OMIPs Directory

OMIP-050

A 28‐color/30‐parameter Fluorescence Flow Cytometry Panel to Enumerate and Characterize Cells Expressing a Wide Array of Immune Checkpoint Molecules

Cytometry PART A, Volume 93, Issue 11, 1094-1096 (2018)
Leonard Nettey, Amber J. Giles, Pratip K. Chattopadhyay

Description: This 28‐color/30‐parameter panel catalogs, enumerates and characterizes cells expressing molecules that regulate T‐cell responses (“checkpoint” molecules). The primary purpose is to measure combinatorial expression of PD‐1, CTLA‐4, TIM‐3, CD244/2B4, TIGIT, BTLA, CD137/4‐1BB, GITR, OX40, CD27, and CD278/ICOS, many of which are targets of the immunotherapy drugs in preclinical or clinical development. This panel is optimized for cryopreserved healthy human peripheral blood mononuclear cells (PBMCs) and cryopreserved mononuclear cells isolated from tumor specimens (tissue MNCs). CD45 is used to distinguish leukocytes from stromal cells and debris often present in tissue digests. CD3, CD4, and CD8 identify T‐cells, whose differentiation status is then assessed with CD45RO, CD95, CCR7, CD27, CD57, and CD28. CD25, HLA‐DR, and CD69 mark activated T‐cells. CD69 is also used to identify tissue‐resident lymphocytes in combination with CD103. CXCR3 and CXCR6 mediate lymphocyte trafficking toward chemokines often found in the tumor microenvironment. Regulatory T‐cells are defined here by antibodies against CD25 and CD127. In sum, this panel comprehensively characterizes cells expressing checkpoint markers.

Species: Human

Tissue: Peripheral blood, Tumor

Cell Subset: T cell, Ctl, T helper, T memory, Tissue resident memory (trm), Treg

Keyword: Activation, Cryopreserved pbmc, Immune checkpoint

Machine: BD FACSymphony A5

Panel details

Marker Species Clone Fluorophore Purpose Co-expressions groups Antigen Density
CD186 Human 13B 1E5 Brilliant Blue 515 Trafficking T cell, T helper Low
TIGIT Human MBSA43 Brilliant Blue 660-P2 co-inhibitory T cell, CTL, Inhibition Low
CD57 Human NK-1 Brilliant Blue 790-P Differentiation CTL, T cell High
CD69 Human FN50 Brilliant Violet 750 Activation T cell, Activation, CTL High
CD27 Human O323 PE Differentiation T cell, T memory Med
CCR7 Human 150503 Brilliant Ultraviolet 395 Lineage T memory, T cell Low
Dead cells Human Live/Dead Fix Blue Viability
CD278 Human DX29 Brilliant Violet 421 co-stimulatory T cell, CTL, Activation Low
CD95 Human Dx2 Brilliant Violet 480 Differentiation T cell, CTL, T memory Low
CD183 Human G025H7 Brilliant Violet 650 Trafficking T cell, T helper Med
CD134 Human L106 Brilliant Violet 711 co-stimulatory T cell, Activation, T helper Low
PD-1 Human EH12.2H7 Brilliant Violet 785 co-inhibitory T cell, CTL, Activation Low
CD25 Human 2A3 Brilliant Ultraviolet 563 Differentiation T cell, Treg Low
CD4 Human SK3 Brilliant Ultraviolet 737 Lineage T cell, T helper, T memory, Treg High
CD45RO Human UCHL1 APC Differentiation T cell, T memory Med
CD272 Human J168-540 PE-CF594 co-inhibitory T cell, CTL, Inhibition Low
CD127 Human A019D5 PE-Cy5 Differentiation T cell, T memory, Treg Low
CTLA-4 Human L3D10 PE-Cy7 co-inhibitory T cell, T helper, Activation, Treg Low
CD8a Human RPA-T8 Brilliant Violet 570 Lineage T cell, CTL, T memory High
HLA DR Human G46-6 APC-R700 Activation T cell, CTL, Activation Med
CD28 Human L293 Brilliant Blue 700 Differentiation T cell, T memory Med
CD103 Human Ber-ACT8 Brilliant Violet 605 Differentiation T cell, CTL, Inhibition Med
TIM-3 Human 7D3 Brilliant Ultraviolet 661 co-inhibitory T cell, T helper Low
CD244 Human C1.7 PE-Cy5.5 co-inhibitory CTL, T cell, Activation Med
CD3 Human UCHT1 Brilliant Ultraviolet 496 Lineage T cell, T helper, CTL, Treg, T memory High
CD45 Human HI30 Brilliant Ultraviolet 805 Lineage T cell, T helper, Treg, CTL, T memory High
CD357 Human 108-17 APC-Fire 750 co-stimulatory T cell, T helper, Activation Med
CD137 Human 4B4-1 Brilliant Blue 630-P2 co-stimulatory T cell, CTL, Activation Med