CD1 Presentation by Human Dendritic Cells as a Target for Immune Evasion

This information is current as Martin J. Raftery, Florian Winau, Stefan H. E. Kaufmann, of September 26, 2021. Ulrich E. Schaible and Günther Schönrich J Immunol 2006; 177:6207-6214; ; doi: 10.4049/jimmunol.177.9.6207 http://www.jimmunol.org/content/177/9/6207 Downloaded from

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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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD1 Antigen Presentation by Human Dendritic Cells as a Target for Immune Evasion1

Martin J. Raftery,* Florian Winau,† Stefan H. E. Kaufmann,† Ulrich E. Schaible,† and Gu¨nther Scho¨nrich2*

In contrast to MHC molecules, which present peptides, the CD1 molecules have been discovered to present lipid Ags to T cells. CD1-restricted T lymphocytes have been recently associated with resistance to virus infection. The mechanisms underlying ac- tivation of CD1-restricted T cells in the course of virus infection are not defined. In this study, we wanted to investigate the interaction of HSV with the antiviral CD1 Ag presentation system in human dendritic cells (DC). In response to low titers of HSV, the surface expression of CD1b and CD1d on human DC was up-regulated. These phenotypic changes enhanced the capacity of infected DC to stimulate proliferation of CD1-restricted T lymphocytes. High titers of HSV, however, lead to strong down- Downloaded from regulation of all surface CD1 molecules. This modulation of surface expression was associated with intracellular accumulation, colocalization with viral proteins, and disruption of the CD1 recycling machinery. Finally, even at low titers HSV interfered with the capacity of infected DC to stimulate the release of important cytokines by CD1d-restricted NKT cells. Thus, we demonstrate both the existence of a CD1 pathway allowing human DC to react to viral infection, as well as its blockage by a human herpesvirus. The Journal of Immunology, 2006, 177: 6207–6214. http://www.jimmunol.org/ ajor histocompatibility class molecules display pep- Recent studies provide evidence that the CD1 Ag presentation tides that are recognized by T cells (1). The distantly system has an antiviral role. Patients with NKT cell deficiencies M related CD1 proteins constitute an evolutionarily dis- associated with disseminated viral infection have been described tinct family of Ag-presenting molecules (2). Humans express five (18, 19). In HIV-infected patients, a selective loss of NKT cells different CD1 molecules (CD1a-e). According to their sequence occurs (20–23). Moreover, insights gained from CD1d-deficient homology, CD1 proteins can be further divided into group I (CD1 mice suggest that CD1d-restricted T lymphocytes are required for a-c), group II (CD1d), and CD1e representing an intermediate protection against some viral infections (24–27) but not others form. Group I members are expressed by thymocytes, activated (28–32), possibly depending on the virulence of the virus strain 3 monocytes, B cells, and dendritic cells (DC) whereas CD1d has a used (32). In addition, CD1d-restricted T cells have been reported by guest on September 26, 2021 broader distribution (3). Unlike MHC class I, the CD1 molecules to play an essential role in virus-induced immunopathogenesis (33, function independently of the TAP molecules (4, 5). By trafficking 34). In a number of infection models, therapeutic activation of through endosomal compartments, CD1 proteins pick up Ags in a NKT cells by ␣GalCer helps to clear the virus (30, 35) or improves similar fashion to MHC class II molecules (6). disease outcome (24, 33). Stimulation of CD1-restricted T cells CD1-restricted T cells recognize lipids, glycolipids, or lipopep- has been demonstrated in a transgenic mouse model of hepatitis B tides of either foreign or self origin as Ags (7). Activation of T virus infection (36) and in patients with (37–39). Fi- cells by CD1-mediated lipid Ag recognition was first demonstrated nally, the discovery that can interfere with CD1 Ag pre- in the context of infection with Mycobacterium tuberculosis (8). The CD1b molecule presents various lipid Ags, including li- sentation (40–44) is indirect evidence for a role of CD1-restricted poarabinomannan (LAM), derived from mycobacteria (9). Besides T lymphocytes in antiviral immune defense. So far, the mecha- natural endogenous Ags (10, 11) and bacterial Ags (11–14), CD1d nisms leading to activation of CD1-restricted T cells in the course molecules can also present ␣-galactosylceramide (␣GalCer), a for- of viral infection remain unclear. eign lipid derived from a marine sponge (15, 16). NKT cells ex- DC, which link innate and adaptive immunity (45), can be in- press a CD1d-restricted invariant AgR and strongly respond fected by a panoply of viruses (46) including HSV (47–54). This to ␣GalCer (7, 17). ubiquitous pathogen is a member of the herpesvirus family, per- sists for the lifetime of its host, and causes life-threatening illness in immunocompromised individuals (55). HSV-infected DC lose *Institute of Virology, Charite´Medical School, Berlin, Germany; and †Max-Planck- their capacity to efficiently stimulate MHC-restricted T cells (47– Institute for Infection Biology, Berlin, Germany 54). On the molecular level, inhibition of MHC class I Ag pre- Received for publication March 14, 2006. Accepted for publication July 31, 2006. sentation is mediated by HSV-encoded infected cell protein (ICP) The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance 47, which blocks TAP function (56, 57). Numerous other findings with 18 U.S.C. Section 1734 solely to indicate this fact. document the ingenious viral strategies to block the MHC I path- 1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB421). way (58). In sharp contrast, knowledge on the interplay between 2 Address correspondence and reprint requests to Dr. Gu¨nther Scho¨nrich, Institute of viruses and the CD1 Ag presentation system is virtually lacking. In Virology, Charite´Medical School, Humboldt University Berlin, Schumannstrasse 20/ this study, we analyzed presentation of CD1-restricted Ags by 21, D-10117 Berlin, Germany. E-mail address: [email protected] HSV-infected human DC. We show that DC respond to the HSV- 3 Abbreviations used in this paper: DC, dendritic cell; LAM, lipoarabinomannan; ␣GalCer, galactosylceramide; ICP47, infected cell protein 47; MOI, multiplicity of encoded TAP blocker ICP47 by enhancing CD1 presentation and infection. that this cellular response, in turn, is counterregulated by the virus.

Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 6208 VIRAL IMMUNE EVASION OF CD1 Ag PRESENTATION

Materials and Methods Ϫ20°C for 5 min. Thereafter, slides were washed three times before being Cells and virus stained with mAbs against CD1a (clone HI149), CD1b (clone 4A7.6.5), CD1d (CD1d42), MHC class II (clone L243), or rabbit anti-HSV followed U373 cells were maintained in Eagle’s MEM supplemented with 10% heat- by isotype-specific Alexa 488- or Cy5-coupled Abs, or FITC-coupled goat inactivated FCS, 100 IU penicillin, 100 ␮g/ml streptomycin, and 4.5 mM anti-rabbit Abs. Slides were analyzed on a Leica TCS SP confocal glutamine. Recombinant virus ICP47⌬, which lacks the MHC class I microscope. blocker ICP47, and ICP47⌬ rescue virus were gifts from R. L. Hendricks (University of Illinois, Chicago, IL) (59). HSV type I strain F, from which Western blot the recombinant viruses were derived, was obtained from American Type Culture Collection (ATCC). Viruses were propagated in Vero E6 cells, the Cells infected for 18 h were lysed for 1 h and cleared of debris by cen- supernatant from lytic cells being cleared of debris by centrifugation and trifugation for 1 h. A total of 50 ␮g (U373) or 150 ␮g (DC) was treated subsequently frozen in liquid nitrogen. After 1 h incubation, cells were with peptide-N-glycosidase F (Roche) for 18 h before being loaded onto a washed three times with PBS before resuspension in medium. 15% polyacrylamide gel. After electrophoresis, transfer onto a polyvinyli- dene difluoride membrane and blocking membranes were stained with anti- CD1 transfectants CD1d mAb (clone Nor3.2), stripped, and restained with anti-␤-actin mAb (clone AC-15). Secondary Abs coupled to HRP were used at a concentra- For generation of CD1b- and CD1d-transfected cell lines, cDNA derived tion of 1/5000. from immature DC was amplified by PCR using appropriate primer sets and cloned into pEF6 (Invitrogen Life Technologies). Similarly, the ICP47⌬ gene was amplified from HSV type I strain F genomic DNA and T cell assays cloned into pIRES (BD Biosciences). The constructs were transfected into A total of 5 ϫ 104 APC (immature DC or U373) was irradiated with 50 Gy cell lines by electroporation. (immature DC) or 100 Gy (U373) and pulsed with 10 ng/ml pp65-peptide, 4 Generation of DC 10 ␮g/ml LAM, or 1–5 ␮g/ml ␣GalCer for 30 min before 5 ϫ 10 Ag- Downloaded from specific T cells/well were added. Cells were cultured in triplicate in com- Human DC were generated from monocytes isolated from buffy coat prep- plete medium in 96-well round-bottom plates for 3 days at 37°C and 5% arations supplied by the German Red Cross (Berlin, Germany). Monocytes 3 ␮ CO2 before [ H]thymidine was added for 18 h (1 Ci/well; Amersham). were isolated from PBMC by negative selection with Ab-coupled magnetic Thereafter, cells were harvested and liquid scintillation measurements were beads (Miltenyi Biotec), and cultured in RPMI 1640 supplemented with performed using a Top Count scintillation counter (Packard Instrument). 10% heat-inactivated FCS, 100 IU penicillin, 100 ␮g/ml streptomycin, 4.5 The specific NKT cell response shown in Figs. 3 and 4 was calculated by mM glutamine, 500 U/ml GM-CSF, and 200 U/ml IL-4. After 2 days of subtracting baseline proliferation (APC plus NKT cells) from proliferation culture, DC were used for experiments. Lipoteichoic acid from Staphylo- in the presence of Ag (APC plus NKT cells plus Ag). The baseline pro- http://www.jimmunol.org/ coccus aureus was provided by Cayla Sas/InvivoGen and used at concen- liferation was in every case below 2000 cpm. LCD4.7, a LAM-reactive T tration of 1 ␮g/ml. cell line, was derived from lesions of a leprosy patient (62). The CD1d- ␣ Antibodies restricted NKT cells have been described elsewhere (12). GalCer was supplied by the Kirin Brewery. In separate assays, supernatant was col- For phenotypic analyses of cell markers by flow cytometry, the following lected from the reactions and frozen at Ϫ20°C before being tested by reagents were used: human CD1a mAb clone HI149 was obtained from ELISA for IFN-␥, IL-4, and IL-10 (ImmunoTools). ImmunoTools; human CD1b mAb clone 4A7.6 (60) and human CD1c clone L161 were purchased from Immunotech; human CD1d mAb clone CD1d42 was obtained from BD Biosciences; clone Nor3.2 was obtained Results from Chemicon International; human CD1d mAb clone CD1d55 (61) was Altered expression of the CD1 Ag presentation system as a by guest on September 26, 2021 a gift from S. A. Porcelli (Albert Einstein College of Medicine, New York, response of human DC to HSV infection NY); and human MHCI mAb clone W6/32 was a gift from G. Moldenhauer (German Cancer Research Center, Heidelberg, Germany). Human ␤-actin HSV modulates both DC phenotype and function of MHC mole- mAb clone AC-15 was supplied by Acris. Isotype-matched control Abs cules. It is not known, however, if HSV alters the expression pat- were purchased from BD Biosciences/BD Pharmingen. Rabbit anti-HSV tern of the MHC-related CD1 proteins, molecules which are was obtained from DAKOPATTS. largely restricted to DC. We assessed surface expression of CD1 Flow cytometry molecules after infection of cell lines transfected with CD1 or endogenously CD1-expressing immature DC (Fig. 1A). At a mul- Cells in suspension were washed once with ice-cold FACSwash solution (PBS with 1% FCS and 0.02% sodium azide) before being resuspended tiplicity of infection (MOI) of 1, the surface levels of CD1a and with the first Ab in ice-cold blocking solution (PBS with 10% heat inac- CD1c molecules remained unchanged. In contrast, CD1d and tivated FCS and 0.2% sodium azide) for 1 h. The cells were then washed CD1b expression both on transfected cells and on DC was en- in ice-cold FACSwash solution and the staining was repeated with PE- hanced after infection with live HSV. Morphological changes of coupled anti-mouse secondary Ab. Subsequently, cells were washed in ice-cold FACSwash and then resuspended in 100 ␮l of PBS with 0.3% infected DC were observed by measuring the forward scatter/side formaldehyde. Flow cytometry was performed on a FACSCalibur (BD scatter profile (Fig. 1B, left panel). Moreover, double staining for Biosciences). For double-staining of CD1b and HSV Ags, the same pro- CD1b and HSV Ags confirmed up-regulation of CD1b on the sur- cedure was followed except with both primary Abs (CD1b-specific mAb face of HSV-infected DC at low MOI (Fig. 1B, right panel). In- and polyclonal antiserum against HSV Ags) or appropriate control Ab terestingly, the small percentage of cells expressing very high lev- (isotype-matched irrelevant Ab) given in the first phase, followed by Cy5- conjugated anti-mouse IgG and FITC-coupled anti-rabbit IgG as els of viral Ags showed strongly down-regulated CD1b expression. secondary Abs. No correlation between this population and apoptosis-induced Endocytosis assay DNA degradation could be seen (data not given). To analyze the cellular CD1 expression in more detail, we used confocal micros- Cells were stained with the relevant mAb in normal culture medium on ice copy. In DC, redistribution of CD1b was associated with the pres- for 1 h. For analysis of endocytosis, cells were stained with a FITC-coupled A secondary Ab and were subsequently incubated for2hat37°C. Cells were ence and colocalization of HSV-encoded Ags (Fig. 2 ). In DC then stripped of cell surface-bound Ab by incubation with a 300 mM gly- infected with live (MOI ϭ 1) but not UV-inactivated HSV, CD1b cine/1% FCS solution at pH 2 for 3 min before neutralization and fixation. lost its characteristic localization in the MHC class II compartment To analyze endocytosis with unlabeled mAb, cells were stained as above and partially relocalized to the cell surface (Fig. 2B). Control stain- but incubated for4hateither 4°C or 37°C. Cells were then stained for ings (Fig. 2, C and D) show the specificity of the reagents used. remaining cell surface-bound mAb with a PE-labeled secondary Ab. Taken together, these data demonstrate that some HSV proteins Confocal microscopy colocalize with CD1b molecules. Furthermore, at low MOIs, DC U373 transfectants grown on coverslips or immature DC (1 ϫ 105) cen- also increase surface expression of CD1d and CD1b in response to trifuged onto slides were fixed/permeabilized by acetone/methanol (1:1) at HSV infection. The Journal of Immunology 6209 Downloaded from

FIGURE 1. CD1 surface expression on human cells 18 h after infection with UV-inactivated (uvHSV) or live HSV at a MOI of 1. A, CD1 expres- http://www.jimmunol.org/ sion was analyzed on U373 cells transfected with the relevant CD1 mol- ecules (left panel) or immature DC (right panel). FACS histograms show CD1 surface expression as indicated (black curves; mean fluorescence in- FIGURE 2. Redistribution of CD1b to the cell surface upon HSV in- tensity (MFI) is given in the upper right corner). As a control, cells were fection, colocalization of viral proteins with CD1 molecules, and loss of stained with an isotype-matched Ab (gray filled curves). B, The forward characteristic colocalization of CD1b with MHC class II molecules. Im- scatter/side scatter profile (left panels) or double staining for HSV Ags and mature DC were infected with UV-inactivated (upper panels) or live HSV CD1b (right panels) of immature DC is shown. In the right panels, the (lower panels) for 18 h before being stained and analyzed by confocal x-axis represents staining with an irrelevant isotype-matched Ab (control) microscopy for (A) CD1b (blue fluorescence, Cy5) and HSV (green fluo- y rescence, FITC) or (B) MHC class II (blue fluorescence, Cy5) and CD1b

or a specific mAb (CD1b) whereas the -axis depicts staining with a poly- by guest on September 26, 2021 clonal antiserum against HSV Ags. Representative results of at least three (green fluorescence, Alexa 488). Colocalized molecules in the overlay ap- ␮ independent experiments are shown. pear light blue. Scale bar represents 10 M. Controls (C and D) demon- strate the specificity of the reagents used. Infected cells were stained as for (A) except that a normal rabbit serum was used instead of rabbit anti-HSV (C). Moreover, uninfected cells were stained as for (B) except that either an Activation of CD1-restricted T cells by HSV-infected human DC IgG1 isotype control (D, upper row) or an IgG2a isotype control (D, lower Next, we analyzed the functional consequences of CD1 up-regu- row) was used instead of human CD1b mAb and human MHC class II mAb, respectively. lation on low titer HSV-infected DC. To this end, HSV-infected immature DC pulsed with lipid Ag presented by CD1b (LAM) or CD1d (␣GalCer) were used to stimulate corresponding CD1-re- and CD1d-restricted T cells less efficiently than the rescue virus stricted human T cell lines (Fig. 3). As expected from previous (the ICP47 gene has been reintroduced into ICP47⌬) or wild-type studies, a T cell line recognizing peptide revealed reduced Ag pre- virus. However, the capacity of ICP47⌬-infected DC to activate sentation through MHC class I molecules (data not shown). In CD1-restricted T cells was still higher than that of uninfected DC. marked contrast, HSV-infected DC stimulated CD1b-restricted T These findings suggested that besides other unknown mechanisms, lymphocytes more efficiently than control DC (Fig. 3A). A similar ICP47 is contributing to increased CD1 Ag presentation by increase was seen for CD1d-restricted T lymphocytes (Fig. 3B). HSV-infected DC. Thus, immature DC respond to HSV infection by decreasing their capacity to induce proliferation of MHC class I-restricted T cell Decreased CD1 surface expression on cells infected with lines while simultaneously increasing the proliferation of CD1b ICP47-negative HSV and CD1d-restricted T cell lines. In additional experiments, we determined whether the absence of ICP47 influences CD1 surface expression. Indeed, immature DC Impaired response of the human CD1 Ag presentation system to infected with ICP47⌬ showed reduced levels of CD1d and CD1b ICP47-negative HSV on the surface as compared with cells infected with rescue virus As viral proteins can have multiple functions, we hypothesized that (Fig. 5A). The ICP47-associated changes in CD1d expression on ICP47, the only known HSV-encoded MHC class I blocker (56, infected DC was difficult to assess due to the low CD1d baseline 57), also interferes with Ag presentation through the MHC class expression on uninfected DC. For this reason, we used CD1d- I-like CD1 molecules. We anticipated that in the absence of ICP47, expressing U373 transfectants (U373-CD1d cells). Fig. 5B shows the HSV-induced activation of CD1-restricted T cells would be reduced CD1d levels on U373-CD1d cells infected with ICP47⌬ in further enhanced. Therefore, the stimulatory capacity of DC after comparison to cells infected with the rescue virus confirming the infection with ICP47⌬, a rHSV lacking ICP47, was determined results obtained with DC. These data imply that ICP47 enhances (Fig. 4). Surprisingly, DC infected with ICP47⌬ stimulated CD1b- the density of CD1 molecules on the surface of HSV-infected DC. 6210 VIRAL IMMUNE EVASION OF CD1 Ag PRESENTATION

FIGURE 5. Lack of CD1 up-regulation in cells infected with ICP47-

negative HSV. A, Immature DC or (B) U373-CD1d cells were infected Downloaded from (MOI ϭ 1) with mutant HSV lacking ICP47 (ICP47⌬) or the rescued virus FIGURE 3. Ag-specific activation of CD1-restricted T cells by HSV- able to express ICP47 (Rescue) 18 h before FACS analysis. In parallel, infected immature DCs. Immature DC were left uninfected or infected with immature DC treated with the corresponding UV-inactivated virus were uv-inactivated (uvHSV) or live HSV (MOI ϭ 1). After pulsing with (A) analyzed in the same way. As negative controls, isotype-matched Abs with LAM (CD1b presentation) or (B) ␣GalCer (CD1d presentation), the cor- an irrelevant specificity were used (gray filled curves). The respective MFI responding T cell clones were added. Activation of T cells was measured values are depicted in the upper right corner of the FACS histograms. The by [3H]thymidine incorporation during proliferation and is shown as cpm. results shown are representative for three independent experiments. http://www.jimmunol.org/ Error bars are mean values Ϯ SD of triplicates. The probability was de- .(p Ͻ 0.05 ,ء) termined by using the unpaired Student t test

CD1b (U373-CD1b) or CD1d (U373-CD1d) with an ICP47 ex- Enhanced CD1 Ag presentation by ICP47-expressing cells pression construct (Fig. 6A). ICP47 induced up-regulation of As a next step, we examined whether ICP47 up-regulates CD1 CD1b or CD1d on U373-CD1b or U373-CD1d cells, respectively. expression independently of viral infection. For this purpose, we transiently supertransfected U373 astroglioma cells expressing by guest on September 26, 2021

FIGURE 6. Enhancement of CD1 expression and presentation by ICP47. U373-CD1b cells or U373-CD1d cells were transiently supertrans- fected with pIRES-ICP47, a bicistronic expression construct driving both FIGURE 4. Reduced capacity of ICP47-negative HSV for activation of the expression of GFP and ICP47. As a control, cells were supertransfected CD1-restricted T lymphocytes. Immature DC were infected (MOI ϭ 1) with the vector expressing only GFP (Empty vector). A, The cells were with mutant HSV lacking ICP47 (ICP47⌬), rescue virus (rescue; the ICP47 stained for CD1b or CD1d and analyzed by FACS. The x-axis represents gene has been reintroduced into ICP47⌬) or the corresponding UV-inac- CD1 expression whereas the y-axis depicts GFP. The results shown are tivated virus (uvICP47⌬ and uvRescue, respectively). After pulsing with representative for three independent experiments. B, The cells were pulsed (A) LAM or (B) ␣GalCer, the corresponding T cells were added. Activation with (left panel) LAM or (right panel) ␣GalCer and the corresponding T of T cells was measured by [3H]thymidine uptake and is shown as cpm. cells were added. Activation of T cells was measured by [3H]thymidine Data are given as mean values and SDs for triplicate determinations. Error uptake and is shown as cpm. Error bars are mean values Ϯ SD of tripli- bars are mean values Ϯ SD of triplicates. The probability was determined cates. The probability was determined by using the unpaired Student t test .(p Ͻ 0.0005 ,ءءء ;p Ͻ 0.05 ,ء) .(p Ͻ 0.05 ,ء) by using the unpaired Student t test The Journal of Immunology 6211

Thus, ICP47 alone is sufficient for increasing CD1 surface expres- sion. Finally, we analyzed the effect of ICP47-induced up-regula- tion of CD1 surface molecules on T cell activation. CD1-restricted T lymphocytes were stimulated with CD1-expressing U373 cells after ICP47 transfection. In the presence of ICP47, the CD1b- expressing U373 cells stimulated LAM-specific T cells with higher efficiency than control cells transfected with the empty vector (Fig. 6B). Similarly, CD1d-expressing U373 cells presented ␣GalCer more efficiently to CD1d-restricted T cells when transfected with the ICP47 gene (Fig. 6B). Thus, host cells respond to the HSV- encoded MHC-I blocker ICP47 by enhancing CD1 Ag presentation.

Viral interference with CD1 Ag presentation in human DC So far, we analyzed the reaction of the cellular CD1 Ag presen- tation system to infection with HSV. However, members of the FIGURE 8. HSV disruption of CD1 trafficking. A, Immature DCs in- herpesvirus family are able to interfere with DC function. To vi- fected with a MOI of 100 were stained with FITC-coupled CD1a mAb and sualize potential viral evasion mechanisms cell lines transfected cultured for 2 h before being stripped by acid wash of cell surface Ab and with CD1 or endogenously CD1-expressing immature DC were fixed. Remaining FITC-positive cells represent endocytosed CD1a. B, Im- Downloaded from infected with a high MOI and surface expression of CD1 mole- mature DC or transfected U373 cells were infected with a MOI of 100 and cules was determined. Indeed, CD1 molecules were down-regu- stained with unlabeled mAb against CD1a or CD1d before being cultured lated on both transfected U373 cells and immature DC at a MOI of for 4 h either on ice or at 37°C. Cells were then stained with a PE-coupled 100 (Fig. 7A). In transfected cell lines, the total amount of CD1 secondary reagent and analyzed by FACS. The difference in positive cells at 37°C was taken from those on ice to give the quantity of CD1 molecules protein remained unchanged (MOI ϭ 1) or was slightly decreased endocytosed. The results shown are representative for three independent

ϭ http://www.jimmunol.org/ (MOI 100) postinfection (Fig. 7B, left panel). In contrast, at experiments. both low and high MOIs the total amount of CD1d protein was found to be diminished in DC after infection with live HSV as compared with UV-inactivated HSV (Fig. 7B, right panel). At low MOI, this block was not reflected by flow cytometry, where an the total amount of CD1 protein in DC indicating viral interference increased density of CD1d was detected on DC (see Fig. 1A) due with the human CD1 Ag presentation system. to the effect of HSV-encoded ICP47 and other unknown mecha- Additional experiments revealed that HSV inhibits internaliza- nisms. Thus, a high viral load reduces both surface expression and tion of CD1 molecules. After infection with HSV at a high MOI (100), DC were less able to internalize CD1a as measured by up- take (Fig. 8A) or reduction in cell surface Ab staining (Fig. 8B). In by guest on September 26, 2021 contrast, maturation induced by lipoteichoic acid from Staphylo- coccus aureus, a ligand of TLR2, which HSV has been shown to stimulate (63), did not interfere with internalization. However, this viral effect on cell function was not observed with transfected cells as both CD1a and CD1d internalization were not reduced by in- fection. Collectively, this observation emphasizes the unique na- ture of DC endogenously expressing CD1 molecules and reveal viral interference with the trafficking of CD1 molecules in DC. Finally, the capacity of HSV-infected DC to stimulate the re- lease of important cytokines characteristic for NKT cells (IL-4, IL-10, and IFN-␥) was assessed. DC infected with UV-inactivated virus induced more IFN-␥ or IL-4 than uninfected DC whereas DC infected with live HSV at a low (1) or high (100) MOI strongly inhibited IFN-␥ or IL-10 release by NKT cells (Fig. 9A). IL-4 secretion was not affected by DC that had been infected with a low MOI, most likely because IL-4 production by NKT cells is more rapid than other cytokines (64). To investigate the immediate effect of HSV, DC were mixed with NKT cells directly after infection (Fig. 9B). In this case, the inhibition of cytokine release by HSV FIGURE 7. Reduced amounts of CD1 molecules in human cells in- was less pronounced. Thus, at low and high titers, HSV interferes fected with a high titer of HSV. A, CD1 surface expression on human cells with the capacity of DC to stimulate cytokine release, particularly after infection with UV-inactivated (uvHSV) or live HSV at a high MOI IFN-␥, by NKT cells. (100). After 18 h, CD1 expression was analyzed by FACS on (left panel) U373 cells transfected with the relevant CD1 molecules or (right panel) Discussion immature DC endogenously expressing CD1 molecules (black curves; CD1-restricted T cells have been shown to contribute to the anti- mean fluorescence intensity (MFI) is given in the upper right corner). As a control, cells were stained with an isotype-matched Ab (gray filled viral immune response against HSV in mice (25). The underlying curves). B, U373 cells transfected with CD1d or immature DCs were in- mechanisms and potential viral countermeasures, however, are not fected with low (1) or high (100) MOIs of HSV for 18 h before being defined. Here, we have demonstrated two novel findings. First, analyzed by Western blot for CD1d expression. Representative results of human DC respond to HSV infection by enhancing CD1 Ag pre- three independent experiments are shown. sentation. Second, this evolutionary pressure has forced the virus 6212 VIRAL IMMUNE EVASION OF CD1 Ag PRESENTATION

FIGURE 9. Impaired cytokine se- cretion by NKT cells stimulated with HSV-infected DC at early and late time points postinfection. A, Imma- ture DC were infected for 18 h with UV-inactivated (uvHSV) or live HSV (MOI ϭ 1orMOIϭ 100) or left un- infected. After pulsing with 1–5 ␮g/ml, ␣GalCer NKT cells were added. Secre- tion of cytokines characteristic of NKT cells (IFN-␥, IL-4, and IL-10) was de- termined by ELISA after 3 days of cul- ture. B, Immature DC were pulsed with 1–5 ␮g/ml ␣GalCer and mixed with NKT cells directly after infection and cytokines were measured by ELISA af- ter 3 days of culture. Data are given as mean values and SDs for duplicate

determinations. Downloaded from

to develop immune evasion mechanisms that target the CD1 Ag complexes by interfering with translocation of peptides into the presentation machinery and modulate the function of NKT cells. endoplasmic reticulum, causes up-regulation of MHC I-like CD1

Many studies have shown that HSV infection impairs the MHC molecules by an unknown mechanism. The capacity of ICP47⌬- http://www.jimmunol.org/ presentation pathway of DC (47–52). We confirmed that HSV- infected DC to activate CD1-restricted T cells was still higher than infected DC have a reduced capacity for presentation of conven- that of uninfected DC. This finding implies that additional mech- tional peptides to MHC class I-restricted T cells (data not shown). anisms are involved in up-regulation of CD1 Ag presentation on In sharp contrast, DC infected with the same MOI (1) had a higher HSV-infected DC. Similarly, regulation of murine CD1d tran- density of CD1b and CD1d molecules on the cell surface and could scripts by microbial signals and proinflammatory cytokines has more efficiently induce proliferation of CD1-restricted T cells than recently been demonstrated (65). We conclude that DC try to coun- control DC. Confocal microscopy analysis suggests that the ele- teract virus-induced paralysis of Ag presentation through MHC vated density of CD1b on low MOI HSV-infected DC is due to molecules by activating CD1-restricted T cells. redistribution of CD1b from the MHC class II compartment to the Besides blocking the biosynthesis of MHC class I complexes, by guest on September 26, 2021 cell membrane. Although the increase in CD1d surface expression numerous other ingenious strategies are used by viruses to inter- was only moderate HSV-infected DC induced strong proliferation fere with the MHC I Ag presentation (58). The Kaposi’s sarcoma- of NKT cells. This result is in accordance with the observation that associated herpesvirus encodes 2 proteins, K3 and K5, which en- even low levels of CD1d molecules on monocytic cells are asso- hance endocytosis of MHC class I complexes. As a consequence, ciated with potent Ag-presenting capacity (61). The observed up- the density of MHC class I molecules on the cell surface is down- regulation of CD1b and CD1d molecules at least in part must be regulated. These viral proteins also trigger internalization of CD1 due to a posttranslational mechanism as it occurred in transfected proteins (our own unpublished data and Ref. 43). Moreover, the cells expressing CD1 molecules under the control of a cellular protein encoded by HIV down-regulates surface expression of promoter (ef1␣). Such a mechanism could allow presentation of MHC class I molecules and also CD1 molecules (our own unpub- CD1-bound viral Ags or self Ags even during viral shutoff of host lished data and Refs. 40, 42, 44). Thus, viral immunoevasins, protein synthesis. which target the endocytic pathway, can interfere with Ag presen- Indeed, we could identify a posttranslational pathway that en- tation through CD1 molecules. The quantity of CD1 molecules on ables DC to enhance Ag presentation through CD1b and CD1d cells infected with live ICP47⌬ was still lower than on cells in- molecules in response to HSV. DC infected with mutant HSV fected with UV-inactivated ICP47⌬. This emphasizes that in the lacking ICP47, the only known HSV-encoded MHC class I blocker absence of ICP47 low titers of HSV can effectively interfere with (56, 57), were surprisingly less efficient stimulators of CD1-re- CD1 expression. Given the demonstrated importance of CD1 mol- stricted T cells. The CD1 molecules are TAP independent, and ecules in viral infections, it is likely that HSV has also developed thus should not be affected by ICP47, which is known to function mechanisms that target the associated Ag presentation pathways by blocking TAP. However, the density of CD1b and CD1d on thereby preventing maximal activation of CD1-restricted T cells. cells infected with ICP47⌬ was diminished compared with control In our study, several findings show the existence of viral evasion cells infected with rescue virus (the ICP47 gene has been reintro- of the CD1-Ag presentation system. At a low MOI, double staining duced into ICP47⌬). This finding indicates that viral interference for HSV Ag and CD1b revealed a small population of DC that with MHC class I molecules at the level of the endoplasmic retic- carried a high HSV Ag load and, simultaneously, showed a ulum can promote CD1 expression on the cell surface. In fact, strongly diminished surface expression of CD1b. Accordingly, af- transfected cell lines (U373-CD1b or U373-CD1d cells) tran- ter infection with a high MOI on both DC and transfected U373 siently supertransfected with an ICP47 expression construct cells the expression of all CD1 molecules was reduced. The ob- showed increased density of CD1b or CD1d, respectively. Accord- served intracellular colocalization of viral proteins and CD1 mol- ingly, these cells were capable of stimulating proliferation of ecules together with the reduction in CD1 endocytosis in DC im- CD1b- or CD1d-restricted T cell lines more efficiently than control plies the immobilization or intracellular redistribution of CD1 by cells. Thus, ICP47, which prevents biosynthesis of MHC class I viral proteins. Moreover, the total amount of CD1d protein in DC The Journal of Immunology 6213 infected with a high MOI of live virus was diminished in compar- 7. Van, R., I, D. M. Zajonc, I. A. Wilson, and D. B. Moody. 2005. T-cell activation ison to DC infected with UV-inactivated HSV. In addition, inter- by lipopeptide . Curr. Opin. Immunol. 17: 222–229. 8. Beckman, E. M., S. A. Porcelli, C. T. Morita, S. M. Behar, S. T. Furlong, and nalization of CD1a was found to be impaired in DC infected with M. B. Brenner. 1994. 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