OMIP-118: A 38-Marker Spectral Flow Cytometry Panel to Assess Human Regulatory T Cell Phenotype and Lineage...

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Human regulatory T cells (Tregs) are vital for immune tolerance, playing critical roles in cancer, autoimmunity, and infectious diseases.
• Understanding Treg heterogeneity and plasticity is essential for developing biomarkers and therapeutics, but current tools are limited.
• Phenotypic and functional diversity within the Treg lineage contributes to various disease states, necessitating advanced characterization methods.

Purpose of the Study:

• To optimize a 38-parameter spectral flow cytometry panel for comprehensive identification, phenotyping, and lineage plasticity assessment of human Tregs.
• To develop a robust tool for deep single-cell level characterization of Treg heterogeneity in peripheral blood.
• To facilitate the discovery of Treg-based biomarkers and evaluate Treg-directed therapies.

Main Methods:

• Development and optimization of a 38-parameter spectral flow cytometry panel, including a 13-marker PBMC backbone and a 25-marker Treg phenotyping module.
• Incorporation of intracellular targets alongside surface markers for comprehensive Treg analysis.
• Evaluation of antibody binding in pre-fixation and post-fixation/permeabilization settings, identifying antibodies suitable for cost-saving overnight staining.

Main Results:

• The panel successfully identifies and characterizes human Tregs, revealing up to 14 discrete Treg phenotypes in healthy donors via dimensionality reduction and clustering.
• Identification of antibodies suitable for overnight post-fixation staining, reducing titers and costs compared to traditional methods.
• Demonstration of the panel's capability for deep phenotypic characterization of Treg heterogeneity in peripheral blood.

Conclusions:

• The optimized 38-parameter spectral flow cytometry panel provides a powerful tool for deep phenotyping of human Treg heterogeneity.
• This panel enables detailed assessment of Treg differentiation, activation, and lineage plasticity.
• The findings advance the study of Tregs in disease and the development of Treg-based immunotherapies.