Simvastatin promotes Th2-type responses throughthe induction of the chitinase family member Ym1in dendritic cellsMeenakshi Arora*, Li Chen*, Melissa Paglia*, Iain Gallagher†, Judith E. Allen†, Yatin M. Vyas‡§, Anuradha Ray*§,and Prabir Ray*§¶

Departments of *Medicine, Division of Pulmonary, Allergy, and Critical Care Medicine and §Immunology, University of Pittsburgh Schoolof Medicine, Pittsburgh, PA 15213; †Institute of Immunology & Infection Research, University of Edinburgh, Edinburgh EH9 3JT, United Kingdom;and ‡Department of Pediatrics, Division of Hematology and Oncology, Children’s Hospital of Pittsburgh, Pittsburgh, PA 15213

Edited by William E. Paul, National Institutes of Health, Bethesda, MD, and approved March 31, 2006 (received for review September 28, 2005)

Statins, best known for their lipid-lowering actions, also possessimmunomodulatory properties. Recent studies have shown a Th2-biasing effect of statins, although the underlying mechanism hasnot been identified. In this study, we investigated whether sim-vastatin can exercise a Th2-promoting effect through modulationof function of dendritic cells (DCs) without direct interaction withCD4� T cells. Exposure of DCs to simvastatin induced the differ-entiation of a distinct subset of DCs characterized by a highexpression of B220. These simvastatin-conditioned DCs up-regu-lated GATA-3 expression and down-regulated T-bet expression incocultured CD4� T cells in the absence of additional simvastatinadded to the coculture. The Th2-biased transcription factor profileinduced by simvastatin-treated DCs also was accompanied byincreased Th2 (IL-4, IL-5, and IL-13) and decreased Th1 (IFN-�)cytokine secretion from the T cells. The Th2-promoting effect ofsimvastatin was found to depend on the chitinase family memberYm1, known to be a lectin. Anti-Ym1 antibody abolished theTh2-promoting effect of simvastatin-treated DCs. Also, simvastatinwas unable to augment Ym1 expression in DCs developed fromSTAT6�/� or IL-4R��/� mice. Thus, modulation of Ym1 productionby DCs identifies a previously undescribed mechanism of Th2polarization by statin.

statin � T cells � IL-4R� � STAT6 � GATA-3

Dendritic cells (DCs) are professional antigen-presenting cellsthat have the ability to stimulate naı̈ve T cells and initiate a

primary immune response. DCs also play a critical role in mediatingthe differentiation of CD4� T cells into Th1- and Th2-polarizedsubsets (1). T cell differentiation along the Th1 lineage is regulatedby specific transcription factors such as T-bet, which plays anessential role in IFN-� production (2). The master regulator of Th2differentiation is GATA-3 as we and others described in refs. 3–7.

Statins are the most potent cholesterol-lowering drugs that targetthe enzyme 3-hydroxy-3-methyl-gutaryl-CoA reductase (8). Recentin vitro findings indicate that statins also have potent immunoregu-latory activity (9). These new effects of statins have been describedas potential treatment options against autoimmune diseases (10–12). Oral administration of atorvastatin prevented paralysis in micevia suppression of Th1 and augmentation of Th2 responses in astudy of experimental autoimmune encephalomyelitis (11). Thereare likely to be several molecular mechanisms through which statinsexert their immunomodulatory effects (13), but these mechanismshave yet to be elucidated. Recent studies have suggested that theTh2-biasing effects of statins may be induced via direct effects ofstatins on T cells (14) and antigen-presenting cells (11, 14, 15).However, whether statins can indeed promote Th2 differentiationvia direct effects on antigen-presenting cells has not been shown.Because DCs are the key antigen-presenting cells that activate naı̈veT cells (16), we addressed the influence of simvastatin on thedifferentiation and function of DCs. We show that simvastatin hasa direct effect on DC function, which instructs DCs to drive T cell

differentiation toward the Th2 lineage. Treatment of DCs withsimvastatin up-regulated expression of the molecule Ym1 on DCs.Ym1 is a member of a family of mammalian proteins that sharehomology to chitinases of lower organisms (17, 18). Chitinases havebeen recently associated with the development of allergic airwaysdisease (19). Ym1 does not have enzyme activity but has beencharacterized as a lectin with specific binding affinity for heparin�heparan sulfate (18). Antibody-mediated neutralization of Ym1abolishes the Th2-polarizing effect of simvastatin-treated DCs.Also, DCs generated from STAT6�/� mice or IL-4R��/� micefailed to up-regulate Ym1 production upon simvastatin treatment.Our studies show that simvastatin-induced augmentation of Th2responses depends on Ym1 production by DCs, which requires theIL-4R��STAT6 signaling axis.

Results and DiscussionSimvastatin Induces High Expression of B220. To determine the effectof simvastatin on DCs, bone marrow cells were cultured withgranulocyte�macrophage colony-stimulating factor for 6 days, fol-lowed by purification of CD11c� cells (�95% purity). Phenotypi-cally, the CD11c� cells were essentially B220� (Fig. 1A). TheCD11c� DCs then were grown in the presence or absence of 1 �Msimvastatin for 2 days, and multicolor flow cytometric analysis wasperformed. Simvastatin treatment was found to induce the differ-entiation of a distinct subset of DCs characterized by high expres-sion of B220 (Fig. 1A). The control DCs and simvastatin-treatedDCs displayed typical DC-type morphology, as observed by lightand electron microscopy (Fig. 1B). Although the control DCsadhered well to glass, the simvastatin-treated DCs adhered less welland were somewhat rounder. Overall, the morphology of thesimvastatin-treated DCs was similar to the description of murineand human pDCs as we and others have described (ref. 20; Fig. 1B).

To further characterize the different DC subsets, the expressionof additional cell surface molecules was examined. DCs generatedin the presence or absence of simvastatin expressed high levels ofCD11b and DEC-205 but no Gr1 or CD19 (Fig. 1C). Importantly,the B220� DCs were not B cells because they lacked the lineagemarker for B cells, CD19. These phenotypic characteristics clearlyshowed that the DCs that developed in the presence of simvastatinwere different from classical CD11chi DCs or CD11cloB220hi plas-macytoid DCs.

We then examined the effect of different doses of simvastatin onthe expression of B220 on DCs. B220 expression was augmented bysimvastatin in a dose-dependent fashion leveling off at a dose of 5�M (Fig. 1D). This up-regulation of B220 was confirmed to be a

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: DC, dendritic cell.

¶To whom correspondence should be addressed. E-mail: rayp@pitt.edu.

© 2006 by The National Academy of Sciences of the USA

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statin-specific effect, because it could be reversed by the addition ofmevalonate (Fig. 1D).

To address whether simvastatin treatment affects DC viability,DCs were cultured with different concentrations of simvastatin, andcell death was determined by using propidium iodide. Simvastatintreatment did not induce cell death in DCs at lower doses (1 �M),but appreciable cell death was observed at higher doses of simva-statin (10 �M) (data not shown). Therefore, all further experimentswere performed by using 1 �M simvastain.

Simvastatin Promotes Th2 Development and Inhibits Th1 Develop-ment in Vitro. DCs are instrumental in the differentiation of naı̈ve Tcells into Th1 or Th2 effector cells. To determine whether CD4�

T cells respond differently to simvastatin-treated DCs, naı̈ve CD4T cells from DO11.10 TCR transgenic mice were incubated withcontrol or simvastatin-treated DCs for 5 days in the presence ofOVA peptide, which is specific for the DO.11 TCR, and withoutfurther addition of statin. The simvastatin-treated DCs were ex-tensively washed before coculture to remove any adhering statin. Atthe end of the 5-day culture period, supernatants were harvested forestimation of cytokine production, and nuclear extracts were pre-pared from the cells to investigate the expression of the transcrip-tion factors T-bet, which is Th1-specific (21), and GATA-3, whichis Th2-specific (3, 4). As shown in Fig. 2, as expected, untreated DCsinduced T cells to secrete a mixture of both Th1-type (IFN-�) andTh2-type cytokines (IL-4, IL-5, and IL-13). When CD4� T cellswere cocultured with simvastatin-treated DCs, production of Th2cytokines was augmented, whereas IFN-� secretion was inhibited(Fig. 2A). The cytokine secretion profile correlated with increasedexpression of GATA-3 and decreased expression of T-bet by the T

cells cultured with simvastatin-treated DCs. Used as a negativecontrol, incubation of either DC type with T cells in the absence ofantigenic peptide gave no detectable response (data not shown).Because Th1 cells are more prone to apoptosis compared with Th2cells, we also investigated T cell proliferation. Simvastatin-pretreated DCs were found to be as efficient as control DCs ininducing proliferation of antigen-specific or allogeneic (data notshown) T cells (Fig. 2C). Taken together, our results demonstratethat pretreatment of DCs with simvastatin alters DC function that,in turn, influences T cell differentiation.

The impact of DCs on T cell polarization is influenced by manyfactors, including the specific DC subset, the activation status ofDCs, and the cytokine microenvironment. Because some studiessuggest a role for the costimulatory molecules CD80 and CD86 inT cell polarization, we investigated whether simvastatin modulatedexpression of these molecules on DCs. As reported in ref. 14,simvastatin treatment did not impact the expression of thesemolecules on DCs (Fig. 3A). Simvastatin pretreatment did notmodulate CD40 expression either, which is important for Th1polarization (Fig. 3A). Analysis of other DC cell surface moleculessuch as OX40L, ICOSL, and ICAM-1, which are known to influ-ence Th polarization, also was not modulated by simvastatin (datanot shown). As reported, when using lovastatin (22), simvastatin-treated DCs were found to have a similar ability as control DCs toendocytose antigen (Fig. 6, which is published as supporting infor-mation on the PNAS web site).

T helper differentiation is largely determined by the cytokinemicroenvironment, and Th1 and Th2 cytokines are known tocross-regulate each other (7). For example, although IL-4 pro-motes Th2 differentiation but inhibits Th1 differentiation, IFN-�

Fig. 1. Phenotype and morphology of simvastatin-treated DCs. (A) Bone marrow progenitors were grown in the presence of granulocyte�macrophagecolony-stimulating factor (10 ng�ml) for 6 days, and CD11c� DCs were purified. CD11c� DCs were cultured further in the presence or absence of simvastatin (1�M) for 2 days. Phenotypic analysis then was performed by flow cytometry. Thin lines indicate staining with isotype controls. (B) Light and scanning electronmicroscopy of DCs cultured in the presence or absence of simvastatin. (C) Expression of CD11b, Gr1, CD19, and DEC-205 on DCs cultured in the presence or absenceof simvastatin. (D) Effect of different doses of simvastatin on the expression of B220. The numbers denote percentages of cells that are specifically stained. Theexperiments were repeated at least four times with similar results.

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promotes Th1 differentiation but inhibits Th2 differentiation (7).In this context, GATA-3 and T-bet, as well as STATs, play animportant role in cross-regulation between Th1 and Th2 cells.Because IL-12 produced by antigen-presenting cells promotesTh1 differentiation, we assessed the level of IL-12p70 andIL-12p40 in culture supernatants of simvastatin-treated andcontrol DCs. Although IL-12p70 was low in either supernatant,IL-12p40 was readily detectable, but its secretion was lower fromsimvastatin-treated DCs (Fig. 7, which is published as supportinginformation on the PNAS web site). Inhibition of p40 inductionby simvastatins, in turn, would inhibit IL-12 production by DCsthat would down-regulate IFN-� production by T cells. We nextinvestigated whether inhibition of IFN-� production by simvas-tatin-treated DCs played a role in the promotion of Th2 devel-opment. Toward this end, simvastatin-treated DCs were cocul-tured with DO.11 T cells and antigen in the presence or absenceof recombinant IFN-�. Although addition of IFN-� enhancedT-bet expression in the CD4� T cells, it did not inhibit GATA-3expression, even at the higher dose of the cytokine (250 units�ml). In fact, for reasons presently unclear, IL-13 secretion wasgreater at the higher dose of IFN-�. These results suggested thatthe increase in Th2 differentiation by simvastatin-treated DCswas a dominant Th2-skewing effect of the DCs that was notsubject to inhibition by IFN-�.

We examined whether the Th2-skewing effect of simvastatin-treated DCs was subject to inhibition by IL-12. As shown in Fig. 3C,both GATA-3 expression and Th2-cytokine production were sig-nificantly reduced in the presence of IL-12 in a dose-dependentfashion. Thus, statin is unable to stimulate DCs to induce Th2skewing in the presence of IL-12. Collectively, our data suggest thatIL-12-induced signaling pathways, such as STAT4 but not IFN-�-induced pathways (such as STAT1), block the ability of statin topromote Th2 differentiation.

Ym-1 Induces the Development of Th2-Type Responses. To furtherelucidate the mechanism underlying Th2 polarization by simvasta-tin, we performed microarray analysis of RNA samples isolatedfrom DCs cultured with or without simvastatin. RNA was hybrid-ized to Codelink Uniset Mouse Expression Bioarray (GE Health-care), which identifies 10,012 unique murine genes. RNA was

isolated from the two sets of DCs in three independent experi-ments. For data analysis, SCOREGENE (23) was used for compari-sons between the processed arrays and for calculating fold changein transcript levels of genes by using various statistical methods. Thegenes whose expression was consistently increased or decreasedwere selected and were further filtered based on Student’s t test.The microarray analysis identified 408 genes that were eithersignificantly repressed or induced by simvastatin (P � 0.05). Inter-estingly, the Ym1 gene, which was previously shown to be up-regulated by Th2 cytokines in macrophages (24), was up-regulatedby simvastatin treatment. Increased expression of Ym1 at theprotein level in simvastatin-treated DCs was confirmed by immu-noblot analysis of cell lysates by using an anti-Ym1 antibody (Fig.4). In previous studies, stimulation of bone marrow-derived DCswith IL-4 was shown to induce Ym1 expression (25). Because Ym1is known to be a secreted protein (24, 25), we also checked the levelof Ym1 in the culture supernatants by immunoblotting techniques.As expected from our analysis of cell lysates, a higher level of Ym1was detected in the culture supernatant of simvastatin-treated DCs.To determine whether Ym1 is expressed on the DC plasmamembrane in addition to being secreted by DCs, we performedimmunofluorescence studies. As shown in Fig. 8, which is publishedas supporting information on the PNAS web site, Ym1 does notappear to be expressed on the DC plasma membrane. Ym1 hasbeen shown to be induced in macrophages in a STAT6-dependentmanner (26). Therefore, we investigated Ym1 induction by simva-statin in STAT6-deficient DCs. The induction of Ym1 appeared tobe strictly regulated by STAT6, because we did not detect anysecretion of Ym1 in the absence of STAT6 (Fig. 4). Becausemultiple groups have shown that Ym1 is up-regulated by the Th2cytokines IL-4 and IL-13 (24, 26), we further investigated whetherthe absence of Ym1 influences the Th2-inducing ability of statin-treated DCs. Statin-treatment of STAT6-deficient DCs did notenhance IL-13 production in CD4� T cells, suggesting that Ym1might play an important role in the ability of simvastatin-pretreatedDCs to promote Th2 polarization. Activation of STAT6 is down-stream of IL-4�IL-13 signaling mediated by the common IL-4R�subunit in the IL-4 and IL-13 receptor complexes. To investigate theinvolvement of IL-4R� in Ym1 up-regulation by statin, IL-4R�-deficient DCs were treated with statin and analyzed for Ym1

Fig. 2. DCs treated with simvastatin polarize naı̈ve CD4� T cells toward the Th2 phenotype in vitro. (A) DCs were treated with simvastatin (1 �M) or left untreatedfor 2 days. Cells were then washed and cultured with naı̈ve DO11.10 CD4� T cells and OVA peptide (5 �g�ml) for 5 days. Nuclear extracts were analyzed byimmunoblotting with antibodies against GATA-3, T-bet, or CREB-1. Each blot was scanned by densitometry, and the ratio of GATA-3 or T-bet to CREB-1 wascalculated and presented. *, P � 0.001; **, P � 0.001; ***, P � 0.01; ****, P � 0.01, relative to an untreated group. (B) Cytokines in culture supernatants asdetermined by ELISA. Data are the mean � SEM of individual wells and are representative of three independent experiments. (C) Similar proliferation of DO11.10TCR transgenic T cells induced by simvastatin-treated or control DCs. The experiment was repeated two times with similar results.

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production. As shown in Fig. 4C, no Ym1 was detected from theDCs lacking IL-4R�, indicating that simvastatin does requireIL-4R� to up-regulate Ym1. Also, similar to STAT6-deficient DCs,DCs lacking IL-4R� did not promote Th2 differentiation uponsimvastatin treatment (Fig. 4C). Because both IL-4R� and STAT6participate in signaling by IL-4 and IL-13 and DCs do not produceeither IL-4 or IL-13, we examined Ym1 induction by simvastatin inthe presence of neutralizing anti-IL-4 or anti-IL-13 antibody toexclude the possibility of any IL-4 or IL-13 contamination in ourcultures. As shown in Fig. 4D, the level of simvastatin-induced Ym1did not change in the presence of either of the neutralizingantibodies. It is unclear how statin utilizes the IL-4R��STAT6 axisfor up-regulation of Ym1 expression, and it will be interesting to see

possible effects of statins on positive (JAKs) and negative regula-tors (SOCS and SHP-1) of STAT6.

Because Ym1 is a secreted molecule and the effect of simvastatinon Th2 polarization is detected even after washing off any adheringstatin from the DCs, we investigated whether simvastatin-treatedDCs continue to make Ym1, even after removal of statin from theculture. DCs were treated with simvastatin for 2 days, extensivelywashed to remove statin, and then cultured for another 3 days in theabsence of statin. Ym1 expression then was examined in the celllysates. A high level of Ym1 still was observed in these DCs ascompared with a low, but detectable, basal level of Ym1 in theuntreated cells (Fig. 4E).

To further confirm a direct association between Ym1 up-

Fig. 3. (A) Effect of simvastatin on the expression of costimulatory molecules on DCs. Thin lines indicate staining with isotype controls. (B) Addition of IFN-�did not reverse Th2 polarization induced by simvastatin-treated DCs. Although IFN-� did not inhibit GATA-3 expression, it augmented T-bet expression insimvastatin-treated DCs. (C) Addition of IL-12 significantly reduced both GATA-3 expression and Th2 cytokine production in a dose-dependent fashion. Culturesupernatants in B and C were assayed for cytokine production by ELISA.

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regulation by simvastatin and the observed Th2-promoting effectof the statin-treated DCs, different doses of the anti-Ym1antibody were added to DC-T cell cocultures. As shown in Fig.5 A and B, both GATA-3 expression and Th2 cytokine produc-tion were reduced significantly in the presence of anti-Ym1 butnot control antibody in the cocultures. These data indicate thatYm1 plays an important role in the ability of simvastatin-pretreated DCs to promote Th2 polarization and provides amechanism for the observed Th2 skewing of T cells by simvas-tatin-treated DCs.

To determine whether Ym1 up-regulation by simvastatin-pretreated DCs is the mechanism by which DCs polarize naı̈veT cells toward Th2, irrespective of the source�origin of DCs, wecocultured simvastatin-treated splenic DCs with naı̈ve T cells.Similar to bone marrow DCs, splenic DCs also enhanced Th2development upon simvastatin treatment. Splenic DCs thenwere cocultured with naı̈ve T cells in the presence of a anti-Ym1or control antibody. Addition of anti-Ym1 to the culturescompletely abrogated the development of Th2 cells in a dose-dependent manner (Fig. 5 C and D).

Fig. 4. Increased STAT6-dependent Ym1 expression in DCs treated with simvastatin. Cell lysates (A) and culture supernatants (B) of control and simvastatin-treated DCs were subjected to immunoblotting with anti-Ym1 antibody. The experiment was repeated three times with similar results, and shown is arepresentative experiment. Ym1 secretion was undetectable when DCs were isolated from Stat6�/� mice (B) or IL4R��/� mice (C). (D) Addition of neutralizingantibodies to IL-4 (Upper) or IL-13 (Lower) to DC culture did not change the level of Ym1 induction by simvastatin. (E) Ym1 expression in DCs that were pretreatedwith simvastatin for 2 days, washed to remove statin, and then cultured for another 3 days.

Fig. 5. Anti-Ym1 antibody reverses the ability of DCs treated with simvastatin to drive Th2 polarization. Bone marrow DCs were treated with simvastatin for2 days and then cocultured with CD4� T cells from DO11.10 mice in the presence of different doses of anti-Ym1 or control antibody (rabbit IgG) for 5 days. GATA-3levels in nuclear extracts (A) was assessed and culture supernatants were analyzed by ELISA for IL-5 and IL-13 (B). *, P � 0.005; **, P � 0.005; ***, P � 0.005; ****,P � 0.005. Results are representative of three independent experiments. (C and D) Similar results were obtained in the case of splenic DCs. *, P � 0.001; **, P �0.001; ***, P � 0.005; ****, P � 0.05.

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Because the concentration of statin used in our study corre-sponds to therapeutic doses (1–10 �M), we were curious whetherour in vitro results might reflect similar effects of simvastatin in vivo.Mice were fed a diet containing simvastatin for 4 weeks, andexpression of Ym1 in lung DCs and the effect of these DCs, in turn,on CD4� T cells were studied. The results of this experiment (Fig.9, which is published as supporting information on the PNAS website) support a Th2-promoting effect of simvastatin in vivo. It wouldbe important to determine whether statin intake over a longerperiod further consolidates the Th2 response.

In summary, we have shown that simvastatin instructs DCs todrive T cell differentiation toward the Th2 lineage. Our studiesprovide a mechanism for the recently described Th2-promotingeffects of statins (11) via up-regulation of Ym1 production by DCs.Although Ym1 expression has been shown to be induced by Th2cytokines in macrophages and DCs (24–26), we show that Ym1produced by DCs also exerts effects on T cells to promote Th2differentiation. Interestingly, 10% of transcripts induced in macro-phages by helminths encode Ym1 (27), and these macrophagespromote Th2 differentiation (28). Ym1 is a lectin with heparin-binding characteristics (18). T cell differentiation is regulated atmany levels by many factors (6, 7, 29–31). Although heparan sulfateproteoglycan expressed by T cells has been associated with cytokineand chemokine binding to T cells and with cell adhesion (32, 33),its role in T cell differentiation is presently unclear. It will beinteresting to see whether the Th2-inducing property of Ym1, asidentified in this study, is mediated by interactions with heparansulfate expressed on T cells.

Materials and MethodsAnimals and Reagents. Male 6- to 8-week-old BALB�c mice, STAT-6�/� mice, and IL-4R��/� mice were purchased from The JacksonLaboratory. DO11.10 T cell TCR-transgenic mice were bred andmaintained in the Department of Laboratory Animal Resources atthe University of Pittsburgh. All studies with animals were ap-proved by the Institutional Animal Care and Use Committee.

Recombinant granulocyte�macrophage colony-stimulating fac-tor was purchased from PeproTech (Rocky Hill, NJ). Mevalonatewas obtained from Sigma. Simvastatin was a gift from Merck.Antibodies were purchased from BD Biosciences and SouthernBiotech (Birmingham, AL). Anti-Ym1 was generated as describedin ref. 25. Anti-DEC-205 (biotin) was a gift of Dr. Robert Hendricks(University of Pittsburgh).

Bone Marrow-Derived Dendritic Cell Generation. Femurs of micewere aseptically removed and cleared from surrounding muscle.Bone marrow cells were isolated by flushing femurs, and the cellswere grown in a medium supplemented with 10 ng�ml granulocyte�

macrophage colony-stimulating factor. At day 6, nonadherent cellswere collected, and CD11c� cells were purified by positive selectionwith MACS CD11c microbeads (Miltenyi Biotec, Auburn, CA)according to the manufacturer’s instructions.

Isolation and Purification of Spleen Dendritic Cells. Spleen DCs wereisolated by minor modifications of ref. 34; see also SupportingMethods, which is published as supporting information on thePNAS web site).

Phenotypic Evaluation of DCs. Cell surface-antigen expression onDCs was evaluated by flow cytometry as described in ref. 20.

Light and Scanning Electron Microscopy. For light microscopy, cyto-spin preparations of cells were used. Cells were stained withHema-3 reagents (Fisher Scientific). For scanning electron micros-copy, cells were allowed to settle onto glass coverslips in PBS for 1 hat 37°C, fixed, and analyzed as described in ref. 20.

T Cell Proliferation Assay. CD4� T cells were isolated by positiveselection and cocultured with �-irradiated DCs. After 72 h, thecultures were pulsed with 1 �Ci [3H]thymidine (1 Ci � 37 GBq) for16 h before harvesting.

Preparation of Nuclear Extracts and Immunoblot Analysis. Nuclearextracts were prepared as described in ref. 3 and analyzed byimmunoblotting techniques.

Cytokine Assays. Cytokine concentrations were measured by ELISAwith commercially available kits for IL-4, IL-5, IFN-�, and IL-10(R & D Systems).

RNA Isolation, Microarray, and Data Analysis. Total RNA was iso-lated from DCs incubated with or without simvastatin and was usedto probe Codelink arrays. Probe synthesis and hybridization wereperformed, and the hybridized chip was scanned by using GenePix400 B array scanner. The scanned images were analyzed by usingdifferent statistical methods.

Statistical Analysis. All results are expressed as the mean � SEM.Student’s t tests were performed, and differences between thegroups were considered significant if P � 0.05.

We thank members of the Center for Biologic Imaging at the Universityof Pittsburgh for help with electron microscopy, M. Yarlagadda fortechnical assistance, T. Richards and N. Kaminski for help with mi-croarray analysis, and Winifred Huang for assistance with fluorescencemicroscopy. This work was supported by National Institutes of HealthGrants R01 HL60207, R01 HL69810, R01 HL77430, and R01 AI48927and a grant from the Wellcome Trust.

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