Supplementary information CD4 T cells are required for both development and maintenance of disease in a new model of reversible colitis

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1 Supplementary information CD4 T cells are required for both development and maintenance of disease in a new model of reversible colitis rasseit and Steiner et al.

2 .. Supplementary Figure 1 % of initial body weight H + H days after CD4 T cell transfer % weight change after treatment H + H days post anti-cd4 treatment C Histopathological score H + H naïve active disease anti-cd4

3 .. Supplementary Figure 2 colon homogenates pg/g tissue pg/g tissue TNFα IL6 d28 post infection perm. colonization IFNγ d28 post infection perm. colonization IL17 IL1α Cxcl9 G-CSF TNFα IFNγ IL17 1 d28 post infection perm. colonization d28 post infection perm. colonization pg/ml serum 6 IL6 2 IL1α Cxcl9 G-CSF 4 5 pg/ml d post infection 7d post infection 28d post infection perm.colonization 1d post infection 7d post infection 28d post infection perm.colonization 1d post infection 7d post infection 28d post infection perm.colonization 1d post infection 7d post infection 28d post infection perm.colonization C H Helicobacter typhlonius infection day1 day7 day28 + permanent H colonization

4 Supplementary Figure 3 12 % of initial body weight anti-cd4 Histopathological Score C TCRαβ 1.4 active disease active disease d11 p.d. d29 p.d..5 d11 p.d. d29 p.d. CD4 Ki67

5 . Supplementary Figure 4 Helicobacter + Helicobacter active disease active disease 11d p.d. 11d p.d. Ifng Il1a Il1b Il6 Trem1 Lcn2 Il33 Tnfa Il17a Il17e Il17f Il23p19 Il12a Il12b Il18 Il5 Il7 Il1 Il22 Il22ra Tgfb1 Tgfb3 Trem2 Ccl2 Ccl3 Cxcl1 Cxcl1 Cxcl2 Ccl2 Cxcl13 Reg3b Reg3g S1a9 Ptprc Ptx Tnfsf11 Mylk Pro inflammatory nti inflammatory Chemokines IEC related response fold induction Others pg/g tissue TNFα IFNγ IL17 IL6 IL1α ns naive H + anti-cd4 H + active disease H anti-cd4 H ns 3 ns 25 ns 3 ns naive H + anti-cd4 H + active disease H anti-cd4 H 2 1 naive H + anti-cd4 H + active disease H anti-cd4 H naive H + anti-cd4 H + active disease H anti-cd4 H 2 1 naive H + anti-cd4 H + active disease H anti-cd4 H

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7 . Supplementary Figure 6 % of initial body weight isotype control anti-cd4l Histopathological score days anti-cd4l naive isotype control anti-cd4l

8 .. Supplementary Figure 7 Thy1.2 + isotype control Thy1.2 DN anti-thy1.2 Thy1.1 DN 6 Thy1.1 CD4 CD25 C % weight change after treatment Thy1.2 anti-cd4 Thy1.1 anti-thy days post treatment D Histopathological score isotype control Thy1.1 anti-thy1.2 isotype control Thy1.2 anti-cd4

9 Supplementary information CD4 T cells are required for both development and maintenance of disease in a new model of reversible colitis rasseit and Steiner et al. Figure Legends Supplementary Figure 1: Disease kinetics of T cell transfer colitis in Helicobacter typhlonius positive (H + ) and Helicobacter typhlonius negative (H ) lymphopenic mice. () Percent body weight loss. () Percent body weight change in H + and H - mice with active disease treated with anti-cd4 antibodies (C) Colonic histopathology scores of H. typhlonius positive and H. typhlonius negative Rag -/- mice transferred with colitogenic CD4 + CD45R hi T cells during active disease (active disease) or 11 days post depletion of colitogenic CD4 T cells (anti-cd4). Supplementary Figure 2: Infection of lymphopenic mice with Helicobacter typhlonius does not promote local and systemic immune responses. () Protein expression levels of pro-inflammatory cytokines, Cxcl9, G-CSF and GM-CSF in colonic tissue homogenates or () serum of naïve Helicobacter-free mice (H - ), mice infected with H. typhlonius by oral gavage of 5x1 8 live bacteria at different time points (d1, d7, d28), naïve mice permanently colonized with H. typhlonius or H. typhlonius-colonized mice (H + ) with active colitis. (C) Presence of H. typhlonius in fecal pellets as assessed by PCR in the different groups. Supplementary Figure 3: Relapsing-remitting colitis. Helicobacter typhlonius positive Rag -/- mice were transferred with colitogenic CD4 T cells and subsequently depleted of CD4 T cells during active colitis on days 1, 13 and 16 post transfer. Long-term remission was monitored until day 3 post first anti-cd4 treatment (i.e. day 4 post transfer). () Weight loss. () Histopathological score of mice with active disease, 11 days post CD4 T cell depletion (p.d.) or during relapse phase on day 29 p.d. (=day 39 post T cell transfer) (C) Representative FCS plots illustrating CD4 T cell frequencies of CD45 + cells and Ki67 expression of CD4 T cells in the peripheral blood of mice with active disease, 11 days p.d. or during relapse phase on day 29 p.d.. Data shown are representative for 2 independent experiments with n=4. Supplementary Figure 4: Comparison of colonic gene expression profiles in Helicobacter typhlonius positive and Helicobacter typhlonius negative mice with active disease or in remission from colitis. () Heat-map of colonic gene expression profiles of H. typhlonius positive (H + ) and H. typhlonius negative (H + ) Rag -/- mice with active colitis and 11 days post anti-cd4 depletion (p.d). RN values were normalized to the endogenous control genes Gapdh and β2-microglobulin and presented as fold induction/reduction over CD4 + CD45R lo T cell transferred and subsequently anti- CD4 treated controls. () ead-based multiplex assay for protein levels of TNFα, IFNγ, IL17, IL6 and IL1α in colon homogenates. Data with n=4-6 mice per group are shown. ns: not significant.

10 Supplementary Figure 5: Increased proliferation rate of colonic epithelial cells in response to mucosal healing. Immunohistochemistry for Ki67 on paraffin embedded tissue sections from colonic tissue of mice with active disease or upon induction of remission from colitis (days 1, 3, 5 and 11 post anti-cd4 depletion (p.d.)). Photographs are representative for n=3 mice per group. Scale bar: 1µm. Supplementary Figure 6: locking CD4L-CD4 interactions in mice with established disease does not promote remission from colitis: H. typhlonius positive (H + ) Rag -/- mice with active colitis ( 1-15% weight loss associated with clinical signs of colitis) were either treated with an isotype control antibody or anti-cd4l at days 13 and 14 and clinical and histopathological parameters of colitis were assessed. () Percent body weight loss. () Colonic histopathological scores of naïve, isotype control- and anti-cd4l-treated mice. Data are representative of 2 independent experiments with n=3-5 mice per treatment group. Supplementary Figure 7: Colitogenic CD4 T cells are critical for maintenance of disease in the preserved presence of CD4+ innate lymphoid cells. Thy1.1 H + Rag -/- mice were transferred with colitogenic Thy1.2 CD4 + CD45R hi T cells to induce active colitis. Selective depletion of CD4 T cells to induce remission from colitis was achieved by anti-thy1.2 antibody treatment. Representative FCS plots of Thy1.1- and Thy1.2 positive cells in the mesenteric lymph nodes of () isotype control treated or () anti-thy1.2 treated mice. CD4 surface expression of transferred colitogenic T cells (green) or resident Thy1.1 innate lymphoid cells (blue) that co-express CD25 are shown. DN: double negative cells. Cells were gated as singlet, live, lineage- (CD11c, Ly6G, CD19) and autofluorescence negative cells. (C) Percent body weight change in mice with active disease treated with anti-thy1.2 or anti CD4 antibodies. (D) Colonic histopathological scores of mice with active disease treated with isotype control antibodies, anti-thy1.2 or anti-cd4 as a control. Data are representative of two independent experiments with n=3-5 per group.

11 Supplementary materials and methods Culture of Helicobacter typhlonius and infection of mice H. typhlonius was purchased from the culture collection of the University of Göteborg (CCUG) and cultured according to the provided protocol under CCUG T ( Rag -/- mice were gavaged with 5x1 8 CFU H. typhlonius. Genomic DN was extracted from colonic content as previously described (Turnbaugh et al., 29). PCR conditions for Helicobacter spp. based on 16S rrn sequences were adapted from previously described protocols (Fox, 1994; attles, 1995, Shames,1995) using the following primer sequences (PHFwd: 5 -GCT TG CG GGT TC C-3 ; PHRev: 5 -GT TTT CC CCT C CC-3 ). Immunhistochemistry for Ki67 Immunohistochemistry was performed on paraffin embedded tissue sections from colonic tissues of mice using primary antibodies against Ki67 (Dako, clone TEC-3) and isotype matched control (rat-antimouse IgG2a) followed by secondary biotinylated rabbit-anti-rat antibody staining (Dako, E468).