Correlation of protein and RNA expression changes during Th17 and iTreg cell differentiation.

<p>Venn diagram showing the comparison of DE proteins with corresponding transcripts and DE transcripts with encoded detected proteins in comparison of Th17 and Th0 cells (a) or iTreg versus Thp cells (b). Scatterplot of proteins that were observed in proteomic and transcriptomic comparison of Th17 versus Th0 cell (a) or iTreg versus Thp cells (b). The lists of detected proteins and transcripts in Th17 versus Th0 cells and in iTreg versus Thp cells are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s009" target="_blank">S4 Data</a>. Ahr, aryl hydrocarbon receptor; Cnot2, CCR4-NOT transcription complex subunit 2; Coa6, cytochrome c oxidase assembly factor 6; DE, differentially expressed; Eno3, enolase 3; Foxo1, forkhead box O1; Foxp3, forkhead box P3; Gimap5, GTPase IMAP family member 5; Il17f, interleukin 17F; Isg15, interferon-stimulated gene 15; iTreg, induced regulatory T; Psmb5, proteasome subunit beta 5; Rorc, retinoic acid receptor–related orphan receptor C; Th0, T cell receptor–activated helper T; Th17, T helper 17; Thp, naïve CD4+ T.</p

Validation of protein expression changes with different technologies.

<p>(a) Heatmap showing the log fold change values (in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s010" target="_blank">S5 Data</a>) of proteins and mRNA DE in Th17 and iTreg cells in comparison with Th0 cells and Th17 compared with iTreg cells. (b) Flow cytometry analysis demonstrating the expression of surface molecules CD69 and CD101 in murine Th0, iTreg, and Th17 cells. One replicate is shown. (c) Immunoblot analysis of DE proteins in iTreg and Th17 cells compared to Th0 cells. Representative blots from 2–3 independent experiments are shown. DE, differentially expressed; ENO3, enolase 3; FOXO1, forkhead box O1; iTreg, induced regulatory T; NFATC2, nuclear factor of activated T cells 2; PSMB5, proteasome subunit beta 5; SMYD3, SET and MYND domain containing 3; Th0, T cell receptor–activated helper T; Th17, T helper 17; Thp, naïve CD4+ T; VIM, vimentin.</p

VIM is highly expressed in iTreg cells and influences TGFβ-induced Foxp3 expression.

<p>(a) mRNA expression of Vim from RNA-seq data generated in the present study. WT Thp cells were cultured under Th0, iTreg, and Th17 polarizing condition for 3 d. RNAs were isolated and processed for RNA-seq. Data shown are median FPKM values from 3 independent experiments (in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s011" target="_blank">S6 Data</a>) with the SEM. Statistical analysis was performed by using paired Student <i>t</i> test. ***: <i>p</i> < 0.01. (b) Immunoblot analysis of Thp cells cultured to Th0 and iTreg with and without 1 μM LY2109761 for 3 d. VIM, Foxp3, and loading control <b>β</b>-actin were shown. Representative of 3 independent experiments is shown. (c) Thp cells cultured to Th0 and iTreg for 3 d. Vim, Foxp3, and loading control <b>β</b>-actin were shown. Representative blots of 3 independent experiments are shown. (d) Flow cytometry analysis of WT and Vim-deficient CD4+ T cells cultured with TCR activation (Th0) and with cytokines (IL2+ TGFβ1, TGFβ1, IL2) for 3 d. Representative intracellular cytokine staining for Foxp3 was shown on left panel, and percentage of Foxp3 expression cells detected from 4 independent experiments (in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s011" target="_blank">S6 Data</a>) was shown on right panel. CTRL, control; Foxp3, forkhead box P3; FPKM, fragments per kilobase of transcript per million mapped reads; IL2, interleukin 2; iTreg, induced regulatory T; SEM, standard error of the mean; TCR, T cell receptor; TGFβ, transforming growth factor beta; Th0, T cell receptor–activated helper T cell; Th17, T helper 17; Thp, naïve CD4+ T; VIM, vimentin; WT, wild-type.</p

DE proteins in Th17 and iTreg cell differentiation.

<p>(a) Volcano plot of the comparison of proteome from Th17 versus iTreg cell. Green and red dots indicate statistically DE proteins. The complete lists of DE proteins in Th17 and iTreg cells are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s008" target="_blank">S3 Data</a>. (b) KEGG pathway analysis was performed on up-regulated (highly expressed in Th17) and down-regulated (highly expressed in iTreg) proteins in Th17 versus iTreg comparison. The pathways presented in the plot are significantly (Benjamini-Hochberg adjusted <i>p</i> < 0.05) enriched; size and color of dot indicate the number of proteins detected for that pathway and adjusted <i>p</i>-value respectively. The lists of pathways and proteins are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s008" target="_blank">S3 Data</a>. (c) Transcriptional regulatory network of Th17 and iTreg cells is shown. The TF annotation for DE proteins in comparison of Th17 and iTreg cells was obtained using IPA. The lists of TFs are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s008" target="_blank">S3 Data</a>. Hierarchical layout of String network is displayed. The red and blue nodes indicate proteins highly expressed in Th17 and iTreg cells, respectively. The size of nodes indicates the degree of connectivity of the nodes. Aebp2, adipocyte enhancer–binding protein 2; Ahr, aryl hydrocarbon receptor; Arid1a, AT-rich interaction domain 1A; Bach, BTB domain and CNC homology; Bcl10, B cell chronic lymphocytic leukemia/lymphoma 10; Brd8, bromodomain-containing 8; C1qbp, complement C1q binding protein; Carm1, coactivator-associated arginine methyltransferase 1; Cbfb, core binding factor beta; Ccar1, cell cycle and apoptosis regulator 1; Ccnh, cyclin H; Ccnt1, cyclin T1; Cdc5l, cell division cycle 5–like protein; Cops2, constitutive photomorphogenesis 9 signalosome subunit 2; Ctla4, cytotoxic T lymphocyte–associated protein 4; Cxxc1, CXXC finger protein 1; DE, differentially expressed; Dnmt3a, DNA methyltransferase 3 alpha; Etv6, ETS Variant 6; fdr, false discovery rate; Foxk1, forkhead box K1; Foxo1, forkhead box O1; Foxp1, forkhead box P1; Foxp3, forkhead box P3; Fus, Fus RNA binding protein; Gabpa, GA-binding protein transcription factor subunit alpha; Gatad2a, GATA zinc finger domain containing 2A; Gtf2a1, general transcription factor IIA subunit 1; Gtf2e1, general transcription factor IIE subunit 1; H2afx, histone 2A family member X; Hcfc1, host cell factor C1; Hdac1, histone deacetylase 1; Hexim1, hexamethylene bisacetamide inducible 1; Hmgb1, high-mobility group box 1; Hnrnpd, heterogeneous nuclear ribonucleoprotein D; Hnrnpk, heterogeneous nuclear ribonucleoprotein K; IKZF4, IKAROS family zinc finger 4; Il2, interleukin 2; Il2ra, interleukin 2 receptor alpha; Il2rg, interleukin 2 receptor gamma; Il17f, interleukin 17f; IPA, Ingenuity Pathway Analysis; Irf3, interferon regulatory factor 3; Irf4, interferon regulatory factor 4; ITGAE, integrin subunit alpha E; iTreg, induced regulatory T; Jak3, Janus kinase 3; KEGG, Kyoto Encyclopedia of Genes and Genomes; Khdrbs1, KH RNA binding domain containing, signal transduction–associated 1; Lag3, lymphocyte activating 3; Lgals3, galectin 3; Lgals7, galectin 7; Max, MYC-associated factor X; Mecp2, methyl-CpG binding protein 2; Mta2, metastasis-associated 1 family member 2; Mybbp1a, MYB binding protein 1A; Nfkbia, nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha; Nfyb, nuclear transcription factor Y beta; Nmi, N-myc and STAT interactor; NR3C1, nuclear receptor subfamily 3 group C member 1; OXPHO, oxidative phosphorylation; Phb1, prohibitin 1; Phb2, prohibitin 2; Phf5a, PHD finger protein 5a; Pml, promyelocytic leukemia; Pqpb1, polyglutamine-binding protein 1; Psmb5, proteasome subunit beta 5; Psmd9, proteasome 26S subunit non-ATPase 9; Rbbp7, retinoblastoma binding protein 7; Rbm39, RNA binding motif 39; Rorc, retinoic acid receptor–related orphan receptor C; Runx1, runt-related transcription factor 1; Runx3, runt-related transcription factor 3; Ruvbl1, RuvB-like AAA ATPase 1; Ruvbl2, RuvB-like AAA ATPase 2; Sap130, Sin3A-associated protein 130; Satb1, special AT-rich sequence-binding protein 1; Sqstm1, sequestosome 1; Sirt2, sirtuin 2; Smarca4, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 4; Smarca5, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A member 5; Smarcb1, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1; Smarcc2, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily C member 2; Smarcd, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily D; Smarce1, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily E member 1; Smyd3, SET And MYND domain containing 3; Srsf2, serine- and arginine-rich splicing factor 2; Stat1, signal transducer and activator of transcription 1; Stat2, signal transducer and activator of transcription 2; Stat3, signal transducer and activator of transcription 3; Stat6, signal tranducer and activator of transcription 6; Supt16h, SPT16 homolog; Tbl1xr1, transducin beta–like 1 X-linked receptor 1; Tcea1, transcription elongation factor A polypeptide 1; Tceb3, transcription elongation factor B polypeptide 3; Tcf7, transcription factor 7; TF, transcription factor; Tgfbr-I, transforming growth factor beta receptor type 1; Th17, T helper 17; Thoc1, THO complex 1; Traf6, tumor necrosis factor receptor–associated factor 6; Trrap, transformation/transcription domain–associated protein; Ube2v1, ubiquitin conjugating enzyme E2 V1; Ubtf, upstream binding transcription factor; Vim, vimentin; Yy1, yin yang 1.</p

Protein Exp changes during Th17 and iTreg cell differentiation.

<p>(a) LC-MS/MS log2 median intensity values of cytokine IL17F, TF Rorc in Th0 and Th17, and TF Foxp3 in Th0 and iTreg cells from 3 replicates. Data shown are median values from 3 technical replicates (in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s007" target="_blank">S2 Data</a>) with the SEM. Dotted line showing the minimum of LC-MS/MS signal intensity detections. (b) Number of DE proteins of Th17 and iTreg in comparison to Th0 cells. The complete lists of DE proteins can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s007" target="_blank">S2 Data</a>. (c) Volcano plots of statistical significance against fold-change of proteins between cell types (in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s007" target="_blank">S2 Data</a>). Blue and red dots indicate statistically differentially abundant proteins. (d) Functional groups of significantly DE proteins in Th17 or iTreg cells compared to Th0 cells. Functional groups were annotated by using IPA (Qiagen Bioinformatics. <a href="http://www.qiagenbioinformatics.com/" target="_blank">www.qiagenbioinformatics.com</a>). The complete lists of DE proteins are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s007" target="_blank">S2 Data</a>. DE, differentially expressed; Exp, expression; Foxp3, forkhead box P3; IKZF3, IKAROS family zinc finger 3; IKZF4, IKAROS family zinc finger 4; IL17F, interleukin 17F; IPA, Ingenuity Pathway Analysis; iTreg, induced regulatory T; LC-MS/MS, liquid chromatography–tandem mass spectrometry; Rorc, retinoic acid receptor–related orphan receptor C; Satb1, special AT-rich sequence-binding protein 1; SEM, standard error of the mean; Tcf7, transcription factor 7; TF, transcription factor; Th0, T cell receptor–activated helper T; Th17, T helper 17; Vim, vimentin.</p

Th17 and iTreg cell proteome.

<p>(a) Illustration of experimental and proteomic workflow in the study. (b) Representative flow cytometry plots showing the expression of murine IL17 in Th0 and Th17 cells cultured for 3 d and Foxp3 in Th0 and iTreg cells cultured for 7 d followed by restimulation with anti-CD3/CD28 and polarizing cytokines for additional 3 d. Percentages of positive cells were indicated. (c) Pearson’s correlation plots showing the correlation value of biological triplicates for Th17, iTreg, Th0 paired with Th17, and Th0 paired with iTreg cells. (d) Cumulative protein abundances plotted against ranked proteins. The number of proteins in each quantile was shown on the lists and in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s006" target="_blank">S1 Data</a>. (e) Pie chart with percentages of proteins identified across different cell compartments in Th17 and iTreg cells. The complete lists of proteins are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s006" target="_blank">S1 Data</a>. (f) Venn diagram with quantified proteins across Th0, Th17, and iTreg cells. The complete lists of proteins are in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004194#pbio.2004194.s006" target="_blank">S1 Data</a>. Actb, β-actin; Actg1, γ-actin 1; Ahr, aryl hydrocarbon receptor; Aldoa, aldolase A; Atp5b, ATP synthase subunit beta; Chd4, chromodomain helicase DNA-binding protein 4; Coro1a, coronin 1A; Ddx5, DEAD-box helicase 5; Eif4a1, eukaryotic translation initiation factor 4a1; Eno1, enolase 1; Fasn, fatty acid synthase; Foxp3, forkhead box P3; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Gimap4, GTPase IMAP family member 4; Hist1h1e; histone cluster 1 H1 family member E; Hk1, hexokinase 1; Hprt, hypoxanthine phosphoribosyltransferase; Hspd1, heat shock protein family D1; IL2, interleukin 2; Il4r, interleukin 4 receptor; IL6, interleukin 6; Il17 A, interleukin 17 A; IL17f, interleukin 17f; iTreg, induced regulatory T; Lck, lymphocyte cell-specific protein tyrosine kinase; LC-MS/MS, liquid chromatography–tandem mass spectrometry; Mif, macrophage migration inhibitory faction; Myh9, myosin heavy chain 9; Ncl, nucleolin; Phb, prohibitin; Pkm, pyruvate kinase M; Ppia, peptidylprolyl isomerase A; Rac2, ras-related C3 botulinum toxin substrate 2; Ran, Ras-related nuclear protein; Rorc, retinoic acid receptor–related orphan receptor C; Runx3, runt-related transcription factor 3; Satb1, special AT-rich sequence-binding protein 1; Serpinb5, Serpin Family B Member 5; Slc25a2, solute carrier family 25 member 2; Stat1, signal transducer and activator of transcription 1; Stat3, signal transducer and activator of transcription 3; Stat5a, signal transducer and activator of transcription 5A; Stip1, stress-induced phosphoprotein 1; TGFβ, transforming growth factor β; Th0, T cell receptor–activated helper T; Th17, T helper 17; Uba1, ubiquitin 1; Uba52, ubiquitin 52; Vim, vimentin; Wdr1, WD repeat domain 1; Zap70, zeta chain of T cell receptor–associated protein kinase 70.</p