Supporting Information - PNAS · 11/6/2014 · a concentration of 1 μg/mL and a final volume of...
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Supporting Information Zanin-Zhorov et al. 10.1073/pnas.1414189111 SI Materials and Methods Cell Purification. The whole blood was incubated (20 min, 22 °C) with a RosetteSep human CD4 + T-cell enrichment mixture or a CD4 + CD127 - T-cell enrichment mixture (StemCell Technologies). The remaining unsedimented cells were loaded onto Ficoll-Paque Plus (Amersham Bioscience), isolated by density centrifugation, and washed with PBS. Tregs were isolated from the CD4 + CD127 - population by using the human CD25-positive selection kit from Stem Cell Technologies. Umbilical cord blood (UCB) CD25 + and CD25 - CD4 + T cells were isolated from frozen UCB units (National Placental Blood Program, New York Blood Center) by positive selection using directly conjugated anti-CD25 magnetic microbeads. Cells were cultured with anti-CD3/CD28 mAb- coated Dynabeads for 18–21 d and split every 2–3 d. Recombi- nant IL-2 (300 IU/mL; Chiron) was added on day 3 and main- tained for culture duration. The expanded cells were used for Western blot analysis. Cell Activation. CD4 + T cells were cultured in complete RPMI 1640 (Invitrogen) containing antibiotics and 10% heat- inactivated FBS. Cells were cultured at a final concentration of 2 × 10 6 cells/mL (cytokine secretion) or 2 × 10 5 cells/mL (proliferation) on anti-CD3 mAb (5 μg/mL) and anti-CD28 mAb (5 μg/mL) (eBioscience) precoated 24-well plates. For Th17- skewing activation, IL-1β (50 ng/mL) and TGF-β (5 ng/mL) (R&D Systems Inc.) were added to the culture. Cytokine se- cretion was determined by ELISA after 48 h by using Human IL-10 and IFN-γ Cytoset (Biosource), IL-17 and IL-2 (R&D Systems Inc.), and IL-21 (eBioscience). Proliferation was assessed by CFSE dilution after 72 h. Treg Suppression Assay. Human CD4 + CD127 - CD25 + regulatory T cells (Tregs) were purified using the Stem Cell Technologies human regulatory T-cell kit. Tregs were stained with Cell Pro- liferation Dye eFluor 660 (eBioscience) at 5 μM, and total PBMCs were stained with CFSE (eBioscience) at 5 μM as per the manu- facturer’s instructions. Tregs were then incubated with KD025 at the indicated concentrations for 5 h before being washed three times in complete RPMI to remove the excess drug. The 3 × 10 5 Tregs and total PBMCs from the same donor were then placed in culture in a U-bottom 96-well plate with anti-CD3 (OKT3) at a concentration of 1 μg/mL and a final volume of 200 μL. Cells were incubated for 4 d at 37° and 5% CO 2 . Cells were then stained with Viability Dye eFluor 780 and CD4 and analyzed by flow cytometry. All FACS plots displayed were gated on CD4 + Viability Dye - CFSE + and eFluor 660 - for analysis of only the target CD4 cell population. Percent of inhibition was calculated by comparing ac- tivated PBMC alone to those wells in which PBMCs were cultured in the presence of Tregs. CFSE Labeling. We added CFSE to the cell suspensions (1–5 × 10 6 cells/mL) at a final concentration of 0.5 μM, 37 °C, for 30 min; we stopped the reaction with FCS at a final concentration of 10%. We washed cells twice with PBS and resuspended them with complete RPMI media. Flow Cytometry. For cytokine intracellular staining, cells were stimulated for 4 h with Cell Stimulation Mixture (00-4975-03; eBioscience) (which contains phorbol 12-myristate 13-acetate, ionomycin, and protein transport inhibitor), fixed, and per- meabilized. Cells were stained with antibodies to human or mouse CD4 (Clone RPA-T4 or RM4-5), IL-17 (Clone eBio64DEC17 or eBio17B7), IL-21 (Clone eBio3A3-N2 or FFA21), RORγt (Clone BD2), and IFN-γ (Clone 4S.B3) (eBioscience). Foxp3 expression was tested by using an anti-Foxp3 antibody (Clone 236A/E7) and a staining kit (eBioscience) Samples were analyzed in a guava easyCyte Flow Cytometer machine (EMD Millipore). Western Blot. Total cell lysates and nuclear and cytoplasmic extracts were prepared and analyzed for protein content. Sample buffer was then added, and after boiling, the samples, containing equal amounts of proteins, were separated on a SDS/PAGE gel and transferred to a nitrocellulose membrane. The membranes were blocked and probed with the specific antibodies overnight. Anti-ROCK1 (HPA007567) and anti-ROCK2 (HPA007459) were purchased from Sigma-Aldrich, and anti-beta actin antibodies (4970S) were purchased from Cell Signaling. Anti-pSTAT1 (#9171), pSTAT3 (#4113), pSTAT4 (#5267), pSTAT5, pSMAD2/3, pSTAT6 (#9361), Lamin B1 (#9087), pMLC (#3674), total MLC (#3672), total STAT3 (#4904), and Tbet (#5214) were purchased from Cell Signaling Technologies. Anti-IRF4 (sc-48338) and anti- RORγt (14-6988) were obtained from Santa Cruz Biotechnology Inc. and eBioscience, respectively. Immunoreactive protein bands were visualized using HRP-conjugated secondary antibodies and an enhanced ECL system. ChIP Assay. Peripheral blood CD4 + T cells were stimulated by anti-CD3/28 mAbs, IL-1β (50 ng/mL), and TGF-β (5 ng/mL) for 48 h in the absence or presence of 10 μM KD025. Cells were harvested at 48 h, and ChIP assays were performed. For ChIP, cells were cross-linked with 1% formaldehyde for 10 min, quenched with 0.125 M glycine, washed with cold PBS, and lysed in buffer A (50 mM Hepes, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% TX100, 0.1% Na-Deoxycholate, 0.1% SDS, and 1× protease inhibitor). Lysates were sonicated, and after centrifugation, supernatants were incubated overnight at 4 °C with protein G-Dynal beads that preconjugated with desired antibodies. The immunoprecipitates on dynal beads were washed with buffer B (20 mM Tris, pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% TX100, and 0.1% SDS) and then with Tris EDTA (TE) buffer before being eluted with buffer C (1% SDS in TE) at 65 °C for 1 h, and the eluates were then incubated at 65 °C for 6 h or overnight for cross-link reversal before being purified with ChIP DNA clean and concentrator (Zymo Research). Binding of factors were determined by real-time PCR with designed primer sets. IL21-1, IL21 promoter; IL21-2, distal IL21 site was predicted to be high for H3K27Ac. IL17A4, IL17A promoter; IL17A5, distal IL17A site was predicted to be high for H3K27Ac. The control site is an irrelevant site that is not active in CD4+ T cells. Planar Lipid Bilayers. Planar lipid bilayers containing anti-CD3 antibodies (5 μg/mL) and ICAM-1 (250 molecules/mm 2 ) were prepared in parallel-plate flow cells as described previously (1). The flow cell containing the bilayers was warmed up to 37 °C, cells were injected in 500 μL of Hepes-buffered saline containing 1% human serum albumin, and images were collected on a cus- tom automated Nikon inverted fluorescence microscope. Microscopy. All TIRF imaging was performed on the custom automated Nikon inverted fluorescence microscope using the 100×/1.45 N.A. TIRF objective from Nikon. TIRF illumination was set up and aligned according to the manufacturer’s in- structions as previously described (2). Briefly, cells interacted with the bilayers for 30 min at 37 °C, fixed with 2% PFA, per- meabilized with 0.05% Triton-X 100, blocked and stained with Zanin-Zhorov et al. www.pnas.org/cgi/content/short/1414189111 1 of 12
Transcript of Supporting Information - PNAS · 11/6/2014 · a concentration of 1 μg/mL and a final volume of...
Supporting InformationZanin-Zhorov et al. 10.1073/pnas.1414189111SI Materials and MethodsCell Purification. The whole blood was incubated (20 min, 22 °C)with a RosetteSep human CD4+ T-cell enrichment mixture or aCD4+CD127− T-cell enrichment mixture (StemCell Technologies).The remaining unsedimented cells were loaded onto Ficoll-PaquePlus (Amersham Bioscience), isolated by density centrifugation, andwashed with PBS. Tregs were isolated from the CD4+CD127−
population by using the human CD25-positive selection kit fromStem Cell Technologies. Umbilical cord blood (UCB) CD25+
and CD25−CD4+ T cells were isolated from frozen UCB units(National Placental Blood Program, New York Blood Center) bypositive selection using directly conjugated anti-CD25 magneticmicrobeads. Cells were cultured with anti-CD3/CD28 mAb-coated Dynabeads for 18–21 d and split every 2–3 d. Recombi-nant IL-2 (300 IU/mL; Chiron) was added on day 3 and main-tained for culture duration. The expanded cells were used forWestern blot analysis.
Cell Activation. CD4+ T cells were cultured in complete RPMI1640 (Invitrogen) containing antibiotics and 10% heat-inactivated FBS. Cells were cultured at a final concentrationof 2 × 106 cells/mL (cytokine secretion) or 2 × 105 cells/mL(proliferation) on anti-CD3 mAb (5 μg/mL) and anti-CD28mAb (5 μg/mL) (eBioscience) precoated 24-well plates. For Th17-skewing activation, IL-1β (50 ng/mL) and TGF-β (5 ng/mL)(R&D Systems Inc.) were added to the culture. Cytokine se-cretion was determined by ELISA after 48 h by using HumanIL-10 and IFN-γ Cytoset (Biosource), IL-17 and IL-2 (R&DSystems Inc.), and IL-21 (eBioscience). Proliferation was assessedby CFSE dilution after 72 h.
Treg Suppression Assay. Human CD4+CD127−CD25+ regulatoryT cells (Tregs) were purified using the Stem Cell Technologieshuman regulatory T-cell kit. Tregs were stained with Cell Pro-liferation Dye eFluor 660 (eBioscience) at 5 μM, and total PBMCswere stained with CFSE (eBioscience) at 5 μM as per the manu-facturer’s instructions. Tregs were then incubated with KD025 atthe indicated concentrations for 5 h before being washed threetimes in complete RPMI to remove the excess drug. The 3 × 105
Tregs and total PBMCs from the same donor were then placed inculture in a U-bottom 96-well plate with anti-CD3 (OKT3) ata concentration of 1 μg/mL and a final volume of 200 μL. Cells wereincubated for 4 d at 37° and 5% CO2. Cells were then stained withViability Dye eFluor 780 and CD4 and analyzed by flow cytometry.All FACS plots displayed were gated on CD4+ Viability Dye−
CFSE+ and eFluor 660− for analysis of only the target CD4 cellpopulation. Percent of inhibition was calculated by comparing ac-tivated PBMC alone to those wells in which PBMCs were culturedin the presence of Tregs.
CFSE Labeling.We added CFSE to the cell suspensions (1–5 × 106
cells/mL) at a final concentration of 0.5 μM, 37 °C, for 30 min;we stopped the reaction with FCS at a final concentration of10%. We washed cells twice with PBS and resuspended themwith complete RPMI media.
Flow Cytometry. For cytokine intracellular staining, cells werestimulated for 4 h with Cell Stimulation Mixture (00-4975-03;eBioscience) (which contains phorbol 12-myristate 13-acetate,ionomycin, and protein transport inhibitor), fixed, and per-meabilized. Cells were stained with antibodies to human ormouseCD4 (Clone RPA-T4 or RM4-5), IL-17 (Clone eBio64DEC17 or
eBio17B7), IL-21 (Clone eBio3A3-N2 or FFA21), RORγt (CloneBD2), and IFN-γ (Clone 4S.B3) (eBioscience). Foxp3 expressionwas tested by using an anti-Foxp3 antibody (Clone 236A/E7) anda staining kit (eBioscience) Samples were analyzed in a guavaeasyCyte Flow Cytometer machine (EMD Millipore).
Western Blot. Total cell lysates and nuclear and cytoplasmicextracts were prepared and analyzed for protein content. Samplebuffer was then added, and after boiling, the samples, containingequal amounts of proteins, were separated on a SDS/PAGE geland transferred to a nitrocellulose membrane. The membraneswere blocked and probed with the specific antibodies overnight.Anti-ROCK1 (HPA007567) and anti-ROCK2 (HPA007459)were purchased from Sigma-Aldrich, and anti-beta actin antibodies(4970S) were purchased from Cell Signaling. Anti-pSTAT1(#9171), pSTAT3 (#4113), pSTAT4 (#5267), pSTAT5, pSMAD2/3,pSTAT6 (#9361), Lamin B1 (#9087), pMLC (#3674), total MLC(#3672), total STAT3 (#4904), and Tbet (#5214) were purchasedfrom Cell Signaling Technologies. Anti-IRF4 (sc-48338) and anti-RORγt (14-6988) were obtained from Santa Cruz BiotechnologyInc. and eBioscience, respectively. Immunoreactive protein bandswere visualized using HRP-conjugated secondary antibodies andan enhanced ECL system.
ChIP Assay. Peripheral blood CD4+ T cells were stimulated byanti-CD3/28 mAbs, IL-1β (50 ng/mL), and TGF-β (5 ng/mL) for48 h in the absence or presence of 10 μM KD025. Cells wereharvested at 48 h, and ChIP assays were performed. For ChIP,cells were cross-linked with 1% formaldehyde for 10 min,quenched with 0.125 M glycine, washed with cold PBS, and lysedin buffer A (50 mM Hepes, pH 7.5, 140 mM NaCl, 1 mM EDTA,1% TX100, 0.1% Na-Deoxycholate, 0.1% SDS, and 1× proteaseinhibitor). Lysates were sonicated, and after centrifugation,supernatants were incubated overnight at 4 °C with proteinG-Dynal beads that preconjugated with desired antibodies. Theimmunoprecipitates on dynal beads were washed with buffer B(20 mM Tris, pH 8.1, 500 mM NaCl, 2 mM EDTA, 1% TX100,and 0.1% SDS) and then with Tris EDTA (TE) buffer beforebeing eluted with buffer C (1% SDS in TE) at 65 °C for 1 h, andthe eluates were then incubated at 65 °C for 6 h or overnight forcross-link reversal before being purified with ChIP DNA cleanand concentrator (Zymo Research). Binding of factors weredetermined by real-time PCR with designed primer sets. IL21-1,IL21 promoter; IL21-2, distal IL21 site was predicted to be highfor H3K27Ac. IL17A4, IL17A promoter; IL17A5, distal IL17Asite was predicted to be high for H3K27Ac. The control site is anirrelevant site that is not active in CD4+ T cells.
Planar Lipid Bilayers. Planar lipid bilayers containing anti-CD3antibodies (5 μg/mL) and ICAM-1 (250 molecules/mm2) wereprepared in parallel-plate flow cells as described previously (1).The flow cell containing the bilayers was warmed up to 37 °C,cells were injected in 500 μL of Hepes-buffered saline containing1% human serum albumin, and images were collected on a cus-tom automated Nikon inverted fluorescence microscope.
Microscopy. All TIRF imaging was performed on the customautomated Nikon inverted fluorescence microscope using the100×/1.45 N.A. TIRF objective from Nikon. TIRF illuminationwas set up and aligned according to the manufacturer’s in-structions as previously described (2). Briefly, cells interactedwith the bilayers for 30 min at 37 °C, fixed with 2% PFA, per-meabilized with 0.05% Triton-X 100, blocked and stained with
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rabbit polyclonal antibodies to ROCK1 and ROCK2 for 20 min,and then incubated with fluorescently tagged goat anti-rabbitFab2 (Invitrogen). Controls included the use of nonimmunespecies-matched IgG. Measurement of signaling was done aspreviously described (1).
Collagen-Induced Arthritis in Mice.The 8-wk-old male DBA/1J micewere obtained from Jackson Laboratories, and the study wasperformed in accordance with institutional guidelines and withapproval by the Institutional Animal Care and Use Committee ofNew York University. The mice were immunized via a 0.1-mLintradermal injection at the base of the tail with 100 μg chickentype II collagen (Chondrex, LLC) emulsified with an equal vol-ume of complete Freund’s adjuvant containing 4 mg/mL heat-denatured Mycobacterium (Chondrex) (day –50). KD025 wasadministered intraperitoneally to mice with established mildarthritis on day 0 (25 d after immunization; clinical score, 6–8).
Mice (n = 10 per group) were dosed intraperitoneally with 50,100, and 200 mg/kg of KD025 or vehicle (0.4% methylcellulose)once a day for 28 d. Clinical scores were monitored every otherday. On day 28, mice were killed and spleens were isolated andanalyzed by FACS and Western blot analysis.
Evaluation for Clinical Arthritis. Clinical signs of arthritis wereevaluated to determine arthritis incidence. Each paw was eval-uated and scored individually using a 0–4 scoring system. Thepaw scores were summed to yield individual mouse scores, witha maximum score of 16 for each animal. The scoring system wasas follows: a paw score of 0, no signs; 1, mild swelling confined tothe tarsal bones or ankle joint; 2, mild swelling extending fromthe ankle to the tarsal bones; 3, moderate swelling extendingfrom the ankle to the metatarsal joints; and 4, severe swellingencompassing the ankle, foot and digits, or ankylosis of the limb.
1. Fooksman DR, et al. (2010) Functional anatomy of T cell activation and synapse for-mation. Annu Rev Immunol 28:79–105.
2. Varma R, Campi G, Yokosuka T, Saito T, Dustin ML (2006) T cell receptor-proximalsignals are sustained in peripheral microclusters and terminated in the central supra-molecular activation cluster. Immunity 25(1):117–127.
R² = 0.7202
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Fig. S1. PK analysis of KD025 in a single ascending dose/multiple ascending dose phase 1 clinical study. The maximum concentration (Cmax) (A) and area underthe curve during 24 h (AUC0–24) (B) of KD025 observed after administration are linear and dose proportional at doses 40–500 mg.
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System Organ ClassPreferred Term
PlaceboN = 16
40 mgN = 6
80 mgN = 6
120 mgN = 6
160 mgN = 6
240 mgN = 6
320 mgN = 6
400 mgN = 6
500 mgN = 6
Gastrointes nal Disorders 0 0 1 (16.7%) 0 0 0 0 0 0
Lip blister 0 0 1 (16.7%) 0 0 0 0 0 0
General Disorders and Administra on Site Condi ons
2 (12.5%) 0 0 0 1 (16.7%) 0 0 0 0
Fa gue 2 (12.5%) 0 0 0 0 0 0 0 0
Edema peripheral 0 0 0 0 1 (16.7%) 0 0 0 0
Infec ons and infesta ons 1 (6.3%) 0 0 0 0 0 1 (16.7%) 1 (16.7%) 0Furuncle 0 0 0 0 0 0 1 (16.7%) 0 0
Upper respiratory tractinfec on
1 (6.3%) 0 0 0 0 0 0 1 (16.7%) 0
Injury, Poisoning and Procedural Complica ons
0 0 0 0 0 0 1 (16.7%) 0 1 (16.7%)
Mouth injury 0 0 0 0 0 0 1 (16.7%) 0 0
Thermal burn 0 0 0 0 0 0 0 0 1 (16.7%)
Inves ga ons 0 0 0 0 0 1 (16.7%) 0 0 0
Blood crea nephosphokinase increased
0 0 0 0 0 1 (16.7%) 0 0 0
Musculoskeletal and Connec ve Tissue Disorders
2 (12.5%) 0 1 (16.7%) 0 0 0 0 0 0
Back pain 1 (6.3%) 0 1 (16.7%) 0 0 0 0 0 0
Muscular weakness 2 (12.5%) 0 0 0 0 0 0 0 0
Musculoskeletal pain 1 (6.3%) 0 0 0 0 0 0 0 0
Pain in extremity 1 (6.3%) 0 0 0 0 0 0 0 0
Nervous System Disorders 3 (18.8%) 0 1 (16.7%) 0 1 (16.7%) 0 0 0 1 (16.7%)
Dizziness 2 (12.5%) 0 0 0 0 0 0 0 0
Headache 1 (6.3%) 0 1 (16.7%) 0 0 0 0 0 1 (16.7%)
Paraesthesia 0 0 0 0 1 (16.7%) 0 0 0 0
Somnolence 1 (6.3%) 0 0 0 0 0 0 0 0
Respiratory, Thoracic and Medias nal Disorders
1 (6.3%) 1 (16.7%) 0 0 0 0 0 0 0
Nasal conges on 1 (6.3%) 1 (16.7%) 0 0 0 0 0 0 0
Skin and Subcutaneous Tissue Disorders
1 (6.3%) 1 (16.7%) 0 0 0 0 0 0 0
Derma s contact 0 1 (16.7%) 0 0 0 0 0 0 0
Hyperhidrosis 1 (6.3%) 0 0 0 0 0 0 0 0
Fig. S2. Summary of adverse effects observed in a phase 1 clinical trial.
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Fig. S3. Oral administration of KD025 has no effect on frequencies of IL-17, IL-21, and IFN-γ–producing CD4+ T cells. Human PBMCs were purified from healthyhuman subjects before and after oral administration of KD025 (400 mg) and stimulated for 4 h with phorbol 12-myristate 13-acetate and ionomycin, fixed,and permeabilized. Cells were stained with antibodies to CD4, IL-17, IL-21, and IFN-γ and analyzed by flow cytometry. Percent of inhibition was calculated as1 – (percentage day 15/percentage day 0). Each dot represents the individual patient.
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Actin
Rock1
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Fig. S4. ROCK1 and ROCK2 are expressed and recruited to the IS in human CD4+ T cells. The percentage of CD4+ T cells was defined by flow cytometry afterpurification (A). Whole cell lysates were prepared from resting or stimulated (by immobilized anti-CD3/28 mAbs) peripheral blood CD4+ T cells and analyzed byWestern blot for expression of ROCK1 and ROCK2 (B). CD4+ T cells were introduced into bilayers containing anti-CD3 (5 μg/mL) and ICAM-1 at 250 molecules/m2,fixed at 30 min, permeabilized, stained with anti-ROCK1 or ROCK2 antibodies, and imaged by TIRFM. The panels show representative images (C ). APC,allophycocyanin.
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actin
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Fig. S5. Selective ROCK2 inhibitor KD025 inhibits phosphorylation of MLC. CD4+ T cells were treated with 10 μM of KD025 for 1 h and then stimulated byimmobilized anti-CD3/28 mAbs (A) or introduced into bilayers containing anti-CD3 and ICAM-1, fixed, and imaged by TIRF microscopy (B). Whole cell lysateswere prepared and analyzed by Western blot for phosphorylation of MLC. Actin was used as a loading control (A). The panels show representative images (B).cSMAC mean fluoresence was quantified in cells (each dot represents an individual cell). One representative experiment of two is shown.
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Fig. S6. KD025 inhibits cytokine secretion in previously stimulated CD4+ T cells. CD4+ T cells were stimulated by immobilized anti-CD3/28 mAbs for 5 d, treatedwith KD025, and then restimulated with anti-CD3/28 mAbs for an additional 48 h. The supernatants were analyzed for IL-21, IL-17, and IFN-γ by ELISA. The dataare presented as percent of inhibition, and the average of three different experiments is shown (A). CD4+ T cells were treated with KD025 at 0.1 to 10 μM for1 h and then stimulated by immobilized anti-CD3/28 mAbs in combination with IL-1β (50 ng/mL) and TGF-β (5 ng/mL). IL-2 and IL-17F secretion was analyzed byELISA after 48 h (B and D). The average of five (B) or three (D) different experiments is shown. Proliferation of cells was defined by CFSE dilution after 4 d (C).The average of three different experiments is shown. The percentage of IL-17F– and IL-17E–producing T cells was determined by intracellular staining byFACS (D).
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Fig. S7. ROCK2 but not ROCK1 inhibition leads to down-regulation of STAT3 phosphorylation and reduces protein levels of RORγt and IRF4. Peripheral bloodCD4+ T cells were transfected with siRNA targeting ROCK1 and ROCK2 or with control siRNA (A, C–E) or treated with KD025 (B) and then stimulated by anti-CD3/28 mAbs, IL-1β (50 ng/mL), and TGF-β (5 ng/mL) for 48 h. Whole cell extracts were prepared and analyzed by Western blot by using antibodies againstpSTAT3 (A), IRF4 (A), RORγt (A), pSTAT1 (B and C), pSTAT4 (B and D), pSTAT6 (B), and pMLC (E). One representative of three different experiments is shown.
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a
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Fig. S8. KD025 down-regulates TGF-β–induced MLC phosphorylation in a SMAD2/3-independent manner. Human CD4+ T cells were treated with KD025 andthen activated by TGF-β (5 ng/mL) for 1 h. Whole cell lysates were analyzed by Western blot for MLC phosphorylation. One representative of three differentexperiments is shown (A). IL-10 secretion was analyzed by ELISA after 48 h. The average of five different experiments is shown (B).
Fig. S9. Purification of human Tregs. CD4+CD25highCD127lo T cells were purified from healthy donors by positive selection by MACS (about 80% of Foxp3+).
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asample age gender ethnicity DAS
score1 64 F Hispanic 5.982 34 F Hispanic 3.813 42 F Hispanic 2.235 35 F Hispanic 6.156 43 M Hispanic 6.237 F Hispanic 3.198 45 F Hispanic 3.69 42 F Hispanic 6.7510 32 F Hispanic 1.911 53 F Hispanic 6.7812 31 F Hispanic 2.44
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RA
Healthy
Fig. S10. CD4+ T cells purified from RA patients secrete higher amount of IFN-γ compared with healthy controls upon stimulation ex vivo. The clinical anddemographic details of studied RA patients are as follows: DAS, disease activity score; F, female; M, male (A). Peripheral blood CD4+ T cells from RA patientswere stimulated by immobilized anti-CD3/28 mAbs. IL-21, IL-17, and IFN-γ secretion was analyzed by ELISA after 48 h (B). The P value was calculated by t test.Whole cell lysates were analyzed by Western blot for STAT4 phosphorylation (C) and Tbet expression (D). One representative of three different experiments isshown (C and D).
Zanin-Zhorov et al. www.pnas.org/cgi/content/short/1414189111 10 of 12
Fig. S11. The effect of KD025 on CD4+ T cells during the progression of CIA in mice. The treatment with indicated doses of KD025 began on day 25 afterimmunization with collagen. After 28 d, mice were killed, and flow cytometry analysis of spleenocytes and lymph node cells was performed. Data are shown asaverage percentage (n = 10) ± SD of total CD4+ T cells.
Zanin-Zhorov et al. www.pnas.org/cgi/content/short/1414189111 11 of 12
Molecular Target Target Iden fica on
Kinases1 ROCK1, Abl, Akt1, Akt2, AuroraA, Cdk1, Cdk2, CHK1, CK2, c-kit, Erk1, Erk2, p38α, p38β,
p38δ, p38γ, DMPK, DRAK, ErbB4, Flt1, Flt4, IKKα, KDR, MAP, MLCK, MRCKα, MRCKβ,
MSK1, MSSK1, p70S6K, PDGFα, PHK2, PKA, PKCα, PKGα, PKGβ, PRK1, PRK2, Pyk2, RSK2,
Src
Cell Surface
Receptors2
Adrenergic: α1, α1, β
Dopamine
(nonselec ve)
Muscarinic
(nonselec ve)
Nico nic: TTX (+), TTX
(-)
Norepinephrine
Transporter
Opioid
Serotonergic
(nonselec ve)
Na+ Type 2
Glutamate: AMPA, KA, NMDA
Glycine (spinal)
Glycine (central)
Calcium: Type L, Type N
GABA
P2X
P2Y
Potassium: ATP sensi ve, Ca++ ac vated, I [Kr]hERG
1 – Inhibi on at 10 μM < 40 % to 0 %
2 – No inhibi on was observed with KD025
Activity of KD025 against ATP-dependent Kinases and Cell Surface Receptors
Fig. S12. Activity of KD025 against ATP-dependent kinases and cell-surface receptors.
Zanin-Zhorov et al. www.pnas.org/cgi/content/short/1414189111 12 of 12
