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Co-inhibitory receptor (immune checkpoint) expression analysis in human T cell subsets

Zachary R. Healy, David M. Murdoch Cytometry PART A, Volume 89, Issue 10, 889–892 (2016)

PURPOSE: This panel was optimized to quantify inhibitory receptor expression on CD4 and CD8 T cells from differentiation and activation subsets. Six inhibitory (i.e., immune checkpoint) receptors (PD-1, TIM-3, LAG-3, CD160, BTLA, CTLA-4) were chosen based upon previously published observations suggesting their role in modulating CD4 and CD8 T cell activation in response to persistent antigen exposure [1-3]. Furthermore, given the important observations that inhibitory receptor expression varies by differentiation and prior antigen experience, markers of T cell differentiation status and prior antigen experience (CCR7, CD45RA, CD28, CD127, KLRG1) were also included [4-6]. This panel was developed and optimized for use in cryopreserved human peripheral blood mononuclear cells (PBMCs), although it has also been applied in fresh PBMCs as well as other bodily fluids (e.g., malignant ascites) (Table 1).


MACHINE: BD Fortessa

Marker Target Clone Fluorochrome Purpose
Dead cells Human Zombie Aqua Viability
CD14 Human M5E2 Brilliant Violet 510 Dump/exclusion
CD19 Human HIB19 Brilliant Violet 510 Dump/exclusion
CD3 Human SK7 BUV395 Phenotyping
CD4 Human SK3 BUV496 Phenotyping
CD8 Human SK1 BUV805 Phenotyping
ccr7 Human G043H7 Brilliant Violet 785 Maturation/Activation
CD45RA Human HI100 BB515 Maturation/Activation
CD28 Human CD28.2 APC-H7 Maturation/Activation
CD127 Human A019D5 Brilliant Violet 650 Maturation/Activation
KLRG1 Human SA231A2 Alexa Fluor 647 Maturation/Activation
PD-1 Human EH12.2H7 Brilliant Violet 711 Inhibitory Receptors
TIM-3 Human F38-2E2 Brilliant Violet 605 Inhibitory Receptors
Lag-3 Human 3DS223H PE-Cy7 Inhibitory Receptors
CD160 Human BY55 PerCP-Cy5.5 Inhibitory Receptors
CD272 Human MIH26 Brilliant Violet 421 Inhibitory Receptors
CTLA-4 Human BNI3 PE-CF594 Inhibitory Receptors