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Evaluation of Human Conventional T Cells, Donor‐Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining

One Dintwe, Shamiska Rohith, Katharine V. Schwedhelm, M. Juliana McElrath, Erica Andersen‐Nissen, Stephen C. De Rosa Cytometry PART A, Volume 95, Issue 7, 722-725 (2019)

PURPOSE: A 26‐color staining panel was developed to profile human antigen‐specific T cells in an intracellular cytokine staining (ICS) assay using peptide pools to various antigens of interest. In addition to multiple functional markers, the panel includes differentiation/activation markers and markers to assess γδ, mucosal‐associated invariant T, and NK T cells as well as conventional NK cells. Panel optimization was performed using previously cryopreserved PBMC from healthy adults, and then, expression of key functional markers in the panel was cross‐validated against a validated ICS assay used in the HIV Vaccine Trials Network (HVTN). The panel is currently being used to evaluate the responses to tuberculosis and malaria vaccine candidates in volunteers from different geographic areas.

CELL TYPE: Cryopreserved PBMC


Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue Viability
CD4 Human RPA-T4 BUV805 T cell lineage
IFN-gamma Human B27 V450 Function
CD14 Human MfP9 BB660-P2 Monocyte
CD3 Human UCHT1 BUV395 T cell lineage
CD40L Human PE-Cy7 Function
CD56 Human CMSSB PE-Cy5.5 NK,NKT
CD8 Human RPA-T8 BUV563 T cell lineage
ccr7 Human G043H7 Brilliant Violet 785 Differentiation
TNF alpha Human Mab11 Brilliant Violet 750 Function
IL-4 Human MP4-25D2 APC Function
IL-2 Human MQ1-17H12 BB700 Function
Perforin Human B-D48 FITC Function
IL-22 Human 22URTI PE Function
CD45RA Human HI100 BUV496 Differentiation
CD183 Human 1C6/CXCR3 PE-Cy5 T helper
CCR6 Human Brilliant Violet 605 T helper
KLRG1 Human REA261 PE-Vio615 Differentiation
IL-13 Human JES10-5A2 APC Function
Granzyme A Human CB9 Alexa Fluor 700 Function
TCR V alpha 7.2 Human REA179 APC-Vio770 MAIT
CD26 Human M-A261 Brilliant Violet 711 MAIT
HLA-DR Human G46-6 BUV661 Activation
IL-17A Human N49-653 BUV737 Function
TCR gamma/delta Human 11F2 Brilliant Violet 480 ?d T
CD16 Human 3G8 Brilliant Violet 570 NK,NKT
CD161 Human DX12 Brilliant Violet 650 MAIT