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918 Functional heterogeneity of CD4+ tumor-infiltrating lymphocytes
  1. Arianna Draghi1,
  2. Christopher A Chamberlain1,
  3. Shawez Khan2,
  4. Katja Harbst3,4,
  5. Mario Presti1,
  6. Martin Lauss4,5,
  7. Michael D Crowther1,
  8. Agnete WP Jensen1,
  9. Anne-Christine K Rasmussen2,
  10. Benedetta Albieri1,
  11. Torben Lorentzen8,
  12. Mads H Andersen1,
  13. Göran Jönsson4,5,
  14. Inge Svane1 and
  15. Marco Donia1
  1. 1National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev, Denmark
  2. 2National Center for Cancer Immune Therapy(CCIT-DK), Herlev, Denmark
  3. 3Lund University, Lund, Sweden
  4. 4Lund University Cancer Centre, Lund University, Lund, Sweden
  5. 5Division of Oncology and Pathology, Department of Clinical Sciences Lund, Faculty of Medicine, Lund University, Lund, Sweden
  6. 8Department of Gastroenterology, Unit of Surgical Ultrasound, Copenhagen University Hospital, Herlev, Denmark
  • Journal for ImmunoTherapy of Cancer (JITC) preprint. The copyright holder for this preprint are the authors/funders, who have granted JITC permission to display the preprint. All rights reserved. No reuse allowed without permission.


Background Immunotherapy has demonstrated impressive results in numerous metastatic solid tumors. However, while most studies have focused on immune responses of CD8+ tumor-infiltrating lymphocytes (TILs), the functional role of tumor-reacting CD4+ TILs remains largely unclear.

Methods To characterize the complete functional repertoire of actively tumor-reacting CD4+ TILs, we generated and tested a workflow allowing accurate reproduction of the TIL-tumor cell interaction of eighteen individual patients with three tumor histologies. Via bulk and single-cell RNA sequencing (scRNAseq) of ex vivo-expanded CD4+ TILs post-coculture with autologous tumor cells, we obtained a complete transcriptomic characterization of tumor-reacting CD4+ TILs following recognition of naturally presented tumor antigens.

Results Recognition of autologous tumor antigens induced detectable transcriptomic and secretome changes in tumor-reacting CD4+ TILs, and allowed the generation of a transcriptomic signature of actively tumor-reacting CD4+ TILs (TR4S) used to identify multiple subpopulations of functionally distinct tumor-reacting CD4+ TILs. The results were then validated on an atlas of public scRNAseq data from fresh tumor tissues of twelve distinct tumor histologies, confirming the presence of multiple tumor-reacting CD4+ TIL subpopulations activating distinct functional programs.

Conclusions In conclusion, we characterized the functional landscape of tumor-reacting CD4+ TILs on a transcriptomic level. These data highlighted the functional heterogeneity of actively tumor-reacting CD4+ TILs, and warrant further investigation into the role of functionally distinct tumor-reacting CD4+ TIL subpopulations in the tumor microenvironment.

Ethics Approval All patients were enrolled in clinical trials performed at the National Center for Cancer Immune Therapy (CCIT-DK), Department of Oncology, Copenhagen University Hospital, Herlev, Denmark. All procedures were performed in compliance with clinical protocols approved by the Ethics Committee of the Capital Region of Denmark (Reference H-19076238 and H-20070020) and national regulations for biomedical research, including appropriate data protection of human participants (Reference P-2020-65 and P-2021-303). Written informed consent was provided by all patients before obtaining any samples.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See

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