A 37‐Color Spectral Flow Cytometric Panel to Assess Transcription Factors and Chemokine Receptors in Human Intestinal Lymphoid Cells

ABSTRACT We have developed a 37‐color spectral flow cytometry panel to assess the phenotypical differentiation of innate and adaptive immune lymphoid subsets within human intestinal tissue. In addition to lineage markers for identifying innate lymphoid cells (ILC), TCRγδ, MAIT (mucosal‐associated invariant T), natural killer (NK), CD4 + and CD8 + T cells, we incorporated markers of differentiation and activation (CD45RA, CD45RO, CD25, CD27, CD38, CD39, CD69, CD103, CD127, CD161, HLA‐DR, CTLA‐4 [CD152]), alongside transcription factors (Bcl‐6, FoxP3, GATA‐3, Helios, T‐bet, PU.1 and RORγt) and chemokine receptors (CCR4, CCR6, CCR7, CXCR3, and CXCR5). Additionally, Granzyme B and Ki‐67 were included to assess cytotoxicity and proliferation potential of the different subsets. This panel is currently used for in‐depth immunophenotyping in endoscopic biopsies and peripheral blood mononuclear cells (PBMC) from inflammatory bowel disease (IBD) patients. Distinguished from other OMIP papers, the comprehensive detection of both transcription factors and chemokine receptors facilitates the efficient assessment of several subsets, particularly CD4 + T helper cells, and its potential application extends to both tissue and circulation.