Accumulating evidence demonstrates that the silencing of tumor suppressor genes by aberrant DNA methylation contributes to the initiation and progression of ovarian cancer (OC), while the systemic methylation profile and the key driver methylation events need to be further explored. Here, by analysing public databases and our resources, we identified the hypermethylation of ZNF154 promoter as a key driver of OC malignancy, which was mediated by the DNA methyltransferase complex DNMT1/UHRF1. Using CRISPR/dCas9-TET1CD, a tool for targeted demethylation, we successfully decreased the methylation level of ZNF154 promoter and reactivated ZNF154 expression, which in turn inhibited the proliferation, migration, and invasion of OC cells. Mechanistically, ZNF154 interacted with KAP1 and directly bound to the ROMO1 promoter, transcriptionally repressing ROMO1 expression, thereby reducing MMP2 and phosphorylated ERK to impede OC progression. Clinically, ZNF154 hypermethylation was correlated with its reduced expression and poor prognosis in OC patients. These findings underscore a pivotal role of aberrant ZNF154 methylation in OC pathogenesis and highlight its potential as both a therapeutic target and a prognostic biomarker for OC patients.

Metastasis is the main cause of treatment failure in nasopharyngeal carcinoma (NPC). Our previous study developed a transcriptomics-based gene signature (AK4, CPAMD8, DDAH1, and CRTR1) to predict metastasis in NPC and identify candidates that could benefit from induction chemotherapy (IC). Of these, adenylate kinase 4 (AK4) is a potent oncogene involved in the malignant progression of a variety of tumors. This study investigated the expression and mechanism of action of AK4, a member of the AK family of enzymes, in NPC. Quantitative real-time PCR, western blotting, and immunohistochemistry revealed that AK4 was upregulated in NPC and correlated with metastasis and chemoresistance. Stable ectopic overexpression of AK4 in NPC cell lines conferred resistance to taxol-induced apoptosis, promoted the migration, invasion, and EMT phenotype, and induced IL-1β secretion by activating the NLRP3 signaling pathway; knockdown of AK4 had the opposite effects. Mechanistically, AK4 co-localized with NNT, upregulated NLRP3 and IL-1β, and consequently altered NPC cell metastasis and chemoresistance. AK4 may play a role in the development of NPC and represent a potential therapeutic target.

The efficacy of human epidermal growth factor receptor 2 (HER2)-targeting antibody-drug conjugates has underscored the critical need for precise HER2 diagnostics in breast cancer treatment. Despite the clinical importance, variability in immunohistochemical (IHC) staining protocols and interobserver inconsistencies challenge the reliability of HER2 status assessment, which is critical for guiding patient treatment strategies. To investigate the factors affecting HER2 interpretation consistency, tissue microarrays from 1063 breast carcinoma cases underwent 3 distinct IHC protocols, and a novel artificial intelligence (AI) model was developed to standardize HER2-stained images. A total of 5 sets of tissue microarrays (Nordi QC, protocol 1, protocol 2, protocol 1 AI, and protocol 2 AI) were independently reviewed by 8 pathologists. The Fleiss Kappa value and overall agreement rate measured interobserver agreement, with logistic regression analyzing the impact of variables on diagnostic accuracy. Our results showed that the Nordi QC protocol had the highest interobserver agreement (Kappa 0.754). AI-based image normalization notably enhanced consistency, particularly for HER2 low cases, aligning scores toward the Nordi QC standard. Logistic regression analysis indicated that both staining protocol and AI-based image standardization significantly influenced diagnostic accuracy (P < .001). The American Society of Clinical Oncology/College of American Pathologists 2018 binary criteria demonstrated the highest HER2 interobserver consistency (Kappa > 0.95). Compared with the American Society of Clinical Oncology/College of American Pathologists 2023 criteria, the newly proposed null, ultra-low/low, positive criteria, merging HER2 low and ultra-low categories, demonstrated improved reliability and agreement, especially in distinguishing the challenging HER2-ultra-low cases, which showed an exceedingly low interobserver agreement (Kappa < 0.20) across all protocols. Overall, variability in IHC staining protocols and HER2 classification criteria significantly affect the diagnostic consistency among pathologists. The integration of an AI model for image standardization and the adoption of the null, ultra-low/low, positive criteria may refine diagnostic precision and bolster clinical decision-making in breast cancer treatment.

BACKGROUND. EBV is associated with nasopharyngeal carcinoma (NPC), but the existence of a NPC protective antibody against EBV-associated antigens remains unclear. METHODS. Patients with NPC and matched controls were identified from prospective cohorts comprising 75,481 participants in southern China. ELISA and conditional logistic regression were applied to assess the effects of gp42IgG on NPC. The expression of HLA-II, the gp42 receptor, in nasopharyngeal atypical dysplasia and its effect on EBV infection of epithelial cells were evaluated. FINDINGS. gp42-IgG titers were significantly lower in patients with NPC compared with controls across various follow-up years before NPC diagnosis (P < 0.05). Individuals in the highest quartile for gp42-IgG titers had a 71% NPC risk reduction compared with those in the lowest quartile (ORsQ4vsQ1= 0.29, 95% CIs = 015 to 0.55, P < 0.001). Each unit antibody titer increase was associated with a 34% lower risk of NPC (OR = 0.66, 95% CI = 0.54-0.81, Ptrend< 0.001). The protective effect of of gp42-IgG was observed in patients diagnosed 5 years or more, 1-5 years, and less than 1 year after blood collection (P < 0.05). HLA-II expression was detected in 13 of 27 specimens of nasopharyngeal atypical dysplasia, and its overexpression substantially promoted epithelial cell-origin EBV infection. CONCLUSION. Elevated EBV gp42-IgG titers can reduce NPC risk, indicating that gp42 is a potential EBV prophylactic vaccine target. TRIAL REGISTRATION. NCT00941538, NCT02501980, ChiCTR2000028776, ChiCTR2100041628. FUNDING. Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD0501003), National Natural Science Foundation of China (82030046, 82073625, 81860601, 82373655), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2019BT02Y198), and Central Financial Transfer Payment Projects of the Chinese Government, Cancer Research Grant of Zhongshan City.

Nasopharyngeal carcinoma (NPC), a squamous cell carcinoma originating in the nasopharynx, is a leading malignancy in south China and other south and east Asia areas. It is frequently associated with Epstein-Barr virus (EBV) infection, while there are also some NPC patients without EBV infection. Here, it is shown that the EBV+ (EBV positive) and EBV- (EBV negative) NPCs contain both shared and distinct genetic abnormalities, among the latter are increased mutations in TP53. To investigate the functional roles of NPC-associated genetic alterations, primary, orthotopic, and genetically defined NPC models were developed in mice, a key tool missed in the field. These models, initiated with gene-edited organoids of normal nasopharyngeal epithelium, faithfully recapitulated the pathological features of human disease. With these models, it is found that Trp53 and Cdkn2a deficiency are crucial for NPC initiation and progression. And latent membrane protein1 (LMP1), an EBV-coding oncoprotein, significantly promoted the distal metastasis. Further, loss of TGFBR2, which is frequently disrupted both in EBV- and EBV+ NPCs, dramatically accelerated the progression and lung metastasis of NPC probably by altering tumor microenvironment. Taken together, this work establishes a platform to dissect the genetic mechanisms underlying NPC pathogenesis and might be of value for future translational studies.

Numerous clinical studies speculated the association between multiple myeloma (MM) and inflammatory diseases; however, there is limited validation of these claims via establishing a causal relationship and revealing the underlying mechanism. This exploratory study employed bidirectional Mendelian randomization (MR) analysis to investigate the causal relationships between MM and inflammatory diseases, including atherosclerosis, asthma, ankylosing spondylitis, Alzheimer's disease (AD), Parkinson's disease (PD), sarcoidosis, inflammatory bowel disease, nonalcoholic fatty liver disease, type II diabetes, and schizophrenia (SZ). Transcriptomic and genome-wide Bayesian colocalization analyses were further applied to reveal the underlying mechanism. A significant and previously unrecognized positive association was identified between genetic predisposition to MM and the risk of SZ. Two independent case reports showed that treatment-resistant psychosis is due to underlying MM and is resolved by treating MM. From our MR analyses, various statistical methods confirmed this association without detecting heterogeneity or pleiotropy effects. Transcriptomic analysis revealed shared inflammation-relevant pathways in MM and SZ patients, suggesting inflammation as a potential pathophysiological mediator of MM's causal effect on SZ. Bayesian colocalization analysis identified rs9273086, which maps to the protein-coding region of HLA-DRB1, as a common risk variant for both MM and SZ. Polymorphism of the HLA-DRB1 allele has been implicated in AD and PD, further highlighting the impact of our results. Additionally, we confirmed that interleukin-6 (IL-6) is a risk factor for SZ through secondary MR, reinforcing the role of neuroinflammation in SZ etiology. Overall, our findings showed that genetic predisposition to MM, HLA-DRB1 polymorphism, and enhanced IL-6 signaling are associated with the increased risk of SZ, providing evidence for a causal role for neuroinflammation in SZ etiology.

Small cell cervical carcinoma (SCCC) is the most common neuroendocrine tumor in the female genital tract, with an unfavorable prognosis and lacking an evidence-based therapeutic approach. Until now, the distinct subtypes and immune characteristics of SCCC combined with genome and transcriptome have not been described. We performed genomic (n = 18), HPV integration (n = 18), and transcriptomic sequencing (n = 19) of SCCC samples. We assessed differences in immune characteristics between SCCC and conventional cervical cancer, and other small cell neuroendocrine carcinomas, through bioinformatics analysis and immunohistochemical assays. We stratified SCCC patients through non-negative matrix factorization and described the characteristics of these distinct types. We further validated it using multiplex immunofluorescence (n = 77) and investigated its clinical prognostic effect. We confirmed a high frequency of PIK3CA and TP53 alterations and HPV18 integrations in SCCC. SCCC and other small cell carcinoma had similar expression signatures and immune cell infiltration patterns. Comparing patients with SCCC to those with conventional cervical cancer, the former presented immune excluded or 'desert' infiltration. The number of CD8+ cells in the invasion margin of SCCC patients predicted favorable clinical outcomes. We identified three transcriptome subtypes: an inflamed phenotype with high-level expression of genes related to the MHC-II complex (CD74) and IFN-alpha/beta (SCCC-I), and two neuroendocrine subtypes with high-level expression of ASCL1 or NEUROD1, respectively. Combined with multiple technologies, we found that the neuroendocrine groups had more TP53 mutations and SCCC-I had more PIK3CA mutations. Multiplex immunofluorescence validated these subtypes and SCCC-I was an independent prognostic factor of overall survival. These results provide insights into SCCC tumor heterogeneity and potential therapies. (c) 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

目的为了探索杂交捕获二代测序技术在子宫内膜癌分子亚型的临床应用。方法收集中山大学附属肿瘤医院经病理确诊的子宫内膜癌112例，均进行基于1 021个基因panel的杂交捕获二代测序检测，分析各分子亚型的分子变异谱及与临床病理特征之间的关系。结果DNA聚合酶ε（POLE）突变型8例（7.1%），错配修复功能缺陷型34例（30.4%），TP53突变型26例（23.2%），非特异性分子谱型44例（39.3%），4个亚型肿瘤突变负荷中位数分别为252.0、38.4、5.8及5.4 Muts/Mb。子宫内膜癌各分子亚型之间与患者临床分期、肌层浸润深度及是否有脉管内癌栓与淋巴结转移等临床病理特征无统计学差异。PTEN（75.5%）、PIK3CA（66.7%）、ARID1A（55.9%）、TP53（40.2%）、NF1（29.4%）为子宫内膜癌最常见的突变。结论对子宫内膜癌进行二代测序，可同时获悉其分子分型各项指标，有效筛查Lynch综合征、各亚型分子变异谱、为免疫治疗及靶向治疗提供指导，并有助于分子遗传特征的进一步积累及探索。

Supplementary Data from Dopamine Signaling Promotes Tissue-Resident Memory Differentiation of CD8+ T Cells and Antitumor Immunity

Abstract Background Existing treatments for cholangiocarcinoma have poor efficacy. However, chimeric antigen receptor‐T (CAR‐T) cells are emerging as a potential therapeutic strategy. Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR‐T cell infiltration and function. This study aimed to improve the function of CAR‐T cells through knock down immune checkpoints and immunosuppressive molecular receptors. Methods We evaluated the expression of epidermal growth factor receptor (EGFR) and B7 homolog 3 protein (B7H3) antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry. Subsequently, we engineered CAR‐T cells targeting EGFR and B7H3 antigens. We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR‐T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR‐T cells for antitumor activity both in vitro, using tumor cell lines and cholangiocarcinoma organoid models, and in vivo, using humanized mouse models. Results We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues. EGFR‐CAR‐T and B7H3‐CAR‐T cells demonstrated specific anti‐tumor activity. We found an abundance of programmed cell death protein 1 (PD‐1), T cell immunoglobulin and mucin domain‐containing protein 3 (Tim‐3), and T cell immunoglobulin and ITIM domain (Tigit) on infiltrated CD8+ T cells in the cholangiocarcinoma microenvironment. We then decreased the expression of these 3 proteins on the surface of CAR‐T cells, named PTG‐scFV‐CAR‐T cells. Furthermore, we knocked‐down the expression of transforming growth factor beta receptor (TGFβR), interleukin‐10 receptor (IL‐10R), and interleukin‐6 receptor (IL‐6R) of PTG‐scFV‐CAR‐T cells. Those cells, named PTG‐T16R‐scFV‐CAR‐T cells, potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoid model. Finally, the PTG‐T16R‐scFv‐CAR‐T cells showed greater inhibitory effect on tumor growth in vivo, and were superior in prolonging the survival of mice. Conclusions Our results revealed that PTG‐T16R‐scFV‐CAR‐T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long‐term efficacy both in vitro and in vivo. This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.

Background: This study was to evaluate the effectiveness and safety of the preoperative regional trans-arterial immunotherapy combined with intravenous chemotherapy in patients with locally advanced cervical cancer (LACC). Methods: Trans-arterial programmed cell death receptor 1 (PD-1) monoclonal antibody infusion combined with intravenous paclitaxel-cisplatin chemotherapy was adopted as neoadjuvant therapy in six patients with primary LACC (stage IIB-IVA) who were willing to undergo surgical resection, and treatment effectiveness was evaluated according to the RECIST 1.1 criteria. If the local tumor was regressive and assessed as operable, surgery was performed, and postoperative adjuvant therapy was selected based on the histopathological findings. Results: One patient was discontinued the neoadjuvant therapy from the study due to an allergic reaction during the 2 nd course and underwent surgical resection directly. Five patients achieved partial response after neoadjuvant therapy with an objective response rate of 100% and a surgical conversion rate of 100%. All patients had R0 resection. Serious adverse events did not occur during neoadjuvant therapy. The six patients were followed up for 15-30 months (median, 23.5 months) and none of them relapsed. Conclusions: Trans-arterial PD-1 monoclonal antibody infusion combined with intravenous paclitaxel-cisplatin as neoadjuvant therapy in patients with LACC showed a higher objective response rate, surgical conversion rate, and safety with satisfactory near-term outcomes. Further investigation is required to substantiate the outcome of regional trans-arterial infusion immunotherapy combined with intravenous chemotherapy as an alternative treatment modality for LACC.

Supplementary Figure from Dopamine Signaling Promotes Tissue-Resident Memory Differentiation of CD8+ T Cells and Antitumor Immunity

Aim: Metastasis is the primary cause of treatment failure in nasopharyngeal carcinoma (NPC); however, the current tumour-node-metastasis staging system has limitations in predicting distant metastasis and guiding induction chemotherapy (IC) application. Here, we established a transcriptomics-based gene signature to assess the risk of distant metastasis and guide IC in locoregionally advanced NPC.Methods: Transcriptome sequencing was performed on NPC biopsy samples from 12 pairs of patients with different metastasis risks. Bioinformatics and qPCR were used to identify differ-entially expressed genes (DEGs), while univariate and multivariate analyses were used to select prognostic indicators for the gene signature. A signature-based nomogram was established in a training cohort (n = 191) and validated in an external cohort (n = 263).Results: Eleven DEGs were identified between metastatic and non-metastatic NPC. Four of these (AK4, CPAMD8, DDAH1 and CRTR1) were used to create a gene signature that effec-tively categorised patients into low-and high-risk metastasis groups (training: 91.1 versus 70.4%, p < 0.0001, C-index = 0.752; validation: 88.4 versus 73.9%, p = 0.00057, C-index = 0.741). IC with concurrent chemoradiotherapy (CCRT) improved distant metastasis-free survival in low-risk patients (94.4 versus 85.0%, p = 0.043), whereas patients in the high-risk group did not benefit from IC (72.6 versus 74.9%, p = 0.946).Conclusions: Our transcriptomics-based gene signature was able to reliably predict metastasis in locoregionally advanced NPC and could be used to identify candidates that could benefit from IC thorn CCRT.(c) 2021 Elsevier Ltd. All rights reserved.

Kelch superfamily involves a variety of proteins containing multiple kelch motif and is well characterized as substrate adaptors for CUL3 E3 ligases, which play critical roles in carcinogenesis. However, the role of kelch proteins in lung cancer remains largely unknown. In this study, the non-small cell lung cancer (NSCLC) patients with higher expression of a kelch protein, kelch domain containing 3 (KLHDC3), showed worse overall survival. KLHDC3 deficiency affected NSCLC cell lines proliferation in vitro and in vivo. Further study indicated that KLHDC3 mediated CUL2 E3 ligase and tumor suppressor p14ARF interaction, facilitating the N-terminal ubiquitylation and subsequent degradation of p14ARF. Interestingly, Gefitinib-resistant NSCLC cell lines displayed higher KLHDC3 protein levels. Gefitinib and Osimertinib medications were capable of upregulating KLHDC3 expression to promote p14ARF degradation in the NSCLC cell lines. KLHDC3 shortage significantly increased the sensitivity of lung cancer cells to epidermal growth factor receptor (EGFR)-targeted drugs, providing an alternative explanation for the development of Gefitinib and Osimertinib resistance in NSCLC therapy. Our works suggest that CRL2KLHDC3 could be a valuable target to regulate the abundance of p14ARF and postpone the occurrence of EGFR-targeted drugs resistance.

Intrinsic and acquired anti-HER2 resistance remains a major hurdle for treating HER2-positive breast cancer. Using genome-wide CRISPR/Cas9 screening in vitro and in vivo, we identify FGFR4 as an essential gene following anti-HER2 treatment. FGFR4 inhibition enhances susceptibility to anti-HER2 therapy in resistant breast cancer. Mechanistically, m6A-hypomethylation regulated FGFR4 phosphorylates GSK-3β and activates β-catenin/TCF4 signaling to drive anti-HER2 resistance. Notably, suppression of FGFR4 dramatically diminishes glutathione synthesis and Fe2+ efflux efficiency via the β-catenin/TCF4-SLC7A11/FPN1 axis, resulting in excessive ROS production and labile iron pool accumulation. Ferroptosis, a unique iron-dependent form of oxidative cell death, is triggered after FGFR4 inhibition. Experiments involving patient-derived xenografts and organoids reveals a synergistic effect of anti-FGFR4 with anti-HER2 therapy in breast cancer with either intrinsic or acquired resistance. Together, these results pinpoint a mechanism of anti-HER2 resistance and provide a strategy for overcoming resistance via FGFR4 inhibition in recalcitrant HER2-positive breast cancer.

Checkpoint immunotherapy is a promising treatment option for advanced cervical cancer. To aid in selecting patients for this treatment, we identified potential predictors of the response to anti-PD-1 combination therapy. We simultaneously characterized CD8+, FoxP3+, PD-L1+, CD68+, CD31+, PANCK+, and PANCK−PD-L1+ cells at the invasive margin (IM) of tumor by multispectral imaging of tissue sections from 37 patients with advanced cervical cancer in our previous trial cohort. The densities of each cell and cell-to-cell topography were compared between the responder and non-responder groups and evaluated for their predictive value in clinical response and survival. CD8+ T cells, PD-L1+ cells, and PANCK−PD-L1+ immune cells showed higher densities at the IM in the responders than in the non-responders (P = 0.022, 0.0094, and 0.049, respectively). A higher density of CD8+ T cells at the IM was related to prolonged progression-free survival (PFS; P = 0.031). A higher ratio of CD68+/CD8+ cells was found in the non-responder group (P = 0.003) and related to poor PFS (P = 0.016). A higher density of PANCK−PD-L1+ immune cells within 20, 30, and 45 µm of PANCK+ tumor cells was correlated with better clinical response (P = 0.017, 0.017, and 0.02, respectively). Multiparametric immune profiling of CD8+ T cells, PD-L1+ cells, CD68+ macrophages and PANCK−PD-L1+ immune cells at the invasive margin may help identify patients with cervical cancer who may benefit from anti-PD-1 combination therapy. ClinicalTrials. gov identifier: NCT03816553, January 25, 2019.

Abstract Background The prognosis of non-small cell lung cancer (NSCLC) with brain metastases (BMs) had been researched in some researches, but the combination of clinical characteristics and serum inflammatory indexes as a noninvasive and more accurate model has not been described. Methods We retrospectively screened patients with BMs at the initial diagnosis of NSCLC at Sun Yat-Sen University Cancer Center. LASSO-Cox regression analysis was used to establish a novel prognostic model for predicting OS based on blood biomarkers. The predictive accuracy and discriminative ability of the prognostic model was compared to Adjusted prognostic Analysis (APA), Recursive Partition Analysis (RPA), and Graded Prognostic Assessment (GPA) using concordance index (C-index), time-dependent receiver operating characteristic (td-ROC) curve, Decision Curve Analysis(DCA), net reclassification improvement index (NRI), and integrated discrimination improvement index (IDI). Results 10-parameter signature's predictive model for the NSCLC patients with BMs was established according to the results of LASSO-Cox regression analysis. The C-index of the prognostic model to predict OS was 0.672 (95% CI = 0.609 ~ 0.736) which was significantly higher than APA,RPA and GPA. The td-ROC curve and DCA of the predictive model also demonstrated good predictive accuracy of OS compared to APA, RPA and GPA. Moreover, NRI and IDI analysis indicated that the prognostic model had improved prediction ability compared with APA, RPA and GPA. Conclusion The novel prognostic model demonstrated favorable performance than APA, RPA, and GPA for predicting OS in NSCLC patients with BMs.

AbstractTissue-resident memory CD8+ T (TRM) cells have been associated with robust protective antitumor immune responses and improved prognosis of patients with cancer. Therefore, therapeutic strategies that modulate either the production or activity of TRM cells could be effective for treating cancer. Using a high-throughput drug screen, we showed that the neurotransmitter dopamine drives differentiation of CD8+ T cells into CD103+ TRM cells. In murine syngeneic tumor xenograft models and clinical human colon cancer samples, DRD5 served as the major functional dopamine receptor on CD8+ T cells and positively correlated with TRM cell density. DRD5 deficiency led to a failure of CD8+ T cells to accumulate in tissues, resulting in impaired TRM cell formation, reduced effector function, and uncontrolled disease progression. Moreover, dopamine treatment promoted the antitumor activity of CD8+ T cells and suppressed colorectal cancer growth in immunocompentent mouse models, and ex vivo preconditioning with dopamine enhanced the in vivo efficacy of chimeric antigen receptor (CAR)-T cells. Finally, in a patient with colorectal cancer cohort, dopamine expression was positively associated with patient survival and CD8+ T-cell infiltration. These findings suggest that dopaminergic immunoregulation plays an important role in the differentiation of CD8+ cells into CD103+ TRM cells and thereby modulates TRM-elicited antitumor immunity in colorectal cancer.Significance:Identification of an immunostimulatory function of dopamine signaling by promoting tissue-resident memory T-cell differentiation and sustaining T-cell effector functions reveals potential therapeutic strategies and prognostic biomarkers for colorectal cancer.

Background In the tumor microenvironment, tumor cells are able to suppress antitumor immunity by competing for essential nutrients, including amino acids. However, whether amino acid depletion modulates the activity of CD8 + tumor-infiltrating lymphocytes (TILs) is unclear. Method In this study, we evaluated the roles of amino acids and the Rag complex in regulating mammalian target of rapamycin complex 1 (mTORC1) signaling in CD8 + TILs. Results We discovered that the Rag complex, particularly RagD, was crucial for CD8 + T-cell antitumor immunity. RagD expression was positively correlated with the antitumor response of CD8 + TILs in both murine syngeneic tumor xenografts and clinical human colon cancer samples. On RagD deficiency, CD8 + T cells were rendered more dysfunctional, as demonstrated by attenuation of mTORC1 signaling and reductions in proliferation and cytokine secretion. Amino acids maintained RagD-mediated mTORC1 translocation to the lysosome, thereby achieving maximal mTORC1 activity in CD8 + T cells. Moreover, the limited T-cell access to leucine (LEU), overshadowed by tumor cell amino acid consumption, led to impaired RagD-dependent mTORC1 activity. Finally, combined with antiprogrammed cell death protein 1 antibody, LEU supplementation improved T-cell immunity in MC38 tumor-bearing mice in vivo. Conclusion Our results revealed that robust signaling of amino acids by RagD and downstream mTORC1 signaling were crucial for T-cell receptor-initiated antitumor immunity. The characterization the role of RagD and LEU in nutrient mTORC1 signaling in TILs might suggest potential therapeutic strategies based on the manipulation of RagD and its upstream pathway.