doi:1.138/nature1554 a TNF-α + in CD4 + cells [%] 1 GF SPF 6 b IL-1 + in CD4 + cells [%] 5 4 3 2 1 Supplementary Figure 1. Effect of microbiota on cytokine profiles of T cells in GALT. Frequencies of TNF-α (a) or IL-1 (b) producing T cells from spleen, pooled axillary and inguinal lymph nodes (), mesenteric lymph nodes (), Peyer s patches () and small intestinal lamina propria () of GF and SPF RR mice, measured by flow cytometry. Data represent the percentage of cytokine producing cells in the gated CD4 + population. Each circle represents an individual mouse and bars depict mean ± s.e.m. n = 6-11 mice per group. Data were pooled from 4 independent experiments. WWW.NATURE.COM/NATURE 1
CD4 + cells [%] 1 GF SPF 6 *** CD8 + cells [%] 3 1 TCRγδ cells [%] 3 1 1 3 3 B cells [%] 6 CD11b + cells [%] 1 CD11c + cells [%] 1 NKp46 + cells [%] 3 1 Gr1 + cells [%] 3 1 FoxP3 + in CD4 + cells [%] 15 1 5 Supplementary Figure 2. Immune cell composition in lymphoid organs of GF and SPF RR mice. 2 WWW.NATURE.COM/NATURE
Frequencies of CD4 + T cells, CD8 + T cells, TCRγδ + T cells, B2 + B cells, CD11b + macrophages, CD11c + dendritic cells, NKp46 + NK cells, Gr1 + granulocytes and Foxp3 + Tregs from spleen,,, and small intestinal of GF and SPF RR mice determined by flow cytometry. Data represent frequencies of positive cells in the live gate with the exception of Foxp3 + cells where CD4 + gate was used. Each circle represents an individual mouse and bars are shown as mean ± s.e.m. n = 3-9 mice per group. Data were pooled from 4 independent experiments. ***P =.7 (Mann-Whitney U test). Vα8.3 high Vβ4 high in CD4 + cells [%] 1 GF SPF 6 Vα8.3 low Vβ4 low in CD4 + cells [%] 1 * 6 Supplementary Figure 3. TCR high and TCR low transgenic T cells in lymphoid tissues of GF and SPF RR mice. Frequencies of Vα8.3/Vβ4 high (TCR high ) and Vα8.3/Vβ4 low (TCR low ) T cells from spleen,,, and small intestinal of GF and SPF RR mice were determined by flow cytometry. Data represent frequencies of positive cells in the CD4 + gate. Each circle represents an individual mouse and bars are shown as mean ± s.e.m. n = 5-7 mice per group. Data were pooled from 4 independent experiments. *P =.16 (Mann-Whitney-U-test). WWW.NATURE.COM/NATURE 3
a [ 3 H] incorporation [c.p.m.], 15, 1, 5, GF SPF Ex-GF.1 1 1 1 rmog [µg/ml] [ 3 H] incorporation [c.p.m.] 15, 1, 5,..3.5.8 1. 1.3 anti-cd3 [µg/ml] b rmog 6, * * 2,5 2, ** rmog IFN-γ [pg/ml] 4, 2, IL-17 [pg/ml] 1,5 1, 5 c IFN-γ [pg/ml] 8, 6, 4, 2, GF SPF Ex-GF anti-cd3 * IL-17 [pg/ml] GF SPF Ex-GF anti-cd3 2,5 * * 2, 1,5 1, 5 GF SPF Ex-GF GF SPF Ex-GF Supplementary Figure 4. Reduced cytokine production by T cells from GF RR mice. (a) Proliferation of splenic T cells from GF, SPF and Ex-GF RR mice cultured with increasing concentrations of rmog or anti-cd3 antibody was measured by 3 [H]-thymidine incorporation. n = 3-6 per group. Data were pooled from 3 individual experiments. (b - c) Cytokine production of GF, SPF and Ex-GF RR mice splenocytes. cells stimulated for 48 h with either µg/ml rmog (b) or 1 µg/ml anti-cd3 antibody (c). IFN-γ and IL-17 levels in the supernatants were measured by ELISA. Bars depict mean ± s.e.m. n = 3-5 per group. *P <.5; **P <.1 (unpaired t test). 4 WWW.NATURE.COM/NATURE
Antibiotics Untreated 1.4% 2.4%.6% 5.6% 13.9% 1.4% 1.5% 2.% 1.1% 2.2% CFSE CFSE low CD4 + T cells [%] 24 Untreated 16 12 8 4 Antibiotics ** Supplementary Figure 5. Antibiotics treatment suppresses T cell activation induced by commensal flora. CD4 + T cells from Wt mice were labeled with CFSE and transferred into untreated or antibiotics treated SPF Wt mice. Shown are the frequencies of CFSE low cells in the CD4 + gate. Each circle represents an individual mouse and bars depict mean ± s.e.m. n = 6-12 per group. Data were pooled from 3 individual experiments. **P =.87 (Mann-Whitney U test). WWW.NATURE.COM/NATURE 5
2.4 Wt 2. RR x MOG -/- OD 5 nm 1.6 1.2.8.4. Day Day 7 Day 14 Supplementary Figure 6. Immunization of SPF RR x MOG -/- mice induces anti-mog antibodies. Similar levels of anti-mog antibodies in Wt (n = 7) and RR x MOG -/- (n = 6) mice immunized with rmog in CFA. MOG specific IgG2a antibodies were measured by sandwich ELISA in sera collected on days, 7 and 14 after immunization. Values shown are the absorbances at 5 nm. Each circle represents an individual mouse and bars depict mean ± s.e.m. Data were pooled from 2 individual experiments. 6 WWW.NATURE.COM/NATURE
a 12.6%.3% Healthy 1.1% 5.6% 36.5% 1.4%.2% Sick 8.9% SSC CD19 26.1% 63.3% CD45 CD4 b B2 DAPI CD4 DAPI Supplementary Figure 7. T and B cells infiltrate the CNS of RR mice before onset of clinical EAE. (a) CNS infiltrating cells from healthy (top) and sick (bottom) RR mice. Mice were perfused with PBS and CNS mononuclear infiltrates were isolated by percoll gradient. Distributions of cells stained with immune cell markers were determined by flow cytometry. Numbers in quadrants indicate percentage of stained cells. Data represent 3 animals analyzed in 2 independent experiments. (b) Immunofluorescent staining of brain sections from healthy RR mouse for infiltrating B (B2 + ) and T (CD4 + ) cells (CD4, green; B2, red; DAPI (cell nuclei), blue). Representative staining of >3 mice is shown. Scale bars, 25 µm. WWW.NATURE.COM/NATURE 7
a IgH MOG -GFP B cells RR or Wt mice Day Day 14 b Serum Serum IHC RR Wt GFP B2 PNA c OD 5 nm IgG1 a.5 Day.4.3.2.1 Day 14 OD 5 nm.5.4.3.2.1 IgG2a a. 1 5 1 4 1 3 1 2 Fold dilution 1 1. 1 5 1 4 1 3 1 2 Fold dilution 1 1 OD 5 nm.5.4.3.2.1. 1 6 IgG1 b 1 5 1 4 1 3 1 2 1 1 Fold dilution OD 5 nm.5.4.3.2.1. 1 6 IgG2a b 1 5 1 4 1 3 1 2 1 1 Fold dilution Supplementary Figure 8. Homing behavior of MOG specific B cells. (a) Purified IgH MOG -GFP B cells 8 WWW.NATURE.COM/NATURE
were transferred into adult RR or Wt mice (day ). Isotype switched serum anti-mog antibody levels of the donor (a allotype) and recipients (b allotype) were measured before (day ) and on day 14 after transfer. The distribution of transferred B cells (green) was documented 14 days after transfer via immunofluorescence in cln sections. (b) MOG specific B cells home to the GC of brain draining cervical lymph nodes of RR but not Wt mice. B2 (red) and PNA (blue) label GC and naïve B cells, respectively. Arrow heads indicate transferred B cells. Dotted lines define boundaries of GCs. Representative immunofluorescence staining from two independent experiments consisting of 4-5 mice per group is shown. PNA (blue) and B2 (red) label GC and naïve B cells, respectively. Magnification: x. (c) Transgenic MOG specific B cells do not spontaneously isotype switch after transfer into Wt mice. Error bars represent s.e.m. n = 3-5 mice per group. Data were pooled from 2 independent experiments. Supplementary Table 1: Activated MOG-specific T cells induce EAE in both GF and SPF mice. TCR transgenic T cells were activated in vitro with rmog for 2 days and transferred into GF and SPF mice and observed for clinical signs of EAE. Experiment Recipient EAE affected/total Day of onset (Mean ± SD) Experiment-1 SPF 4/4 4 ± GF 4/4 6.25 ±.5 Experiment-2 SPF 3/3 5 ± GF 4/4 5.75 ±.5 WWW.NATURE.COM/NATURE 9