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Originally published online as doi:10.1189/jlb.0307181 on September 26, 2007

Published online before print September 26, 2007
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(Journal of Leukocyte Biology. 2008;83:56-63.)
© 2008 by Society for Leukocyte Biology

Human cytomegalovirus-derived protein UL18 alters the phenotype and function of monocyte-derived dendritic cells

Claudia S. Wagner*,1, Lilian Walther-Jallow*, Eva Buentke{dagger}, Hans-Gustaf Ljunggren*, Adnane Achour* and Benedict J. Chambers*

* Center for Infectious Medicine, Department of Medicine, Karolinska University Hospital Huddinge, and
{dagger} Cancer Center Karolinska, Department of Oncology and Pathology, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden

1 Correspondence: Center for Infectious Medicine, F59, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, 141 86 Stockholm, Sweden. E-mail: claudia.wagner{at}ki.se

ABSTRACT

Human cytomegalovirus (HCMV) encodes the MHC class I-like molecule UL18, which binds with high affinity to the leukocyte Ig-like receptor-1 (LIR-1), an inhibitory receptor commonly expressed on myeloid cells and subsets of NK and T cells. The exact role of UL18 is not known, in particular in relation to its proposed role in HCMV immune escape. Given the ubiquitous expression of LIR-1 on dendritic cells (DCs), we hypothesized that UL18 may affect DC function. To study the effects of UL18 on DC, we made use of UL18 fusion proteins. We demonstrate that UL18 fusion proteins inhibit the chemotaxis of DCs. Furthermore, UL18 interfered with CD40 ligand-induced maturation of DCs, resulting in reduced allogeneic T cell proliferation. Finally, we demonstrate that UL18 proteins up-regulate the expression of the maturation marker CD83 on immature monocyte-derived DCs and induce cytokine production. The capacity of UL18 to affect the function and the phenotype of DCs suggests a novel role for this HCMV-derived protein.

Key Words: MHC class I homologue • host/pathogen interaction • cell trafficking • cell surface molecules • immune modulation

INTRODUCTION

Dendritic cells (DCs) are important constitutes of the immune system, as they bridge innate and adaptive immunity. In their immature state, DCs passively sample antigens from their surroundings. In case of infection, DCs temporarily enhance their capacity to sample antigen, a process followed by up-regulation of costimulatory molecules and chemokine receptors and migration to lymphatic organs [1 ]. There, they encounter T cells and initiate appropriate immune responses. Cytokine and chemokine production by DCs also influences the immune system by activating or inhibiting cells of the adaptive and innate immune systems [2 ].

Human cytomegalovirus (HCMV) is a widespread herpes virus, causing primary infections, which are usually mild, followed by latency throughout life. However, reactivation of latent virus or primary infection can lead to life-threatening diseases in immunocompromised individuals [3 ]. Many gene products of HCMV, such as chemokine receptor homologues, a viral IL-10 homologue, and modulators of cell cycle and apoptosis, are dedicated to antagonize the immune system [4 ]. The virus also possesses several proteins capable of down-modulating MHC class I expression [5 ] as well as multiple strategies to impair NK cell recognition [6 ].

As DCs play a crucial role in the initiation of adaptive T cell responses, they are an important target for viral immune evasion strategies [7 8 ]. HCMV-infected monocyte-derived DCs or bone marrow progenitor-derived Langerhans-type DCs have impaired expression of costimulatory molecules, possess reduced antigen-presenting capacity, are incapable of up-regulating the lymph node homing receptor CCR7, and have altered cytokine production upon LPS or CD40 ligand (CD40L) stimulation [9 10 11 12 13 14 ]. Soluble CD83 released by HCMV-infected, mature DCs is one factor responsible for the related reduction in T cell proliferation [15 ]. Controversially that the activation of freshly isolated blood DCs was not impaired by the virus [16 ].

The HCMV-derived MHC class I homologue UL18 has been proposed as a decoy molecule for NK cells [17 18 ]. However, these results have been subject to controversy, as UL18 deletion of HCMV triggers equal or less cytotoxicity by NK cells compared with the parental virus [19 20 ]. A recent report demonstrated that UL18 inhibits NK cells expressing the leukocyte Ig-like inhibitory receptor 1 (LIR-1/Ig-like transcript 2/CD85j/leukocyte Ig-like receptor B1) but may activate other NK cells [21 ]. UL18 has also been suggested to activate T cells [22 23 ]. Thus, the exact function(s) of UL18 is/are still not established. The only receptor for UL18 identified so far is LIR-1 [24 25 ], a cell surface-expressed, type 1 transmembrane protein, which contains ITIMs in its cytoplasmic tail [26 ]. The natural ligands for LIR-1 are a broad spectrum of MHC class I molecules. It is interesting that they bind to this receptor with 1000-fold lower affinity than UL18 [26 27 28 ]. LIR-1 is expressed on the surfaces of a subset of NK and T cells, on all DC subsets as well as monocytes and B cells [25 ]. As LIR-1 is not a ubiquitous NK receptor, cell types other than NK cells may instead be the main targets for UL18. It has been speculated that UL18 could modulate myeloid cells or B cells through LIR-1 interactions [4 29 30 ], but so far, no system to evaluate these possibilities has been devised [4 ].

As DCs express LIR-1 and play a central role in combating CMV infection, we hypothesized that the virus may use the high-affinity LIR-1-ligand UL18 to change DC performance. In this study, we set out to determine if and how UL18 protein in isolation could affect DC function. Our results indicate that UL18 can substantially alter the phenotype and the function of monocyte-derived DCs.

MATERIALS AND METHODS

Cell cultures
Cells were cultured in RPMI 1640 (Invitrogen, Carlsbad, CA, USA) supplemented with L-glutamine, penicillin, streptomycin, and 10% heat-inactivated FCS (Integro, Zaandam, the Netherlands), referred to as "complete medium" below.

CD14+ monocytes were isolated from healthy PBMC by positive selection and T cells by negative selection (RosetteSepTM, StemCell Technologies, Vancouver, Canada), according to the manufacturer’s instructions. In addition, purified monocytes were allowed to adhere for 2 h and then washed with PBS before addition of complete medium supplemented with recombinant human cytokines IL-4 (6.5 ng/ml, R&D Systems, Minneapolis, MN, USA) and GM-CSF (250 ng/ml, PeproTech, Rocky Hill, NJ, USA). After 6 days, immature CD1a+ DCs were obtained (>95%), and less than 8% were CD83+ or CD86+.

Maturation was achieved by culturing immature DCs for an additional 20 h in the presence of 100 ng/ml LPS (Sigma-Aldrich, St. Louis, MO, USA) or by coculturing DCs for 16 h with 20 µg/ml polyinosinic:polycytidylic acid [poly(I:C); Sigma-Aldrich] or CD40L-expressing L cells [31 ], kindly provided by Dr. Thorbald van Hall (Leiden University Medical Center, The Netherlands), at a ratio of 10:1. Enriched T cells were >98% pure and were kept overnight in complete medium supplemented with IL-2 (80 U/ml, PeproTech).

Production of UL18 fusion proteins
Dr. David Cosman (Amgen Inc., Seattle, WA, USA) kindly provided the plasmid encoding for UL18-Fc fusion proteins. UL18 proteins were produced in 293FT cells (Invitrogen), as described previously [24 32 ], with some modifications [33 ]. The DNA was prepared with an Endofree plasmid maxi kit (Qiagen, Valencia, CA, USA), and cells were transfected with Lipofectamine 2000 (Invitrogen). The transfected cells were kept for 72 h in Advanced DMEM (Invitrogen), supplemented with 1.8% FCS, penicillin, and streptomycin. Proteins were purified from cell culture supernatant using a HiTrapTM Protein A HP column (Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer’s instructions. Recombinant human β2microglobulin (β2m), produced as described previously [34 ] and purified by size exclusion chromatography, was added in a tenfold molar excess to UL18 proteins to facilitate proper complex formation following acid elution. UL18 proteins were separated from unbound human β2m aggregates and medium contaminants through size exclusion chromatography on a Superdex 200 10/300 GL column (Amersham Biosciences). In the control protein UL18mut, which does not bind to LIR-1, the cysteine residue C255 in the {alpha}3 region of UL18 was substituted by alanine. Comparative circular dichroism, used to assess the correct fold of the fusion proteins, demonstrated that UL18mut displayed a fold similar to wild-type UL18 [33 ]. The stability of UL18mut was not affected by the C255A mutation [33 ].

Antibodies for cell surface molecule expression
The following mAb were used: anti-CD83 FITC, anti-CD40 FITC, anti-HLA-ABC FITC (clone W6/32), anti-HLA-DR PerCP, anti-CD86 allophycocyanin (APC), anti-CD3 APC, and anti-CCR7 Alexa Fluor 647 (all from BD Biosciences, San Diego, CA, USA); anti-LIR-1/CD85j PE (clone HP-F1, Beckman Coulter, Fullerton, CA, USA); anti-CD1a PE and anti-CD14 PE (Dako, Glostrup, Denmark); and FITC-conjugated (Fab')2 goat anti-human IgG1 (anti-hIgG1), Fc{gamma} fragment-specific for UL18 detection (Jackson ImmunoResearch, West Grove, PA, USA).

Maturation analysis and cytokine detection
To achieve directed binding of UL18, 96-well flat-bottom plates (BD Biosciences) were coated for 2 h at 37°C with purified F(ab')2 goat anti-hIgG1, Fc{gamma} fragment-specific (Jackson ImmunoResearch) at 15 µg/ml in PBS. Plates were washed twice and incubated with UL18, UL18mut, or hIg (Baxter Healthcare, Deerfield, IL, USA) at 15 µg/ml for an additional 2 h at 37°C. DCs were added to coated wells and incubated for 20 h. LPS was used in control wells. Supernatants were saved and frozen at –20°C for cytokine detection, whereas cells were washed and stained for maturation markers. Supernatants were also collected from cultures incubated for 2 h, 8 h, and 15 h. Flow cytometry was performed using a FACSCalibur (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). To quantify the levels of IL-6, IL-10, IL-12 p70, TNF-{alpha}, and MIP-1{alpha} secreted from DCs, a Bio-Plex assay (Invitrogen) was performed, and the results were analyzed using a Luminex reader (Luminex, Austin, TX, USA) as described previously [35 ]. Samples were analyzed in duplicates and presented as cytokine concentrations in pg/ml.

ELISA
Purified LIR-1 proteins consisting of the complete extracellular region (domains D1–D4) were immobilized on PolySorp plates (Nunc, Wiesbaden, Germany). UL18 and UL18mut Fc-fusion proteins were added at 4 ug/ml to test their relative binding affinity to LIR-1. Binding was detected using HRP-labeled mouse anti-hIgG (Fc-specific, Southern Biotech, Birmingham, AL, USA). Trimethyoxybenzoic acid substrate (TMB) (Sigma, Stockholm, Sweden) was used for enzymatic development.

Migration assays
Chemotaxis was assessed in Transwell cell culture chambers with 8 µm pore size (BD Biosciences) using 250 µg/ml RANTES (PeproTech). DCs (0.5x106) were incubated with UL18, UL18mut, or hIg at 15 µg/ml for 10 min and subsequently cross-linked with anti-human Fc F(ab')2 for 5 min before transfer to the Transwell. After 2 h, the number of migrated DCs was determined by flow cytometry at a constant, low flow rate for 1 min 15 s.

T cell proliferation assays
CD3+ T cells were purified as described above and labeled with 5 µM CFSE (Molecular Probes Leiden, The Netherlands) in PBS containing 0.5% FCS the following day. DCs were matured by exposure to CD40L-expressing L cells in the presence of UL18 or control proteins and cross-linking anti-human Fc F(ab')2 antibody. Alternatively, instead of CD40L, poly(I:C) was used as maturation stimulus, or cells were left untreated and subsequently washed three times. DCs (0.2x106) were added to 0.6 x 106-labeled allogeneic T cells and cocultured for 5 days prior to measurement of proliferation by intensity of CFSE of CD3-labeled T cells using flow cytometry.

Statistical analysis
Data were analyzed by ANOVA using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Results were considered significant at a P < 0.05.

RESULTS

UL18 fusion proteins stain monocyte-derived DCs uniformly
To study potential effects of UL18 on DCs, we made use of UL18-Fc fusion proteins (termed UL18 throughout the article). We have recently produced soluble UL18-Fc fusion proteins substituted in different regions to determine the structural basis underlying the high binding affinity of UL18 to LIR-1 [33 ]. We identified an additional disulfide bond between C240 and C255 in the {alpha}3 domain of UL18, which is not present in classical MHC class I molecules. The disruption of this additional disulfide bridge by alanine substitution of C255 (UL18mut) or C240 affected binding of the β2m subunit to UL18 and impaired the binding to LIR-1 in ELISA assays and in flow cytometry assays using LIR-1-expressing NKL cells [33 ]. Here, wild-type UL18 and UL18mut proteins were used to determine if UL18 contact can influence DCs. Binding of the UL18 proteins to the surface of monocyte-derived DCs was assessed (Fig. 1 ). All DCs expressed LIR-1, the receptor for UL18, as demonstrated previously [25 ] and illustrated in Figure 1A . To ensure that UL18 binding was mediated by UL18 only and not via the Fc portion of the fusion protein, FcRs on DCs were blocked with human serum prior to staining. Furthermore, hIg and UL18mut, which had lost the ability to bind to LIR-1, were used as control proteins (Fig. 1B) . Almost no background staining was observed with these controls, verifying that the UL18 proteins could be used to study the effect of UL18 on DCs in functional assays. To confirm that UL18mut does not recognize LIR-1, the ability of UL18 and UL18mut to bind to plate-bound LIR-1 proteins was assessed in an ELISA (Fig. 1C) . Whereas UL18 clearly recognizes LIR-1, no interaction could be detected with UL18mut (Fig. 1C) .


Figure 1
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  		Figure 1. UL18 fusion proteins bind to DCs. (A) LIR-1 expression on DCs, which were stained with anti-LIR-1 PE (black, solid line) or isotype control (filled, gray histogram) and assessed by flow cytometry. (B) UL18 fusion protein binds to DCs homogeneously in flow cytometry assays (solid, black line). As a control for specific binding, DCs were also stained with secondary antibody (anti-human Fc) alone (filled gray), in combination with hIg (dotted gray), or UL18mut (dashed, dark gray line). (C) UL18 proteins but not UL18mut bind to immobilized LIR-1 in an ELISA. Statistical analysis of five independent experiments was performed with paired t-test; ***, P < 0.001. Error bars indicate SD. A 405 nm, Absorbance at 450 nm.

UL18 induces expression of the maturation marker CD83 on DCs
It has been demonstrated that antibody cross-linking of LIR-1 inhibits the function of DCs [36 ]. Conversely, the engagement of LIR-1 via UL18 activates T cells [22 ]. To assess the effects of UL18 proteins on immature DCs, cells were incubated for 20 h with LPS or plate-bound UL18 or a combination of UL18 and LPS. UL18mut and hIg were used as controls. Plates were precoated with anti-human Fc (Fab')2 antibody to achieve a directional binding of UL18 proteins. DCs were then analyzed by flow cytometry for expression of the maturation markers CD40, CD83, CD86, and HLA-DR as well as the lymph node homing receptor CCR7 (Fig. 2A and 2B ). It is surprising that UL18 increased the number of CD83-expressing DCs in all experiments (P<0.01 for UL18 compared with UL18mut or hIg, Fig. 2B ) but displayed little or no effect on the expression of the other markers tested (P>0.05, Fig. 2B ). All protein batches for UL18 and UL18mut were produced in the same way, excluding potential endotoxin contaminations as a reason for the observed differences. The presence of UL18 during LPS stimulation did not alter the maturation effect of LPS on DCs (data not shown), in line with previous observations using anti-LIR-1 antibodies [36 ].


Figure 2
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  		Figure 2. UL18 induces expression of CD83 but not of CD40, CD86, HLA-DR, and CCR7 on immature DCs, which were cultured for 20 h in untreated wells, wells coated with the indicated proteins, or with added LPS as a positive control. Anti-human Fc (Fab')2 antibody was used to achieve directional coating of the Fc-fusion proteins. (A) Expression of maturation markers in immature DCs following stimulation with LPS (gray, solid line), anti-human Fc (Fab')2 alone (gray, solid histogram), or in combination with UL18 (black, solid line) or UL18mut (black, dashed line). (B) Summary of four independent experiments assessing surface maturation markers of untreated DCs, DCs treated with LPS, or anti-human Fc (Fab')2 alone (Ab) or in combination with UL18, UL18mut, or hIg. Numbers in percentages or mean fluorescence intensity (MFI) values are provided relative to the expression levels of the protein of interest in untreated DCs. Statistical analysis of non-normalized data was performed by ANOVA; **, P < 0.01. Error bars indicate SD.

The production of cytokines by DCs is increased in the presence of UL18
To determine the effect of UL18 on cytokine profile, supernatants of DCs cultured in plates coated with UL18 or controls (as described above) were tested for their content of TNF-{alpha}, IL-6, IL-12, IL-10, and MIP-1{alpha} (Fig. 3 ). UL18 proteins induced significantly increased amounts of IL-10 when compared with UL18mut or hIg controls after 20 h of cultures (P<0.05). IL-6 secretion was increased significantly by UL18 when compared with UL18mut (P<0.05) and elevated when compared with hIg. UL18 did not affect the other cytokines significantly, although levels of IL-12 and TNF-{alpha} were elevated (mean 286 pg/ml IL-12 for UL18 vs. 24 pg/ml for UL18mut and 72 pg/ml for hIg; mean 3200 pg/ml TNF-{alpha} for UL18 vs. 118 pg/ml for UL18mut and 573 for hIg; Fig. 3 ). MIP-1{alpha} secretion was also elevated by the presence of UL18 (mean 3714 pg/ml for UL18 vs. 45 pg/ml for UL18mut and 37 pg/ml for hIg; Fig. 3 ). In contrast, LPS induced significantly higher levels of all these cytokines as well as MIP-1{alpha}. In summary, UL18 induced elevated cytokine production in DCs after 20 h.


Figure 3
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  		Figure 3. UL18 increases production of IL-10 by DCs. Supernatants of immature DCs incubated for 20 h with plate-bound UL18, hIg, UL18mut, or LPS were tested for the presence of TNF-{alpha}, IL-6, IL-12, IL-10, and MIP-1{alpha} using a Bio-Plex. The Box-and-Whisker plots display data range, interquartile range, and median. Statistical analysis on data derived from at least four independent experiments was performed by ANOVA; *, P < 0.05.

To investigate if UL18 influences the cytokine profiles of DCs at earlier time-points, DC supernatants were analyzed after 2 h, 8 h, and 15 h incubation in protein-coated wells as described above. TNF-{alpha} and MIP-1{alpha} levels did not differ in UL18-coated wells compared with control proteins hIg or UL18mut at any of the assessed time-points (Fig. 4 ). There was a trend for more IL-6 production of DCs incubated with UL18 for 15 h compared with control proteins; however, the difference did not reach statistical significance (Fig. 4) . At earlier times, IL-6 production was below the detection limit of our method in UL18 and control protein-coated wells, and only the LPS-treated DCs produced measurable cytokine amounts (mean 230 pg/ml IL-6 after 2 h, 11,442 pg/ml after 8 h). After 2 h of incubation, IL-10 and IL-12 amounts were below the detection limit for all samples. Similarly, IL-10 and IL-12 levels were too low in supernatants from UL18 or control protein-coated wells after 8 h and 15 h, and only LPS-induced cytokines could be detected then (mean 117 pg/ml IL-10 after 8 h and 411 pg/ml after 15 h; mean 194 pg/ml IL-12 after 8 h and 475 pg/ml after 15 h). Therefore, no differences in cytokine production of DCs treated with UL18, hIg, or UL18mut control could be observed at time-points earlier than 20 h.


Figure 4
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  		Figure 4. UL18 does not influence cytokine profile during the first 2–15 h of coincubation. Supernatants of immature DCs incubated for 2 h, 8 h, or 15 h with plate-bound UL18, hIg, UL18mut, or LPS were tested for the presence of TNF-{alpha}, IL-6, IL-12, IL-10, and MIP-1{alpha} in a Bio-Plex assay. Graphs display cytokine amounts of IL-6 production after 15 h of incubation with the indicated proteins as well as amounts of TNF-{alpha} and MIP-1{alpha} after 2 h, 8 h, and 15 h. IL-6 (15 h) and TNF-{alpha} (2 h) produced by DCs treated with hIg were below the detection limit of this assay. The Box-and-Whisker plots display data range, interquartile range, and median. Statistical analysis on data derived from four experiments was performed by ANOVA.

The migration of DCs is inhibited in the presence of UL18
The effects of UL18 on chemotaxis of DCs were then studied. The inflammatory chemokine RANTES binds to several receptors on immature DCs and can trigger multiple, distinct biochemical pathways [37 ]. In particular, with regard to DCs, RANTES mediates signals via the phosphotyrosine kinase pathway [38 ], a cascade, which potentially represents a target for UL18 interference through LIR-1 signaling. The chemotactic attraction of DCs toward RANTES was tested in a transwell system by comparing the number of migrated cells in the presence or absence of cross-linking UL18. DCs treated with cross-linking antibody alone or in combination with UL18mut or hIg were used as controls (Fig. 5 ). The numbers of cells migrating toward RANTES varied in these experiments (DCs treated with cross-linking antibody only: mean 3675, range 2584–5642; UL18 and cross-linking antibody: mean 1833, range 920–3208; UL18mut and cross-linking antibody: mean 3842, range 2275–6337). By paired analysis, we demonstrated that UL18 reduced the migration of DCs significantly (P<0.05 for UL18 compared with UL18mut or hIg, Fig. 5 ). When compared with UL18mut, UL18 reduced migration by 30–67%, suggesting that UL18 had an inhibitory effect on DCs and was able to impair migration.


Figure 5
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  		Figure 5. UL18 inhibits the migration of immature DCs toward RANTES. DCs were incubated for 2 h in Transwell chambers in the presences or absence of cross-linked UL18 or control proteins hIg or UL18mut. RANTES was present in the lower chamber of Transwells where indicated. The amount of migrated DCs treated with the indicated proteins is provided relative to the number of migrated DCs treated with cross-linking antibody only (Ab). Summarized are five independent experiments. Statistical analysis was performed on raw data using one-way ANOVA; *, P < 0.05; **, P < 0.01. Error bars indicate SD.

The presence of UL18 during CD40L-induced maturation impairs induction of T cell proliferation
As anti-LIR-1 antibody cross-linking on DCs has been shown to affect T cell activation [36 ], we tested whether UL18 could interfere with CD40L-induced maturation of DCs and subsequently alter the induction of T cell responses. CD40-CD40L interactions result in intracellular tyrosine phosphorylation events, involving the Src kinase family [39 40 ]. UL18 engagement of LIR-1 may thus interfere with CD40L-induced signaling. DCs were matured through coculture with CD40L-expressing L cells in the presence of UL18, UL18mut, or hIg in combination with cross-linking anti-human Fc (Fab')2 antibody or antibody alone. These DCs were then cocultured with CFSE-labeled allogeneic T cells, and the extent of T cell proliferation was determined by flow cytometry. Treatment of DCs with UL18 resulted in a significant reduction (P<0.05 for UL18 compared with UL18mut or hIg; Fig. 6A and 6B ). The number of divisions (Fig. 6A) and the proportion of proliferating T cells (mean 6.2% for UL18, 8.9% for UL18mut, and 9.6% for hIg; Fig. 6B ) diminished in the presence of UL18 during DC maturation.


Figure 6
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  		Figure 6. UL18 decreases the ability of CD40L-matured DCs to stimulate allogeneic T cell proliferation. DCs were matured through CD40L (A and B) or poly(I:C) (C) before coculture with CFSE-marked T cells for 5 days. In addition, DCs were incubated with hIg, UL18mut, or UL18 and cross-linking antibody during maturation. The percent of proliferating T cells is displayed in the graphs. (A) One representative example of five independent experiments summarized in B is shown. (C) Summary of three different experiments. Statistical analysis was performed with ANOVA; *, P < 0.05.

As T cell proliferation in MLRs can be suppressed by IL-10 [15 ], we examined if the enhanced production of IL-10 by the DCs could be the reason for the reduced T cell proliferation in the cultures. Adding anti-IL10 antibody during CD40L-induced DC maturation and in the cocultures did not overcome the UL18-induced inhibition of proliferation (data not shown).

In addition, the presence of UL18 had no effect when poly(I:C)-stimulated (Fig. 6C) or untreated DCs (data not shown) were used for coculture with allogeneic T cells. In summary, UL18 was able to alter CD40L but not poly(I:C)-induced DC maturation, resulting in reduced capability to stimulate T cell proliferation.

DISCUSSION

The results presented here demonstrate that the HCMV protein UL18 binds to DCs and has the capacity to alter them phenotypically by up-regulating the maturation marker CD83. Functional alterations in DCs attributed to UL18 are impairment of DC migration, enhancement of cytokine production at later time-points, and interference with induction of T cell proliferation. Therefore, UL18 may be used by HCMV to confound immune responses and thus, could serve as a strategy to avoid immune recognition.

Upon ligand binding, the cytoplasmic tail of LIR-1 becomes phosphorylated by Src kinases and recruits tyrosine phosphatases, such as Src homology 2-containing tyrosine phosphatase 1 (SHP-1), which are involved in negative signaling [25 41 ]. LIR-1 signaling does not depend on coligation with activating receptors, but ligand engagement alone is sufficient to induce signaling [40 ]. Ligation of LIR-1 via antibodies on the surface of DCs has been reported to suppress Ca2+ mobilization, cytokine production, and induction of antigen-specific T cell proliferation [35 ]. In T cells, LIR-1 signaling suppresses antigen-specific proliferation, cytokine production, and cytotoxicity [42 43 44 ]. Conversely, LIR-1 engagement by UL18 has been shown to result in the stimulation of T cells [22 ]. Therefore, it was important to determine the outcome of the UL18-LIR-1 interaction in DCs. Our results suggest that UL18 is capable of inhibiting DC activities. The high binding affinity of UL18 to LIR-1 when compared with classical MHC class I molecules [28 ] may allow for a different magnitude of LIR-1 engagement, far beyond the LIR-1-MHC class I interaction. The competition of UL18 with endogenous MHC-I ligands for LIR-1 binding may also disturb the natural signaling balance in DCs and other LIR-1-expressing cells, impairing immunosurveillance.

Immature DCs incubated with UL18 in the absence of other stimuli specifically up-regulated CD83, a marker, which reflects the state of DC activation [45 ]. Recombinant, soluble CD83 binds to immature and mature DCs and can inhibit maturation as well as the ability of mature DCs to stimulate allogeneic or antigen-specific T cell responses [46 ]. It is interesting that HCMV infection of mature monocyte-derived DCs results in loss of surface CD83, increased release of soluble CD83, and inhibition of noninfected DCs in their capacity to stimulate T cell proliferation [15 ]. In view of these data, one may speculate that the virus up-regulates CD83 expression on DCs through UL18, present on infected cells, to block maturation of surrounding DCs. Another potential consequence of the up-regulation of CD83 by UL18 could be incomplete maturation of DCs, resulting in poor stimulation of T cells or presentation of antigen.

The recruitment of immature DCs to sites of infection by inflammatory chemokines, such as MIP-1-{alpha}/CCL3, MIP-1β/CCL4, and RANTES/CCL5 [47 ], is crucial for the initiation of immune responses. RANTES signals both via G-protein-coupled chemokine receptor-dependent and -independent pathways, involving activation of the Src kinase family and mobilization of cytosolic Ca2+ [48 49 50 ]. In monocyte-derived, immature DCs, RANTES triggers tyrosine phosphorylation cascades following binding to CCR-1, -3, and -5 [38 ]. This enhanced the likelihood for inhibition of signaling through concomitant cross-linking of LIR-1 with UL18 proteins, as observed in this study. Additional evidence that chemotaxis can be regulated by ITIM-containing immune receptors comes from a study in which the inhibitory Ly49A receptor, signaling via SHP-1, negatively regulates the migration capacity of a rat NK cell line [51 ]. Reduced motility of DCs to/from sites of infection may prevent efficient immunosurveillance, in turn, reducing immune responses. In line with this notion, HCMV infection of immature monocyte-derived DCs prevents migration toward inflammatory cytokines [52 ] and impairs migration to secondary lymphoid organs upon DC maturation [53 ]. UL18 expression on other cell types could be used by HCMV to impair migration of uninfected DCs.

Along with a more direct, inhibitory effect of UL18 on maturation, the enhanced IL-10 production in the presence of UL18 could impair DC trafficking during HCMV infection. As reported previously, IL-10 in the presence of other proinflammatory stimuli, induces the expression of unresponsive, inflammatory chemokine receptors on DCs [54 ]. Furthermore, IL-10 can reduce the chemotaxis of DCs toward CCL19, a ligand for CCR7, which plays a key role in lymph node homing of activated DCs [55 ].

However, the level of IL-10 up-regulation by UL18 observed after 20 h was less in magnitude than the effect on proinflammatory cytokines and may not be physiologically relevant. As a trend for more IL-6 upon contact with UL18 became already apparent after 15 h and remained after 20 h, the dominating effect of UL18 on cytokine production may be proinflammatory. Induction of mediators such as MIP-1{alpha} may attract immature DCs to sites of infection, increasing the possibility of infection. Thereafter, other HCMV genes would be able to manipulate infected DCs and prevent migration of mature DCs to lymphoid organs [53 ]. As UL18 did not influence MIP-1{alpha} production between 2 h and 5 h of incubation, it is unlikely that endogenous cytokine production influenced the outcome of our migration experiments. Opposing effects of UL18, such as reducing migration shortly after contact with DCs but potentially increasing proinflammatory mediators at later time-points, may be temporally effective in the course of immune responses or spatially separated, depending where and when DCs meet UL18. Generally, HCMV needs some degree of immune activation to ensure viral dissemination, but at the same time, it disturbs the normal immunosurveillance mechanisms to avoid eradication. For example, the differentiation of latently infected DC progenitors into mature DCs reactivates the virus [56 ], whereas trafficking and function of infected immature and mature DCs are profoundly disturbed [9 10 13 14 15 52 53 ].

DC maturation can be induced through different stimuli, including proinflammatory cytokines, pathogen-derived products, such as superantigens, endotoxins, bacterial DNA, viral double-stranded RNA [mimicked by poly(I:C)], and/or CD40L, expressed by activated T cells [57 ]. Apparently, LIR-1 signaling interferes only with certain pathways, as TLR-mediated activation is not affected by LIR-1 cross-linking in monocyte-derived DCs [36 ]. Similarly, TLR-induced maturation was not affected by UL18 in our experiments (data not shown). UL18 also did not influence the capacity of DCs to induce T cell proliferation when the TLR3 agonist poly(I:C) was used as a maturation stimulus. In contrast, CD40 ligation on DCs involves tyrosine phosphorylation and signal transduction via the Src kinase family [39 40 ], potentially allowing interference through LIR-1 signaling, as observed in our T cell proliferation experiments. As some T cells also express LIR-1, it was important to prevent direct contact of UL18 proteins with T cells during DC-T cell coincubation. Theoretically, UL18 may inhibit T cell proliferation, in analogy to the published inhibition of IFN-{gamma} production of T cells upon contact with UL18 proteins [23 ]. We tried to minimize that risk by including an extensive washing step for DCs, which had been incubated with UL18 proteins. As UL18 did not inhibit T cell proliferation when no stimulus or poly(I:C) was used during DC maturation, a potential, direct inhibition of T cells through UL18 is not likely to play a decisive role.

In infected cells, UL18 is expressed late during the infectious cycle [58 ] and is not easily detectable by surface staining [19 59 ]. Nevertheless, UL18-specific T cells can be found in healthy CMV-seropositive individuals [60 61 ], and the protein has been detected in immunohistochemical organ stainings of patients with high viremia [22 ], suggesting a role of this protein in in vivo settings. Even if most UL18 was expressed predominantly intracellularly [23 ], DCs could meet this protein during phagocytosis of infected apoptotic cells, when being infected themselves or through cell-to-cell transport via microvesicles. In our study, we concentrated on isolated UL18 proteins to identify any direct effects of this molecule on DCs, without confounding contributions of other CMV evasion mechanisms. However, care should be taken when directly translating results obtained with purified proteins to the complex setting of viral infection. We have shown previously that UL18 proteins can inhibit T cells upon direct contact but that the effect of UL18 is activating in the context of HCMV infection [23 ]. The temporal expression of UL18 during viral infection and its potential interaction with other HCMV or host gene products cannot be addressed in our system. This study should be used as a starting point for further investigations with the task to determine the interplay of UL18 with other immuno-evasins and its role in the infectious cycle under physiological conditions.

In conclusion, our data suggest that UL18 can modulate DCs, affecting their phenotype and impairing functions such as migration capacity and T cell stimulation, which may enhance the ability of HCMV to evade immune defenses.

ACKNOWLEDGEMENTS

This work was supported by grants from the Swedish Research Council, Karolinska Institutet Research Fund, Tobias Foundation, Clas Groschinsky Foundation, and Swedish Foundation for Strategic Research. We thank Prof. A. Scheynius for help in the initiation of this work and U. Johansson and A. Sköld for technical assistance.

Received March 22, 2007; revised August 8, 2007; accepted September 2, 2007.

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