Three of these four significantly different cell subsets were enriched for GPR1517,18, demonstrating that trafficking molecule expression by blood leukocytes facilitates complex disease differentiation. patterns of cell localization in disease. Our findings highlight the importance of gut tropic leukocytes in blood circulation and reveal that blood-based immune signatures differentiate clinically relevant subsets of IBD. test (CD remission vs. HC, t?=?12.43, df?=?4412; CD remission vs. UC remission, t?=?14.12, df?=?4406; UC flare vs. HC, t?=?6.994, df?=?4403; UC flare vs. UC remission, t?=?8.621, df?=?4397). Sample sizes: CD flare?=?13; CD remission?=?11; UC flare?=?10; UC remission?=?10; HC?=?12. c Features distinguished all CD and UC. Statistics: BH FDR-corrected unpaired two-tailed Students test using Morpheus (see the Methods section; CCR9+GPR15+CD56+ B cells, t?=?2.58; 47+CCR1+CD56+ plasmablasts, t?=?2.74). Sample sizes: CD?=?23, UC?=?18. d Features differentiating CD and UC recognized by hypothesis-driven assessments. Statistics: unpaired two-tailed Students test (Basophils [% of live singlets]: all CD vs. UC, t?=?2.57, df?=?42; CD vs. UC flare, t?=?3.34, df?=?21; CD flare vs. HC, t?=?2.79, df?=?23; CD flare vs. remission, t?=?2.87, df?=?22; all UC vs. HC, t?=?3.88, df?=?30; UC flare vs. HC, t?=?4.02, df?=?20; UC flare vs. remission, t?=?6.91; df?=?18. Basophils [median pCREB]: all CD vs. UC, t?=?2.53, df?=?42; CD vs. UC flare, t?=?3.17; df?=?21. pDCs [% Rabbit polyclonal to ANAPC10 of DCs]: all CD vs. UC, t?=?2.61, df?=?42; CD vs. UC flare, t?=?2.97, df?=?21; UC flare vs. remission, S18-000003 t?=?4.03; df?=?18. 47+ mDCs [% of mDCs]: all CD vs. UC, t?=?2.07, df?=?39; CD vs. UC flare, t?=?3.30, df?=?19; CD flare vs. remission, t?=?2.33, df?=?21. Effector memory CD4 T cells [median pCREB]: all CD vs. UC, t?=?2.27, df?=?42; CD vs. UC flare, t?=?3.13, df?=?21; CD flare vs. remission, t?=?2.92; df?=?22. IgD?CD27? B cells [% of CD19+CD20+]: all CD vs. UC, t?=?2.15, df?=?42; CD vs. UC flare, t?=?2.77, df?=?21; UC flare vs. remission, t?=?3.47, df?=?18; UC flare vs. HC, t?=?5.05, df?=?20). Sample sizes: all CD?=?24; CD flare?=?13; CD remission?=?11; all UC?=?20; UC flare?=?10; UC remission?=?10; HC?=?12 (23, 13, 10, 18, 8, 10, and 12, respectively, for 47+ mDCs). Center lines?=?mean; whiskers?=?standard deviation. Source data are provided as a Source Data file Table 1 Summary of demographic and clinical characteristics of the patients patients)?Left-sided73?Pan colonic123?Proctitis10Biopsies collected per patient (test (cohort 1 age, t?=?0.5036, df?=?42; cohort 2 age, t?=?0.3607, df?=?10; cohort 1 age at onset, t?=?1.496, df?=?42; cohort 2 age at onset, t?=?0.5421, df?=?10; cohort 1 disease duration, t?=?1.155, df?=?42; cohort 2 disease duration, t?=?0.1947, df?=?10; cohort 2 biopsies collected per patient, t?=?2.712, df?=?10) and two-sided Fishers exact test (disease status; sex; reported extra-intestinal manifestations; tissue state). Sample sizes are shown in the table for each comparison. (a?=?median [range]; CD?=?Crohns S18-000003 disease; UC?=?ulcerative colitis; HC?=?healthy control) We analyzed viably cryopreserved leukocytes from blood and tissue by CyTOF using panels with surface and intracellular antigens (Supplementary Table?3; Supplementary Figs?1, 2). We used four trafficking molecules to identify gut tropic cells: 47, a pan-gut-trafficking molecule and target of the therapeutic antibody vedolizumab13; CCR1, a trafficking molecule recognized in GWAS studies and a marker of activity in CD15,16; CCR9, a lymphocyte trafficking S18-000003 molecule associated with small intestine tropism13; and GPR15, a T cell?trafficking molecule that we and others showed to be important for trafficking to the colon13,17,18. While our CyTOF panels included phosphoproteins and functional markers, we found in pilot studies that cell activation was unnecessary to resolve differences in phospho-signaling between sample groups. Trafficking receptor expression patterns in tissue and blood shed light on local and peripheral immune responses since little is known about leukocyte trafficking to the gut in human, especially in the context of disease. Blood leukocytes demonstrate increased heterogeneity in CD We conducted targeted analysis of CyTOF data by manually gating and calculating median protein expression levels to compile 2208 parameters per sample, as well as unbiased analysis using viSNE, CITRUS, and Spade algorithms. Coefficients of variance (CVs) for each parameter were used as a proxy for disease group populace diversity19, supporting clinical observations that CD includes more heterogenous disease manifestations than UC (Fig.?1b). Samples.