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30‐Parameter Flow Cytometry Panel to Assess T Cell Effector Functions and Regulatory T Cells

Thomas Liechti, Mario Roederer Cytometry PART A, Volume 95, Issue 11, 1129-1134 (2019)

PURPOSE: 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 (Tregs), we included CD25, CD39, and the canonical Tregs 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.



Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue viability
Granzyme B Human GB11 FITC Cytotoxic molecule
IL-8 Human G265-8 BB630-P2 Effector function
IL-13 Human JES10-5A2 BB660-P2 Effector function
IFN-gamma Human B27 BB700 Effector function
CD137 Human 4B4-1 BB790-P Co-stimulatory
CD28 Human CD28.2 PE Differentiation
TCR gamma/delta Human B1 PE-Cy5 ?dT
FoxP3 Human PCH101 PE-Cy5.5 Treg
IL-22 Human 22URTI PE-Cy7 Effector function
IL-21 Human 3A3-N2.1 Alexa Fluor 647 Effector function
CD107a Human H4A3 Alexa Fluor 700 Degranulation
CD3 Human SK7 APC-H7 T cell lineage
ccr7 Human 150503 BUV395 Differentiation
CD4 Human SK3 BUV496 T cell lineage
CD25 Human 2A3 BUV563 Treg
CD39 Human TU66 BUV661 Differentiation
CD95 Human Dx2 BUV737 Differentiation
CD8 Human SK1 BUV805 T cell lineage
IL-2 Human MQ1-17H12 Brilliant Violet 421 Effector function
CD40L Human TRAP1 Brilliant Violet 480 Co-stimulatory
CD45RO Human UCHL1 Brilliant Violet 570 Differentiation
IL-17A Human BL168 Brilliant Violet 605 Effector function
Ki-67 Human B56 Brilliant Violet 650 Proliferation
CD69 Human FN50 Brilliant Violet 711 Activation
TNF alpha Human Mab11 Brilliant Violet 750 Activation
CD27 Human L128 Brilliant Violet 786 Differentiation
Perforin Human B-D48 Alexa Fluor 594 cytotoxic molecule