Novel Regulation of CD80/CD86-induced Phosphatidylinositol 3 ...

Novel Regulation of CD80/CD86-inducedPhosphatidylinositol 3-Kinase Signaling by NOTCH1 Proteinin Interleukin-6 and Indoleamine 2,3-DioxygenaseProduction by Dendritic Cells*�

Received for publication, September 16, 2013, and in revised form, January 10, 2014 Published, JBC Papers in Press, January 10, 2014, DOI 10.1074/jbc.M113.519686

Chandana Koorella‡, Jayakumar R. Nair‡, Megan E. Murray‡, Louise M. Carlson‡, Stephanie K. Watkins§1,and Kelvin P. Lee‡2

From the ‡Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263 and the §National Cancer Institute-Frederick, National Institutes of Health, Frederick, Maryland 21702

Background: Engagement of CD80/CD86 on dendritic cells by CD28 on T cells induces dendritic cell production of IL-6and IDO.Results: The NOTCH pathway modulates activation of the PI3K pathway downstream of CD80/CD86 ligation and regulatesIL-6 and IDO production.Conclusion: Cross-talk between NOTCH and PI3K pathways modulates dendritic cell production of IL-6 and IDO.Significance: Elucidating the molecular mechanism of NOTCH-PI3K cross-talk will have broad implications in human disease.

Dendritic cells (DC) play a critical role in modulating antigen-specific immune responses elicited by T cells via engagement ofthe prototypic T cell costimulatory receptor CD28 by the cog-nate ligands CD80/CD86, expressed on DC. Although CD28 sig-naling in T cell activation has been well characterized, it has onlyrecently been shown that CD80/CD86, which have no demon-strated binding domains for signaling proteins in their cytoplas-mic tails, nonetheless also transduce signals to the DC. Func-tionally, CD80/CD86 engagement results in DC production ofthe pro-inflammatory cytokine IL-6, which is necessary for fullT cell activation. However, ligation of CD80/CD86 by CTLA4also induces DC production of the immunosuppressive enzymeindoleamine 2,3-dioxygenase (IDO), which depletes local poolsof the essential amino acid tryptophan, resulting in blockade ofT cell activation. Despite the significant role of CD80/CD86 inimmunological processes and the seemingly opposing roles theyplay by producing IL-6 and IDO upon their activation, howCD80/CD86 signal remains poorly understood. We have nowfound that cross-linking CD80/CD86 in human DC activates thePI3K/AKT pathway. This results in phosphorylation/inactiva-tion of its downstream target, FOXO3A, and alleviatesFOXO3A-mediated suppression of IL-6 expression. A secondevent downstream of AKT phosphorylation is activation of thecanonical NF-�B pathway, which induces IL-6 expression. Inaddition to these downstream pathways, we unexpectedly foundthat CD80/CD86-induced PI3K signaling is regulated by previ-ously unrecognized cross-talk with NOTCH1 signaling. Thiscross-talk is facilitated by NOTCH-mediated up-regulation of

the expression of prolyl isomerase PIN1, which in turn increasesenzyme activity of casein kinase II. Subsequently, phosphataseand tensin homolog (which suppresses PI3K activity) is inacti-vated via phosphorylation by casein kinase II. This results in fullactivation of PI3K signaling upon cross-linking CD80/CD86.Similar to IL-6, we have found that CD80/CD86-induced IDOproduction by DC at late time points is also dependent upon thePI3K 3 AKT 3 NF-�B pathway and requires cross-talk withNOTCH signaling. These data further suggest that the same sig-naling pathways downstream of DC CD80/CD86 cross-linkinginduce early IL-6 production to enhance T cell activation, fol-lowed by later IDO production to self-limit this activation. Inaddition to characterizing the pathways downstream of CD80/CD86 in IL-6 and IDO production, identification of a novelcross-talk between NOTCH1 and PI3K signaling may providenew insights in other biological processes where PI3K signalingplays a major role.

Antigen-specific activation of T cells is critically dependentupon costimulatory signals provided by their interaction withantigen-presenting cells, in particular dendritic cells (DC).3CD28 engagement on T cells by its natural ligands CD80 andCD86 expressed on DC plays a central role in T cell activationby delivery of costimulation (1). The biological consequences ofCD28 ligation in T cells are well described and result in prolif-eration, augmented metabolic efficiency, and effector function(2– 4). However, it has only recently been appreciated that

* This work was supported, in whole or in part, by National Institutes of HealthGrants R01 CA140622, R01 CA121044, R01 AI10015, and T32 CA085183.This work was also supported by the Multiple Myeloma ResearchFoundation.

� This article was selected as a Paper of the Week.1 Present address: Dept. of Surgery, Oncology Institute, Loyola University Chi-

cago, Maywood, IL 60559.2 To whom correspondence should be addressed. Tel.: 716-845-4106; Fax:

716-845-2993; E-mail: [email protected].

3 The abbreviations used are: DC, dendritic cell; PC, plasma cell; MM, multiplemyeloma; PIP3, phosphatidylinositol 1,4,5-phosphate; BMDC, bone mar-row derived dendritic cell; FOXO3A, Forkhead Box O3a; NF-�B, nuclear fac-tor �B; PTEN, phosphatase and tensin homolog; NICD, NOTCH intracellulardomain; NRR, negative regulatory region; PIN1, peptidyl-prolyl isomerase1; CK, casein kinase; IDO, indoleamine 2,3-dioxygenase; PHB, prohibitin;Mo-DC, monocyte-derived DC; N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 289, NO. 11, pp. 7747–7762, March 14, 2014Published in the U.S.A.

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CD80/CD86 are not merely ligands but are also capable of sig-naling. In this context, it has been shown that cross-linkingCD80/CD86 by CD28 induced DC production of IL-6 (5),which is required for full T cell activation (6, 7). Conversely, ithas also been shown that ligation of CD80/CD86 by CTLA4 (areceptor belonging to the CD28 superfamily) results in the pro-duction of indoleamine 2,3-dioxygenase (IDO), which catabo-lizes the essential amino acid tryptophan to L-kynurenine,resulting in inhibition of T cell activation (8 –10).

In comparison with T cell CD28 signaling, substantially lessis known about CD80/CD86 signaling, especially in human DC.Constraining the understanding of CD80/CD86 signaling isthat these membrane proteins have cytoplasmic tails that donot have demonstrated binding domains for other signalingmolecules (with the exception of the binding of the prohibitinadaptor proteins to the CD86 tail in B cells (11)). Previous stud-ies have demonstrated that p38 MAPK and NF-�B are activatedupon CD80/CD86 ligation in murine splenic DC IL-6 produc-tion (5) and in another study that the transcription factorFOXO3A regulated IL-6 production (12). However, whetherp38 MAPK, NF-�B, and FOXO3A regulate or coordinate witheach other in an integrated signaling pathway in CD80/CD86-induced DC IL-6 production is unknown.

In addition to playing a central role in T cell activation, IL-6 isalso a major growth/survival factor for both normal and malig-nant plasma cells (e.g. multiple myeloma (MM)) (13, 14). Weand others have previously found that normal plasma cells (PC)and myeloma cells express CD28 and that activation of PC/MMCD28 by CD80/CD86� DC transduced a major pro-survivalsignal to the PC/MM (15, 16). Furthermore, we found thatPC/MM CD28-mediated CD80/CD86 cross-linking alsoinduced DC IL-6 production (15, 16), similar to what has beenreported for T cells. Paralleling these observations, it has beenreported that the NOTCH-JAGGED receptor-ligand pair isalso involved in myeloma-induced stromal IL-6 production(17). Thus, the importance of DC IL-6 production for both Tcell activation and PC/MM survival led us to characterize howCD80 and CD86 were inducing IL-6 production, whetherNOTCH1 signaling was involved, and whether IDO productionwas regulated through the same pathways.

EXPERIMENTAL PROCEDURES

Mice, Cell Cultures, and Flow Cytometric Analysis—FemaleC57BL/6J (WT) mice were purchased from The Jackson Labo-ratory at 5– 6 weeks of age. Upon receipt, animals were housedat the Division of Laboratory Animal Resources (Roswell ParkCancer Institute) in a pathogen-free facility. All animal experi-ments were approved by the Roswell Park Cancer InstituteInstitutional Animal Care and Use Committee. Murine bonemarrow mononuclear cells were differentiated as describedpreviously (15) to obtain BMDC and were analyzed by flowcytometry for CD40, CD80, CD86, CD11b, CD11c, MHC I, andMHC II (all antibodies were conjugated to phycoerythrin andpurchased from BioLegend) expression using FACSCalibur II(15). Data were analyzed using the FCS Xpress software.

Antibodies and Reagents—Antibodies for detecting p85,NOTCH1intracellularfragment(NICD),JAGGED2,phosphor-ylated AKT (Thr-308), phosphorylated and total amounts of

FOXO3A and PTEN, and PIN1 were purchased from Cell Sig-naling Technology. Pan-AKT antibody was purchased fromR&D Systems, and the IDO antibody was purchased from Mil-lipore. The anti-NRR1 antibody that blocks NOTCH1 signalingwas obtained from Genentech under a material transfer agree-ment. The �-secretase inhibitor DAPT, PI3K inhibitor LY-294002, and NF-�B inhibitor Bay-11-7082 were purchasedfrom Calbiochem and used at 50 �M. The AKT inhibitor II usedat 2.5 �M and the casein kinase II inhibitor IV used at 50 �g/mlwere both purchased from Calbiochem. All inhibitors wereadded to DC cultures for 2 h before the addition of CD28-Ig.CD28-Ig was purified from spent medium of COS-7 cells trans-fected with plasmids expressing CD28-Ig (gift from Peter S.Linsley, AVI Biopharma, Inc.) and was used at 10 �g/ml.

Culture and Flow Cytometry of Human Mo-DC—Monocyteswere purified from normal human blood obtained under pro-tocols approved by the Institutional Review Board of RoswellPark Cancer Institute, as described previously (16). They weredifferentiated to human DC in RPMI 1640 media with GM-CSF(10 ng/ml, Sigma) and IL-4 (1000 units/ml, R&D Systems) for 7days and were analyzed for expression of CD14, CD11b, CD11c,CD80, CD83, CD86, CD1a, MHC I, and MHC II (all conjugatedto PE, Beckman Coulter). Cells were stained with anti-NOTCH1 antibody (clone A6, Thermo Scientific) or anti-JAGGED2 (N-19, Santa Cruz Biotechnology). Goat anti-mousesecondary antibody-PE (Jackson ImmunoResearch) was usedto detect NOTCH1, and goat anti-rabbit FITC was used todetect JAGGED2.

T Cell Proliferation Assay—T cell proliferation assay was per-formed as described previously (18). Briefly, normal human Tcells (2 � 105 cells/well) were cultured with human DC (7 � 105

cells/well) that were immature or matured with LPS (10 ng/ml)for 72 h. This was followed by addition of [3H]thymidine (pur-chased from PerkinElmer Life Sciences) for 18 h to assess T cellproliferation.

DC-Jurkat Cocultures—DC (7 � 105 cells/well) were cul-tured in medium alone, with 50 �M DAPT, or were trans-fected with scramble siRNA/NOTCH1 siRNA. After 24 h,DC were further cultured alone or with Jurkat cells (2 � 105

cells/well) that were untransfected or transfected withscramble/JAGGED2 siRNA.

ELISA for IL-6, IL-23, and IFN-�—Cell culture supernatantsfrom DC cultured in medium, CD28-Ig, or the indicated inhib-itors were collected after 4 h and were assayed by ELISA todetect IL-6 as described previously (16). IL-23 and IFN-� wasdetected using supernatants of cells cultured for 48 h. Briefly,ELISA plates coated with the IL-6 capture antibody wereblocked with 1% BSA and incubated with culture supernatants.This was followed by incubation with the IL-6 detection anti-body, and streptavidin-HRP was added. IL-6 was measuredbased on a colorimetric reaction at 450 nm. ELISA reagents forIL-6 were purchased from R&D Systems, and reagents for IL-23and IFN-� were purchased from eBioscience.

Quantitative-Real Time PCR—Total RNA that was isolatedusing TRIzol LS reagent (Invitrogen), as per the manufacturer’sinstructions, was used to generate cDNA (iScript cDNA syn-thesis, Bio-Rad), as per the manufacturer’s protocol. Geneexpression was determined using CK II-specific primers (for-

Cross-talk between NOTCH1 and CD80/CD86 Signaling in DC

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ward, 5�-AGCGATGGGAACGCTTTGTCC-3�, and reverse,5�-CATCCCAAGGGGGTTGGCAGC-3�) using quantitative-PCR (Bio-Rad). Data are represented as fold change relative tountreated DC, normalized to actin.

Reverse Transcriptase PCR (RT-PCR)—Extraction of RNAand synthesis of cDNA were performed as described above.Gene expression was determined using gene-specific primersas follows: HES-1: forward, 5�-AAACCCTCAGCACTT-GCTC-3�, and reverse, 5�-TCACCTCGTTCATGCACTC-3�;CK-II: forward, 5�-AGCGATGGGAACGCTTTGTCC-3�, andreverse, 5�-CATCCCAAGGGGGTTGGCAGC-3�); FOXO3A:forward, 5�-CGGCGGCGGGCTGTCTC, and reverse, 5�-AGTGGGCGATGGCTGGGATGG; and PIN1: forward, 5�-CTGATCAACGGCTACATCCA-3�, and reverse, 5�-TCA-AATGGCTTCTGCATCTG-3� using RT-PCR (EppendorfAG). Data are represented as relative change in mRNA expres-sion to actin.

Western Blot—Western blot analysis was performed as de-scribed previously (19). Briefly, cells were lysed in RIPA buffer,and the amount of protein was quantified using Bradford rea-gent (Bio-Rad). SDS-PAGE was performed, and membraneswere probed with the indicated antibodies. Secondary antibod-ies were obtained from Promega.

siRNA Knockdown—DC were transfected with siRNA forp85, AKT1, NOTCH1, JAGGED-2, or PIN1 (ON-TARGETSMARTpool, Thermoscientific) using the human dendritic celltransfection kit from Lonza as per the manufacturer’s instruc-tions. Scramble siRNA was used as control. After 24 h, DC werecultured �/� CD28-Ig (10 �g/ml) for an additional 24 h. Cellsupernatants were collected to perform ELISA for IL-6, and celllysates were used to perform Western blots to detect the per-centage of knockdown of the protein of interest. Jurkat cellswere transfected with siRNA for JAGGED2 or scramble siRNAusing the Lipofectamine kit (Invitrogen) as per the manufactu-rer’s instructions. After 24 h, a second transfection was per-formed to sustain the knockdown of JAGGED2, and the cellswere further cultured for 24 h. The transfected cells were thencocultured with DC. Densitometry was performed to quantifythe percentage of knockdown by using the ImageJ software.

Casein Kinase II Assay—DC were lysed in RIPA buffer con-taining 0.5% Nonidet P-40, followed by brief sonication. Thelysates were used to assay enzyme activity by measuring the CKII substrate peptide (RRRDDDSDDD, provided in the enzymeactivity kit, Millipore) phosphorylation by estimating the trans-fer of �-phosphate of [�-32P]ATP by the enzyme. The activitywas measured using a luminescence counter (Microbeta Triluxfrom PerkinElmer Life Sciences), and data were represented asunits/min/mg.

EMSA for NF-�B Activity—EMSA was performed as de-scribed previously (19). Briefly, cells were lysed in RIPA buffer,and the lysates were incubated in a mixture containing BufferD, dIdC, and BSA. This was followed by incubation with 32P-labeled primer containing consensus NF-�B-binding sites (5�-GATCCAACGGCAGGGGAATTCCCCTCTCCTTA-3�). Forsupershift assay, anti-p50 or anti-p65 (Santa Cruz Biotechnol-ogy) antibodies were added to the lysates. Samples were run ona 4% polyacrylamide gel and transferred to a filter paper. Thedried gel was exposed to an x-ray film overnight at �80 °C.

IDO Activity Assay—DC (1 � 105 cells/well) were culturedwith IFN-� (1000 units/ml) in media containing 100 mM tryp-tophan for 2.5 h followed by addition of CTLA4-Ig (50 �g/ml)or CD28-Ig (50 �g/ml) and the indicated inhibitors. After 48 h,cell culture supernatants were assayed for L-kynurenine levelsas a readout of IDO activity that was measured based on a col-orimetric reaction at 490 nm.

Statistical Analysis—Student’s t test was performed for sta-tistical analysis using a two-tailed, equal variance test.

RESULTS

Ligation of CD80 and/or CD86 on DC Induces IL-6 Expres-sion via Activation of PI3K/AKT Pathway—In these studies, weexamined DC differentiated from primary human monocytestreated with GM-CSF � IL-4. These Mo-DC expressed charac-teristic DC markers and were immature as indicated by theabsence of the maturation marker CD83 and also by low/me-dium expression of other maturation markers CD40 and MHCII (Fig. 1A). They were capable of driving T cell proliferation(the functional hallmark of DC), and this was significantlyincreased if they were matured by the addition of LPS (Fig. 1B).

To elucidate the mechanism of CD80/CD86 signaling in DC,we used the recombinant fusion protein CD28-Ig (the extracel-lular domain of CD28 fused to the Fc part of IgG1), which spe-cifically binds CD80 and CD86 and elicits IL-6 production inmurine DC (20). We found that CD28-Ig significantly up-reg-ulated IL-6 production by human Mo-DC compared with con-trol IgG (Fig. 1C). Addition of anti-CD80 or anti-CD86 mAbsimilarly induced IL-6 production, demonstrating that eitherCD80 or CD86 activation is sufficient to elicit full IL-6 produc-tion in Mo-DC (in contrast to what we (15) and others (5) havefound for murine DC) (Fig. 1C). Previously, it was reported inmurine DC that ligation of CD80/CD86 by CD28-Ig slightlyup-regulated IL-23 and significantly increased IFN-� produc-tion (5). However, we found that human Mo-DC did not pro-duce either of the cytokines (Fig. 1C). In considering whatdownstream pathway(s) might be involved, previous studiesreported activation of PI3K upon CD86 activation in IgG1 pro-duction by murine B cells (21). Based on this, we investigatedthe role of PI3K in CD80/CD86-induced DC IL-6 productionby inhibiting PI3K activity with the small molecule inhibitorLY294002. We found that CD28-Ig markedly induced phos-phorylation of p85, the regulatory subunit of PI3K which indi-cates PI3K activity, when compared with Mo-DC cultured inmedium (Fig. 1D, inset). Whereas CD28-Ig significantly up-reg-ulated DC IL-6 production compared with vehicle control orcontrol IgG, inhibiting PI3K activity significantly inhibited this(Fig. 1D). Similarly, siRNA-mediated silencing of the p85 regu-latory subunit of PI3K (90% knockdown by densitometry com-pared with scrambled siRNA, Fig. 1E, inset), significantlydecreased DC IL-6 production (Fig. 1E). Of note, inhibitingPI3K activity did not affect the phenotype of Mo-DC (Fig. 1F)when compared with Mo-DC cultured in medium (Fig. 1A).Mo-DC-induced T cell proliferation was reduced upon cultur-ing Mo-DC with the PI3K inhibitor LY294002 or siRNA p85(Fig. 1G). Although the decrease we observed was not signifi-cant, it showed a trend reflecting reduced T cell proliferation.To confirm that the effect we saw on IL-6 production was not




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limited to human Mo-DC, we cultured murine BMDC withLY294002 and/or murine CD28-Ig, and found the effect ofblocking PI3K activity on IL-6 production in human DC wasrecapitulated (Fig. 1H).

Downstream of PI3K activation, phosphatidylinositol 1,4,5-phosphate (PIP3) produced by PI3K recruits AKT to the plasmamembrane, where it is phosphorylated/activated by PDK1 (22).As predicted, we found that blocking PI3K activity decreasedAKT phosphorylation irrespective of the presence of CD28-Ig(Fig. 1I). As seen for LY294002, inhibiting AKT activation bythe small molecule inhibitor AKT II caused significant down-regulation of CD28-Ig-induced DC IL-6 production (Fig. 1J).siRNA-mediated silencing of AKT1 (95% knockdown, Fig. 1K,inset) also significantly reduced CD28-Ig-mediated inductionof IL-6 in DC (Fig. 1K). Downstream of AKT activation, it hasbeen previously reported that the transcriptional factorFOXO3A, which has been reported to regulate DC IL-6 pro-duction (12), is directly phosphorylated by AKT (23). Phosphor-ylated FOXO3A is shuttled out of the nucleus and retained inthe cytoplasm, preventing it from repressing its transcriptionaltargets, including IL-6 (23). We found that whereas CD28-Igincreased phosphorylation of FOXO3A compared with con-trol, blocking AKT activity completely inhibited this effect (Fig.1L), and blocking PI3K activity also reversed CD28-Ig-medi-ated phosphorylation of FOXO3A (Fig. 1M).

Involvement of FOXO3A and NF-�B Downstream of PI3K/AKT in IL-6 Production—It has been previously shown thatCTLA4-Ig (a soluble fusion protein consisting of an IgG tailfused to the extracellular domain of CTLA4, a member of theCD28 superfamily that also binds CD80 and CD86) down-reg-ulated TLR7-induced IL-6 production via up-regulation ofnuclear FOXO3A, which in turn suppressed IL-6 transcriptionin murine DC (12). To test if FOXO3A is important in CD28-Ig-induced IL-6 production via CD80/CD86 engagement, weused BMDC from FOXO3A�/� or FOXO3A�/� mice that have50 and 100% less FOXO3A mRNA expression versus WT (Fig.2A), and we compared them with wild type DC for IL-6 induc-tion. DC from FOXO3A�/� and FOXO3A�/� mice producedsignificantly higher amounts of IL-6 compared with WT DCupon CD28-Ig addition (Fig. 2B), suggesting that FOXO3A wassuppressing CD80/CD86-induced IL-6 production. However,BMDC from FOXO3A�/� and FOXO3A�/� mice producedminimal IL-6 in the same 24-h period without CD28-Ig (Fig.

2B). This suggests that in the absence of CD80/CD86 activa-tion, the reduction in FOXO3A expression, which would sup-press its ability to negatively regulate IL-6 production, is notsufficient to induce DC IL-6 production and that another mol-ecule downstream of AKT was positively inducing IL-6 produc-tion. This led us to examine whether other pathways down-stream of AKT are up-regulating IL-6 expression. One pathwayactivated by AKT is NF-�B (21, 23), which also induces IL-6production in murine DC (5). Cross-linking CD80/CD86 byCD28-Ig induced NF-�B signaling (as measured by electromo-bility gel shift assays), which was markedly decreased uponinhibiting PI3K or AKT activity (Fig. 2C, left panel). Supershiftassays for canonical NF-�B signaling demonstrated increasedp50 and p65 activity with CD28-Ig that was abrogated by inhib-iting PI3K or AKT activity (Fig. 2C, right panel). The nonca-nonical NF-�B signaling pathway was not activated, as evi-denced by the lack of RELB induction by CD28-Ig whencompared with the positive control (the myeloma cell line U266treated with a CD28-activating mAb (16)) (Fig. 2D). Consistentwith these findings, the NF-�B inhibitor Bay-11-7082 signifi-cantly reduced CD28-Ig-mediated up-regulation of DC IL-6production (Fig. 2E). Collectively, our data suggest that theCD80/CD863 PI3K3 p-AKT3 p-FOXO3A pathway de-re-presses the negative regulation of IL-6 production by FOXO3A,whereas the CD80/CD863 PI3K3 p-AKT3NF-�B pathwaypositively induces IL-6 expression and that both are necessaryfor the maximal IL-6 response.

Cross-talk between NOTCH and CD80/CD86 Signaling inDC IL-6 Production—In addition to playing a critical role in Tcell activation, IL-6 produced by DC is an important stromalfactor for plasma cell and multiple myeloma growth/survival(13, 14, 24). We have previously reported that the CD28-ex-pressing normal PC and MM cells induced DC IL-6 productionin a CD80/CD86-dependent manner (15, 16). Paralleling theseobservations, it has been reported that JAGGED2 on MM cellsinduced IL-6 in NOTCH1-expressing stromal cells (17), and wehave found that MM induced DC IL-6 production in aNOTCH-dependent fashion (16).

Upon ligation of NOTCH receptors by one of its ligands(belonging to the JAGGED or Delta-like family) a conforma-tional change is induced in the receptor negative regulatoryregion (NRR) that enables the first stage cleavage by a disinteg-rin and metalloprotease (ADAM) proteases. This is followed by

FIGURE 1. A, flow cytometric analysis of Mo-DC cultured in medium for expression of CD1a, CD11b, CD11c, CD14, CD40, CD80, CD83, CD86, MHC I, and MHC II.B, T cell proliferation assay was performed by determining the amount of thymidine incorporated by proliferating T cells when cultured with DC �/� LPS. C,ELISA for IL-6 of cell culture supernatants of Mo-DC �/� control IgG, CD28-Ig, anti-CD80 antibody, or anti-CD86 antibody. ELISA for IL-23 and IFN-� of cellculture supernatants of Mo-DC cultured �/� CD28-Ig. D, Western blots were performed to detect phosphorylated p85 and total p85 using cell lysates fromMo-DC cultured in medium or with CD28-Ig (inset). ELISA for IL-6 of cell culture supernatants from Mo-DC cultured with vehicle control (VC, DMSO), control IgG,or the PI3K inhibitor LY294002 �/� CD28-Ig. E, Western blots were performed to detect p85 and actin using cell lysates from Mo-DC transfected with scramblesiRNA or p85 siRNA (inset). ELISA for IL-6 of cell culture supernatants of Mo-DC cultured with scramble siRNA, p85 siRNA �/� CD28-Ig, is shown. F, flowcytometric analysis of Mo-DC cultured with the PI3K inhibitor LY294002 for expression of CD1a, CD1b, CD11c, CD14, CD40, CD80, CD83, CD86, MHC I, andMHCII. G, T-cell proliferation assay was performed by determining the amount of thymidine incorporated by proliferating T cells when cultured withDC cultured in medium �/� the PI3K inhibitor LY294002 or siRNA p85 and further cultured with T cells. H, ELISA for IL-6 of cell culture supernatants from murineBMDC (VC, DMSO), control IgG, or the PI3K inhibitor LY294002 �/� CD28-Ig. I, Western blots were performed to detect phosphorylated and total AKT usinglysates from Mo-DC cultured with vehicle control (VC, DMSO), control IgG, or the PI3K inhibitor LY294002 �/� CD28-Ig. J, ELISA for IL-6 of cell culturesupernatants of Mo-DC cultured with vehicle control (VC, DMSO), control IgG, or the AKT inhibitor AKT II �/� CD28-Ig. K, Western blots were performed todetect total AKT and actin using lysates from DC transfected with scramble siRNA or AKT1 siRNA (inset). ELISA for IL-6 of cell culture supernatants of Mo-DCcultured with scramble siRNA, AKT1, siRNA �/� CD28-Ig. L, Western blots were performed to detect phosphorylated and total FOXO3a using cell lysates fromMo-DC cultured with vehicle control (VC, DMSO), control IgG or the AKT inhibitor AKT II �/� CD28-Ig. M, Western blots were performed to detect phospho-rylated and total FOXO3a using cell lysates from Mo-DC cultured with vehicle control (VC, DMSO), or the PI3K inhibitor. Results shown are representative ofthree experiments. ns, not significant; *, p � 0.05; **, p � 0.01; ***, p � 0.001.




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a second stage cleavage of the receptor’s cytoplasmic region by�-secretase, which releases the NICD to translocate to thenucleus where it transcribes its target genes, including HES-1(25, 26). We have found that human Mo-DC coexpressNOTCH1 and JAGGED2 (Fig. 3A), and these DC exhibit con-stitutive NOTCH signaling as evidenced by expression of NICD(Fig. 3B). To investigate if NOTCH signaling induces IL-6 inde-pendent of CD28-Ig-triggered CD80/CD86 signaling, we firstcultured DC with soluble JAGGED2 (sJAG2) and found no fur-ther increase in NICD expression, in contrast to the effect ofsJAG2 on activating NOTCH1 in the positive control macro-phage cell line RAW (Fig. 3B) (27). Consistent with this, sJAG2induced no more IL-6 than DC cultured alone, in contrast to asignificant induction by CD28-Ig (Fig. 3C). This suggests that inDC, NOTCH signaling does not independently induce IL-6. Toinvestigate if there was an interaction between NOTCH andCD80/CD86 signaling, we blocked NOTCH signaling using the�-secretase inhibitor DAPT. Although CD28-Ig significantlyup-regulated DC IL-6 production, blocking NOTCH signaling(as demonstrated by a decrease in the NOTCH target geneHES-1 (see Fig. 4C)) significantly inhibited this effect (Fig. 3D).Importantly, DAPT treatment did not cause significant altera-tions in Mo-DC as evidenced by the phenotype (Fig. 3E, com-pared with Mo-DC cultured in medium, Fig. 1A). BecauseDAPT is a pan-NOTCH inhibitor, we investigated the specificrole for NOTCH1 by using an anti-NRR1 antibody that specif-ically blocks NOTCH1 signaling by binding to the NRR regionand inhibiting cleavage by ADAM proteases (28), and we foundthat the anti-NRR1 mAb also significantly inhibited CD28-Ig-mediated IL-6 induction (Fig. 3F). Because both DAPT andanti-NRR1 antibody prevents NOTCH1 cleavage but does notblock NOTCH1 binding to JAGGED2, these data also suggestthat potential signaling through JAGGED2 (or other NOTCH1ligands) is not involved. Similarly, siRNA-mediated silencing ofNOTCH1 (95% knockdown of NOTCH1 signaling as measuredby NICD, Fig. 3G, inset) significantly reduced CD28-Ig-medi-ated IL-6 production by DC (Fig. 3G). Furthermore, siRNAknockdown of JAGGED2 expression (85% knockdown ofJAGGED2, Fig. 3H, inset) completely abrogated CD28-Ig-in-duced IL-6 production (Fig. 3H). To eliminate the possibilitythat these findings were limited to human DC, we inhibitedNOTCH signaling using DAPT in murine BMDC and foundthat CD28-Ig-mediated up-regulation of IL-6 production wassignificantly abrogated (Fig. 3I). These data indicate thatNOTCH1 and JAGGED2 play a key role in regulating CD80/CD86-induced DC IL-6 production and also suggest that otherNOTCH receptor and ligand family members are not involved.

Because the above experiments were performed using ahomogeneous population of DC, we investigated if NOTCHsignaling is necessary for CD80/CD86-mediated DC IL-6 pro-

duction in the more biologically relevant interaction betweenDC and T cells. CD28� Jurkat human T cells also expressNOTCH1 and JAGGED2, similar to Mo-DC (Fig. 3J). DC orJurkat cultured alone in medium only produce basal amounts ofIL-6 (Fig. 3K). Coculturing DC with Jurkat significantly inducedIL-6 production, which was significantly reduced uponsiRNA-mediated silencing of JAGGED2 (80% reduction, Fig.3K, inset) in Jurkat or NOTCH1 in DC (90% reduction in NICDexpression). Silencing both NOTCH1 on DC and JAGGED2 onT cells did not further reduce IL-6 production (Fig. 3K), sug-gesting that they are not interacting with other NOTCH recep-tors and ligands in the induction of IL-6. Similar to our previousobservations in myeloma-DC cocultures, blocking NOTCHsignaling in DC with DAPT significantly reduced Jurkat-in-duced IL-6 production (Fig. 3K). Taken together, our data sug-gest that DC production of IL-6 upon cross-linking CD80/CD86 during DC:T cell engagement is dependent uponNOTCH signaling.

Ligation of CD80/CD86 Does Not Affect NOTCH Signaling—To examine the mechanism of cross-talk between NOTCH sig-naling and CD80/CD86 signaling, we first examined the effectof inhibiting NOTCH signaling on DC CD80 and CD86 expres-sion, and we found no marked effects (Fig. 4A). Alternatively, itis possible that CD80/CD86 signaling is modulating NOTCH1or JAGGED2 expression. However, CD28-Ig treatment had nomajor effect in DC NOTCH1 or JAGGED2 expression (Fig. 4B).Furthermore, CD28-Ig treatment had no effect on constitutiveNOTCH signaling as measured by expression of the NOTCH1target gene HES-1 expression (Fig. 4C). However, inhibitingNOTCH signaling with DAPT decreased HES-1 expressionirrespective of the presence of CD28-Ig, suggesting the lack ofany direct effect of CD80/CD86 activation on NOTCH1 signal-ing in DC (Fig. 4C).

Cross-talk between NOTCH and CD80/CD86 SignalingInvolves the PI3K-AKT-FOXO3A Pathway—Because of theabsence of any effect on surface receptor expression orNOTCH signaling, we examined whether cross-talk betweenthe two pathways involved molecules downstream of CD80/CD86 activation. We first evaluated if NOTCH signaling regu-lated CD80/CD86-induced PI3K signaling by inhibitingNOTCH signaling with DAPT. Although CD28-Ig up-regu-lated phosphorylation of AKT and FOXO3A, DAPT reversedthis effect (Fig. 5A). Interestingly, DAPT alone inhibited basalAKT phosphorylation, suggesting there is cross-talk betweenNOTCH and PI3K signaling even in the absence of CD80/CD86activation. We also found that DAPT down-regulated basalNF-�B (the second downstream target of AKT) activation (Fig.5B, left). Although CD80/CD86 ligation up-regulated NF-�Bactivation, DAPT abrogated CD28-Ig-mediated induction ofcanonical NF-�B signaling (Fig. 5B, right). These data indicate

FIGURE 2. A, cell lysates of BMDC from wild type mice (WT), FOXO3A heterozygotes (F�/�), or knock outs (F�/�) were analyzed for FOXO3A mRNAexpression by RT-PCR. B, cell culture supernatants of murine BMDC from wild type, FOXO3A�/�, or FOXO3A�/� mice cultured �/� CD28-Ig were usedto perform ELISA for IL-6. C, whole cell lysates of DC cultured alone, with LY294002, or AKT II �/� CD28-Ig were analyzed by electromobility gel shiftassay with primers containing consensus NF-�B-binding sites. The arrow (left panel) indicates NF-�B dimers. Supershift EMSA was performed to detectp50 and p65 subunits as represented by the arrow (right panel). Actin was used as loading control. D, whole cell lysates of Mo-DC cultured �/� CD28-Igwere used to perform EMSA to detect RELB subunit as represented by the arrow. U266 myeloma cells treated with anti-CD28 activating antibody wereused as positive control for RELB expression. Actin was used as the loading control. E, cell culture supernatants of Mo-DC cultured �/� vehicle control(VC), control IgG, or the NF-�B inhibitor Bay-11-7082 were assayed for IL-6 by ELISA. Results shown are representative of three experiments. ns, notsignificant; **, p � 0.01; ***, p � 0.001.




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that NOTCH signaling regulates the PI3K3 p-AKT3NF-�Bpathway downstream of CD80/CD86 in DC.

NOTCH Signaling Modulates PI3K/AKT Activity by Regulat-ing Casein Kinase II Activity—We next examined the mecha-nism by which NOTCH signaling might regulate the PI3K path-way. PTEN sequesters the PI3K substrate PIP3 and inhibitsPI3K activity. PTEN phosphorylation prevents it from bindingto PIP3 and allows for full PI3K activity (29). We initiallyhypothesized that NOTCH signaling down-regulated PTENexpression through HES-1-mediated inhibition of PTEN tran-scription, as reported previously in leukemic T cells (30). How-ever, there was no change in total PTEN levels with DAPTtreatment under any condition (Fig. 6A). However, comparedwith untreated DC, DAPT treatment with or without CD28-Ig

decreased PTEN phosphorylation (which inactivates PTEN).CD28-Ig alone did not affect PTEN phosphorylation, suggest-ing that this was independent of CD80/CD86 signaling. Takentogether, these data suggest that NOTCH signaling induces thephosphorylation/inactivation of PTEN, which permits fullPI3K activity upon CD80/CD86 activation with subsequentinduction of IL-6 production.

Candidate molecules regulating PTEN phosphorylation inDC include casein kinase II (CK II), as it plays a predominantrole in phosphorylating/inactivating PTEN in a number ofother systems (31, 32). Consistent with this, inhibiting CK IIactivity (see Fig. 6G) with the chemical inhibitor CK II-IVdecreased PTEN phosphorylation versus untreated DC (Fig.6B). As seen in Fig. 6A, CD28-Ig alone had no effect on PTEN

FIGURE 3. A, flow cytometric analysis of Mo-DC for expression of NOTCH1 and JAGGED2. B, cell lysates from Mo-DC cultured in medium (med) or solubleJAGGED2 (sJAG2) were used to perform Western blots for NICD. As a positive control, RAW cells were cultured in medium, with LPS or soluble JAGGED2.Densitometric analysis was performed using ImageJ software. C, ELISA for IL-6 from cell supernatants of Mo-DC was cultured in control IgG, sJAG2 �/�CD28-Ig. D, ELISA for IL-6 from cell supernatants of Mo-DC cultured alone or with vehicle control (VC), control IgG, DAPT �/� CD28-Ig. E, flow cytometricanalysis of Mo-DC treated with DAPT for expression of CD1a, CD14, CD11b, CD11c, CD40, CD80, CD83, CD86, MHC I, and MHC II. F, ELISA for IL-6 from cellsupernatants of Mo-DC cultured alone, with control Ig or anti-NRR1 antibody. G, Western blotting was performed using cell lysates from Mo-DC transfectedwith scramble/NOTCH1 siRNA (inset). ELISA for IL-6 from cell supernatants of Mo-DC cultured alone, with scramble siRNA, or NOTCH1 siRNA. H, Western blottingwas performed using cell lysates of Mo-DC transfected with scramble/JAGGED2 siRNA (inset). ELISA for IL-6 from cell supernatants of Mo-DC cultured alone,with scramble siRNA, or JAGGED2 siRNA. I, ELISA for IL-6 of cell culture supernatants from murine BMDC cultured alone or with vehicle control, control IgG,DAPT �/� CD28-Ig. J, flow cytometric analysis of Jurkat for expression of NOTCH1 and JAGGED2. K, Western blots were performed using cell lysates from Jurkatcells transfected with scramble/JAGGED2 siRNA (inset). ELISA for IL-6 from cell supernatants of Mo-DC cultured with vehicle control, DAPT, scramble siRNA,siRNA NOTCH1�/� CD28-Ig, and Jurkat cells that were untransfected or transfected with JAGGED2 siRNA. Results shown are representative of threeexperiments. ns, not significant; *, p � 0.05; **, p � 0.01; ***, p � 0.001.

FIGURE 4. A, flow cytometric analysis of Mo-DC cultured alone, with control Ig, or DAPT for expression of CD80 and CD86. B, flow cytometric analysis of Mo-DCcultured alone, with control Ig, or CD28-Ig for NOTCH1 and JAGGED2 expression. C, RNA from Mo-DC cultured alone, with vehicle control (VC), DAPT, or CD28-Igwas analyzed by RT-PCR for expression of HES-1 and actin. Densitometric analysis was performed using ImageJ software. Results shown are representative ofthree experiments.




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phosphorylation and also did not affect CK II-mediated inhibi-tion of PTEN phosphorylation (Fig. 6B). However, CD28-Ig-induced up-regulation of IL-6 was completely abrogated byinhibiting CK II activity (Fig. 6C).

To investigate how NOTCH signaling might be regulatingCK II, we first examined the effect of inhibiting NOTCH sig-naling on CK II expression, and we found that CK II mRNA asassessed by RT-PCR (Fig. 6D), quantitative-PCR (Fig. 6E), orprotein as analyzed by Western blots (Fig. 6F) remainedunchanged. However, DC treated with DAPT exhibit a signifi-cantly lower CK II enzymatic activity versus DC treated withvehicle control (Fig. 6G). Unexpectedly, given our findings thatCD28-Ig does not affect the (in)activation of PTEN, CD28-Igsignificantly up-regulated CK II activity. However, DAPT com-pletely blocked CD28-Ig-mediated up-regulation of CK IIactivity to levels similar to the CK II inhibitor (Fig. 6G). Thissuggests that although CD80/CD86-mediated signaling can up-regulate CK II activity (and thus may sustain PI3K signaling),the initiation of CD80/CD86 signaling cannot occur without aprior NOTCH1-mediated alleviation of the signaling blockadeby PTEN.

NOTCH1 Target Gene PIN1 Regulates Casein Kinase IIEnzyme Activity—To elucidate how NOTCH1 up-regulates CKII activity, we investigated the role of the prolyl isomerase PIN1.PIN1 has been previously reported to be a NOTCH1 gene targetand up-regulates CK II activity in human breast cancer cells,but it has not been studied in DC (33). First, we tested if PIN1 isa target of NOTCH1 in human DC and found that siRNA-mediated inhibition of NOTCH1 signaling (95% knockdown ofNICD, Fig. 7A) markedly reduced PIN1 mRNA compared withscramble siRNA control (Fig. 7B). This observation was

reflected in PIN1 protein levels as well (Fig. 7C). We next exam-ined if PIN1 is involved in CD28-Ig-mediated production of DCIL-6, and we found that siRNA-mediated silencing of PIN1(90% knockdown of PIN1, Fig. 7D, inset) significantly reducedIL-6 production by DC compared with the scrambled siRNAcontrol (Fig. 7D). This was consistent with a significant reduc-tion in CK II activity upon silencing PIN1 (Fig. 7E). Altogether,these data suggest that NOTCH1 signaling induces transcrip-tion of PIN1, which in turn up-regulates casein kinase IIactivity.

IDO Production by DC upon Cross-linking CD80/CD86—Al-though engagement of CD80/CD86 by CD28-Ig induces IL-6 inmurine DC, CTLA4-Ig induces IDO, an enzyme that catabo-lizes tryptophan to L-kynurenine and blocks T cell activation(8). This raised the previously unexplored question of whetherthe pathways downstream of CD80/CD86 that are inducing DCIL-6 production are also inducing IDO production in humanDC. We found that CTLA4-Ig induced IDO protein in Mo-DC,at a later time point (24 h) than seen for IL-6 (4 h), and thatmaximal expression occurs in combination with IFN-� treat-ment (Fig. 8A). Cross-linking CD80/CD86 by CTLA4-Ig, anti-CD80, or anti-CD86 mAb also significantly increases IDOactivity (as measured by production of the L-kynurenine metab-olite) at 48 h in the presence of IFN-� (Fig. 8B). We next exam-ined if CTLA4-Ig-mediated IDO production is dependent uponthe downstream PI3K/AKT pathway. CTLA4-Ig increased IDOactivity, which was significantly inhibited by blocking PI3K sig-naling with LY294002 (Fig. 8C). As predicted, inhibition of theactivity of the downstream targets of PI3K, AKT (Fig. 8D), andNF-�B (Fig. 8E) also significantly decreased CTLA4-Ig-inducedIDO activity. Similar to IL-6 production, we found that inhib-

FIGURE 5. Cell lysates from Mo-DC cultured alone, with vehicle control (VC), control IgG, or DAPT �/� CD28-Ig were used to perform Western blots forphosphorylated and total AKT and FOXO3A (A) and EMSA to detect NF-�B dimers as represented by the arrow (left panel) (B). Supershift EMSA was performedto detect p50 and p65 subunits as represented by the arrow (right panel). Actin was used as a loading control. Results shown are representative of threeexperiments.




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iting NOTCH signaling with DAPT or the anti-NRR1 blockingantibody significantly decreased CD80/CD86-induced IDOactivity (Fig. 8F). The cross-talk between NOTCH and CD80/CD86 signaling in DC IDO production also involves CK II, asCK II inhibition significantly decreased CTLA4-Ig-inducedIDO activity (Fig. 8G). Interestingly, we found that cross-link-ing CD80/CD86 with CD28-Ig also induces active IDO in thepresence of IFN-� (Fig. 8H) and that CTLA4-Ig can also induceDC IL-6 production (Fig. 8I), which is in contrast to what hasbeen reported in murine DC (5, 8). These data suggest that DCIDO production is dependent on the PI3K signaling pathwaydownstream of CD80/CD86, which cross-talks with NOTCHsignaling. Additionally, regardless of its binding partner, CD80/CD86 can induce both IL-6 and IDO in Mo-DC.

DISCUSSION

Antigen-specific activation of T cells is critically dependentupon the costimulatory signals provided by DC involving theCD28-CD80/CD86 interaction. T cell activation is regulatednot only by the T cell-intrinsic signals provided by CD28 butalso by cytokines such as IL-6 produced by DC upon CD80 andCD86 activation (6, 7, 34). In the B cell lineage, we have previ-

ously demonstrated that these same molecular interactionsbetween PC and DC induce DC IL-6 production, whichenhances normal PC immunoglobulin production and malig-nant PC survival (15, 16). However, a thorough understandingof the molecular pathways involved in CD80/CD86-inducedDC IL-6 production is lacking. To decipher signaling down-stream of CD80/CD86, we used CD28-Ig, which binds both theligands specifically and induces DC IL-6 production (5, 20).IL-6 induced by anti-CD80 and anti-CD86 antibodies in DCwas comparable with the effect of CD28-Ig, which supports theconclusion that CD28-Ig is functioning by activating CD80/CD86 and argues against off-target effects. We also found thatengagement of either CD80 or CD86 was sufficient to inducehuman DC IL-6 production, which is in contrast to findings byus (15) and others (5) in murine DC that both CD80 and CD86are essential. However, our findings are consistent with otherreports that describe the redundancy of CD80 and CD86 inhuman DC production of IDO (10). Why there is a differencebetween murine and human CD80 and CD86 is not known, butalignment of the human versus murine protein sequence showsthat CD80 is homologous across species but CD86 is not.4 Wehave also found by in silico analysis that the murine and humanCD80/CD86 cytoplasmic tails contain potential binding motifsfor different kinases (data not shown), suggesting that they maybe distinctly phosphorylated and initiate different signalingpathways upon activation. This is consistent with our observa-tion that although PI3K plays a dominant role in human DCIL-6 production (blocking PI3K activity completely abrogatesIL-6 production), in murine DC blocking PI3K activity resultsin significant yet incomplete down-regulation of IL-6 produc-tion, suggesting that a second pathway involving p38 MAPK(activated by MAPK kinases) activation may be regulating IL-6production in murine DC (5).

The most proximal signaling molecules that activate PI3Kdownstream of CD80/CD86 activation in human DC are cur-rently unknown. In B cells, it has been reported that CD86engagement activates the membrane adaptor proteins prohib-itins (PHB) (11). Although PHB interacts with PIP3 (36) andAKT (37), it is unknown if PHB interacts with PI3K. BecausePHB are expressed by Mo-DC (38) and they up-regulate NF-�Bsignaling upon CD86 engagement in B cells (11) (similar to ourfindings reported here), we speculate that PHB may play a rolein PI3K activation in DC. Downstream of this, we have foundthat PI3K activates AKT with subsequent phosphorylation ofFOXO3A preventing it from suppressing IL-6 transcription.Simultaneously, activation of NF-�B by AKT induces DC IL-6production.

4 L. Boise, unpublished data.

FIGURE 6. A, Western blots were performed to detect phosphorylated and total PTEN using cell lysates from Mo-DC cultured alone or with vehicle control (VC),control IgG, or DAPT �/� CD28-Ig. B, Western blotting was performed to detect phosphorylated and total PTEN using cell lysates from Mo-DC cultured aloneor with vehicle control, control IgG, or the CK II inhibitor-IV �/� CD28-Ig. C, ELISA for IL-6 from cell supernatants of Mo-DC cultured with vehicle control, controlIgG, or CK II inhibitor-IV �/� addition of CD28-Ig. D, Mo-DC were cultured alone or with vehicle control, control IgG, or DAPT �/� CD28-Ig. RNA was analyzedby RT-PCR. E, Mo-DC were cultured alone or with vehicle control, control IgG, or DAPT. RNA was analyzed by quantitative-PCR for mRNA expression of CK II withactin as a loading control. F, Mo-DC were cultured alone or with vehicle control, control IgG, or DAPT �/� CD28-Ig, and cell lysates were analyzed by Westernblot to detect CK II expression. Densitometric analysis was performed using ImageJ software. G, cell lysates of Mo-DC cultured alone or with vehicle control,control IgG, DAPT, or the CK II inhibitor CK II-IV �/� CD28-Ig were used to assay CK II enzyme activity. Results shown are representative of three experiments.ns, not significant; *, p � 0.05; **, p � 0.01.

FIGURE 7. A, Western blots were performed to detect NICD and actin using celllysates from DC transfected with scramble siRNA or NOTCH1 siRNA. B, RT-PCRwas performed to detect PIN1 and actin mRNA using RNA extracted from DCtransfected with scramble siRNA or NOTCH1 siRNA. C, Western blots wereperformed to detect PIN1 and actin using cell lysates from DC transfectedwith scramble siRNA or NOTCH1 siRNA. D, Western blots were performed todetect PIN1 and actin using cell lysates from DC transfected with scramblesiRNA or PIN1 siRNA (inset). ELISA for IL-6 from cell supernatants of Mo-DCcultured alone, with scramble siRNA, or PIN1 siRNA �/� CD28-Ig. E, Mo-DCwere cultured alone, with scramble siRNA, or PIN1 siRNA �/� addition ofCD28-Ig. Cell lysates were analyzed for enzyme activity assay for CK II. Resultsshown are representative of three experiments. ns, not significant; **, p �0.01.




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FIGURE 8. A, Western blots were performed to detect IDO and actin using cell lysates from Mo-DC cultured with IFN-� �/� CTLA4-Ig for 18, 24, or 48 h. B, Mo-DCwere cultured with IFN-� followed by addition of CTLA4-Ig, anti-CD80, or anti-CD86. Cell culture supernatants were assayed for L-kynurenine levels to estimateIDO activity. Mo-DC cultured with IFN-� and treated with the PI3K inhibitor LY294002 (C), AKT inhibitor AKT II (D), NF-�B inhibitor Bay-11-7062 (E), NOTCHsignaling inhibitor DAPT or anti-NRR1 blocking antibody (F), or CK II inhibitor CK II-IV �/� CTLA4-Ig (G). Cell culture supernatants were assayed for L-kynureninelevels to estimate IDO activity. Mo-DC were cultured with or without IFN-� followed by addition of CTLA4-Ig or CD28-Ig. Cell culture supernatants were usedto assay L-kynurenine levels to estimate IDO activity (H) or perform ELISA for IL-6 (I).




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In addition to IL-6 production, CD80/CD86 activation byCTLA4-Ig induces production of immunosuppressive trypto-phan catabolizing IDO (8). The literature is inconclusivewhether CD80/CD86-induced IDO induction utilizes the samesignaling pathways as CD80/CD86-induced IL-6 production.In CD8�� splenic DC from nonobese diabetic mice, CD80/CD86 engagement by CTLA4-Ig down-regulated PI3K signal-ing by increasing PTEN expression, which enhanced FOXO3A-mediated production of IDO (39). How CTLA4-Ig up-regulatesPTEN expression was not determined, however. One possibil-ity is that CD80/CD86 ligation induces IFN-� (39), which pro-duces nitric oxide (NO) in murine DC (40). Nonobese diabeticmice express high amounts of nitric oxide (NO) (41) that tran-scribes p53 (42) and EGR-1 (43). Separately, it has beenreported that p53 and EGR-1 induce PTEN transcription (44,45). Although these data have not been reported in DC, theysuggest a potential role for CD80/CD86-induced NO in PTENup-regulation in nonobese diabetic mice. However, in DCunder homeostatic conditions, NO is nearly absent (46), sug-gesting that direct regulation of PTEN expression by activatedCD80/CD86 via NO may be lacking.

Instead, we have found in human DC that PI3K signalingdownstream of CD80/CD86 activation is being modulated byNOTCH1 regulation of PTEN. Although total PTEN expres-sion is unaffected, PTEN is phosphorylated/inactivated byNOTCH1 signaling (initiated upon ligation of DC NOTCH1 byJAGGED2 expressed on a neighboring DC), which up-regulatesPIN1 expression resulting in increased CK II activity. Thisallows for full PI3K activation downstream of CD80/CD86 liga-tion (summarized in Fig. 9). NOTCH1 has been reported toregulate PI3K signaling, but the mechanisms are distinct in var-ious cell types. In activated T cells, NOTCH1 forms a complexwith p56lck and PI3K that activates downstream AKT signaling(47). DC, however, do not express p56lck (48), and such a molec-

ular interaction seems unlikely in our system. In T cell acutelymphoblastic leukemia, activating mutations of NOTCH1 up-regulate HES-1 expression that subsequently inhibits PTENexpression (30). PTEN phosphorylation by CK II was also pre-viously reported in primary T cell acute lymphoblastic leuke-mia (49); however, this was independent of NOTCH signaling.In pancreatic cancer cells, RhoA/ROCK1 activation down-stream of NOTCH signaling results in PTEN phosphorylation(50), but it is not known whether CK II is also involved orwhether this pathway also occurs in nontransformed cells.Here, in human DC we propose that NOTCH13 PIN13 CKII3 phospho-PTEN allows for very rapid signaling cross-talkwith the PI3K pathway, which may be essential to initiate thevery early events required for DC-mediated activation of Tcells.

Consistent with this idea, we found that DC IL-6 and IDOproduction (which is dependent upon the same signaling path-ways downstream of CD80/CD86 ligation in the presence ofIFN-�) is temporally regulated. CD80/CD86-induced IL-6 pro-duction occurs early (4 h), although IDO production/activationoccurs late (24 h). This suggests that CD80/CD86 activationinitially induces DC IL-6 production to promote T cell activa-tion, but the same pathway later induces IDO production (inconjunction with evidence of a successfully activated T cell,namely IFN-� production) to self-limit this activation.

Because the DC in our study have constitutive NOTCH sig-naling that is not inducibly regulated, this raises the question ofthe relevance of NOTCH in modulating physiological CD80/CD86 signaling. However, NOTCH1-CD80/CD86 cross-talkmay be particularly relevant in vivo, where unlike in our in vitroexperiments there is not a high density of DC interacting withone another to drive NOTCH1 signaling. One such setting asdemonstrated by us is the T cell/DC interaction, where CD28�

T cells expressing JAGGED2 engage NOTCH1 on CD80/CD86� DC inducing IL-6 production. The second setting maybe in the PC/MM-DC interaction, where CD28� PC/MM cellsexpressing JAGGED2 engage NOTCH1 on CD80/CD86� DC,inducing production of IL-6 to support PC/MM survival (16).Intriguingly, our data also demonstrate that blocking NOTCHsignaling inhibits basal AKT and FOXO3A phosphorylation,implying that NOTCH signaling can regulate the PI3K pathwayindependent of CD80/CD86 activation. In DC, such regulationof PI3K signaling by NOTCH may modulate PI3K-mediatedregulation of DC maturation, antigen presenting capability, andblockade of DC IL-12 production (35, 51). Thus, the modula-tion of PI3K signaling by NOTCH1, regardless of its initiatingsource, may be a general regulatory pathway for many differentbiological responses.

Acknowledgments—We thank Dr. Arthur A. Hurwitz (NCI-Frederick,National Institutes of Health) for helping us with experiments with theFOXO3A heterozygous and FOXO3A homozygous knock-out mice(originally generated by Dr. Stephen M. Hedrick, University of Cali-fornia, San Diego, La Jolla, CA).

REFERENCES1. Lanier, L. L., O’Fallon, S., Somoza, C., Phillips, J. H., Linsley, P. S., Oku-

mura, K., Ito, D., and Azuma, M. (1995) CD80 (B7) and CD86 (B70) pro-

FIGURE 9. Engagement of NOTCH1 on DC by its ligand JAGGED2expressed on a neighboring DC initiates NOTCH1 signaling resulting inthe transcription of the prolyl isomerase PIN1. Subsequently, caseinkinase II activity is up-regulated, which inactivates PTEN by phosphorylation.This allows for full activation of PI3K downstream of CD80/CD86 ligation.Downstream of PI3K, AKT is activated, which in turn phosphorylates/inacti-vates FOXO3A and inhibits IL-6 suppression. Simultaneous activation ofNF-�B downstream of AKT allows for IL-6 production by DC.




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vide similar costimulatory signals for T cell proliferation, cytokine pro-duction, and generation of CTL. J. Immunol. 154, 97–105

2. Sharpe, A. H., and Freeman, G. J. (2002) The B7-CD28 superfamily. Nat.Rev. Immunol. 2, 116 –126

3. Frauwirth, K. A., Riley, J. L., Harris, M. H., Parry, R. V., Rathmell, J. C., Plas,D. R., Elstrom, R. L., June, C. H., and Thompson, C. B. (2002) The CD28signaling pathway regulates glucose metabolism. Immunity 16, 769 –777

4. Bour-Jordan, H., and Blueston, J. A. (2002) CD28 function: a balance ofcostimulatory and regulatory signals. J. Clin. Immunol. 22, 1–7

5. Orabona, C., Grohmann, U., Belladonna, M. L., Fallarino, F., Vacca, C.,Bianchi, R., Bozza, S., Volpi, C., Salomon, B. L., Fioretti, M. C., Romani, L.,and Puccetti, P. (2004) CD28 induces immunostimulatory signals in den-dritic cells via CD80 and CD86. Nat. Immunol. 5, 1134 –1142

6. Gajewski, T. F., Renauld, J. C., Van Pel, A., and Boon, T. (1995) Costimu-lation with B7-1, IL-6, and IL-12 is sufficient for primary generation ofmurine antitumor cytolytic T lymphocytes in vitro. J. Immunol. 154,5637–5648

7. Holsti, M. A., McArthur, J., Allison, J. P., and Raulet, D. H. (1994) Role ofIL-6, IL-1, and CD28 signaling in responses of mouse CD4� T cells toimmobilized anti-TCR monoclonal antibody. J. Immunol. 152,1618 –1628

8. Grohmann, U., Orabona, C., Fallarino, F., Vacca, C., Calcinaro, F., Falorni,A., Candeloro, P., Belladonna, M. L., Bianchi, R., Fioretti, M. C., and Puc-cetti, P. (2002) CTLA-4-Ig regulates tryptophan catabolism in vivo. Nat.Immunol. 3, 1097–1101

9. Fallarino, F., Grohmann, U., Hwang, K. W., Orabona, C., Vacca, C., Bian-chi, R., Belladonna, M. L., Fioretti, M. C., Alegre, M. L., and Puccetti, P.(2003) Modulation of tryptophan catabolism by regulatory T cells. Nat.Immunol. 4, 1206 –1212

10. Munn, D. H., Sharma, M. D., and Mellor, A. L. (2004) Ligation of B7–1/B7–2 by human CD4� T cells triggers indoleamine 2,3-dioxygenase ac-tivity in dendritic cells. J. Immunol. 172, 4100 – 4110

11. Lucas, C. R., Cordero-Nieves, H. M., Erbe, R. S., McAlees, J. W., Bhatia, S.,Hodes, R. J., Campbell, K. S., and Sanders, V. M. (2013) Prohibitins and thecytoplasmic domain of CD86 cooperate to mediate CD86 signaling in Blymphocytes. J. Immunol. 190, 723–736

12. Dejean, A. S., Beisner, D. R., Ch’en, I. L., Kerdiles, Y. M., Babour, A., Arden,K. C., Castrillon, D. H., DePinho, R. A., and Hedrick, S. M. (2009) Tran-scription factor FOXO3 controls the magnitude of T cell immune re-sponses by modulating the function of dendritic cells. Nat. Immunol. 10,504 –513

13. Uchiyama, H., Barut, B. A., Mohrbacher, A. F., Chauhan, D., and Ander-son, K. C. (1993) Adhesion of human myeloma-derived cell lines to bonemarrow stromal cells stimulates interleukin-6 secretion. Blood 82,3712–3720

14. Kawano, M., Hirano, T., Matsuda, T., Taga, T., Horii, Y., Iwato, K., Asaoku,H., Tang, B., Tanabe, O., and Tanaka, H. (1988) Autocrine generation andrequirement of BSF-2/IL-6 for human multiple myelomas. Nature 332,83– 85

15. Rozanski, C. H., Arens, R., Carlson, L. M., Nair, J., Boise, L. H., Chanan-Khan, A. A., Schoenberger, S. P., and Lee, K. P. (2011) Sustained antibodyresponses depend on CD28 function in bone marrow-resident plasmacells. J. Exp. Med. 208, 1435–1446

16. Nair, J. R., Carlson, L. M., Koorella, C., Rozanski, C. H., Byrne, G. E.,Bergsagel, P. L., Shaughnessy, J. P., Jr., Boise, L. H., Chanan-Khan, A., andLee, K. P. (2011) CD28 expressed on malignant plasma cells induces aprosurvival and immunosuppressive microenvironment. J. Immunol. 187,1243–1253

17. Houde, C., Li, Y., Song, L., Barton, K., Zhang, Q., Godwin, J., Nand, S.,Toor, A., Alkan, S., Smadja, N. V., Avet-Loiseau, H., Lima, C. S., Miele, L.,and Coignet, L. J. (2004) Overexpression of the NOTCH ligand JAG2 inmalignant plasma cells from multiple myeloma patients and cell lines.Blood 104, 3697–3704

18. Cejas, P. J., Carlson, L. M., Zhang, J., Padmanabhan, S., Kolonias, D., Lind-ner, I., Haley, S., Boise, L. H., and Lee, K. P. (2005) Protein kinase C �IIplays an essential role in dendritic cell differentiation and autoregulates itsown expression. J. Biol. Chem. 280, 28412–28423

19. Bahlis, N. J., King, A. M., Kolonias, D., Carlson, L. M., Liu, H. Y., Hussein,

M. A., Terebelo, H. R., Byrne, G. E., Jr., Levine, B. L., Boise, L. H., and Lee,K. P. (2007) CD28-mediated regulation of multiple myeloma cell prolifer-ation and survival. Blood 109, 5002–5010

20. Linsley, P. S., Brady, W., Grosmaire, L., Aruffo, A., Damle, N. K., andLedbetter, J. A. (1991) Binding of the B cell activation antigen B7 to CD28costimulates T cell proliferation and interleukin 2 mRNA accumulation. J.Exp. Med. 173, 721–730

21. Kin, N. W., and Sanders, V. M. (2006) CD86 stimulation on a B cell acti-vates the phosphatidylinositol 3-kinase/AKT and phospholipase C�2/protein kinase C�� signaling pathways. J. Immunol. 176, 6727– 6735

22. Cantley, L. C. (2002) The phosphoinositide 3-kinase pathway. Science 296,1655–1657

23. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Ander-son, M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) AKTpromotes cell survival by phosphorylating and inhibiting a Forkhead tran-scription factor. Cell 96, 857– 868

24. Minges Wols, H. A., Underhill, G. H., Kansas, G. S., and Witte, P. L. (2002)The role of bone marrow-derived stromal cells in the maintenance ofplasma cell longevity. J. Immunol. 169, 4213– 4221

25. Kidd, S., Lieber, T., and Young, M. W. (1998) Ligand-induced cleavage andregulation of nuclear entry of NOTCH in Drosophila melanogaster em-bryos. Genes Dev. 12, 3728 –3740

26. Schroeter, E. H., Kisslinger, J. A., and Kopan, R. (1998) NOTCH-1 signal-ling requires ligand-induced proteolytic release of intracellular domain.Nature 393, 382–386

27. Tsao, P. N., Wei, S. C., Huang, M. T., Lee, M. C., Chou, H. C., Chen, C. Y.,and Hsieh, W. S. (2011) Lipopolysaccharide-induced NOTCH signalingactivation through JNK-dependent pathway regulates inflammatory re-sponse. J. Biomed. Sci. 18, 56

28. Wu, Y., Cain-Hom, C., Choy, L., Hagenbeek, T. J., de Leon, G. P., Chen, Y.,Finkle, D., Venook, R., Wu, X., Ridgway, J., Schahin-Reed, D., Dow, G. J.,Shelton, A., Stawicki, S., Watts, R. J., Zhang, J., Choy, R., Howard, P.,Kadyk, L., Yan, M., Zha, J., Callahan, C. A., Hymowitz, S. G., and Siebel,C. W. (2010) Therapeutic antibody targeting of individual NOTCH recep-tors. Nature 464, 1052–1057

29. Das, S., Dixon, J. E., and Cho, W. (2003) Membrane-binding and activationmechanism of PTEN. Proc. Natl. Acad. Sci. U.S.A. 100, 7491–7496

30. Palomero, T., Sulis, M. L., Cortina, M., Real, P. J., Barnes, K., Ciofani, M.,Caparros, E., Buteau, J., Brown, K., Perkins, S. L., Bhagat, G., Agarwal,A. M., Basso, G., Castillo, M., Nagase, S., Cordon-Cardo, C., Parsons, R.,Zúñiga-Pflücker, J. C., Dominguez, M., and Ferrando, A. A. (2007) Muta-tional loss of PTEN induces resistance to NOTCH1 inhibition in T-cellleukemia. Nat. Med. 13, 1203–1210

31. Torres, J., and Pulido, R. (2001) The tumor suppressor PTEN is phosphor-ylated by the protein kinase CK2 at its C terminus. Implications for PTENstability to proteasome-mediated degradation. J. Biol. Chem. 276,993–998

32. Kim, K. Y., Shin, H. K., Lee, J. H., Kim, C. D., Lee, W. S., Rhim, B. Y., Shin,Y. W., and Hong, K. W. (2004) Cilostazol enhances casein kinase 2 phos-phorylation and suppresses tumor necrosis factor-�-induced increasedphosphatase and tensin homolog deleted from chromosome 10 phosphor-ylation and apoptotic cell death in SK-N-SH cells. J. Pharmacol. Exp. Ther.308, 97–104

33. Rustighi, A., Tiberi, L., Soldano, A., Napoli, M., Nuciforo, P., Rosato, A.,Kaplan, F., Capobianco, A., Pece, S., Di Fiore, P. P., and Del Sal, G. (2009)The prolyl-isomerase PIN1 is a NOTCH1 target that enhances NOTCH1activation in cancer. Nat. Cell Biol. 11, 133–142

34. June, C. H., Bluestone, J. A., Nadler, L. M., and Thompson, C. B. (1994)The B7 and CD28 receptor families. Immunol. Today 15, 321–331

35. Fukao, T., Tanabe, M., Terauchi, Y., Ota, T., Matsuda, S., Asano, T., Kad-owaki, T., Takeuchi, T., and Koyasu, S. (2002) PI3K-mediated negativefeedback regulation of IL-12 production in DCs. Nat. Immunol. 3,875– 881

36. Ande, S. R., and Mishra, S. (2009) Prohibitin interacts with phosphatidy-linositol 3,4,5-triphosphate (PIP3) and modulates insulin signaling.Biochem. Biophys. Res. Commun. 390, 1023–1028

37. Han, E. K., Mcgonigal, T., Butler, C., Giranda, V. L., and Luo, Y. (2008)Characterization of AKT overexpression in MiaPaCa-2 cells: prohibitin is




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

an AKT substrate both in vitro and in cells. Anticancer Res. 28, 957–96338. Liu, K., Li, Y., Prabhu, V., Young, L., Becker, K. G., Munson, P. J., and

Weng, N. (2001) Augmentation in expression of activation-induced genesdifferentiates memory from naive CD4� T cells and is a molecular mech-anism for enhanced cellular response of memory CD4� T cells. J. Immu-nol. 166, 7335–7344

39. Fallarino, F., Bianchi, R., Orabona, C., Vacca, C., Belladonna, M. L.,Fioretti, M. C., Serreze, D. V., Grohmann, U., and Puccetti, P. (2004)CTLA-4-Ig activates forkhead transcription factors and protects dendriticcells from oxidative stress in nonobese diabetic mice. J. Exp. Med. 200,1051–1062

40. Lu, L., Bonham, C. A., Chambers, F. G., Watkins, S. C., Hoffman, R. A.,Simmons, R. L., and Thomson, A. W. (1996) Induction of nitric oxidesynthase in mouse dendritic cells by IFN-�, endotoxin, and interactionwith allogeneic T cells: nitric oxide production is associated with dendriticcell apoptosis. J. Immunol. 157, 3577–3586

41. Weinberg, J. B., Granger, D. L., Pisetsky, D. S., Seldin, M. F., Misukonis,M. A., Mason, S. N., Pippen, A. M., Ruiz, P., Wood, E. R., and Gilkeson,G. S. (1994) The role of nitric oxide in the pathogenesis of spontaneousmurine autoimmune disease: increased nitric oxide production and nitricoxide synthase expression in MRL-lpr/lpr mice, and reduction of sponta-neous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine. J. Exp. Med. 179, 651– 660

42. Forrester, K., Ambs, S., Lupold, S. E., Kapust, R. B., Spillare, E. A., Wein-berg, W. C., Felley-Bosco, E., Wang, X. W., Geller, D. A., Tzeng, E., Billiar,T. R., and Harris, C. C. (1996) Nitric oxide-induced p53 accumulation andregulation of inducible nitric oxide synthase expression by wild-type p53.Proc. Natl. Acad. Sci. U.S.A. 93, 2442–2447

43. Cibelli, G., Policastro, V., Rössler, O. G., and Thiel, G. (2002) Nitric oxide-induced programmed cell death in human neuroblastoma cells is accom-panied by the synthesis of Egr-1, a zinc finger transcription factor. J. Neu-

rosci. Res. 67, 450 – 46044. Stambolic, V., MacPherson, D., Sas, D., Lin, Y., Snow, B., Jang, Y., Benchi-

mol, S., and Mak, T. W. (2001) Regulation of PTEN transcription by p53.Mol. Cell 8, 317–325

45. Virolle, T., Adamson, E. D., Baron, V., Birle, D., Mercola, D., Mustelin, T.,and de Belle, I. (2001) The Egr-1 transcription factor directly activatesPTEN during irradiation-induced signalling. Nat. Cell Biol. 3, 1124 –1128

46. Paolucci, C., Burastero, S. E., Rovere-Querini, P., De Palma, C., Falcone, S.,Perrotta, C., Capobianco, A., Manfredi, A. A., and Clementi, E. (2003)Synergism of nitric oxide and maturation signals on human dendritic cellsoccurs through a cyclic GMP-dependent pathway. J. Leukocyte Biol. 73,253–262

47. Sade, H., Krishna, S., and Sarin, A. (2004) The anti-apoptotic effect ofNOTCH-1 requires p56lck-dependent, AKT/PKB-mediated signaling inT cells. J. Biol. Chem. 279, 2937–2944

48. Liu, P., Aitken, K., Kong, Y. Y., Opavsky, M. A., Martino, T., Dawood, F.,Wen, W. H., Kozieradzki, I., Bachmaier, K., Straus, D., Mak, T. W., andPenninger, J. M. (2000) The tyrosine kinase p56lck is essential in coxsacki-evirus B3-mediated heart disease. Nat. Med. 6, 429 – 434

49. Silva, A., Jotta, P. Y., Silveira, A. B., Ribeiro, D., Brandalise, S. R., Yunes,J. A., and Barata, J. T. (2010) Regulation of PTEN by CK2 and NOTCH1 inprimary T-cell acute lymphoblastic leukemia: rationale for combined useof CK2- and �-secretase inhibitors. Haematologica 95, 674 – 678

50. Vo, K., Amarasinghe, B., Washington, K., Gonzalez, A., Berlin, J., andDang, T. P. (2011) Targeting NOTCH pathway enhances rapamycin an-titumor activity in pancreas cancers through PTEN phosphorylation. Mol.Cancer 10, 138

51. Bhattacharyya, S., Sen, P., Wallet, M., Long, B., Baldwin, A. S., Jr., andTisch, R. (2004) Immunoregulation of dendritic cells by IL-10 is mediatedthrough suppression of the PI3K/AKT pathway and of I�B kinase activity.Blood 104, 1100 –1109




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Stephanie K. Watkins and Kelvin P. LeeChandana Koorella, Jayakumar R. Nair, Megan E. Murray, Louise M. Carlson,

Production by Dendritic Cellsby NOTCH1 Protein in Interleukin-6 and Indoleamine 2,3-Dioxygenase

Novel Regulation of CD80/CD86-induced Phosphatidylinositol 3-Kinase Signaling

doi: 10.1074/jbc.M113.519686 originally published online January 10, 20142014, 289:7747-7762.J. Biol. Chem.

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