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Updated: Jun 30, 2026
High-Dimensionality Flow Cytometry for Immune Function Analysis of Dissected Implant Tissues
Published on: September 15, 2021
High-dimensional single-cell mass cytometry analysis of IL-4 treated human macrophages reveals heterogeneity and a
Blanca E Callejas1, Jared Schlechte2,3, James A Sousa1
1Department of Physiology and Pharmacology, Gastrointestinal Research Group, Inflammation Research Network and Host-Parasite Interactions Group, Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, 3280 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.
Autologous cell therapy is a patient-embraced approach to inflammatory disease. Murine models demonstrate that alternatively activated (or regulatory) macrophages can reduce the severity of disease, and human macrophages (M0) educated with interleukin-4 (M(IL4)) limit murine colitis. M0 and M(IL4) from healthy donors and individuals with Crohn's disease (active or remission) were assessed by qPCR, characterized by a time-of-flight mass cytometry (CyTOF) comparison of 16 myeloid marker molecules expression, and subtypes of M(IL4)s tested in the murine dinitrobenzene sulfonic acid (DNBS) model of colitis and in vitro co-culture with T cells. Most macrophage preparations responded to IL-4 by increasing CD206, CCL18 and RAMP1 mRNA expression and produced mediators that promoted epithelial repair in a wound assay with the human CaCo2 epithelial line. Single-cell clustering by FlowSOM defined 8 distinct subpopulations (meta-clusters) of macrophages, the proportions of which were unaffected by biological sex, age or cryopreservation: meta-cluster 4 (CD206highPD-L1highHLA-DRhigh) represented 20 to 40% of M0s, with a higher proportion in controls compared with active Crohn's disease. IL-4 treatment significantly expanded meta-cluster 4 in all M0s. Transfer of M(IL4)-CD206high (ie, predominantly meta-cluster 4) into rag1-/- mice significantly reduced DNBS-induced colitis, and to a greater extent than M(IL4)-CD206low from the same individual. IL-10 production was increased in M(IL4)-CD206high-T-cell co-cultures. Having confirmed the prohealing effect of the human M(IL4), the predominant meta-cluster M(IL4)-CD206high was found to have superior anticolitic effect. Reduced numbers of M0-CD206high and M(IL4)-CD206high cells in some individuals with Crohn's disease suggest that their absence may contribute to more severe inflammation or reduced healing capacity.
Autologous cell therapy is a patient-embraced approach to inflammatory disease. Murine models demonstrate that alternatively activated (or regulatory) macrophages can reduce the severity of disease, and human macrophages (M0) educated with interleukin-4 (M(IL4)) limit murine colitis. M0 and M(IL4) from healthy donors and individuals with Crohn's disease (active or remission) were assessed by qPCR, characterized by a time-of-flight mass cytometry (CyTOF) comparison of 16 myeloid marker molecules expression, and subtypes of M(IL4)s tested in the murine dinitrobenzene sulfonic acid (DNBS) model of colitis and in vitro co-culture with T cells. Most macrophage preparations responded to IL-4 by increasing CD206, CCL18 and RAMP1 mRNA expression and produced mediators that promoted epithelial repair in a wound assay with the human CaCo2 epithelial line. Single-cell clustering by FlowSOM defined 8 distinct subpopulations (meta-clusters) of macrophages, the proportions of which were unaffected by biological sex, age or cryopreservation: meta-cluster 4 (CD206highPD-L1highHLA-DRhigh) represented 20 to 40% of M0s, with a higher proportion in controls compared with active Crohn's disease. IL-4 treatment significantly expanded meta-cluster 4 in all M0s. Transfer of M(IL4)-CD206high (ie, predominantly meta-cluster 4) into rag1-/- mice significantly reduced DNBS-induced colitis, and to a greater extent than M(IL4)-CD206low from the same individual. IL-10 production was increased in M(IL4)-CD206high-T-cell co-cultures. Having confirmed the prohealing effect of the human M(IL4), the predominant meta-cluster M(IL4)-CD206high was found to have superior anticolitic effect. Reduced numbers of M0-CD206high and M(IL4)-CD206high cells in some individuals with Crohn's disease suggest that their absence may contribute to more severe inflammation or reduced healing capacity.
