Establishment of a mechanism-based in vitro coculture assay for evaluating the efficacy of immune checkpoint inhibitors

Myeong Joon Kim, Kyeong Hee Hong, Bo Ryeong Lee, Sang Jun Ha

Research output: Contribution to journalArticlepeer-review


Cancer immunotherapy, which blocks immune checkpoint molecules, is an effective therapeutic strategy for human cancer patients through restoration of tumor-infiltrating (TI) cell function. However, evaluating the efficacy of immune checkpoint inhibitors (ICIs) is difficult because no standard in vitro assay for ICI efficacy evaluation exists. Additionally, blocking a particular immune checkpoint receptor (ICR) is insufficient to restore T cell functionality, because other ICRs still transduce inhibitory signals. Therefore, limiting inhibitory signals transduced via other ICRs is needed to more accurately assess the efficacy of ICIs targeting a particular immune checkpoint. Here, we introduce a newly developed in vitro coculture assay using human peripheral blood mononuclear cells (hPBMCs) and engineered human cancer cell lines. We enriched CD8+ T cells from hPBMCs of healthy donors through low-dose T cell receptor stimulation and cytokine (human IL-2 and IL-7) addition. These enriched CD8+ T cells were functional and expressed multiple ICRs, especially TIM-3 and TIGIT. We also established immune checkpoint ligand (ICL) knockout (KO) cancer cell lines with the CRISPR-Cas9 system. Then, we optimized the in vitro coculture assay conditions to evaluate ICI efficacy. For example, we selected the most effective anti-TIM-3 antibody through coculture of TIM-3+CD8+ T cells with PD-L1-/-PVR-/- cancer cells. In summary, we developed a mechanism-based in vitro coculture assay with hPBMCs and ICL KO cancer cell lines, which could be a useful tool to identify promising ICIs by providing reliable ICI efficacy information.

Original languageEnglish
Pages (from-to)2777-2789
Number of pages13
JournalCancer Immunology, Immunotherapy
Issue number11
Publication statusPublished - 2022 Nov

Bibliographical note

Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.

All Science Journal Classification (ASJC) codes

  • Immunology and Allergy
  • Immunology
  • Oncology
  • Cancer Research


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