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Translational Therapeutics

Persistence and enrichment of dominant T cell clonotypes in expanded tumor-infiltrating lymphocytes of breast cancer



Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has shown promising results in cancer treatment, including breast cancer. However, clonal dynamics and clinical significance of TIL expansion ex vivo remain poorly understood.


We investigated T cell receptor (TCR) repertoire changes in expanded TILs from 19 patients with breast cancer. We compared TCR repertoire of TILs at different stages of expansion, including initial (2W TILs) and rapid expansion (REP TILs), and their overlap with formalin fixed paraffin embedded (FFPE) and peripheral blood. Additionally, we examined differences in TCR repertoire between CD4+ and CD8+ REP TILs.


In descending order of proportion, average of 60% of the top 10% clonotypes of FFPE was retained in 2W TIL (60% in TRB, 64.7% in TRA). Among the overlapped clonotypes between 2W TILs and REP TILs, 69.9% was placed in top 30% of 2W TIL. The proportion of clonotypes in 2W TIL and REP TIL showed a significant positive correlation. CD4+ and CD8+ T cells show similar results in diversity and CDR3 length.


Our study traces the changes in TILs repertoire from FFPE to 2W TIL and REP TIL and confirmed that clonotypes with high frequencies in TILs have a high likelihood of maintaining their priority throughout culture process.

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Fig. 1: Clonal expansion and correlation of proportion in overlapped clones between FFPE and 2W TIL.
Fig. 2: Clonal expansion and correlation of proportion in overlapped clones between 2W TIL and REP TIL.
Fig. 3: Clonal expansion and correlation of proportion in overlapped clones between TCR clones in PBMC and other groups.
Fig. 4: Comparison between CD4+ and CD8+ T cells.
Fig. 5: Clonal fractions according to the in vitro reactivity.

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Data are available on reasonable request.


  1. Restifo NP, Dudley ME, Rosenberg SA. Adoptive immunotherapy for cancer: harnessing the T cell response. Nat Rev Immunol. 2012;12(4):269–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Diaz-Cano I, Paz-Ares L, Otano I. Adoptive tumor infiltrating lymphocyte transfer as personalized immunotherapy. Int Rev Cell Mol Biol. 2022;370:163–92.

    Article  CAS  PubMed  Google Scholar 

  3. Scheper W, Kelderman S, Fanchi LF, Linnemann C, Bendle G, de Rooij MAJ, et al. Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers. Nat Med. 2019;25(1):89–94.

    Article  CAS  PubMed  Google Scholar 

  4. Lee HJ, Kim YA, Sim CK, Heo SH, Song IH, Park HS, et al. Expansion of tumor-infiltrating lymphocytes and their potential for application as adoptive cell transfer therapy in human breast cancer. Oncotarget. 2017;8(69):113345–59.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Zhang C, Ding H, Huang H, Palashati H, Miao Y, Xiong H, et al. TCR repertoire intratumor heterogeneity of CD4(+) and CD8(+) T cells in centers and margins of localized lung adenocarcinomas. Int J Cancer. 2019;144(4):818–27.

    Article  CAS  PubMed  Google Scholar 

  6. Hendry S, Salgado R, Gevaert T, Russell PA, John T, Thapa B, et al. Assessing tumor-infiltrating lymphocytes in solid tumors: a practical review for pathologists and proposal for a standardized method from the international immunooncology biomarkers working group: part 1: assessing the host immune response, TILs in invasive breast carcinoma and ductal carcinoma in situ, metastatic tumor deposits and areas for further research. Adv Anat Pathol. 2017;24(5):235–51.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Poncette L, Bluhm J, Blankenstein T. The role of CD4 T cells in rejection of solid tumors. Curr Opin Immunol. 2022;74:18–24.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Park IA, Rajaei H, Kim YA, Lee H, Lee H, Seo JH, et al. T cell receptor repertoires of ex vivo-expanded tumor-infiltrating lymphocytes from breast cancer patients. Immunol Res. 2020;68(5):233–45.

    Article  PubMed  Google Scholar 

  9. Bolotin DA, Poslavsky S, Mitrophanov I, Shugay M, Mamedov IZ, Putintseva EV, et al. MiXCR: software for comprehensive adaptive immunity profiling. Nat methods. 2015;12(5):380–1.

    Article  CAS  PubMed  Google Scholar 

  10. Bagaev DV, Vroomans RMA, Samir J, Stervbo U, Rius C, Dolton G, et al. VDJdb in 2019: database extension, new analysis infrastructure and a T-cell receptor motif compendium. Nucleic Acids Res. 2019;48(D1):D1057–62.

    Article  PubMed Central  Google Scholar 

  11. Tickotsky N, Sagiv T, Prilusky J, Shifrut E, Friedman N. McPAS-TCR: a manually curated catalogue of pathology-associated T cell receptor sequences. Bioinformatics. 2017;33(18):2924–9.

    Article  CAS  PubMed  Google Scholar 

  12. Vita R, Mahajan S, Overton JA, Dhanda SK, Martini S, Cantrell JR, et al. The immune epitope database (IEDB): 2018 update. Nucleic Acids Res. 2019;47(D1):D339–d43.

    Article  CAS  PubMed  Google Scholar 

  13. Chen SY, Yue T, Lei Q, Guo AY. TCRdb: a comprehensive database for T-cell receptor sequences with powerful search function. Nucleic Acids Res. 2021;49(D1):D468–d74.

    Article  CAS  PubMed  Google Scholar 

  14. Pasetto A, Gros A, Robbins PF, Deniger DC, Prickett TD, Matus-Nicodemos R, et al. Tumor- and neoantigen-reactive T-cell receptors can be identified based on their frequency in fresh tumor. Cancer Immunol Res. 2016;4(9):734–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. John J, Chen SMY, Woolaver RA, Ge H, Vashisht M, Huang Z, et al. Host-specific differences in top-expanded TCR clonotypes correlate with divergent outcomes of anti-PD-L1 treatment in responders versus non-responders. Front Immunol. 2023;14:1100520.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Labarrière N, Gervois N, Bonnin A, Bouquié R, Jotereau F, Lang F. PBMC are as good a source of tumor-reactive T lymphocytes as TIL after selection by Melan-A/A2 multimer immunomagnetic sorting. Cancer Immunol Immunother. 2008;57(2):185–95.

    Article  PubMed  Google Scholar 

  17. Al-Kadhimi Z, Callahan M, Fehniger T, Cole KE, Vose J, Hinrichs S.Enrichment of innate immune cells from PBMC followed by triple cytokine activation for adoptive immunotherapy.Int Immunopharmacol.2022;113:109387.

    Article  CAS  PubMed  Google Scholar 

  18. Ghaffari S, Torabi-Rahvar M, Aghayan S, Jabbarpour Z, Moradzadeh K, Omidkhoda A, et al. Optimizing interleukin-2 concentration, seeding density and bead-to-cell ratio of T-cell expansion for adoptive immunotherapy. BMC Immunol. 2021;22(1):43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ahrends T, Busselaar J, Severson TM, Bąbała N, de Vries E, Bovens A, et al. CD4(+) T cell help creates memory CD8(+) T cells with innate and help-independent recall capacities. Nat Commun. 2019;10(1):5531.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Ahrends T, Spanjaard A, Pilzecker B, Bąbała N, Bovens A, Xiao Y, et al. CD4(+) T cell help confers a cytotoxic T cell effector program including coinhibitory receptor downregulation and increased tissue invasiveness. Immunity. 2017;47(5):848–61.e5.

    Article  CAS  PubMed  Google Scholar 

  21. John J, Woolaver RA, Popolizio V, Chen SMY, Ge H, Krinsky AL, et al. Divergent outcomes of anti-PD-L1 treatment coupled with host-intrinsic differences in TCR repertoire and distinct T cell activation states in responding versus non-responding tumors. Front Immunol. 2022;13:992630.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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This study was supported by the Asan Institute for Life Sciences, Asan Medical Center, Seoul, Republic of Korea (2018IL0169).

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Authors and Affiliations



HJL and GG designed the study. BH, SYK, YAK, HJL, and JS developed the methodology. Experiments were performed by BH and YAK. Data were analyzed by BH, SYK, and JS. BH, SYK, YAK, HJL, and JS wrote the manuscript with input from all coauthors. DYH, TP, SC, SWJ, JHK, GP, and GG edited the manuscript.

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Correspondence to Gyungyub Gong, Hee Jin Lee or Junyoung Shin.

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Competing interests

HJL is the CEO and owns stocks in NeogenTC Corp.

Ethics approval and consent to participate

This study was approved by the institutional review board of Asan Medical Center (approval no. 2016-0935), and written informed consent was obtained from the patients.

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Ham, B., Kim, S.Y., Kim, YA. et al. Persistence and enrichment of dominant T cell clonotypes in expanded tumor-infiltrating lymphocytes of breast cancer. Br J Cancer 131, 196–204 (2024).

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