Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data
Background T-cell receptor (TCR)-T immunotherapy has emerged as a promising strategy for cancer treatment. However, identifying TCRs that can be used to generate TCR-T cells remains challenging due to tumor heterogeneity, the scarcity of tumor-specific T cells, and the diversity of human leukocyte a...
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| Format: | Article |
| Language: | English |
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BMJ Publishing Group
2025-07-01
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| Series: | Journal for ImmunoTherapy of Cancer |
| Online Access: | https://jitc.bmj.com/content/13/7/e012029.full |
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| author | Yi Li Weiguo Zhang Xuelian Song Xianyao Wang Yanshu Ding Chenfei Yin Tianyuan Ren |
| author_facet | Yi Li Weiguo Zhang Xuelian Song Xianyao Wang Yanshu Ding Chenfei Yin Tianyuan Ren |
| author_sort | Yi Li |
| collection | DOAJ |
| description | Background T-cell receptor (TCR)-T immunotherapy has emerged as a promising strategy for cancer treatment. However, identifying TCRs that can be used to generate TCR-T cells remains challenging due to tumor heterogeneity, the scarcity of tumor-specific T cells, and the diversity of human leukocyte antigens (HLA). To advance TCR-T immunotherapy, it is crucial to develop an efficient and scalable method to identify tumor-specific TCRs.Methods To identify tumor-specific TCRs, epitopes, and their corresponding HLA subtypes, we developed a method for rapidly assembling TCRs identified through the single-cell analysis of T cells from various tumors. For each TCR, only two pairs of oligonucleotides corresponding to the CDR3 regions of TCR-α and β chains needed to be synthesized, enabling the construction of a TCR library quickly in a cost-effective manner. Additionally, we engineered HLA-knockout HEK-293T cells as antigen-presenting cells to express patient-specific HLA-I and neoantigens, and a Jurkat NFAT-GFP reporter cell line for screening antigen-reactive TCRs. The efficacy of our TCR-screening system was validated through a small-scale screening of HPV16-specific TCRs from patients with cervical cancer.Results We successfully developed a TCR assembly method that enables the rapid cloning and construction of TCR libraries within 2 days, significantly accelerating the process and reducing costs. Our antigen-presenting system also allows for flexible expression of patient-specific HLA-I molecules, facilitating personalized screening. The Jurkat reporter cells demonstrated high sensitivity for screening functional TCRs. Using published datasets from patients with HPV16-positive cervical cancer, we successfully used our system to isolate a human papillomavirus (HPV)-specific TCR. Through deletion, alanine scanning, and mass spectrometry analysis, we determined that this TCR specifically recognized an 8-mer peptide (MHGDTPTL) from HPV-E7 presented by HLA-B*15:18. Moreover, T cells expressing this TCR were able to effectively kill HPV-positive cells.Conclusions We developed an integrated antigen-presenting, TCR assembly, and TCR reporter system for screening tumor-specific TCRs using single-cell sequencing datasets. By using this system, we have successfully identified a functional, HPV-specific TCR, demonstrating the potential of our approach for the efficient screening of tumor-specific TCRs to advance TCR-T immunotherapy. |
| format | Article |
| id | doaj-art-96c76fa7eb6d493d9c519ec2d3e27681 |
| institution | Kabale University |
| issn | 2051-1426 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | BMJ Publishing Group |
| record_format | Article |
| series | Journal for ImmunoTherapy of Cancer |
| spelling | doaj-art-96c76fa7eb6d493d9c519ec2d3e276812025-08-20T03:58:11ZengBMJ Publishing GroupJournal for ImmunoTherapy of Cancer2051-14262025-07-0113710.1136/jitc-2025-012029Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing dataYi Li0Weiguo Zhang1Xuelian Song2Xianyao Wang3Yanshu Ding4Chenfei Yin5Tianyuan Ren6National Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, ChinaNational Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, ChinaNational Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, ChinaNational Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, ChinaNational Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, ChinaNational Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, ChinaNational Key Laboratory of Immunity and Inflammation, Suzhou Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Suzhou, Jiangsu, ChinaBackground T-cell receptor (TCR)-T immunotherapy has emerged as a promising strategy for cancer treatment. However, identifying TCRs that can be used to generate TCR-T cells remains challenging due to tumor heterogeneity, the scarcity of tumor-specific T cells, and the diversity of human leukocyte antigens (HLA). To advance TCR-T immunotherapy, it is crucial to develop an efficient and scalable method to identify tumor-specific TCRs.Methods To identify tumor-specific TCRs, epitopes, and their corresponding HLA subtypes, we developed a method for rapidly assembling TCRs identified through the single-cell analysis of T cells from various tumors. For each TCR, only two pairs of oligonucleotides corresponding to the CDR3 regions of TCR-α and β chains needed to be synthesized, enabling the construction of a TCR library quickly in a cost-effective manner. Additionally, we engineered HLA-knockout HEK-293T cells as antigen-presenting cells to express patient-specific HLA-I and neoantigens, and a Jurkat NFAT-GFP reporter cell line for screening antigen-reactive TCRs. The efficacy of our TCR-screening system was validated through a small-scale screening of HPV16-specific TCRs from patients with cervical cancer.Results We successfully developed a TCR assembly method that enables the rapid cloning and construction of TCR libraries within 2 days, significantly accelerating the process and reducing costs. Our antigen-presenting system also allows for flexible expression of patient-specific HLA-I molecules, facilitating personalized screening. The Jurkat reporter cells demonstrated high sensitivity for screening functional TCRs. Using published datasets from patients with HPV16-positive cervical cancer, we successfully used our system to isolate a human papillomavirus (HPV)-specific TCR. Through deletion, alanine scanning, and mass spectrometry analysis, we determined that this TCR specifically recognized an 8-mer peptide (MHGDTPTL) from HPV-E7 presented by HLA-B*15:18. Moreover, T cells expressing this TCR were able to effectively kill HPV-positive cells.Conclusions We developed an integrated antigen-presenting, TCR assembly, and TCR reporter system for screening tumor-specific TCRs using single-cell sequencing datasets. By using this system, we have successfully identified a functional, HPV-specific TCR, demonstrating the potential of our approach for the efficient screening of tumor-specific TCRs to advance TCR-T immunotherapy.https://jitc.bmj.com/content/13/7/e012029.full |
| spellingShingle | Yi Li Weiguo Zhang Xuelian Song Xianyao Wang Yanshu Ding Chenfei Yin Tianyuan Ren Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data Journal for ImmunoTherapy of Cancer |
| title | Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data |
| title_full | Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data |
| title_fullStr | Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data |
| title_full_unstemmed | Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data |
| title_short | Integrated system for screening tumor-specific TCRs, epitopes, and HLA subtypes using single-cell sequencing data |
| title_sort | integrated system for screening tumor specific tcrs epitopes and hla subtypes using single cell sequencing data |
| url | https://jitc.bmj.com/content/13/7/e012029.full |
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