A Systems Immunology Approach Identifies the Collective Impact of Five Mirs in Th2 Inflammation Supplementary Information

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A systems immunology approach identifies the collective impact of five miRs in Th2 inflammation

Supplementary information

Ayşe Kılıç, Marc Santolini, Taiji Nakano, Matthias Schiller, Mizue Teranishi,Pascal Gellert, Yuliya Ponomareva, Thomas Braun, Shizuka Uchida, Scott T. Weiss, Amitabh Sharma and Harald Renz

Corresponding Author: Harald Renz, MD Institute of Laboratory Medicine Philipps University Marburg 35043 Marburg, Germany Phone +49 6421 586 6235 [email protected] 1 Kılıç A et. al. 2018 A

C D PBS OVA chronic

PAS

(cm H2O*s/ml (cm

Raw Sirius Red Sirius

Supplementary figure 1: Characteristic phenotype of the acute and chronic allergic airway inflammatory response in the OVA-induced mouse model. (a) Total and differential cell counts determined in BAL and cytospins show a dominant influx of eosinophils. (b) Serum titers of OVA-specific immunoglobulins are elevated. (c) Representative measurement of airway reactivity to increasing methacholine (Mch) responsiveness measured by head-out body plethysmography. (d) Representative lung section staining depicting airway inflammation and mucus production (PAS, original magnification x 10) and airway remodeling (SiriusRed; original magnification x 20). Data are presented as mean ± SEM and are representative for at least 3 independent experiments with n=8-10 animals per group. Statistical analyses have been performed with one-way ANOVA and Tukey’s post-test and show *p<0.05, **p<0.01 and ***p<0.001. 2 Kılıç A et. al. 2018

lymphocytes A lung

activated CD4+-cells CD4+-T-cell-subpopulations SSC

FSC ST2 live CD69

CD4 CXCR3

spleen Naive CD4+- T cells DAPI FSC

single cells

SSC CD62L

CD4 CD44 FSC

FSC-W

Supplementary figure 2. Gating strategy for the isolation of live CD4+ T cell subsets using FACS-sorting. Representative dot-plots, depicting the gating strategy for the isolation of activated Th2 cells as CD4+ CD69+ T1/ST2+ CXCR3- from inflamed lung tissue. (A) Activated/tissue resident Th1 and Th2 cells were sorted from the CD4+CD69+ fraction and based on surface expression of CXCR3+ (Th1) and T1/ST2+ (Th2). (B) Naïve CD4+ T cells were isolated from spleens of sham immunized and sham challenged mice by sorting CD4+CD62L+CD44- cells. (C) 3 Kılıç A et. al. 2018

A B mRNA-mRNA network miRNA-mRNA network

PLAUR CTNNA1 CDK6 KIF3A MAP3K5 LTB ETV6 GNB4 LILRB4 DMXL2 DDX26B EZH2 CTSH EPAS1 NAV2 ARHGAP29 CENPE PICALM CYP11A1 FURIN RORA TYMS RHOQ CNN3 DSTN BUB1 TNFSF10 LNX2 hsa-mir-921hsa-mir-181a ECT2 TNFRSF1A SEC24D NCAPG CD44 PER1 PTPN4 IPCEF1 PTPRJ HIST2H2AA3 AHR NPNT hsa-mir-182 BCL9 PITPNM2 TIGIT CCR8 E2F8 IKZF2 TIPARP LFNG TPX2 NEB CD38 IPMK FRMD4B hsa-mir-509-3p ERN1 DAPK1 RAB31 hsa-mir-363hsa-mir-374b hsa-mir-181c ACTN1 ID2 TEC HIST1H2AG TMEM62 PDE2A ALDOA CREB3L2 GNPTAB hsa-mir-595 hsa-mir-603 ACP2 CAMK2G MPZL1 LEF1 CTLA4 PBX3 TUBA4A KIF23 ATRNL1 DUSP2 CD9 AKAP8 RACGAP1 TANK PPP4R1 hsa-mir-520e IMPA2 hsa-mir-665 hsa-mir-17 ZBTB32 CREM LZTFL1 NCKAP1 MTMR10 IL2RB PTGS2 ARL14 PDLIM5 hsa-mir-302c PALLD HIST1H4J hsa-mir-30b CRIM1 HSF2 HIST2H3A HIST1H2BO MFNG MAP3K5 ARMCX2 PRDM1 BCAR3 IL4 DNAJB14 ELL2 GPM6B ACVR2A HIST1H2AA GNG2 CD2AP IL10 BTG2 GAD1 EVI5 TAF7 AHR BCL3 ANLN GEM GNPDA1 hsa-mir-25 hsa-mir-24 hsa-mir-146a DGKZ RPL13A BACH2 CD80 KLF12 hsa-mir-96 hsa-mir-93 hsa-mir-148b PTPN3 STAMBPL1 hsa-mir-943 MYO1Dhsa-mir-604 PCGF5 PTPN6 CYTH3 hsa-mir-593hsa-let-7b OLR1 hsa-mir-578 hsa-mir-18a BCL2L11 RALGDS NCKAP1 COBLL1 MKI67 hsa-mir-32 USP28 ID3 HIST1H3A hsa-let-7d PLP2 SVIL CNN3 PCYT1A EPAS1 VCL IL13 ARL5A HSF2 hsa-mir-136CD2AP HMMR NFKBIZ hsa-mir-23b ALDH3A2 GRPhsa-mir-98 OSBPL3 BCL3 PRDM1 ITGB8 hsa-mir-181dMPZL1 IL1R2 SAMSN1HIST1H3C CHKA IL6 IL13 APBB1IP HIST2H2BF CDK1 PIP4K2A PLAGL1 ELMO2 USP24 PHF20L1 RPL13 STX11 HIST1H3E CSF1 ATXN1 hsa-mir-504 PLEKHA1 IKZF3 TIAM1 hsa-mir-657 TNFRSF1B ACTN1 UBE2D1 TNFSF11 FYN MBP ETFB S100A11 INHBA H2AFZ PLCD4 SLC7A8 PDE5A hsa-mir-335 TNFRSF1B MMP13 MCM6 UBE2D1 SLCO3A1 ATP2B4 IL24 HLF SPP1 RAB11FIP1 PHF20 CBLB HIST1H2BF INPP5A CYCS IGF2R DUSP4 PLCL1 hsa-mir-19a TIMP1 IL1R1 hsa-mir-150 RAPH1 DYNLT3 TRPS1 AQP9 hsa-mir-92a hsa-mir-27b PLP2 INPP4A RAB39B hsa-mir-20b CCNB1 ANLN PDLIM5 ALDOA STRBP PACSIN1 HIST1H4C GOT1HIST1H2AC IL10RA ZBTB10 LEF1 CYFIP1 MYLIP KIAA1598 DGCR2 IKBKE CD200R1 HIST1H2BC HIST1H3F RACGAP1 IFNG TXNDC17 hsa-mir-874PTPN4 BCAR3 hsa-mir-380 TAF4B GSN TRIM35 NCF4 ARHGEF12 GAD1 hsa-mir-29b hsa-mir-22 CDKN2C HIST1H2BI MYO1C ELK3 hsa-let-7c KSR1 INADL hsa-mir-498 F2R GNPTAB HIST1H2BN INHBA C14orf1 IER3 ANXA11 AURKB VDR FRMD4B hsa-mir-378 ADAM19STAT5A hsa-mir-422a SLC20A1 HIST1H2BL VCL ARHGEF3 HIST1H2AM GEM RGS1 CEP68 CA5B ECT2 CRTAM ADAM9 hsa-mir-545 NOTCH1 MYLIP ATP1B1 HIST1H2BK TBX21 ICOS CAMK2G ADARB1 hsa-mir-484 SETX INPP5A EBI3 HIST1H3B PPARG CASC5 GZF1 hsa-mir-138 BOLL HIST4H4 CCR5 PLEK CBX7 hsa-mir-581 RRM2 HIST1H3J NT5E IL6 ENTPD1 STAT5A hsa-mir-7 MID2 hsa-mir-554hsa-mir-26b NRP1 PTP4A3 LY9 ANXA2 hsa-mir-200c PTH RAPGEF6 NR3C1 hsa-let-7i N4BP1 PRKCH FHL3 RNF19A EGLN3 HMGB2 GRAMD3 HIST1H2AE IFI16 HIST1H3H ACP2 PTPN13 hsa-mir-106a UTRN ACLY HIST1H4F IRF8 BACH2 hsa-mir-31 C14orf1 FAM46A GLRX SEC23A hsa-mir-29a NFIL3 MID2 GAPDH hsa-mir-200bhsa-mir-557 FNDC3A GLUD1 RNF128 RYK hsa-mir-21 PTPRS IRF5 MAF AP1S2 IGF1R LPXN RPS6KA5 RAB39B LY75 GNG2 HIST1H2BB CTNNA1 EZH2 ARNT2 hsa-mir-198 ATP1B1 GNAQ CTSB LTB4R CD44 ACVR2A CAPG DGKD ARHGEF12 NFIL3 hsa-let-7g hsa-mir-200a MAF FOXP3 CCR1 EVI5 hsa-mir-221 hsa-mir-141 RNF130 NCAPG2 CREM ATP2B4 NFKBIZ C21orf91 SEC23A NEB hsa-mir-203 HIST1H4D RAB9A SOCS2 hsa-mir-505 FAM46Ahsa-mir-29c HSPA8 STAT3 DLG2 CD200R1 TASP1 hsa-mir-20a HIST2H4B GATA3 TOP2A hsa-mir-154 IGFBP7 CDK6 PBX3 PLEKHA1 HIST1H2AH hsa-mir-27a RUNX2 CASP6 CASP4 GNA15 SNX9 hsa-mir-185 PLEKHB2 ELK3 GRIPAP1 LNX2 UHRF2 KLF12 PLEK TMEM62 PDE5A PLCD4 BMPR2 DUSP16 DSTN HIST1H2BG PLOD2 SNX9 DDX11 PTGER2 DLGAP5 FUNDC2 HIST1H2AJ MYO1E PHF20 PTPRC HLF PLCXD2 PTPN3 RUNX2 FLNB PLCL1 NDUFV2 hsa-mir-610 hsa-mir-520h IGFBP7 N4BP2 RIOK3hsa-mir-132 PALLD IKZF4 MAD2L1 S100A6 HIST1H2AB CCNA2 RPS6KA5 hsa-mir-493 HAVCR2 NOTCH1 CBLB GALNT3 ARNT2 CCNE2 BATF TPI1 MAML3 hsa-mir-487a PRICKLE3 HIST1H2BM H2AFZ CLCN5 ITPR1 DLG4 CCL1 hsa-mir-520f ADAM9 BMPR2 ADAM19 HMGB2 PTPLAD1 CCNE2 TNFSF11 ATXN7L1 HIST1H3D SEC24D STAT3 NEK6 hsa-mir-26a hsa-mir-383 KIF23 RPLP2 INADL AHNAK CSF1 GATA3 NEK6 hsa-let-7a ZDHHC23 HIC1 TAF4B hsa-mir-500 ZNF318 IL10hsa-mir-30e RGS1 UBASH3B ARL14 NCAPH ETV6 FFAR2 GALC CASP1 IKZF2 OTUB2 CYP1A1 AMMECR1 ERO1L DAPK1 FNTB IL5 IL1RN ATP2B1 NR3C1 HIST2H2AC LDHB hsa-mir-16 IKZF4 CLDND1 BZW2 RNH1 KIF3A NDUFB1 HIST1H2BE CHKA LAMC1 hsa-mir-922 SMURF1 ZDHHC21 hsa-mir-520g BMPR1A SVIL TIAM1 ZFYVE16 RNF128 FES GNAQ hsa-mir-650 hsa-mir-100 target ATXN1 hsa-mir-575 BAZ1A PPARG CLCF1 ERF HIST1H2AD HIST1H3I hsa-mir-30a HIST2H4A hsa-mir-187 DLG2 KIF11 hsa-mir-891b ITPR1 AGPAT4 IGF1R HS3ST3B1 ANXA4 FURIN ETV5 hsa-mir-34a INTS8hsa-mir-149 miRNA PTPN13 DAPP1 RNF130 hsa-mir-192hsa-mir-15b hsa-mir-23a SCN1B

hsa-mir-617hsa-mir-744hsa-mir-95hsa-mir-133bhsa-mir-585 hsa-mir-620 hsa-mir-99b hsa-mir-639hsa-mir-663 hsa-mir-652 hsa-mir-637 hsa-mir-638 hsa-mir-765hsa-mir-220b hsa-mir-675 hsa-mir-181b hsa-mir-625 COBLL1 FES hsa-mir-597 t t es es h mRNA w ig Lo H miRNA Degree

Supplementary Figure 3. (A) The connectivity of mRNA/genes coding proteins in interactome in early Th2 cells. (B) The connectivity of miRNA and their targets in miRNA-mRNA network (mirDIP). We applied force-directed layout in Cytoscape for visualization of the networks.

4 Kılıç A et. al. 2018

mRNA-mRNA network miRNA-mRNA network

RAD50 TRIM35 CYP1B1 CHRAC1 EZR hsa-mir-922 RAB8A PTPN3 GLB1 CYP1A1 hsa-mir-30b A CAPN2 SPN STK25 RXRB XRN2 CUL4A NCOR2 B TAF4 hsa-mir-551b BACE2 VCL BRPF1 PSIP1 FOXN2 SRRT IL5 PRMT8 MAPKAPK2 DYRK2 CHCHD3 ACIN1 hsa-mir-138 PITPNM2 STUB1 CSNK1G3 PUM1 U2AF2 VEZT CHRM2 ATG16L1CCDC22 KIF2A PTPRC ATRIP GAPVD1 GATA3 LPHN2 hsa-mir-203 RBM28 FRMD4B NFIL3 NR3C1 CRY2 USP28 RNF130 SERINC3 FOXP4 NEDD8 HMGXB3 RRAS2 LCOR SDC1 ENAH TARS2 DYRK1B PCYT1A hsa-mir-649 IL1R2 ELL2 PACSIN1 hsa-mir-891b GRM5 FOXJ3 TIMP1 KLC1 SIAH2 PPARG GPR45 hsa-mir-575 TERF2 CDON DYRK2 ABCC1 RALY DUSP4 RAB31 CHKA ESYT2 DCAF6 FGF2 SRF SLC25A27 CD79A RANBP3 SLC48A1 OVGP1 TPBG BACH2 CD84 MAPK8IP3 hsa-mir-607MLF1 hsa-mir-593 RPS15A RALGPS2 PTK2B hsa-mir-665 PTPLAD1 GNPDA1 THOC1 ELF2 ZNF277 hsa-mir-363 FOXJ3 MVP GLUD1 DSTN HPSE TOM1 ITPR1 ABL2 XRCC4 ELK4 FUNDC2MYO1E FBXW11 IL22 FURIN SLK PEX13 MKNK1 DVL1 NCOA3 GRN MAPRE1 hsa-mir-27a hsa-mir-548a-3p IL10RA BRF1 CLSTN1 RPS5 RBPJ H2AFZ IPPK DENND4A RTN3 PEX10 ITPR1 TSC1 ZFAND3 CNOT10 SETD7 GAPDH CA13 PTPN12 PIP4K2A JPH1 TCF12 HIPK2 CWC15 KIF16B SP1 KSR1 PTPN4 hsa-mir-30ePPP1R2 ITGB8 GJB3 hsa-mir-516a-5p hsa-mir-195 PCID2 RNLS PLCD1 C1orf94 IMPA2 PTPN9 HLA-A ADAM9 ME1 hsa-mir-220c KLRD1 hsa-mir-520hPRDM2 POGZ ID2 SUFU CPSF3 RHOQ ID3 IL9R hsa-mir-610 hsa-mir-452 SFN RGS13 IL12RB1RPL18 GOT1 ARMC8 EPN2 BCAR3 MIPOL1 PRICKLE3 TREX1 hsa-mir-24 ZNF653 KLHL6 MYO1D SVIL GMNN EFR3A RUNX2 INHBA SMAD3 RPS13 GSN REV1 ATR SAP130 CTNNA1 GATA3 SUV420H1hsa-mir-22 IER3PRKCD PYGM BCL2L11 PHF20L1 CAMKK2 BNC1 RYK ERO1L CRMP1 PLEKHO1 IL6ST SLC4A2 PROS1 UTRN TAF8 PRDX6 RPL13 GNAQ BMPR1A CLDND1 IGFBP7 MAF CD2AP PDCD10 ACBD5 IL1RL1 IGF1R IRF3 hsa-mir-378 PTK2B UBE2D1 GTF2I RBM6 COIL SERINC3 TASP1 IFI16 RAB11FIP1 TNFRSF1A AKAP8L MED1 FURIN SDC4 N4BP1 hsa-mir-383 IL24 PLK2 MSC INADL SLC44A1 DDX19B MKRN2 LRP12 MEF2A ERAP1 TESK1 USP15MYLIP ITGAD TPI1 PLOD2 PHF3 SERTAD2 TSC22D1 SUCLG2 CHD7PCNA TXK RPL19 AMPH PLCD4 SMAD3 R3HDM1 PALLDMYD88 LY75RPL31 NCKAP1 INPPL1 CTSH hsa-mir-181b ATP2B1 PRDM1 FBXW11 TRIM8 VASP MAPKAPK5 TSC2 EMP1 hsa-mir-21 NDUFB1 PKD2 ARNT2 USP7 ATG4B IGF2R IPO9 EIF3KSMARCD1 CA5B LY75 ASH1L ZNF281 EHMT1 PPP2R5E RBM41 ETV6 CBX7 RABGGTA TMEM62 PIK3AP1 EML2 YWHAZ hsa-mir-380FKBP3 CAP2 DUSP19 FEZ2RREB1 RXRA EDN1 FLRT2 CCR1 NUP153 APBB1IPC19orf66 IKZF4 RAPGEF1 AMPH DMXL2 GNG2 USP9X PKN2 ZBTB44 MDK ATP1B1 RPAP2 RPL9 RAB20 VPS37A PAIP2 hsa-mir-107 EZH2 INTS4 APBB3 DAPK1 ERF RAPGEF6 TIAM1 hsa-mir-425 IGF2R GTF2I POGZ KHSRP RRM2 CD200R1 STAR SFRP4 RASSF5 PPARG CABYR DLG4 PUM2 MPI ADNP hsa-mir-580 DUSP2 SERPINA3TRIM44 USP9X FBXL5 SMYD3 RAB3GAP1 FNDC3A CNTNAP4 MKNK1 PLCD4 PITPNM2 RALGDS CENPT FOXP3 ATXN7L1 PALLD ATP2B4 DAPK1 EXOC6 H1F0 PDE1B HIVEP2 CASP6 XPO7 C1GALT1 GALC SUFU SPINT1 PTP4A3EHMT1 ITGB2CCDC90B GATA2 ZNF318 YTHDC2 TRAK2 LDHB THRAP3 RNF128 PRKAR2A HIP1 TKT MOCOS DHX9 NEB STXBP4 MLLT10 CNOT6 ERCC4 PARD6G ATXN7L2 RGS1 NEU1 TNFSF8 ELL2 BATF YLPM1 MLF1 HSD3B1 ITM2A hsa-mir-214 SLC20A1 TEC CNN3 hsa-mir-374b RAF1 RAPGEF1 MEF2A KHDRBS1 SRXN1 MAF LAMC1 RAB9A CD5 FARP2 PILRA SMARCD3 ADORA2B hsa-let-7b GLDC TRIM44 NUP153 RPE VAV1 CYCS RORA ADCY3 TUBA4A SAT1 BMPR2 ITGA2 IL1R1 F11R SETX LIMK1 PPAP2B ATXN7L2 hsa-mir-204 ATP1B1 SAP130 NRF1 MAML3 GNG2PBX3 SPHK2 FBXO4 NCK1MMP25 QTRT1 MED26 HLF TTBK1 SP1 RCOR1 SEMA4D ELK3 ANXA2SMAD1 LLGL1 MEOX2 SLC7A8 FRMD4B RASSF5 MAP2K7 ZBTB11 NEK7 FOXF2AHNAK MYCBP2 KIF2A PARP4 UBE2F CLCF1 ASB13 SELL FAU RAB11FIP4 S100A16 HAVCR2 DLG2 TIPARP ZMYND11 ARHGEF7 RAF1 ABL2 STAT3RPS29 NOTCH1 DUSP16 hsa-mir-936 TAOK2 LIMK1 NCLN LFNG IL10 RNF31 FYN CD74 PICALM hsa-mir-30c GAPVD1 ITGA2 KIF13A DUSP16 TAF7RGS19 CYP11A1 DIS3 SFPQ CCL1 HIP1 EBAG9 ZBTB10 VDR LSM1 GNG4 PLCL1 IFNG DTYMKEPB41 TNFRSF14 XRCC4 hsa-mir-106b hsa-mir-106a MKL2 EIF4G3 MIPOL1 DIP2C KLHL3 MPP7 CDK10RNF111 GPATCH8 hsa-mir-505 hsa-mir-181c RSBN1 UBE2F hsa-mir-216a TOP3A RPS6KA1 DGKZ AKAP6WDR82 hsa-mir-422a hsa-let-7c hsa-mir-494 EP400 NRF1 RPL17 APLP1 TOP2A CNKSR3 RAB11FIP4 JPH1 ENY2 NUMA1 ZRSR2 CDK11AIRAK3 TIGIT PLAUR CSGALNACT1 DCP2 hsa-mir-301aEPAS1 RAB39B CCR8 BCL3 IL23R LGALS1 CTNNA1 NCAPHMAX IL19 CTCF CYP1B1 hsa-mir-200c hsa-mir-135b HOOK1 PBX2 CD38 MBP SH3PXD2A TRIM24 BCL3 FANCLTXNDC17 LGMN CUL4B hsa-mir-34a UGP2 CUL4B INPP4AGNPAT TSC22D4 NDOR1 DGKI RAG1 DICER1 LYN GCNT1 PTPRJ CDK6hsa-mir-154 ACVR2A GRAMD3 CA8 CNOT1 TRIM25 ARSB NOTCH1 FLOT1 LAG3 DAXX hsa-let-7g ARMC8 EIF5 VTI1A UPF3A CD2AP PIK3CB TIAM1 CLK2 SMG1 PIK3AP1 CHST11 SETBP1 hsa-mir-26a ARL5A CENPJ IRF2BP1ATXN1 RPTOR LEF1 ADORA2B ARHGEF12 DLG2 ITGB1BP1 USP15 SSFA2 NCK1 ARHGEF2hsa-let-7e SMAD2 ASH1L ATG4D ATP2B4 CNOT2MIF4GD SH2D1A DUSP14 PBX3 ETV5 SMARCD1 UGCG FFAR2 MYO1C LITAF APLP1 hsa-mir-20b MAX hsa-mir-103 ALDH3A2 GATA2 NDUFV2 CEP68 IL2RB KLF12 WHSC1 EPB41 hsa-mir-27b INHBA RPL23 NUMA1 CDKN2C IKBKE GNA15 MCPH1 B3GALT2 hsa-mir-19bADARB1 AP1S2 GATM PSMD7 GCC2 SVIL HBS1L ERC1 NDUFA10 hsa-mir-340 TAF4 TLE4 RBBP9 SH3RF2 ABCB8 ENTPD1 TNKS2 SH3PXD2A MIA3 NCAPG2 UNKL TAGLN RNF146 MAFKFGF2 S100A1 POLR3F LTB RGS1 hsa-mir-132 CDC27 CAMK2N1 SLC2A3 SH2D1A MAP3K14 HOOK1 GAD1 PDLIM5 ENY2 ARL6IP6MAPK8 PSME2 FBXW2 ADAM9 ZFYVE16 HMCN1 hsa-mir-211 FRS2 STAT5A RPL10 TINF2 RPSA PTPRJ ETV6 RC3H1 IL1R1 RPS12 C14orf1 MUS81 PTPN1 PTPN9 hsa-let-7aHAVCR2 CNN3 hsa-mir-152 CRMP1 EXOSC5 AMMECR1 RUNX2 hsa-mir-200a MYO10 SPRED2 RPL12 GAS7 BRPF1 RGS3 TWISTNB TRAF3IP3 PHOX2A TRA2A CA1 MSH3 F2RL1 NLRP3 SETDB2 TADA2B GALNT3 TBX21 C2orf42 KPNA1 SMAD2 QRICH1 AQR ZMYND11 FNTBSDCCAG3 RPL30 CENPOPRKCH INTS7 SRGAP3 DUSP19 COX7A2L CPSF7 PBRM1 FBXW8 CHKA FGF18 COIL MED18 BAZ1A hsa-mir-33b FN1 MID2 BCL7B PRPF38B ZRANB1hsa-mir-520f hsa-mir-552 GRSF1 GZF1 KIF2C UTRN BCL9 ARHGAP29 BNIP3 FN1 PLB1 MPRIP hsa-mir-765 UHRF2 RNF146 CEP55 NFX1 RNF216 DENND5A RAB39B RGS9 hsa-mir-554 SLCO3A1 PHACTR4 SF3B1 UBE2H NYNRINSMURF1 SUOX ZNF276 PTPN13 CREB3L2 GAS7 RPS16SMC4 RPS10 CAPG FRS2 IFITM2 hsa-mir-25 DNAJB9 STX1A POLI DYNLT3 LXN RPL35A VCL ELK3 ATG4D ETNK1 PER1 PLEKHO1 MYH9 MTOR PHC1 BTLA ERGIC1 CYTH3 hsa-mir-19a STAT3 PLEKHA1 FGFR1OP2 LTN1 GRIPAP1 CBLB CDK4 MKL2 STT3B ZBTB38 C1GALT1 PPM1F UHRF2 GNB4 TAF4B LSR UBR1 HIST1H3H hsa-mir-181dNCKAP1 NEK6 ICOS hsa-mir-921 MAPRE1 AHR PUM1HIST1H3B STAG1 PTGER2 MYO1C FBXW7 DEK MED13 PSME1 MCL1 FNBP4EPAS1 FES JAG1 CA2 STAG1 LEF1 CTCF APBB3 ST6GAL1 P2RY1 CBFA2T2 UXT MID2 IFNG RXRA CA5B BOLL SLK ST3GAL4 CRTAM ARNT2 ZRANB1NEK6 MAP2K7 SCN1B hsa-mir-23a FOXN2 OGDH TSC1 MECR PFKL SUB1 CASP4 CLTA IFNA4 UBR1 RNF130 USP34 GTF3A RNPC3 BCAS2TRRAP ZFYVE16 ARFGEF1 DDX11 IKZF4 MTMR2 RNF111 DCP2 GAS2 IKZF2 SUV39H1 MPHOSPH8CCL19 CST3 hsa-mir-200b hsa-mir-625 RANBP3 WBP11 ZNF281 LPXN RNF128 SDC1 MTMR10 ARHGEF3 YWHAZ PTPN4 JAG1 SCMH1 ATXN1hsa-mir-206 BMPR2 PJA2 KLKB1 KPNA1 EIF5 CDK6 NCKAP5 LAMC1 hsa-mir-874 THOC1 INPP5A AXIN1 MAP3K12AKAP8 CYP17A1MEPCE TRIM8 CD80 SEC11AANKRD17 FRMD6 STX11 CD44 PRDM2HSPA2 TRPS1 SUV39H1 PDE2A hsa-mir-488 TMOD4 STAMBPL1 EZH2 ATRN BTG2 LYRM2 ADAM19 MRPL30 ENAH CMPK1ARHGEF7 PANK4 FKBP3 IL6ST PLCXD2 STOM ADAM19 NCOA3 ANXA2 RAPH1 STARD13 TYMS STAT5A CCL1 ASXL2 VEZT TNKS2 FUCA2 UCHL3 CYFIP1 IL13 CLCN5 PHF20 BZW2 CHD7 MPZL1 ICAM2RPS21 BCL2L15 hsa-mir-623 NEDD8 ARFGEF1 SUSD2 RASA3 CD27 NRBP1 DNAJB14 CDK2NR3C1 hsa-mir-143hsa-mir-657 DNAJB14 MEOX2 RGS3 CSNK1E CCR7 SERF2 RPL26 PCGF2 ELMOD2 FOXF2 hsa-mir-504 SMURF1 MAFK SMAD1 USP28 TANK NKIRAS1 SETD1A CPNE1 KHDRBS1 POLD2 AKAP6 CMAS KIFC1 VRK2 EIF3F TDG PLP2 RASL11A CTDP1 hsa-mir-760 PLP2 CCR3 SEC63 TNFSF10 TIMP3 TOR1AIP2 VAMP4 ADCY3 IGF1R N4BP2 CRY1 RNH1 RPLP2 HSD17B7 EPS15L1 ANKRD17 SIN3A QPCTL RPS7IL1RN CAMK2D RAB11FIP1 FBXL5 hsa-mir-23b ZMYM4 LAT NPY RAG1 OLR1 VAT1 BCL2L11 SKP1ARHGEF2 CASP1 NEK1 PTPN3hsa-mir-18b HSF2hsa-mir-15b NPY PHF20L1 DDX28 MRAS CA2 PTPN12RPL8 MARK3 RAPH1 SOCS2 OLR1 CSF1 PDLIM5 TAGLN CBLB PRDM4 COX7A2L HIST1H4C PLEKHA1 COBLL1 NAV2 hsa-mir-148b RRAS2 ACTN1 TERF2 PRR5L USE1 PTGS2 SMARCA5 IGFBP7 SIN3A FBXW7 S100A11 hsa-let-7i ABCC1 RBM12 PAN3 STRBP GCC2 PARD3 ATXN7L3 SRSF2 RPS23LRP8 FBXL6 EDN1 FNBP4 IGBP1 MLXIP TAF4B RIPK1 MPHOSPH9 SCGB1A1 GNPTAB AXL hsa-mir-32 AHR PDSS2 FBXL12 FUT8 AMBRA1 ZMIZ2 AURKB HSF2 DUSP4 DLG4 DAG1 ACTR6 SSFA2 RNF19A CSF1 NUP107 DNAJC5B ACVR1 CD44 MFNG DTL EPN1 PDCD10PRDM1 ACVR1 DEK PICK1 HIPK1 GRP RTN3 hsa-mir-192 AKR1B1KLHDC10 S100A6 DSTN NRP1 B4GALT5 LRP12 FDFT1 CABIN1 SENP2 IL17RB GIGYF2 MRPS24 FBXL2 KIF3A RPS6KA1 MARK3 F11R hsa-mir-302b PKN2 GSR PDE2A IPCEF1 F2R CD200R1 TRAM1 EGLN3 hsa-mir-146a PRDM4 IPPK MGRN1 RHOH CTLA4 GIT1 KAT2ADUSP2 DHRS3 VAV1 hsa-mir-620 PSMD7 EIF4G3 EML2 HIST1H3C MLECRPL13A CCDC101 RASSF8 IKZF2hsa-mir-98 CDK12 KLRG1 BMPR1A CSTA AQP9 EYA1 GNPTAB hsa-mir-155 ZNF644 NRBP1 QRICH1 NDUFB6 AHDC1 HIPK1 IPMK MAP3K5NRP1 AURKC C5 MPZL1hsa-mir-326 IL10 RNF24 WHSC1 PTBP2 IGBP1 KSR1 GLUD1 LZTFL1 SF3B1 hsa-mir-603 STK38 STAB1 DDX46 FAM57A ZYG11B MPHOSPH9 BTG2 GLRX IRF8 LRRFIP1 CDO1 MLKL ALDOA NPNT TLE4 INPP5A GABARAP CCR5 SMC5 SOCS2 STXBP1 PTPN5 ZBTB32 XAB2 IMPDH1 PLCL1 H2AFZ BRWD1 SETD7 CRIM1 GNB2L1 SLC25A46 GOLGA1 MRAS AGTPBP1 BCAR3 RASL11B CCL17 ING2 hsa-mir-449b TIMP3 SLC20A1 HIVEP2 LTB4R SETX ARL14 STK24 HIRA STARD13 CD9 hsa-mir-30a FGL2 MCL1 GTF3C2 ALDOA DDX19B PAIP2 HSF1 ORMDL3 KRTAP11-1 PRKAR2A CBFA2T2 PTPN13 ADPGK EP400 hsa-mir-93 GPRASP1 RBM38 ASAP1 NT5E hsa-mir-448 hsa-mir-150 USP34 NDUFA3 TAF1D RPL27A hsa-mir-372 CDON KLK3 ADH5 FUT8 RNF138 CHST15 SERPINF1 ARL6 ARL5A NFS1 PHF3 BAZ1A NEB hsa-mir-29c CAMK2D RIOK3 hsa-mir-517ahsa-mir-887hsa-mir-567hsa-mir-523 hsa-mir-518e ZNF592 GTF2A1 ZDHHC3 RPS9 SRF hsa-mir-487a MAP3K12 hsa-mir-182 TMEM43 ETS2 ARHGAP29 PCGF5 TDG ST6GALNAC2 LGALS3 KIAA2026 MAST1 IL13 KIF3ASF3B4 PTPN6 DYNLT3 hsa-mir-29a TMEM62 DHRS7 PTDSS1 ANKRD12 ARL14 MAP3K5 ATF7 CSK PPFIA1 RAB11B hsa-mir-17 HS3ST3B1 ENTPD1 PRPF39 CNOT1 DVL1 SAMSN1 HMOX2 ZDHHC23 C21orf91hsa-let-7d LAMA5 DIAPH3 BACH2 CHRM2 KLHL3 PLK2 DET1 ZNF605 CRY2 HSPA8 PHF20 DGKI SMARCA5 DYRK1B MAP3K14 SPP1 TPD52 UBP1 hsa-mir-26bNEO1 ARHGEF12hsa-let-7f ANXA4 DAG1 TIPARP DET1 CCR8 TMEM159 CAPNS1 ZMYM4 hsa-mir-18a ATXN2 USP22 LY9 IKZF3 BOLL N4BP1 GOLGA1 ARL6IP6 SH3BGRL KLF12 AGTPBP1 hsa-mir-222 PRSS8 FHL3 MYO10 GYPC PJA1 MYNN ELF2 LCORL NBN STX1A PER1 HDAC7 PLXNC1 WBP11 CARM1 ANKRD12 PLCB4 PIGF DGCR2 PTPLAD1 B4GALT5 RCOR1 DPM1 KIAA1598 DMXL2 RB1 IL9 ENTPD6 NPAT GAD1 hsa-mir-379 BZW2 EIF5B THADA OSBPL3 hsa-mir-29b PJA1 CGGBP1 PCYT2 UBE2D1 hsa-mir-328 DICER1 LILRB4 RPS28 ASTE1 CDC27 ETNK1 hsa-mir-636 GPM6B hsa-mir-99b hsa-mir-766 hsa-mir-220b FES MYH9 USP14 DGKD ERN1 hsa-mir-571 SDC4 NFIL3 hsa-mir-181a hsa-mir-545 LIN9 ACVR2A VASP hsa-mir-194 USP24 XPC HN1L CEP55 ZCCHC7 CXCL10 FBXW2 GAPDHS KDSR CCR4 PPFIBP1 NEO1 BNC1 LCOR ARG1 ADAP1 CNOT6 MYO1D RB1 PCGF2 WDR33 TASP1 HSPB1 IKBKB PLCB4 hsa-mir-7 PPP4R1 FRMD6 hsa-mir-497 IMPDH1 TAOK2 HLF PCSK1 hsa-mir-585hsa-mir-541hsa-mir-608hsa-mir-572 hsa-mir-632 hsa-mir-629 hsa-mir-661 hsa-mir-637 hsa-mir-675 hsa-mir-647 NPAT GRM5 COBLL1 HIC1 MKRN2 RBM12 TCF12 ATRNL1 OSBPL3hsa-mir-149 FGF18 PKD2 LRP8

t t es es h mRNA w ig Lo H miRNA Degree

Supplementary figure 3. (A) The connectivity of mRNA/genes coding proteins in Interactome in stable Th2 cells. The module comprised of 4104 edges between 1059 genes ecoding proteins. (B) The connectivity of miRNA and their targets in miRNA-mRNA network (mirDIP). The network consisted of the connection between 511 genes and 142 miRNAs. We applied force-directed layout in Cytoscape for visualization of the networks.

5 Kılıç A et. al. 2018

CTCF 212:5' ucucugaggguUUACUGUGAa 3' mCtcf 3’-UTR WT ::||||||| 213:5' ucccugaggguUUACUGUGAa 3' hCTCF 3’-UTR WT ::||||||| 3' cgucuugaaucGGUGACACUu 5' miR-27b ::||||||| 213:5' ucucugaggguUU------a 3' hCTCF 3’-UTR ΔmiR-27b

637:5' aggaauggugaaccACAUUCCu 3' mNcoa3 3’-UTR WTT NCOA3 ||||||| 763:5' gggaauagugaaacACAUUCCu 3' hNCOA3 3’-UTR WT ||||||| 3' ggugugugaaggaaUGUAAGGu 5' hsa-miR-206 ||||||| 763:5' gggaauagugaaac------u 3' hNCOA3 3’-UTR ΔmiR-206

ELK3 116:5' gUUCAUUUGGUUUGCACUUUu 3' mElk3 3’-UTR WT ||||||| 100:5' guuaauuugGUUUGCACUUUu 3' hELK3 3’-UTR WT ||: ||||||| 3' uagacgugaCAGUCGUGAAAu 5' hsa-miR-106b ||: ||||||| 100:5' guuaauuugGUUU------u 3' hELK3 3’-UTR ΔmiR-106b

AHR 1091:5' uuaagcagagcacuCAUUUCAc 3' mAhr 3’-UTR WT ||||||| 1041:5' uuaagcaaagcaccCAUUUCAa 3' hAHR 3’-UTR WT ||||||| 3' gaucaccaggauuuGUAAAGUg 5' hsa-miR-203 ||||||| 1041:5' uuaagcaaagcacc------a 3' mAHR 3’-UTR ΔmiR-203 (1)

2021:5' acaacuGUCUAAAACAUUUCAc 3' mAhr 3’-UTR WT | |||: |||||||||| 2395:5' aaAUUUGUCUGAAACAUUUCAu 3' hAHR 3’-UTR WT | | |||: |||||||||| 3' gaUCACCAGGAUUUGUAAAGUg 5' hsa-miR-203 | | |||: |||||||||| 2395:5' aaAUUU------u 3' mAHR 3’-UTR ΔmiR-203(2)

ELF2 458:5' acaGGUAUCUAG-AAUGUGAa 3' mElf2 3’-UTR WT ||||| | ||||||| 470:5' auaGGUAUUUUAGAAUGUGAa 3' hELF2 3’-UTR WT ::| ::| ||||||| 3' ccaUUAGGGACCGUUACACUa 5' hsa-miR-23b ::| ::| ||||||| 470:5' auaGGUAUUUUAG------a 3' hELF2 3’-UTR ΔmiR-23b

Supplementary figure 3. Conservation of miRNA-seed sequences among mouse and human. Alignment of the conserved miR target site in wildtype human and mouse target 3’-UTRs. To assess sequence specificity deletion of the seed sequence was performed and is highlighted in red color. 6

Supplementary Table 1: Genes upregulated in early Th2 cells versus naive T cells

early Th2 vs naive Gene Symbol Gene Name (log2-fold) Il5 interleukin 5 7.5 Il10 interleukin 10 6.9 Il13 interleukin 13 6.8 Il3 interleukin 3 6.3 Il1rl1 interleukin 1 receptor-like 1 6.2 Il9 interleukin 9 6.2 Gp49a glycoprotein 49 A 5.8 Mt2 metallothionein 2 5.6 Il4 interleukin 4 5.5 Hamp hepcidin antimicrobial peptide 5.4 Cxcr6 chemokine (C-X-C motif) receptor 6 5.4 Penk preproenkephalin 5.0 Tigit T cell immunoreceptor with Ig and ITIM domains 5.0 Gzmb granzyme B 4.9 Ctla4 cytotoxic T-lymphocyte-associated protein 4 4.9 Lilrb4 leukocyte immunoglobulin-like receptor, subfamily B, 4.8 member 4 Ifitm3 interferon induced transmembrane protein 3 4.7 Ccr4 chemokine (C-C motif) receptor 4 4.6 Il17f interleukin 17F 4.4 Perp PERP, TP53 apoptosis effector 4.3 Gcnt1 glucosaminyl (N-acetyl) transferase 1, core 2 4.2 Maf avian musculoaponeurotic fibrosarcoma (v-maf) AS42 4.1 oncogene homolog Ly6a lymphocyte antigen 6 complex, locus A 4.1 Fgl2 fibrinogen-like protein 2 4.0 Ccr2 chemokine (C-C motif) receptor 2 4.0 Sytl3 synaptotagmin-like 3 4.0 Ptprj protein tyrosine phosphatase, receptor type, J 4.0 Ttc39c tetratricopeptide repeat domain 39C 4.0 Ccr8 chemokine (C-C motif) receptor 8 3.9 Atp2b4 ATPase, Ca++ transporting, plasma membrane 4 3.8 Icos inducible T-cell co-stimulator 3.8 Cyp11a1 cytochrome P450, family 11, subfamily a, polypeptide 1 3.7 Cysltr2 cysteinyl leukotriene receptor 2 3.7 Osbpl3 oxysterol binding protein-like 3 3.7 Tnfrsf8 tumor necrosis factor receptor superfamily, member 8 3.7 Areg amphiregulin 3.7 Cpd carboxypeptidase D 3.6 Mid2 midline 2 3.6 Mmp13 matrix metallopeptidase 13 3.6 Ccl1 chemokine (C-C motif) ligand 1 3.6 Cysltr1 cysteinyl leukotriene receptor 1 3.6 Vdr vitamin D receptor 3.6 7

Il21 interleukin 21 3.6 Prdm1 PR domain containing 1, with ZNF domain 3.6 Itgb8 integrin beta 8 3.6 Il1r2 interleukin 1 receptor, type II 3.5 Havcr2 hepatitis A virus cellular receptor 2 3.5 Olr1 oxidized low density lipoprotein (lectin-like) receptor 1 3.4 Nek6 NIMA (never in mitosis gene a)-related expressed kinase 6 3.3 Car5b carbonic anhydrase 5b, mitochondrial 3.3 Ero1l ERO1-like (S, cerevisiae) 3.3 Glrx glutaredoxin 3.3 Bmpr2 bone morphogenic protein receptor, type II 3.3 (serine/threonine kinase) Casp4 caspase 4, apoptosis-related cysteine peptidase 3.3 4930506M07Rik RIKEN cDNA 4930506M07 gene 3.3 Pparg peroxisome proliferator activated receptor gamma 3.3 Mt1 metallothionein 1 3.2 Plin2 perilipin 2 3.2 Egln3 EGL nine homolog 3 (C, elegans) 3.1 Entpd1 ectonucleoside triphosphate diphosphohydrolase 1 3.1 Zbtb32 zinc finger and BTB domain containing 32 3.1 Raph1 Ras association (RalGDS/AF-6) and pleckstrin homology 3.1 domains 1 2010002N04Rik RIKEN cDNA 2010002N04 gene 3.0 Slc15a3 solute carrier family 15, member 3 3.0 Spp1 secreted phosphoprotein 1 3.0 Gata3 GATA binding protein 3 3.0 Klrg1 killer cell lectin-like receptor subfamily G, member 1 3.0 Adam19 a disintegrin and metallopeptidase domain 19 (meltrin beta) 3.0 Plek pleckstrin 2.9 Tnfsf10 tumor necrosis factor (ligand) superfamily, member 10 2.9 Ptpn13 protein tyrosine phosphatase, non-receptor type 13 2.9 Asb2 ankyrin repeat and SOCS box-containing 2 2.9 Ptpn4 protein tyrosine phosphatase, non-receptor type 4 2.9 Igfbp7 insulin-like growth factor binding protein 7 2.9 AW112010 expressed sequence AW112010 2.9 Zdhhc2 zinc finger, DHHC domain containing 2 2.8 Nfil3 nuclear factor, interleukin 3, regulated 2.8 Rgs1 regulator of G-protein signaling 1 2.8 Furin furin (paired basic amino acid cleaving enzyme) 2.8 Lgals1 lectin, galactose binding, soluble 1 2.8 Lamc1 laminin, gamma 1 2.8 Tnfsf8 tumor necrosis factor (ligand) superfamily, member 8 2.8 Olfr60 olfactory receptor 60 2.8 Pth parathyroid hormone 2.8 Wisp1 WNT1 inducible signaling pathway protein 1 2.8 Etv6 ets variant gene 6 (TEL oncogene) 2.8 Myo1e myosin IE 2.8 Lag3 lymphocyte-activation gene 3 2.8

Ell2 elongation factor RNA polymerase II 2 2.8 Epas1 endothelial PAS domain protein 1 2.7 Il17a interleukin 17A 2.7 Creb3l2 cAMP responsive element binding protein 3-like 2 2.7 Stom stomatin 2.7 Mmp10 matrix metallopeptidase 10 2.7 Aqp9 aquaporin 9 2.7 Hdc histidine decarboxylase 2.7 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 2.7 Il10ra interleukin 10 receptor, alpha 2.7 Stx11 syntaxin 11 2.6 Ccna2 cyclin A2 2.6 G530011O06Rik RIKEN cDNA G530011O06 gene 2.6

Supplementary Table 2: Genes downregulated in early Th2 cells versus naive T cells

Gene Symbol Gene Name early Th2 vs naive (log2-fold) Dapl1 death associated protein-like 1 -3.9 Ms4a4b membrane-spanning 4-domains, subfamily A, member 4B -3.9 Itm2a integral membrane protein 2A -3.8 Atp1b1 ATPase, Na+/K+ transporting, beta 1 polypeptide -3.7 Actn1 actinin, alpha 1 -3.7 Sell selectin, lymphocyte -3.6 Cmah cytidine monophospho-N-acetylneuraminic acid hydroxylase -3.5 St8sia6 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 -3.4 Tcf7 transcription factor 7, T-cell specific -3.0 Tmem71 transmembrane protein 71 -3.0 Ipcef1 interaction protein for cytohesin exchange factors 1 -2.8 Lef1 lymphoid enhancer binding factor 1 -2.8 Gm16591 predicted gene, 16591 -2.8 Ms4a6b membrane-spanning 4-domains, subfamily A, member 6B -2.6 Qser1 glutamine and serine rich 1 -2.6 Ms4a6c membrane-spanning 4-domains, subfamily A, member 6C -2.6 Parp8 poly (ADP-ribose) polymerase family, member 8 -2.6 E430029J22Rik RIKEN cDNA E430029J22 gene -2.6 Ms4a4c membrane-spanning 4-domains, subfamily A, member 4C -2.5 Gramd3 GRAM domain containing 3 -2.5 Cd55 CD55 antigen -2.5 Sidt1 SID1 transmembrane family, member 1 -2.5 D130062J21Rik RIKEN cDNA D130062J21 gene -2.5 Txk TXK tyrosine kinase -2.4 Fam49a family with sequence similarity 49, member A -2.4 Adamts6 a disintegrin-like and metallopeptidase (reprolysin type) with -2.4 thrombospondin type 1 motif, 6 BC094916 cDNA sequence BC094916 -2.4 Pyhin1 pyrin and HIN domain family, member 1 -2.3 Sepp1 selenoprotein P, plasma, 1 -2.3 Tagap T-cell activation Rho GTPase-activating protein -2.3 Ephx1 epoxide hydrolase 1, microsomal -2.2 Il21r interleukin 21 receptor -2.2 Rapgef4 Rap guanine nucleotide exchange factor (GEF) 4 -2.2 2610019F03Rik RIKEN cDNA 2610019F03 gene -2.2 1700025G04Rik RIKEN cDNA 1700025G04 gene -2.2 Sgk3 serum/glucocorticoid regulated kinase 3 -2.1 Bach2 BTB and CNC homology 2 -2.1 Klhl6 kelch-like 6 (Drosophila) -2.1 A430078G23Rik RIKEN cDNA A430078G23 gene -2.1 Pde5a phosphodiesterase 5A, cGMP-specific -2.0 Treml2 triggering receptor expressed on myeloid cells-like 2 -2.0 Fam101b family with sequence similarity 101, member B -2.0 Atp8b4 ATPase, class I, type 8B, member 4 -2.0

Crtam cytotoxic and regulatory T cell molecule -2.0 Arhgef3 Rho guanine nucleotide exchange factor (GEF) 3 -2.0 St8sia1 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 -2.0 Ccr7 chemokine (C-C motif) receptor 7 -1.9 Itga6 integrin alpha 6 -1.9 Mctp2 multiple C2 domains, transmembrane 2 -1.9 Boll bol, boule-like (Drosophila) -1.9 Trav14-3 T cell receptor alpha variable region 14-3 -1.9 Ifi203 interferon activated gene 203 -1.9 Slc44a2 solute carrier family 44, member 2 -1.9 Gm16452 predicted gene 16452 -1.9 Trav14d-1 T-cell receptor alpha variable region 14D-1 -1.8 Samd9l sterile alpha motif domain containing 9-like -1.8 Ltb lymphotoxin B -1.8 Arhgef18 rho/rac guanine nucleotide exchange factor (GEF) 18 -1.8 Gm4759 GTPase, very large interferon inducible 1 pseudogene -1.8 Clec2i C-type lectin domain family 2, member i -1.8 Cnn3 calponin 3, acidic -1.8 Cd9 CD9 antigen -1.8 Ttyh3 tweety homolog 3 (Drosophila) -1.7 Igf1r insulin-like growth factor I receptor -1.7 Utrn utrophin -1.7 Il1rl2 interleukin 1 receptor-like 2 -1.7 Pacsin1 protein kinase C and casein kinase substrate in neurons 1 -1.7 Gm13892 predicted gene 13892 -1.7 Dgka diacylglycerol kinase, alpha -1.7 B230325K18Rik RIKEN cDNA B230325K18 gene -1.7 LOC547323 similar to RIKEN cDNA A430107P09 gene -1.7 Usp28 ubiquitin specific peptidase 28 -1.7 Pik3ip1 phosphoinositide-3-kinase interacting protein 1 -1.7 Lfng LFNG O-fucosylpeptide 3-beta-N- -1.7 acetylglucosaminyltransferase Cd27 CD27 antigen -1.7 Ctsl cathepsin L -1.7 Dusp2 dual specificity phosphatase 2 -1.7 Snora75 small nucleolar RNA, H/ACA box 75 -1.6 Rassf2 Ras association (RalGDS/AF-6) domain family member 2 -1.6 Il6ra interleukin 6 receptor, alpha -1.6 Per1 period homolog 1 (Drosophila) -1.6 Aff3 AF4/FMR2 family, member 3 -1.6 Gimap4 GTPase, IMAP family member 4 -1.6 Scarna17 small Cajal body-specific RNA 17 -1.6 Zbtb20 zinc finger and BTB domain containing 20 -1.6 Znrf3 zinc and ring finger 3 -1.6 Cblb Casitas B-lineage lymphoma b -1.6 H2-Ob histocompatibility 2, O region beta locus -1.5 Ift80 intraflagellar transport 80 homolog (Chlamydomonas) -1.5

Plekhg2 pleckstrin homology domain containing, family G (with -1.5 RhoGef domain) member 2 Sik1 salt inducible kinase 1 -1.5 Klf2 Kruppel-like factor 2 (lung) -1.5 Gm13969 predicted gene 13969 -1.5 Esyt2 extended synaptotagmin-like protein 2 -1.5 Myo10 myosin X -1.5 Hsd11b1 hydroxysteroid 11-beta dehydrogenase 1 -1.5 Gm614 predicted gene 614 -1.5 Iqcb1 IQ calmodulin-binding motif containing 1 -1.5 Chchd10 coiled-coil-helix-coiled-coil-helix domain containing 10 -1.5 Mef2d myocyte enhancer factor 2D -1.5 Dnajb4 DnaJ (Hsp40) homolog, subfamily B, member 4 -1.5

Supplementary Table 3: Top 100 Genes upregulated in stable Th2 cells versus naive T cells

Gene Symbol Gene Name stable Th2 vs naïve (log2-fold) Il10 interleukin 10 7.4 Il5 interleukin 5 7.2 Il13 interleukin 13 6.5 Il1rl1 interleukin 1 receptor-like 1 6.4 Il9 interleukin 9 6.2 Il4 interleukin 4 5.9 Gp49a glycoprotein 49 A 5.9 Penk preproenkephalin 5.7 Mt2 metallothionein 2 5.6 Cxcr6 chemokine (C-X-C motif) receptor 6 5.5 Tigit T cell immunoreceptor with Ig and ITIM domains 5.3 Ctla4 cytotoxic T-lymphocyte-associated protein 4 5.2 Lilrb4 leukocyte immunoglobulin-like receptor, subfamily B, 4.8 member 4 Maf avian musculoaponeurotic fibrosarcoma (v-maf) AS42 4.7 oncogene homolog Ccr2 chemokine (C-C motif) receptor 2 4.6 Il1r2 interleukin 1 receptor, type II 4.6 Hamp hepcidin antimicrobial peptide 4.6 Fgl2 fibrinogen-like protein 2 4.5 Ccr4 chemokine (C-C motif) receptor 4 4.5 Gzmb granzyme B 4.5 Ly6a lymphocyte antigen 6 complex, locus A 4.4 Sytl3 synaptotagmin-like 3 4.3 Areg amphiregulin 4.3 Perp PERP, TP53 apoptosis effector 4.3 Il3 interleukin 3 4.2 4930506M07Rik RIKEN cDNA 4930506M07 gene 4.2 Itgb8 integrin beta 8 4.2 Ccr8 chemokine (C-C motif) receptor 8 4.2 Atp2b4 ATPase, Ca++ transporting, plasma membrane 4 4.2 Ttc39c tetratricopeptide repeat domain 39C 4.1 Icos inducible T-cell co-stimulator 4.1 Ptprj protein tyrosine phosphatase, receptor type, J 4.1 Vdr vitamin D receptor 4.0 Gcnt1 glucosaminyl (N-acetyl) transferase 1, core 2 3.9 Prdm1 PR domain containing 1, with ZNF domain 3.9 Ifitm3 interferon induced transmembrane protein 3 3.7 Mid2 midline 2 3.6 Tnfrsf8 tumor necrosis factor receptor superfamily, member 8 3.6 Cpd carboxypeptidase D 3.5 Ccr1 chemokine (C-C motif) receptor 1 3.5 Glrx glutaredoxin 3.4 Havcr2 hepatitis A virus cellular receptor 2 3.4 13

Nek6 NIMA (never in mitosis gene a)-related expressed kinase 6 3.4 Osbpl3 oxysterol binding protein-like 3 3.4 Ahr aryl-hydrocarbon receptor 3.3 Bmpr2 bone morphogenic protein receptor, type II 3.3 (serine/threonine kinase) Cysltr2 cysteinyl leukotriene receptor 2 3.3 Lag3 lymphocyte-activation gene 3 3.3 Raph1 Ras association (RalGDS/AF-6) and pleckstrin homology 3.3 domains 1 Cysltr1 cysteinyl leukotriene receptor 1 3.3 Pparg peroxisome proliferator activated receptor gamma 3.3 Cyp11a1 cytochrome P450, family 11, subfamily a, polypeptide 1 3.2 Tnip3 TNFAIP3 interacting protein 3 3.2 Il21 interleukin 21 3.2 Calca calcitonin/calcitonin-related polypeptide, alpha 3.2 AW112010 expressed sequence AW112010 3.2 Il17f interleukin 17F 3.2 Epas1 endothelial PAS domain protein 1 3.2 Asb2 ankyrin repeat and SOCS box-containing 2 3.1 Nfil3 nuclear factor, interleukin 3, regulated 3.1 Olfr60 olfactory receptor 60 3.0 2010002N04Rik RIKEN cDNA 2010002N04 gene 3.0 Ptpn4 protein tyrosine phosphatase, non-receptor type 4 3.0 Neb nebulin 3.0 Gata3 GATA binding protein 3 3.0 Tnfsf8 tumor necrosis factor (ligand) superfamily, member 8 2.9 Ero1l ERO1-like (S, cerevisiae) 2.9 Rgs1 regulator of G-protein signaling 1 2.9 Erc1 ELKS/RAB6-interacting/CAST family member 1 2.9 Adam8 a disintegrin and metallopeptidase domain 8 2.9 Adam19 a disintegrin and metallopeptidase domain 19 (meltrin beta) 2.9 Igfbp7 insulin-like growth factor binding protein 7 2.8 Lgals1 lectin, galactose binding, soluble 1 2.8 Entpd1 ectonucleoside triphosphate diphosphohydrolase 1 2.8 Etv6 ets variant gene 6 (TEL oncogene) 2.8 Ccl1 chemokine (C-C motif) ligand 1 2.8 Ell2 elongation factor RNA polymerase II 2 2.7 Zbtb32 zinc finger and BTB domain containing 32 2.7 Casp4 caspase 4, apoptosis-related cysteine peptidase 2.7 Stx11 syntaxin 11 2.7 Runx2 runt related transcription factor 2 2.7 Klrg1 killer cell lectin-like receptor subfamily G, member 1 2.7 Arnt2 aryl hydrocarbon receptor nuclear translocator 2 2.7 Car5b carbonic anhydrase 5b, mitochondrial 2.7 Egln3 EGL nine homolog 3 (C, elegans) 2.7 Gas2l3 growth arrest-specific 2 like 3 2.7 Tnfsf10 tumor necrosis factor (ligand) superfamily, member 10 2.7 Il10ra interleukin 10 receptor, alpha 2.7

Tmtc2 transmembrane and tetratricopeptide repeat containing 2 2.7 Mt1 metallothionein 1 2.6 Slc15a3 solute carrier family 15, member 3 2.6 Ptpn13 protein tyrosine phosphatase, non-receptor type 13 2.6 Ptpn3 protein tyrosine phosphatase, non-receptor type 3 2.6 Plek pleckstrin 2.6 Wisp1 WNT1 inducible signaling pathway protein 1 2.6 Lamc1 laminin, gamma 1 2.5 Cd200r1 CD200 receptor 1 2.5 Galnt3 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N- 2.5 acetylgalactosaminyltransferase 3 Ier3 immediate early response 3 2.5 Cyfip1 cytoplasmic FMR1 interacting protein 1 2.5 Trps1 trichorhinophalangeal syndrome I (human) 2.5

Supplementary Table 4: Top 100 Genes down regulated in stable Th2 cells versus naive T cells

stable Th2 vs naive Gene Symbol Gene Name (log2-fold) Ms4a4b membrane-spanning 4-domains, subfamily A, member 4B -4.2 Sell selectin, lymphocyte -4.1 Atp1b1 ATPase, Na+/K+ transporting, beta 1 polypeptide -3.7 Itm2a integral membrane protein 2A -3.7 Actn1 actinin, alpha 1 -3.7 Dapl1 death associated protein-like 1 -3.7 Cmah cytidine monophospho-N-acetylneuraminic acid hydroxylase -3.4 Lef1 lymphoid enhancer binding factor 1 -3.4 Tcf7 transcription factor 7, T-cell specific -3.4 St8sia6 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 6 -3.4 Ipcef1 interaction protein for cytohesin exchange factors 1 -3.2 Gm16591 predicted gene, 16591 -3.1 Tmem71 transmembrane protein 71 -3.1 Qser1 glutamine and serine rich 1 -3.1 Parp8 poly (ADP-ribose) polymerase family, member 8 -3.0 Gramd3 GRAM domain containing 3 -2.9 Cd55 CD55 antigen -2.8 Txk TXK tyrosine kinase -2.7 5830405N20Rik RIKEN cDNA 5830405N20 gene -2.7 Klhl6 kelch-like 6 (Drosophila) -2.7 Adamts6 a disintegrin-like and metallopeptidase (reprolysin type) with -2.7 thrombospondin type 1 motif, 6 Arhgef3 Rho guanine nucleotide exchange factor (GEF) 3 -2.7 Tagap T-cell activation Rho GTPase-activating protein -2.7 Gramd1b GRAM domain containing 1B -2.7 Ms4a6b membrane-spanning 4-domains, subfamily A, member 6B -2.5 Boll bol, boule-like (Drosophila) -2.5 Gm13949 predicted gene 13949 -2.5 Ms4a6c membrane-spanning 4-domains, subfamily A, member 6C -2.5 Atp8b4 ATPase, class I, type 8B, member 4 -2.5 Sidt1 SID1 transmembrane family, member 1 -2.5 Pyhin1 pyrin and HIN domain family, member 1 -2.5 Trav14-3 T cell receptor alpha variable region 14-3 -2.5 Sepp1 selenoprotein P, plasma, 1 -2.4 Gimap4 GTPase, IMAP family member 4 -2.4 LOC547323 similar to RIKEN cDNA A430107P09 gene -2.3 Fam49a family with sequence similarity 49, member A -2.3 2610019F03Rik RIKEN cDNA 2610019F03 gene -2.3 E430029J22Rik RIKEN cDNA E430029J22 gene -2.3 BC094916 cDNA sequence BC094916 -2.3 Arhgap15 Rho GTPase activating protein 15 -2.2 D130062J21Rik RIKEN cDNA D130062J21 gene -2.2 Itga6 integrin alpha 6 -2.2

Ephx1 epoxide hydrolase 1, microsomal -2.2 Ifi203 interferon activated gene 203 -2.2 Gm16452 predicted gene 16452 -2.2 Rasgrp2 RAS, guanyl releasing protein 2 -2.2 Gm8721 predicted gene 8721 -2.2 1700025G04Rik RIKEN cDNA 1700025G04 gene -2.2 Ccr7 chemokine (C-C motif) receptor 7 -2.2 Slc44a2 solute carrier family 44, member 2 -2.2 Bach2 BTB and CNC homology 2 -2.1 Myo10 myosin X -2.1 Ltb lymphotoxin B -2.1 Ms4a4c membrane-spanning 4-domains, subfamily A, member 4C -2.1 Utrn utrophin -2.1 Mctp2 multiple C2 domains, transmembrane 2 -2.1 Il1rl2 interleukin 1 receptor-like 2 -2.1 Rassf2 Ras association (RalGDS/AF-6) domain family member 2 -2.0 Rasa3 RAS p21 protein activator 3 -2.0 Dgka diacylglycerol kinase, alpha -2.0 Fam101b family with sequence similarity 101, member B -2.0 Gm13892 predicted gene 13892 -2.0 Sgk3 serum/glucocorticoid regulated kinase 3 -2.0 Il6ra interleukin 6 receptor, alpha -2.0 Tmsb10 thymosin, beta 10 -2.0 Fam65b family with sequence similarity 65, member B -2.0 Treml2 triggering receptor expressed on myeloid cells-like 2 -2.0 Aff3 AF4/FMR2 family, member 3 -2.0 Ikbke inhibitor of kappaB kinase epsilon -2.0 Ly9 lymphocyte antigen 9 -2.0 Pde5a phosphodiesterase 5A, cGMP-specific -1.9 Rapgef4 Rap guanine nucleotide exchange factor (GEF) 4 -1.9 A430078G23Rik RIKEN cDNA A430078G23 gene -1.9 Usp28 ubiquitin specific peptidase 28 -1.9 Il21r interleukin 21 receptor -1.9 Samd9l sterile alpha motif domain containing 9-like -1.9 St8sia1 ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 -1.9 Crtam cytotoxic and regulatory T cell molecule -1.9 Trav14d-1 T-cell receptor alpha variable region 14D-1 -1.9 Spnb2 spectrin beta 2 -1.9 Cd9 CD9 antigen -1.8 Smc6 structural maintenance of chromosomes 6 -1.8 Tle4 transducin-like enhancer of split 4, homolog of Drosophila -1.8 E(spl) Ift80 intraflagellar transport 80 homolog (Chlamydomonas) -1.8 Gm4759 GTPase, very large interferon inducible 1 pseudogene -1.8 Gm13969 predicted gene 13969 -1.8 Arhgef18 rho/rac guanine nucleotide exchange factor (GEF) 18 -1.8 Cnn3 calponin 3, acidic -1.8 5033414D02Rik RIKEN cDNA 5033414D02 gene -1.8 17

Znrf3 zinc and ring finger 3 -1.8 Snora34 small nucleolar RNA, H/ACA box 34 -1.8 Ecm1 extracellular matrix protein 1 -1.8 Apbb1ip amyloid beta (A4) precursor protein-binding, family B, member -1.7 1 interacting protein Sesn1 sestrin 1 -1.7 Gm6907 predicted gene 6907 -1.7 Per1 period homolog 1 (Drosophila) -1.7 Cd52 CD52 antigen -1.7 Esyt2 extended synaptotagmin-like protein 2 -1.7 Dnajb4 DnaJ (Hsp40) homolog, subfamily B, member 4 -1.7 Pvr poliovirus receptor -1.7 Prkcq protein kinase C, theta -1.7

Table 5: Top 10 KEGG*-pathways enriched in early Th2 cells versus naive CD4+ T cells

term fold p-value Genes enrichment mmu05321:Inflammatory bowel 9.5 2.58E-10 MAF; IL4; IL6; IL18RAP; IL5; TBX21; IL13; RORA; IL21; IL10; disease (IBD) STAT3; IL17A; IL12RB1; IL17F; IFNG mmu05310:Asthma 9.4 3.54E-04 IL4; IL3; IL5; IL9; IL13; IL10 mmu04060:Cytokine-cytokine 4.9 2.20E-13 IL1R2; IL9R; CSF1; CCR1; BMPR2; TNFRSF8; IL13; receptor interaction TNFRSF4; IL10; IL17RB; TNFRSF1B; IL17A; IL12RB1; IL10RA; IFNG; CXCR6; IL4; IL3; IL2RB; IL6; IL5; IL18RAP; IL9; IL21; TNFSF8; CCR8; ACVR2A; TNFSF10; CCR4; CCR3; CCR2; BMPR1A mmu04630:Jak-STAT signaling 4.7 2.46E-07 IL4; IL3; IL2RB; IL6; IL9R; IL5; SOCS2; STAT5A; IL9; IL13; pathway IL24; IL21; IL10; STAT3; CISH; IL12RB1; IL10RA; IFNG mmu04672:Intestinal immune 4.5 2.46E-02 IL4; IL6; IL5; ICOS; IL10 network for IgA production mmu04640:Hematopoietic cell 3.6 6.76E-03 IL4; IL1R2; IL3; IL6; IL9R; IL5; CD44; CSF1 lineage mmu05203:Viral carcinogenesis 3.2 1.10E-05 CDK1; HIST1H2BB; RBL2; DDB1; STAT5A; HIST1H2BH; CDK6; MAPKAPK2; STAT3; HIST2H4; CCR8; HIST1H2BM; HIST2H2BB; CCR4; GSN; CCR3; CREB3L2; HIST1H4F; RBPJ; CCNA2 mmu05202:Transcriptional 3.2 4.52E-04 MAF; IL3; IL1R2; IL6; IL2RB; ARNT2; PPARG; GZMB; NUPR1; misregulation in cancer ID2; CDKN2C; ETV6; ETV5; RUNX2 mmu04350:TGF-beta signaling 3.1 2.51E-02 ACVR2A; INHBA; ID2; ZFYVE16; IFNG; BMPR2; BMPR1A pathway mmu05133:Pertussis 3.0 4.63E-02 IL6; LY96; HC; IRF8; CASP1; IL10 mmu05200:Pathways in cancer 1.7 3.47E-02 PTGER2; IL6; PTGS2; EPAS1; STAT5A; PPARG; ARNT2; EGLN3; CDK6; CTNNA1; ARHGEF12; STAT3; RAD51; HIF1A; GNAQ; GNG2; GNB4; LAMC1

*KEGG- Pathway analysis was performed using DAVID Bioinformatics Resources 6.8.

Table 6: Top 10 KEGG*-pathways enriched in stable Th2 cells versus naive CD4+ T cells

Term fold enrichment p-Value Genes mmu05310:Asthma 11.7 1.25E-04 IL4; IL3; IL5; IL9; IL13; IL10 mmu05321:Inflammatory bowel disease MAF; IL4; IL18RAP; IL5; TBX21; IL13; RORA; 10.3 2.66E-09 (IBD) IL21; STAT3; IL10; IL12RB1; IL17F; IFNG IL4; IL3; IL2RB; IL5; SOCS2; SOCS3; STAT5A; mmu04630:Jak-STAT signaling pathway 5.5 5.98E-08 IL9; IL13; IL24; IL21; IL10; STAT3; CISH; IL12RB1; IL10RA; IFNG IL1R2; CSF1; CCR1; BMPR2; TNFRSF8; IL13; mmu04060:Cytokine-cytokine receptor IL10; IL17RB; IL12RB1; IL10RA; CXCR6; IFNG; 5.0 4.53E-11 interaction IL4; IL3; IL2RB; IL18RAP; IL5; IL9; IL21; TNFSF8; CCR8; ACVR2A; TNFSF10; CCR4; CCR3; CCR2 mmu05330:Allograft rejection 4.2 3.07E-02 IL4; IL5; IFNG; GZMB; IL10 mmu04640:Hematopoietic cell lineage 3.3 3.28E-02 IL4; IL1R2; IL3; IL5; CD44; CSF1 mmu04512:ECM-receptor interaction 3.2 3.89E-02 CD44; ITGB8; ITGAV; NPNT; LAMC1; SPP1 CCL1; LYN; CCR1; CXCL2; STAT3; CCR8; mmu04062:Chemokine signaling pathway 3.1 9.17E-04 TIAM1; CCR4; CCR3; CXCR6; CCR2; GNG2; GNB4 mmu05202:Transcriptional misregulation MAF; IL1R2; IL3; IL2RB; ID2; CDKN2C; PPARG; 3.1 2.75E-03 in cancer ARNT2; GZMB; ETV6; RUNX2 mmu05146:Amoebiasis 2.8 3.81E-02 IL1R2; GNA15; GNAQ; IFNG; LAMC1; IL10; VCL

*KEGG- Pathway analysis was performed using DAVID Bioinformatics Resources 6.8.

Table 1: Top 10 pathways formed by IDEAL miRNAs in the molecular interactome Immediate targets of the IDEAL miRNAs and their first neighbors in the disease module were subjected to pathway enrichment analysis using the Enrich method (see Methods section). The selection was done according to a high combined score.

miRNA Term Overlap 1 Adjusted Genes p-value 2 Regulation of IL10;SMAD2;HSPA8;SMAD3;VDR;MAX; nuclear GATA3;NR3C1;RUNX2;SP1;CDK4;SIN3 miR-27b 13/81 2.78E-08 SMAD2/3 A;CDK2 signaling mTOR signaling IKBKB;CDK2;TSC2;TSC1;SFN;RAF1;YW miR-206 8/69 5.57E-05 pathway HAZ; MTOR TRIF-mediated MEF2A;IKBKB;MAPK8;IRF3;FBXW11;RI miR-106b TLR3/TLR4 9/97 7.39E-04 PK1;MAP2K7; IKBKE;SKP1 signaling IL-5 Signaling LYN;STAT5A;PRKCD;STAT3;PTK2B; miR-203 7/68 1.31E-04 Pathway YWHAZ;VAV1 EGFR1 SMAD2;STAT5A;SMAD3;STAT3;ASAP1; miR-23b Signaling 16/171 4.0399E-08 PTPN12;VAV1;ELK4;SP1;SIN3A;PTK2B; Pathway PTPN6; RAF1;RALGDS;GIT1

1 Overlap: number of genes in Th2 disease module overlapping with genes annotated to respective pathway 2 Adjusted p-value: p-value was calculated using the Fisher exact test and adjustment was done using Bonferroni correction.