As carriers of tumor antigen-specific T-cell receptors (TCRs), cytotoxic CD8+ T cells (CTLs) are the most crucial anti-tumor effector lymphocytes. These TCRs specifically recognize tumor neoantigens presented by MHC-I molecules on the target cell membrane. Upon antigen recognition, CTLs execute their cytotoxic functions by secreting granzyme B and perforin, increasing the synthesis and release of cytokines such as interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) and inducing tumor cell apoptosis via the FasL/Fas pathway (17, 18). IFN-γ, TNF-α, and IL-2 are key cytokines secreted by activated T lymphocytes and play significant roles in TILs therapy. When T cells are cultured in vitro during TILs therapy, the proportion of CD8+ T cells targeting tumor antigens is further enriched through continuous artificial tumor antigen presentation.

Among the CD8+ T cell subtypes, Th1 cells, the most typical cytotoxic T cells, are known for secreting high levels of perforin, granzyme B, IFN-γ, and TNF-α and exhibiting potent cytotoxicity (19). Research indicates that Tc1 cells are the predominant subtype found in TILs in patients with malignant melanoma, ovarian cancer, lung cancer, or breast cancer (18). Tc2 cells, which primarily produce type II cytokines, also express high levels of granzyme B and exhibit cytotoxicity comparable to Tc1 cells, making them significant in adoptive transfer anti-tumor therapies (19, 20). In a study of the cytokine expression profiles of CD8+ T cells in the TILs from eight cervical cancer patients, Sheu et al. found that 67% of the CD8+ T cells were Tc2 cells (21). Tc22 cells, another subset within the CD8+ T cell population, secrete granzyme B and possess substantial cytotoxic activity, highlighting their distinct anti-tumor potential in adoptive cell immunotherapy.

CD4+ T cells are a key subset of T lymphocytes that recognize antigens presented by antigen-presenting cells, such as dendritic cells. Specifically binding to MHC-II molecules on the antigen-presenting cells through TCRs, CD4+ T cells primarily play a supportive and regulatory role in immune responses. CD4+ T cells enhance immune responses and anti-tumor effects by secreting cytokines and interacting with other immune cells. CD4+ T cells secrete cytokines such as IFN-γ and TNF, which directly limit tumor growth and modulating the immunogenicity and vascularization of the tumor microenvironment (TME) (22).

Within TILs, CD4+ T cells play crucial roles in supporting and regulating immune responses. Based on their function differentiation, CD4+ T cells can be divided into different subsets, including T helper 1 (Th1), Th2, Th9, Th17, T follicular helper (Tfh), and regulatory T (Treg) cells. Correspondingly, these subsets secrete cytokines and exert distinctive effects on tumor immunity. Th1 cells primarily produce IFN-γ and TNF-α, which activate other immune cells, including CD8+ T cells, macrophages, and B cells (23), and promote cell-mediated immune responses and sustain anti-tumor activity. Th2 cells can either exhibit anti-tumor immune responses or inhibit anti-tumor cell activity, sometimes promoting tumor growth (22). Research suggests that in the immune response to HPV infection, CD4+ T cells tend to favor Th2 responses. The imbalance between Th1 and Th2 cells in HPV-infected individuals might limit immune function, leading to cancer cell proliferation and metastasis. This Th1/Th2 imbalance may be associated with the development and poor prognosis of cervical cancer (24). Th9 cells secrete IL-9, stimulate dendritic cells to uptake and present antigens, and activate CD8+ T cells (23). Th17 cells have high metabolic activity and can transform into Th1 cells. Th17 cells secrete IL-17, IL-22, and other cytokines, activate immune cells and enhance anti-tumor effects (25). Tfh cells mainly promote humoral immunity by facilitating germinal center (GC) responses and differentiation of memory B cells and plasma cells (26). Treg cells exert immunosuppressive effects and play a dual regulatory role in high-risk HPV infections. They form protective physiological defenses against persistent inflammatory stimulation induced by HPV, reducing immune clearance of pathogens and the resulting tissue damage caused by immune imbalance. On the other hand, Treg cells consume IL-2 and release inhibitory cytokines such as TGF-β and IL-10, leading to T-cell apoptosis and promoting tumor growth. Therefore, elevated Treg levels are associated with increased risk of cervical cancer (22, 27).

CD4+ T cells also have immune memory capabilities, enabling them to persist and quickly respond when re-exposure to the same antigen (22). This allows them to maintain long-lasting immune responses against tumors and mount rapid attacks when needed. Although CD4+ T cells were traditionally thought to kill tumors primarily by regulating and coordinating with other immune cells, recent research has found that CD4+ effector T cells can independently eliminate established tumors as effectively as CTLs. It has been shown that only a small number of CD4+ effector T cells (about 1% of tumor-infiltrating immune cells) are located at the tumor invasion edge, where they interact with CD11c+MHC-II+ antigen-presenting immune cells and indirectly eliminate tumors. In contrast, a large number of CTLs infiltrate into the tumor core, directly targeting and killing MHC-I-expressing tumor cells (28).

T cells are the primary cells in TILs, and combining CD4+ T-cell and CD8+ T-cell therapies may lead to a more effective immune response against the target. Clinical efficacy may be related to the CD4+/CD8+ T-cell ratio in the infusion. Research data indicate that CD4+/CD8+ T-cell ratio is associated with clinical response in adoptive immunotherapy targeting B-cell maturation antigen (BCMA) in multiple myeloma (29). However, a group of clinical trials related to HPV-associated cancers suggested that the CD4+/CD8+ T-cell composition did not affect outcomes, as clinical responses were observed in infusion products with different CD4+/CD8+ T-cell ratios (30). The cytokines produced by TILs can also activate and proliferate NK cells, generating an anti-tumor immune response. During the preparation of TILs therapy, identifying the cell subsets with the most potent anti-tumor activity can be prioritized for further optimization in cell production, which may help enhance and sustain comprehensive immune response, thereby harnessing the full potential of anti-tumor immunity.

The tumor microenvironment (TME) refers to a heterogeneous and continuously dynamic assembly of immune cells, stromal cells, vasculature, and extracellular matrix (ECM) that collectively shape the local microenvironment influencing tumor cell proliferation, invasion, and metastasis (31). In cervical cancer, the immune microenvironment exhibits marked heterogeneity across patients, with its composition and functional states being closely linked to pathological subtypes, molecular profiles, and therapeutic responses. Significant differences exist between the immune microenvironments of cervical squamous cell carcinoma (CSCC) and cervical adenocarcinoma (CAde): CSCC exhibits higher infiltration of cytotoxic CD8+ T cells, natural killer (NK) cells, and pro-inflammatory macrophages (M1 phenotype), whereas CAde is dominated by regulatory T cells (Tregs), immunosuppressive macrophages (M2 phenotype), and cancer-associated fibroblasts (CAFs) (32–34). Single-cell RNA sequencing (scRNA-seq) studies further reveal that in CSCC, T cells undergo a phenotypic shift from cytotoxic to exhausted states, whereas CAFs in CAde drive immunosuppression through inflammatory regulation (34, 35). Additionally, germinal center B cells within tertiary lymphoid structures are associated with improved prognosis (36). scRNA-seq analyses demonstrate that CSCC is enriched with cytotoxic CD8+ T cells, effector memory CD8+ T cells, and pro-inflammatory NK cells, whereas CAde is predominantly infiltrated by naïve T cells and immunosuppressive Tregs (34). CAFs in CAde participate in immune modulation through the secretion of inflammatory cytokines, whereas CAFs in CSCC are linked to tumor epithelial-mesenchymal transition (EMT) (34, 35). Furthermore, therapeutic interventions differentially remodel the immune microenvironment of cervical cancer. For instance, concurrent chemoradiotherapy (CCRT) may transiently enhance local CD8+ T cell infiltration, while systemic therapies can induce systemic expansion of immunosuppressive monocytic myeloid-derived suppressor cells (Mo-MDSCs) and Tregs in peripheral blood (37, 38).

The typical process of TILs preparation includes the following steps: (1) Obtaining tumor tissue: Fresh tumor specimens are obtained via surgical resection or biopsy, followed by meticulous dissection to remove necrotic debris and blood clots under aseptic conditions. This procedure demands meticulous precision and technical expertise to ensure procurement of sufficient viable tumor specimens while minimizing trauma to the patient. (2) Performing the pre-rapid expansion protocol (pre-REP): Following surgical resection, tumor specimens are promptly transferred to the laboratory for processing. The enzymatic digestion protocol involves sequential treatment of tumor fragments with a cocktail of collagenase, DNase, and hyaluronidase to achieve tissue disaggregation. Tumor-infiltrating lymphocytes (TILs) are subsequently isolated through advanced cell sorting methodologies, including flow cytometry-based sorting (e.g., CD3⁺CD8⁺ surface marker selection) or magnetic bead-based isolation (41). Alternatively, the tissue explant migration method entails aseptically mincing tumor specimens into fragments (1–3 mm³) that are evenly distributed in culture dishes. These fragments are maintained in IL-2-supplemented medium (1000–6000 IU/mL) under standardized conditions (37°C, 5% CO₂) for 1–3 weeks, allowing spontaneous migration of lymphocytes from the tumor periphery into the culture medium, followed by collection of migratory TIL populations (42). The isolation process must be conducted under sterile conditions to ensure TILs purity and activity. (3) Performing the rapid expansion protocol (REP): The isolated TILs are cultured in a medium containing a high dose of IL-2, in which they expand efficiently (approximately 95,652-fold) while retaining strong anti-tumor cytotoxic function (43). Several weeks are required in this process, during which culture conditions, such as temperature, gas composition, and nutrients in the medium, are fine-tuned to optimize TILs growth and activity. The expanded TILs undergo rigorous quality-control tests to ensure their safety and efficacy. Only cells that pass these tests can be used in the subsequent treatment process ( Figure 2 ).

Comparative analyses demonstrate that the tissue explant migration method induces significantly less cellular stress than enzymatic digestion protocols, better preserving TIL viability and functional integrity. This approach exhibits particular suitability for cervical carcinoma specimens characterized by low-fibrosis soft tissue architecture. However, its clinical implementation requires optimization of tumor fragment dimensions (typically 1–3 mm³) to maximize lymphocyte migration efficiency, with extended ex vivo expansion timelines (3–4 weeks) representing a notable limitation. To reconcile processing efficiency with cellular fitness, hybrid protocols incorporating partial enzymatic pretreatment followed by explant culture of residual tissue fragments have shown promise in preliminary studies.

After surgical excision, the tumor tissue is surgically removed before TILs are isolated from the excised tumor tissue, selected, and cultured. Using the rapid expansion protocol (REP), the TILs are expanded in vitro in the presence of high-dose interleukin-2 (IL-2). The patient undergoes lymphodepletion and receives a high dose of IL-2 before reinfusion of the TILs. After the TILs enter the patient’s body, CD4+ T cells bind to major histocompatibility complex (MHC) II, assisting and regulating the immune response. Cytotoxic CD8+ T cells (CTLs) bind to MHC I and secrete cytokines such as granzyme B, perforin, IFN-γ, and TNF-α as well as mediate tumor cell apoptosis through FasL/Fas interaction.

Before TILs infusion, patients undergo an NMA lymphodepletion regimen, commonly referred to as “lymphodepletion,” through chemotherapy or total body irradiation (TBI). The main purpose of lymphodepletion is to eliminate the endogenous lymphocytes, which can significantly enhance the sustained activity of the adoptively transferred T cells. Lymphodepletion also enhances the effect of TILs through two other potential mechanisms. One mechanism is by increasing the number of host homeostatic cytokines, including IL-7 and IL-15, while decreasing the number of endogenous lymphocytes that compete for these trophic cytokines (44, 45). The second mechanism is by enhancing the activation of antigen-presenting cells (APCs), which play a crucial role in regulating adoptively transferred T cells (46). In a clinical study in which infused TILs were labeled with radionuclides and tracked in vivo by imaging, patients who had undergone cyclophosphamide lymphodepletion had significantly more TILs migration to the tumor than those who had not undergone this pretreatment (47). Currently, cyclophosphamide + fludarabine (FC) chemotherapy is widely used for lymphodepletion. A recent study comparing different lymphodepletion regimens found that cyclophosphamide (60 mg/m²) combined with fludarabine (125 mg/m²) provided the best lymphodepletion effect and bone marrow recovery with minimal side effects (48). After lymphodepletion, patients receive the cultured TILs along with a high dose of IL-2.

IL-2 is an important immunomodulatory cytokine that stimulates the growth and survival of effector T cells (49), thereby promoting the growth and activity of TILs. Rosenberg et al. discovered that lymphocytes grown in the presence of IL-2 could lyse fresh syngeneic or autologous tumor cells without affecting normal cells. Moreover, in vitro experiments have shown the antitumor activity of TILs is greatly enhanced when cultured in a medium supplemented with high-dose IL-2 (50). Although there is no standardized dose for IL-2, most trials use high-dose IL-2 (600,000 IU/kg or 720,000 IU/kg) administered every eight hours until the patient can no longer tolerate the dose (51, 52). The toxicity of IL-2 is dose-dependent and includes high fever, capillary leak syndrome, and other adverse effects. To reduce the toxicity of high-dose IL-2 without compromising T-cell activity, researchers have explored low-dose IL-2 regimens. Clinical trials have reported sizable tumor regression (30%–63%) in 5 out of 25 patients with 10 different cancer diagnoses treated with TILs and low-dose IL-2 (200,000 IU/kg for 14 days), with no severe IL-2-related adverse effects reported (53). In a Danish pilot trial (54) of six patients with metastatic melanoma treated with TILs, low-dose IL-2 (200,000 IU/kg for 14 days) considerably decreased the treatment-related toxicity, with no grade 3 to 4 IL-2-related AEs reported. Objective clinical responses were observed in two of six treated patients, who had ongoing complete responses lasting over 30 months and over 10 months, and two other patients had stable disease lasting four months and five months. These results suggest that low-dose IL-2 administration in TILs therapy can induce an immune response while significantly reducing toxicity.

However, such promising results were not obtained in a phase II clinical trial (55) in which 12 patients with unresectable metastatic melanoma received TILs therapy with an even lower IL-2 dose (125,000 IU/kg × 12 days). Although no unexpected AEs were observed, only three patients experienced partial clinical responses, and none achieved complete remission. These results suggest that low-dose IL-2 might be associated with lower T-cell activity. When the findings of these previous studies are considered together, they indicate that there is currently not sufficient evidence to conclude that low-dose IL-2 regimens can produce the same sustained response as high-dose IL-2 regimens. Large-scale clinical trials are now needed to identify the optimal dosing regimen.

TILs infusion must be performed under strictly sterile conditions to ensure cell purity and activity. In most clinical trials, TILs therapy is administered intravenously, although some studies have reported other routes, such as intrapleural, intraperitoneal, or intratumoral administration (56, 57). Once infused back into the patient’s body, these specially cultured and expanded immune cells begin to seek out and attack cancer cells. Ideally, TILs effectively recognize and kill tumor cells to achieve the treatment goal.

The application of TILs in cancer therapeutics traces back to the 1980s. In 1982, Steven Rosenberg (58), widely considered as the father of ACT, first isolated TILs from a murine tumor model and expanded them in vitro in the presence of IL-2. He then reinfused these cells into a tumor-bearing host, leading to a significant anti-tumor response. This study laid the foundation for TILs therapy in treating advanced tumors. In 1986, Rosenberg and his team (50) found that with the combination of cyclophosphamide, TILs, and IL-2, 50% to 100% of mice bearing colon adenocarcinoma were cured of advanced hepatic or pulmonary metastasis. Based on their findings, they concluded that infiltrating lymphocytes are composed of tumor antigen-specific immune cells capable of sustained tumor cell killing. In 1994, a clinical trial of TILs therapy for metastatic melanoma showed promising results, with 10% to 30% of patients achieving complete remission and an overall objective response rate of up to 50% (59).

In 2015, Rosenberg et al. (60) reported the first clinical trial of TILs therapy for cervical cancer. This trial with TILs of nine patients diagnosed with advanced recurrent or metastatic cervical cancer resulted in complete regression in two patients and partial remission in one patient. Remarkably, the complete responses persisted for 22 and 15 months respectively, These groundbreaking results marked the first positive outcome of TILs therapy in cervical cancer, offering a potential alternative treatment for patients with advanced disease who had previously undergone chemotherapy or chemoradiotherapy. Nevertheless, the objective response rate (ORR) in the TILs treatment protocol established by Rosenberg’s laboratory was approximately 33.3%, indicating the need for further optimization and improvement in TILs therapy for cervical cancer.

In 2017, Rosenberg et al. (61) published the results from a phase II single-arm clinical trial (NCT01585428) evaluating TILs therapy in patients with metastatic HPV-associated carcinomas. Among eighteen HPV-positive cervical cancer patients, 27.8% (5/18) achieved objective tumor responses, including two complete remission. Follow-up reports revealed that the two patients who had achieved complete remission survived for over five years. In 2019, another clinical trial of TILs therapy for HPV-associated carcinomas was published involving 29 patients, including 18 with cervical cancer. Of these, two had achieved complete remission and three partial remission, with an ORR of 28% (15).

At the 2019 American Society of Clinical Oncology (ASCO) Annual Meeting, Iovance presented compelling clinical data from the C-145–04 study. Among 27 patients with recurrent, metastatic, or persistent cervical cancer, a single infusion of TILs resulted in an ORR of 44% and a complete response rate of 11%. The disease control rate (DCR) was 85%,with the primary adverse events (AEs) being chills, anemia, and diarrhea (62) and no serious adverse effects were reported, In a Chinese translational investigation of adjuvant TILs following concurrent chemoradiotherapy in patients with cervical cancer (NCT 4443296), TILs from 20 of the 27 patients were successfully expanded, with a feasibility of 74.1%. Of the 12 patients who received TILs following concurrent chemoradiotherapy (CCRT), 9 (75.0%) attained complete responses, with a disease control duration of 9 to 22 months (63). Another report (NCT 04766320) detailed the use of TILs in combination with PD-1 antibody therapy in a patient with advanced cervical cancer who achieved complete remission 10 weeks after drug infusion with only mild and controllable adverse effects (40).

The results of these studies indicate that TILs therapy has demonstrated effectiveness and safety in treating advanced cervical cancer in the US and Europe. However, its exploration in China remains nascent. Currently, Zhilin Biotech’s ZLT-001, Grit Biotechnology’s GT101 and GT201, and Jun Sai Biotech’s GC101 and GC203 have received clinical trial approval and are currently undergoing corresponding trials. If found effective, they could offer new treatment options for patients with metastatic or recurrent cervical cancer ( Table 1 ).

Genetically modified TILs (GM-TILs) refer to engineered TILs modified via gene-editing technologies—such as CRISPR/Cas9, TALEN, or viral vector transduction—to enhance their antitumor activity, persistence, or resistance to immunosuppressive signals within the tumor microenvironment (TME). This strategy aims to overcome limitations of conventional TIL therapy, including T cell exhaustion, restricted antigen recognition, and immunosuppressive TME influences.

Currently, GM-TIL applications in cervical cancer primarily focus on two strategies: CRISPR/Cas9-mediated knockout of immunosuppressive targets and cytokine signaling enhancement. The first approach is exemplified by GT316, which employs CRISPR-based dual knockout of undisclosed immunomodulatory targets (identified via genomic screening) to reduce T cell exhaustion and amplify antitumor efficacy. In a Phase I trial (NCT06145802), a heavily pretreated cervical cancer patient achieved a complete response (CR) lasting 32 weeks following infusion of 1.0×10^10 GT316 cells, with no dose-limiting toxicity observed. This response correlated with TIL expansion and elevated serum IFN-γ levels post-treatment, potentially linked to reduced TIL exhaustion phenotypes, though the precise targets require further validation (64). The second strategy, represented by GT201, involves engineering TILs to express membrane-bound IL-15 (mbIL-15), thereby enhancing persistence and reducing reliance on exogenous IL-2. In a Phase I trial (NCT05430360), a refractory cervical cancer patient infused with 5.3×10^9 GT201 cells achieved a partial response (PR) lasting 30 weeks, with detectable functional TILs persisting for one month post-infusion (65, 66). Both strategies demonstrated enhanced cytotoxicity in preclinical models (64, 65), and early clinical data suggest that targeting T cell exhaustion (e.g., GT316) or cytokine signaling (e.g., GT201) may overcome barriers such as IL-2 deficiency or T cell dysfunction in the cervical cancer TME (64, 66, 67). Additionally, alternative strategies—including PD-1 or TGFBR2 knockout—have shown promise in melanoma or ovarian cancer models (67–69), though data in cervical cancer remain unreported. Their applicability may depend on cervical cancer-specific TME features, such as elevated TGF-β levels (68, 69).

Despite preliminary validation of GM-TIL safety in early trials, significant limitations persist: small sample sizes (single-case reports), unclear mechanisms of genetic modifications (64, 65), and tumor heterogeneity potentially compromising therapeutic generalizability. Future directions should prioritize HPV-specific targeting (e.g., E6/E7 TCR-T), multiplex gene editing (e.g., PD-1 KO combined with IL-15 expression), and optimization of TIL expansion protocols to minimize exhaustion phenotypes (70, 71). Rigorous validation through cervical cancer-specific preclinical models and randomized trials will be critical to establish efficacy (72, 73).

For patients with recurrent or metastatic cervical cancer, traditional treatment options are limited. Although TILs therapy has advantages, it may not be effective for all patients. A combination of different treatment can synergistically enhance the efficacy of cancer therapy.

Current studies have identified critical prognostic biomarkers associated with cervical cancer TILs therapy. CD8+ TILs density serves as a pivotal indicator, with higher infiltration levels significantly correlating with prolonged recurrence-free survival (RFS) and overall survival (OS) (106). Conversely, FasL overexpression in tumor cells negatively correlates with CD45+ TILs infiltration (OR=9, p=0.01), suggesting FasL-mediated TILs apoptosis may drive immune evasion (106). PD-L1 expression (positive in 68% of cases) and its Combined Positive Score (CPS) exhibit dual implications: PD-L1 positivity may predict responsiveness to immune checkpoint inhibitors, yet its co-occurrence with TILs exhaustion phenotypes underscores the need for combinatorial strategies (107). Epigenetic markers such as ERBB3 promoter methylation reduce TILs infiltration by suppressing immune-related RNA pathways, offering novel avenues for patient stratification (108). These biomarkers guide both prognosis and therapeutic optimization: high CD8+ TILs density and low ERBB3 methylation may identify candidates for adoptive TILs therapy, while FasL inhibitors could counteract immunosuppressive microenvironments (106, 108). However, challenges persist due to TILs functional heterogeneity (e.g., dynamic equilibrium between exhausted and activated subsets) and limited multicenter validation (107). Future integration of spatial transcriptomics and standardized AI-driven quantification is essential to enhance prognostic precision and advance personalized therapies (109).

Compared to other solid tumors (e.g., colorectal cancer, glioblastoma), cervical cancer TILs therapy demonstrates unique advantages driven by HPV-associated antigen immunogenicity and microenvironmental plasticity. Persistent HPV infection induces stable expression of E6/E7 viral antigens, providing TILs with highly specific targets to overcome tumor heterogeneity through polyclonal T cell responses (110–113). Preclinical studies reveal that HPV antigen-specific TILs combined with anti-PD-1 therapy significantly enhance CD8+ T cell infiltration while reducing immunosuppressive cells (e.g., Tregs, MDSCs), achieving higher tumor regression rates than breast or lung cancer models (111, 113). In contrast, TILs in other solid tumors (e.g., MC38 colorectal cancer) are prone to exhaustion due to antigen heterogeneity and immunosuppressive pathways (e.g., KLF5/COX2, MIF/CD74), necessitating genetic editing (e.g., Cbl-b knockout) or metabolic reprogramming (e.g., PGC-1α overexpression) to restore functionality (114–116). Additional advantages of cervical cancer TILs include: (1) viral origin of HPV antigens enhancing antigen presentation efficiency (110, 117); (2) synergy with mRNA vaccines to convert immunologically ‘cold’ tumors (110); and (3) activation of non-classical immune subsets (e.g., γδ T cells) for coordinated antitumor activity (117). Future priorities include HPV multi-epitope TIL therapies combined with metabolic modulation, though efficacy in HPV-negative cervical cancer remains unresolved (115).