrates should therefore be regarded as tools for short-term, reversible metabolic programming during ex vivo expansion or transient in vivo windows, rather than maintenance drugs, in order to achieve a balance between long-term persistence and immediate cytotoxic activity.

PD-1 binds to PD-L1 to suppress T-cell activity, while IFN-γ and TNF-α in the tumor microenvironment increase PD-L1 expression, aiding tumor immune escape. Immune checkpoint inhibitors, including PD-1 and PD-L1 inhibitors, can block the interaction between PD-1 and PD-L1, restore T cell activity, and enable them to recognize and attack tumor cells (85). Combining immune checkpoint inhibitors with anti-inflammatory drugs or other targeted therapies, such as chemotherapy or radiotherapy, can enhance the efficacy of immunotherapy (86, 87).

The application of immune checkpoint inhibitors (ICIs) has advanced cancer immunotherapy, yet their efficacy and safety remain constrained. Some patients develop immune-related adverse events, including colitis, pneumonitis, and myocarditis. Others exhibit primary or acquired resistance, which arises from intrinsic tumor characteristics and adaptive changes within the tumor microenvironment, ultimately leading to the establishment of an immunosuppressive TME (88). Although combinations with chemotherapy, radiotherapy, or anti-inflammatory agents may enhance therapeutic efficacy, additive toxicities hinder clinical optimization. Future strategies must therefore achieve a more refined balance between efficacy and safety, rather than relying on a single immunological pathway.

Regulation of the function of immunosuppressive cells can effectively enhance the antitumor activity of T cells, thereby improving the overall efficacy of immunotherapy.

In the tumor microenvironment, regulatory T cells (Tregs) suppress the activity of effector T cells via multiple mechanisms. Early clinical trials (NCT00888927) shows that pharmacological approaches such as PI3Kδinhibitors, anti-CTLA-4 antibodies, CCR4 antagonists, and metabolic targeting (e.g., CD36 inhibition) can reduce Treg numbers or function, thereby enhancing effector T cell responses (89–91). Other metabolic pathways such as fatty acid oxidation, glycolysis, and amino acid metabolism are also potential targets for regulating Tregs and improving the efficacy of immunotherapy.

Although these strategies may indirectly restore antitumor immune responses, systemic depletion of Tregs can disrupt immune tolerance and trigger severe autoimmune reactions (92). Therefore, future studies should prioritize the selective targeting of tumor-infiltrating Tregs and the development of delivery systems that minimize systemic toxicity without compromising self-tolerance.

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive myeloid cells that proliferate widely in cancerous and chronic inflammatory environments.

Chemotherapy drugs such as gemcitabine and 5-fluorouracil have been shown to reduce the number of MDSCs (93). Targeted small-molecule drugs like SRA737 (a CHK1 inhibitor) combined with low-dose gemcitabine significantly reduce MDSC numbers, while boosting the expression of IFNβ and chemokines CCL5 and CXCL10, enhancing T cell antitumor responses (94).

MDSCs accumulate in the tumor microenvironment via chemokines, thereby hindering T cell function. Blocking CXCR1/2 (e.g., with SX-682) can reduce MDSC migration in head and neck cancer mouse models, thereby enhancing the effectiveness of NK cell immunotherapy and anti-PD-1 treatment (95–97).

Reducing the immunosuppressive activity of MDSCs can restore T-cell function and delay tumor progression. This can be achieved by inhibiting key signaling pathways such as JAK/STAT or NF-κB, or by targeting the secretion of suppressive molecules (94). PDE5 inhibitors such as sildenafil, tadalafil, and vardenafil have been shown to weaken MDSC function by reducing the secretion of inhibitory molecules (98, 99). STAT3 inhibitors, such as JSI-124 can further reduce MDSC activity (100). Reducing MDSC activity can help restore T-cell function and enhance the antitumor immune response.

Current strategies targeting MDSCs primarily involve reducing their numbers, blocking their recruitment, and inhibiting their immunosuppressive functions, thereby restoring T cell activity and enhancing antitumor immunity. However, these agents often lack specificity and may simultaneously affect normal myeloid cells, leading to bone marrow suppression and increased risk of infection (101). Moreover, the high degree of heterogeneity of MDSCs across different tumor types and patients, coupled with the absence of standardized biomarkers for their identification, has significantly limited the clinical translation of these approaches.

M2-type tumor-associated macrophages (TAMs) suppress T-cell function and promote tumor progression, making their reprogramming into M1-type macrophages an attractive therapeutic approach. Agents such as TLR agonists (3M-052, CpG ODN), anti-CD40 antibodies, and low-dose metformin can induce M1 polarization, enhance macrophage and T-cell antitumor activity, and increase T-cell infiltration into the tumor microenvironment (102–105).

Targeting TAMs with small-molecule inhibitors or nanotechnology can effectively inhibit their tumor-promoting functions and enhance the efficacy of therapies, such as immune checkpoint inhibitors.

This indicates that reprogramming TAMs not only directly enhances T cell function but also significantly improves the tumor microenvironment, offering synergistic benefits for immunotherapy. However, the inherent plasticity of tumor-associated macrophages (TAMs) often renders these effects transient, and the systemic administration of Toll-like receptor (TLR) agonists may trigger a cytokine storm (106). Recently, antibody-mediated and nanomedicine-based strategies have been explored to improve TAM targeting, yet issues such as delivery efficiency, off-target uptake, and long-term safety remain unresolved (107). Although TAM reprogramming is conceptually sound, it must be refined through targeted delivery systems and combinatorial therapies to achieve durable clinical benefits.

Future directions may also involve the integration of nanotechnology or biomaterial-based delivery systems to precisely modulate the TME, thereby enhancing therapeutic specificity and durability.

PD1 plays a critical role in maintaining an exhausted state. By using PD1 or PD-L1 inhibitors, exhausted T cells can regain some of their functions (8), particularly their proliferative capacity and cytotoxicity. Importantly, such blockade preferentially activates progenitor-like exhausted T cells, which display superior proliferative and functional recovery compared to terminally differentiated exhausted subsets (108–110). In contrast, terminally differentiated exhausted T cells respond less to PD1 blockade but retain significant cytotoxic potential (108–110). These findings highlight that PD1 inhibition does not fully reverse exhaustion but can augment effector capacity within specific subsets. Sustained use of PD-1 blocking agents has the potential to exacerbate autoimmune responses and destabilize immune homeostasis, emphasizing the necessity of carefully balancing immune reactivation and safety considerations.

Epigenetic regulation is a promising strategy for reversing T cell exhaustion, enhancing T cell function, and boosting the efficacy of immunotherapy. DNMT3A-driven de novo DNA methylation promotes dysfunction in CD8+ T cells, while treatment with DNA methyltransferase inhibitors such as 5-aza-2-deoxycytidine restores function when combined with PD1 blockade (111). Deleting the DNA demethylase TET2 significantly extends the lifespan of CAR-T cells and enhances their tumor-killing capacity (112). TET2 deletion prevents T cell exhaustion and promotes T cell memory formation. Early clinical trials (NCT01029366) shows that CRISPR-Cas9–mediated CD5 knockdown enhances cytotoxicity, proliferation, and survival, effects associated with increased Ras/ERK and PI3K/AKT/mTOR signaling and reduced exhaustion markers (113).

Currently, early clinical trials (NCT03179943) are evaluating the use of epigenetic drugs such as DNA methyltransferase inhibitors and histone deacetylase inhibitors (114) in combination with immune checkpoint blockers, aiming to enhance the durability of the treatment. However, the systemic nature of epigenetic therapies has raised concerns about non-targeted effects, hematotoxicity, and unexpected immune activation (115). Moreover, the optimal timing of use, dosage, and patient selection criteria have not yet been determined. Therefore, careful clinical design is still needed to maximize the benefits of epigenetic regulation and minimize long-term risks.

Table 2 presents details on preclinical research and clinical.