Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 1

RORt supports in vitro T cell development and TH17 differentiation.

(a) Flow cytometer analyzing the expression of CD24 and TGF by CD8+ thymocytes as in Fig.1a. (b) Flow

cytometer analysis of CD4 and CD8 in ex vivo development of untransduced GFP- cells from CD4-CD8-RORt-

/- thymocytes transduced with retrovirus expressing various RORt shown in Fig. 1c. (c) Flow cytometer

analysis of various RORt expression in CD4-CD8-RORt-/- thymocytes transduced with retrovirus expressing

various RORt and ex vivo development for three days. (d) Flow cytometer analysis of RORt expression in

WT and RORt-/- CD4+ T cells differentiated under TH17 priming conditions for three days. (e) Flow cytometer

analysis of IL-17A+ cells in untransduced GFP- cells from RORt-/- CD4+ T cells transduced with retrovirus

expressing various RORt as shown in Fig. 1e. (f) Immunoblot analysis of various RORt expression in cells shown in Fig. 1e. (g) Flow cytometer analysis of ex vivo thymocyte development (top panel) and TH17

differentiation of RORt-/- cells retrovirally expressing indicated RORt mutant. (h) Immunoblot of various RORt

expression in RORt-/- CD4+ T cells transduced with retrovirus expressing indicated RORt. Full-length gel imagines (f,h), Supplementary Figure 8. Data are from one experiment representative of three experiments (a-h).

Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 2

RORtM cannot induce genes encoding molecules critical for TH17 differentiation.

(a) Flow cytometer identifying IL-17+ cells among naive RORt-/- CD4+ T cells retrovirally expressing indicated

RORt polarized under TH17 conditions for three days. (b) Immunoblot analysis of RORt and RORt-Flag

Nature Immunology: doi:10.1038/ni.3832

expression in cells shown in a. (c) ChIP-seq mapped RORt DNA-binding peaks at Il1r, Il17re, Rorc and Ccr6

loci in RORt-/- CD4+ T cells retrovirally reconstituted with EV-GFP, RORt or RORtM and polarized under TH17

conditions. Full-length gel imagines (b), Supplementary Figure 8. Data are from one experiment

representative of three experiments (a,b), or pooled from three experiments (c).

Nature Immunology: doi:10.1038/ni.3832

Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 3

Generation of RortM/M mice.

(a) Strategy for generation of RORtM/M mice. To ensure that RORtM/M is expressed under the control of

endogenous RORt gene locus, a knock-in strategy was used to replace wild type (WT) exon 4 containing the coding sequence for S92L93 without disrupting other DNA sequence. Exon 4 containing S92L93 was replaced by exon with S92A/L93A point mutations via gene targeting vector. A neomycin resistant gene (Neo) for selection of homologous recombination was inserted into the intron between exon 3 and exon 4, so it did not disrupt coding sequence. Neo was also flanked by Frt sites which allow deletion of Neo by flp recombinase

(Neo). A diphtheria toxin (DTA) gene cassette was added to select against cells without homologous recombination. (b) PCR analysis of WT allele (left panel), Neo containing allele resulted from recombination

(middle panel), and Neo deleted allele (Neo) after crossing with Frt mice (right panel). (c) Sequencing

analysis of expression of RORt with S92A/L93A mutations in thymocytes of RORtM/M mice. Figures b and c are representative of nine mice.

Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 4

RORtM supports thymocyte development in vivo.

(a) Flow cytometer analysis of RORt expression in thymocytes of indicated mice (n=3 per genotype). (b)

Flow cytometer analysis of CD44 and CD25 in CD4-CD8-CD3- thymocytes from indicated genotypes of mice

(n=5 per genotype). (c) Total number of CD4+, CD8+ and CD4+CD8+ thymocytes averaged from mice of each

indicated genotype of mice (n=5). **P < 0.01 (ANOVA). Data are from one experiment representative of three

experiments (a,b), or pooled from three experiments (c; plotted as in Fig.4a).

Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 5

RortM/M mice have defective TH17 differentiation.

(a) Flow cytometer analysis of surface CD62L and CD44 on CD4+ and CD8+ T cells from indicated genotypes

of mice (n=5 per genotype). (b) Quantification of results shown in c. (c) Flow cytometer analysis of IL-4+ cells

among naive CD4+ cells differentiated under TH2 priming conditions for three days. Right panel is the

quantification of the results (n=5 per genotype). (d) Flow cytometer analysis of Foxp3+ cells in naive CD4+ cells

Nature Immunology: doi:10.1038/ni.3832

differentiated under Treg priming conditions for three days. Right panel is the quantification of the results (n=5

per genotype). *P < 0.05 (ANOVA). Data are from one experiment representative of three experiments (a and

left panels of c,d) , or pooled from three experiments (mean ± s.e.m (in b) or plotted as in Fig. 4a (in right

panels of c, d)).

Nature Immunology: doi:10.1038/ni.3832

Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 6

RortM/M mice are resistant to EAE.

(a and b) Gating strategy for flow cytometric analysis CNS-infiltrated lymphocytes (CD45+) at the peak of the

disease after EAE induction in mice described in Fig. 5d (a) and 6b (b). (c) Flow cytometer analysis of

indicated cytokines in CD4+ T cells from draining lymph nodes (LN) (top panel) or spleens (bottom panel) of

indicated EAE-induced mice described in Fig. 6a (n=5 per genotype). (d) qPCR analysis of Foxp3 mRNA

levels in lymphocytes obtained from EAE-induced WT and RORtM/M mice (n=5 per genotype). (e) Flow

cytometer analysis of ILC3 cells in mesenteric lymph nodes (mLN) of indicated genotypes of mice (n=3 per

genotype). (f) Number of ILC3 cells from mLN shown in e. Each symbol (d) represents an individual mouse.

Data are from one experiment representative of three experiments (a,b,c,e), or pooled from five mice (mean ±

s.e.m (in d), or plotted as in Fig. 4a (in f)).

Nature Immunology: doi:10.1038/ni.3832

Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 7

RORtM interferes with the ubiquitination of RORt at Lys69.

(a) Immunoprecipitation (IP) detection of interaction between various RORt and SRC1 in HEK 293T cells

transfected with indicated expression plasmids. (b) RORt-luciferase reporter activity in HEK 293T cells

transfected with RORt expression plasmid in the presence (black bars) or absence (open bars) of SRC1 for

24 hrs. (c) Immunoblot of ubiquitinated various RORt at K69 in HEK 293T cells transfected with expression

plasmids for indicated RORt-K69, together with HA-tagged ubiquitin. RORt-K69, RORt have all lysines mutated to arginines except lysine 69. Indicated amino acids, 91-95, 151-155, 156-160, 161-165, were

mutated to alanines in RORt-K69. (d) Flow cytometer analysis of RORt protein levels in WT and RORtM/M CD4+ T cells differentiated under TH17 conditions for three days, followed by treatment with protein synthesis

inhibitor cycloheximide (CHX) for indicated times. (e) Degradation rate of WT and RORtM based on data

shown in d. (f) Immunoprecipitation (IP) detection of interaction between various RORt and SRC1 in HEK 293T cells transfected with indicated expression plasmids. ** P < 0.05 (ANOVA). Full-length gel imagines (c,f), Supplementary Figure 8. Data are from one experiment representative of three experiments (a,c,d,f), or pooled from three experiments (b,e; mean ± s.e.m).

Nature Immunology: doi:10.1038/ni.3832

Supplementary Figure 8

Full-length images of immunoblot analyses.

Nature Immunology: doi:10.1038/ni.3832

Supplementary Table 1: Quantitative RT-PCR primers

Il17a Int 1 forward ACATGAGTGCCGACAAACAACGGGT

reverse AGTCAGTGGGCTGCTGAATGACCCC

Il23r Pro forward GCATTTGCTTCAGGCTTTTACCTATG

reverse GGCAGCTCACTTTCAGTAATCTGGG

Bcl2l1 forward TCAGAGCAAAAGCAAGAGCA

reverse GAGGAGGAGGGACGTTTTGT

2310007L24Rik forward AAACCAAGTCACCCCAGCTT

reverse ATGGGGTTTCCCAGACTACC

Hbb forward GCTCTGGGTACTCCCTCTGA

reverse GCAAATGTGTTGCCAAAAAG

il17a forward TTTAACTCCCTTGGCGCAAAA

reverse CTTTCCCTCCGCATTGACAC

Ifng forward ATGAACGCTACACACTGCATC

reverse CCATCCTTTTGCCAGTTCCTC

Csf2 forward GGCCTTGGAAGCATGTAGAGG

reverse GGAGAACTCGTTAGAGACGACTT

Il3 forward GGGATACCCACCGTTTAACCA

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reverse AGGTTTACTCTCCGAAAGCTCTT

Il1rn forward GCTCATTGCTGGGTACTTACAA

reverse CCAGACTTGGCACAAGACAGG

Lrmp forward

TTTGGACGTGACAAGAGGGTG reverse

ACTCTGGTCCTCGTTCATTCTG

Gzmb forward

CCACTCTCGACCCTACATGG reverse

GGCCCCCAAAGTGACATTTATT

Il9 forward

ATGTTGGTGACATACATCCTTGC reverse

TGACGGTGGATCATCCTTCAG

Il10 forward

GCTCTTACTGACTGGCATGAG reverse

CGCAGCTCTAGGAGCATGTG

Maf forward

GGAGACCGACCGCATCATC reverse

TCATCCAGTAGTAGTCTTCCAGG

Il22 forward

ATGAGTTTTTCCCTTATGGGGAC reverse

GCTGGAAGTTGGACACCTCAA

Ccl4 forward

TTCCTGCTGTTTCTCTTACACCT reverse

CTGTCTGCCTCTTTTGGTCAG

Nature Immunology: doi:10.1038/ni.3832

Ccl5 forward

GCTGCTTTGCCTACCTCTCC reverse

TCGAGTGACAAACACGACTGC

Casp1 forward

ACAAGGCACGGGACCTATG reverse

TCCCAGTCAGTCCTGGAAATG

Ccl3 forward

TTCTCTGTACCATGACACTCTGC reverse

CGTGGAATCTTCCGGCTGTAG

Icos forward

ATGAAGCCGTACTTCTGCCAT reverse

CGCATTTTTAACTGCTGGACAG

Il7r forward

GCGGACGATCACTCCTTCTG reverse

AGCCCCACATATTTGAAATTCCA

Stat4 forward

TGGCAACAATTCTGCTTCAAAAC reverse

GAGGTCCCTGGATAGGCATGT

Lagls3 forward

AGACAGCTTTTCGCTTAACGA reverse

GGGTAGGCACTAGGAGGAGC

Lag3 forward

CTGGGACTGCTTTGGGAAG reverse

GGTTGATGTTGCCAGATAACCC Il6st forward

CCGTGTGGTTACATCTACCCT

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reverse CGTGGTTCTGTTGATGACAGTG

Ikzf3 forward

CTGAATGACTACAGCTTGCCC reverse

GCTCCGGCTTCATAATGTTCT

Il4 forward

GGTCTCAACCCCCAGCTAGT reverse

GCCGATGATCTCTCTCAAGTGAT

Nature Immunology: doi:10.1038/ni.3832