Panel Design And Optimization For Assessment Of T Cell Phenotype In Patients With Melanoma

The advent of immune checkpoint blockade (ICB) treatment has dramatically transformed outcomes for patients with advanced melanoma, however, many patients still fail to respond or experience severe treatment-related toxicity. Currently, there are no biomarkers available to predict these events and allow rationalized treatment choices. Spectral flow cytometry enables robust, high-dimensional assessment of patient immunophenotype and hence is an ideal tool for immune monitoring and biomarker discovery in clinical cohorts. 

We present a high-dimensional approach for blood T cell profiling using three multi-parameter cytometry panels: (1) a panel for absolute cell counts, (2) a 27-color spectral panel assessing resting T cell markers, and (3) a 20-color spectral panel evaluating intracellular cytokine expression. We applied these optimized panels to a cohort of patients with advanced melanoma before and during treatment with combination PD-1/CTLA-4 blockade, as well as age-matched healthy controls. Blood mononuclear cells were cryopreserved and stained across 24 separate batches, and batch effects assessed with a single-donor sample. 

Our optimized spectral flow cytometry approach found that response to ICB was associated with distinct T cell profiles before and after one cycle of ICB. Non-responders had significantly fewer circulating naive T cells and more highly immunosuppressive regulatory T cells at baseline. However, responders showed greater Ki67 upregulation across multiple T cell subsets after one cycle of ICB as compared to non-responders. Batch effects varied between markers but did not affect manual gating, highlighting spectral flow cytometry as a suitable method for longitudinal immunophenotyping.

Key topics discussed in this webinar will include:

• ­­The design and optimization of large panels for the Cytek Aurora™ spectral flow cytometer
• How to track and limit the impact of batch effects in longitudinal studies
• The benefit of including absolute cell counts in routine immunophenotyping
• Immune monitoring for biomarker discovery in a cohort of advanced melanoma patients

Who should attend:

• Anyone considering moving from conventional to spectral flow cytometry 
• Those interested in panel design and optimization, or exploring the impact of batch effects
• Scientists using flow cytometry for immune monitoring and biomarker discovery 
• Those interested in optimizing immune checkpoint blockade treatment of solid cancers

For Research Use Only. Not intended for use in diagnostic procedures.