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The Journal of Immunology, 2007, 179, 7767 -7776
Copyright © 2007 by The American Association of Immunologists, Inc.
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Human Cytomegalovirus Differentially Controls B Cell and T Cell Responses through Effects on Plasmacytoid Dendritic Cells1

Stefania Varani*,{dagger}, Madeleine Cederarv*, Sari Feld*, Charlotte Tammik*, Giada Frascaroli{dagger},{ddagger}, Maria P. Landini{dagger} and Cecilia Söderberg-Nauclér2,*

* Department of Medicine, Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden; {dagger} Department of Clinical and Experimental Medicine, Section of Microbiology, University of Bologna, Bologna, Italy; {ddagger} Department of Virology, University of Ulm, Ulm, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Plasmacytoid dendritic cells (PDCs), the main producers of type I IFN in response to viral infection, are essential in antiviral immunity. In this study, we assessed the effect of human CMV (HCMV) infection on PDC function and on downstream B and T cell responses in vitro. HCMV infection of human PDCs was nonpermissive, as immediate-early but not late viral Ags were detected. HCMV led to partial maturation of PDCs and up-regulated MHC class II and CD83 molecules but not the costimulatory molecules CD80 and CD86. Regardless of viral replication, PDCs secreted cytokines after contact with HCMV, including IFN-{alpha} secretion that was blocked by inhibitory CpG, suggesting an engagement of the TLR7 and/or TLR9 pathways. In the presence of B cell receptor stimulation, soluble factors produced by HCMV-matured PDCs triggered B cell activation and proliferation. Through PDC stimulation, HCMV prompted B cell activation, but only induced Ab production in the presence of T cells or T cell secreted IL-2. Conversely, HCMV hampered the allostimulatory ability of PDCs, leading to decreased proliferation of CD4+ and CD8+ T cells. These findings reveal a novel mechanism by which HCMV differentially controls humoral and cell-mediate immune responses through effects on PDCs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Like other viruses causing persistent infection, human CMV (HCMV)3 has developed strategies to subvert the host immune system and escape immune surveillance, enabling the virus to coexist with its host. Among these tactics, a generalized immunosuppression during viral infection inhibits the immune response against the virus itself as well as unrelated pathogens (1). HCMV-induced suppression of the immune system is transitory but substantial. In transplant recipients, HCMV infection leads to more severe disease and poor clinical outcome due to an increased incidence of secondary opportunistic infections (2, 3).

Beside immunosuppression, HCMV-infected individuals often develop other immune dysfunctions, such as serological abnormalities. For example, natural and anti-CD13-specific autoantibodies have been found in bone marrow transplant patients undergoing HCMV infection, and anti-CD13 Abs have been associated with the development of chronic graft-vs-host disease (4, 5). Nonorgan-specific autoantibodies associated with HCMV infection have also been detected in solid organ transplant recipients and may contribute to the development of acute and chronic allograft rejection (6, 7). In addition, hypergammaglobulinemia, cryoglobulinemia, and autoantibody production are features of HCMV-induced mononucleosis and postperfusion syndrome (8, 9).

How HCMV manipulates the immune response is not fully understood. Virus-induced subversion of the immune system most likely results from diverse immunological abnormalities. Is there a key mechanism that might explain these events? We hypothesized that HCMV controls cells, such as dendritic cells (DCs), that link innate and adaptive immunity and trigger the adaptive immune response.

In humans, two major subsets of DCs have been identified that function differently in both the innate and adaptive immune responses: myeloid DCs (MDCs), such as interstitial DCs and Langerhans cells, and plasmacytoid DCs (PDCs). MDCs are professional APCs with a strong capacity to prime naive T cells and to induce and regulate T cell responses through secretion of IL-12. PDCs resemble MDCs in Ag presentation, maturation, and trafficking properties, but differ by being the main producers of type I IFNs in response to viral DNA or RNA stimulation (10).

PDCs respond to viruses through a unique set of TLRs, including TLR7 and TLR9 (10). In most cell types, pathogen-induced release of IFN type I by PDCs initiates a cascade of events leading to restriction of viral replication (11), activation of NK cells (12), and differentiation of PDCs into mature DCs that can trigger adaptive T cell-mediated immunity (13). Virus-activated PDCs also play a critical role in the differentiation of B cells into Ig-secreting plasma cells (14).

The effect of HCMV on MDCs has been extensively investigated in monocyte-derived DCs (Mo-DCs). Infection of MDCs by HCMV is permissive and lytic (15); it inhibits cell maturation, blocks cytokine secretion, obstructs migration in response to chemokines, and impairs the allostimulatory ability (16, 17, 18). However, little is known about how HCMV infection affects the function of PDCs. In this study, we investigated the interaction of HCMV with PDCs and the effects of such interaction on the innate and adaptive immunological responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Culture conditions

Human fibroblasts were cultured in DMEM supplemented with 10% FCS (Invitrogen Life Technologies). HUVEC (Cambrex) were cultured in EGM-2 medium containing growth factors (Cambrex). B cells, T cells, PDCs, and Mo-DCs were cultured in RPMI 1640 supplemented with 10% FCS. Two mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin were added when not supplied in medium. F(ab') 2 of rabbit anti-human IgG plus IgA plus IgM Abs were from Jackson ImmunoResearch Laboratories. Completely and partially phosphorothioate-modified oligodeoxynucleotides (ODNs), CpG-A ODN 2216 (19), CpG-C ODN M352 (20, 21), and inhibitory CpG ODN 2088 (22), were from Metabion. IL-2 was from R&D Systems.

Cell isolation

PBMCs from healthy donors were isolated by density gradient centrifugation with Lymphoprep (Axis-Shield PoC AS). PDCs were isolated from PBMCs with PDC-specific blood DC Ag (BDCA)-4 Abs according to the manufacturer’s protocol (BDCA-4 isolation kit; Miltenyi Biotec). The purity of isolated PDCs was >95% as assessed by FACS (BDCA-2+ and BDCA-4+ cells). MDCs were isolated from PBMCs with BDCA-1 Dendritic Cell Isolation kit (Miltenyi Biotec). The purity of isolated MDCs was >95% as assessed by FACS (BDCA-1+ cells). B cells were purified with CD19 microbeads (Miltenyi Biotec). The purity of isolated B cells was >95%, with <1% CD3+ cells. CD4+ T cells were purified with CD4 microbeads (Miltenyi Biotec) and the purity of isolated CD4+ cells was >98%. Mo-DCs were differentiated in vitro from PBMCs as described (18). After gating in FACS to exclude debris, 70–90% of the cells were of the MDC phenotype (CD1a+, HLA-DR+, and CD14–).

HCMV infection of DCs

PDCs or MDCs were incubated with either of two endotheliotropic strains of HCMV, TB40/E (cultured in HUVEC with a final passage in fibroblasts) and VR1814 (cultured in HUVEC). DCs were infected at a multiplicity of infection (MOI) of 5 PFU/cell for 4 h or overnight at 37°C. HCMV was removed by washing, and fresh medium was added. Infection was detected with an indirect immunofluorescence assay as described (18). In addition, an anti-pp28 Ab, clone P2G11 (23), was used at a dilution 1/20 in PBS. At 1 day postinfection (p.i.), BDCA-2 and HCMV immediate-early Ag (IE) in PDCs were simultaneously detected with PE-conjugated anti-BDCA-2 Ab (Miltenyi Biotec) and anti-IE (Argene) by double immunofluorescence staining as described (18). To confirm HCMV infection, the RT-PCR assay for IE and pp150 were performed as described (24, 25).

B cell culture

For CFSE-based proliferation assays, B cells were stained with CFSE (CellTrace CFSE Cell Proliferation kit; Molecular Probes) according to the manufacturer’s instructions, incubated for 5 or 7 days, and harvested for FACS analysis. For coculture experiments, B cells (1 x 105/well) were cultured with or without PDCs (1 x 104 cells/well) and with or without HCMV (TB40/E; MOI of 5) or influenza virus (strain A/New Caledonia/20/99, H1N1, 70 µl/well, viral titer 1/40). Experiments were also performed with purified ultracentrifuged TB40/E (pelleted at 23,000 rpm at 4°C for 1 h and dissolved in sucrose buffer). Cultures were stimulated with anti-Ig (10 µg/ml), CpG-C ODN M352 (3 µg/ml), and IL-2 (50U/ml) when needed. In other coculture experiments, B cells (1 x 105/well) and CD4+ T cells (1 x 105/well) were cultured with PDCs (1 x 104 cells/well) and with or without HCMV (TB40/E; MOI of 5). After 3 days, supernatants were collected, and cells were harvested for FACS analysis. After 7 days, cells were collected and analyzed by FACS for plasma cell development. Ig production was measured in supernatants after 13 days of incubation by IgM and IgG ELISA (Bethyl Laboratories).

Flow cytometry analysis

For cell surface staining, we used Abs against CD1a, CD3, CD4, CD8, CD14, CD19, CD20, CD38, CD80, CD86, HLA-DR, and mouse isotype controls (BD Pharmingen); CD83 (Immunotech); and BDCA-2 and BDCA-4 (Miltenyi Biotec). All were mouse mAbs conjugated to FITC, PE, or PerCP. Total levels of TLR7 and TLR9 were determined by PDC fixation and permeabilization with Cytofix/Cytoperm kit (BD Pharmingen) and staining with anti-TLR7 and anti-TLR9 (Nordic BioSite). Data were acquired and analyzed with a FACSCalibur (BD Biosciences) and CellQuest software. Dead cells were excluded by staining with propidium iodide. Ag expression was measured as the percentage of positive cells and as the mean channel fluorescence intensity (MFI) of the respective Ab compared with the isotype-matched control. The difference in MFI for different samples was calculated; a difference >10 channels between two different samples was considered a positive or negative change, based on variations among controls.

Allogeneic MLR

The stimulatory ability of mock-infected and HCMV-infected PDCs was assessed in an allogeneic MLR. PDCs were mock infected, infected with TB40/E (MOI: 5), or incubated with UV-inactivated TB40/E. After 1 day, PDCs were washed three times in PBS and irradiated (3000 cGy). Proliferation of allogeneic responder PBMCs was assessed by [3H] thymidine incorporation as described (25). The stimulation index was calculated for each experiment as the ratio of cpm of responder PBMCs plus PDCs to the cpm of responder PBMCs alone. For CFSE-based proliferation assays, responder PBMCs were labeled with CFSE and mixed with irradiated PDCs. After 6 days, cells were harvested for FACS analysis. The cell division index was calculated, based on a fixed number (usually 3500) of CFSEbright CD4+ or CD8+ cells, with the following formula (using CD4+ cells as an example): number of CD4+CFSEdim cells with allostimulation/number of CD4+CFSEdim cells without allostimulation, as adapted from a previously described method (26).

Quantification of cytokines and chemokines

PDCs were mock infected, infected with TB40/E or VR1814, or incubated with UV-inactivated TB40/E or virus-free supernatant overnight. The virus was removed, and cells were washed and seeded (106 cells/ml) in fresh medium. Supernatants were collected 24 h after infection from the same wells, clarified by centrifugation, and frozen at –80°C until tested. IL-6, IL-10, IL-12, TNF-{alpha}, CCL3, and CCL5 levels were measured with Quantikine (R&D Systems) and IFN-{alpha} levels with an ELISA kit (PBL Biomedical Laboratories).

Statistical analysis

Data are expressed as mean ± SD or mean ± SEM. An unpaired, two-tailed t test was used to determine statistically significant differences (p < 0.05).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
HCMV infects PDCs with low efficiency

Mo-DCs and PDCs were infected with either of two endotheliotropic strains of HCMV, TB40/E and VR1814, at an MOI of 5 overnight. VR1814 infected Mo-DCs with high efficiency, but infected only 3–12% of PDCs, as detected by HCMV IE expression, at 1 day p.i. (Fig. 1, A and B) and 1–10% at day 3 p.i. (data not shown). No late viral Ags (pp28 or pp150) were detected at day 1, 3, or 5 p.i. (data not shown). Double immunofluorescence analysis of the PDC-specific marker BDCA-2 and viral IE confirmed nuclear expression of IE in PDCs but not in other cells possibly contaminating the PDC culture (data not shown). Increased amount of virus (MOI = 25) did not change the infection rate (5–10% of IE, positive cells at day 1 p.i.) whereas lower virus concentration (MOI = 1) resulted in <0.1% of IE-positive PDCs at day 1 p.i. We therefore chose to infect PDCs with an MOI of 5 in all following experiments.


Figure 1
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  		FIGURE 1. HCMV infects PDCs with low efficiency. A, Expression of IE viral products was analyzed in PDCs by indirect immunofluorescence. Mock-infected and VR1814-infected PDCs were spotted, fixed, and stained with anti-HCMV IE mAb. IE was detected as green nuclear staining at day 1 p.i. in VR1814-infected PDCs (II) but not in mock-infected cells (I). Evans blue was used as unspecific contrast staining. B, Mo-DCs and PDCs were infected with VR1814 or TB40/E (MOI of 5) overnight, spotted at 1 day p.i., fixed, and stained for anti-IE. The percentage of IE-positive cells was then calculated. Values are means ± SD of three independent experiments. C, Total RNA was isolated from mock-infected and HCMV-infected PDCs, and cDNA was synthesized and used as templates for HCMV-specific primer pairs for the major IE (MIE) and the late pp150 (UL32) genes in a nested PCR assay. cDNA samples from uninfected (neg) and HCMV-infected (pos) fibroblasts were included as negative and positive controls, respectively. The final PCR products were 218 bp (MIE) and 150 bp (p150). D, Viability of PDCs cultured with medium or different HCMV strains (TB40/E and VR1814), determined by trypan blue staining. Results of six donors in separate experiments (means ± SEM) are shown.

 
To investigate the permissiveness of infection in PDCs, we analyzed mRNA expression of IE and late viral genes in an RT-PCR assay. Both IE and pp150 viral mRNA were detected in TB40/E- and VR1814-infected cultures at day 1 and 3 p.i. (Fig. 1C). Uninfected PDC samples were negative for HCMV mRNA.

PDCs died rapidly, and few viable cells remained after 3–5 days of culture in medium alone, as reported (27). However, PDC cultures infected with TB40/E had greater viability than mock-infected cells at day 1 and 3 p.i. VR1814 appeared to be toxic for PDCs, with greatly reduced survival at day 1 p.i. (Fig. 1D).

HCMV induces activation and partial maturation of PDCs

Exposure to viruses such as influenza A, HIV, HSV, and murine CMV induces secretion of IFN-{alpha} and TNF-{alpha}, which mediates PDC maturation (27, 28, 29, 30, 31). We tested whether PDCs matured after encountering HCMV. After 24 h of infection with VR1814 or TB40/E, purified PDCs developed dendrites and clustered together, implying maturation. Neither mock-infected cultures nor PDCs treated with virus-free supernatants obtained by filtration of the viral stock through 0.1-µm pore filters underwent morphological changes (Fig. 2A and data not shown). PDCs cultured with infectious virus (TB40/E or VR1814) also exhibited immunophenotypic changes typical of mature PDCs, such as up-regulation of CD83 and MHC class II molecules and a slight but not significant down-regulation of the characteristic PDC marker BDCA-2 (Fig. 2, B and C). However, the levels of costimulatory molecules (CD80 and CD86) were unchanged in four of seven cases after infection and increased only slightly in the others, suggesting that the HCMV-induced activation of PDCs leads to a partial maturation of these cells. Conversely, a significant increase in the expression of CD80 and CD86 molecules was found when PDCs were incubated with CpG-A, a strong activator of PDCs (19) (Fig. 2B). The virus-induced changes were not seen in cultures treated with virus-free supernatant, indicating that intact viral particles are required to activate PDCs (data not shown).


Figure 2
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  		FIGURE 2. HCMV activates PDCs. A, HCMV alters PDC morphology. PDCs were incubated overnight with medium or TB40/E at an MOI of 5 and examined by light microscopy and Evans blue staining of cytospin preparations. At 24 h, mock-infected PDCs showed plasmacytoid morphology, whereas TB40E-infected PDCs formed clusters, developed dendrites, and had larger cytoplasm. B and C, HCMV induces partial maturation of PDCs. PDCs were mock-infected (mock inf), stimulated with CpG-A (3 µg/ml), or infected with TB40/E or VR1814 (MOI of 5) overnight. The stimuli were removed, cells were washed, and fresh medium was added. After 24 h, expression of CD83, HLA-DR, BDCA-2, CD80, and CD86 was measured by FACS. B, Values are means ± SEM of seven donors in independent experiments. *, p < 0.05. Y-axis values indicate MFI. C, Open histograms depict cells stained with isotype-matched control Ab. Representative data from one of seven experiments are shown.

 
PDCs respond to viruses through a restricted and unique set of TLRs, including TLR7 and TLR9 (10). Because TLR expression may itself be modulated in response to viral exposure (32), we investigated TLR expression in PDCs exposed to HCMV. TLR7 levels were not affected, but expression of TLR9 significantly increased after viral treatment (data not shown).

Increased cytokine and chemokine secretion in HCMV-activated PDCs

Next, we analyzed cytokine and chemokine production by PDCs 1 day after exposure to HCMV. After overnight incubation, the virus was removed and the cells were washed and seeded in fresh medium for 6 h. We performed this washing procedure to eliminate the viral inoculum that contains IFN-{alpha} (204 ± 34 pg/ml) and probably other cytokines that would interfere with the determination of soluble factors produced by PDCs. TB40/E-infected PDCs produced IL-6, IL-10, TNF-{alpha}, CCL3, and high amounts of IFN-{alpha} (Fig. 3A), as determined by ELISA of culture supernatants. Interestingly, VR1814-infected and mock-infected PDCs secreted similar amounts of IL-10 and CCL3; IL-12 and CCL5 were not detected. UV-treated viral stocks also induced cytokine production, indicating that viral replication is not required to trigger cytokine secretion by PDCs. However, PDCs infected with virus-free supernatant did not secrete cytokines, suggesting that intact viral particles are necessary to stimulate production of soluble factors. Thus, HCMV activates and induces partial maturation of PDCs, resulting in the secretion of cytokines that are critical for humoral and cell-mediated immunity.


Figure 3
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  		FIGURE 3. Regulation of cytokine production in PDCs upon HCMV infection. A, HCMV induces cytokine/chemokine secretion by PDCs. PDCs were mock infected (mock), infected with TB40/E (TB40) or VR1814, incubated with TB40/E inactivated by UV (TB40-UV), or incubated with virus-free supernatant (TB40-sup) overnight. The virus was removed and the cells were washed and seeded (106 cells/ml) in fresh medium. The concentrations of IL-6, IL-10, IFN-{alpha}, TNF-{alpha}, and CCL3 in supernatants were measured by ELISA at 24 h p.i. Values are means ± SEM of independent experiments performed with cells obtained from three donors. B, HCMV activates IFN-{alpha} secretion from PDCs by engaging TLR7 and/or TLR9. PDCs were preincubated with or without increasing doses of the TLR7 and TLR9 agonist CpG ODN 2088 (0.3–30 µg/ml; iCpG) and cultured overnight with TB40/E (MOI of 5; TB40), CpG ODN 2216 (3 µg/ml; CpG-A) or medium alone (mock inf). The virus and stimulatory or inhibitory CpGs were removed, and the cells were washed and seeded (106 cells/ml) in fresh medium. Supernatants were collected 24 h after infection from the same wells, and the concentration of IFN-{alpha} was measured by ELISA. Values are means ± SEM of separate experiments performed with cells obtained from ten donors. **, p < 0.01.

 
Stimulation of TLR7 and/or TLR9 is required for viral PDC activation

PDCs respond to several herpesviruses by engaging TLR9 (29, 30, 33). To determine whether HCMV induces a similar response, we cultured purified PDCs with HCMV in the presence or absence of CpG 2088, a competitive inhibitor of TLR7 and TLR9 (22, 34, 35). The activity of inhibitory CpG was first confirmed by coincubation with the stimulatory CpG ODN 2216 (CpG-A; final concentration 3 µg/ml), which induces high-level production of IFN-{alpha} in PDCs (19). Treatment of PDCs with stimulatory CpG and the same amount of inhibitory CpG partially prevented IFN-{alpha} secretion (13713 ± 5430 pg/ml vs 1786 ± 1055 pg/ml; IFN-{alpha} secretion by PDCs stimulated with 3 µg/ml CpG-A and PDCs stimulated with 3 µg/ml CpG-A plus 3 µg/ml CpG 2088, respectively, results are mean values ± SD of three independent experiments), whereas the release of IFN-{alpha} was completely abolished by treatment of PDCs with stimulatory CpG and 3.3 times that amount of inhibitory CpG in the same donors (data not shown). Similarly, CpG 2088 significantly inhibited TB40/E-induced secretion of IFN-{alpha} in a dose-dependent fashion (Fig. 3B). This result suggests that HCMV induces IFN-{alpha} secretion from PDCs in vitro by engaging the TLR7 and/or the TLR9 pathways.

Soluble factors produced by HCMV-infected PDCs contribute to B cell activation and proliferation

HCMV infection induces a strong B cell activation, both in vitro (36) and in the natural host (4, 5, 7, 8, 9). Since TB40/E-stimulated PDCs secrete IFN-{alpha} and other cytokines, such as IL-6 and IL-10, that are critical for B cell activation and differentiation (14, 20), we investigated whether soluble factors produced by HCMV-infected PDCs could induce B cell activation in vitro.

Purified B cells were stimulated by ligation of the B cell receptor with anti-Ig or left unstimulated and incubated for 3 days with virus-free supernatant from mock-infected or TB40/E-infected PDCs. The levels of the activation markers CD38 and CD86 were significantly higher after stimulation with supernatants from HCMV-infected than from mock-infected PDCs (Fig. 4A). No difference was observed in the absence of anti-Ig stimulation.


Figure 4
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  		FIGURE 4. Soluble factors secreted by HCMV-infected DCs contribute to B cell activation. A and B, PDCs were mock infected or infected with TB40/E (MOI of 5) for 4 h, washed, and resuspended in complete medium for 18 h. Supernatants were collected and filtered through 0.1-µm filters to eliminate viral particles. B cells (2.5 x 105 cells/well) were cultured in 200 µl of supernatant from PDC cultures in 96-well round-bottom plates. B cells were unstimulated or stimulated by B cell-receptor ligation (anti-Ig, 10 µg/ml). A, After 3 days, B cells were harvested, stained for the B cell markers CD19, CD20, CD38, and CD86, and analyzed by FACS. Values are means ± SD. *, p < 0.05 (n = 4 donors in independent experiments). B, For proliferation assay, B cells were labeled with 1 µM CFSE, incubated with PDC supernatant for 5 days, harvested, and analyzed by FACS. Data from one representative of three donors in independent experiments are shown. C, MDCs were mock infected or infected with TB40/E (MOI of 5) for 4 h, washed, and resuspended in complete medium for 18 h. Supernatants were collected and filtered. B cells (2.5 x 105 cells/well) were cultured in 200 µl of supernatant from MDC cultures. B cells were unstimulated or stimulated by B cell-receptor ligation (anti-Ig, 10 µg/ml). After 3 days, B cells were harvested, stained for the B cell markers CD19, CD20, CD38, and CD86, and analyzed by FACS. Values are means ± SD. *, p < 0.05 (n = 4 donors in independent experiments).

 
Next, we determined whether soluble factors produced by HCMV-infected PDCs could induce B cell proliferation. Unstimulated or anti-Ig-stimulated B cells were labeled with CFSE and incubated for 5 or 7 days with virus-free supernatant from mock-infected or TB40/E-infected PDC cultures. Individually, neither B cell receptor ligation nor soluble factors produced by HCMV-infected PDCs affected B cell proliferation. However, in the presence of supernatant from HCMV-infected PDCs, B cell receptor stimulation induced B cell division after 5 and 7 days of culture (Fig. 4B and data not shown).

To understand whether B cell activation was induced by soluble factors specifically produced by PDCs upon HCMV infection or whether other DC subsets may be involved in production of cytokines that stimulate B cell activation, we incubated B cells with virus-free supernatants obtained from uninfected and TB40/E-infected purified MDCs for 3 days. As for PDCs, the levels of the activation markers CD38 and CD86 were significantly higher in anti-Ig stimulated B cells after incubation with supernatants obtained from HCMV-infected MDCs as compared with mock-infected MDCs (Fig. 4C). Conversely, soluble factors produced by HCMV-infected MDCs in combination with B cell receptor stimulation failed to induce B cell proliferation after 5 days of culture in three different donors (data not shown). Thus, factors released by different DC subsets can induce B cell activation upon HCMV infection, but only soluble mediators produced by HCMV-stimulated PDCs specifically induce B cell proliferation.

HCMV triggers B cell activation and Ab production in a T cell dependent way

To investigate whether B cell receptor ligation and HCMV stimulation of PDCs induce differentiation of B cells into Ig-secreting plasma cells in a T cell independent manner, we coincubated B cells and PDCs isolated from PBMCs with TB40/E, or CpG ODN M352 (CpG-C) with or without anti-Ig for 72 h. In concordance with published data (20, 21), CpG-C alone induced B cell activation as shown by up-regulation of CD86 (MFI: 23.3 ± 11.6 vs 55.0 ± 6.3; B cells in medium alone and B cells stimulated with 3 µg/ml CpG-C, respectively, results are mean values ± SD of three independent experiments). The activation marker CD86 was significantly up-regulated when HCMV or CpG-C was added to anti-Ig-stimulated B cells in culture with PDCs (Fig. 5A). This up-regulation appeared to be mediated by PDCs because anti-Ig activated B cells stimulated by the virus but not by PDCs did not exhibit a significant increase in CD86 expression levels. We also found an up-regulated expression of CD19 in the presence of the virus, but the level of expression of this marker was not significantly affected by the presence of PDCs. In experiments with purified HCMV particles, up-regulation of CD86 on B cells was comparable to that obtained with nonpurified virus (data not shown), indicating that B cell activation was not induced by soluble factors in the viral inoculum. In contrast, coincubation of anti-Ig-stimulated B cells and PDCs infected with influenza virus failed to induce B cell activation (data not shown), suggesting that the stimulation of B cells by PDCs is an HCMV-specific phenomenon. Furthermore, the secretion of IL-6 and TNF-{alpha} was enhanced when B cells were stimulated with anti-Ig and HCMV in the presence of PDCs (Fig. 5B).


Figure 5
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  		FIGURE 5. Costimulation of B cells with HCMV, PDCs, and anti-Ig triggers B cell activation at different time points and Ab production in a T cell dependent manner. Purified B cells were incubated with different combinations of anti-Ig (10 µg/ml), TB40/E (MOI = 5), and CpG-C (3 µg/ml). B cells were incubated alone (1 x 105 cells/well) or cocultured with PDCs (1 x 104 cells/well) or MDCs (1 x 104 cells/well). A, After 72 h, CD19 and CD86 expression was analyzed by FACS. Values are means ± SEM of four donors in independent experiments. *, p < 0.05. B, After 72 h, supernatants were collected from B cell/PDC cocultures, and IL-6, IL-10, and TNF-{alpha} levels were measured by ELISA. Values are means ± SEM of three donors in independent experiments. *, p < 0.05. C, After 7 days, cells were collected and CD38 was analyzed by FACS. Values are means ± SEM of four donors in independent experiments. *, p < 0.05. D, B cells (1 x 105 cells/well) were incubated with or without PDCs and/or CD4+ T cells (1 x 105 cells/well) and stimulated with different combinations of anti-Ig (10 µg/ml), TB40/E (MOI = 5), and CpG-C (3 µg/ml). After 13 days, IgM and IgG were measured in the supernatant by ELISA. Values are means ± SEM of four donors in independent experiments. *, p < 0.05.

 
Next, B cells were analyzed for plasma cell differentiation (CD38high and CD20low B cell phenotype) and Ab production after 7 and 13 days of culture, respectively. CpG-C, an ODN that combines B cell activation with high IFN-{alpha} induction in PDCs, was used as a positive control to generate plasma cells as described (20). When CpG-C or HCMV was added to anti-Ig-stimulated B cells in culture with PDCs, the B cell activation marker CD38 was significantly up-regulated (Fig. 5C). The up-regulation of CD38 appeared to be mediated by PDCs because B cells stimulated by the virus in the absence of PDCs did not exhibit a significant increase in CD38 expression levels.

No plasma cells were detected at day 7 and no Ab secretion was observed at day 13 in HCMV-infected cultures. However, low levels of IgG were detected in two of four donors when the T cell cytokine IL-2 was added to the culture (Fig. 5D). As expected, plasma cells were detected at day 7 and Ab secretion was observed at day 13 when CpG-C- and anti-Ig-stimulated B cells were cocultured with PDCs (Fig. 5D and data not shown).

We next exposed B cells to three different stimuli: HCMV, PDCs, and CD4+ T cells. Supernatants were obtained from cultures at day 13 for measurement of IgM and IgG levels. Although B cells incubated with T cells and PDCs produced low levels of Abs in the absence of virus, the addition of HCMV to the cocultures resulted in a significant Ab production (Fig. 5D). Thus, HCMV prompts B cell activation through PDC engagement. Differentiation of B cells to Ig-secreting cells upon virus stimulation required the presence of T cells or T cell-produced cytokines in our experimental setting.

Next, we analyzed the effect of MDCs on B cell activation in a coculture system. Anti-Ig-stimulated B cells were cocultured with or without MDCs in the presence of the virus or not. After 3 days, a significant down-regulation of CD86 was detected when anti-Ig stimulated B cells were incubated with MDCs, indicating an inhibitory effect of MDCs on B cell activation induced by anti-Ig (Fig. 5A). HCMV stimulation induced a significant B cell activation that did not appear to be mediated by MDCs because B cells stimulated by the virus in the presence or absence of MDCs exhibited similar increased CD86 expression levels (Fig. 5A). Conversely, MDCs induced a significant up-regulation of CD38 on anti-Ig stimulated B cells in the presence of the virus at day 7 but did not induce Ab production at day 13 (Fig. 5C and data not shown).

HCMV interferes with allogeneic T cell proliferation induced by PDCs

Although immature PDCs only exhibit a weak T cell-stimulatory capacity, PDCs matured by HSV, HIV, or influenza virus can expand allogeneic naive T cells. Thus, activated PDCs can present Ags and induce considerable expansion of T cell populations, although less efficiently than MDCs (13, 37, 38). To study whether HCMV affects the ability of PDCs to serve as APCs, we tested the stimulatory activity of uninfected and HCMV-infected PDCs in an allogeneic MLR assay at 24 h after infection. Surprisingly, infection with TB40/E clearly impaired the allostimulatory activity of PDCs. Furthermore, inhibition of T cell stimulation in HCMV-infected PDC cultures was not dependent on HCMV replication because incubation with UV-inactivated virus exerted similar effects (Fig. 6A).


Figure 6
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  		FIGURE 6. PDCs in contact with HCMV exhibit reduced immunostimulatory ability. A, PDCs were mock infected (mock), infected with TB40/E (TB40; MOI of 5), or incubated with UV-inactivated TB40/E (TB40-UV). At 1 day p.i., PDCs were washed three times, irradiated, and added as stimulator cells to allogeneic PBMCs (responder cells). Responder cells (105/triplicate well) were stimulated with different doses of PDCs. Y-axes depict PBMC proliferation based on [3H] thymidine incorporation (cpm) during the last 16 h of a 6-day culture. The stimulation index was calculated as described in Materials and Methods. *, p < 0.05 (n = 3 donors in independent experiments). B–C, For CFSE-based proliferation assays, responder PBMCs were labeled with CFSE and mixed with irradiated mock-infected PDCs (mock), TB40/E-infected PDCs (TB40), or PDCs incubated with UV-inactivated TB40/E (TB40-UV) at a ratio of 25,000 PDCs to 100,000 PBMCs. After 6 days, cells were harvested for FACS analysis. B, PDC-stimulated MLR cultures were evaluated by 2-color plot of CD4 or CD8 expression vs CFSE. 3500 CD4+ or CD8+ and CFSEbright events were collected (right-hand box); the percentage of CD4+ or CD8+ CFSEdim events (left-hand box) was then determined. Quadrant gates of one of four experiments are shown. C, CD4+ and CD8+ lymphocytes were selected by gating and analyzed for CFSE fluorescence. The cell division index (CDI) was calculated as described in Materials and Methods. Values are means ± SEM of four donors in independent experiments. *, p < 0.05.

 
To further characterize the biology of the allogeneic response, we used a cell-permeant fluorescent dye to label responder PBMCs in an allogeneic MLR. This approach enabled us to identify and track both dividing alloreactive cells (CFSEdim) and nondividing cells (CFSEbright) by FACS. The number of proliferating CFSEdim blasts was reduced when PDCs treated with TB40/E or UV-inactivated virus were used as stimulator cells as compared with mock-infected PDCs. The inhibition of PBMC proliferation was 22 ± 5% with TB40/E-infected PDCs and 50 ± 24% with PDCs incubated with UV-inactivated virus. In addition, both CD4+ and CD8+ T cell proliferation was lower in the presence of HCMV-infected PDCs or PDCs incubated with UV-inactivated virus than in the presence of mock-infected PDCs (Fig. 6, B and C). Thus, HCMV hampers the ability of PDCs to stimulate T cells.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
PDCs are a central cell type in the immune system. By producing type I IFN, these cells possess a dual role in antiviral responses by directly inhibiting viral replication and by determining the initiation and orchestration of downstream B and T cell responses (11, 13, 14, 31). In this study, we describe the effect of HCMV infection of PDCs at an immunophenotypical and functional level. Interestingly, the virus-PDC interaction differentially influences B and T cell functions.

We analyzed the susceptibility of human MDCs and PDCs to two endotheliotropic and leukotropic HCMV strains (TB40/E and VR1814). As in previous reports (15, 39), these viruses infected MDCs with high efficiency and PDCs with low efficiency; only 1–12% of PDCs were IE-positive at day 1 p.i. Although late viral RNA was detected in HCMV-infected PDC cultures, the infection appeared to be mainly nonpermissive because no late viral Ags were detected. We cannot exclude that contaminating leukocytes such as circulating MDCs or other HCMV-permissive cells in our cultures could have influenced the positive RT-PCR finding for pp150.

Although unstimulated PDCs have minimal expression of costimulatory molecules and low MHC class II expression, virus-activated PDCs augment cell surface expression of MHC class II and costimulatory molecules and develop into efficient APCs for the adaptive immune response (13, 28, 37). We found that TB40/E and VR1814 activated human PDCs, resulting in a typical DC-like morphology, up-regulation of CD83 and MHC class II molecules, and increased intracellular expression of TLR9, the last effect possibly inducing a synergism with the virus in PDC activation. However, in contrast to CpG-A stimulated PDCs, HCMV-stimulated PDCs were not fully mature, as slightly increased CD80 and CD86 levels were observed only in three of seven donors.

Viral infection leads to maturation of PDCs in an autocrine manner by induced cytokine production, particularly type I IFN and TNF-{alpha} (27, 28, 29, 30, 31), and HCMV stimulates IFN-{alpha} and IL-6 secretion in PDCs after 24–84 h of infection (39, 40). We found that PDCs began secreting IFN-{alpha}, TNF-{alpha}, IL-6, and IL-10 within 24 h after exposure to HCMV, regardless of whether they supported viral replication. Type I IFN released by PDCs may inhibit viral infection of neighboring cells (11) and promote the differentiation of activated CD4+ into Th1 helper CD4+ cells and naive CD8+ cells into cytotoxic CD8+ T cells (41, 42). Together with IL-6, type I IFN would be involved in the B cell activation and differentiation into Ig-secreting plasma cells (14, 20). In contact with HCMV, PDCs also produced a β-chemokine, CCL3, and therefore would be able to attract Th1-type cells, B cells, NK cells, and immature MDCs to sites of viral infection (43, 44).

Recent evidence suggests that herpes viruses activate PDCs by engaging TLR9, which results in an instant type I IFN response that is independent of viral replication (29, 30, 33). In our study, UV-inactivated HCMV and untreated virus stimulated IFN-{alpha} production to a similar extent, suggesting that HCMV replication is not required for effective stimulation of PDCs. HCMV-induced activation of PDCs was prevented in a dose-dependent fashion by an inhibitory CpG that blocks TLR7 and TLR9 activation (22, 34, 35). Thus, our findings suggest that HCMV induces IFN-{alpha} secretion from PDCs in vitro through engagement of the TLR7 and/or TLR9 pathways. In support to our observations, murine CMV has been shown to interact with TLR9 for PDC activation in mice (29).

PDCs can also act as APCs and trigger adaptive immunity by directing T cell responses (45). Until they mature, PDCs have little capacity to stimulate T cells (46). Surprisingly, HCMV-exposed PDCs were even less efficient than mock-infected PDCs at inducing allogeneic T cell proliferation despite the increased expression MHC class II molecules. The reduced immunostimulatory capacity affected both CD4+ and CD8+ T cell subsets. These observations may be explained by a lack of enhanced expression of costimulatory molecules on PDCs after HCMV infection. Failure to receive costimulation after Ag presentation may render T cells anergic and functionally incapable of proliferating (47, 48).

HCMV subverts the stimulatory potential of MDCs against alloantigens during in vitro (17, 49) and in vivo (25) infection. The impaired immunostimulatory activity caused by HCMV may depend on release of soluble CD83 into the culture supernatant of HCMV-infected MDCs (50). It has been shown that HCMV-infected MDCs have a reduced capacity of stimulating T cell response up to 80–85% (17, 49). In this study, we found that HCMV inhibits the PDC immunostimulatory ability by ~50%. Nevertheless, the ability of HCMV to simultaneously undermine the immunostimulatory properties of different APC subsets may enable the virus to evade the early host immune response and may contribute to the profound immunosuppression often observed in HCMV-infected patients (2, 3).

In contrast to its inhibitory effect on T cell proliferation, HCMV prompted B cell activation through the activation of PDCs and the production of soluble factors secreted by PDCs. In contrast to previous findings (14), virus-induced IL-6 secretion by PDCs was not dependent on CD40 ligation.

A T cell-independent pathway for plasma cell development that involves costimulation of B cells with PDCs, anti-Ig, and CpG-C, a TLR9 activator, has been recently discovered (20). In our experiments, HCMV possibly activated TLR9 but did not induce plasma cell differentiation or Ab production in a T cell-independent manner since T cells or the T cell produced cytokine IL-2 were required to trigger Ab secretion. A plausible explanation for our findings may be that significantly lower levels of cytokines such as IL-10 and TNF-{alpha} were produced when HCMV was added to the coculture as stimulator instead of CpG-C (Fig. 5B). In support of this hypothesis, TNF-{alpha} appears to contribute to PDC maturation (38), and IL-10 is a crucial factor for terminal differentiation of B cells (51, 52).

One can wonder how HCMV may simultaneously hamper T cell proliferation via effect on PDCs and induce a T cell-dependent differentiation of B cells to Ig-producing plasma cells. As a possible explanation, the virus-induced impairment of T cell functionality may be limited to proliferation and not affect T cell function in a general manner. Consistent with this hypothesis, we found that Th cells induce differentiation of Ab-secreting cells in the presence of HCMV (Fig. 5D). In addition, soluble factors produced by HCMV-stimulated PDCs, such as IFN-{alpha}, IL-6, and IL-10, are critical for B cell activation (14, 20) and may be of additional help in triggering in vivo differentiation of B cells to Ig-secreting plasma cells.

PDCs are not the only cells that play a fundamental role in B cell activation and differentiation and it has been previously demonstrated that MDCs can take part in stimulation of a B cell response (53). We cannot exclude that MDCs may contribute to B cell activation upon HCMV infection, but the effect exerted by MDCs in stimulating B cells was ambiguous in our in vitro infection model. In fact, soluble factors produced by HCMV-infected MDCs induced B cell activation, but not cell division. In addition, MDCs impaired B cell activation induced by B cell receptor ligation after 3 days of coculture, but stimulated B cell activation in the presence of the virus at day 7.

The B cell activation caused by HCMV through effects on PDCs could contribute to the nonspecific polyclonal B cell activation by HCMV in vitro (36) and abnormal humoral features in HCMV-infected patients, such as hypergammaglobulinemia, cryoglobulinemia, and autoantibody production (4, 5, 7, 8, 9). However, the significance of B cell hyperactivation for viral pathogenesis is unclear. Hyperactivation of humoral immunity may impede the development of specific B cell responses and may be another mechanism of immune evasion. B cell hyperactivation may also be a simple epiphenomenon that does not affect viral efficiency, but may have clinical relevance. Autoantibodies may contribute to the development of graft-vs-host disease in infected bone marrow transplant patients and graft rejection in solid organ recipients (4, 6).

In summary, contact with HCMV activated PDCs via TLR7 and/or TLR9 and resulted in opposite consequences for the two arms of adaptive immunity. HCMV-activated PDCs secreted soluble factors that stimulated B cell activation and proliferation. Conversely, HCMV inhibited the allostimulatory ability of these professional APCs, leading to depressed proliferation of CD4+ and CD8+ T cells and hampered T cell responses. Thus, through effects on PDCs, HCMV controls the humoral and cell-mediated immune responses in a novel and opposite manner. Our findings emphasize the importance of functional interactions between cells of the innate and adaptive immune system during HCMV infection.


   Acknowledgments
 
We thank Prof. G. Jahn (University of Tübingen, Tübingen, Germany) for providing the TB40/E strain of HCMV, Prof. G. Gerna (University of Pavia, Pavia, Italy) for supplying the VR1814 strain of HCMV, and Prof. A. Linde (Karolinska Institutet, Stockholm, Sweden) for providing the influenza virus. We also thank Eicke Latz for scientific advice and Stephen Ordway for editorial assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interests.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Heart-Lung Foundation (199941305, 200241138, 20030055, and 20050266), King Gustaf V’s 80-Year Anniversary Foundation, Swedish Research Council (K2004-16X-12615-07A), Konung Gustaf V’s och Drottning Victorias Foundation, Groschinsky’s Foundation, Pfizer, Swedish Cancer Foundation (060253), and Children’s Cancer Foundation (05/100). C.S.-N. is a fellow of the Royal Swedish Academy of Science, Sweden. Back

2 Address correspondence and reprint requests to Dr. Cecilia Söderberg-Nauclér, Center for Molecular Medicine, L8:03, Karolinska Hospital, Stockholm, Sweden. E-mail address: cecilia.naucler{at}ki.se Back

3 Abbreviations used in this paper: HCMV, human CMV; DC, dendritic cell; MDC, myeloid dendritic cell; PDC, plasmacytoid dendritic cell; Mo-DC, monocyte-derived DC; ODN, oligodeoxynucleotide; BDCA, blood dendritic cell antigen; MOI, multiplicity of infection; p.i., postinfection; IE, immediate-early Ag; MFI, mean channel fluorescence intensity. Back

Received for publication June 13, 2006. Accepted for publication September 20, 2007.


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A.-C. Iversen, B. Steinkjer, N. Nilsen, J. Bohnhorst, S. H. Moen, R. Vik, P. Stephens, D. W. Thomas, C. A. Benedict, and T. Espevik
A Proviral Role for CpG in Cytomegalovirus Infection
J. Immunol., May 1, 2009; 182(9): 5672 - 5681.
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