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

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

PURPOSE: 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.

CELL TYPE: Cryopreserved PBMC and mononuclear cells isolated from tumor specimens (tissue MNCs)


Marker Target Clone Fluorochrome Purpose
Dead cells Human Live/Dead Fix Blue Viability
CD186 Human 13B 1E5 BB515 Trafficking
CD137 Human 4B4-1 BB630-P2 co-stimulatory
TIGIT Human MBSA43 BB660-P2 co-inhibitory
CD28 Human L293 BB700 Differentiation
CD57 Human NK-1 BB790-P Differentiation
CD278 Human DX29 Brilliant Violet 421 co-stimulatory
CD95 Human Dx2 Brilliant Violet 480 Differentiation
CD8 Human RPA-T8 Brilliant Violet 570 Lineage
CD103 Human Ber-ACT8 Brilliant Violet 605 Differentiation
CD183 Human G025H7 Brilliant Violet 650 Trafficking
CD134 Human L106 Brilliant Violet 711 co-stimulatory
CD69 Human FN50 Brilliant Violet 750 Activation
PD-1 Human EH12.2H7 Brilliant Violet 785 co-inhibitory
ccr7 Human 150503 BUV395 Lineage
CD3 Human UCHT1 BUV496 Lineage
CD25 Human 2A3 BUV563 Differentiation
CD366 Human 7D3 BUV661 co-inhibitory
CD4 Human SK3 BUV737 Lineage
CD45 Human HI30 BUV805 Lineage
CD45RO Human UCHL1 APC Differentiation
HLA-DR Human G46-6 APC-R700 Activation
CD357 Human 108-17 APC-Fire 750 co-stimulatory
CD27 Human O323 PE Differentiation
CD272 Human J168-540 PE-CF594 co-inhibitory
CD127 Human A019D5 PE-Cy5 Differentiation
CD244 Human C1.7 PE-Cy5.5 co-inhibitory
CTLA-4 Human L3D10 PE-Cy7 co-inhibitory