Supp. Fig. 1 a APC b APC ICOS ICOS TCR CD28 mir P TCR CD28 P T cell Tolerance Roquin WT SG Icos mrna T cell Autoimmunity Roquin M199R SG Icos mrna www.nature.com/nature 1
Supp. Fig. 2 CD4 + CD44 low CD4 + CD44 high Spleen LNs Spleen LNs MFI of ICOS 12 9 6 3 4 3 2 1 Rc3h1 san/san Rc3h1 san/san Rc3h1 +/+ Icos +/+ Icos +/- Icos +/+ www.nature.com/nature 2
Supp. Fig. 3 a B22 + splenocytes b Total splenocytes 4.1% 59.3%.5% GL7 CXCR5 29.6% 1.6% Fas CD4 www.nature.com/nature 3
Supp. Fig. 4 a Cell number (x 1 6 ) 7 6 5 4 3 2 1 Spleen LNs Weight (g) b.6.5.4.3.2.1 Spleen LNs. c 6 LN cell number (X1 6 ) 5 4 3 2 1 n=3 ICOS MFI r s =.8461 P<.1 Rc3h1 san/san Cd28 +/- Rc3h1 san/san Icos +/+ Rc3h1 san/san Icos +/- Rc3h1 san/san Icos -/- Rc3h1 +/+ Icos +/+ www.nature.com/nature 4
Supp. Fig. 5 a pr-roquin WT -IRES-GFP GFP (Roquin) ICOS MFI Percentage Hi 1 42.25 25.3% Low 2 59.69 3.8% Nil 3 69.25 43.9% huicos pr-ires-gfp Hi Low 4 5 ICOS MFI 2.5 94.69 Percentage 23.4% 27.6% Nil 6 63.15 49% huicos b Roquin level High Low Nil Calculation 42.25 (#1) 2.5 (#4) 59.69 (#2) 94.69 (#5) 69.25 (#3) 63.15 (#6) Relative expression 21% 7% 11% Protein expression (%) (normalised to empty vector) 1 5 High Low Nil Roquin level www.nature.com/nature 5
Supp. Fig. 6 GFP (Roquin) huicos FL huicos 3 UTR Hi Low Nil Hi Low Nil Roquin WT Empty vector hucd4 www.nature.com/nature 6
Supp. Fig. 7 a Protein expression (%) (normalised to empty vector) b Protein expression (%) (normalised to empty vector) 1 5 GFP (Roquin) 1 5 GFP (Roquin) huicos Roquin WT Roquin M199R * ** Hi Low Nil hucd4 Roquin WT Roquin M199R * * Hi Low Nil www.nature.com/nature 7
Supp. Fig. 8 YFP MS2 MS2 YFP (12) 5 LTR huicos FL IRES hucd4 3 LTR www.nature.com/nature 8
Supp. Fig. 9 a MTR1: 2238-2284 3 UTR-F3 (1851-2613) MTR2: 2515-2561 b Within MTR1 hsa-mir-338 hsa-mir-13 hsa-mir-17 hsa-mir-11 Within MTR2 hsa-mir-149 www.nature.com/nature 9
Supp. Fig. 1 a 3 b 3 Mature mir-11 levels (%) 2 1 Rc3h1 +/+ * * Rc3h1 san/san Pri-miR-11 levels (%) 2 1 Rc3h1 +/+ * * Rc3h1 san/san www.nature.com/nature 1
Supp. Fig. 11 Endogenous Icos mrna (%) 1 75 5 25 mir-13 mir-338 * NS Negative control mirna-13 Endogenous Icos mrna (%) 1 75 5 25 Negative control mirna-338 www.nature.com/nature 11
Supp. Fig. 12 a mmu-mir-11a ENSMUSESTT3722641 3999 b c d Endogenous neuropilin 1 mrna (%) 3 2 1 Rc3h1 +/+ * * Rc3h1 san/san Endogenous neuropilin 1 mrna (%) 25 2 15 1 5 Empty vector * * * Roquin WT Roquin M199R Endogenous neuropilin 1 mrna (%) 1 75 5 25 Negative control * * mirna-11 www.nature.com/nature 12
Supplementary Table 1 Conserved regions within huicos mrna predicted by LAGAN Region Starts Ends Length (bp) Homology #1 1(5) 853 (863) 863 75.2% #2 96 (919) 1149 (1161) 248 7.2% #3 122 (1242) 1329 (1348) 11 71.8% #4 141 (1397) 1812 (1798) 417 74.1% #5 1943 (1866) 213 (256) 191 8.6% #6 2185 (213) 2386 (2331) 29 72.7% #7 2446 (2351) 2595 (2498) 151 71.5% Conserved Regions - BC2821 (AK3827) www.nature.com/nature 13
Legends to Supplementary Figures Supplementary Figure 1. Model by which ICOS repression by Roquin inhibits autoimmunity. a, In wild-type mice, a significant proportion of Icos transcripts induced by TCR and CD28 signaling undergo degradation enhanced by Roquin acting with the mirna machinery, limiting the amount of ICOS expressed on the cell surface. After ligation by ICOSL consitutively expressed by APCs, ICOS transduces a signal that provides co-stimulation for T cell activation and differentiation while maintaining selftolerance due to limits imposed on its expression by Roquin. b, In sanroque mice, the degradation of Icos transcripts is impaired due to the M199R mutation in Roquin, resulting in overexpressed ICOS on the cell surface. Exaggerated signaling transduced by uncontrolled expression of ICOS contributes to autoimmunity. APC: antigen presenting cells; P: processing-body; SG: stress granule; mir: microrna. Supplementary Figure 2. Expression of ICOS in sanroque (Rc3h1 san/san ) ICOS +/- mice. Dots plots showing ICOS expression on naïve (CD44 low, left) and antigen-experienced (CD44 high, right) splenic or lymph node CD4 + T cells from Rc3h1 san/san Icos +/+ (red), Rc3h1 san/san Icos +/- (blue) and Rc3h1 +/+ Icos +/+ (black) mice. Each symbol represents one mouse. Lines are drawn through the mean values for each group. Supplementary Figure 3. Flow cytometric strategy for gating GC B cells and T FH cells in splenocytes. www.nature.com/nature 14
a, GC B cells were gated as Fas + GL7 high in B22 + population. b, T FH cells were gated as the CXCR5 high CD4 + population. Supplementary Figure 4. Lymphadenopathy and splenomegaly are not corrected in sanroque (Rc3h1 san/san ) Cd28 +/- mice. a, b, Total white blood cell numbers (a) and weight (b) of spleen and combined lymph nodes (LNs) of mice with the indicated genotypes. c, Correlation of total cell numbers in lymph nodes with ICOS MFI of naïve (CD44 low ) CD4 + T cells from mice with the indicated genotypes. Nonparametric Spearman correlation coefficient (r s ) and P value were calculated using the GraphPad Prism software. All mice were 12 to 14-week old C57BLxCBA H-2 kk. Each symbol represents one mouse. Supplementary Figure 5. Quantification of Roquin-mediated repression. a, Retroviral titres were determined to consistently achieve a transduction efficiency of 5-6% of NIH3T3 cells. In cells transduced with Roquin (thus GFP + ), the half with higher GFP expression was defined as the Roquin Hi population, while the half with lower GFP expression was defined as Roquin Low. b, The mean of fluorescent intensity (MFI) of ICOS in each population was normalized to the counterpart population from cells transduced with empty vector. Supplementary Figure 6. Roquin represses hucd4 expression through the 3 UTR of huicos mrna. Experiment depicted in Fig. 2e. HuCD4 expression was measured by flow cytometry in cells transduced with the retroviruses indicated. www.nature.com/nature 15
Supplementary Figure 7. Roquin M199R impairs the repression of both huicos and hucd4 in NIH3T3 cells a, b, In the experiment shown in Fig. 2f and g, the repression on huicos (a) or hucd4 (b) by Roquin WT and RoquinM 199R was compared. Data shown are mean values ± S.D. with n = 3. Statistically significant differences are marked by asterisks: Student s t-test, * P <.5, ** P <.1. Supplementary Figure 8. Plasmid expressing the transcript of huicos FL fused to 24 MS2 Binding Sites. Plasmid expressing Icos FL mrna fused to 24 binding sites for the MS2 coat protein. These MS2-binding sites allow identification of the localisation of huicos FL mrnas upon co-expression of an MS2-YFP-NLS fusion protein. Supplementary Figure 9. MiRNA target regions within huicos 3 UTR-F3 predicted by MiRanda. a, Position of MTR1 and MTR2 within 3 UTR-F3 of huicos nucleotide sequence BC2821. b, Sequences within MTR1 and MTR2 recognised by the indicated mirnas as predicted by MiRanda. Supplementary Figure 1. MiRNA-11 expression in sanroque (Rc3h1 san/san ) and wild-type (Rc3h1 +/+ ) CD4 + T cells. a, b, Naïve (CD44 low ) CD4 + T cells were sorted from spleen and lymph nodes of sanroque and wild type C57BL/6 mice. Levels of mature mirna-11 (a) were quantified using TaqMan MicroRNA assays. Levels of pri-mirna-11 (b) were quantified by measuring the levels of the transcript ENSMUSESTT3824221 www.nature.com/nature 16
using real-time RT-PCR. Data shown are mean values ± S.D. with n = 3. Statistically significant differences are marked by asterisks: Student s t-test, ** P <.1. Supplementary Figure 11. Icos mrna is repressed by ectopic expression of mirna-13 but not mirna-338. Endogenous Icos mrna levels assessed by real-time RT-PCR in EL4 cells transfected with mirna precursor negative control (1#) and hsa mirna-13 or mirna-338 precursor (Ambion) for 24 hours. The level of Icos mrna in cells transfected with mirna precursor negative control was assigned 1% and the other mrna levels were calculated relative to that value. Data shown are mean values ± S.D. with n = 3. Statistically significant differences are marked by asterisks: Student s t-test, * P <.5, NS = no significant difference. Supplementary Figure 12. Roquin represses neuropilin 1 mrna through mirna-11. a, Sequence within neuropilin 1 mrna 3 UTR (Ensembl Transcript ID: ENSMUSESTT3722641) recognised by mirna-11 as predicted by MiRanda. b, Naïve (CD44 low ) CD4 + T cells were sorted from spleen and lymph nodes of sanroque (Rc3h1 san/san ) and wild-type (Rc3h1 +/+ ) mice. Endogenous levels of neuropilin 1 mrna were measured using real-time RT-PCR. The level of neuropilin 1 mrna in wild-type mice was assigned 1% and the mrna level in sanroque mice was calculated relative to that value. c, Endogenous neuropilin 1 mrna levels assessed by real-time RT-PCR in EL4 cells transfected with empty vector, Roquin WT or Roquin M199R and harvested 16 hours later. The level of neuropilin 1 mrna in cells transfected with empty vector was assigned 1% and the other mrna levels were www.nature.com/nature 17
calculated relative to that value. d, Endogenous neuropilin 1 mrna levels assessed by real-time RT-PCR in EL4 cells transfected with mirna precursor negative control (#1) and hsa mirna-11 precursor (Ambion) for 24 hours. The level of neuropilin 1 mrna in cells transfected with mirna precursor negative control was assigned 1% and the other mrna level was calculated relative to that value. Data shown are mean values ± S.D. with n = 3. Statistically significant differences are marked by asterisks: Student s t-test, * P <.5, ** P <.1. www.nature.com/nature 18