bims-tuinly 2026-05-10 papers

bims-tuinly

Biomed News

on Tumor-infiltrating lymphocytes therapy

Issue of 2026–05–10
twenty-two papers selected by
Pierpaolo Ginefra, Ludwig Institute for Cancer Research

Culture and Expansion of Tumor-Infiltrating Lymphocyte From Biopsy Tissue Samples of Gastric Cancer Are Feasible.
Correlation between recognition of autologous tumor organoids by tumor-infiltrating lymphocytes from metastatic epithelial cancers and clinical response.
Tumor-Immune-On-Chip to Evaluate Pathophysiological Feature of T Lymphocytes Expanded from Patient Tumors and Lymph Node Tissues.
Artificial intelligence-powered H&E-based quantification of spatial tumor-infiltrating lymphocyte distribution identifies prognostic immune niches in colorectal cancer.
IL-12 augments adoptive cell therapy by reshaping CD8+ T dynamics and broadening antitumor immunity.
Prognostic value of tumor-infiltrating lymphocytes and common subtypes in patients with gastroenteropancreatic neuroendocrine neoplasms: a systematic review and meta-analysis.
Concordance and Prognostic Impact of Tumor-Stroma Ratio and Tumor-Infiltrating Lymphocytes in Preoperative Biopsies and Matched Surgical Specimens in Oral Squamous Cell Carcinoma.
The Correlation Between PD-L1 Expression in Metaplastic Breast Cancer and Clinical-Pathological Features and Prognosis.
Single-cell profiling uncovers a hypoxia-vascular-immune axis underlying poor immunotherapy response in acral versus cutaneous melanoma.
Tumor reactivity assessment using clonal expression reveals tumor reactive CD8+ T cell heterogeneity across solid tumors.
Advances in cell therapy for solid tumours: European perspective and future directions.
Prognostic Significance of Selected Tumor Stroma Parameters in Patients with HER2-Positive Breast Cancer Treated with Adjuvant Trastuzumab.
Construction and experimental verification of a prognostic model based on tumor-infiltrating lymphocytes related genes (TILRGs) in diffuse large B-cell lymphoma.
Physically Inactivated Tumor Organoids Enable Rapid and Personalized Enrichment of Cytotoxic T Cells for Solid Tumor Immunotherapy.
AI-driven spatial analysis of tumor-infiltrating lymphocytes predicts chemo-immunotherapy response in triple-negative breast cancer.
ATG5-mediated inducible autophagy sustains CAR-T cell durability under solid tumor stress.
Real-World, Evidence-Based, Retrospective Study of Patients Infused with Commercially Released Lifileucel for Advanced Melanoma.
Turep: Detecting cross-cancer tumor-reactive T cells in single-cell and spatial transcriptomics data.
Deep learning based on CD3 histological slides for prediction of colon cancer outcome: analysis of three international stage III colon cancer cohorts.
Immunotherapy innovations in triple-negative breast cancer: targeting checkpoints, combinations, and biomarkers.
Unique Pathways that drive CD8+ T cell dysfunction and immune evasion in lung adenocarcinoma.
Transcription factor collaboration enables precise T cell state engineering.

J Immunother. 2026 May 08.

Culture and Expansion of Tumor-Infiltrating Lymphocyte From Biopsy Tissue Samples of Gastric Cancer Are Feasible.

Bitna Park, Hanna Park, Min-Jung Seo, Sung W Kim, In-Pyo Baek, Young W Kim, Hark K Kim.

  Adoptive transfer of tumor-infiltrating lymphocytes (TILs) achieves promising clinical results in solid tumors. TIL trials use surgically resected tissue to isolate TILs. However, certain tissues are difficult to surgically resect, and a minimally invasive approach might be advantageous if it could achieve similar TIL yields. We tested a minimally invasive approach to culture and expand TILs using endoscopic biopsy tumors from patients with metastatic gastric cancer (GC). TILs were successfully cultured from all of the intestinal GC biopsies, while TIL populations had slow growth from diffuse GCs. Initial TIL growth was faster in intestinal GC biopsies (n=9) than in diffuse GC biopsies (n=4) (P=0.021). All 4 biopsy TILs tested were expanded >1010 by our rapid expansion protocol. Biopsy TILs demonstrated equivalent expansion and FACS profiles to TILs cultured from gastrectomy. Thus, clinical-grade TILs can be successfully cultured and expanded from endoscopic biopsy tumors in patients with metastatic GC.

Keywords:  Biopsy; rapid expansion protocol; tumor-infiltrating lymphocyte

DOI:  https://doi.org/10.1097/CJI.0000000000000607
J Immunother Cancer. 2026 May 06. pii: e014644. [Epub ahead of print]14(5):

Correlation between recognition of autologous tumor organoids by tumor-infiltrating lymphocytes from metastatic epithelial cancers and clinical response.

Alexandra M Gustafson, Aarushi Bhasin, Billel Gasmi, Brian D Bui, Catherine Marie Ade, Ken-Ichi Hanada, Hyunmi K Halas, Jared J Gartner, Todd D Prickett, Jon Salazar, Maria R Parkhust, Paul Robbins, Nicholas D Klemen, Mei Li Kwong, Stephanie L Goff, Steven A Rosenberg, James C Yang.

   BACKGROUND: Neoantigen-specific tumor-infiltrating lymphocytes (TILs) have the ability to mediate responses in patients with metastatic epithelial cancers. While some identified factors are associated with response, there is a need for ways to determine which patients will benefit from adoptive cell therapy with TIL. Patient-derived tumor organoids (PDTO) retain the genetic makeup of the tumor from which they are derived, and are a useful tool in personalized medicine to test the effectiveness of anticancer therapies.
METHODS: Tumor organoids were grown from patients with metastatic epithelial cancers treated on a TIL protocol. TIL recognition of autologous PDTO by enzyme-linked immunosorbent spot assays and 4-1BB upregulation, as well as objective Response Evaluation Criteria in Solid Tumors (RECIST) responses to treatment, were measured in a cohort of treated patients. The most common histologies were colorectal (20 patients, 67%), pancreatic (4 patients, 13%), and breast (2 patients, 7%) cancer.
RESULTS: Recognition of PDTO was driven by major histocompatibility complex (MHC) class I-restricted reactivity and upregulation of MHC in response to interferon-γ was not associated with response. There were no responders in the 9 patients who lacked PDTO recognition on their initial TIL screening compared with seven responses in 18 patients with initial reactivity (p=0.059). After TIL were selected and expanded for administration, 15 infusion products showed organoid recognition, with 8 of these patients achieving objective responses (53%), while only 1 of 15 patients without organoid recognition responded (7%; p=0.014).
CONCLUSIONS: TIL reactivity against autologous tumor organoid appears to be associated with objective clinical response in patients with metastatic epithelial cancers.

Keywords:  Colorectal Cancer; Escape/evasion; Immunotherapy; Solid tumor; Tumor infiltrating lymphocyte - TIL

DOI:  https://doi.org/10.1136/jitc-2025-014644
Cancer Med. 2026 May;15(5): e71906

Tumor-Immune-On-Chip to Evaluate Pathophysiological Feature of T Lymphocytes Expanded from Patient Tumors and Lymph Node Tissues.

Gyu-Bon Cho, Hyun-Joo Lee, You Jeong Heo, Jihoon Ko, Won-Suk Lee, Sujin Hyung.

  The infiltration and cytotoxicity of T lymphocytes are critical for cancer immunotherapy efficacy; however, the behavior of these immune cells has not been thoroughly investigated. Herein, a Tumor-Immune-On-Chip is established using cells acquired from the tissues of a patient with colorectal cancer to monitor T lymphocytes. Through the Tumor-Immune-On-Chip, the interaction between tumor spheroid and either T lymphocytes expanded from tumors (tumor-infiltrating lymphocytes; TILs) or lymph nodes (lymph node-derived lymphocytes; LN T cells) are investigated. Although initial 24-h analysis showed no statistical differences, extended 48-h observation revealed a significant deviation in T cell-mediated cell death signals between TILs and LN T cells. TILs demonstrated more potent cytotoxic effects than LN T cells after 48 h. The number of tumor-infiltrating CD3+ cells and cleaved caspase-3 expression levels were 4- and 2.1-fold higher, respectively, in TIL co-cultures compared to LN T cell co-cultures. Therefore, this proof-of-concept platform allows us to explore the patient-specific tumor-immune microenvironment, focusing on different types of T lymphocytes and establishing methodology for future clinical applications. ClinicalTrial.gov identifier: NCT02589496.

Keywords:  T lymphocytes; colorectal cancer; immunotherapy; patient‐specific‐tumor microenvironment; tumor‐immune‐on‐Chip

DOI:  https://doi.org/10.1002/cam4.71906
Cancer Immunol Immunother. 2026 May 04. pii: 163. [Epub ahead of print]75(6):

Artificial intelligence-powered H&E-based quantification of spatial tumor-infiltrating lymphocyte distribution identifies prognostic immune niches in colorectal cancer.

Hyun-Hee Koh, Seungeun Lee, Chiyoon Oum, Sanghoon Song, Soo Ick Cho, Sérgio Pereira, Chang Ho Ahn, Jun Yong Kim, Milim Kim, Minsun Jung.

   PURPOSE: The prognostic significance of tumor-infiltrating lymphocytes (TILs) in colorectal cancer (CRC) is well established; however, existing approaches inadequately capture their spatial distribution. We investigated the prognostic implications of TIL spatial distribution in CRC using an artificial intelligence (AI)-based method.
METHODS: A total of 202 patients with stage II-III CRC were included. TIL densities in intratumoral (iTIL) and stromal (sTIL) regions were quantified using AI-based analysis of hematoxylin and eosin (H&E)-stained images. Based on proximity to the tumor-stromal border (TSB), TILs were subclassified into core iTIL, bounding iTIL, bounding sTIL, and outermost sTIL. Immunoscore was calculated from CD3+ and CD8+ T-cell densities in the tumor center and invasive margin.
RESULTS: Correlations between AI-based and pathologist assessments (iTIL: r = 0.57; sTIL: r = 0.70) were comparable to inter-pathologist correlations (iTIL: r = 0.47; sTIL: r = 0.70). In univariate Cox regression analysis, bounding iTIL, bounding sTIL, and outermost sTIL were significantly associated with recurrence-free survival (RFS), whereas core iTIL was not. Composite TIL and TSB scores were developed by incorporating the prognostically significant regions. In multivariable analysis, the TIL score (p = 0.001), TSB score (p < 0.001), and Immunoscore (p < 0.001) independently predicted RFS. In microsatellite instability-high tumors, only the TSB score remained prognostically significant.
CONCLUSION: AI-powered spatial analysis of TILs, particularly the TSB score, demonstrated prognostic performance comparable to conventional Immunoscore, thereby supporting the value of spatial immune profiling and AI-driven analysis of H&E-stained slides for improved risk stratification in CRC.

Keywords:  Artificial intelligence; Colorectal cancer; Spatial analysis; Tumor-infiltrating lymphocyte; Tumor-stromal border

DOI:  https://doi.org/10.1007/s00262-026-04409-9
Cancer Lett. 2026 May 05. pii: S0304-3835(26)00326-5. [Epub ahead of print]652 218563

IL-12 augments adoptive cell therapy by reshaping CD8+ T dynamics and broadening antitumor immunity.

Yue Huang, Xiangming Liu, Yiwei Chen, Huihui Zhang, Junshi Zhang, Yang-Xin Fu, Xuanming Yang, Zaopeng Yang.

Adoptive cell therapy (ACT) against solid tumors is constrained by tumor heterogeneity, immunosuppressive microenvironments, and insufficient T cell potency and persistence. Although IL-12 has long been recognized as a potent enhancer of T-cell immunity, its clinical application has been hindered by systemic toxicity and the lack of a strategy to harness its benefits without chronic exposure. Here, we show that ex vivo IL-12 preconditioning programs tumor-specific T cells with increased antigen sensitivity, yielding superior antitumor activity compared with IL-7/IL-15. Systemic delivery of Pro-IL-12 further rejuvenates exhausted CD8+ tumor-infiltrating lymphocytes (TILs) by restoring IL-2 hypersensitivity, sustaining persistence, and strengthening effector machinery. When combined with ACT, a single dose of Pro-IL-12 reinstated IFN-γ production in exhausted TILs and doubled intratumoral T-cell accumulation via upregulation of IL-2Rα and increased IL-2 sensitivity. Pro-IL-12 markedly improved therapeutic outcomes, inducing durable complete tumor regression, promoting antigen spreading, and establishing long-term immune memory that prevented relapse from antigen-loss tumor. Across multiple xenograft models, including pancreatic cancer, lymphoma and triple negative breast cancer, distinct human CAR-T products paired with Pro-IL-12 enhanced tumor eradication without cytokine release syndrome. Together, these findings establish precision delivery of IL-12 as a translatable strategy that harnesses known IL-12 biology to achieve robust antitumor efficacy without systemic toxicity.

DOI: https://doi.org/10.1016/j.canlet.2026.218563
BMC Cancer. 2026 May 04.

Prognostic value of tumor-infiltrating lymphocytes and common subtypes in patients with gastroenteropancreatic neuroendocrine neoplasms: a systematic review and meta-analysis.

Qiming Zheng, Xinyue Xie, Qingjun Guo, Chiyi Chen, Wentao Jiang.

   BACKGROUND: The incidence of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) has been rising. However, current prognostic evaluation systems have certain limitations. As a key component of the tumor microenvironment, the prognostic value of tumor-infiltrating lymphocytes (TILs) in GEP-NENs remains controversial. This study aims to systematically evaluate the prognostic impact of common TILs subsets, including CD3+, CD4+, CD8+, and FoxP3 + cells, in patients with GEP-NENs to provide evidence-based support.
METHODS: Following the PRISMA guidelines, we systematically searched PubMed, EMBASE, the Cochrane Library, and Web of Science for studies published up to February 2026. Two researchers independently completed the literature screening, data extraction, and quality assessment using the Newcastle-Ottawa Scale (NOS). Meta-analysis was performed using Stata 17.0 software to calculate pooled hazard ratios (HR) with 95% confidence intervals (CI). Heterogeneity testing, subgroup analysis, sensitivity analysis, and GRADE certainty of evidence assessment were also conducted.
RESULTS: A total of 8 studies involving 915 patients were included after corrected literature screening. The meta-analysis demonstrated that a high density of FoxP3+ TILs was significantly associated with worse overall survival (OS) in patients with GEP-NENs (HR = 2.18, 95% CI: 1.36-3.49, P = 0.001). In contrast, infiltration of CD3+, CD4+, and CD8+ TILs showed no significant correlation with OS. The analysis of total TILs was not meta-analyzed as only one study reported this data; qualitatively, it showed a significant association with OS (HR = 1.37, 95% CI: 1.13-1.65). Exploratory subgroup analyses indicated that the prognostic value might be influenced by tumor location and the site of TILs detection. Sensitivity analysis confirmed the stability of the results, and publication bias could not be reliably assessed due to the small number of studies. GRADE assessment showed low-to-moderate certainty of evidence for all pooled estimates.
CONCLUSION: High infiltration of FoxP3+ TILs can serve as a potential prognostic biomarker for poor prognosis in patients with GEP-NENs. The prognostic value of CD3+, CD4+, and CD8+ TILs remains unclear. The evaluation of TILs subsets is influenced by tumor characteristics and may help guide prognostic stratification and immunotherapeutic strategies. However, further validation through multicenter prospective studies and standardized protocols is needed.

Keywords:  Biomarker; Gastroenteropancreatic neuroendocrine neoplasms; Meta-analysis; Prognosis; Tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.1186/s12885-026-16127-7
Diagnostics (Basel). 2026 Apr 17. pii: 1202. [Epub ahead of print]16(8):

Concordance and Prognostic Impact of Tumor-Stroma Ratio and Tumor-Infiltrating Lymphocytes in Preoperative Biopsies and Matched Surgical Specimens in Oral Squamous Cell Carcinoma.

Michal Mozola, Michal Herman, Katerina Brachtlova, Jaroslav Michalek, Jana Zapletalova, Zdenek Bednarik, Michal Hendrych, Richard Pink, Peter Tvrdy, Marketa Hermanova.

  Background/Objectives: Tumor-stroma ratio (TSR) and tumor-infiltrating lymphocytes (TILs) were suggested as prognostic markers in oral squamous cell carcinoma (OSCC). Identification of markers assessable in preoperative biopsies that could guide treatment planning is of great importance. This study aimed to evaluate the concordance and prognostic impact of TSR and TILs in preoperative biopsies and matched resection specimens of OSCC. Methods: This study included 100 patients with OSCC. TSR and stromal TILs were evaluated on hematoxylin and eosin-stained slides of biopsies and paired resection specimens and categorized (into low TSR and high TSR; high TILs and low TILs). The agreement between resections and biopsies, and the prognostic significance and clinicopathological correlations of TSR and TILs, were investigated. Results: For TSR, substantial agreement between preoperative biopsies and surgical specimens (kappa correlation coefficient 0.713) was demonstrated. The assessment of TILs showed poor concordance between biopsies and resections (kappa correlation coefficient 0.372). For both biopsies and resections, Cox regression showed an independent negative prognostic impact of low TSR on disease-free, disease-specific, and overall survival. Independent prognostic value of TILs evaluated in biopsies was not found, and the negative prognostic impact of low TILs on disease-free and overall survival was observed only in the main resection specimens. Conclusions: TSR evaluated in preoperative biopsies was highly concordant with results in main resection specimens and may provide significant information for OSCC prognostication, risk stratification, and treatment decisions. In contrast, TILs evaluated in biopsies showed poor concordance with main resection specimens and failed to demonstrate prognostic significance.

Keywords:  concordance; oral squamous cell carcinoma; pretreatment biopsies; prognostic impact; resection specimens; tumor-infiltrating lymphocytes; tumor–stroma ratio

DOI:  https://doi.org/10.3390/diagnostics16081202
Medicina (Kaunas). 2026 Apr 10. pii: 726. [Epub ahead of print]62(4):

The Correlation Between PD-L1 Expression in Metaplastic Breast Cancer and Clinical-Pathological Features and Prognosis.

Tugba Toyran, Ertuğrul Bayram, Yasemin Aydınalp Camadan, Berksoy Sahin, Kubilay Dalcı, Yusuf Kemal Arslan, Melek Ergin.

  Background and Objectives: Metaplastic breast carcinoma (MBC) is a rare, aggressive malignancy that is often resistant to conventional chemotherapy and characterized by a triple-negative phenotype. While immune checkpoint inhibition shows promise, the prognostic significance and distribution of programmed death-ligand 1 (PD-L1) expression within the heterogeneous architecture of MBC remain poorly understood. This study aimed to evaluate PD-L1 expression and the density of tumor-infiltrating lymphocytes (TILs) to clarify their roles in patient stratification and overall survival (OS). Materials and Methods: We retrospectively analyzed 48 MBC cases diagnosed between 2010 and 2025. PD-L1 expression was quantified using the Combined Positive Score (CPS) with the 22C3 antibody clone across diverse histological components. The density of stromal TIL density was assessed following internationally standardized guidelines. Clinical outcomes and clinicopathological parameters, including metastasis, lymphovascular invasion (LVI), and histological subtype, were correlated with biomarker status using Kaplan-Meier survival analysis and Cox proportional hazards regression models. Results: PD-L1 positivity (CPS ≥1) was identified in 72.9% of cases, one of the highest rates documented in literature. Notably, an inverse relationship was observed with PD-L1-negative tumors, which exhibited significantly higher rates of distant metastasis (46.2% vs. 17.1%; p = 0.039). Multivariate analysis confirmed that low density of TILs (HR = 9.66; p = 0.016), metastasis (HR = 4.40; p = 0.023), and the presence of LVI (HR = 3.84; p = 0.047) were strong independent predictors of mortality. While PD-L1 status alone did not directly dictate overall survival, mean overall survival was markedly reduced in the low TILs cohort (32.2 months) compared to the high TILs group (114.2 months). Conclusions: The high prevalence of PD-L1 expression supports routine screening for immunotherapy eligibility in MBC. Our findings suggest that PD-L1-negative cases represent a high-risk biological subset driven by alternative immune evasion mechanisms. Integrating TIL density with conventional pathological parameters provides a more robust prognostic framework, enabling personalized therapeutic strategies for this challenging malignancy.

Keywords:  PD-L1 expression; combined positive score; immunotherapy; metaplastic breast carcinoma; prognosis; triple-negative breast cancer; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.3390/medicina62040726
J Transl Med. 2026 May 07.

Single-cell profiling uncovers a hypoxia-vascular-immune axis underlying poor immunotherapy response in acral versus cutaneous melanoma.

Qian Dong, Yiming Zhang, Fuchu He, Aihua Sun.

   BACKGROUND: Acral melanoma (AM), accounting for ~50% of melanomas in Asian populations, exhibits far poorer immunotherapy response (anti-PD-1 ORR < 20%) than cutaneous melanoma (CM, ~43.7%). While the tumor microenvironment (TME) plays a pivotal role in immunotherapy resistance, previous studies mainly focused on immune cells, neglecting stromal components like pericytes-creating a critical knowledge gap in understanding AM's therapeutic refractoriness.
METHODS: We integrated multiple single-cell transcriptomic datasets from 31 acral melanoma, 13 cutaneous melanoma and 35 normal skin samples. Comprehensive bioinformatics analyses including cell type annotation, differential expression, gene functional enrichment, and cell-cell interaction analysis, were performed to delineate the tumor microenvironment differences between acral and cutaneous melanoma. Key findings were validated using spatial transcriptomics, bulk RNA-seq datasets, and functional assays including qPCR and western blot.
RESULTS: AM had a more immunosuppressive TME, enriched in collagen-secreting RGS5+/COL3A1+ pericytes that co-localized with KDR+/PODXL+ endothelial cells, associated with vascular abnormalities and hypoxia. These collagen-secreting RGS5+/COL3A1+ pericytes (enriched in AM) were significantly negatively correlated with the infiltration ratio of CD8+ tumor-infiltrating lymphocytes (TILs); moreover, in immunotherapy cohorts, high expression of the collagen-secreting pericyte marker RGS5 was associated with poorer response to immune checkpoint blockade (ICB) treatment. We also identified a hypoxia-related NECTIN2-TIGIT immunosuppressive axis: hypoxia upregulated NECTIN2 on multiple cells, interacting with TIGIT on T cells to potentially exacerbate dysfunction.
CONCLUSIONS: This study provides insights into vascular-stromal features associated with immunotherapy resistance in AM, highlighting RGS5+/COL3A1+ pericytes and the NECTIN2-TIGIT axis as targets, and guides development of effective combination immunotherapies for AM.

Keywords:  Acral melanoma; Pericyte; Single-cell RNA-seq; Tumor microenvironment

DOI:  https://doi.org/10.1186/s12967-026-08201-2
Front Immunol. 2026 ;17 1815974

Tumor reactivity assessment using clonal expression reveals tumor reactive CD8+ T cell heterogeneity across solid tumors.

David Monteiro, Jack Denebeim, Anne E Dodson, Ashish Yeri, Mitali Ghose, Meghan Travers, Sophie Capobianco, Conor Calnan, Gustavo J Martinez, Charles H Yoon, Karrie Wong, Micah J Benson, Dipen Sangurdekar.

   Introduction: Tumor infiltrating lymphocytes (TIL) drive the anti-tumor activity of a broad class of immunotherapies. In situ TIL are composed of T cells that recognize tumor antigens (Tumor Reactive T cells, or TRTs) as well as bystander T cells with specificity for other antigens. TRT clonotypes are associated with a unique and tumor-driven exhausted transcriptional state, enabling single-cell RNA sequencing (scRNA-seq)-based predictive models for TRTs using experimentally validated clone labels.
Methods: In this study, a clonotype-level CD8+ TRT classifier (TRACE) was built using an aggregated dataset of validated tumor reactive clonotypes and associated scRNA-seq data from multiple publications that overcomes the limitations of training on a single dataset, donor, or indication. TRACE does not require dataset manipulation for training or prediction, enabling it to be easily applied to new test datasets as they emerge.
Results: TRACE exhibited robust performance on held-out TIL and PBMC clones - achieving a mean Matthews correlation coefficient of 0.84 and F1-score of 0.85 - comparable to or outperforming other TRT prediction methods. We experimentally confirmed the reactivity of TRACE-identified TRT clones by co-culturing ex vivo expanded TIL with an autologous melanoma tumor cell line. Finally, we applied TRACE to evaluate the frequency of TRTs across hundreds of patient samples from multiple tumor atlases spanning lung, colorectal, and pancreatic cancer. TRACE scores were observed to be significantly higher in exhausted CD8+ T cells in tumors but not in exhausted cells in normal adjacent or non-cancer samples, suggesting specificity towards identifying tumor-antigen experienced T cells.
Conclusion: TRACE is a tumor reactivity scoring algorithm released with open model weights that can be applied to tissue or blood single-cell RNAseq datasets. Its application should be of general interest for characterizing the fraction of TRTs in TIL and for establishing correlations with clinical response to immunotherapies.

Keywords:  AI-driven biomarker discovery; cancer immunotherapy; computational immunology; machine learning; multi-modal single-cell analysis; tumor microenvironment (TME); tumor-infiltrating lymphocytes (TIL); tumor-reactive T cells (TRT)

DOI:  https://doi.org/10.3389/fimmu.2026.1815974
Lancet Reg Health Eur. 2026 May;64 101590

Advances in cell therapy for solid tumours: European perspective and future directions.

Victor Moreno, Fiona Thistlethwaite, Ramón Yarza, Willemijn S Tak, Kok Haw Jonathan Lim, John B A G Haanen.

  Cell therapy has revolutionised the landscape of cancer treatment, with therapies such as Chimeric Antigen Receptor T cells (CAR-T cells), showing remarkable efficacy in haematological malignancies, and approaches such as Tumour Infiltrating Lymphocytes (TILs) and T-cell receptor-engineered T cells (TCR-T) showing increasing promise in solid tumours. The recent US FDA approvals of lifileucel (a TIL therapy for advanced melanoma) and afamitresgene autoleucel (a TCR therapy targeting MAGE-A4 in synovial sarcoma) mark the first regulatory recognition of cell therapies for solid tumours and signal a new era for oncology. Europe has played a central role in these advances, leading pivotal phase 3 trials and pioneering hospital-exemption-based manufacturing programmes. However, the continent still faces major challenges, including fragmented regulatory frameworks, high manufacturing costs, and inequitable patient access across member states. Emerging innovations such as gene-edited, allogeneic, and iPSC-derived cell products promise to address current limitations by improving scalability, safety, and time-to-treatment. This Series paper examines the latest advancements in cell therapy, focussing on the European experience, while comparing global trends. We discuss challenges specific to Europe, such as regulatory frameworks, manufacturing scalability, and disparities in access. Emphasis is placed on emerging innovations like gene-edited and allogeneic therapies, as well as future directions for integrating cell therapies into mainstream oncology. We conclude with recommendations for overcoming barriers related to cost, toxicity management, and equitable access across Europe.

Keywords:  Advanced therapy medicinal products (ATMPs); Cell therapy; Chimeric antigen receptor T cells (CAR-T); European Medicines Agency (EMA); Gene; Immunotherapy; Solid tumors; T-cell receptor therapy (TCR-T); Tumor infiltrating lymphocytes (TILs)

DOI:  https://doi.org/10.1016/j.lanepe.2026.101590
Cancers (Basel). 2026 Apr 14. pii: 1243. [Epub ahead of print]18(8):

Prognostic Significance of Selected Tumor Stroma Parameters in Patients with HER2-Positive Breast Cancer Treated with Adjuvant Trastuzumab.

Aleksandra Ambicka, Aleksandra Grela-Wojewoda, Joanna Niemiec, Katarzyna Mularz, Agnieszka Harazin-Lechowska, Janusz Ryś, Agnieszka Adamczyk.

   BACKGROUND: While the prognostic and predictive value of tumor cell-derived features such as grade, estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) status, and Ki67 index is well established in breast cancer, less is known about the prognostic role of tumor stroma. This study aimed to evaluate stromal parameters in HER2-positive breast cancer patients treated with adjuvant trastuzumab.
MATERIAL AND METHODS: The study included 224 patients (T ≥ 1, N ≥ 0, M0) who underwent radical treatment followed by adjuvant chemotherapy, hormone therapy (if ER/PR-positive), and trastuzumab. The following histological and immunohistochemical parameters were analyzed: stroma type, tumor-infiltrating lymphocytes (TILs), eosinophils, neutrophils, central area of fibrosis, necrosis, and programmed cell death protein ligand 1 (PD-L1) expression in tumor and stromal cells.
RESULTS: Low TILs percentage (≤50%) was associated with lower tumor grade (G2) (p = 0.013) and ER/PR positivity (p = 0.001). Tumors lacking PD-L1 expression had a lower percentage of TILs (p < 0.001), less frequently exhibited tumor-associated neutrophilia (p = 0.019), and more often presented with desmoplastic stroma (p < 0.001). The following parameters were associated with prognosis: TILs percentage, stroma type, and PD-L1 expression. High TILs percentage (>50%) was an independent positive prognostic factor.
CONCLUSIONS: In patients with HER2-positive breast cancer treated with adjuvant trastuzumab, the percentage of TILs, stroma type, and PD-L1 expression are prognostically relevant. Specifically, a TILs percentage >50% independently predicts favorable outcomes. Routine evaluation of stromal features may provide additional prognostic information and support treatment planning.

Keywords:  HER2-positive breast cancer; PD-L1; desmoplasia; eosinophils; neutrophils; prognostic factors; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.3390/cancers18081243
SLAS Technol. 2026 May 01. pii: S2472-6303(26)00038-5. [Epub ahead of print] 100425

Construction and experimental verification of a prognostic model based on tumor-infiltrating lymphocytes related genes (TILRGs) in diffuse large B-cell lymphoma.

Jing Zhao, Kunquan Su, Shoubin Zhong.

   PURPOSE: Our research objective is to construct a prognostic model of tumor-infiltrating lymphocytes related genes (TILRGs) for predicting the survival of cases with diffuse large B-cell lymphoma (DLBCL).
METHODS: Based on the clinical data of DLBCL patients in GEO database, the TILRGs that were significantly associated with survival prognosis were screened. Cox regression analysis and LASSO analysis were used to construct prognostic risk models for TILRGs. Kaplan-Meier (K-M) curve and ROC curve were used to assess the predictive performance of the model. CIBERSORT algorithm and GDSC database were applied to quantify tumor immune infiltration and chemotherapy drugs. The levels of key genes were detected via qRT-PCR. Additionally, K-M curves were plotted to analyze the relationship between LCP2 expression and patient prognosis.
RESULTS: A prognostic risk model consisting of 20 TILRGs was constructed. This model has high accuracy in predicting the survival of DLBCL patients, and reveals significant differences in tumor immune infiltration and chemotherapy drugs between two groups. Low expression of LCP2 in DLBCL patients is obviously related to serum LDH, IPI score, stage and poor prognosis. Further analysis revealed that LCP2 overexpression inhibited the growth of DLBCL cells.
CONCLUSION: In the current study, an accurate and reliable prognostic risk model was constructed based on 20 TILRGs. Low expression of LCP2 indicates poor survival of DLBCL cases and is a potential prognostic indicator.

Keywords:  Diffuse large B-cell lymphoma (DLBCL); Tumor-infiltrating lymphocytes (TILs); immunoinfiltration; prognosis

DOI:  https://doi.org/10.1016/j.slast.2026.100425
Adv Healthc Mater. 2026 May 06. e03992

Physically Inactivated Tumor Organoids Enable Rapid and Personalized Enrichment of Cytotoxic T Cells for Solid Tumor Immunotherapy.

Yu Zhang, Haoran Zhao, Junhong Zeng, Zitian Wang, Xiaoyong Dai, Yongde Cai, Ruirui Qiao, Qingwen Wang, Peter E Lobie, Shaohua Ma.

  Adoptive T cell therapy holds great promise for the treatment of solid tumors but remains constrained by tumor heterogeneity, inefficient neoantigen targeting, and the complexity of T cell manufacturing. Here, we present a patient-specific, broadly applicable platform using physically inactivated tumor organoids (PIOs) to generate tumor-specific cytotoxic T cells ex vivo. Derived from droplet-engineered tumor organoids (DEOs), PIOs preserve the full antigenic repertoire of the patient's tumor without requiring synthetic peptides, antigen-presenting cells, or neoantigen prediction. Using matched tumor tissue and PBMCs from colorectal and liver cancer patients, we show that PIOs activate and expand tumor-specific T cells with enhanced infiltration, selective cytotoxicity, and robust secretion of IFN-γ and IL-2. Multi-round PIO stimulation achieves 80-400-fold expansion of CD8+CD137+ T cells within two weeks. Transcriptomic and epigenetic profiling suggest that PIOs modulate T cell programs linked to migration and persistence. This work redefines tumor organoids as immunotherapeutic materials and establishes a rapid, cost-effective platform for personalized T cell manufacturing. Our findings provide a new translational route for adoptive cell therapy in solid tumors using patient-derived materials.

Keywords:  adoptive T cell therapy; microfluidics droplet‐engineered tumor organoid; neoantigen‐independent activation; personalized immunotherapy; tumor‐infiltrating lymphocytes (TILs)

DOI:  https://doi.org/10.1002/adhm.202503992
Front Immunol. 2026 ;17 1797753

AI-driven spatial analysis of tumor-infiltrating lymphocytes predicts chemo-immunotherapy response in triple-negative breast cancer.

Hai Liang, Hejie Zhu, Liwei Wang, James Yu, Ru Feng, Xiaoshuang Yan, Liang Shi, Jiayi Xiao, Huirong Zhou, Peilin Chen, Hao Liu, Linfeng Zheng.

   Introduction: Triple-negative breast cancer (TNBC) is an aggressive subtype with limited therapeutic options due to lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. While neoadjuvant immunotherapy shows promise, the impact of spatial organization of tertiary lymphoid structures (TLS) and tumor-infiltrating lymphocytes (TILs) on treatment efficacy remains incompletely characterized. This exploratory study aims to establish an AI-based quantitative framework for analyzing tumor-immune spatial interactions and develop a predictive model for chemo-immunotherapy response.
Methods: We developed an integrated AI pipeline for automated analysis of hematoxylin-eosin (HE)-stained breast cancer samples. The framework incorporates: 1) TLS detection and classification, 2) TIL quantification, and 3) spatial relationship mapping between lymphocytes and tumor cells. Biomarkers associated with Miller-Payne (MP) response grades were identified using multivariate statistics, enabling construction of a random forest prognostic model.
Results: The TLS recognition model demonstrated substantial agreement with pathologists (κ=0.73), while the TIL classifier achieved 0.92 accuracy. Analysis of 32 HE images from triple-negative breast cancer (TNBC) cases treated with neoadjuvant chemo-immunotherapy revealed significant associations: stromal lymphocyte density and percentage positively correlated with MP grades (p<0.05); average cell counts in 2-cell and 3-cell lymphocyte aggregates showed no significant correlations; shorter mean minimum distances between lymphocytes and tumor cells within 10-30 μm radius range were inversely associated with MP grades (p<0.05). The spatial-feature-based prediction model achieved an AUC of 0.81 (95% CI: 0.76-0.86).
Conclusions: This study established an AI-driven HE analysis pipeline that precisely quantified spatial determinants of tumor-immune interactions. Lymphocyte spatial organization, particularly proximity to tumor cells and lymphocyte aggregation patterns, serves as a critical predictor of chemo-immunotherapy response beyond density metrics. The validated MP-grade prediction model demonstrates translational potential for clinical decision-making in TNBC management, although external validation in larger cohorts is warranted.

Keywords:  Miller-Payne; artificial intelligence; tertiary lymphoid structures; triple-negative breast cancer; tumor-infiltrating lymphocytes

DOI:  https://doi.org/10.3389/fimmu.2026.1797753
Front Immunol. 2026 ;17 1720544

ATG5-mediated inducible autophagy sustains CAR-T cell durability under solid tumor stress.

Sang-Eun Jung, Minji Lim, Hyungwoo Jeong, Youngchae Moon, Hyungseok Seo.

  Autophagy functions as a context-dependent stress adaptation pathway in T cells; however, its role in sustaining chimeric antigen receptor (CAR)-T cell function within solid tumor environments remains insufficiently defined. In this study, we investigated whether ATG5-mediated autophagy regulation contributes to CAR-T cell functional durability under tumor-associated stress conditions. ATG5 overexpression (OE) CAR-T cells did not increase basal autophagy activity but instead selectively enhanced autophagy flux in response to inducible stimuli. Under tumor-mimicking immunosuppressive conditions, ATG5 OE CAR-T cells maintained cytotoxic activity during prolonged antigen exposure and exhibited preserved effector cytokine production together with reduced oxidative stress. Consistent with these in vitro findings, ATG5 OE CAR-T cells exhibited enhanced antitumor efficacy in vivo under IR-preconditioned settings, characterized by improved tumor control and survival, which was associated with sustained effector function of tumor-infiltrating CAR-T cells. Collectively, these findings demonstrate that reinforcing inducible autophagy capacity through ATG5 promotes the maintenance of CAR-T cell function under tumor-associated challenges, highlighting a targeted strategy to enhance CAR-T cell persistence in solid tumor immunotherapy.

Keywords:  ATG5 overexpression; CAR-T cell therapy; CAR-T persistence; autophagy; tumor microenvironment

DOI:  https://doi.org/10.3389/fimmu.2026.1720544
Transplant Cell Ther. 2026 May 04. pii: S2666-6367(26)00329-5. [Epub ahead of print]

Real-World, Evidence-Based, Retrospective Study of Patients Infused with Commercially Released Lifileucel for Advanced Melanoma.

Lilit Karapetyan, Justin Moser, Barbara T Ma, Christopher Otteni, Elizabeth Cathcart, Kimberly Loo, Jay Jacoby, Jamie K Chau, James W Smithy, Nikhil Nayee, Anna C Pavlick, Ahmed Tawashi, Amritha Seshaadri, Min Yi, Friedrich Graf Finckenstein, Brian Gastman, Alexander N Shoushtari.

   INTRODUCTION: Lifileucel is a tumor-derived autologous T-cell therapy approved by the US FDA for patients with advanced melanoma previously treated with an immune checkpoint inhibitor and, for BRAF V600-mutated disease, a BRAF ± MEK inhibitor. The real-world effectiveness of commercially available lifileucel has not yet been characterized.
METHODS: We conducted a retrospective study across four US centers of adults with metastatic melanoma treated with standard-of-care lifileucel per the US prescribing information. The primary objective was to evaluate real-world effectiveness based on treating physician-assessed objective response rate (ORR). Secondary objectives included progression-free survival (PFS) and overall survival (OS). Preconditioning lymphodepletion was administered per institutional practice, followed by lifileucel infusion and up to six doses of interleukin-2 (IL-2; 600,000 IU/kg).
RESULTS: Forty-three patients were included (median age 59 years [range, 28-79]; 54% male); 44% had BRAF V600 mutations. Melanoma subtypes included cutaneous non-acral (70%), acral (9%), mucosal (19%), and unknown primary (2%); liver and treated brain metastases were present in 30% (n=13) and 28% (n=12), respectively. Patients received a median of three prior systemic anticancer therapies (range, 1-7). Prior to lymphodepletion, 44% received bridging therapy, which predominantly reflected continuation of prior targeted therapy. Following lifileucel infusion, patients received a median of five IL-2 doses (range, 1-6). Among 41 response-evaluable patients, physician-assessed ORR was 44% (n=18), including complete response in 5% (n=2) and partial response in 39% (n=16). ORR was 52% among patients who received ≤2 prior lines of therapy (n=23) and 33% among those who received ≥3 prior lines (n=18). ORR was 58% among patients receiving ≤3 IL-2 doses (n=12) and 38% among those receiving ≥4 doses (n=29). With a median follow-up of 5.7 months (95% CI, 1-15), median PFS and OS were 4.4 (95% CI, 2.8, 8.9) and 10.2 months (95% CI, 6.1, NR), respectively.
CONCLUSION: In this multicenter real-world cohort, commercially available lifileucel demonstrated meaningful clinical activity in patients with advanced melanoma, with outcomes comparable to pivotal trial results. Fewer prior lines of therapy and normal LDH were associated with improved outcomes, supporting earlier consideration of TIL therapy in appropriate patients.

Keywords:  Tumor-infiltrating lymphocyte therapy; adverse event; lifileucel; melanoma; response; survival

DOI:  https://doi.org/10.1016/j.jtct.2026.04.044
bioRxiv. 2026 Apr 24. pii: 2026.04.21.719961. [Epub ahead of print]

Turep: Detecting cross-cancer tumor-reactive T cells in single-cell and spatial transcriptomics data.

Wendao Liu, Chia-Hao Tung, Eva M Sevick-Muraca, Zhongming Zhao.

Tumor-infiltrating lymphocytes are essential for anti-tumor immunity, yet distinguishing tumor-reactive T cells from non-reactive bystander cells remains a significant challenge. Existing signatures, often derived from single cohorts, lack robustness in cross-cancer prediction. We present Turep, a deep learning method designed for robust, cross-cancer prediction of tumor-reactive T cells using single-cell or spatial transcriptomics data. By integrating paired single-cell RNA and T cell receptor sequencing data from seven human malignancies, we identified a pan-cancer tumor-reactive gene signature and leveraged generative data augmentation to address data imbalance. Turep consistently outperformed existing biomarkers, achieving a mean area under the receiver operating characteristic curve of 0.870 across cancer types. In validation across diverse cohorts, we found that Turep-predicted tumor-reactive T cell proportions could predict clinical response to immunotherapy. Furthermore, extending Turep to spatial transcriptomics revealed that tumor-reactive T cells preferentially resided in spatial niches where target cells exhibited elevated antigen presentation. Overall, Turep provides a powerful, generalizable tool for identifying tumor-reactive T cells and their spatial architectures, facilitating personalized cancer immunotherapy strategies.

DOI: https://doi.org/10.64898/2026.04.21.719961
Oncoimmunology. 2026 Dec 31. 15(1): 2668797

Deep learning based on CD3 histological slides for prediction of colon cancer outcome: analysis of three international stage III colon cancer cohorts.

Julie Lécuelle, Caroline Truntzer, Debora Basile, Luigi Laghi, Luana Greco, Alis Ilie, David Rageot, Titouan Huppé, Jean-François Emile, Fréderic Bibeau, Julien Taïeb, Valentin Derangère, Come Lepage, François Ghiringhelli.

  Prognostic stratification in stage III colon cancer remains poor, despite treatment advances. Tumor-infiltrating lymphocytes, particularly CD3+ T cells, are potential prognostic markers, but manual assessment is labor-intensive and not robust. This study aimed to develop a deep learning model for automated analysis of CD3-stained histological slides to improve prognostic prediction. A total of 1737 patients from three international cohorts (PETACC08, PRODIGE-13, and HARMONY) were analyzed. The deep learning model (VGG19) identified tumor core (TC) and invasive margin (IM) regions on CD3-stained slides. Features from VGG19 and UNI models were used to cluster patients using hierarchical classification. Prognostic performance was evaluated using disease-free survival (DFS) across training, internal validation, and external validation sets. Deep learning classifiers identified distinct patient clusters with significantly different DFS based on TC and IM. For both IM and TC analysis, patients in the favorable group had a better DFS in all sets (IM: p < 0.001, p = 0.04, p = 0.02; TC: p = 0.002, p = 0.01, p = 0.12, respectively). Combining classifiers enhanced prognostic accuracy in all sets (p < 0.001, p = 0.01, p = 0.06, respectively). The model outperformed traditional clinical variables and CD3 enumeration, which demonstrated variability across cohorts. Automated deep learning analysis of CD3-stained slides enables robust and reproducible prognostic stratification in stage III colon cancer, independently of staining and scanning variations. This approach holds promise for guiding personalized treatment strategies.ClinicalTrials.gov Identifiers: NCT00265811, NCT00995202.

Keywords:  Tumour infiltrating lymphocytes; biomarkers; colonic neoplasms; deep learning; prognosis

DOI:  https://doi.org/10.1080/2162402X.2026.2668797
Oncol Rev. 2026 ;20 1802090

Immunotherapy innovations in triple-negative breast cancer: targeting checkpoints, combinations, and biomarkers.

Zi-Xin Wang, Ju-Hang Chu, Ya-Ru Wang, Lu-Yao Huang, Ming-Ping Qian.

  Triple-negative breast cancer (TNBC), an aggressive subtype lacking estrogen receptor (ER), progesterone receptor (PR), and HER2 expression, accounts for 10-20% of breast cancers and is characterized by high metastatic potential and poor survival outcomes. Despite advancements in chemotherapy, the 5-year survival rate for metastatic TNBC remains below 30%, underscoring the need for innovative therapeutic approaches. This review comprehensively examines recent breakthroughs in TNBC immunotherapy, focusing on immune checkpoint inhibitors (ICIs), combination strategies, and biomarker-driven therapy. Landmark trials such as KEYNOTE-355 and IMpassion130 have demonstrated that combining PD-1/PD-L1 inhibitors with chemotherapy improves survival in PD-L1-positive metastatic TNBC. Beyond monotherapy, combination therapies-including dual checkpoint inhibition, PARP inhibitors in BRCA-mutated tumors, and antibody-drug conjugates (ADCs) -show promise in overcoming resistance and enhancing antitumor immunity. Emerging targets further expand therapeutic possibilities, though their paradoxical roles as biomarkers and immunosuppressive mediators require precision-based approaches. Biomarkers like PD-L1, tumor-infiltrating lymphocytes (TILs), tumor mutational burden (TMB), and circulating tumor DNA (ctDNA) are critical for patient stratification and predicting immunotherapy response. Despite progress, challenges persist, including tumor heterogeneity, resistance mechanisms, and access to advanced therapies. Future directions emphasize next-generation ICIs, optimized combination regimens, and AI-driven biomarker integration to achieve durable, personalized treatments. This review underscores the potential of immunotherapy to redefine TNBC management while highlighting the imperative for continued innovation to address unmet clinical needs.

Keywords:  biomarker-driven therapy; combination therapy; immune checkpoint inhibitors; immunotherapy; triple-negative breast cancer

DOI:  https://doi.org/10.3389/or.2026.1802090
Am J Respir Cell Mol Biol. 2026 Mar 19. pii: aanag053. [Epub ahead of print]

Unique Pathways that drive CD8+ T cell dysfunction and immune evasion in lung adenocarcinoma.

Jinjin Chen, Fen Wen, Chengwei Chen, Yao Wang, Jian Sun, Yunhao Sun.

  WNT ligands have been critically implicated in many aspects of lung cancer progression. This study explores the impacts of Wnt family member 10A (WNT10A) in progression and immune response in lung adenocarcinoma (LUAD) and investigates the underpinning mechanisms. Expression, prognosis, and immune correlations of WNT10A in LUAD were explored using TCGA-LUAD, GEO datasets, and tissue microarrays. In vitro, WNT10A was knocked down in LUAD cell lines (H441 and H1299), followed by RNA sequencing and functional assays. Tumor-bearing mouse models with WNT10A knockdown were used to assess tumor growth and immune response. The functions of WNT10A on CD8 + T cell cytotoxicity were further explored through co-culture experiments and flow cytometry. FRAT1 overexpression, Wnt activators (BML-284), and CXCL12 manipulations were employed to verify mechanistic links. High WNT10A expression showed a trend toward poor overall survival, reduced CD8 + T cell infiltration, and unfavorable prognosis in LUAD patients. WNT10A loss in LUAD cells reduced growth, mobility, and inhibited Wnt signaling. In vivo, WNT10A loss prolonged animal survival in immunocompetent mice, reduced tumorigenic activity of mouse 3LL cells, and enhanced CD8 + T cell activity and tumor cell apoptosis. Mechanistically, WNT10A promoted FRAT1-mediated suppression of GSK3β, facilitating CTNNB1 activation and CXCL12 expression, which restricted CD8 + T cell function. WNT10A silencing synergized with anti-PD-1, reducing tumor burden and boosting CD8 + T cell infiltration/function. Generally, WNT10A promotes LUAD progression and CD8 + T cell dysfunction through FRAT1-mediated Wnt signaling and CXCL12 activation. Focusing on WNT10A could offer a compelling approach to boosting T cell-driven anti-tumor responses for the treatment of LUAD.

Keywords:  CD8-Positive T-Lymphocytes; Immune Evasion; Lung Adenocarcinoma; Tumor Microenvironment; Wnt Signaling Pathway

DOI:  https://doi.org/10.1093/ajrcmb/aanag053
bioRxiv. 2026 Apr 22. pii: 2026.04.20.718569. [Epub ahead of print]

Transcription factor collaboration enables precise T cell state engineering.

Rachel E Savage, Christian D McRoberts Amador, Conrad T Hock, Ruochi Zhang, Hung-Che Kuo, Aretha R Gao, Max A Horlbeck, Charles A Gersbach, Jason D Buenrostro.

Transcription factors (TFs) collaborate to regulate gene expression programs that define cell fate. In CD8 + T cells, this coordinated regulation underlies exhaustion, a dysfunctional state that constrains immunity in chronic infection and cancer. Here, we screen for cell state-specific TFs by performing pooled overexpression screens of 3,548 TF and TF isoforms in primary T cells across multiple CD8 + T cell states. We identify 82 regulators that collaborate with exhaustion-specific programs and profile their effects using perturb-SHARE-seq, connecting perturbations to changes in chromatin accessibility and gene expression across 702,314 single cells. We identify 38 reproducible regulatory programs and construct a map of 12,616 TF-program connections that shape CD8 + T cell states, nominating KLF2 as predictive of positive response to CAR-T therapy. Using seq2PRINT, a deep learning framework that predicts functional TF interactions, we identify RUNX as a "master collaborator", a TF that broadly collaborates with other factors, and uncover a RUNX2:KLF2 interaction that specifies exhaustion-associated programs. Mutation of the RUNX2:KLF2 protein interface attenuates KLF2-mediated repression of exhaustion, while synthetic tethering of RUNX2 to KLF2 leads to an amplification of the phenotype. More broadly, we identify the collaborative action of RUNX as a driver in CD8 + T cell states, and show that tethering TFs enables the rational engineering of cell state identity for cell and gene therapies.

DOI: https://doi.org/10.64898/2026.04.20.718569