Immunology JoVE (Open Access) Citable · DOI

In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function

Name: In Vitro Tumor Cell Rechallenge For Predictive Evaluation of Chimeric Antigen Receptor T Cell Antitumor Function
Uploaded: 2026-06-17
Duration: 8 min 4 s
Description: The field of chimeric antigen receptor (CAR) T cell therapy is rapidly advancing with improvements in CAR design, gene-engineering approaches and ...

DOI: 10.3791/59275-v

What you'll learn

✓ Set up and execute recursive tumor cell-T cell co-cultures for CAR T cell evaluation
✓ Harvest samples and prepare cells for flow cytometric phenotypic analysis
✓ Assess CAR T cell functional capacity through repetitive antigen challenge
✓ Interpret CD4+ and CD8+ CAR T cell phenotype changes post-rechallenge

Protocol

Here, we describe an in vitro co-culture method to recursively challenge tumor-targeted T cells, which allows for phenotypic and functional analysis of antitumor T cell activity.

Difficulty

advanced

Total time

~3–5 days (depends on culture duration and rechallenge cycles)

Model organism

Human CAR T cells (ex vivo engineered); tumor cell lines (specific line not specified in abstract)

Biosafety

BSL-2

Steps

Set up tumor cell and CAR T cell co-culture

Establish initial co-culture of tumor-targeted T cells with tumor cells at defined ratios and conditions. This step initiates the recursive challenge format for evaluating antitumor function.

▶ 00:54

Perform recursive tumor cell rechallenge cycles

Execute repetitive rounds of tumor cell rechallenge to model repeated antigen exposure and assess T cell persistence and functionality across multiple challenge cycles.

▶ 02:37

Harvest co-culture samples for analysis

Collect and process co-cultured cells from the rechallenge assay, preparing samples for downstream flow cytometric characterization.

▶ 04:12

Perform flow cytometric phenotypic and functional analysis

Conduct multiparameter flow cytometry to assess CAR T cell phenotype markers, activation status, and functional capacity following repetitive tumor challenge.

▶ 06:52

Interpret CD4+ and CD8+ CAR T cell phenotype patterns

Analyze representative flow data showing phenotypic differences in CD4+ and CD8+ CAR T cells in response to recursive antigen challenge, identifying markers of persistence and function.

▶ 08:34

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