Supplementary Information

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1 Supplementary Information Vedolizumab is associated with changes in innate rather than adaptive immunity in patients with inflammatory bowel disease Sebastian Zeissig, Elisa Rosati, C. Marie Dowds, Konrad Aden, Johannes Bethge, Berenice Schulte, Wei Hung Pan, Neha Mishra, Maaz Zuhayra, Marlies Marx, Maren Falk-Paulsen, Anne Strigli, Claudio Conrad, Dörthe Schuldt, Anupam Sinha, Henriette Ebsen, Sabin-Christin-Kornell, Susanna Nikolaus, Alexander Arlt, Dieter Kabelitz, Mark Ellrichmann, Ulf Lützen, Philip Rosenstiel, Andre Franke, Stefan Schreiber 1

2 Fig. S1 A B Total T cells All patients Remission No remission C Week Week D Figure S1: Relative frequency and activation of lamina propria T cells in patients treated with infliximab (A) or vedolizumab (B- D). (A) Relative frequency of lamina propria CD3+ T cells in individual patients (indicated by individual symbols) before and two weeks after treatment with infliximab. (B- C) Immunohistochemical quantification (B) and representative images (C) of lamina propria CD3+ T cells. In B, data are shown for all patients (left column), patients who achieved clinical remission (middle column), and patients who failed to achieve clinical remission (right column). The upper row shows relative T cell abundance expressed as percentage of lamina propria mononuclear cells. The middle row shows absolute T cell counts per high power field (HPF) and the lower row shows absolute mononuclear cell counts per HPF. The size bar in (C) represent 5 μm. (D) Relative frequency of lamina propria CD9+ and CD5+ T cells in individual patients before and weeks after treatment with vedolizumab. Lines show the median of all patients at the respective time point. Statistical significance was determined using the Wilcoxon rank sum test.

3 Fig. S Figure S: Changes in the relative frequency of lamina propria cells upon treatment with vedolizumab. Relative frequency of the indicated immune cell subsets in individual patients before and at weeks of treatment with vedolizumab. Lines show the median of all patients at the respective time point. Statistical significance was determined using the Wilcoxon rank sum test. 3

4 Fig. S3 A B Figure S3: Changes in the relative frequency of intraepithelial (A) and peripheral blood (B) activated T cells upon treatment with vedolizumab. Relative frequency of CD9 + T cell subsets in individual patients (indicated by individual symbols) before and at weeks of treatment with vedolizumab. Lines show the median of all patients at the respective time point. Statistical significance was determined using the Wilcoxon rank sum test.

5 Fig. S4 A Time (weeks) Time (weeks) B Time (weeks) Ulcerative colitis Crohn's disease TRAV38 /DV8 TRAV13 1 TRAV9 TRAV1 TRAV1 3 TRAV9/DV5 TRAV17 TRAV1 TRAV1 1 TRAV7 TRAV TRAV3 TRAV13 TRAV8 TRAV1 TRAV 1 TRAV TRAV TRAV38 1 TRAV5 TRAV3/DV TRAV4 TRAV1 TRAV5 TRAV/DV4 TRAV8 3 TRAV39 TRAV1 1 TRAV4 TRAV TRAV41 TRAV3/DV7 TRAV3 TRAV8 1 TRAV TRAV19 TRAV34 TRAV35 TRAV1 TRAV8 4 TRAV4 TRAV18 TRAV11 TRAV8 7 TRAV7 TRAV9 1 TRAV8 Ulcerative colitis Time (weeks) Ulcerative colitis Crohn's disease TRBJ1 TRBJ1 1 TRBJ 7 TRBJ 1 TRBJ 5 TRBJ 3 TRBJ TRBJ1 5 TRBJ TRBJ 4 TRBJ1 3 TRBJ1 TRBJ Crohn's disease TRAJ43 TRAJ3 TRAJ TRAJ33 TRAJ1 TRAJ4 TRAJ4 TRAJ37 TRAJ54 TRAJ39 TRAJ TRAJ5 TRAJ53 TRAJ9 TRAJ17 TRAJ3 TRAJ49 TRAJ8 TRAJ48 TRAJ5 TRAJ TRAJ13 TRAJ34 TRAJ15 TRAJ45 TRAJ4 TRAJ31 TRAJ3 TRAJ57 TRAJ47 TRAJ8 TRAJ7 TRAJ44 TRAJ1 TRAJ1 TRAJ1 TRAJ TRAJ9 TRAJ11 TRAJ38 TRAJ3 TRAJ5 TRAJ41 TRAJ5 TRAJ7 TRAJ4 TRAJ3 TRAJ18 TRAJ4 TRAJ Fig. S4: Colonic Vα (A), Jα (B) and Jβ (C) gene usage at different time points before and during vedolizumab treatment. The relative abundance of each gene is shown for each sample. Different patients are divided by black lines. For visualization purposes, genes are hierarchically clustered using the Ward.D agglomeration method. Time (weeks) C Time (weeks)

6 Fig. S5 A TCR αv gene usage B TCR βv gene usage UC patients Controls CD patients CD patients UC patients..1. Controls C TRAV1 3 TRAV9/DV5 TRAV TRAV1 1 TRAV1 TRAV17 TRAV9 TRAV1 TRAV1 TRAV34 TRAV39 TRAV4 TRAV8 4 TRAV8 TRAV8 7 TRAV9 1 TRAV18 TRAV7 TRAV11 TRAV35 TRAV1 TRAV3 TRAV8 3 TRAV4 TRAV1 1 TRAV3/DV7 TRAV5 TRAV TRAV41 TRAV TRAV19 TRAV/DV4 TRAV8 1 TRAV4 TRAV5 TRAV3 TRAV13 TRAV7 TRAV 1 TRAV38 1 TRAV TRAV TRAV1 TRAV8 TRAV3/DV TRAV38 /DV8 TRAV13 1 TCR αj gene usage D TRBV8 TRBV18 TRBV9 1 TRBV7 TRBV7 9 TRBV7 7 TRBV5 5 TRBV1 1 TRBV5 1 TRBV3 1 TRBV1 1 TRBV5 8 TRBV7 4 TRBV1 TRBV 7 TRBV11 TRBV7 TRBV4 3 TRBV5 TRBV TRBV 3 TRBV4 TRBV1 4 TRBV7 1 TRBV1 3 TRBV11 3 TRBV11 1 TRBV3 1 TRBV1 5 TRBV 4 TRBV TRBV1 TRBV5 4 TRBV TRBV13 TRBV4 1 TRBV 1 TRBV7 3 TRBV4 1 TRBV TRBV9 TRBV7 8 TRBV3 TRBV 5 TRBV19 TRBV15 TRBV1 3 TRBV5 1 TRBV 1 TRBV7 TCR βj gene usage TRAJ3 TRAJ49 TRAJ TRAJ43 TRAJ4 TRAJ TRAJ3 TRAJ9 TRAJ5 TRAJ41 TRAJ5 TRAJ7 TRAJ TRAJ11 TRAJ3 TRAJ1 TRAJ38 TRAJ1 TRAJ8 TRAJ1 TRAJ7 TRAJ4 TRAJ18 TRAJ4 TRAJ9 TRAJ1 TRAJ33 TRAJ39 TRAJ3 TRAJ13 TRAJ17 TRAJ TRAJ44 TRAJ5 TRAJ47 TRAJ34 TRAJ54 TRAJ45 TRAJ4 TRAJ3 TRAJ31 TRAJ48 TRAJ57 TRAJ37 TRAJ TRAJ53 TRAJ4 TRAJ5 TRAJ15 TRAJ8 UC patients Controls CD patients CD patients UC patients TRBJ1 4 TRBJ1 TRBJ 4 TRBJ TRBJ1 3 TRBJ 7 TRBJ1 1 TRBJ 1 TRBJ1 TRBJ 3 TRBJ TRBJ 5 TRBJ1 5 Controls E P=.4 P=. P=. P= Diagnosis Healthy Ulcerative colitis Crohn's disease Fig. S5: Colonic TRAV (A), TRBV (B), TRAJ (C), and TRBJ (D) gene usage in IBD patients and controls. The relative abundance for each gene is shown for IBD patients before treatment and for healthy individuals. Every row represents one individual. For visualization purposes, genes were hierarchically clustered using the Ward.D agglomeration method. E. V genes which showed significant differences in usage between IBD patients and healthy individuals. TRBV5 1 TRAV38 /DV8

7 Fig. S A P=.9 P=.7 P=.9 P=.8 Gini index B..1. TCR α TCR α TCR β Diagnosis Healthy Ulcerative colitis Crohn's disease.4.3 Gini index. Diagnosis Ulcerative colitis Crohn's disease.1. C TCR β.4 Gini index Weeks Diagnosis Ulcerative colitis Crohn's disease Fig. S: Colonic TCR Gini index at baseline (A) and during vedolizumab treatment (B-C). A. TCRα and TCRβ Gini inequality index in IBD patients and controls at baseline. B-C. TCRα (B) and TCRβ (C) Gini index in individual patients during vedolizumab treatment. Statistical significance was determined using the Wilcoxon rank sum test. 7

8 Fig. S7 Fig. S7: Genes downregulated in expression in response to infliximab (IFX) or vedolizumab (VDZ) among patients who achieved clinical remission. Venn diagram of genes downregulated in response to either IFX or VDZ treatment in patients who achieved clinical remission. 8

9 Fig. S8 A B C D E F G H Fig. S8: Vedolizumab does not affect the relative abundance of T and B cell populations. Relative abundance of the indicated populations among colonic leukocytes as determined by RNAbased cell deconvolution using CIBERSORT. Individual patients are shown by individual symbols. Lines show the median of all patients at the respective time point. Statistical significance was determined using the Wilcoxon rank sum test. 9

10 Fig. S9 A B C D E F Fig. S9: Infliximab and the relative abundance of macrophage populations. Relative abundance (A-B, D-E) of the indicated macrophage populations among colonic leukocytes and M1/M macrophage ratio (C, F) as determined by RNA-based cell deconvolution using CIBERSORT. Data are shown for all infliximab-treated patients (A-C) as well as for patients who did or did not achieve clinical remission in response to infliximab (D-F). Individual patients are shown by individual symbols. Lines show the median of all patients at the respective time point. Statistical significance was determined using the Wilcoxon rank sum test. 1

11 Fig. S1 A B Fig. S1: Effects of vedolizumab (A) and infliximab (B) on the relative abundance of the indicated myeloid populations. Relative abundance of the indicated myeloid populations among colonic leukocytes as determined by RNA-based cell deconvolution using CIBERSORT in patients treated with infliximab (A) or vedolizumab (B). Individual patients are shown by individual symbols. Lines show the median of all patients at the respective time point. Statistical significance was determined using the Wilcoxon rank sum test. 11

12 Table S1. Overview of patients and analyses. All patients used in the study are listed with a patient ID and subdivided by disease and medication. Disease CD UC Medication Vedolizumab Infliximab Vedolizumab Infliximab Patient ID Immunophenotyping TCR Endo microscopy RNAseq Indiumlabelling 1 x x x x x x 3 x x x x x 4 x x x x x 5 x x 7 x x x 8 x 9 x 1 x x x 11 x x 1 x 13 x x 15 x x x 1 x 17 x 18 x x x 19 x x x 1 x x x x 3 x x x 4 x x x 5 x x 7 x x 8 x 9 x x 3 x x x 31 x x 3 x x x 33 x 34 x x x 35 x 3 x 37 x 38 x Total

13 Table S. Median frequencies and interquartile ranges of lamina propria immune cell subpopulations at two and six weeks of treatment with vedolizumab. No significant differences were detected at the two time points compared to week. Week Week P-value Week P-value αβ T cells (% of total) 3.9 (1.4-4.).4 ( ).94 (n = 7) 3.7 ( ).94 (n = 7) CD4 + T cells 57.7 ( ) 5.4 ( ).9 (n = 7) 53.1 ( ).3 (n = 7) CD8 + T cells.9 ( ) (1.-.7).59 (n = 7).7 (11.4-3).9 (n = 7) CD9 + T cells 77.5 ( ) 8.7 (7-8.9).81 (n = 7) 4. ( ).94 (n = 7) CD9 + T cells (% of CD4 + T cells) 84. ( ) 84.5 ( ).35 (n = 7).1 (.-8.).9 (n = 7) CD5 + T cells 8.3 (.8-1.) 8.8 ( ) 1 (n = 4) 8.5 (.8-1.5).3 (n = 5) Central memory T cells 1. (1.5-3.) 3.4 (.4-5.8).5 (n = ) 7. ( ). (n = ) Effector memory T cells 7.9 ( ) 77.4 ( ).5 (n = ) 75. ( ) 1 (n = ) B cells (% of total) 1.3 (.5-1.).8 (.8-.8).81 (n = 5).9 (.8-.3) 1 (n = ) Memory B cells 3 (% of B cells) 1.7 ( ).9 (1.9-.8) 1 (n = 5) 5. ( ).84 (n = ) Plasma cells 4 (% of total). (.-.4).1 (.1-.8).81 (n = 5).1 (.1-.4).5 (n = ) NK cells 5 (% of total). (.-.7). (.1-.5).13 (n = 5). (.1-.4).1 (n = ) 1 CD45RA - CCR7 + CDL + ;; CD45RA - CCR7 - CDL - ;; 3 CD19 + CD7 + CD38 lo ;; 4 CD19 + CD7 + CD38 hi ;; 5 CD3 - CD5 + 13

14 Table S3. Relative frequencies of T cells among epithelial/intraepithelial cells of the colon before (week ) and after weeks of treatment with vedolizumab or infliximab. Median and interquartile range is shown. P values are based on the Wilcoxon signed rank test. Cell type Vedolizumab Infliximab (n = 1) αβ T cells (% of total) CD4 + T cells CD8β + T cells CD9 + T cells CD9 + T cells (% of CD4 + T cells) CD9 + T cells (% of CD8 + T cells) γδ T cells (% of total) CD9 + T cells (% of γδ T cells) Week Week P-value Week Week P-value (n = 8). (n = 8).5 (n = 8)..94 (n = 7) (n = 1).5 (n = 1) (n = 7).74 (n = 8).4 (n = 8)

15 Table S4. Relative frequencies of immune cell populations in peripheral blood mononuclear cells before (week ) and after weeks of treatment with vedolizumab or infliximab. Significant differences between time points are shown in bold (based on Wilcoxon signed rank test). αβ T cells (% of total) Cell type Vedolizumab Infliximab (n = 1) CD4 + T cells CD8 + T cells CD9 + T cells CD9 + T cells (% of CD4 + T cells) CD9 + T cells (% of CD8 + T cells) CD5 + T cells Week Week P-value Week Week P-value effector memory 1.3 T cells 1 B cells (% of total) memory B cells 9.1 (% of B cells) plasma cells.4 (% of total) NK cells. (% of total) (n = ).74 (n = 8).84 (n = ).44 (n = ).84 (n = ) 1 (n = ) 1 CD45RA - CCR7 - CDL - ;; CD19 + CD7 + CD38 lo ;; 3 CD19 + CD7 + CD38 hi ;; 4 CD3 - CD (n = 1).5 (n = 1) 1 (n = 1). (n = 1).11 (n = 1)..84 (n = 8).1.3 (n = 1).3 (n = 1) 1 (n = 1).85 (n = 1) 15

16 For Table S5-S1 please refer to original Excel files, which can be found at the Gut Reviewer Center and can also be accessed and downloaded via: Table S5: TCR sequences shared between different individuals. The table provides a list of clonotypes which were found in at least individuals (either IBD patients or healthy controls). Information on nucleotide (column B) and amino acid (column C) sequence is provided. The number of individuals in which the sequence was found is shown in column D. Columns E and F show the relative representation of this TCR in healthy controls and IBD patients, respectively. Table S: Genes differentially expressed in response to vedolizumab treatment. Significantly (P<.5) upregulated and downregulated genes in response to vedolizumab treatment (week vs. week ) in all patients, irrespective of remission status. lfcse indicates the standard error of log fold change. padj is the P value adjusted using the Benjamini-Hochberg method. Table S7. Genes differentially expressed in response to vedolizumab treatment in patients who achieved clinical remission. The table provides a list of significantly (P<.5) upregulated and downregulated genes in response to vedolizumab treatment (week vs. week ) of patients who achieved remission status at week. lfcse indicates the standard error of log fold change. padj is the P value adjusted using the Benjamini-Hochberg method. Table S8. Overlapping differentially expressed genes in all patients and patients that achieved clinical remission. The table provides a list of genes significantly (P<.5) upregulated and downregulated in response to vedolizumab treatment (week vs. week ) and overlapping between all patients and those patients who achieved remission until week. lfcse indicates the standard error of log fold change. padj is the P value adjusted using the Benjamini-Hochberg method. 1

17 Table S9. Genes regulated in response to infliximab treatment. The table provides a list of genes significantly (P<.5) upregulated and downregulated in response to infliximab treatment (week vs. week ). lfcse indicates the standard error of log fold change. padj is the P value adjusted using the Benjamini-Hochberg method. Table S1. Downregulated genes indicating unifying or overlapping regulation in response to infliximab or vedolizumab. The table provides a list of genes significantly (P<.5) upregulated and downregulated in response to infliximab treatment (week vs. week ). lfcse indicates the standard error of log fold change. padj is the P value adjusted using the Benjamini-Hochberg method. 17