A novel co-culture assay to assess anti-tumor CD8(+) T cell cytotoxicity via luminescence and multicolor flow cytometry

Veronica Olivo Pimentel, Ala Yaromina, Damienne Marcus, Ludwig J. Dubois*, Philippe Lambin

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

14 Citations (Web of Science)


T cell immunotherapies have shown great promise in patients with advanced cancer disease, revolutionizing treatment. T cell cytotoxicity is crucial in its efficacy, therefore developing ex vivo methods testing tumor and T cell interactions is pivotal. Increasing efforts have been made in developing co-culture assays with sophisticated materials and platforms aiming to mimic the tumor microenvironment (TME), but its complexity makes it difficult to develop the ideal model. In this study, we developed a simple co-culture assay, reproducible in any lab, but respecting the multicellular nature of the TME. Our goal is to combine in a single assay well-established techniques such as a luciferase assay for target cell viability analysis, a CD107a degranulation assay, and multicolor flow cytometry for the detection of cytokines and cytotoxicity markers. Cell suspensions of whole spleens and tumors containing splenic or tumor-infiltrating effector T cells of mice bearing Lewis lung carcinoma (LLC) or CT26 colon carcinoma tumors treated with radiation alone or in combination with immunotherapies were used for co-culture. LLC and CT26 cell lines transduced with the firefly luciferase gene were used as target cells. We demonstrated that splenocytes and tumor-infiltrating T cells derived from mice treated with combination therapy were able to kill approximately 50% of target cells after 48 h of co-culture. This effect was tumor cell-specific and dependent on CD8(+) T cells evidenced by in vitro CD8(+) T cell depletion. Flow cytometry demonstrated increased expression of CD107a and production of granzyme B, IFN gamma, and TNF alpha by CD8(+) T cells. Our co-culture assay is therefore suitable as proof of principle for in vivo therapeutic studies testing immunotherapies, and specifically to assess the involvement of cytotoxic CD8(+) T cells in treatment response in LLC and CT26 tumor models. We also propose this assay as an ex vivo platform for high-throughput screening of immunomodulating agents to be tested in these two murine tumor models. This assay can be adapted to other tumor models after optimizations.

Original languageEnglish
Article number112899
Number of pages14
JournalJournal of Immunological Methods
Publication statusPublished - Dec 2020


  • Co-culture assay
  • Ex vivo
  • Cytotoxic T cells
  • Tumor cells
  • Flow cytometry
  • Luciferase assay

Cite this