TLR and Nucleotide-Binding Oligomerization Domain-like Signals Differentially Regulate Exogenous Presentation This information is current as of September 26, 2021. Claudia S. Wagner and Peter Cresswell J Immunol 2012; 188:686-693; Prepublished online 9 December 2011; doi: 10.4049/jimmunol.1102214 http://www.jimmunol.org/content/188/2/686 Downloaded from

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Material 4.DC1 http://www.jimmunol.org/ References This article cites 62 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/188/2/686.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

TLR and Nucleotide-Binding Oligomerization Domain-like Receptor Signals Differentially Regulate Exogenous

Claudia S. Wagner and Peter Cresswell

The effect of (DC) maturation on MHC class II-restricted Ag presentation is well studied, but less is known about the effects of DC maturation on MHC class I-restricted cross-presentation. We investigated the ability of mature DCs to present Ags from cells infected with HSV-1. Pretreatment with pure LPS increased cross-presentation in a manner dependent on both MyD88 and Toll/IL-1R domain-containing adaptor inducing IFN-b, whereas a similar dose of a less pure LPS preparation inhibited cross- presentation. The difference could not be attributed to differences in uptake or phenotypic maturation. The likely contaminant responsible for shutting down cross-presentation is peptidoglycan (PGN). Addition of PGN to pure LPS abrogated its ability to Downloaded from enhance cross-presentation. Direct activation of DCs with PGN inhibited cross-presentation through nucleotide-binding oligo- merization domain-like receptor signaling. These results demonstrate that different maturation stimuli can have opposite impacts on the ability of DCs to cross-present viral Ags. The Journal of Immunology, 2012, 188: 686–693.

endritic cells (DCs) are specialized in the presentation of Much attention has been focused on the effects on cross-

exogenous Ags by MHC class II and I molecules. The presentation of maturation stimuli given with or after Ag expo- http://www.jimmunol.org/ D latter process is referred to as cross-presentation and, for sure (11, 14–16). How and to what extent activation prior to Ag most Ags, involves their transport into the cytosol and subsequent exposure regulates cross-presentation is less clear. Cross- translocation of derived peptides into phagosomes, early endo- presentation was inhibited in vivo after systemic administration somal compartments, or the endoplasmic reticulum for loading of TLR ligands (8) as well as after prolonged treatment of DCs onto MHC class I molecules (1). with certain TLR ligands in vitro (8, 17, 18). Besides decreased After activation with microbial products or inflammatory Ag uptake (8, 17, 18), inhibition of Ag access to the cytosol has mediators, DCs undergo a maturation process that regulates key also been proposed to explain the inability of mature DCs to functions, including migration, Ag processing, and expression of mediate cross-presentation (17), but the precise mechanism is not costimulatory molecules, all required for efficient priming (2, well characterized. A common rationale for the inhibition of by guest on September 26, 2021 3). Ag uptake is significantly reduced in mature DCs (4–10), and cross-presentation upon maturation is that presentation needs to be presentation of new Ags on MHC class II is impaired. Regulation restricted to Ags acquired during the initial stimulus (19). How- of MHC class II presentation during maturation has been inten- ever, there are examples in which pretreatment of DCs with cer- sively studied and includes changes in proteolysis, redistribution tain TLR ligands did not alter or even enhanced cross-presentation of peptide-loaded MHC class II molecules to the cell surface, of subsequently acquired Ag (18, 20–25). This may have the stabilization of surface complexes, and reduction of MHC class II benefit of allowing priming of CD8-positive T cells to pathogens synthesis (reviewed in Ref. 11). In contrast, MHC class I synthesis during an ongoing infection. Continuous Ag presentation in ma- is increased during maturation (12, 13). ture DCs has been reported for MHC class II-restricted presen- tation in certain cases (24, 25). TLR ligands are commonly chosen as activating stimuli for Ag Department of Immunobiology, Yale University School of Medicine, New Haven, presentation studies, reflecting the important role of TLR signal- CT 06520; and Howard Hughes Medical Institute, New Haven, CT 06520 ing in regulating DC maturation (26). TLRs are found on the Received for publication July 29, 2011. Accepted for publication November 9, 2011. cell surface and in endosomal compartments, sensing conserved This work was supported by the Howard Hughes Medical Institute. C.S.W. was structures of a broad range of microbial and viral ligands (27). supported by Vetenskapsra˚det and Deutsche Forschungsgemeinschaft Grant WA 2710/1-1. MyD88 is an intracellular adaptor protein that couples all TLRs Address correspondence and reprint requests to Dr. Peter Cresswell, Yale University except TLR3 to signaling pathways that induce inflammatory cyto- School of Medicine, Howard Hughes Medical Institute, Department of Immunobiol- kines. TLR4 is unique in that activation by this receptor involves se- ogy, 300 Cedar Street, Room TAC S669, New Haven, CT 06520. E-mail address: quential involvement of MyD88 and Toll/IL-1R domain-containing [email protected] adaptor inducing IFN-b (TRIF) (28). The nucleotide-binding oligo- The online version of this article contains supplemental material. merization domain (Nod), leucine-rich repeat-containing receptors Abbreviations used in this article: DC, dendritic cell; DKO, double-knockout; gB, (NLRs) are cytosolic pathogen sensors (29, 30). Two members of glycoprotein B; gD, glycoprotein D; iE-DAP, g-D-glutamyl-meso-diaminopimelic acid; KO, knockout; MDP, muramyl dipeptide; NLR, nucleotide-binding oligomer- the NLR family, the Nod-like receptors 1 and 2 (Nod1, Nod2), rec- ization domain, leucine-rich repeat-containing receptor; Nod, nucleotide-binding ognize specific structures in peptidoglycan (PGN) (31), whereas oligomerization domain; Nod1, nucleotide-binding oligomerization domain-like re- ceptor 1; Nod2, nucleotide-binding oligomerization domain-like receptor 2; Pen/ TLR2 signaling in response to PGN is mediated by linked lipo- Strep, penicillin/streptomycin; PGN, peptidoglycan; TRIF, Toll/IL-1R domain- teichoic acid and lipoproteins (32, 33). Signal transduction via containing adaptor inducing IFN-b; UT, untreated; VV-OVA, vaccinia virus- Nod1 and Nod2 results in an NF-kB–mediated proinflammatory expressed OVA. response (34). Besides bacterial recognition, Nod receptors are Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 involved in many processes, including autophagy induction, an- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102214 The Journal of Immunology 687 tiviral responses, and initiation of adaptive T cell responses (35, cubation. Necrotic or apoptotic HeLa cells were resuspended to 2.5 3 106/ml 36). in DC medium. Plaque assays with both cell preparations confirmed that no The field of cross-presentation is dominated by studies of the infectious virus could be recovered from either. For direct infection of DCs, HSV was added at a multiplicity of infection of 3 to DCs in medium model Ag OVA, and the vast majority of maturation studies use containing 10 mg/ml gentamicin instead of Pen/Strep. After 1 h of infec- OVA in soluble or immune-complexed forms. Bone marrow- tion, DCs were washed three times and kept in acyclovir containing DC derived DCs require an additional maturation stimulus during medium for 12 h before addition of T cells as described below. or shortly after Ag uptake to cross-present soluble OVA (15–17, Ag presentation assays 20). In this study, we have used apoptotic and necrotic HSV-1– infected cells as an Ag source, which have the advantage of Matured or untreated DCs were harvested from 24 wells, washed, and fed with apoptotic or necrotic infected HeLa cells at a 4:1 (DC:HeLa) ratio for 5 providing sufficient intrinsic immunostimulatory signals for to 6 h in 96-well round bottom plates. Subsequently, 1 3 105 gB- or gD- cross-presentation (37–40). Untreated and matured DCs were specific hybridoma T cells were cocultured with 1 3 104 Ag-loaded DCs compared for their ability to take up and present HSV-derived for 20 h. For coculture with the RR1/ICP6-specific CTL, Vero cells were Ags. We found that maturation induced by TLR2 and TLR4 used instead of HeLa, and 5 3 104 DCs were cocultured with 1 3 105 CTL signaling strongly inhibited subsequent MHC class II presenta- for 20 h. IL-2 in coculture supernatants was measured by ELISA according to the manufacturer’s protocol (BD Biosciences). tion, whereas cross-presentation was only inhibited after TLR2 triggering. PGN signaling through Nod receptors inhibited cross- Flow cytometry presentation but did not affect MHC class II presentation. Reg- Staining for cell-surface markers was performed with the following Abs and ulation of cross-presentation by intracellular innate immune appropriate isotype controls (all BD Biosciences) for 20 min at 4˚C. Anti– receptors may reflect the involvement of critical cytosolic pro- H-2Kb (AF6-88.5), anti–I-A/I-E (M5/114.15.2), anti-CD40 (3/23), anti- Downloaded from cesses in this phenomenon. CD86 (GL1), anti-CD70 (FR70), and all PE-conjugated and allophyco- cyanin-conjugated anti-CD11c (HL3). Data was collected on an FACS- Calibur (BD Biosciences) and analyzed with FlowJo software (Tree Star). Materials and Methods Mice profile C57BL/6 (B6) were obtained from The Jackson Laboratory (Bar Harbor, DCs were treated in the same manner as for Ag presentation assays to mimic ME). MyD88 knockout (KO) mice, TRIF KO mice, and TRIF/MyD88 cytokine levels in T–DC cocultures. After 22 h of maturation, DCs were http://www.jimmunol.org/ double-KO (DKO) mice were provided by R. Mezhitov (Yale University washed extensively and plated in 96-well plates, either with or without the School of Medicine). Animals were housed and used according to Yale’s addition of HSV-infected HeLa cells. After 24 h, supernatants were har- institutional guidelines. vested, and IL-2, IL-10, IL-12(p70), IFN-g, and TNF-a were measured with the Bio-Plex Pro Mouse Cytokine Assay on a Bio-Plex System (Bio- Cells Rad) according to the manufacturer’s instructions. Alternatively, super- natants of matured DCs cocultures with infected HeLa were used for Bone marrow-derived DCs were prepared from femurs/tibiae of mice be- transfer experiments and added to T–DC cocultures during Ag presentation tween 6 and 12 wk of age and cultured for 5–7 d in 24-well plates in RPMI assays with untreated DCs. 1640 (Life Technologies, Rockville, MD) with 10% FCS (Thermo Fisher Scientific, Waltham, MA), 50 mM 2-ME (Sigma-Aldrich), 2 mM L-glu- assays tamine, 100 U/ml penicillin, 100 mg/ml streptomycin (penicillin/ by guest on September 26, 2021 streptomycin [Pen/Strep]), 12 mM HEPES, nonessential amino acids Untreated or matured DCs were allowed to phagocytose apoptotic HeLa (all Life Technologies), and 20 ng/ml GM-CSF (R&D Systems). Medium cells labeled with the fluorescent membrane binding dye PKH26 (Sigma- was replenished on day 3. The HSV-1 glycoprotein B (gB)498–505-specific Aldrich) for 4 h at a 4:1 (DC/HeLa) ratio in a total volume of 250 ml MHC class I-restricted hybridoma HSV-2.3.2E2 was a generous gift from complete DC medium as was used for Ag presentation assays. As control G. Belz (Walter and Eliza Hall Institute of Medical Research) and cultured for uptake, one sample with untreated DCs and PKH26-labeled HeLa cells described (41, 42). The HSV glycoprotein D (gD)290–302-specific MHC was kept on ice during the 4-h uptake. Samples were then washed two class II-restricted hybridoma F1 was provided by A. Brooks (University of times in cold PBS-1% FBS, labeled on ice with anti-CD11c allophyco- Melbourne) and cultured as HSV-2.3.2E2. The RR1/ICP6822–829–specific cyanin, and fixed in 2% paraformaldehyde after two additional washes. H-2Kb–restricted CTL clone 1D11 and corresponding stimulator cell line Alternatively, untreated or matured DCs were fed in 24-well plates with B6/T350 RR1822–829 were provided by R. Bonneau (Pennsylvania State fixed bacteria (E. coli-expressing DsRed) at a DC/bacteria ratio of 1:25. University) and cultured as described (43). The B3Z CD8+ T cell hy- Samples were centrifuged for 1 min at 420 3 g at the onset of a 25-min bridoma specific for OVA257–264 was a gift from N. Shastri (University uptake. As a control in this experiment, one sample was pretreated for 20 of California, Berkeley) and kept in RPMI 1640 with 10% FBS, 2 mM min with 2.5 mg/ml cytochalasin D, and the drug was also present during L-glutamine, Pen/Strep, 50 mM 2-ME, and 1 mM pyruvate. HeLa and Vero uptake of the E. coli DsRed. DCs were then harvested and washed three cells were grown in DMEM with 10% FCS, L-glutamine, and 20 mg/ml times over a cold 2-ml FBS cushion, stained with anti-CD11c, and fixed in gentamicin. 2% paraformaldehyde. Phagocytosis was assessed by flow cytometry, gating on CD11-positive cells. Maturation of DCs Statistical analysis DCs were stimulated on day 5 or 6 of culture for 22 h or shorter times as indicated in supplemental figures. Stimuli were added to the medium in 24- Statistical analysis of ELISA data was done using ANOVA with Bonfer- well plates without disturbing DC clusters. Control cells were left untreated. roni’s post hoc test. Stimuli used were 0.1 and 1 mg/ml LPS (Escherichia coli 055:B5; catalog number L288; Sigma-Aldrich), ultrapure LPS (E. coli 0111:B4; Invivo- Gen, San Diego, CA), ultrapure PGN (insoluble and soluble sonicated Results form from E. coli K12; InvivoGen) at 2 mg/ml (except for Fig. 7), 10 mg/ LPS enhances cross-presentation while eliminating MHC class ml g-D-glutamyl-meso-diaminopimelic acid (iE-DAP), or muramyl di- II-restricted presentation peptide (MDP; InvioGen). There are conflicting data concerning the ability of mature DCs to Preparation of infected cells cross-present exogenous Ags. To determine whether DCs are ca- HeLa cells were infected with HSV-1 (F strain) at a multiplicity of infection pable of cross-presenting ligands from virally infected cells, we of 5:1 for 1 h in culture medium. Cells were then washed and kept in used HSV-1–specific T cell hybridomas as a readout, as they are medium containing 0.2 mM acyclovir (Calbiochem) for 10 h. Residual virus less influenced by costimulation than primary T cells. DCs were was UV inactivated with 200 mJ/cm2 in a Stratalinker 1800. HeLa-HSV were cultured for an additional 6 h before use as apoptotic bodies. To matured for 22 h with 0.1 or 1 mg/ml LPS, either conventionally produce necrotic bodies, HeLa-HSV were subjected to three rounds of isolated or highly purified, or left untreated, and subsequently freeze/thaw after residual virus inactivation and used without further in- allowed to phagocytose apoptotic HSV-1–infected HeLa cells. 688 Nod RECEPTOR SIGNALS INFLUENCE CROSS-PRESENTATION

They were then cocultured with the H2-Kb–restricted HSV-1– explained by a differential capacity to take up exogenous material specific hybridoma HSV-2.3.2E2, recognizing an from gB (Fig. 1C). Although the DCs stimulated with either concentration (Fig. 1A), or assessed for their ability to present Ags on MHC of impure LPS exhibited decreased phagocytosis compared with class II using the HSV-1 gD-specific hybridoma F1 (Fig. 1B). DCs untreated DCs, they were virtually identical in taking up PKH26- matured with 1 mg/ml dose of the less pure LPS lost the ability to labeled apoptotic HeLa cells (Fig. 1C, upper panel)orE. coli cross-present, whereas the lower dose enhanced cross-presentation expressing the fluorescent protein DsRed (Fig. 1C, lower panel). (Fig. 1A). The responses at the different doses could not be Notably, internalization was inhibited by cytochalasin D, indi- cating that the assay reflects genuine phagocytosis (Fig. 1C). The different response of DCs to high and low doses of LPS was not seen when the pure LPS was used. DCs pretreated with either dose of pure LPS cross-presented better than untreated DCs (Fig. 1A). MHC class II presentation was shut off independently of the LPS source or dose (Fig. 1B). The inhibition of MHC class II pre- sentation was faster than that of cross-presentation and could al- ready be detected after 2 h of LPS pretreatment (Supplemental Fig. 1A,1B). Both LPS concentrations induced a strong mature phenotype, and both sources resulted in the same degree of phe- notypic maturation (Fig. 1D, Supplemental Fig. 1C) and induced

comparable reduction in phagocytosis (see below). Similar results Downloaded from were obtained using freeze-thawed infected HeLa cells, mimick- ing the process of necrotic death (data not shown). MyD88 and TRIF signaling pathways are both involved in regulation of Ag presentation during LPS-induced maturation

LPS can signal through two different pathways. The MyD88- http://www.jimmunol.org/ dependent pathway is initiated after LPS binding to surface TLR4, whereas a second pathway involves TRIF signaling from the endosome (28). To investigate which pathway is involved in the regulation of cross-presentation, we prepared DCs from MyD882/2, TRIF2/2, or DKO mice and compared their ability to present HSV-1 Ags after 22 h of maturation induced by the less pure LPS. The enhancement of cross-presentation that was seen in wild-type DCs pretreated with 0.1 mg/ml LPS was clearly attenuated in both MyD882/2 and TRIF2/2 DCs, and in the DKO cells, the en- by guest on September 26, 2021 hancement was eliminated (Fig. 2A). Unexpectedly, however, we found that high-dose (1 mg/ml) LPS pretreatment abolished cross- presentation even in the MyD882/2/TRIF2/2 DCs, despite the complete absence of phenotypic maturation (Fig. 2C). Loss of either MyD88 or TRIF reduced phenotypic maturation after LPS treatment, with the reduction in the TRIF2/2 DCs being slightly greater than in MyD882/2 DC (Fig. 2C). Elimination of either adaptor protein alleviated to some extent the LPS-induced block in MHC class II presentation (Fig. 2B). As expected, MHC class II presentation by DKO DCs treated with either dose of LPS was essentially identical to that of untreated DCs (Fig. 2B).

FIGURE 1. Pure LPS enhances gB cross-presentation. DCs were acti- PGN both blocks cross-presentation and overrides its vated for 22 h with 0.1 or 1 mg/ml LPS (Sigma-Aldrich) or pure LPS LPS-induced enhancement (InvivoGen), fed apoptotic HSV-1–infected HeLa cells for 5 h, and finally A possible explanation for the observation in Fig. 2A that cross- cocultured with the MHC class I-restricted gB-specific hybridoma (A)or the MHC class II-restricted gD-specific hybridoma (B). IL-2 was measured presentation was eliminated in a MyD88/TRIF-independent man- by ELISA after 20 h. One representative of three experiments is shown. ner at the higher concentration of the less pure LPS is that the Error bars depict SD. C, Phagocytic capacity of LPS-matured DCs. DCs preparation was contaminated with a component that inhibits were matured with the indicated amounts of LPS (Sigma-Aldrich) for 22 h cross-presentation independently of TLR signaling. LPS prepa- or left untreated. DCs were then fed for 4 h with PKH26-labeled infected rations are often contaminated with lipoproteins and/or PGNs. To apoptotic HeLa cells (upper panel)orfixedE. coli-expressing DsRed determine whether PGN could inhibit cross-presentation and/or (lower panel). As controls, DCs were left on ice or treated with cytocha- override the LPS-mediated enhancement of gB cross-presen- + lasin D. Samples were gated on CD11c cells. Numbers indicate percent tation, DCs were matured for 22 h in the presence of either uptake. One representative of three experiments is shown. D, DCs prepared stimulus or with a combination of both. Subsequently, DCs were as in A and B were labeled with anti-CD11c and anti-H2Kb (MHC I), anti– tested for the ability to take up and process HSV-1 Ags for cross- I-A/I-E (MHC II), anti-CD86, anti-CD40, or anti-CD70 and analyzed by flow cytometry gating on CD11c+ cells. Black histograms represent un- presentation. DCs matured with 2 mg/ml PGN alone completely treated DCs, DCs treated with 0.1 mg/ml LPS (Sigma-Aldrich) are in or- lost the ability to cross-present (Fig. 3). PGN also eliminated ange, 1 mg/ml LPS (Sigma-Aldrich) in red, 0.1 mg/ml LPS (InvivoGen) in cross-presentation when added together with either 0.1 mg/ml of light blue, and 1 mg/ml LPS (InvivoGen) in dark blue. *p , 0.05, **p , the less pure or 1 mg/ml of the pure form LPS, which, when added 0.01, ***p , 0.001. alone, enhanced cross-presentation as before. The Journal of Immunology 689 Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Disruption of LPS signaling and the effect on modulation of Ag presentation. DCs were prepared from WT, MyD88 KO, TRIF KO, or MyD88/TRIF DKO mice, activated for 22 h with 0.1 or 1 mg/ml LPS, then fed apoptotic HSV-1–infected HeLa cells for 5 h, and finally cocultured with the MHC class I-restricted gB-specific hybridoma (A) or the MHC class II-restricted gD-specific hybridoma (B). IL-2 was measured by ELISA after 20 h. One representative of three experiments is shown. Error bars depict SD. C, DCs prepared as in A and B were labeled with anti-CD11c and anti-H2Kb (MHC I), anti–I-A/I-E (MHC II), anti-CD86, anti-CD40, or anti-CD70 and analyzed by flow cytometry gating on CD11c+ cells. Shaded histograms represent UT DCs, black lines DCs treated with 0.1 mg/ml LPS, and gray lines 1 mg/ml LPS. *p , 0.05, **p , 0.01, ***p , 0.001.

PGN-mediated signaling through Nod receptors can inhibit course, exclude the possibility that TLR2 signaling by PGN might cross-presentation also be capable of inhibiting cross-presentation. To further investigate the effect of PGN on cross-presentation, We also examined the effects of these PGN preparations on we compared highly purified PGN, detected solely through Nod MHC class II-restricted presentation. In wild-type DCs, this was receptors, termed PGN (Nod), with PGN that still contains TLR2- dramatically reduced by PGN (TLR2+Nod), whereas there was stimulating activity [PGN (TLR2+Nod)]. Cross-presentation was hardly any effect when the PGN preparation only capable of Nod abrogated when DC maturation was induced with either PGN triggering was used (Fig. 4B, left panel). Although still show- preparation prior to Ag uptake (Fig. 4A, left panel). A time course ing a statistically significant difference to untreated DCs, MHC for PGN pretreatment showed a progressive inhibition, with clearly class II presentation by PGN (TLR2+Nod) was largely restored in 2/2 decreased cross-presentation after 12 h and almost complete in- the MyD88 DCs (Fig. 4B, right panel), arguing that TLR2 hibition after 18 h (Supplemental Fig. 1D). That activation by the signaling is mainly responsible for inhibition of MHC class II pre- Nod signaling pathway is capable of inhibiting cross-presentation sentation. Consistent with this, in wild-type cells PGN (TLR2+Nod) was confirmed by using DCs from MyD882/2 mice, in which the treatment induced greater phenotypic maturation, including in- TLR2 signal induced by the PGN(TLR2+Nod) preparation would creased upregulation of MHC class II cell surface expression, be absent (27) (Fig. 4A, right panel). This experiment does not, of than did PGN (Nod) (Fig. 4C). 690 Nod RECEPTOR SIGNALS INFLUENCE CROSS-PRESENTATION

FIGURE 3. PGN inhibits cross-presentation in the presence of LPS. DCswereactivatedfor22hwith0.1or2mg/ml PGN alone or in combination with 0.1 mg/ml LPS (Sigma-Aldrich) or 1 mg/ml pure LPS (InvivoGen) as indicated, then fed apoptotic HSV-1–infected HeLa cells for 5 h, and finally cocultured with the MHC class I-restricted gB-spe- cific hybridoma. IL-2 was measured by ELISA after 20 h. One repre- sentative of two experiments is shown. Error bars depict SD. *p , 0.05, ***p , 0.001. Downloaded from

Differences in Ag uptake could not explain the results. PGN (TLR2+Nod) reduced uptake to a similar extent as LPS, and PGN (Nod)-treated DCs were comparable to untreated DCs in the phagocytosis of apoptotic HeLa cells (Fig. 5A). PGN-treated DCs did not have a general defect in MHC class I presentation as direct http://www.jimmunol.org/ presentation after DC infection with HSV-1 was not compromised (Fig. 5B). Neither was the ability to stimulate T cells after pulsing with the antigenic peptide gB498–505 (Fig. 5C). These experiments also rule out a possible role for a loss of a costimulatory signal or induction of an inhibitory molecule by the DCs that might affect hybridoma stimulation. Secreted are also unlikely to be responsible for the observed inhibition of cross-presentation (Fig. 5D, Supplemental Fig. 2). The cytokine profile of DCs (IFN-g, FIGURE 4. PGN can inhibit cross-presentation through Nod signaling. TNF-a, IL-2, IL-10, and IL-12) was assessed under conditions DCs were prepared from WT or MyD88 KO mice and activated for 22 h by guest on September 26, 2021 mimicking the situation in T cell cocultures. PGN (Nod)-matured with 2 mg/ml PGN (TLR2+Nod) or PGN (Nod). Matured DCs were fed DCs induced slightly more TNF-a and IL-12 than untreated DCs, apoptotic HSV-infected HeLa cells for 5 h and cocultured with the MHC class I-restricted gB-specific hybridoma (A) or the MHC class II-restricted but overall cytokine levels were low (Supplemental Fig. 2). DCs gD-specific hybridoma (B). IL-2 was measured by ELISA after 20 h. One cultured with infected HeLa cells showed a remarkable increase in representative of three is shown. Error bars depict SD. C, DCs prepared as IL-10 production, with Nod-activated cells producing comparable in A and B were labeled with anti-CD11c and anti-H2Kb (MHC I), anti–I- levels and TLR-stimulated DCs producing less IL-10 than un- A/I-E (MHC II), anti-CD86, anti-CD40, or anti-CD70 and analyzed by treated (UT) (Supplemental Fig. 2). To directly test whether se- flow cytometry gating on CD11c+ cells. Shaded histograms represent UT creted cytokines interfered with the outcome of cross-presentation, DCs, dashed black lines PGN (Nod), and gray lines PGN (TLR2+Nod). these supernatants were added to DC–T cocultures during Ag pre- **p , 0.01, ***p , 0.001. sentation assays (Fig. 5D). Supernatant of PGN-stimulated DCs had no influence, whereas supernatant of LPS-treated DCs enhanced Cross-presentation of HSV-RRI/ICP6 is also affected by PGN- cross-presentation to some extent, although not reaching statistical induced maturation significance. To test whether PGN (Nod)-mediated effects are specific for gB, we Nod1 and Nod2 signaling interferes with cross-presentation assessed cross-presentation for another HSV protein, HSV-RRI/ Nod1 and Nod2 recognize different structural motifs in PGN. Nod1 ICP6, which encodes a cytosolic ribonucleotide reductase. Pre- recognizes the dipeptide iE-DAP, found in PGN of all Gram- treatment with 2 mg/ml PGN (Nod) significantly reduced re- negative and only certain Gram-positive bacteria (44, 45). Nod2 sponses, and 10 mg/ml resulted in strong inhibition (Fig. 7A), has a broader detection range, recognizing MDP that is found in whereas gDT presentation on MHC class II remained unaffected PGN from both Gram-positive and Gram-negative bacteria (46, (Fig. 7B). Both concentrations of PGN (Nod) induced a compa- 47). To confirm that PGN regulated cross-presentation through rable level of mild phenotypic maturation (Supplemental Fig. 3B). Nod signaling, DCs were pretreated with synthetic iE-DAP or MDP. Both abrogated the ability to cross-present HSV-1–gB (Fig. Discussion 6A). Similar to pure PGN, iE-DAP signaling had little effect on Presentation of exogenous Ags is differently regulated for MHC MHC class II presentation of HSV-gD (Fig. 6B). Treatment with classes I and II during maturation. Our results demonstrate that, MDP, however, also reduced MHC class II presentation to a depending on the nature of the activation stimulus, cross- greater extent (Fig. 6B) and induced stronger phenotypic matu- presentation can be either enhanced or inhibited in a manner ration than pure PGN or iE-DAP (Fig. 6C). No differences in cell independent of regulation of Ag uptake. In addition to plasma viability were observed after treatment with any of these stimuli as membrane and endosomal TLRs, cytosolic receptors can contrib- assessed by flow cytometry (data not shown). ute to regulation of cross-presentation. The Journal of Immunology 691 Downloaded from

FIGURE 6. Nod receptor signaling interferes with cross-presentation. DCs were activated for 22 h with PGN, iE-DAP, or MDP, then fed apo- ptotic HSV-infected or uninfected HeLa for 5 h, and finally cocultured with the MHC class I-restricted gB-specific hybridoma (A) or the MHC class II- restricted gD-specific hybridoma (B). IL-2 was measured by ELISA after

20 h. One representative of three experiments is shown. Error bars depict http://www.jimmunol.org/ SD. C, DCs prepared as in A and B were labeled with anti-CD11c and anti- H2Kb (MHC I), anti–I-A/I-E (MHC II), anti-CD86, anti-CD40, or anti- CD70 and analyzed by flow cytometry, with gating on CD11c+ cells. Shaded histograms represent untreated, black lines DAP-treated, and gray lines MDP-treated DCs. *p , 0.05, **p , 0.01, ***p , 0.001.

ditions that induced the same strong phenotypic maturation and decreased uptake to a similar degree, as well as eliminating MHC class II-restricted Ag presentation, but had opposite effects on by guest on September 26, 2021 cross-presentation. A change in Ag uptake was thus ruled out as the major determining factor in the difference in the ability to cross-present. We have also been unable to observe any changes in phagosomal acidification upon PGN treatment that could have explained the block in cross-presentation (data not shown). FIGURE 5. Other functions of PGN treated DCs are normal. A, Uptake Both enhancement of cross-presentation and downregulation of of PKH26 labeled apoptotic cells. DCs were left untreated or matured for 22 h with the indicated stimuli, then fed infected apoptotic PKH26 labeled MHC class II-restricted presentation by prior LPS treatment of DCs HeLa cells for 4 h and analyzed by flow cytometry as in Fig. 1C. Percent were codependent on MyD88 and TRIF, consistent with the in- uptake in CD11c+ cells was normalized to uptake in CD11c+ UT DCs. volvement of TLR4 (28). Based on the increased response toward Numbers after stimuli indicate concentration in micrograms per milliliter. Summary of three experiments; error bars depict SD. B, Direct presenta- tion on MHC class I. UT or DCs matured for 22 h were directly infected with HSV overnight and cocultured with the gB-specific hybridoma. Numbers after stimuli indicate concentration in micrograms per milliliter. C, UT or DCs matured for 22 h with the indicated stimuli (PGN, 2 mg/ml; pure LPS, 0.1 mg/ml) were pulsed with no gB498–505 peptide, 0.5, or 5 nM peptide for 1 h, washed, and cocultured with the gB-specific hybridoma. D, Effect of cytokine release by PGN-matured DCs. DCs were matured for 22 h or left untreated, washed extensively, and recultured with HeLa-HSV for supernatant collection for additional 20 h. Supernatants were added to DC–T cocultures during Ag presentation assays. For B–D, one represen- tative of two experiments is shown. *p , 0.05, **p , 0.01. FIGURE 7. PGN inhibits cross-presentation of a second HSV Ag. DCs were activated for 22 h with the indicated stimuli (numbers give concen- A common explanation for downregulation of cross-presentation tration in micrograms per milliliter) and then fed apoptotic HSV-infected Vero cells (A) or HSV-infected HeLa cells (B) as described in the Materials during DC maturation is inhibition or decrease in Ag uptake (8, 17, and Methods section. The RR1822–829-expressing stimulator cell line 18). However, more recent literature suggests that certain forms of (Stim) was used as positive control in A. DCs were subsequently cocul- are still functional in mature DCs (22, 24, 25). In our tured with an MHC class I-restricted RR1/ICP6-specific T cell line (A)or hands, mature DCs could still take up apoptotic cells or bacteria, the MHC class II restricted gD-specific hybridoma (B). IL2 was measured albeit in reduced amounts compared with untreated DCs. By by ELISA; error bars show SD. One experiment of at least two is shown. comparing less pure with highly purified LPS, we defined con- **p , 0.01, ***p , 0.001. 692 Nod RECEPTOR SIGNALS INFLUENCE CROSS-PRESENTATION directly infected or peptide-pulsed matured DCs compared with MHC class II presentation was observed with synthetic MDP. UT DCs, it is likely that at least some of the increase in cross- Perhaps we were unable to achieve sufficiently high cytosolic presentation observed with LPS-treated DCs was due to costim- concentrations of MDP to observe this. How PGN degradation ulation. However, costimulation could not explain inhibition of products reach the cytosol to meet their respective receptors is not cross-presentation by the different PGN preparations or less pure fully clarified (49), similar to the situation regarding the mecha- LPS. Phenotypic maturation is inversely correlated with the ability nism for translocation of phagosomal Ags for cross-presentation to present newly acquired Ags on MHC class II, but in our (1). Nevertheless, cross-presentation was highly susceptible to experiments, there was no correlation between the degree of signaling mediated by cytosolic Nod receptors. Pretreatment with phenotypic maturation and the ability to cross-present Ags on pure PGN as well as both synthetic ligands for Nod1 and Nod2 MHC class I. impaired cross-presentation. Unusually high amounts of LPS have often been used experi- Why should a cytosolic receptor be involved in regulating cross- mentally for DC stimulation. Concentrations have ranged up to 10– presentation of exogenous Ags? Nod receptors have been shown 20 mg/ml (17, 21, 23) even though LPS can mediate effects at to be important for immune responses against multiple bacteria nanogram/milliliter levels (20, 48). In the experiments reported in (reviewed in Refs. 35, 36), including bacteria that directly gain this study, inhibition of cross-presentation by 1 mg/ml LPS oc- access to the cytosol such as Listeria monocytogenes (60, 61) or curred only when using the less pure LPS, and it is possible that Shigella flexneri (62). There is evidence that Nod receptor acti- impurities could have contributed to the inhibitory effects of high- vation occurs in vicinity to cellular membranes. Close proximity dose LPS described in the literature (17, 21). Although we cannot to endosomal and phagosomal membranes positions Nod recep- be certain of the exact nature of the impurity in LPS responsible tors at sites that potentially have the highest concentration of Downloaded from for eliminating cross-presentation PGN is a likely candidate. In translocated ligands liberated during degradation of bacterial cell analyzing the problem, we unexpectedly found that PGN signaling wall components in the respective organelle or ligands that are via cytosolic Nod receptors can inhibit cross-presentation of HSV- directly injected via bacterial secretion systems (29, 30). In terms 1 Ags without affecting MHC class II presentation. of Ag presentation, it may make sense that a DC invaded by a Under physiological conditions, DCs often meet a combination bacterium ceases to shuttle exogenous Ags into the cytosol for the

of TLR and NLR ligands. It is thus not surprising that cross cross-presentation–dependent MHC class I pathway. Ags already http://www.jimmunol.org/ talk occurs not only between different TLRs but also between in the cytosol could be presented via the classical MHC class I TLR and Nod receptors as seen in this study. Synergistic or an- route, which is still functional in activated DCs (8, 17). Unlike for tagonistic effects may occur (49). Nod ligands injected into mice MHC class II presentation, most protein Ags or Ag fragments concomitant with OVA resulted in enhanced cross priming, which must first be transported for further processing into the cytosol for was in part due to upregulation of costimulatory molecules (50). cross-presentation to occur. Nod receptor triggering may thus act TLR triggering through signals delivered together with antigenic as a feedback loop that signals that sufficient foreign material is material during phagocytosis or endocytosis enhances cross- present in the cytosol to induce CD8-positive T cell priming. The presentation and cross priming (16, 51, 52). This is somewhat precise mechanism by which the inhibition of cross-presentation different from our in vitro experiments, which examined how occurs remains to be determined, but a potentially exciting ex- by guest on September 26, 2021 maturation prior to Ag encounter modulates the ability to cross- planation is that Ag translocation from the phagosome into the present. We clearly saw that Nod ligation abrogated cross- cytosol is shut down, either by inhibiting translocation directly or presentation of HSV Ags even in the presence of an LPS signal by eliminating the translocation machinery from the phagosomal that would otherwise enhance it. Cross-presentation of vaccinia membrane. virus-expressed OVA (VV-OVA) was also decreased by DC pre- treatment with either PGN (Nod) or PGN (TLR2+Nod) (Supple- Acknowledgments mental Fig. 3A). However, the effect was not as profound as We thank Noah Palm for helpful discussions and Nancy Dometios for observed for HSV-gB or HSV-ICP6 (Figs. 4, 7). This may either manuscript preparation. argue for an additional role of signaling events triggered by HSV- infected cells not present in VV-infected cells, or it may be Disclosures explained by the nature of the OVA Ag. The fact that OVA is The authors have no financial conflicts of interest. soluble and expressed in the cytosol, whereas gB is a transmem- brane protein does not appear to be relevant, because cross- presentation of RRI/ICP6, also a cytosolic soluble molecule, is References inhibited by PGN (Nod) (Fig. 7). Depending on the delivery form, 1. Amigorena, S., and A. Savina. 2010. Intracellular mechanisms of antigen cross presentation in dendritic cells. Curr. Opin. Immunol. 22: 109–117. OVA is predominantly cross-presented through either the vacuolar 2. Mellman, I., and R. M. Steinman. 2001. Dendritic cells: specialized and regu- or the cytosolic pathway (53). Although VV-OVA is thought to lated antigen processing machines. Cell 106: 255–258. follow the cytosolic route for cross-presentation (54 and data not 3. Guermonprez, P., J. Valladeau, L. Zitvogel, C. The´ry, and S. Amigorena. 2002. Antigen presentation and T cell stimulation by dendritic cells. Annu. Rev. shown), a certain degree of endosomal loading that is not affected Immunol. 20: 621–667. by maturation could still take place. The high Ag expression 4. Garrett, W. S., L. M. Chen, R. Kroschewski, M. Ebersold, S. Turley, generated by VV-driven OVA compared with naturally expressed S. Trombetta, J. E. Gala´n, and I. Mellman. 2000. Developmental control of endocytosis in dendritic cells by Cdc42. 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