Fig 2: TCR- and Cytokine-Activated MAIT Cells Possess Distinct Transcriptional Profiles(A–C) Venn diagrams showing genes that are significantly differentially modulated (p < 0.05, fold change > 4) in TCR (T)-, cytokine (C)-, or TCR and cytokine (TC)-treated CD8+ MAIT cells compared with untreated (UT) MAIT cells of three healthy individuals. The cytokine (C) stimulation consisted of a cocktail of 4 cytokines: IL-12 (2 ng/mL), IL-18 (50 ng/mL), IL-15 (25 ng/mL), and TL1A (100 ng/mL). Genes with significantly altered expression levels (A) are divided into two sets: those are that are upregulated upon stimulation (B) and those that are downregulated upon stimulation (C).(D) Heatmap showing 1,594 significantly differentially expressed transcripts (p < 0.05, fold change > 4) between TCR/C/TC-stimulated and UT CD8+ MAIT cells among the same three healthy individuals.(E) Visualization of the CD8+ MAIT cell transcripts elicited by differential stimulations in the subspace of the first principle components (PCs). Each colored circle represents a sample and is color coded in accordance with the conditions with which cells were stimulated, as illustrated on the right-hand side of the graph.(F–K) Volcano plots to visualize differentially expressed transcriptional profiles of activated CD8+ MAIT cells stimulated in different ways. Each point represents a single gene, and genes expressed at significantly higher or lower levels between the compared conditions are depicted, respectively, in the upper-right or upper-left corner of each plot. Genes discussed in the text are highlighted in blue (tissue repair associated) or in red (inflammation associated). The gene expression of untreated MAIT cells was compared to (F) T-, (G) C-, or (H) TC-stimulated MAIT cells. Further, gene expression in those cells was also compared directly between the different stimulation conditions: (I) T- to C- stimulation, (J) T- to TC-stimulation, and finally (K) C- to TC-stimulation.Data were acquired from three donors in one experiment.See also Figure S4 and Tables S1, S2, and S3.

Fig 3: Atsttrin inhibits TL1A activity. A, B. Atsttrin inhibits TL1A-activated gene expressions of βigH3 and C1qTNF3 in THP-1 cells. Total RNA was extracted from THP-1 cells treated with 100 ng/ml of TL1A in the presence of various dose of Atsttrin, and then was reverse-transcribed to cDNA, expression level of βigH3 and C1qTNF3 was examined by quantitative real time PCR. C. Atsttrin inhibits TL1A-enhanced osteoclastogenesis. RAW264.7 cells were treated with 100 ng of TL1A and 35 ng/ml of RANKL in the presence of various dose of Atsttrin (as indicated), TRAP staining was then performed. D. Quantitative assay of osteoclastogenesis. Cells, treated as described in C, were washed twice with 0.9% sodium chloride; osteoclast activity was determined using 50 mM PNPP as substrate, the absorbance was measured at 540 nm. *p<0.05, **p<0.01, ***p<0.001. E, F. Atsttrin prevented body weight loss and reduced bleeding score in DSS-induced colitis. Mice challenged with 3% DSS were treated with either PBS or Atsttrin, and the body weight (E) and the bleeding score (F) were monitored daily.

Fig 4: Gut-Derived MAIT Cells Show a Broadly Similar Response Pattern toward Innate and Adaptive Stimuli Compared with Their Blood-Derived CounterpartsRepresentative plots showing the percentage of cells positive for the indicated effector molecules as a proportion of CD8+ MAIT cells.(A–C) Proportions of blood-derived (n = 32) CD8+ MAIT cells producing IFN-γ (A), TNF-α (B), or GrB (C) following overnight stimulation with combinations of suboptimal concentrations of IL-12 and IL-18, TL1A, and αCD3/CD28 beads as indicated.(D–F) Proportions of gut-derived (n = 13) CD8+ MAIT cells producing IFN-γ (D), TNF-α (E), or GrB (F) stimulated in the same way as in (A)–(C).(G and H) Expression of IFN-γ, TNF-α, and GrB by blood-derived (G, n = 7) or gut-derived (H, n = 6) CD8+ MAIT cells 20 h after coculture with THP1 cells alone or THP1 cells incubated with 25 fixed E. coli bacteria per cell.Data were acquired from multiple donors as indicated in 3–5 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests (A–F), two-way ANOVA (G), or Wilcoxon tests (H). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.001.See also Figure S3.

Fig 5: TL1A Enhances the Activation of MAIT Cells Suboptimally Stimulated with IL-12 and IL-18CD8+ T cells were enriched from healthy peripheral blood mononuclear cells (PBMCs) and stimulated overnight with different combinations of cytokines: IL-12 at 2 ng/mL, IL-18 at 50 ng/mL, IL-15 at 25 ng/mL, and TL1A from 0.01 to 100 ng/mL as indicated.(A–C) Proportions of CD8+ MAIT/CD161+ or CD161− cells producing IFN-γ (A), TNF-α (B), or CD69 (C) following overnight stimulation with suboptimal concentrations of IL-12 and IL-18, plus varying concentrations of TL1A.(D) Representative histograms showing the expression of IFN-γ, TNF-α, GrB, and CD69 by MAIT cells after stimulation with different combinations of cytokines.(E–H) Frequency of MAIT cells expressing IFN-γ (E), TNF-α (F), GrB (G), and CD69 (H) upon stimulation with the indicated cytokines.Data were acquired from seven donors in 2–3 experiments. Error bars represent means ± SEM. Differences among conditions were analyzed by Friedman tests with Dunn’s multiple comparison tests. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.See also Figure S1.