P152 ANALYSIS OF HUMAN COLONIC TISSUE AND PERIPHERAL BLOOD BY MASS CYTOMETRY REVEALS IMMUNE SIGNATURES DIFFERENTIATING INFLAMMATORY STATES IN UC, CD, AND CONTROLS. (7th February 2019) - Mitsialis, Vanessa
Abstract: Intro: Inflammatory bowel disease (IBD), consisting of ulcerative colitis (UC) and Crohn's disease (CD), leads to chronic intestinal inflammation associated with significant morbidity. The underlying mucosal dysregulation is not entirely understood, and many patients are refractory to available medical therapy. Defining inflammatory and disease-specific immune signatures would improve our understanding of IBD and help identify novel biomarkers and targets for treatment. We used mass cytometry (CYTOF) to profile colonic lamina propria (LP) and peripheral blood (PB) mononuclear (MN) cells of patients with IBD as well as non-IBD controls. Methods: LPMCs (n=87) and PBMCs (n=88) were obtained from non-IBD controls (LP n=18, PB n=27), uninflamed UC (UC-; LP n=13, PB n=6), inflamed UC (UC+; LP n=19, PB n=21), uninflamed CD (CD-; LP n=24, PB n=9), and inflamed CD (CD+; LP n=13, PB n=25) patients, cryopreserved and thawed in batches. Cells were stimulated with PMA/ionomycin, stained with a panel of CYTOF antibodies, and data analyzed with both automated clustering and manual gating. Results: LP differences distinguish inflamed IBD (IBD+) from uninflamed IBD (IBD-) and from non-IBD: IBD LP is enriched with HLADR+CD38+ ("activated") T cells (26.4% IBD+ > 13.9% IBD- > 5.9% non-IBD, out of total CD45+ cells [p=5.6e-07]). Tregs (1.10% IBD+ > 0.6% IBD- > 0.26% non-IBD [p=0.00067]) and IL-1b+ macrophages (0.31% IBD+, 0.07% IBD-, 0.23% non-IBD [p=2.1e-07]) were increased in IBD+. Additionally, IBD- had fewer DCs than IBD+ and non-IBD (4.6% IBD+, 2.4% IBD-, 5.8% non-IBD [p=0.00024]). LP differences distinguish UC+ from CD+: UC+ LP has fewer T cells compared to CD+ (UC+ 59% vs. CD+ 69% [p=0.013]) with a trend towards more B cells. Among T cells, CD4+ are more abundant in UC+ than in CD+ and non-IBD (UC+ 19% vs. CD+ 14% [p=0.026] vs. 14% non-IBD [p=0.024]). There are more "activated" CD4+ T cells in UC+ that express IL-17 over IFNg with the opposite being true in CD+ (ratio 1.55 IL-17:IFNg in UC+ vs. 0.27 IL17:IFNg in CD+ [p=0.0017]). CD+ LP has greater IL-1b+ CD123+DCs compared to UC+ and non-IBD (0.026% UC+, 0.082% CD+, 0.034% non-IBD [p=0.011]). The unique PB difference distinguishing CD+ from UC+ and non-IBD was an increase in IL-1b+ monocytes in CD+ (0.24% UC+, 0.69% CD+, 0.29% non-IBD [p=0.009]). Conclusions: CYTOF analysis of LP and PB reveals differences associated with colonic inflammation as well as immune characteristics that differentiate UC and CD. Whereas differences in the LP involve populations of activated T cells, Tregs, and innate immune cells, significant differences in the PB are restricted to innate cells. Certain signatures, such as greater abundance of IL-1b+ monocytes in the PB of CD+ patients, could potentially be used to distinguish inflammatory disease states in the periphery and identify new avenues of diagnostic and treatment approaches. Figure 1. VISNE density heatmaps demonstrate global differences in T cell population abundance in LP across IBD disease types and inflammation status in a representative subset of patients included in the abstract (a). Area defined by asterisk (*) is diminished in controls but significantly more abundant in inflamed tissue (both UC and CD). This area corresponds to both CD4+ and CD8+ T cells that co-express HLADR and CD38 (as demonstrated by the marker heatmaps in b). Manual analysis of HLADR+CD38+ T cells as a percent of total CD45+ cells reveals that this population is most abundant in inflamed IBD > uninflamed IBD > control tissue (p=5.6e-07).