16-Color Panel to Measure Inhibitory Receptor Signatures from Multiple Human Immune Cell Subsets

About the Presenter

Anna Belkina, MD, PhD
Associate Director of the Flow Cytometry Core Facility
Assistant Professor of Pathology and Laboratory Medicine
Boston University School of Medicine

Anna Belkina is the associate director of the Flow Cytometry Core Facility and an assistant professor of pathology and laboratory medicine at Boston University School of Medicine. She received her MD from Russian State Medical University in Moscow and her PhD degree from Boston University School of Medicine investigating the epigenetic regulation of inflammatory responses driven by bromodomain proteins. Anna’s research is focused on the intersection of immunology and computational biology. Her current efforts include investigating the immune landscape of chronic inflammatory diseases and developing computational techniques to assess high-parameter single cell cytometry data. Anna is an active member of ISAC and was named an ISAC SRL Emerging Leader in 2015.

Webinar Summary

This webinar will walk you through the development and optimization of a 16-color human flow cytometry panel for combinational analysis of the Inhibitory Receptors PD-1, TIGIT, CD160, LAG-3, TIM-3 and the activation marker CD137 from CD4+ T cells, CD8+ T cells, NK cells, iNKT cells, and gamma delta T cells. A flow cytometry panel that measures 5-plex IR signatures from multiple populations of immune cells would be useful for both mechanistic studies of exhaustion as well as translational research efforts for a wide span of chronic diseases such as cancer and HIV, especially in the context of scarce sample material.

This panel was thoroughly optimized for use on a 4-laser, 16-detector BD FACSARIA II SORP. In order to gain optimal performance from using multiple polymer fluorophores, panel design was preceded by instrument calibration and optimization based on previous publications. The selection of reagents was defined by predicted marker distribution on cells and levels of their expression and fluorophore "brightness," as well as predicted spillover spread within the fluorophore matrix and reagent availability from common suppliers. When multiple clones were available for particular antigens of interest, selections were made after thorough literature review. Finally, FMO tests were performed to verify specificity of observed populations. Several reagents to limit spillover spread in the channels that appeared to be more vulnerable to this issue.

The conclusion will include some data from a large-scale study, where this panel has been employed, to successfully map an IR-specific phenotype in a cohort of aviremic HIV+ individuals and link it to disease.

Learning Objectives

• Recall basic principles of high-parameter panel design and prerequisite instrument optimization.
• Review major human immune subset mapping with surface markers.
• Demonstrate the process of panel development and optimization.
• Evaluate the resulting panel design with real life application.

Who Should Attend

High-parameter flow cytometry practitioners including primary researchers and core facility staff, as wellas scientists who want to start building their own extensive multicolor flow cytometry panels.

CMLE Credit: 1.0