Supplementary Figure 4: Pathogenic mechanisms of TRM cells in T cell transfer colitis.
(a) Functional annotation analysis of previously published whole genome transcript analysis in WT and DKO NKT cells (Mackay et al. Science 352, 459–463) with Ingenuity Pathway Analysis (Qiagen). Using the “Diseases and Functions” mode the function “Recruitment” (subordinate to the function “Cell Movement”) was analyzed. Boxes are sized according to the respective -log(p) value and colored according to the z-score (right) assigned to the respective function. Fisher’s Exact test. (b) Expression of Cxcl3 mRNA in LPMCs from Rag1-/- mice transferred with WT, Hobit KO and DKO cells as determined by qPCR. n = 27 from three independent experiments. (c) Correlation of CD69 mRNA expression with mRNA expression of CCL3 (n = 37) and CCL4 (n = 34) as determined by qPCR in intestinal tissue from patients with IBD. Spearman’s R and regression lines are indicated; * P < 0.05. (d) mRNA expression of CCL3 and CCL4 in CD3+CD69+ vs. CD3+CD69- T cells isolated from colon lamina propria as determined by qPCR. Pairwise normalization to the expression in CD3+CD69+ T cells was performed. n = 6. (e, f) Expression of IL-2 in mRNA from LPMCs of RAG1–/– mice after transfer of WT, Hobit KO and DKO cells as determined by qPCR (e) and in supernatants from anti-CD3/CD28-stimulated LPMCs as determined by multiplex assay (n = 20 from two independent experiments). (g) Left panels: mRNA expression of Gzmb and Prf1 in LPMCs from the colon of Rag1-/- mice after transfer of WT, Hobit KO and DKO cells as determined by qPCR (n = 27 from three independent experiments). Right panel: Granzyme B concentration in the supernatants of anti-CD3/CD28-stimulated LPMCs from such mice as determined by ELISA (n = 30 from three independent experiments). (h) Culture of organoids in pure crypt culture medium (CCM) or with 25 % supernatant from WT T cells or from DKO T cells. Left panels: Representative bright-field and fluorescence microscopy images of organoid architecture and dead dye (red)/nuclei dye (blue) intensity, respectively; right panels: quantification of cell death. n = 24. (i) Immunohistochemistry staining for EpCAM (red) and TUNEL (green) in colon tissue from Rag1-/- mice after transfer of WT, Hobit KO or DKO cells. Left: Representative images, white arrows highlight EpCAM+TUNEL+ apoptotic epithelial cells. Right: Quantification of EpCAM+TUNEL+ cells per high power field (HPF). n = 21 from two independent experiments. Bars: 50 µm (upper row), 25 µm (lower row). (d, i) Center values – mean; error bars – s.e.m., (b, e-h) Boxplots – median with upper and lower quartile, whiskers – minimum and maximum. (b, d-i) * P < 0.05, ** P < 0.01, *** P < 0.001, one-way ANOVA with Newman-Keuls post-hoc testing (b, e-g, i) or paired (d) or unpaired (h) two-tailed student’s t-test.
