Detection of clinically relevant immune checkpoint markers by multicolor flow cytometry

Area of Science:

• Immunology
• Cancer Research
• Flow Cytometry

Background:

• Checkpoint inhibitor immunotherapies are increasingly important in cancer treatment.
• Accurate phenotyping of patient immune cells is crucial for these therapies.
• Existing assays may require optimization for clinical samples.

Purpose of the Study:

• To develop and validate an 8-color flow cytometry panel for immune cell phenotyping.
• To analyze immune checkpoint marker expression on T-lymphocytes.
• To assess the utility of the panel for cryopreserved human peripheral blood mononuclear cells (PBMCs).

Main Methods:

• An 8-color flow cytometry panel was designed for lineage and immune checkpoint markers.
• Peripheral blood mononuclear cells (PBMCs) from healthy human donors were used for validation.
• Flow cytometry data was acquired on a BD LSR Fortessa.
• Luminex multiplex soluble immunoassay was used for soluble immune checkpoint quantification.

Main Results:

• Significant inter-donor variation in immune cell markers was observed at baseline and during activation.
• Expression of immune checkpoint markers on stimulated CD4+ and CD8+ T-cells increased over time.
• Soluble isoforms of LAG-3, TIM-3, CTLA-4, and PD-1 were upregulated following stimulation.

Conclusions:

• The 8-color flow cytometry panel effectively identifies and evaluates immune checkpoints on T-lymphocytes.
• The panel is suitable for analyzing cryopreserved human PBMCs, crucial for clinical sample handling.
• This assay supports the characterization of immune checkpoint expression in immunotherapy research.