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
Nature Immunology: doi:10.1038/ni.3832
reverse CGTGGTTCTGTTGATGACAGTG
Ikzf3 forward
CTGAATGACTACAGCTTGCCC reverse
GCTCCGGCTTCATAATGTTCT
Il4 forward
GGTCTCAACCCCCAGCTAGT reverse
GCCGATGATCTCTCTCAAGTGAT
Nature Immunology: doi:10.1038/ni.3832