International Immunology, Vol. 14, No. 9, pp. 1027±1037 ã 2002 The Japanese Society for Immunology

Differential regulation of maturation and of human -derived dendritic cells mediated by MHC class II

Anna E. Lokshin1, Pawel Kalinski2, R. Rita Sassi1, Robbie B. Mailliard2, Jan MuÈ ller-Berghaus2, Walter J. Storkus2, Xiaojun Peng1, Adele M. Marrangoni1, Robert P. Edwards1 and Elieser Gorelik3

1Department of Obstetrics/Gynecology and Reproductive Sciences, 2Department of Surgery, and 3Department of Pathology, University of Pittsburgh and University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213, USA

Keywords: apoptosis, dendritic cells, homotypic aggregation, maturation, MHC class II,

Abstract

+ Antigen-driven interaction of dendritic cells (DC) with CD4 Th cells results in the exchange of bidirectional activating signals. Cross-linking of TCR by MHC class II-bound antigen activates Th cells, resulting in their up-regulation of CD40 ligand. Here we show that MHC class II molecules, in addition to their passive role in DC±Th cell interaction, can also actively induce DC maturation. Cross-linking of MHC class II molecules on human monocyte-derived DC results in the up- regulation of the surface expression of CD83, CD80, CD86, CD54, CD1a and CD40 molecules, the typical DC maturation-associated markers. It also promotes a rapid homotypic aggregation of DC paralleled by the up-regulation of such adhesion molecules as VLA-4, tissue transglutaminase, CD54 and CD11c. The impact of MHC class II cross-linking upon DC was context dependent. The outcome of MHC class II signaling depends on the maturation status of DC. While the cross-linking of MHC class II on immature DC promoted their maturation, the dominant effect upon the DC that were previously matured was the induction of DC apoptosis. Our current observations indicate that, in addition to the previously reported negative impact of MHC class II-mediated signaling on DC function, it also promotes DC maturation, participating in the enhancement of DC stimulatory function. Importantly, MHC class II-induced DC maturation and apoptosis are mediated by different signaling pathways, sensitive to different sets of inhibitors. This opens the possibility of differential regulation of each of these events in immunotherapy.

Introduction Dendritic cells (DC) are the most potent antigen-presenting The MHC class II molecules are constitutively expressed by cells (APC) and can play a critical role in the initiation of T cell- the various APC, such as DC, macrophages and B cells, and mediated immunity. Immature DC reside in most non- have a crucial role in generating antigen-speci®c T cell lymphoid tissues, and have high capability of antigen capture responses. MHC class II are heterodimeric glycoproteins and processing. Following their activation, DC migrate to the consisting of two non-covalently associated polymorphic draining lymphoid organs, where they mature and acquire the chains, a (32±34 kDa) and b (29±32 kDa). MHC class II ability to present antigenic peptides to T lymphocytes chains consist of extracellular, transmembrane and cytoplas- [reviewed in (1,2)]. DC maturation is manifested by an mic domains, and are expressed by the various APC, such as increase in the expression of co-stimulatory molecules, such DC, macrophages and B cells [reviewed in (6)]. The as CD80, CD86 and CD40, and a decreased capacity to extracellular portion of MHC class II molecules that consists process antigen. DC present antigenic peptides complexed of a1 and b2 domains forms a groove containing nanomeric with MHC class I and II molecules that are recognized by peptide that is recognized by TCR of CD4+ T cells. In addition, CD8+ or CD4+ T cells respectively (3±5). CD4 co-receptor binds the same MHC class II molecule (7,8).

Correspondence to: A. Lokshin, Lab 320, MWRI, 204 Craft Avenue, Pittsburgh PA, 15213, USA. E-mail: [email protected] Transmitting editor: D. Green Received 10 February 2002, accepted 4 June 2002 1028 MHC class II-mediated maturation and apoptosis of human DC Engagement of TCR and CD4 molecules by MHC class II form (Ser473 pAkt) were from Cell Signaling (Beverly, MA). results in a well-documented signal transduction cascade Monoclonal anti-Lyn, -Src, -Bcl-2, phosphotyrosine (PY99), as inside CD4+ T cells leading to T cell activation [reviewed in well as rabbit anti-Bax, and goat anti-caspase-3 and anti-g- (9)]. To verify whether the TCR±MHC class II interaction may actin antibodies were from Santa Cruz Biotechnology result in a signal transduction also into APC, several studies (Santa Cruz, CA). PKC inhibitor, H-7, mitogen-activated were performed using antibody-mediated cross-linking of kinase kinase (MEK) inhibitor, PD 98059, and MHC class II molecules on B lymphocytes as a model. Cross- phosphatidylinositol (PI)-3 kinase inhibitor, wortmannin, were linking of MHC class II molecules by speci®c antibodies leads from Biomol (Plymouth Meeting, PA). Caspase inhibitor Z-Val- to either proliferation or apoptosis of B cells (10,11). These two Ala-Asp(OMe)-CH2F (zVAD-fmk) was obtained from BD outcomes were regulated by two separate MHC class II- PharMingen (San Diego, CA) and zVAD±FITC was purchased initiated pathways, with proliferation requiring new from Promega (Madison, WI). Mitochondrial dye, DiOC6, was transcription and activation of Src family tyrosine kinases, from Molecular Bioprobes (Eugene, OR). and apoptosis depending on serine/threonine phosphatases, and cytoskeletal mobility (10±17). Activation and translocation Generation and culture of DC of isoenzymes of the protein kinase C (PKC) family and Peripheral blood mononuclear cells (PBMC) were obtained intracellular calcium were shared between the two pathways from venous blood drawn from normal healthy volunteers at (10±17). Furthermore, MHC class II was shown to mediate the Pittsburgh Central Blood Bank. PBMC were isolated by aggregation of B lymphocytes via a protein tyrosine kinase centrifugation on a Ficoll-Diatrizoate density gradient (ICN, (PTK)-dependent pathway that preceded activation of PKC Aurora, OH). Subsequently, the cells were separated on a and involved the LFA-1 molecules (13,14,18,19). The intracy- Percoll (Sigma) gradient (1.076, 1.059 and 1.045 g/ml). toplasmic region of the HLA-DR chain was requisite for the were further puri®ed by a 45-min adherence principal signaling pathway initiated via MHC class II (6,16). step. Non-adherent cells were harvested, and monocytes (5 These results imply that MHC class II molecules may function 3 105 cells/ml) were cultured for 6 days in 24-well plates simultaneously both as ligands and receptors which send (Costar, Cambridge, MA) in IMDM with 10% FCS (Hyclone, signals into the cells expressing these molecules. Logan, UT) supplemented with granulocyte macrophage Recently, several groups reported the induction of apopto- colony stimulating factor (GM-CSF; 1000 U/ml) (gift from sis after MHC class II ligation on human monocytes and DC Immunex, Seattle, WA) and IL-4 (800 U/ml) (Genzyme, Boston, (20±24). According to these reports, in these cells, MHC class MA). At day 6, the cultures consisted of uniformly HLA-DR+, II cross-linking with speci®c antibodies led to caspase- and CD83± and CD40high immature DC, without detectable CD3+ Fas-independent forms of apoptosis within 6±24 h (20,22). cells. MHC class II ligation on human DC has been reported to stimulate tyrosine phosphorylation (25,26), suggesting the Treatment procedures possibility of signal transduction from MHC class II molecules The viability of the cells preceding treatment was con®rmed to into DC. Recent data suggested that HLA-DR, -DQ and -DP be >90% by Trypan blue exclusion. DC were treated with 500 molecules transmit signals to monocytes via MAP kinases and ng/ml of anti-MHC class II antibody, isotype-matched control lead to distinct monokine activation patterns, which may affect IgG1 or anti-CD45 antibody for the indicated time periods. T cell responses in vivo (16). While it has been reported that Based on the results of preliminary dose±response experi- MHC class II may participate in the regulation of the cytokine ments (data not shown), this concentration of anti-MHC class II production by DC (27), its impact on DC maturation has not mAb was chosen as optimally stimulating. When the effects of been addressed. Here we report that ligation of MHC class II different inhibitors were evaluated, drugs were added to the by speci®c antibody transduces a maturation-inducing signal cells 1 h before anti-MHC or control antibodies. into DC, suggesting an additional role of MHC class II±TCR Flow cytometry binding during the antigen-driven cognate interaction of Th cells and DC. Our data indicate that the induction of DC Cells were pelleted and washed with FACS buffer (PBS, 1% maturation and apoptosis are regulated by different signaling BSA and 0.1% sodium azide). Cells were incubated with cascades, and preferentially affect DC at different stages of optimal concentrations of FITC- or phycoerythrin-conjugated maturation. primary antibodies for 30 min at 4°C. For all experiments, isotype control IgG1 or irrelevant control anti-CD45 mAb were included. Cells were then washed, ®xed with 2% paraform- Methods aldehyde, and ¯ow cytometric analysis was performed using a FACScan apparatus and CellQuest software from Becton Reagents Dickinson Immunocytometry Systems (San Jose, CA). Mouse anti-HLA-DR,DP,DQ mAb (TuÈ39, mouse IgG2a, k), anti-CD45 mAb, isotype-matched mouse IgG1 and all Cell death assays ¯uorochrome-conjugated antibodies for ¯ow cytometry were Immature DC were stimulated with a cytokine maturation purchased from PharMingen (San Diego CA). A series of 15 cocktail (MC) consisting of tumor necrosis factor (TNF)-a anti-MHC class II antibodies with different speci®cities was (10 ng/ml; Sigma, St Louis, MO), rhIL-1b (10 ng/ml; Genzyme), obtained from Terra Nova Biotechnology (St John's, rhIL-6 (1000 IU/ml; Novartis, Basel, Switzerland) and prosta-

Newfoundland, Canada). Neutralizing anti-Fas mAb ZB4, glandin E2 (1 mg/ml; Sigma). Immature and mature DC were polyclonal rabbit antibodies to Akt and its phosphorylated incubated with 500 ng/ml of anti-MHC class II antibody or MHC class II-mediated maturation and apoptosis of human DC 1029

Fig. 1. Stimulation of DC maturation by anti-MHC class II mAb. Monocyte-derived DC were cultured with TuÈ39 antibody (MHC class II) or mouse control isotype IgG2a (Control) (each at 500 ng/ml) for 24 h. Expression of CD86, CD80, CD83 and CD1a was analyzed by ¯ow cytometry using FITC- or phycoerythrin-conjugated mAb. Cells stained with FITC- or phycoerythrin-conjugated mouse IgG (IgG) served as control for non-speci®c antibody binding. Similar results were obtained in ®ve independent experiments.

IgG1 control in the presence or absence of inhibitors for 8 h. Apoptosis was evaluated using the Annexin V binding assay. Fig. 2. Anti-MHC class II mAb enhances adhesive properties of Cells (5 3 105) were washed in PBS and 100 ml of FITC- human DC. DC were incubated with 500 ng/ml of TuÈ39 antibody conjugated Annexin V (5 mg/ml) in a calcium-containing buffer (MHC class II) or mouse IgG2a (Control). (A) Homotypic clustering was detected after 3 h of incubation. (B) Expression of adhesion- were added according to the manufacturer's instructions related molecules (VLA-4, tTG, CD54 and CD11c) was analyzed by (Clontech, Palo Alto, CA). After incubation for 10 min at room ¯ow cytometry after 24 h of incubation. temperature, 400 ml of calcium-containing buffer was added and the samples were immediately analyzed by ¯ow cytometry as described above. To assess loss of mitochondrial potential, bated with 20 ml of Protein A/G±agarose beads (Calbiochem) DC were stained with 40 nM mitochondrial potential-sensitive for 1 h at 4°C with shaking. Total cell extracts containing 100 mg dye DiOC6 for 15 min at 37°C according to the manufacturer's total protein were separated on 12% SDS±PAGE, transferred protocol and changes of DiOC6 staining were analyzed by ¯ow to nitrocellulose membranes (Hybond-C; Amersham) and cytometry. When caspase inhibitor, zVAD-fmk, was used, DC immunoblotted with speci®c antibodies as indicated. were preincubated with 50 mM of this compound 1 h before were visualized by enhanced chemiluminescence addition of antibodies. After 4 h of incubation, more zVAD-fmk using Western blotting Luminol reagent (Santa Cruz was added to a ®nal concentration of 100 mM and incubation Biotechnology). continued for a further 4 h. Immunoprecipitation and immune complex kinase assay Caspase activity assay Lyn and Src proteins were immunoprecipitated from total cell Total caspase activity was measured in DC treated with or lysates. Immunoprecipitations were conducted for 1 h at 4°C without anti-MHC class II mAb using FITC±VAD-fmk caspase using speci®c antibodies immobilized on Protein A/G±agarose substrate (Promega) according to manufacturer's instructions. beads (Santa Cruz). Immunoprecipitates were incubated for Activated caspases covalently bind this substrate allowing for 20 min at 24°C with exogenous substrates in protein kinase

¯ow cytometric detection. DC were incubated with 10 nM buffer (50 mM HEPES, pH 7.4, 10 mM MgCl2, 10 mM MnCl2, FITC±VAD-fmk for 20 min at 37°C, washed with PBS and 50 mM ATP and 250 mCi/ml [g-32P]ATP; 4500 Ci/mmol). subjected to ¯ow cytometric analysis. [g-32P]ATP incorporation was analyzed by SDS±PAGE and autoradiography. Western blot analysis and immunoprecipitation Lysates of DC (106 cells/sample) treated with anti-MHC class II Cytokine release or control IgG1 were prepared in a lysis buffer (50 mM Tris, pH DC were grown in 24-well plates at 1 3 106 cells/well and 7.5, 150 mM NaCl, 4 mM EDTA, 1% Triton X-100, 20 mg/ml treated with or without anti-MHC class II antibody, isotype aprotinin, 10 mM leupeptin and 10 mM pepstatin A). To deplete control IgG1 or MC for 24±48 h at 37°C. Following treat- lysates from anti-MHC class II IgG, they were further incu- ment supernatants were collected and stored at ±80°C. IL-6, 1030 MHC class II-mediated maturation and apoptosis of human DC IL-12p70, IFN-g and TNF-a concentrations were measured using ELISA kits (R & D Systems, Minneapolis, MN) according to the manufacturer-provided protocols. The detection limits for the kits were: IL-1b (>30 pg/ml), TNF-a (>50 pg/ml), IL-6 (>50 pg/ml) and IL-12 p70 (>10 pg/ml).

Statistics For each experiment DC from at least ®ve donors were evaluated. All experiments were performed in triplicate, unless otherwise indicated, and mean values 6 SD are presented. Comparisons between the values were performed using a two- tailed Student's t-test. A value of P < 0.05 was considered statistically signi®cant.

Results

Effect of MHC class II cross-linking on DC adhesion Immature DC were generated by culturing peripheral blood monocytes with GM-CSF and IL-4 for 6 days. These cells had Fig. 3. Increased sensitivity of mature DC to apoptosis following typical DC morphology and were characterized by the MHC class II cross-linking. Immature and mature human monocyte- absence of monocyte marker, CD14, and lineage-related derived DC were treated with 500 ng/ml of TuÈ39 antibody for 8 h. antigens, CD3, CD56, CD19 and CD16 (not shown). They DC were stained with Annexin V±FITC and analyzed by ¯ow expressed CD80 (B7-1), CD86 (B7-2) and CD1a (Fig. 1), as cytometry. The results of one of ®ve representative experiments are presented. well as MHC class I and II molecules (data not shown), all molecules that are involved in antigen presentation and T cell activation by APC (1,2). However, these DC expressed very examined. To test the impact of MHC class II triggering on low amounts of CD83, which is a marker of mature DC, and expression of DC maturation markers, day 6 immature DC could, therefore, be characterized as immature (Fig. 1). To were exposed to 500 ng/ml of TuÈ39 for 24 h or to control examine the direct effects of MHC class II triggering on DC, isotype IgG1. Treatment of DC with TuÈ39 antibody resulted in day 6 immature DC were exposed to 500 ng/ml of TuÈ39 anti- up-regulation of CD83, CD80, CD86 and CD1a as compared MHC class II mAb (for various time intervals. The most to control (Fig. 1). Prolonged (up to 4 days) exposure of DC to noticeable and immediate response of DC was a rapid (after anti-MHC class II antibody further potentiated maturation 3±4 h) and robust cluster formation (Fig. 2A), indicating an effects (data not shown). It is possible that just cross-linking of increase in homotypic aggregation. No unclustered cells any molecules expressed on DC surface would non-speci®c- could be observed in anti-MHC class II antibody-treated DC ally up-regulate maturation markers. To address this concern, cultures following their resuspension by gentle pipetting. In we incubated DC with anti-CD45 antibody, which is abundant contrast, IgG1 or anti-CD45 antibody-treated DC cultures had on DC. However, engagement of CD45 did not result in DC 92.2 6 14.3 and 93.0 6 11.5% of non-clustered cells maturation (see below in Fig. 4). respectively. In order to investigate mechanisms of MHC To investigate whether anti-MHC class II mAb stimulated class II-mediated homotypic aggregation of DC we analyzed cytokine production by DC, we measured the concentration of the expression of adhesion molecules that are involved in cell± proin¯ammatory cytokines, IL-1b, TNF-a and IL-6, as well as cell and cell±substrate binding of DC, such as CD11c (integrin IL-12 p70, in cell culture medium in the presence or absence a chain) (28), CD54 (ICAM-1) (29,30) and VLA-4 (a b x 4 1 of TuÈ39 or MC. TuÈ39 did not up-regulate the secretion of any of integrin) (31,32). Flow cytometric analysis revealed that anti- these cytokines (data not shown). It is of note that incubation of MHC class II antibody-treated DC had a higher surface DC with MC also did not stimulate cytokine production (data expression of all three adhesion molecules as compared to not shown) untreated or anti-CD45 mAb-treated DC. Up-regulation of VLA-4 was most profound (Fig. 2B). Tissue transglutaminase (tTG) serves as a ligand for VLA-4 in non-hematopoietic cells, Engagement of MHC class II on DC triggers apoptotic cell promoting cell adhesion and spreading (33,34). In our death experiments, MHC class II-mediated homotypic clustering of MHC class II-mediated apoptosis in DC has been well DC was accompanied by a substantial increase in surface tTG documented (20±24). Before we explored the relationship expression (Fig. 2B). between maturation and apoptosis, we ®rst re-evaluated MHC class II-mediated apoptosis in our experimental system. In Effect of MHC class II cross-linking on expression of DC agreement with these data, in our experiments the percentage surface maturation markers of apoptotic DC increased from 10.6 6 3.45% in IgG1-treated Homotypic clustering of DC closely correlates with their state cells to 41.1 6 13.41% in immature DC and from 14.5 6 6.92 to of maturation (35). However, to this end, the effects of MHC 72.8 6 17.12% in mature DC treated with TuÈ39 (Fig. 3). No class II cross-linking on DC maturation have not been apoptotic changes could be detected in anti-CD45 antibody MHC class II-mediated maturation and apoptosis of human DC 1031

Fig. 4. Regulation of MHC class II-mediated maturation and apoptosis in human DC. Human monocyte-derived DC were treated with 500 ng/ ml of anti-MHC class II mAb in the presence or absence of inhibitors (see Methods). DC incubated with 500 ng/ml of mouse IgG or anti-CD45 mAb served as controls. (A) Apoptosis measured as percent of Annexin V+ cells. (B and C) Expression of maturation markers CD83 and CD86 presented as MFI after subtraction of non-speci®c antibody binding. Each point presents the mean of three representative experiments. *P > 0.95. or isotype-matched mouse IgG-treated DC (Fig. 4A). In involving the downstream executioners caspase-3, -6 and -7 agreement with published observations, neutralizing anti-Fas (40). Caspase-10 is a novel caspase homologous to caspase- antibody, ZB4, did not abrogate MHC class II-induced 8 whose physiological role is yet unknown (41). It has been apoptosis (Fig. 4A), suggesting that MHC class II-induced demonstrated that caspase-10 mutations result in abnormal apoptosis is Fas± independent. However, contrary apoptosis of DC underlying a unique disorder of DC home- to the published observations (20,22,24), in our experimental ostasis (38). Western blot demonstrated cleavage of caspase- system, the broad-spectrum caspase inhibitor/pseudosub- 3, -9 and -10, but not of caspase-8, in response to stimulation strate zVAD-fmk substantially inhibited MHC class II-induced with TuÈ39 (Fig. 5B). apoptosis in DC (Fig. 4A), indicating that activation of It has been documented that MHC class II ligation in mouse caspases is required for MHC class II-induced cell death in splenic DC results in mitochondrial permeability transition, DC. When DC were loaded with FITC-conjugated zVAD-fmk, indicating that MHC class II-mediated apoptosis in DC utilized MHC class II ligation resulted in increased intracellular the mitochondrial pathway (22). In agreement with these ¯uorescence indicative of caspase activation (Fig. 5A). To observations in our experiments, MHC class II cross-linking ascertain the input of individual caspases in total caspase resulted in pronounced loss of mitochondrial dye DiOC6 activity, Western blot analysis was performed using speci®c staining, indicating altered mitochondrial permeability transi- antibodies against caspase-3, -8, -9, and -10, known to be tion (Fig. 5C). However, in contrast to the cited publication, activated during different stages of apoptotic pathways (36± bongkrekic acid, which is an inhibitor of the mitochondrial 38). Caspase-9 is the initiating caspase in mitochondrial permeability transition, reduced apoptosis as measured by apoptotic pathway that is death receptor independent (39,40). Annexin V binding (Fig. 4A) in human monocyte-derived DC, Once activated, caspase-9 can initiate a caspase cascade implicating the mitochondrial pathway in the MHC class II- 1032 MHC class II-mediated maturation and apoptosis of human DC

Fig. 5. Effect of anti-MHC class II antibody on caspase activity and mitochondrial membrane potential. Immature and mature DC were treated with isotype control IgG (Control) or anti-MHC class II mAb for 6 h (A and C) or for indicated time intervals (B). (A) The level of caspase activity was analyzed using the zVAD±FITC substrate by ¯ow cytometry. (B) Expression of caspases was analyzed by Western blot using speci®c antibodies. Jurkat cells treated with 50 ng/ml of anti-Fas antibody for 6 h were used as a positive control for caspase-8 cleavage. (C) Mitochondria permeability transition was assessed by DiOC6 staining and analyzed by ¯ow cytometry. mediated apoptotic cell death of human DC. We next examined the effects of MHC class II cross-linking on expression of anti-apoptotic protein, Bcl-2, and pro-apoptotic protein, Bax, in human DC. Interestingly, increased expression of both proteins was observed following 24 h of anti-MHC class II antibody treatment (Fig. 6). In addition, TuÈ39 up- regulated the expression of p53 protein in human DC (Fig. 6).

MHC class II-induced maturation and apoptosis of human DC are regulated via different signaling pathways We next proceeded to ascertain the regulation of maturation and apoptosis of human DC following MHC class II cross- linking. To accomplish this task, the speci®c inhibitors of several signal transduction pathways were employed. DC Fig. 6. Anti-MHC class II antibody up-regulates the expression of were pretreated with either protein kinase A (PKA)/PKC apoptosis-regulating proteins. Mature DC were treated with anti- MHC class II mAb for the indicated time periods. Expression levels inhibitor, H-7, broad spectrum tyrosine kinase inhibitor, of Bcl-2, Bax and p53 were analyzed by Western blot analysis with genistein, PI-3 kinase inhibitor, wortmannin or MEK kinase speci®c antibodies. Expression of g-actin serves as a loading inhibitor, PD 98059, for 1 h before stimulating with anti-MHC control. class II. The selected concentrations of inhibitors have been demonstrated to inhibit corresponding enzymes in DC or monocytes (12,42). At selected concentrations, neither in- induced maturation of DC by testing the expression of hibitor alone affected the viability of DC (data not shown). We maturation-associated molecules, CD83, CD80, CD86, evaluated the effect of kinase inhibitors on MHC class II- CD11c and CD40. We have observed that MHC class II- mediated apoptosis. MHC class II-induced apoptosis de- mediated up-regulation of different maturation-associated pended on the MEK kinase/ERK pathway, since it was proteins was inhibited by a distinct set of inhibitors. completely inhibited by PD 98059 (Fig. 4A). Additionally, PI- However, contrary to Annexin V binding, the up-regulation of 3 kinase inhibitor, worthmannin, substantially inhibited MHC all these markers required tyrosine phosphorylation since it class II-induced apoptosis in DC (Fig. 4A). We further was completely abrogated by genistein (Fig. 4B and C, and examined the effect of these inhibitors on the MHC class II- data not shown). We chose to present here the effect of the MHC class II-mediated maturation and apoptosis of human DC 1033 Table 1. Effects of different maturation agents and anti- MHC class II antibodies on apoptosis and maturation of human DC

Description CD86 (MFI) Annexin+ (%)

IgG control 21.2 6 14.87 17.3 6 7.44 MC 161.2 6 21.08 19.9 6 9.44 TNF-a 133.8 6 16.25 18.5 6 7.26 LPS 76.9 6 19.40 30.2 6 11.21 DP + DQ + DR (TuÈ39), IgG2a 74.6 6 17.24 47.3 6 14.23a DP monomorph, IgG2a 76.5 6 21.40a 44.3 6 14.46a DPB1:55±57:DEE, IgG2a 58.2 6 14.25a 18.6 6 8.24 DPB1:85±87:GPM, IgG2a 66.0 6 15.21a 39.3 6 9.38a DPB1:DPB1:55±56:DE, IgG2a 28.5 6 10.34 43.4 6 12.46a DPB1:84±87:GGPM, IgM 53.0 6 11.14a 38.4 6 14.1a DPB1:8:V/11:L + 35±36:FV, IgG1 27.6 6 13.21 33.5 6 10.18a DPB1:55±57:DED, IgG2b 64.2 6 18.12a 43.0 6 9.34a DPB1:55±56:DE+57±58:DE(DR11), 26.6 6 11.40 36.2 6 12.98a IgG1 DP monomorph, IgG1 59.1 6 12.30a 31.8 6 9.18a DQ monomorph, IgG2a 67.6 6 14.65a 22.2 6 12.66 DQ monomorph, IgG2a 53.0 6 18.92a 36.0 6 11.75a DR monomorph IgG3 60.1 6 15.14a 47.1 6 10.28a DR monomorph, IgG2a 74.1 6 18.16a 45.4 6 15.61a DR4 monomorph, IgG1 64.8 6 14.87a 32.5 6 11.23a DR4Dw14, IgM 66.7 6 20.64a 37.4 6 14.41a Fig. 7. MHC class II cross-linking induces protein tyrosine DC were puri®ed and grown for 6 days as described in Methods. phosphorylation. DC (106 cells/sample) were treated with or without DC were treated with or without indicated anti-MHC class II mAb at anti-MHC class I mAb for indicated time intervals at 37°C and then 500 ng/ml for 24 h and analyzed by ¯ow cytometry. The values subjected to Western blot analysis. (A) MHC class II cross-linking given correspond to one out of at least three representative induces tyrosine phosphorylation of 60-, 32- and 25-kDa proteins experiments. (arrows) as determined by staining with anti-phosphotyrosine (PY99) aP > 0.95. mAb. (B) Activation of Lyn and Src kinases was assayed by immune complex kinase assay and autoradiography. Equal loading of Src and Lyn proteins was con®rmed by staining with appropriate above inhibitors on the expression of two representative antibodies (not shown). Activation of Akt was evaluated using phospho-Akt (Ser473) or pan-Akt antibodies. markers, CD86 and CD83 (Fig. 4B and C). As shown, in addition to the requirement for tyrosine phosphorylation, MHC class II-driven up-regulation of CD86 depended on the PKA or 16 anti-MHC class II mAb with different epitope speci®cities PKC pathway since it was inhibited by H-7. However, the for 24 h and tested for expression of CD86. As shown in above pathway was not involved in MHC class II-mediated up- Table 1, all anti-HLA-DQ and -DR antibodies tested up- regulation of CD83. It is possible that the apoptosis is the regulated CD86 regardless of their epitope speci®city. primary effect of MHC class II cross-linking resulting in However, three out of eight anti-HLA-DP mAb did not affect phagocytosis of dead DC by bystander DC, which may lead the expression of this maturation marker. Next, DC were to their maturation. To test this hypothesis, we examined the incubated with these antibodies for 8 h and tested for effect of MHC class II cross-linking by TuÈ39 antibody in the apoptosis by Annexin V binding. All examined anti-HLA-DR presence of zVAD-fmk and bongkrekic acid, both of which antibodies induced apoptosis. However, one anti-HLA-DP and ef®ciently inhibit MHC class II-mediated apoptosis in DC. one of the two anti-HLA-DQ mAb failed to induce apoptosis in Neither inhibitor affected MHC class II-mediated maturation of human DC (Table 1). DC (Fig. 4B and C). MHC class II ligation drives tyrosine phosphorylation and Effect of different maturation agents and various anti-MHC activation of Akt kinase class II antibodies on maturation and apoptosis of human We next proceeded to investigate whether MHC class II DC ligation triggers signal transduction cascade in human DC. To investigate whether maturation of DC is always paralleled First the changes in protein tyrosine phosphorylation in anti- with apoptosis, we examined Annexin V binding in DC MHC class II antibody-treated DC versus control DC were following incubation with the agents, known to be potent examined by Western blot analysis using speci®c anti- maturation inducers, lipopolysaccharide (LPS) and TNF-a phosphotyrosine antibody, PY99. MHC class II cross-linking and with a cytokine MC consisting of TNF-a (10 ng/ml), rhIL-1b consistently induced tyrosine phosphorylation of three

(10 ng/ml), rhIL-6 (1000 IU/ml) and PGE2 (1 mg/ml) (3). As proteins with apparent mol. wt ~60, 32 and 25 kDa (Fig. 7). shown in Table 1, stimulation of DC with LPS, but not with TNF- Tyrosine phosphorylation of these proteins was completely a and MC, was accompanied by apoptosis. To further prevented by pre-incubation of DC with the PTK inhibitor, examine the effects of MHC class II cross-linking on DC genistein (data not shown). Given that one of the phosphoryl- maturation and apoptosis, DC were incubated with a panel of ated proteins detected in our assay has mol. wt of ~60 kDa, it 1034 MHC class II-mediated maturation and apoptosis of human DC may correspond to activated p53/56lyn or p60Src kinases induced by the in¯ammatory cytokine TNF-a, alone or in (25,43,44). However, the immunoprecipitation of these combination with another in¯ammatory mediator, prostaglan- kinases followed by immune complex kinase assay did not din E2 (50). In contrast, factors such as CD40 ligand, TRANCE, reveal their activation following anti-MHC class I treatment of LPS, SAC and other bacterial products, induce an in¯amma- DC (Fig. 7). Therefore, the nature of proteins that become tory pattern of DC maturation, associated with the production phosphorylated upon MHC class I cross-linking remains of IL-12p70 and other DC-derived cytokines (51±55). unknown and may deserve further investigation. In accordance with previous publications (20±24), cross- We next explored the possibility that engagement of MHC linking of MHC class II molecules on human DC increases class I on human DC could activate serine-threonine kinase binding of Annexin V to the DC cell surface. It has been activity. MHC class II cross-linking on human DC did not observed that DC undergo apoptosis upon interaction with stimulate the activity of PKC and PKA kinases (data not antigen-speci®c T lymphocytes (4,56). MHC class II-mediated shown). However, anti-MHC class II antibody-treated DC apoptosis could be accountable for their death after accom- displayed a higher degree of phosphorylation and hence plishing their antigen-presenting function to CD4+ T lympho- activation of Akt/protein kinase B, a protein kinase that is cytes. The current observations that mature DC are more downstream of PI-3 kinase (Fig. 7). prone to undergo spontaneous apoptosis and that an add- itional activating/maturation-inducing signal, such as MHC class II-triggering, induces their death suggest that the Discussion optimal immunogenic activity of DC is limited to a relatively Data presented in this study indicate that the ligation of MHC narrow time window following their activation. class II molecules induces a signaling cascade in human Several studies reported the caspase independence of monocyte-derived DC, and modulates their maturation status, MHC class II-mediated apoptosis in DC based on the lack of stimulatory function and survival. To the best of our know- inhibition of HLA-DR-mediated apoptosis by zVAD-fmk or ledge, this is the ®rst discovery of MHC class II-mediated zDEVD-fmk (20,22). In our hands, however, zVAD-fmk was induction of maturation in DC. able to inhibit MHC class II-mediated apoptosis. The differ- Cross-linking of MHC class II molecules elicited several ence in the results obtained may be explained by different measurable effects. We observed that anti-MHC class II mAb schedules of zVAD-fmk administration. In our experiments, we potently stimulated homotypic aggregation of DC. Homotypic pre-incubated cells for 30 min before addition of the TuÈ39 aggregation of DC may correlate with their T cell stimulating antibody and then added more inhibitor 4 h later. Our rationale capabilities, as has been demonstrated in syngeneic mixed was that the reported half-life for irreversible caspase inhib- lymphocyte reactions (35). Enhanced homotypic aggregation ition by zVAD-fmk in vitro is <40 min. In agreement with the may result from MHC class II-mediated up-regulation of CD54 demonstrated role of caspase activation in MHC class II-

(ICAM-1), CD11c (integrin ax chain) and especially VLA-4 mediated apoptosis in DC, we have observed activation of (CD49d) molecules that determine adhesive properties of DC caspase-3, -9 and -10, but not caspase-8. (28,29,45,46). VLA-4 directly interacts with multiple cell sur- Inhibition of apoptosis by bongkrekic acid, which is an face proteins on hematopoetic and non-hematopoietic cells, inhibitor of the mitochondrial permeability transition (57), such as ®bronectin and VCAM-1. It has been shown that the a4 implicates the mitochondrial pathway in MHC class II-medi- subunit (CD49d) could also serve as a ligand for VLA-4 itself ated apoptosis. Our experiments revealed depolarization of (47). Thus cells expressing VLA-4 could interact with each mitochondrial membranes following MHC class II cross- other and form aggregates via VLA-4 molecules. Cross-linking linking. Furthermore, anti-MHC class II antibody activated enzyme tTG represents another ligand for VLA-4. tTG plays an caspase-9, which is the initiating caspase in the mitochondrial important role in adhesion and migration of monocytic cells apoptotic pathway that is death receptor independent (39,40). (48), mediating VLA-4 association with ®bronectin and Engagement of MHC class II up-regulated two apoptosis- potentiating integrin-mediated signaling (33,49). Induction of related proteins, Bcl-2 and Bax, that are localized to the tTG expression upon MHC class II cross-linking suggests that mitochondrial membrane [for review, see (58)]. Furthermore, tTG may play a role in MHC class II-mediated homotypic we have detected the up-regulation of expression of tumor aggregation of human DC. suppressor gene, p53. p53 mediates apoptosis through a Ligation of MHC class II by speci®c mAb leads to up- linear pathway involving up-regulation of Bax, release of regulation of several cell surface molecules including co- cytochrome c from mitochondria and activation of caspases stimulatory molecules, CD80 and CD86, that are actively (59,60). Therefore, it is possible that MHC class II cross-linking involved in antigen presentation and T cell activation (1,2). up-regulates p53, which in turn initiates the mitochondrial These phenotypic changes are typical for DC maturation, cascade. These observations further signify the role of which is further con®rmed by induction of expression of CD83, mitochondrial pathway in MHC class II-mediated apoptosis which is a marker of mature DC (1,2). Interestingly, MHC class in DC. II cross-linking does not induce the synthesis of DC Anti-MHC class II-induced increase in anti-apoptotic proin¯ammatory cytokines, such as IL-1b, TNF-a and IL-6, protein, Bcl-2, is somewhat surprising. Since no apoptotic indicating that the MHC class II-induced DC maturation is a cells remain in culture after 24 h, probably due to phagocy- direct phenomenon and not a secondary effect of the tosis by surviving DC [(21) and our unpublished observation], induction of maturation-inducing cytokines. MHC class II- it is possible that only the cells that express suf®cient levels of induced DC maturation occurs in the absence of the induction Bcl-2 to protect them from the apoptotic effects of Bax were of IL-12p70. Such a `silent' pattern of DC maturation is also able to survive. On the other hand, increased expression of MHC class II-mediated maturation and apoptosis of human DC 1035 Bax may be responsible for higher sensitivity of mature DC to Liu, Y. J., Pulendran, B. and Palucka, K. 2000. Immunobiology of induction of apoptosis. Both Fas±Fas ligand independence dendritic cells. Annu. Rev. Immunol. 18:767. 3 Ranieri, E., Herr, W., Gambotto, A., Olson, W., Rowe, D., Robbins, and involvement of mitochondria imply that MHC class II- P. D., Kierstead, L. S., Watkins, S. C., Gesualdo, L. and Storkus, mediated apoptosis occurs via the mitochondrial rather than W. J. 1999. Dendritic cells transduced with an adenovirus vector via death receptor signaling pathway (36±38,61). CD95 ligand encoding Epstein±Barr virus latent membrane protein 2B: a new and MHC class II ligation are likely to present distinct modality for vaccination. J. Virol. 73:10416. pathways for the elimination of DC at different stages of 4 Ingulli, E., Mondino, A., Khoruts, A. and Jenkins, M. K. 1997. In vivo detection of antigen presentation to CD4+ T maturation (22). cells. J. Exp. Med. 185:2133. The results of our study demonstrate the differential regu- 5 Steinman, R. M. 2001. Dendritic cells and the control of immunity: lation of MHC class II-mediated maturation and apoptosis in enhancing the ef®ciency of antigen presentation. Mt Sinai J. Med. human DC. Apoptosis is not required for MHC class II- 68:106. 6 Wade, W. F., Davoust, J., Salamero, J., Andre, P., Watts, T. H. and mediated maturation of DC. Furthermore, MC TNF-a and some Cambier, J. C. 1993. Structural compartmentalization of MHC anti-MHC class II antibodies that induce apoptosis do not class II signaling function. Immunol. Today 14:539. affect maturation and vice versa. Finally, different signal 7 Janeway, C. A., Jr, Mamula, M. J. and Rudensky, A. 1993. Rules transduction pathways regulate these two processes. for peptide presentation by MHC class II molecules. Int. Rev. Tyrosine phosphorylation appears to be critical for MHC Immunol. 10:301. 8 Wang, J. and Reinherz, E. L. 2000. Structural basis of cell±cell class II-mediated maturation, but not for apoptosis. interactions in the immune system. Curr. Opin. Struct. Biol. 10:656. Interestingly, the pattern of tyrosine phosphorylation following 9 Germain, R. N. and Stefanova, I. 1999. The dynamics of T cell cross-linking of class II molecules on human Langerhans cells receptor signaling: complex orchestration and the key roles of was shown to be similar to that induced by contact sensitizers tempo and cooperation. Annu. Rev. Immunol. 17:467. that are known to activate these cells (25). On the contrary, 10 Truman, J. P., Garban, F., Choqueux, C., Charron, D. and Mooney, N. 1996. HLA class II signaling mediates cellular MHC class II-induced apoptosis, but not maturation, depends activation and programmed cell death. Exp. Hematol. 24:1409. on MEK kinase/ERK and PI-3 kinase pathways. Our data agree 11 Truman, J. 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