1. Volume 150, Issue 2, Pages 477-487.e9 (February 2016)
Association Between Response to Etrolizumab and Expression of Integrin αE and Granzyme A in Colon Biopsies of Patients With Ulcerative Colitis 
Gaik W. Tew, Jason A. Hackney, Deena Gibbons, Christopher A. Lamb, Diana Luca, Jackson G. Egen, Lauri Diehl, Jeff Eastham Anderson, Severine Vermeire, John C. Mansfield, Brian G. Feagan, Julian Panes, Daniel C. Baumgart, Stefan Schreiber, Iris Dotan, William J. Sandborn, John A. Kirby, Peter M. Irving, Gert De Hertogh, Gert A. Van Assche, Paul Rutgeerts, Sharon O’Byrne, Adrian Hayday, Mary E. Keir 
Gastroenterology 
Volume 150, Issue 2, Pages e9 (February 2016)
DOI: /j.gastro
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2. Figure 1
Differences in baseline colonic biopsy gene expression between clinical remitters and nonremitters treated with etrolizumab. Distribution of curated (A) T-cell− and (B) neutrophil-associated genes in baseline colonic biopsies of anti−TNF-naïve etrolizumab-treated patients. P value was empirically calculated by counting the number of permuted gene-set statistics that were more extreme than the gene-set statistic calculated for the true sample labels. X-axis represents clinical remitter to nonremitter gene-set statistic.
Gastroenterology  , e9DOI: ( /j.gastro )
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3. Figure 2
Increased granzyme A in αE+ T cells in UC patients. (A) TCRαβ+ CD4+ and CD8+ T cells from colonic biopsies from cohort 2 were sort purified by αE expression. CD4+αE+ T cells from UC patients had increased granzyme A (GZMA) expression compared with sorted CD4+αE− T cells from UC patients and CD4+αE+ T cells from non-IBD controls. (B) GZMA and αE gene expression was significantly correlated in sorted CD4+αE+ T cells, but not sorted CD8+αE+ T cells from UC patients in cohort 2. In cohort 1, GZMA gene expression was significantly correlated with (C) αE gene expression (dotted lines indicate qPCR median cutoff) and (D) the number of αE+ cells in the epithelium and LP in colonic biopsies. (E) Representative immunofluorescence co-staining of granzyme A and αE in a colonic biopsy sample from cohort 2. ∗P < .05; ∗∗∗∗P <
Gastroenterology  , e9DOI: ( /j.gastro )
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4. Figure 3
Clinical remission and mucosal healing by baseline GZMA expression. (A) Clinical remission and (B) mucosal healing by baseline GZMA gene expression. Baseline colonic biopsy qPCR median value was used to categorize patients as GZMAhigh (greater than or equal to median) or GZMAlow (less than median). LD, loading dose; TNF, tumor necrosis factor.
Gastroenterology  , e9DOI: ( /j.gastro )
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5. Figure 4
Effect of etrolizumab on epithelial crypt-associated αE+ cells by biomarker stratification. Epithelial crypt-associated αE+ cells were counted before and after treatment with etrolizumab or placebo in cohort 1. (A) Baseline distribution of epithelial crypt-associated αE+ cells in GZMAhigh or αEhigh (greater than or equal to median) or GZMAlow or αElow (less than median) patients where baseline colonic biopsy qPCR median value was used to categorize patients. (B) Change in epithelial crypt-associated αE+ cells in GZMAhigh or αEhigh and GZMAlow or αElow patients after treatment with etrolizumab (combined dose groups) or placebo. ∗P < .05; ∗∗P < .01. Scr, screening.
Gastroenterology  , e9DOI: ( /j.gastro )
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6. Figure 5
Effect of etrolizumab on colonic tissue gene expression by granzyme A stratification. Colonic biopsy gene expression was assessed by qPCR before and after treatment with etrolizumab (combined dose groups) or placebo in cohort 1. (A) T-cell−associated genes and (B) cytokines and chemokines were reduced in colonic tissue after treatment with etrolizumab in GZMAhigh (greater than or equal to median) but not GZMAlow (less than median) patients relative to placebo. Data are presented as fold (2−ΔΔCt) group median ± median absolute deviation. ∗P < .05 vs placebo. Scr, screening.
Gastroenterology  , e9DOI: ( /j.gastro )
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7. Supplementary Figure 1
Differences in baseline colonic biopsy gene expression between anti−TNF-naïve etrolizumab-treated clinical remitters and nonremitters. Distribution of curated DC-, monocyte-, and B-cell−associated genes in baseline colonic biopsies of anti−TNF-naïve etrolizumab-treated patients. P value is empirically calculated by counting the number of permuted gene-set statistics that were more extreme than the gene-set statistic calculated for the true sample labels. X-axis represents clinical remitter to nonremitter gene-set statistic.
Gastroenterology  , e9DOI: ( /j.gastro )
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8. Supplementary Figure 2
Top differentially expressed genes between anti-TNF−naïve etrolizumab-treated clinical remitters and nonremitters at baseline. Dendrogram showing unsupervised clustering analysis of top differentially expressed genes between anti-TNF−naïve etrolizumab-treated clinical remitters and nonremitters, as measured by qPCR in baseline colonic biopsies from all etrolizumab-treated patients in cohort 1. Each gene is color coded to reflect the leukocyte subsets with the highest expression of the gene of interest based on gene expression database of flow cytometry sort-purified cells.27 Genes shown in gray are not listed in the database or have low expression across all leukocyte subsets.
Gastroenterology  , e9DOI: ( /j.gastro )
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9. Supplementary Figure 3
Sort strategy and qPCR profiling of αE+ and αE− T cells. (A) Biopsies from UC patients and healthy controls enrolled in cohort 2 were sorted based on size and single cells (top row), then live CD45+ TCRαβ+ (middle row) were further sorted into CD4+αEβ7+ and CD4+αEβ7− cell populations and CD8+αEβ7+ and CD8+αEβ7− cell populations (bottom row). Healthy control is shown. (B) qPCR of sort purified cells for the sort parameters αE, β7, CD4, and CD8.
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10. Supplementary Figure 4
Correlation of αE and effector molecule gene expression in flow cytometry sort-purified CD4+αE+ cells in UC patients. Sort-purified CD4+αE+ cells from cohort 2 were evaluated for gene expression of effector molecules. The correlation in gene expression between effector molecules perforin, Fas ligand with granzyme A, and αE in sort purified CD4+αE+ cells in UC patients is shown.
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11. Supplementary Figure 5
Proportion of patients with mucosal healing by biomarker αE stratification. Baseline colonic biopsy qPCR median value was used as cutoff to categorize patients as αEhigh (greater than or equal to median) or αElow (less than median).
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12. Supplementary Figure 6
Distribution of baseline colonic gene expression of granzyme A by dose group and anti-TNF status. Granzyme A gene expression relative to GAPDH expression in baseline colonic biopsies from cohort 1. The dotted line indicates the median.
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13. Supplementary Figure 7
Distribution of baseline gene expression of αE, granzyme B, CXCL1, and CCL5 by biomarker status and treatment group. Baseline colonic biopsy gene expression was assessed by qPCR in cohort 1. Baseline colonic biopsy qPCR median value was used as cutoff to categorize patients as granzyme Ahigh or αEhigh (greater than or equal to median) or granzyme Alow or αElow (less than median). (A) αE, granzyme B, CCL5, and CXCL1 gene expression relative to glyceraldehyde-3-phosphate dehydrogenase (phosphorylating) (GAPDH) expression in baseline colonic biopsy by granzyme A status. (B) CCL5 expression relative to GAPDH in baseline colonic biopsy by αE status. NS, not significant; ∗P < .05; ∗∗P < .01; ∗∗∗P < .001.
Gastroenterology  , e9DOI: ( /j.gastro )
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14. Supplementary Figure 8
Effect of etrolizumab on CCL5 colonic biopsy gene expression by αE stratification. Colonic biopsy gene expression was assessed by qPCR before and after treatment with etrolizumab or placebo in cohort 1. Baseline colonic biopsy qPCR median value was used as cutoff to categorize patients as αEhigh (greater than or equal to median) or αElow (less than median). Decreased CCL5 expression in colonic biopsy in αEhigh patients treated with etrolizumab, but not αElow patients relative to placebo was observed. Data presented as fold (2−ΔΔCt) group median ± median absolute deviation. P values correspond to the comparison between etrolizumab and placebo. ∗P < .05 (vs placebo). SCR, screening.
Gastroenterology  , e9DOI: ( /j.gastro )
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15. Supplementary Figure 9
Baseline and post-treatment histologic score by biomarker stratification. Histologic activity was evaluated using the modified Geboes score30 for biopsies taken at the screening visit and week 10 in cohort 1. Baseline colonic biopsy qPCR median value was used as cutoff to categorize patients as granzyme Ahigh or αEhigh (greater than or equal to median) or granzyme Alow or αElow (less than median). Change in modified Geboes score before and after treatment with etrolizumab or placebo by baseline biopsy (A) αE or (B) granzyme A gene expression levels. ∗P < .05; ∗∗P < .01.
Gastroenterology  , e9DOI: ( /j.gastro )
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