OMIP-060: 30-Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells

Abstract

We developed this comprehensive 28-color flow cytometry panel with the aim to measure a variety of T cell effector functions in combination with T cell differentiation markers (CCR7, CD27, CD28, CD45RO, CD95) in γδ T cells and CD4⁺ and CD8⁺ αβ T cells (Table 1). The effector functions measured in this panel include activation and co-stimulatory molecules (CD69, CD137, and CD154), cytokines (IL-2, IL-13, IL-17A, IL-21, IL-22, TNF, and IFNγ), the chemokine IL-8, cytotoxic molecules (perforin and granzyme B), and the degranulation marker CD107a. In addition, Ki67 enables the identification and analysis of recently activated T cells. To characterize regulatory T cells (T_regs ), we included CD25, CD39, and the canonical T_regs transcription factor FoxP3. We developed and optimized this panel for cryopreserved human peripheral blood mononuclear cells (PBMC) and stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. However, we successfully tested other types of stimulation such as staphylococcus enterotoxin B (SEB) or a mix of immunodominant peptides (CEF peptide pool) from cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza. Published 2019. This article is a U.S. Government work and is in the public domain in the USA.

Keywords: 28-color flow cytometry; T cells; activation markers; chemokines; cytokines; high-dimensional flow cytometry; intracellular cytokine staining; regulatory T cells.