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1 doi: 1.138/nature89 IFN- (ng ml ) Splenocytes NS IFN- (ng ml ) 6 4 Lymph node cells NS Nfkbiz / Nfkbiz / Nfkbiz / Nfkbiz / IL- (ng ml ) 3 1 Splenocytes IL- (ng ml ) *** ** Lymph node cells Supplementary Figure 1 IFN-γ and IL- production in splenocytes and lymph node cells from immunized mice. IFN- and IL- production in splenocytes and lymph node cells after restimulation with anti-cd3 and anti-cd8. Error bars denote mean ± s.e.m. **P <.1; ***P <.5; NS, not significant. 1

2 doi: 1.138/nature NS NS 18 NS TNF- (ng ml ) 1..5 IL-6 (ng ml ) 1 6 * Nfkbiz / None LPS None CpG None LPS None CpG cdcs pdcs cdcs pdcs Supplementary Figure LPS- or CpG DNA-induced TNF- and IL-6 production in or Nfkbiz / DCs. cdcs or pdcs isolated from or Nfkbiz / mice were stimulated with LPS or CpG DNA. Twenty-four hours later, TNF- and IL-6 production was assessed by ELISA. Error bars denote mean ± s.e.m. *P <.5; NS, not significant.

3 doi: 1.138/nature89 Nfkbiz / Rag / Rag / Rag / 1.8%.% 6.% 5.5% CD4 CD3 Supplementary Figure 3 Similar reconstitution efficiency in mice that received or Nfkbiz / CD4 T cells. The populations of CD3! CD4 + T cells in the spleen of Rag / mice reconstituted with or Nfkbiz / CD4 T cells. Representative data from three mice 1 days after immunization. 3

4 doi: 1.138/nature89 5 NS BrdU + (% of CD4 + ) Nfkbiz -/- (5 5.%) (7.6%) Supplementary Figure 4 No difference in proliferation between and Nfkbiz / CD4 + T cells in vivo after MOG immunization. Rag / mice reconstituted with or Nfkbiz / CD4 T cells were administered with BrdU 1 days after MOG immunization, and then the proliferation of CD4 T cells was measured by BrdU incorporation. NS, not significant. 4

5 doi: 1.138/nature Runx1 Ahr Irf4 Socs3 Batf () () () () () 16 Ccr6 3 Ccl 18 S1pr1 4 Il6ra 4 Il6st () () () 3 1 () 3 1 () 3 Il1r.5 Ilrg 1 Il1r1 1.5 Il1rap 1 () () () 1..5 () Nfkbiz / Supplementary Figure 5 The mrna expression level of genes related to cells in Nfkbiz / T cells. The mrna expression of genes related to development (Runx1, Ahr, Irf4, Socs3 and Batf), as well as genes related to the migration of cells (Ccr6, Ccl and S1pr1) and -related cytokine receptors (Il6ra, Il6st, Il1r, Ilrg, Il1r1 and Il1rap), in or Nfkbiz / CD4 T cells activated under - and -polarizing conditions were analyzed by quantitative RT-PCR. CCR6 expression was slightly decreased (but not statistically significant) in Nfkbiz / T cells under -polarizing conditions. The resistance of Nfkbiz / mice to EAE is mainly due to a defect in the early development of cells, although a contribution of a decrease in CCR6-mediated migration of cells to the central nervous system cannot be completely excluded. Error bars denote mean ± s.e.m. ignificant. 5

6 doi: 1.138/nature89 IL-6 min 3 min 6 min 18 min Control IgG phospho-stat3 Nfkbiz / phospho-stat3 Supplementary Figure 6 IL-6-induced phosphorylation of STAT3 in Nfkbiz / T cells. and Nfkbiz / naive CD4 T cells were stimulated with anti-cd3 and anti-cd8 in the presence of IL-6 for the indicated periods, and then stained with anti-phospho-stat3 or control IgG. 6

7 doi: 1.138/nature89 a Thymus Spleen Lymph nodes.6% 9.% 8.4% Nfkbiz / Foxp3.7% 8.1% 7.9% CD5 b Neutral TGF- IL-3 IL-1 TGF- IL-3.9% 43.% 8.8% 19.1% 3.4% 1.1% 49.9% 14.3% 16.9% 3.9% Nfkbiz / Foxp3 Supplementary Figure 7 Targeted disruption of the Nfkbiz gene has no effect on the development of natural T reg and inducible T reg cells. a, CD4 CD5 Foxp3 natural T reg cells in the thymus, spleen or lymph nodes of mice and Nfkbiz / mice. b, Foxp3 expression in or Nfkbiz / CD4 T cells activated under the indicated conditions. There was no defect in the development of inducible T reg cells from Nfkbiz / naive CD4 T cells. Under -polarizing conditions, there was no difference in Foxp3 expression between and Nfkbiz / T cells. 7

8 doi: 1.138/nature89 a Relative Nfkbiz mrna level RPMI IMDM * * None IL-3 IL-3 anti-il None IL-3 IL-3 anti-il b M IB (L) IB (D) LPS-stimulated M 14 bp 818 bp IL-3 14 bp 818 bp c Relative Nfkbiz mrna level RPMI IMDM Supplementary Figure 8 Nfkbiz mrna is highly expressed in cells. a, Quantitative RT-PCR analysis of Nfkbiz mrna expression in CD4 T cells activated under different -polarizing conditions. cells expressed a high level of Nfkbiz mrna when cultured in RPMI containing IL-6 and TGF-. The expression of Nfkbiz mrna was further increased if cultured in the presence of anti-il or in IMDM. The primer set used in Figs 1a, f and g and Supplementary Fig.8a was designed to detect both IB (L) and (S). b, The mrna expression of IB (D) in bone marrow-derived macrophage (M), LPS-stimulated M, cell subsets (top). RT-PCR was performed using a primer set [detecting both (D) and (L) isoforms] designed to amplify the region between nucleotides 141 and 154 of the cdna for IB (L) (bottom). IB (D) expression in cells was found to be under the detection limit of RT-PCR. c, The expression of mrna for IB (L) was analyzed using an IB (L)-specific primer set by quatitative RT-PCR. Error bars (a and c) denote mean ± s.e.m. *P <.5. 9 IL-3 * IL-3 anti-il it reg * 9 Naive IL-3 IL-3 anti-il 8

9 doi: 1.138/nature89 a (kda) Naive 1 Anti-IB it reg 9 IB(L) IB(S) ns b (kda) 75 5 None IL-6 TGF- IL-3 Anti-IB IL-3 IB(L) IB(S) ns Relative expression (IB (L)/ -actin) Anti--actin *** Relative expression (IB (L)/ -actin) Anti--actin ** ** c Relative expression (IB (L)/ -actin) (kda) None RPMI IL-3 IL-3 anti-il- RPMI IL-3 Anti-IB Anti--actin * None * IMDM IL-3 IL-3 anti-il- ** IB(L) IB(S) ns d Relative expression (IB (L)/ -actin) (kda) Myd88 / Ila / Anti-IB Anti--actin *** Rorc / Stat3 flox/ Lck-Cre IB(L) IB(S) ns Supplementary Figure 9 Protein expression level of the IB splicing variants in cells. a, Protein expression level of the IB splicing variants [IB (L) (79 kda), IB (S) (69 kda) and IB (D) (57 kda)] in naive CD4 T cells, cell subsets and inducible T reg (it reg ) cells. Consistent with the results of RT-PCR analysis (Fig. 1a and Supplementary Fig. 8c), IB expression was moderately increased in 9 cells. b, Effects of the cytokines on the protein expression of the IB splicing variants in CD4 T cells. c, Protein expression level of IB in CD4 T cells activated under different -polarizing conditions. cells expressed high levels of IB (L) and (S) when cultured in RPMI containing IL-6 and TGF-. The expression of IB was further increased if cultured in the presence of anti-il or in IMDM. d, Protein expression level of IB in CD4 T cells derived from Myd88 -/-, Ila -/-, Rorc -/- or Stat3 flox/- Lck-Cre mice. ns, non-specific band. The expression of IB in cells was more obvious at the protein level than at the mrna level, suggesting the involvement of the post-transcriptional regulatory mechanisms that were reported in previous studies 1,. Error bars denote mean ± s.e.m. *P <.5; **P <.1; ***P <.5; NS, not significant. 9

10 doi: 1.138/nature89 pmx 1.% 3.8% pmx-ib (L).1% 14.% IL- 4.6% 54.7% 5.6% 33.3% EGFP Supplementary Figure 1 Ectopic expression of IB enhances IL- production in a cell-intrinsic manner. Retroviral expression of IB (L) led to a high production of IL-. EGFP- nontransduced cells did not exhibit IL- production. 1

11 doi: 1.138/nature89 pmx pmx-rela pmx-crel pmx-p5 1.3%.%.% 1.7% pmx pmx-rela pmx-crel pmx-p5 8.8% 9.3% 9.4% 8.8% pmx pmx- IB (L) IL-.6%.5%.5%.6% FSC 19.% 9.% 14.3% 6.9% Supplementary Figure 11 Effects of the NF-B subunits on the ability of IB to induced IL- production. Naive CD4 T cells were co-transduced with an NF-B subunit (RelA, crel or p5)-expressing retrovirus (IRES-EGFP) and IB (L)-expressing retrovirus (IRES-hCD), and then IL- production in EGFP hcd cells was assessed. FSC, forward scatter. 11

12 doi: 1.138/nature89 a Neutral IL-3 IL-3 IL-1 TNF-.% 5.3% 5.7% 1.% 1.%.4%.3% 1.6%.1% 8.% 8.8%.5% Nfkb1 / IL- 3.%.5%.4% 1.5% b IFN- Nfkb1 / Ilf Il Il3r Il Neutral IL-3 Neutral IL-3 Neutral IL-3 Neutral IL-3 Supplementary Figure 1 Normal differentiation of Nfkb1 / naive CD4 T cells into cells. a, Intracellular expression of IFN- and IL- in or Nfkb1 / CD4 T cells activated under - or -polarizing conditions. b, Ilf, Il1, Il3r and Il mrna expression in or Nfkb1 / CD4 T cells activated under - or -polarizing conditions. Error bars denote mean ± s.e.m. 1

13 doi: 1.138/nature89 a pmx pmx-ib (L) 4.4%.3% 6.1% 19.3% Nfkb1 / IL- EGFP b Ilf Il1 Il3r pmx pmx-ib (L) pmx pmx-ib (L) pmx pmx-ib (L) Nfkb1 / Supplementary Figure 13 NF-B p5 is not essential for IB-mediated regulation of development. a, Effects of retroviral expression of IB (L) on IL- production in or Nfkb1 / CD4 T cells activated under -polarizing conditions. b, Effects of retroviral expression of IB (L) on Ilf, Il1 and Il3r mrna expression in or Nfkb1 / CD4 T cells activated under -polarizing conditions. Error bars denote mean ± s.e.m. 13

14 doi: 1.138/nature89 Control Control.9% Control IB(L) 3.5% Control IB(S) 1.6% RORt Control.9% RORt IB(L) 48.6% RORt IB(S) 3.% ROR Control 16.4% ROR IB(L) 38.8% ROR IB(S) 3.9% (IRES-EGFP) (IRES-hCD) (+ anti-tgf-) IL- FSC Supplementary Figure 14 Effects of overexpression of the ROR nuclear receptors and IB in the presence of anti-tgf-. Intracellular expression of IL- in CD4 T cells transduced with IB (IRES-hCD) and RORt or ROR (IRES-EGFP) after activation under -polarizing conditions with anti-tgf-. Even in the presence of anti-tgf-, the ROR nuclear receptors and IB synergistically promoted development. When the anti-tgf- was added to the cultures, it was found that RORt or ROR induced IL- production significantly, possibly because of an inhibition of Foxp3 expression 3. FSC, forward scatter. 14

15 doi: 1.138/nature89 a 3 1 NS Il9 NS Nfkbiz / IL-3 b pmx- pmxpmx RORt ROR 1.6% 51.5% 34.%.5% 1.% 16.% IL-1 Nfkbiz / 9.6% 54.8% 4.3% 3.9% 9.5% 13.% IL-9 Nfkbiz / IL- EGFP Supplementary Figure 15 The defect in the ROR nuclear receptor-induced IL- production in Nfkbiz / T cells is independent of IL-1 or IL-9 expression. a, Il9 mrna expression was normal in Nfkbiz / T cells under -polarizing conditions. Error bars denote mean ± s.e.m. NS, not significant. b, Even when 8 ng ml 1 IL-1 or ng ml 1 IL-9 were added into the cultures, IL- production by RORt or ROR overexpression was impaired in Nfkbiz / CD4 T cell. 15

16 doi: 1.138/nature89 5 *** *** *** *** IL- cells (% of EGFP cells) ** *** Rorc / Rora sg/sg Rorc / Rora sg/sg Rorc / Rora sg/sg pmx pmx- IB (L) pmx- IB (S) Supplementary Figure 16 Effects of retroviral expression of IB on the generation of IL--producing cells in, Rorc / or Rora sg/sg CD4 T cells. Bar graphs represent the frequency of IL--producing cells among EGFP cells (see Fig. 4c for the dot plots). The data are shown as mean ± s.e.m of three independent experiments. **P <.1; ***P <.5. 16

17 doi: 1.138/nature89 a CNS TK pro Luc Luc Il-Luc Il CNS-Luc : ROR response element : IB response element Luc Il 4371-Luc Luc Il 1547-Luc b Relative luciferase activity 1 +1 Il CNS-Luc IB (L) IB (S) IB (D) RORt ROR RORt ROR c Relative luciferase activity Il 4371-Luc IB (L) IB (S) d Relative luciferase activity Il 1547-Luc IB (L) IB (S) RORt ROR RORt ROR RORt ROR RORt ROR Supplementary Figure IB promotes the CNS enhancer activity together with RORt and/or ROR. a, Schematic of a series of reporter constructs containing the regions upstream of the mouse Ila transcriptional initiation site. Il CNS-Luc carries the CNS region upstream of a minimal thymidine kinase promoter (TK pro) linked to the luciferase (Luc) gene. Il 4371-Luc and Il 1547-Luc contain the 4371 bp and the 1547 bp promoter region of the mouse Ila gene, respectively. Il 4371-Luc and Il 1547-Luc lack the CNS region. b, c, d, Effects of the IB variants, RORt and/or ROR on the reporter activity of Il CNS-Luc (b), Il 4371-Luc (c) or Il 1547-Luc (d). Error bars (b-d) denote mean ± s.e.m.

18 doi: 1.138/nature89 a CNS ISE1 ISE ISE TK pro Luc Il CNS-Luc TK pro Luc Il CNS mut1-luc TK pro Luc Il CNS mut-luc TK pro Luc Il CNS mut3-luc : ROR response element : IB response element b Relative luciferase activity 1 Il CNS-Luc Il CNS mut1-luc Il CNS mut-luc Il CNS mut3-luc - IB (S) RORt RORt IB (S) ROR ROR IB (S) RORt ROR RORt ROR IB (S) Supplementary Figure 18 Requirement of ISE1, an IB response element, for IB-mediated CNS enhancer activity. a, Three IB response elements were located in the CNS region (ISE1, -635 to -6339; ISE, to -4786; ISE3, to -4445). Il CNS mut1-luc, Il CNS mut-luc and Il CNS mut3-luc carry a mutation of ISE1, ISE and ISE3, respectively. b, Effects of IB (S), RORt and/or ROR on the reporter activity of Il CNS-Luc, Il CNS mut1-luc, Il CNS mut-luc and Il CNS mut3-luc. Error bars denote mean ± s.e.m. 18

19 doi: 1.138/nature89 a Ilf -154 to kb +6kb +4kb +kb +1 -kb -4kb -6kb -8kb CNS7 Human vs Mouse Relative binding Ilf promoter -154 to -163 Relative binding CNS7-744 to Nfkbiz/ Nfkbiz/ Con IgG Anti-IB b Il1 promoter -31 to -4 +1kb +8kb +6kb Il1 +4kb -31 to -4 +kb +1 -kb -4kb Human vs Mouse Relative binding Con IgG Anti-IB.1 c Il3r Nfkbiz / -1kb +8kb +6kb +4kb +kb to to kb Human vs Mouse Rat vs Mouse Relative binding Il3r promoter -13 to -14 Relative binding Il3r promoter to -143 Nfkbiz/ Nfkbiz/ Con IgG Anti-IB Supplementary Figure 19 Recruitment of IB to the Ilf, Il1 and Il3r promoter in cells. a, rvista alignment plot displaying the sequence similarity between the human and mouse Ilf loci (left). The transcriptional initiation site was designated as +1. Putative IB response sites were found within the proximal promoter region of the Ilf gene (-154 to -163) and the CNS7 region located -7 kb from the transcriptional start site (-744 to -7433). ChIP analysis revealed binding of IB to the CNS7 region, but not the proximal promoter, in cells (right). b, rvista alignment plot displaying the sequence similarity between the human and mouse Il1 loci (left). Recruitment of IB in cells to the conserved proximal promoter region of the Il1 gene (-31 to -4) (right). c, rvista alignment plot displaying the sequence similarity between the human, rat and mouse Il3r loci (left). Two putative IB response sites were found within the promoter region of the mouse Il3r gene (-13 to -14 and to -143). IB was recruited only to the distal element in the mouse Il3r promoter, which is conserved between mice and rats (right). It is also possible that the decreased expression of Il3r mrna in Nfkbiz / T cells is partly due to the impaired expression of IL-1. Error bars denote mean ± s.e.m. 19

20 doi: 1.138/nature89 Supplementary Table 1 Primers used for RT-PCR. Gene Forward primer Reverse primer Gapdh 5 -AACTTTGGCATTGTGGAAGG-3 5 -GGATGCAGGGATGATGTTCT-3 Nfkbiz # 5 -TCCAGAATGTCCCAGTCTCC-3 5 -GAGTCTCAGTTTGGGGTGGA-3 Nfkbiz (LD) 5 -CGACACCGGCTACCTGTC-3 5 -CACCAAGGTTTACCAGATCTTG-3 Nfkbiz (L) 5 -CGCTCAACCTGGCTTACTTC-3 5 -GGGCTCAACTTGAGGGCG-3 Rorc 5 -TGCAAGACTCATCGACAAGG-3 5 -AGGGGATTCAACATCAGTGC-3 Rora 5 -GAACACCTTGCCCAGAACAT-3 5 -AGCTGCCACATCACCTCTCT-3 Ilf 5 -CAAAACCAGGGCATTTCTGT-3 5 -ATGGTGCTGTCTTCCTGACC-3 Il1 5 -CGCCTCCTGATTAGACTTCG-3 5 -GCCCCTTTACATCTTGTGGA-3 Il 5 -TCATCGGGGAGAAACTGTTC-3 5 -CATGTAGGGCTGGAACCTGT-3 Il3r 5 -GGTCTTCTTGGCCATCATGT-3 5 -GCCACTTTGGGATCATCAGT-3 Il9 5 -ATCACGTGTCCGTCCTTTTC-3 5 -TCTTCATGGTCGGCTTTTCT-3 Runx1 5 -TACCTGGGATCCATCACCTC-3 5 -GACGGCAGAGTAGGGAACTG-3 Ahr 5 -AGCAGCTGTGTCAGATGGTG-3 5 -CTGAGCAGTCCCCTGTAAGC-3 Irf4 5 -GCAGCTCACTTTGGATGACA-3 5 -CCAAACGTCACAGGACATTG-3 Socs3 5 -AGCTCCAAAAGCGAGTACCA-3 5 -TGACGCTCAACGTGAAGAAG-3 Batf 5 -CCAGAAGAGCCGACAGAGAC-3 5 -GAGCTGCGTTCTGTTTCTCC-3 Ccr6 5 -TCCAGGCAACCAAATCTTTC-3 5 -GATGAACCACACTGCCACAC-3 Ccl 5 -CGACTGTTGCCTCTCGTACA-3 5 -CACCCAGTTCTGCTTTGGAT-3 S1pr1 5 -TTGCCACCCCAGATCTATTC-3 5 -TTGCCACCCCAGATCTATTC-3 Il6ra 5 -CCAGGTGCCCTGTCAGTATT-3 5 -CTGGACTTGCTTCCCACACT-3 Il6st 5 -ACCAGATTCCTGTGGACGAC-3 5 -AGAATCCACATGCACAACCA-3 Il1r 5 -TGTCAATGTGACGGACCAGT-3 5 -CACGTAGTTGGAGGGTTCGT-3 Ilrg 5 -TTGCCACCCCAGATCTATTC-3 5 -TTGCCACCCCAGATCTATTC-3 Il1r1 5 -TGAAGAGCACAGAGGGGACT-3 5 -CATTGATCCTGGGTCAGCTT-3 Il1rap 5 -TTGCCACCCCAGATCTATTC-3 5 -TTGCCACCCCAGATCTATTC-3 # The primer set designed to detect mrna for both IκBζ (L) and (S) was used in Figs 1a, f and g and Supplementary Fig. 8a. Supplementary Fig. 8b. The primer set designed to detect mrna for IκBζ (L) and (D) was used in The IκBζ (L)-specific primer set used in Supplementary Fig. 8c.

21 doi: 1.138/nature89 Supplementary Table PCR Primers used for ChIP assays. Position of Gene IκBζ response element # Primer Ila -635 to Forward: 5 -CAGATGCATGCAGAACTGACT-3 (ISE1) Reverse: 5 -AAGGCTCTGGAGAGCAGACA-3 Ila to Forward: 5 -AGTCTGGCCCCTACACACAC-3 (outside CNS) Reverse: 5 -ATGGGGGACTTTTGGGATAG-3 Ilf -154 to -163 Forward: 5 -CACTTCCTGAAGGGGAATCA-3 (promoter) Reverse: 5 -GGGTGGGCTTAGAAGAGAGG-3 Ilf -744 to Forward: 5 -CTGAGTTGGGGGCTGTGTAT-3 (CNS7) Reverse: 5 -CATATCGAGGGTGTCGGACT-3 Il1-31 to -4 Forward: 5 -ACCTTGGTGAATGCTGAAAACTGG-3 Reverse: 5 -TGCGTGTGGGGGCAGGGATGGATA-3 Il3r -13 to -14 Forward: 5 -AACACTGACAAGGCAGCTCA-3 Reverse: 5 -GTCAGCAGAGCCCTGACCTA-3 Il3r to -143 Forward: 5 -CCCTCCCTGCTTGATCACTA-3 Reverse: 5 -TCAGCTTGACACAGGATGGA-3 # The numbering is relative to the transcriptional initiation site. 1

22 doi: 1.138/nature89 Supplementary References 1 Muta, T. IκB-ζ: an inducible regulator of nuclear factor-κb. Vitam. Horm. 74, (6). Watanabe, S., Takeshige, K. & Muta, T. A cis-element in the 3'-untranslated region of IκB-ζ mrna governs its stimulus-specific expression. Biochem Biophys Res Commun 356, (7). 3 Zhou, L. et al. TGF-β-induced Foxp3 inhibits cell differentiation by antagonizing RORγt function. Nature 453, 36-4 (8).