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J. Mol. Sci.","IJMS"],"abstract":"<jats:p>Tumor-infiltrating CD8+ T cells are transcriptionally heterogeneous, and robust features linking their clonal dynamics to experimentally defined tumor reactivity remain incompletely characterized. We integrated single-cell RNA sequencing (scRNA-seq) and paired T-cell receptor sequencing (scTCR-seq) data from 31 single-cell immune datasets to characterize CD8+ T-cell states associated with clonal expansion and tumor reactivity. Across cancers, HLA-II-expressing CD8+ T cells were enriched in activated, cytotoxic, proliferative, exhausted, and terminally differentiated states and were preferentially associated with expanded TCR clonotypes. Pseudotime analysis indicated that HLA-II expression occurs along late differentiation states. HLA-DR blockade reduced CD25 upregulation in activated mixed PBMC and purified CD8+ T-cell cultures and attenuated CellTrace CFSE dilution in purified CD8+ T cells. An HLA-II-associated CD8+ T-cell signature showed cancer-type-dependent survival associations, including longer overall survival in several cohorts. To evaluate its contribution to tumor-reactivity prediction, an expansion-pretrained DNN was adapted using experimentally annotated tumor-reactive and non-tumor-reactive clonotypes from six pancreatic ductal adenocarcinoma samples and evaluated in 2225 functionally annotated CD8+ T cells from three held-out samples. Adding six HLA-II genes improved ROC-AUC across three feature panels from 0.540 to 0.781, 0.626 to 0.732, and 0.838 to 0.878, respectively, and improved PR-AUC from 0.570 to 0.739, 0.640 to 0.718, and 0.799 to 0.869. These findings identify HLA-II expression as a feature of activated and clonally expanded CD8+ T-cell states and support the complementary value of HLA-II-associated transcriptional features for prioritizing candidate tumor-reactive TCRs.<\/jats:p>","DOI":"10.3390\/ijms27188338","type":"journal-article","created":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T14:23:56Z","timestamp":1790864636000},"page":"8338","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["HLA-II Expression Marks Activated and Clonally Expanded CD8+ T Cells and Improves Tumor-Reactivity Prediction Across Human Cancers"],"prefix":"10.3390","volume":"27","author":[{"given":"Xue","family":"Mi","sequence":"first","affiliation":[{"name":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"software","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]},{"given":"Jinghua","family":"Zhu","sequence":"additional","affiliation":[{"name":"Department of Medical Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, The Affiliated Cancer Hospital of Nanjing Medical University, Baiziting 42, Nanjing, 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"}]},{"given":"Zhu","family":"Dai","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Ruyue","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"}]},{"given":"Dongmei","family":"Tian","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Guochun","family":"Cao","sequence":"additional","affiliation":[{"name":"Department of Medical Oncology, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, The Affiliated Cancer Hospital of Nanjing Medical University, Baiziting 42, Nanjing, 210009, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Zhongdang","family":"Xiao","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,19]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1297","DOI":"10.1056\/NEJMp2116863","article-title":"The Wild West of Checkpoint Inhibitor Development","volume":"386","author":"Beaver","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"eadf3700","DOI":"10.1126\/sciadv.adf3700","article-title":"TCR-engineered T cell therapy in solid tumors: State of the art and perspectives","volume":"9","author":"Baulu","year":"2023","journal-title":"Sci. Adv."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2112","DOI":"10.1056\/NEJMoa2119662","article-title":"Neoantigen T-Cell Receptor Gene Therapy in Pancreatic Cancer","volume":"386","author":"Leidner","year":"2022","journal-title":"N. Engl. J. Med."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"144","DOI":"10.1038\/s41586-023-06063-y","article-title":"Personalized RNA neoantigen vaccines stimulate T cells in pancreatic cancer","volume":"618","author":"Rojas","year":"2023","journal-title":"Nature"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1016\/j.ejca.2022.05.008","article-title":"Tumour burden and antigen-specific T cell magnitude represent major parameters for clinical response to cancer vaccine and TCR-engineered T cell therapy","volume":"171","author":"Mallet","year":"2022","journal-title":"Eur. J. Cancer"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1016\/j.ccell.2022.03.005","article-title":"Transcriptomic profiles of neoantigen-reactive T cells in human gastrointestinal cancers","volume":"40","author":"Zheng","year":"2022","journal-title":"Cancer Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"239","DOI":"10.1038\/ni1173","article-title":"How T cells \u2018see\u2019 antigen","volume":"6","author":"Krogsgaard","year":"2005","journal-title":"Nat. Immunol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Mi, X., Li, S., Ye, Z., Dai, Z., Ding, B., Sun, B., Shen, Y., and Xiao, Z. (2024). LRMAHpan: A novel tool for multi-allelic HLA presentation prediction using Resnet-based and LSTM-based neural networks. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1478201"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"bbag418","DOI":"10.1093\/bib\/bbag418","article-title":"Mitigating negative data bias to enhance TCR\u2013epitope binding and residue interaction prediction","volume":"27","author":"Mi","year":"2026","journal-title":"Brief. Bioinform."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"958","DOI":"10.1126\/science.286.5441.958","article-title":"A direct estimate of the human alphabeta T cell receptor diversity","volume":"286","author":"Arstila","year":"1999","journal-title":"Science"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"13139","DOI":"10.1073\/pnas.1409155111","article-title":"Diversity and clonal selection in the human T-cell repertoire","volume":"111","author":"Qi","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"116","DOI":"10.1016\/j.molimm.2016.07.002","article-title":"T cell receptor diversity in the human thymus","volume":"76","author":"Vanhanen","year":"2016","journal-title":"Mol. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1038\/s41568-019-0235-4","article-title":"CD8+ T cell states in human cancer: Insights from single-cell analysis","volume":"20","author":"Thommen","year":"2020","journal-title":"Nat. Rev. Cancer"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.immuni.2011.07.010","article-title":"CD8(+) T cells: Foot soldiers of the immune system","volume":"35","author":"Zhang","year":"2011","journal-title":"Immunity"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2287","DOI":"10.1038\/s12276-023-01105-x","article-title":"CD8 T-cell subsets: Heterogeneity, functions, and therapeutic potential","volume":"55","author":"Koh","year":"2023","journal-title":"Exp. Mol. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"684","DOI":"10.1038\/nbt.2938","article-title":"Linking T-cell receptor sequence to functional phenotype at the single-cell level","volume":"32","author":"Han","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"881","DOI":"10.1038\/s41592-021-01201-8","article-title":"High-throughput and single-cell T cell receptor sequencing technologies","volume":"18","author":"Pai","year":"2021","journal-title":"Nat. Methods"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1038\/s41592-024-02243-4","article-title":"Single-cell immune repertoire analysis","volume":"21","author":"Irac","year":"2024","journal-title":"Nat. Methods"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"623","DOI":"10.1038\/nbt.2593","article-title":"Combinatorial tetramer staining and mass cytometry analysis facilitate T-cell epitope mapping and characterization","volume":"31","author":"Newell","year":"2013","journal-title":"Nat. Biotechnol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"abe6474","DOI":"10.1126\/science.abe6474","article-title":"Pan-cancer single-cell landscape of tumor-infiltrating T cells","volume":"374","author":"Zheng","year":"2021","journal-title":"Science"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3188","DOI":"10.1038\/s41467-023-38946-z","article-title":"Neoantigen-specific CD8 T cells with high structural avidity preferentially reside in and eliminate tumors","volume":"14","author":"Schmidt","year":"2023","journal-title":"Nat. Commun."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1038\/s41586-023-05787-1","article-title":"Neoantigen-targeted CD8+ T cell responses with PD-1 blockade therapy","volume":"615","author":"Sennino","year":"2023","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Salvador, R., Correia, B.F., Grosa, D., Martins, T., Soares Baal, S.C., Saraiva, D.P., Crist\u00f3v\u00e3o-Ferreira, S., Pereira, I.L., Rebelo de Almeida, C., and Fior, R. (2025). HLA-DR expression in cytotoxic T lymphocytes: A key to boost the therapeutic potential of T cell-based strategies for breast cancer. Front. Immunol., 16.","DOI":"10.3389\/fimmu.2025.1653970"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"eadh9562","DOI":"10.1126\/scitranslmed.adh9562","article-title":"Transcriptome-based identification of tumor-reactive and bystander CD8+ T cell receptor clonotypes in human pancreatic cancer","volume":"15","author":"Meng","year":"2023","journal-title":"Sci. Transl. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"435","DOI":"10.1038\/s41592-024-02530-0","article-title":"Integrative mapping of human CD8+ T cells in inflammation and cancer","volume":"22","author":"Xue","year":"2025","journal-title":"Nat. Methods"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1038\/s41421-025-00836-7","article-title":"A pan-disease and population-level single-cell TCR\u03b1\u03b2 repertoire reference","volume":"11","author":"Xue","year":"2025","journal-title":"Cell Discov."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1070","DOI":"10.1038\/s41467-024-55059-3","article-title":"A minimal gene set characterizes TIL specific for diverse tumor antigens across different cancer types","volume":"16","author":"Zeng","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1038\/s41587-024-02161-y","article-title":"Prediction of tumor-reactive T cell receptors from scRNA-seq data for personalized T cell therapy","volume":"43","author":"Tan","year":"2025","journal-title":"Nat. Biotechnol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1186\/s13046-025-03517-1","article-title":"Functional tumor-reactive CD8\u2009+\u2009T cells in pancreatic cancer","volume":"44","author":"Sun","year":"2025","journal-title":"J. Exp. Clin. Cancer Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"14049","DOI":"10.1038\/ncomms14049","article-title":"Massively parallel digital transcriptional profiling of single cells","volume":"8","author":"Zheng","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"329","DOI":"10.1016\/j.cels.2019.03.003","article-title":"DoubletFinder: Doublet Detection in Single-Cell RNA Sequencing Data Using Artificial Nearest Neighbors","volume":"8","author":"McGinnis","year":"2019","journal-title":"Cell Syst."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s13059-017-1382-0","article-title":"SCANPY: Large-scale single-cell gene expression data analysis","volume":"19","author":"Wolf","year":"2018","journal-title":"Genome Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"4817","DOI":"10.1093\/bioinformatics\/btaa611","article-title":"Scirpy: A Scanpy extension for analyzing single-cell T-cell receptor-sequencing data","volume":"36","author":"Sturm","year":"2020","journal-title":"Bioinformatics"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"979","DOI":"10.1038\/nmeth.4402","article-title":"Reversed graph embedding resolves complex single-cell trajectories","volume":"14","author":"Qiu","year":"2017","journal-title":"Nat. Methods"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1088","DOI":"10.1038\/s41467-021-21246-9","article-title":"Inference and analysis of cell-cell communication using CellChat","volume":"12","author":"Jin","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"496","DOI":"10.1038\/s41586-019-0969-x","article-title":"The single-cell transcriptional landscape of mammalian organogenesis","volume":"566","author":"Cao","year":"2019","journal-title":"Nature"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/18\/8338\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,10,1]],"date-time":"2026-10-01T14:24:13Z","timestamp":1790864653000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/18\/8338"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,19]]},"references-count":36,"journal-issue":{"issue":"18","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["ijms27188338"],"URL":"https:\/\/doi.org\/10.3390\/ijms27188338","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,19]]}}}