bims-tuinly 2025-11-30 papers

bims-tuinly

Biomed News

on Tumor-infiltrating lymphocytes therapy

Issue of 2025–11–30
eighteen papers selected by
Pierpaolo Ginefra, Ludwig Institute for Cancer Research

RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy.
Enhanced anti-tumor efficacy of tumor-infiltrating lymphocytes by GITR agonist in ovarian cancer.
Tumor-infiltrating lymphocyte aggregation refines prognosis in patients with hepatocellular carcinoma.
Transcriptome profiling of tumor-infiltrating lymphocyte-mediated cytotoxicity against patient-derived lung cancer organoids.
Exploratory Analysis of the Impact of a Single Dose of Trastuzumab on the Immune Microenvironment in HER2-Positive Early-Stage Breast Cancer.
Interplay Between Immune Microenvironment CD8+ Tumor-Infiltrating Lymphocytes and PDL-1 Expression as Prognostic Markers in Invasive Cervical Squamous Cell Carcinoma.
Prognostic value of stromal CD39+CD8+ T cells in predicting platinum sensitivity and survival outcomes in epithelial ovarian cancer.
Clinical characteristics and prognostic impact of HER2-ultralow breast cancer and tumor-infiltrating lymphocytes (TILs).
Tumor-infiltrating T Lymphocytes Recognize Thyroid-specific and Neo-antigens in Follicular Cell-derived Thyroid Cancers.
T Cell Exhaustion in the Cervical Cancer Tumor Microenvironment: PD-1 Overexpression and Co-Expression with TIGIT, Tim-3, LAG-3, and NKG2A.
NeoPAIR-T: Functional Mapping of Neoantigen-TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System.
FXR-mediated antigen-specific CD8+ T cell enhances antitumor immunity in intrahepatic cholangiocarcinoma.
A clinically relevant pan-cancer prognostic signature derived from T-cell activation immune genes: Experimental validation across malignancies with emphasis on breast cancer.
Systemic activation and tissue infiltration of CD8 + CX3CR1 + T cells in non-small cell lung cancer treated with neoadjuvant immune checkpoint blockade.
Multiplex gene-editing strategy to engineer allogeneic EGFR-targeting CAR T-cells with improved efficacy against solid tumors.
CD8+MT+ effector T cells from enlarged tumor-draining lymph nodes enhanced anti-tumor immunity and improved prognosis in colorectal cancer.
In vivo reprogramming of cytotoxic effector CD8 + T cells via fractalkine-conjugated mRNA-LNP.
Dynamics of checkpoint receptors in γδ T cell subsets are associated with clinical response during anti-PD-1 immunotherapies.

bioRxiv. 2025 Oct 12. pii: 2025.10.11.681829. [Epub ahead of print]

RELB Reprograms Exhausted Tumor-Infiltrating Lymphocytes for Improved Adoptive Cell Therapy.

Christian D McRoberts Amador, Rachel E Conover, Michael C Brown, Liliana S Lyniv, Pamela K Noldner, Ying Zhou, Aretha R Gao, Sean R McCutcheon, Scott J Antonia, Charles A Gersbach.

Tumor-infiltrating lymphocytes (TILs) are a promising autologous cell therapy to treat solid tumors. TILs are manufactured by expanding and reinfusing tumor-reactive T cells from tumor biopsies. Efficacy of TIL therapies has been limited by the heterogeneity of expanded TIL products and the high prevalence of dysfunctional exhausted CD8+ T cells (TEX). While a subset of CD8+ TILs co-expressing CD103 and CD39 are enriched for tumor-reactive TILs across multiple cancer types, these cells are often in the TEX state with low proliferative potential. To identify regulators of human TIL proliferation, we screened an open reading frame library encoding for all human transcription factors (TFs). RELB emerged as the dominant driver of human TIL expansion with a skew towards CD8+ cells. TCR diversity was maintained after multiple days of in vitro expansion driven by RELB. Transcriptome profiling of multiple RELB-expressing TIL subtypes revealed a shift towards a memory/costimulatory-like phenotype. Using a HER2-targeting CAR and tumor co-culture model, RELB conferred improved persistence after multiple tumor challenges in vitro and improved solid tumor control in mouse xenografts in vivo. Finally, co-culture of RELB-overexpressing TILs with patient-matched tumor organoids showed an increase in TIL product polyfunctionality, tumor reactivity, and tumor killing. Collectively these results support promoting RELB expression as a strategy for broadly enabling TIL therapy for treating solid tumors.

DOI: https://doi.org/10.1101/2025.10.11.681829
Front Immunol. 2025 ;16 1670841

Enhanced anti-tumor efficacy of tumor-infiltrating lymphocytes by GITR agonist in ovarian cancer.

Daun Jung, Ah-Ra Goh, Ki Yeon Kim, Ji Min Lee, Eun Ji Lee, Sohyun Hwang, Haeyoun Kang, Hyun Park, Hee Jung An.

   Background: Adoptive cell therapy (ACT) using autologous tumor-infiltrating lymphocytes (TILs) is a personalized immunotherapy that has shown promising clinical results in various tumor types. Although TILs are associated with improved survival in patients with ovarian cancer (OC), their therapeutic efficacy remains limited. Therefore, novel strategies to enhance the anti-tumor activity of TILs are needed to improve outcomes in OC treatment.
Methods: Single cells were isolated from tumor tissues of patients with high-grade serous carcinoma (HGSC) and expanded for 14 days in the presence of IL-2 under four different conditions: (1) control (W), (2) PD-1 antagonist (WI), (3) PD-1 antagonist + IL-15 + IL-21 (WIO), and (4) PD-1 antagonist + IL-15 + IL-21 + GITR-agonist (WIOG). Following validation of TIL purity and activation phenotypes by flow cytometry, RNA sequencing was performed to elucidate the underlying mechanisms. In vitro efficacy was assessed using a 7-AAD/Far-Red cytotoxicity assay against autologous tumor cells, and in vivo efficacy was evaluated in NSG mice bearing subcutaneous patient-derived tumor cell xenografts (PDCX).
Results: On day 14, the WIOG group showed a 1.3-fold increase in expansion compared to the control group, along with a high CD8+/Treg ratio (454.6). Furthermore, both CD8+ and CD4+ T cells in the WIOG group exhibited elevated Granzyme B expression. RNA sequencing identified 279 upregulated genes associated with T cell activation (CSF2, TNFRSF4), cytotoxicity (IFNG, GZMB), and anti-apoptosis (BMF, BCL2L1). Compared to the controls, the WIOG group demonstrated a 1.9-fold increase in cytolytic activity in vitro and a 56% reduction in tumor growth in the patient-derived tumor cell xenograft (PDCX) model.
Conclusions: Taken together, we demonstrated that the addition of an agonistic GITR antibody during the early phase of TIL culture increased the CD8+ T cell to Treg cell ratio and enhanced anti-tumor T cell immunity. Enhancing TILs with a GITR agonist may be beneficial for improving the clinical outcomes of TIL-based ACT in OC.

Keywords:  GITR; PDCX; T cell expansion; cancer immunotherapy; ovarian cancer; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.3389/fimmu.2025.1670841
Lab Invest. 2025 Nov 25. pii: S0023-6837(25)00177-1. [Epub ahead of print] 104267

Tumor-infiltrating lymphocyte aggregation refines prognosis in patients with hepatocellular carcinoma.

Zehong Zhang, Yuan'e Lian, Yijuan Wu, Zhongchang Weng, Jiaying Ye, Siqin Zhang, Lianhuang Li, Yannan Bai.

  The prognostic role of tumor-infiltrating lymphocytes (TILs) in hepatocellular carcinoma (HCC) remains elusive. This study explored the association between TIL aggregation in early-stage HCC tissues and patient survival. Individual patient-level data from 353 patients diagnosed with HCC who underwent radical hepatectomy were retrospectively analyzed. TIL aggregation was analyzed using an artificial neural network (ANN) algorithm to define the immune phenotypes (IPs) within hematoxylin and eosin-stained whole-slide images. The association between TIL aggregation and survival was compared among IPs, and an optimized TIL phenotype was validated with clinical variables. Among the eight IP features measured using the ANN algorithm, TIL densityK50, a metric generated by an assigned density of each cell based on its distance with the 50 nearest lymphocytes, was associated with improved disease-free survival (DFS) in univariate (hazard ratio [HR], 0.282; 95% confidence interval [CI],CI 0.181-0.439; P < 0.0001) and multivariate analyses (HR, 0.315; 95% CI, 0.115-0.860; P = 0.024) after adjusting for clinical factors. When TIL densityK50 combined with clinical factors (TIL-Clinical-Integrated model, TCI-model), AUC performance for DFS and overall survival was improved to 0.798 and 0.781 in the training cohort and 0.759 and 0.723 in the validation cohort, respectively. Furthermore, subgroup analyses indicated that its predictive value is particularly strong for very late recurrence and survival up to < 8 years. Abundant TILs in HCC tissue are associated with reduced late recurrence risk and improved survival, suggesting a potential time-dependent prognostic role of TILs in early-stage HCC.

Keywords:  Hepatocellular carcinoma; Tumor microenvironment; Tumor-infiltrating lymphocyte

DOI:  https://doi.org/10.1016/j.labinv.2025.104267
Commun Biol. 2025 Nov 23.

Transcriptome profiling of tumor-infiltrating lymphocyte-mediated cytotoxicity against patient-derived lung cancer organoids.

Ziwen Qin, Hongchang Zhang, Yanling Li, Jianyi Yang, Haozhen Liu, Zhuojue Guan, Qinghua Hou, Haocheng Du, Xiaoqiang Li, Xian Lin, Qumiao Xu, Qiao Li, Junhui Chen, Jixian Liu, Chao Chen.

Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality, and therapies utilizing tumor-infiltrating lymphocytes (TILs) show significant promise. However, molecular signatures defining a productive TIL-mediated response remain poorly characterized. Here, we establish a patient-derived organoid and autologous TIL co-culture platform to show that expanded TILs mediate potent, specific cytotoxicity against NSCLC organoids. This functional response is associated with a crucial shift in T-cell states from proliferative towards effector memory phenotypes and involves activating key signaling networks, including the TNF and IL-17 pathways. Furthermore, T-cell receptor (TCR) analysis confirms the expansion process selectively enriches tumor-associated clonotypes, resulting in a more focused repertoire. This work delineates the transcriptional and clonal signatures of an effective anti-tumor immune response, providing a robust framework to guide next-generation personalized TIL therapies.

DOI: https://doi.org/10.1038/s42003-025-09188-0
Biomedicines. 2025 Nov 14. pii: 2784. [Epub ahead of print]13(11):

Exploratory Analysis of the Impact of a Single Dose of Trastuzumab on the Immune Microenvironment in HER2-Positive Early-Stage Breast Cancer.

Nikita Bastin, Jessica Mezzanotte-Sharpe, Rebecca Alvarez, Savannah C Partridge, Suzanne M Dintzis, Sasha E Stanton, V K Gadi, Laura C Kennedy.

  Background: How the tumor microenvironment (TME) influences treatment response in HER2+ breast cancer following HER2-directed therapy is crucial for individualizing therapies and is currently understudied. The purpose of this exploratory analysis was to elucidate changes in the TME following treatment with trastuzumab. Methods: Fourteen HER2+ early-stage breast cancer patients underwent tissue biopsies before and after a dose of trastuzumab. Samples were evaluated for stromal tumor-infiltrating lymphocytes (TILs) and RNA-based cell and gene expression signatures. Tumor inflammation signature scores were generated to measure whether an adaptive immune response developed to trastuzumab within the tumor. Patients were also stratified as immune responders or non-responders based on changes in TILs. Results: Of the 14 enrolled patients, 13 had samples available for analysis, and 7 had an immune response as assessed by changes in TILs compared to 6 non-responders. Trastuzumab treatment decreased PD-L1 and TGF-Beta signatures and increased CTLA4 gene signatures, although results were not statistically significant, and increased DUSP1 expression. In the TIL responder group, there was increased expression of dendritic cells as well as MARCO expression. Conclusions: These findings, although exploratory in nature, highlight trastuzumab's ability to induce an immune response and suggest that some patients may be more primed to mount an immune response following treatment than others. Patients without a robust response in TILs may benefit from additional agents to favorably modulate the TME for optimized responses to HER2-directed therapy, an area of research which warrants further study.

Keywords:  breast cancer; immune biomarker; trastuzumab; tumor microenvironment; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.3390/biomedicines13112784
Medicina (Kaunas). 2025 Nov 10. pii: 2007. [Epub ahead of print]61(11):

Interplay Between Immune Microenvironment CD8+ Tumor-Infiltrating Lymphocytes and PDL-1 Expression as Prognostic Markers in Invasive Cervical Squamous Cell Carcinoma.

Laura-Andra Petrică, Mariana Deacu, Georgeta Camelia Cozaru, Anca Florentina Mitroi, Gabriela Izabela Bălţătescu, Manuela Enciu, Oana Cojocaru, Anca-Antonela Nicolau, Andrei Radu Baz, Lucian Șerbănescu, Mariana Aşchie.

  Background: Cervical cancer remains a major cause of cancer-related morbidity and mortality worldwide, with limited therapeutic options for advanced disease. As we better understand the fine mechanisms involved in the interaction between tumor cells and the tumor microenvironment, new paths and opportunities will emerge. Recent evidence highlights the prognostic and predictive roles of immune checkpoint markers and tumor-infiltrating lymphocytes (TILs), especially CD8+ TILs, in shaping treatment outcomes. Objectives: This study investigated the immunohistochemical expression of PD-L1 and CD8+ TILs in 48 newly diagnosed, treatment-naive cervical cancer cases and analyzed their associations with clinicopathological features and survival outcomes. Results: In our cohort, we observed that PD-L1 positivity was identified in 68.8% of cases, most frequently in advanced FIGO stages and in tumors with lympho-vascular invasion or with a high proliferation rate evaluated by the Ki-67 index. High levels of intra-tumoral CD8+ TILs were observed in 52.1% of cases and correlated positively with stromal TILs, lower proliferation rates, and absence of vascular invasion. A significant inverse relationship was found between PD-L1 expression and the density of CD8+ TILs (p = 0.047). Survival analysis showed that patients exhibiting a "cold" immunophenotype with low levels of CD8+ TILs and PD-L1-positive tumors had worse outcomes, while high levels of CD8+ TILs played a protective role. Conclusions: Our study highlights the importance of the immunohistochemical assessment of PD-L1 and CD8+ TILs biomarkers, which have a complementary inter-relationship and have a significant prognostic impact on cervical squamous cell carcinoma. PD-L1 positivity marks aggressive disease features, while higher intra-tumoral CD8+ TIL density is protective. Their combined evaluation may improve patient stratification and inform immunotherapy strategies.

Keywords:  CD8+ tumor-infiltrating lymphocytes; Ki-67 expression; PD-L1expression; cervix; invasive squamous cell carcinoma; tumor microenvironment

DOI:  https://doi.org/10.3390/medicina61112007
Sci Rep. 2025 Nov 27. 15(1): 42369

Prognostic value of stromal CD39+CD8+ T cells in predicting platinum sensitivity and survival outcomes in epithelial ovarian cancer.

Hai-Yan Sun, Shan Kang, Jian-Lei Wu, Xue-Ping Li, Yue-Ping Liu, Yan Li.

  This study aimed to investigate the expression levels of CD8+ and CD39+CD8+ T cells in both intratumoral (CK+) and stromal (CK-) regions, in conjunction with clinical parameters, to assess their potential impact on chemotherapy response and prognosis in epithelial ovarian cancer (EOC) patients. Formalin-fixed, paraffin-embedded (FFPE) surgical specimens from 129 patients with EOC were used to construct tissue microarrays (TMAs) for assessing the expression of CD8+ and CD39+CD8+ tumor-infiltrating lymphocytes (TILs). Cytokeratin (CK) immunolabeling was employed to distinguish epithelial tumor regions (CK+) from stromal regions (CK-) in EOC tissues. Independent prognostic factors were identified using univariate and multivariate Cox regression analyses, and these variables were incorporated into nomogram models to predict overall survival (OS). Patients in the platinum-sensitive group exhibited significantly higher expression levels of stromal CD8+ cells and CD39+CD8+ T cells, which were strongly correlated with platinum sensitivity compared with the platinum-resistant group (p < 0.0001 and p = 0.0004, respectively). Multivariate Cox regression analysis in the training cohort indicated that elevated stromal levels of CD39+ T cells and CD39+CD8+ T cells were independently associated with improved OS (HR = 2.587, p = 0.033; HR = 3.090, p = 0.008, respectively). Nomograms were developed to visually predict OS by integrating these biomarkers with relevant clinical indicators identified in the Cox regression analyses. Calibration curves demonstrated excellent concordance between predicted and observed survival outcomes. Elevated stromal levels of CD39+CD8+ T cells represent a promising prognostic biomarker for predicting platinum sensitivity and favorable prognosis in patients with advanced EOC. These findings may provide valuable insights into the tumor microenvironment and its role in modulating therapeutic responses.

Keywords:  CD39+CD8+ t cells; Epithelial ovarian cancer; Nomogram; Platinum-based chemotherapy; Prognosis; Tumor microenvironment

DOI:  https://doi.org/10.1038/s41598-025-26383-5
BMC Cancer. 2025 Nov 28.

Clinical characteristics and prognostic impact of HER2-ultralow breast cancer and tumor-infiltrating lymphocytes (TILs).

Koji Takada, Shinichiro Kashiwagi, Mariko Nishikawa, Asuka Kochi, Chika Watanabe, Haruhito Kinoshita, Kana Ogisawa, Masatsune Shibutani, Tamami Morisaki.

   PURPOSE: HER2 expression is crucial in breast cancer classification and treatment. Traditionally, tumors were categorized as HER2-positive or HER2-negative, but HER2-low (IHC 1 + or 2 + without ISH amplification) has emerged as a new classification. Among HER2-negative cases, HER2-ultralow (≤ 10% faint HER2 staining) and HER2-null (completely HER2-negative) have been proposed. While differences between HER2-low and HER2-zero tumors are studied, little is known about the clinical and prognostic characteristics of HER2-ultralow breast cancer. This study aimed to clarify the clinical characteristics, immune microenvironment, treatment response, and prognosis of HER2-ultralow tumors, with HER2-null and HER2-low tumors analyzed as comparators.
METHODS: A retrospective analysis of 244 HER2-negative breast cancer patients treated with neoadjuvant chemotherapy (NAC) at Osaka Metropolitan University Hospital (2007-2018) classified tumors into HER2-low (41.0%), HER2-ultralow (36.1%), and HER2-null (23.0%). Clinicopathological features, tumor-infiltrating lymphocyte (TIL) counts, pathological complete response (pCR), and prognostic outcomes (disease-free survival [DFS] and overall survival [OS]) were evaluated.
RESULTS: HER2-ultralow tumors showed significantly higher estrogen receptor (ER) positivity compared with HER2-null tumors (51.8% vs. 19.6%, p < 0.001), and also tended to have higher progesterone receptor positivity (p = 0.048). In contrast, HER2-null tumors were associated with younger age (median 50.0 vs. 56.0 years, p = 0.004) and higher TIL density (50.0% vs. 36.8%, p = 0.016). The overall pCR rate was 27.9%. DFS showed no significant differences among the three groups (p = 0.087), but OS was significantly worse in HER2-null compared with Not HER2-null tumors (p = 0.026, HR = 0.454). HER2-ultralow cases demonstrated an intermediate prognosis between HER2-low and HER2-null (OS comparison with HER2-null,>p= 0.101).
CONCLUSION: HER2-ultralow tumors represent a distinct subgroup characterized by higher hormone receptor positivity, whereas HER2-null tumors were associated with younger age, higher TIL density, and poorer survival. These findings emphasize the clinical significance of refining HER2-negative subclassification to distinguish HER2-ultralow, while acknowledging limitations of sample size and retrospective design.

Keywords:  Breast cancer; HER2-low; HER2-ultaralow; Neoadjuvant chemotherapy; Tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.1186/s12885-025-15255-w
J Clin Endocrinol Metab. 2025 Nov 22. pii: dgaf639. [Epub ahead of print]

Tumor-infiltrating T Lymphocytes Recognize Thyroid-specific and Neo-antigens in Follicular Cell-derived Thyroid Cancers.

Breaunna Garza, Jacob Calhoun, Paul Norman, Noah Cline, Katherine M Kichula, Ticiana D J Farias, Sharon Sams, Meher Preethi Boorgula, Thomas Danhorn, Bryan R Haugen, Jena D French.

   BACKGROUND: Thyroid cancers are among the growing list of cancer types that are resistant to immune-checkpoint inhibitor (ICI) monotherapy. While T cell infiltration is common in follicular cell-derived thyroid cancers, tumor mutation burden is low. The antigenic potential of thyroid cancers is unknown.
METHODS: To investigate the anti-tumor T cell response in thyroid cancer, we expanded tumor infiltrating lymphocytes (TIL) from primary thyroid tumors and tumor-involved lymph nodes (TILN). Putative neoantigens and both tissue-associated and tumor-associated antigens (TAA) were identified by targeted sequencing and RNASeq. HLA typing was performed for all patients, and neoantigen-MHC binding potential was predicted for each patient using NetMHCpan 4.1 and NetMHCIIpan 4.0 algorithms. In parallel, TCRβ sequencing was performed to detect T cell clonal expansions in patient-matched primary tumors and TILN. TIL reactivity to TAA and neoantigens was determined in vitro by IFNγ ELISA and flow cytometry.
RESULTS: Tumor-infiltrating T cells were evident in all thyroid tumors and readily expanded ex vivo. Shared clones were present in primary thyroid tumors and matched TILN in all patients tested, constituting 1-10% of the sequenced clones in 6/8 patients. T cell reactivity to thyroid tissue-specific proteins, thyroid peroxidase (TPO) and thyroglobulin (Tg), was observed in 84.6% (11/13) and 69.2% (9/13) patients, respectively. A BrafV600E-specific T cells response was evident in 80% (4/5) BrafV600E+ patients. T cells reactive to gene fusion-derived neoantigens were detected in patients with TPR-NTRK1+ and CCDC6-RET+ thyroid cancer.
CONCLUSIONS: Our studies confirm the presence of tumor antigen-specific T cells in patients with thyroid cancer and encourage further exploration of T cell-targeted immunotherapies for patients with progressive, treatment refractory thyroid cancer.

Keywords:  T cells; immunotherapy; thyroid Cancer; tumor antigen; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.1210/clinem/dgaf639
Cancers (Basel). 2025 Nov 11. pii: 3627. [Epub ahead of print]17(22):

T Cell Exhaustion in the Cervical Cancer Tumor Microenvironment: PD-1 Overexpression and Co-Expression with TIGIT, Tim-3, LAG-3, and NKG2A.

Nadia Tatiana García-Barrientos, Fabiola Solorzano-Ibarra, Ksenia Klimov-Kravtchenko, Jose Manuel Rojas-Diaz, Marcela Sofia Guitron-Aviña, Francisco Javier Ceja-Flores, Jose Alfonso Cruz-Ramos, Pablo Cesar Ortiz-Lazareno, Felipe de Jesús Bustos-Rodriguez, Juan Carlos Vazquez-Limon, Miriam Ruth Bueno-Topete, Martha Cecilia Tellez-Bañuelos, Jesse Haramati, Susana Del Toro-Arreola.

  Objective: T cell exhaustion is a major mechanism of immune evasion in cancer, characterized by the sustained expression of multiple inhibitory receptors. This study aimed to evaluate the expression of immune checkpoints in peripheral and tumor-infiltrating CD8+ T cells from cervical cancer patients. Methods: We enrolled 104 participants: 37 treatment-naïve patients, 36 treated patients, and 31 age-matched healthy donors. Peripheral blood mononuclear cells (PBMCs) were isolated from all participants. Ten cervical biopsies were collected for tumor-infiltrating lymphocyte (TIL) isolation and paraffin fixation. Immune checkpoint expression was analyzed by multiparametric flow cytometry and immunohistochemistry. Results: In peripheral CD8+ T cells, we found a significant upregulation of exhaustion-associated markers PD-1, TIGIT, Tim-3, and LAG-3. In the tumor infiltrating lymphocytes, these same molecules, with the addition of NKG2A, were notably upregulated further. While BTLA and NKG2A showed no systemic changes, NKG2A increased in TILs and BTLA decreased in TILs. The co-expression of PD-1 with TIGIT, Tim-3, LAG-3, and NKG2A was notably enriched between 2- and 6-fold in TILs compared with patient PBMCs. The tumor microenvironment was highly immunosuppressive, characterized by enrichment with PD-1, PD-L1, and TIGIT; TIGIT was notably upregulated in locally advanced versus early-stage tumors. Conclusions: Our findings highlight the strongly immunosuppressive environment of cervical tumors in treatment-naïve patients and the presence of elevated inhibitory checkpoint expression in peripheral blood of both pre- and post-treatment patients. These results underscore the importance of investigating immune regulation within the tumor site itself and suggest that immune checkpoint co-expression may serve as a biomarker of T cell exhaustion and therapeutic resistance. Understanding how treatment alters these pathways could guide rational combination immunotherapies to restore CD8+ T cell function in cervical cancer.

Keywords:  CIN; ICI; LAG-3; PD-1; PD-L1; TIGIT; cervical cancer; cervical dysplasia; checkpoint therapy; chemoradiotherapy

DOI:  https://doi.org/10.3390/cancers17223627
Cells. 2025 Nov 14. pii: 1789. [Epub ahead of print]14(22):

NeoPAIR-T: Functional Mapping of Neoantigen-TCR Pairs Using a CRISPR-Engineered Jurkat Reporter System.

Koji Nagaoka, Yukari Kobayashi, Kazuhiro Kakimi.

  Targeting mutation-derived neoantigens is a promising strategy for personalized immunotherapies. However, identifying true neoantigens and cognate T cell receptors (TCRs) remains challenging because computational prediction of neoantigen peptides is uncertain and most tumor-infiltrating lymphocytes are bystanders rather than tumor-reactive, necessitating functional validation. Here, we developed NeoPAIR-T (Neoantigen-TCR Pairing Assay using reporter T cells), a functional assay based on co-culture of TCR-T reporter cells and autologous antigen-presenting cells (APCs) to screen neoantigen-TCR pairs. Reporter T cells are Jurkat-derived cells engineered to express a luciferase/eGFP dual reporter, providing quantitative readouts of TCR activation, while APCs are immortalized autologous cells transfected with tandem minigenes (TMGs) encoding predicted neoantigens, bypassing peptide synthesis. NeoPAIR-T also includes TCRα-knockout with targeted knock-in of candidate TCRs at the TCRβ locus to prevent mispairing and enables parallel testing of multiple reporter T cell clones co-cultured with the same APCs for efficient identification of functional pairs. Using lung cancer samples, whole-exome and RNA sequencing predicted 63 candidate peptides assembled into three TMGs. Single-cell RNA/TCR sequencing identified eight TCR clonotypes, introduced into reporter T cells and tested in parallel. Co-culture with TMG-expressing APCs revealed two functional neoantigen-TCR pairs validated by peptide assays (EC50: 10-9.2-10-6.7 M). Collectively, NeoPAIR-T streamlines neoantigen-TCR identification for vaccine and TCR-T applications.

Keywords:  CRISPR/Cas9; T-cell receptor; functional screening; neoantigen; personalized immunotherapy; single-cell analysis; tandem minigene

DOI:  https://doi.org/10.3390/cells14221789
J Immunother Cancer. 2025 Nov 28. pii: e012259. [Epub ahead of print]13(11):

FXR-mediated antigen-specific CD8+ T cell enhances antitumor immunity in intrahepatic cholangiocarcinoma.

Shanshan Liu, Zhixing Liang, Lisi Zhu, Yuhao Zheng, Haoyuan Yu, Shusen Ye, Texi Liang, Linzi Huang, Jiayi Ouyang, Yingjiao Cao, Lilin Ye, Linsen Ye.

   BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) offers limited opportunities for surgical treatment, and advanced-stage patients exhibit poor responses to immunotherapy. Therefore, the exploration of new therapeutic strategies is of paramount importance. The farnesoid X receptor (FXR) is a nuclear receptor that has been reported to regulate immune cells in recent years. However, whether FXR can regulate CD8+ T cells to affect tumor development remains unknown.
METHODS: The function of FXR in CD8+ T cell-mediated antitumor immunity was assessed using spontaneous murine ICC models in systemic Nr1h4-knockout, and T cell-specific conditional knockout mice, with immune phenotyping performed by multicolor flow cytometry. The tumor antigen-specific CD8+ T response was tracked by tetramer staining and adoptive transfer of OT-1 T cells. The regulatory mechanism of FXR was confirmed using RNA sequencing, Chromatin Immunoprecipitation sequencing (ChIP-seq), Chromatin Immunoprecipitation polymerase chain reaction (ChIP-PCR), and luciferase reporter assays. The translational therapeutic potential was evaluated by administration of the FXR inhibitor ursodeoxycholic acid (UDCA), both as monotherapy and in combination with anti-programmed death-ligand 1 (PD-L1) blockade, in murine models and ex vivo using patient-derived tumor fragments.
RESULTS: FXR was specifically overexpressed in exhausted CD8+ tumor-infiltrating lymphocytes in both human and murine ICC. FXR ablation enhanced CD8+ T-cell effector function, proliferation, and stem-like progenitor capacity, leading to potent tumor control. Mechanistically, FXR transcriptionally upregulated the exhaustion marker LAG3 by directly binding to its promoter. Pharmacological inhibition of FXR with UDCA reversed T-cell exhaustion, and synergized with anti-PD-L1 therapy to significantly suppress tumor growth and enhance tumor cell apoptosis in murine models and human ICC ex vivo cultures.
CONCLUSIONS: Together, these data identify FXR as an immune checkpoint and support repurposing UDCA for ICC immunotherapy.

Keywords:  Combination therapy; Penile Cancer; T-Lymphocytes; Tumor infiltrating lymphocyte - TIL; Tumor microenvironment - TME

DOI:  https://doi.org/10.1136/jitc-2025-012259
Pharmacol Res. 2025 Nov 20. pii: S1043-6618(25)00469-4. [Epub ahead of print]222 108044

A clinically relevant pan-cancer prognostic signature derived from T-cell activation immune genes: Experimental validation across malignancies with emphasis on breast cancer.

Jia-Ning Zhang, Xi-Rui Zhou, Yu-Tong Wang, Zi-Lu Yi, Lin-Wei Li, Hong Liu.

  T-cell activation is widely recognized as a pivotal anti-tumor mechanism, yet emerging evidence reveals functional heterogeneity among T-cell subtypes within the tumor microenvironment (TME). To address this complexity, we developed a pan-cancer prognostic model to systematically evaluate the roles of T-cell activation-related immune response genes (TCR-IRGs) across malignancies. Pan-cancer transcriptomic data from UCSC Xena were analyzed. Through LASSO Cox regression, a prognostic signature comprising 23 TCR-IRGs was established.The high-risk score was significantly associated with aggressive malignant phenotypes, including enhanced epithelial mesenchymal transition and tumor proliferation. Further investigation focused on PTGER4, a gene within the signature predominantly expressed in T cells, as revealed by single-cell RNA sequencing analysis in breast tissues. Mechanistically, PTGER4 expression correlated with multiple immune checkpoints. In mouse models, PTGER4 overexpression enhanced the efficacy of PD-1 blockade by promoting CD8⁺ T cell infiltration and function, as indicated by increased levels of IFN-γ and Granzyme B. This study establishes a TME-centric prognostic framework, revealing that while the composite TCR-IRGs score generally indicates a higher risk of tumor progression, individual components like PTGER4 may paradoxically mark a T-cell state amenable to reinvigoration by immunotherapy, highlighting the context-dependent utility of immune biomarkers in oncology.

Keywords:  Biomarker; PD-1/PD-L1; PTGER4; Pan-cancer; T-cell activation

DOI:  https://doi.org/10.1016/j.phrs.2025.108044
Br J Cancer. 2025 Nov 22.

Systemic activation and tissue infiltration of CD8 + CX3CR1 + T cells in non-small cell lung cancer treated with neoadjuvant immune checkpoint blockade.

Sisi Dai, Yongcheng Liu, Tingting Guo, Hong Luo, Guanglei Yang, Songliang Yu, Sifan Zhang, Lili Jiang, Jie Liu, Ye Wang, Daxing Zhu, Xuyu Cai.

BACKGROUND: Neoadjuvant immune checkpoint blockade (NA-ICB) shows promise in treating resectable and locally advanced non-small cell lung cancer (NSCLC), yet the specific T cell subtypes that expand and become functionally reactivated remain incompletely characterised.
METHODS: We applied single-cell RNA sequencing, TCR repertoire analysis, and flow cytometry to tumour, paired normal lung tissue, and peripheral blood samples from 26 NA-ICB-treated and 14 treatment-naïve NSCLC patients to investigate responsive T cell subtypes, their tissue origins, migration patterns, and phenotype transitions.
RESULTS: CD8 + CX3CR1 + T cells were significantly enriched in responsive tumours, as evidenced by increased proportions (p = 0.0027) and clonal expansion in scRNA-seq, and elevated protein-level frequencies detected by flow cytometry (p = 0.021). Longitudinal analysis revealed proliferation of these cells in peripheral blood post-treatment. Shared TCR clonotypes were identified across blood and tumour samples. Pseudotime analysis indicated differentiation of these cells into exhausted and cytotoxic NK-like CD8 + T cells upon tumour infiltration.
CONCLUSION: These findings suggest that CD8 + CX3CR1 + T cells may represent circulating cytotoxic precursors associated with effective NA-ICB responses, suggesting their potential as predictive biomarkers and therapeutic targets for adoptive cell therapy.

DOI: https://doi.org/10.1038/s41416-025-03160-9
Nat Commun. 2025 Nov 23.

Multiplex gene-editing strategy to engineer allogeneic EGFR-targeting CAR T-cells with improved efficacy against solid tumors.

Ryan Murray, Md Raihan Chowdhury, Nuria Roxana Botticello-Romero, Kashvi Desai, Shanmuga Reddy Chilakapati, Brian Chong, Yixue Xia, Angelica Messana, Hanna Sobon, Joe Rocha, Faith Musenge, Adam Camblin, Giuseppe Ciaramella, Michail V Sitkovsky, Colby R Maldini, Stephen M Hatfield.

Chimeric Antigen Receptor (CAR) T cells have induced remarkable clinical responses in patients with hematological cancers. However, CAR T-cell therapies against solid tumors have not elicited similar outcomes since immunosuppressive barriers in the tumor microenvironment attenuate anti-tumor activity. Here, we describe a multifaceted approach to engineer allogeneic CAR T-cells resistant to both biochemical (hypoxia-adenosinergic) and immunological (PD-L1 and TGF-β) inhibitory signaling using an adenine base editor and a CRISPR-Cas12b nuclease. The resulting EGFR-targeting CAR T-cell product comprised a combination of six gene edits designed to evade allorejection (B2M, CIITA), prevent graft-versus-host disease (CD3E) and overcome biochemical (ADORA2A) and immunological (PDCD1, TGFBR2) barriers in solid tumor microenvironment of subcutaneously grown EGFR+ human lung tumor xenografts. This combinatorial genetic disruption enhances CAR T cell effector function and anti-tumor efficacy leading to improved tumor elimination and survival in xenograft and humanized mouse solid tumor models. Our strategy confers CAR T cells resistance to multiple clinically relevant inhibitory signaling pathways that are amplified in hypoxic tumor areas and may improve the therapeutic potential of CAR T-cells against solid tumors.

DOI: https://doi.org/10.1038/s41467-025-66737-1
Cell Rep. 2025 Nov 26. pii: S2211-1247(25)01397-X. [Epub ahead of print]44(12): 116625

CD8+MT+ effector T cells from enlarged tumor-draining lymph nodes enhanced anti-tumor immunity and improved prognosis in colorectal cancer.

Tiantongfei Jiang, Xu Guan, Ran Wei, Chongwen Lv, Likun Zan, Zhen Zhang, Shuai Jiao, Jian Ma, Pu Cheng, Jiaqi Zhao, Jinyuan Guo, Zheng Jiang, Haiyi Liu, Shuangmei Zou, Juan Xu, Xishan Wang, Yongsheng Li.

  Despite the critical role of tumor-draining lymph nodes (TDLNs) in immunity, their heterogeneity is poorly understood. We employ single-cell and bulk RNA sequencing (RNA-seq) to dissect the immune landscape of TDLNs in colorectal cancer (CRC) cohorts. We reveal that enlarged TDLNs (L-TDLNs) are enriched with CD8+ effector T (Teff) cells exhibiting a distinct metallothionein (MT)-positive signature. CD8+MT+ Teff cells exhibit heightened cytotoxicity and a stress-adapted phenotype, which are critical mediators of anti-tumor immunity. CD8+MT+ Teff cells originate from L-TDLNs, where they undergo specific differentiation, and subsequently migrate to tumor. Moreover, patients with a high level of CD8+MT+ Teff cells in tumor show a significant survival advantage, in particular those treated with adjuvant chemotherapy. These findings highlight the importance of L-TDLNs in shaping effective anti-tumor immune responses, proposing the CD8+MT+ Teff cells as a prognostic biomarker and potential therapeutic target in CRC.

Keywords:  CP: cancer; CP: immunology; colorectal cancer; single-cell RNA sequencing; tumor microenvironment; tumor-draining lymph node heterogeneity

DOI:  https://doi.org/10.1016/j.celrep.2025.116625
bioRxiv. 2025 Oct 30. pii: 2025.10.29.685358. [Epub ahead of print]

In vivo reprogramming of cytotoxic effector CD8 + T cells via fractalkine-conjugated mRNA-LNP.

Angela R Corrigan, Shin Foong Ngiow, Maura Statzu, Maria Betina Pampena, Jayme M L Nordin, Amie Albertus, Stephen D Carro, Justin Harper, Rachelle L Stammen, Jennifer Wood, Jacob T Hamilton, Houping Ni, Justin Su, Rajesvaran Ramalingam, Vincent H Wu, Mirko Paiardini, Drew Weissman, E John Wherry, Edward F Kreider, Michael R Betts.

Selective in vivo reprogramming of cytotoxic effector CD8 + T (T eff ) cells holds tremendous promise as a therapeutic tool but has not yet been accomplished. Here, we demonstrate that fractalkine-conjugated mRNA lipid nanoparticles (mRNA-LNP) can specifically target and deliver mRNA to CX3CR1 + T eff cells in vitro and in vivo. In mice, fractalkine-conjugated LNP target up to 90% of blood and splenic T eff cells, and delivery of IL-2-encoding mRNA to T eff cells enables robust exogenous IL-2 secretion. In rhesus macaques, fractalkine-conjugated mRNA-LNP target up to ∼100% of peripheral blood T eff cells and delivery of CD62L-mRNA enables transient CD62L expression. Collectively, these data demonstrate the potential of natural receptor ligand-based targeting of mRNA-LNP for effective and efficient transient in vivo modification of T eff cells.

DOI: https://doi.org/10.1101/2025.10.29.685358
EMBO Mol Med. 2025 Nov 27.

Dynamics of checkpoint receptors in γδ T cell subsets are associated with clinical response during anti-PD-1 immunotherapies.

Elisa Catafal-Tardos, Lola Dachicourt, Maria Virginia Baglioni, Marcelo Gregorio Filho Fares da Silva, Davide Secci, Marco Donia, Anders Handrup Kverneland, Inge Marie Svane, Vasileios Bekiaris.

  Gamma delta (γδ) T cells are innate-like lymphocytes with potent anti-tumor properties. Herein, we show that immune checkpoint receptors (ICRs) display differential expression and regulation by the JAK-STAT pathway in Vδ1 and Vδ2 cells and identify constitutive (e.g. TIGIT, PD-1) and inducible (e.g. TIM-3, LAG-3, CTLA-4) ICRs. In melanoma, all γδ T cell subsets downregulated AP-1 transcription factors, but Vδ1 cells specifically expressed high levels of ICR, TOX and inhibitory killer Ig-like receptor (KIR) transcripts, reminiscent of exhaustion. However, patient-derived cells were functionally competent, although induction of LAG-3 and CTLA-4 was impaired. During anti-PD-1 monotherapy, Vδ1 cells specifically bound high levels of therapeutic antibody but only in patients who responded to treatment, revealing a potential new prognostic marker for evaluating the efficacy of IC blockade (ICB) therapy. Finally, expression of KIR genes in Vδ1 cells was downregulated in response to successful ICB therapy. Collectively, our data indicate an intricate relationship between ICRs and γδ T cells and reveal novel approaches by which these cells can be harnessed in order to discern or improve cancer immunotherapy.

Keywords:  Cancer; Immune Checkpoint Receptors; Immunotherapy; Melanoma; γδ T Cells

DOI:  https://doi.org/10.1038/s44321-025-00338-9