Supplemental Information. c-flip Expression in Foxp3-Expressing Cells. Is Essential for Survival of Regulatory T Cells. and Prevention of Autoimmunity

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1 ell Reports, Volume 18 Supplemental Information c-flip Expression in Foxp3-Expressing ells Is Essential for Survival of Regulatory T ells and Prevention of Autoimmunity arlos Plaza-Sirvent, Marc Scuster, Yvonne Neumann, Ulrike Heise, Marina. Pils, Klaus Sculze-Ostoff, and Ingo Scmitz

2 Supplemental Information c-flip expression in Foxp3-expressing cells is essential for survival of regulatory T cells and prevention of autoimmunity arlos Plaza-Sirvent, Marc Scuster, Yvonne Neumann, Ulrike Heise, Marina. Pils, Klaus Sculze-Ostoff and Ingo Scmitz Supplemental ata Inventory Figure S1, related to Figure 1 Figure S2, related to Figure 1 Figure S3, related to Figure 3 Figure S4, related to Figure 4 Figure S5, related to Figure 5 Supplemental Experimental Procedures

3 Plaza(Sirvent et al%c Figure Sm A 3 m %398 69%3 3%38 97%6 3%m9 6%96 69%3 94%3 5%7mE(3 6%8m 56%m 37%m 7%33E(3 3%3m 37%5 59%9 3%344 68%9 3 57%3 3%m4 35%4 7%43 94%3 m3%33 33%3 5%53 58%9 3%m3 59%3 3 6m%6 3%357 53%7 mm%3 99%3 m3%9 98%9 7%43 4m%9 Untreated IL(9 QV B Viable cells H8o m5 m3 5 3 Treg untreated Treg IL(9 Treg QV Treg EX 3 m Hours 3%399 83%9 3 69%9 3%366 43%8 7AA 3%9m m8%9 3%44 33%4 3%9m 59%3 Annexin V anti(3 stimulation 3 m %398 69%3 3%38 97%6 3%39m 3%94 7m%3 95%3 3%3m3 8%m9 63%m 98%7 7%33E(3 3%3m 37%5 59%9 9%59E(3 69%m 3 66%6 3%m5 43%6 5%97 95%6 7%35 96%m 4%45 5m%8 3%358 59%6 3%398 64%3 3%93 67%5 7%83 39%5 9%49 96%5 6%79 95%5 EX Untreated IL(9 QV Viable cells H8o m anti(3 stimulation Treg untreated Treg IL(9 Treg QV Treg EX 3 m Hours 7%95E(3 79%6 3 79%7 3%49 53%9 7AA 3%99 m9%4 3%3m 93%3 3%5m 48%9 EX Annexin V

4 Figure S1, related to Figure 1. Kinetics of Treg viability (A) Representative dot plots and (B) kinetics of Treg cell viability determined by annexin V/7AA staining. ells were purified from 2-Foxp3 reporter mice by magnetic sorting and treated for 16, 24 or 48 r wit IL-2, te caspase inibitor QV, dexametasone (EX) or left untreated. Annexin V - /7AA - cells were considered viable. () Representative dot plots and () kinetics of Treg cell viability determined by annexin V/7AA staining. ells were purified from 2-Foxp3 reporter mice by magnetic sorting, seeded in anti-3-coated plates and additionally stimulated for 16, 24 or 48 r wit IL-2, QV, dexametasone (EX) or left untreated. (B and ) Eac point represents te mean ± SEM of 4 independent experiments. Statistical analyses comparing untreated vs. QV-treated samples were performed by one-tailed Mann- Witney tests; p<.5.

5 Plaza%Sirvent et al)( Figure S9 A m m 95)6 5)55 3)99 m 65)9 93)9 55)6 m)m53 65)8 55)5 99)7 m 57)9 6)m4 76)8 m 59)7 9)55E%3 54)7 m)m57 7)48 66)5 54)9 68)4 56)8 39)3 53)9 3)65E%3 94)5 m)m53 98)7 m)m95 55)3 Untreated IL%9 QV Viable cells o8h B 5mm 8m 6m 4m 9m m Tcon untreated Tcon IL%9 Tcon QV Tcon EX m Hours 68)5 7)44 65)8 9)57 98)5 6m)9 3)56E%3 49)9 m 45)9 m 55)9 7AA Annexin V 3)69 47)5 m)55 58)9 m)m36 88)5 EX anti%3 stimulation m m 5)55 6)88E%3 3m)5 m)m55 95)5 m 6)m4 95)6 3)99 55)5 54)5 m 95)5 65)3 9)69 m 94)3 58)4 56)5 57)9 76)8 m)m5m 93)9 m)m99 94)5 63)7 59)4 37)4 38)5 8)98E%3 96)5 9)84E%3 35)7 Untreated IL%9 QV Viable cells o8h 5mm 8m 6m 4m 9m m anti%3 stimulation Tcon untreated Tcon IL%9 Tcon QV Tcon EX m Hours 68)6 7)m7 68)5 5)77 45)7 96)7 m 6m)3 m)m55 57)5 m)m5m 97)5 7AA Annexin V 8)53 35)6 5)49 37)m m)33 79)6 EX

6 Figure S2, related to Figure 1. Kinetics of Tcon viability (A) Representative dot plots and (B) kinetics of Tcon cell viability determined by annexin V/7AA staining. ells were purified from 2-Foxp3 reporter mice by magnetic sorting and stimulated for te indicated time points wit IL-2, QV, dexametasone (EX) or left untreated. Annexin V - /7AA - cells were considered viable. () Representative dot plots and () kinetics of Tcon cell viability determined by annexin V/7AA staining. ells were purified from 2-Foxp3 reporter mice by magnetic sorting, seeded in anti-3-coated plates and additionally stimulated for te indicated time points wit IL-2, QV, dexametasone or left witout furter stimulation. (B and ) Eac point represents te mean ± SEM of 4 independent experiments. Statistical analyses comparing untreated vs. QV-treated samples were performed by one-tailed Mann- Witney tests; p<.5.

7 Plaza-Sirvent, Figure S3 A B Tymus LN flarδfoxp pl us ym N T + 1 us 1 16 N pl n ee Sp l ym us 2 16 en le 2 16 Kid. Liv. flarδfoxp3 Sp Number of 8+ cells 3 16 T Number of 4+ cells 4 16 n.s. 2 m I 3 T y H 4 16 en Sp le flarδfoxp3 4 Sp le en 31.1 flarδfoxp3 ym T 18.4 n.s. flarδfoxp3 9.4 L/ L+ / freuency (%) us pl N 11.8 L+ / L/ 62 flarδfoxp3 flarδfoxp3 Kid. Liv. N 89.5 G 4+ freuency (%) L+ / L+ / 62 62L Tymus 44 F 4 flarδfoxp Freuency (%) 8 flarδfoxp3 Freuency (%) pl 8 gated E 62 8 gated

8 Figure S3, related to Figure 3. Macroscopic analysis and furter caracterization of te scurfy-like penotype of mice (A) Picture of a 12 days old flar Foxp3 mouse vs. a littermate control mouse in ventral decubitus position. (B) Macroscopic appearance of a 24 days old flar Foxp3 mouse. () Picture of lympoid organs (tymus, spleen and cervical lymp nodes) of a 24 days old flar Foxp3 mouse and a littermate control mouse. () Representative pseudocolor dot plots and (E) freuencies of T cell activation markers 44 and 62L in 8 + cells from spleen and lymp nodes of flar Foxp3 mice and littermate control mice. 62L + /44 - cells are naïve T cells, 62L - /44 + cells are effector-memory T cells and 62L + /44 + cells are central memory T cells (n=7 eac). (F) Representative pseudocolor dot plots and (G) freuencies of 4 + T cells (upper panel) and 8 + T cells (lower panel) in te tymus, spleen and lymp nodes of flar Foxp3 mice and littermate control mice (n=7 eac, except tymus flar Foxp3, n=4). (H) Absolute numbers of 4 + (left panel) and 8 + (rigt panel) of tymus, spleen and periperal from flar Foxp3 mice and littermate control mice (n=4 eac, except tymus flar Foxp3, n=3). (I) Analysis of autoantibodies in sera of flar Foxp3 mice and littermate control mice. Extracts of kidney (Kid.) and liver (Liv.) from Rag-2 -/- mice were separated by SS-PAGE, blotted onto PVF membranes and incubated wit te indicated serum. (E, G and H) Bar graps represent te mean ± SEM. Statistical analyses were performed by twotailed Mann-Witney tests; p<.5, p<.1, p<.1; n.s., not significant.

9 PlazaSirvent et alnm Figure S4 A 15 Tymus Treg cells y7l 1 5 Treg cells y7l 1 5 Treg cells y7l 1 5 day 6 day 8 day 12 day 6 day 8 day 12 day 6 day 8 day 12 B 4 gated 8 gated 62L 44 y7l day 6 day 8 day 12 62L 44 y7l day 6 day 8 day Foxp3 - gated 1N31 2N47 1N69 Treg precursors y7l 2N 1N5 1N N5 N flar Foxp3 2N22 4 Foxp3 freuency y7l flar Foxp3 n.s. 25 GITR 4 Foxp3 E F 4 8 gated 5N54 5N12 5N75 85N2 1N26 85N4 Freuency y7l flar Foxp3 8N79 56N 7N41 2N7 33N1 2N34 Freuency y7l flar Foxp3 4 4N94 8 1N N N

10 Figure S4, related to Figure 4. Analysis of T cell development of Tcon and Treg cells in mice (A) Treg cell freuency kinetics in tymus (left panel), spleen (middle panel) and periperal lymp nodes (rigt panel) from flar Foxp3 and littermate control mice at day 6, 8 and 12 after birt. (flar Foxp3 : day 6 n=4, day 8 n=2, day 12 n=2, controls: day 6 n=7, day 8 n=3, day 12 n=8). (B) Activated (62L - /44 + ) T cell kinetics in 4 + cells (left panel), and 8 + cells (rigt panel) from flar Foxp3 and littermate control mice at day 6, 8 and 12 after born. (flar Foxp3 : day 6 n=4, day 8 n=2, day 12 n=2, controls: day 6 n=7, day 8 n=3, day 12 n=8). () Representative flow cytometric dot plots and percentages Treg cell precursors ( GITR + Foxp3 - ) from te tymus of flar Foxp3 mice and littermate control mice are sown (n=6 flar Foxp3, controls n=32). () Representative flow cytometric dot plots and percentages of Treg cells (4 + Foxp3 + ) in te tymus of flar Foxp3 mice and littermate control mice are sown (n=6 flar Foxp3, controls n=32). (E) Representative flow cytometric dot plots and percentages of , 4-8 +, and cells in te tymus of flar Foxp3 mice and littermate control mice are sown (n=6 flar Foxp3, controls n=32). (F) Representative flow cytometric dot plots and percentages of , , and from double negative (4-8 - ) tymocytes of flar Foxp3 mice and littermate control mice are sown (n=6 flar Foxp3, controls n=32). ( and ) Eac point and bar graps represent te mean ± SEM. Statistical analyses were performed by two-tailed Mann-Witney tests; p<.5 ; n.s., not significant.

11 PlazacSirvent et al(f Figure S5 A Tymus antic95l Neonatal mouse ay: Birt Foxp3 B QV Tymus Neonatal mouse ay: Birt 8 9 1) Foxp3 + of 4 + cells V6 15 1) 5 ) Tymus flar Foxp3 4 Foxp

12 Figure S5, related to Figure 5. Analysis of Treg freuencies after in vivo anti-apoptotic treatment in mice (A) Pseudocolor dot plots of Foxp3 + Treg cells witin cells in tymus, spleen and periperal lymp nodes of flar Foxp3 mice and littermate control mice treated wit 1 µg per injection of anti-95l (clone: MFL3). Mice were injected 3 times in non-consecutive days and te analysis was performed te day after te last injection. ata sown is representative of a single experiment. (B) Representative flow cytometric dot plots of Foxp3 + Treg cells witin cells in tymus, spleen and periperal lymp nodes of flar Foxp3 mice and littermate control mice treated wit 1 µg of QV per injection. Mice were injected day 8, 9 and 1 after birt and te analysis was performed te day 11 after birt. Te bar grap sows te mean ± SEM of Foxp3+ 4+ Treg cells in te different immunological organs. ata sown is representative of 2 independent experiments wit a total of 2 mice analyzed per genotype.

13 Supplemental Experimental Procedures Antibodies used for flow cytometry 3-FIT ( , B Bioscience), 4-PacificBlue (L3T4, BioLegend), 8-PEy7 (53-6.7, Biolegend), 44-AP (IM7, Biolegend), 62L-PerPy5.5 (MEL-14, ebioscience), Foxp3-AlexaFluor488 (FJK-16s, ebioscience), 95-PE (Jo2, B Bioscience), R5-PE (M5-1, Biolegend), TNFR1-PE (55R-286, Biolegend), 2-PE (RPA-2.1, Biolegend) and B22- AP (RA3-6B2, ebioscience). Antibodies used for Western blotting Foxp3 (FJK-16s, ebioscience), cleaved caspase-3 (5A1E, ell Signaling), c-flip (#896537, R& Systems), GAPH (GT239, GeneTex) and β-actin (A-74, Sigma-Aldric). Primers used for uantitative PR (PR) Primers used for PR analysis: UE fwd: 5 -AAGAGAATAAAGGAATTGAATG-3 ; UE rev: 5 -AAAGGATGTGAAATG-3 ; FLIP L fwd: 5 - GAGAAGUUAGA-3; FLIP L rev: 5 -UUUGUAUGAGUUAAGUG-3. FLIP R fwd: 5 -UAGAAGUAAAGUA-3 ; FLIP R rev: 5 -AUGGUAGAUA- 3.