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Human herpesvirus 8 (HHV-8) inhibits monocyte differentiation into dendritic cells and impairs their immunostimulatory activity
Immunology Letters 113 (2007) 40–46
Human herpesvirus 8 (HHV-8) inhibits monocyte differentiation into dendritic cells and impairs their immunostimulatory activity Mara Cirone ∗ , Giuseppe Lucania, Paola Bergamo, Pankaj Trivedi, Luigi Frati, Alberto Faggioni Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Experimental Medicine University of Rome “La Sapienza”, Rome, Italy Received 12 April 2007; received in revised form 12 July 2007; accepted 21 July 2007 Available online 21 August 2007
Abstract Several viruses interfere with the host immune response by infecting dendritic cells and by altering their functional activity. Here, we report that exposure to Human herpesvirus 8 (HHV-8) of human dendritic cell (DC) monocyte precursors resulted in impaired immature DC (iDC) formation as indicated by a reduced CD1a expression. In accordance, the immunostimulatory ability of such iDC was significantly reduced, as indicated by mixed lymphocyte culture (MLR) assays. The immunostimulatory functions of DCs were similarly inhibited by the UV inactivated viral stocks, suggesting that the virus binding is sufficient to determine the observed effect. Furthermore, HHV8 mediated inhibition of the DC allostimulatory function was present in lipopolysaccharide (LPS) matured DCs. A strong reduction of the expression of the costimulatory molecule CD80 on the surface of the virus-exposed cells was observed as well. Impairment of dendritic cell development and function might represent an important strategy used by HHV-8 to escape from the host defense mechanisms. © 2007 Elsevier B.V. All rights reserved. Keywords: Human herpesvirus 8; Dendritic cell; Immunostimulatory activity
1. Introduction Human herpesvirus 8 (HHV-8), also named Kaposi’s sarcoma-associated herpesvirus (KSHV), is a lymphotropic herpesvirus linked to several clinical disorders [1,2]. HHV-8 has been detected in biopsies of all clinical and epidemiological forms of Kaposi’s sarcoma (KS). The virus is present in the spindle-shaped cells that are considered to be the tumor cells of KS, where it establishes a latent infection, and also in the perilesional mononuclear cells where the infection can also be lytic. In addition, HHV-8 has been detected in peripheral blood mononuclear cells of KS patients, including B-cells, T-cells and monocytes. Besides KS, HHV-8 has been also linked to lymphoproliferative disorders such as primary effusion lymphomas (PEL) and multicentric Castelman’s disease (MCD). PEL are latently infected in vivo but can be induced to lytic infection in vitro by treating the cells with phorbol esters or sodium ∗ Corresponding author at: Dipartimento di Medicina Sperimentale, Viale Regina Elena 324, 00161 Roma, Italy. Tel.: +39 06 4463542; fax: +39 06 4454820. E-mail address: [email protected] (M. Cirone).
0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.07.013
butyrate and this treatment is currently used for viral stock preparation. Dendritic cells (DC) play a pivotal role in the initiation and maintenance of immune responses [3]. They are involved in T-lymphocyte activation and in the polarization of immune responses towards Th1 or Th2 cytokine secretion patterns. Triggering of Th1-type immune responses and development of strong cytotoxic activity usually require efficient DCs mainly derived from myeloid precursors, such as blood monocytes. Consistent with an essential role of DC in anti-virus immune response, several viruses have developed strategies to target these cells and impair their function in the attempt of evading and subverting the immune response. In particular viruses that establish persistent infection such as herpesvirus have been shown to inhibit the T-cell-stimulatoty function of DC by interacting with DC or their precursors. Examples are Cytomegalovirus (CMV) that inhibits DC function by directly infecting these cells and/or by secretion of an homolog of IL-10 that affects their maturation [4,5]. Epstein-Barr virus (EBV) which has been shown to inhibit DC function by promoting apoptosis of their monocyte precursors [6] and Herpes Simplex virus that inhibits DC maturation in the presence of LPS [7].
M. Cirone et al. / Immunology Letters 113 (2007) 40–46
We wanted to assess whether HHV-8 is able to interfere with the development of immature-DCs (iDC) from monocyte precursors in the presence of granulocyte macrophage-colonystimulating factor (GM-CSF) and interleukin 4 (IL-4) and to affect their allostimulatory activity. 2. Materials and methods 2.1. Virus preparation For the virus preparation, the HHV-8 positive but EBVnegative PEL cell line BCBL-1 was treated with 20 ng/ml tetradecanoyl phorbol acetate (TPA) for 3–5 days to induce HHV-8 replication. The virus-containing supernatant was centrifuged at 70,000 × g (2 h at 4 ◦ C). The resulting pellet was resuspended in cold PBS in 1/200 of the original volume of medium and filtered through a 0.45 m-pore filter. For HHV8 purification, the concentrated viral stocks were centrifuged through 30–60% sucrose step gradient at 17,000 rpm for 4 h. The band at the gradient junction was collected. The virions were then purified in a second round of gradient centrifugation [8]. Quantitation of viral yields was performed using polymerase chain reaction (PCR) amplification by comparison to the viral copy number of BCBL-1, which is known to have 70 viral copies/cell [9]. Approximately 1 × 108 –1 × 109 ml viral copies were present in the purified viral stocks. 2.2. Generation of monocyte-derived DC and HHV-8 infection To generate monocyte-derived DC, human peripheral blood mononuclear cells were isolated by Ficoll-Paque gradient centrifugation (Pharmacia, Uppsala, Sweden) from buffy coats of healthy volunteers after they had given informed consent to the procedure. CD14+ monocytes were then positively selected using anti-CD14 MAb-conjugated magnetic microbeads (Miltenyi Biotec, Auburn, CA). Purified monocytes (approximately 95% pure, as determined by flow cytometry using Mab directed against CD14) were cultured at a density of 1.5 × 106 cells/3 ml in 12-well plates for 6 days in the following medium: RPMI 1640 (Euroclone) containing 10% heat inactivated fetal calf serum (Gibco), 2 mM l-glutamine, 100 U/ml penicillin G, 100 g/ml streptomycin, 50 mM 2-mercaptoethanol (Sigma), and recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) (20 ng/ml each, Biodesign International). In some experiments, instead of IL-4, recombinant ␣ IFN (5000 U/ml, Alfa Wassermann, Bologna, Italy) was used together with GM-CSF. Cytokines were replenished every other day by adding 20% fresh medium to each well. For DC maturation LPS from Salmonella abortus equi (Sigma) at 1 mg/ml was added 6 days after culture onset and kept for 48 h. HHV-8 infection was performed by incubating 1 × 106 monocytes for 2 h at 37 ◦ C with 100 l of viral stock containing approximately 1 × 108 –1 × 109 viral copies/ml. Cells were then washed in PBS and cultured with the above-mentioned supplements to induce differentiation into iDC as described above.
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For PCR analysis, total DNA was extracted with a DNA extraction kit (Invitrogen, San Diego, CA) from iDC exposed or not exposed to HHV-8 and cultured for 3 and 6 days. One hundred nanogram of DNA was then amplified for HHV-8 DNA sequences by PCR using the following primer set: forward 5 -GCCGAAAGGATTCCACCAT-3 and reverse 5 -TCCGTGTTGTCTACGTCCA-3 from KS330 BamH1 fragment [10]. BC-3 was used as positive control. For detection of HHV-8 antigens by indirect immunofluorescence, iDC exposed for 2 h or not exposed to HHV-8 were subsequently cultured for 6 days. These cells were then air-dried, fixed in cold acetone and methanol (1:1) and incubated with previously characterized human sera from KS patients [11] or with mAbs directed against latent (LANA) or lytic (ORF K8.1) antigens purchased from ABI, USA. The secondary antibodies to rat, mouse or human Ig were purchased from Cappel. 2.3. Mixed lymphocyte reaction MLR was carried out in 96-well flat-bottom microtiter plates by adding three different numbers of irradiated (3000 rad) iDC (previously treated or not treated with HHV-8) to 2 × 105 allogeneic PBMCs. After 6 days at 37 ◦ C, cell proliferation was assessed by uptake of 3 H thymidine (1 Ci/well for 16 h). The cells were harvested onto glass microfiber filters (Packard) and counted using a Canberra Packard scintillation counter. 2.4. Flow cytometry For cell surface staining, after 6 days of culture, iDC were washed in PBS and incubated for 30 min at 4 ◦ C with monoclonal antibodies directed against CD1a, CD86, CD83 (Becton-Dickinson), CD-80 (ImmuneKontakt) and HLADR (Cymbus Biotech Ltd.). The binding of antibodies was revealed by goat FITC-conjugated or PE-conjugated anti-mouse Ig (Cappel). Samples were analyzed using a FACScan cytometer. 3. Results 3.1. Detection of HHV-8 in developing iDCs After concentration and purification of HHV-8 from BCBL1, CD14+ purified monocytes were exposed to the virus. The cell viability was not affected by the virus exposure, as indicated by trypan blue exclusion. To verify if the virus was able to enter into developing DCs, the cells were harvested on days 3 and 6 of culture. After three washes with PBS, DNA was extracted for PCR analysis. Viral DNA was detected in HHV-8-infected iDC in one step PCR both at days 3 and 6 post-infection (Fig. 1a). The expression of HHV-8 latent and lytic antigens in the iDC was assessed in parallel by IFA at day 6. As shown Fig. 1b, HHV-8 treated iDC showed the intracellular speckles typical of LANA while Fig. 1c shows control uninfected DCs. At 6 days postinfection, no positive cells for lytic antigen K8.1 were observed (data not shown), indicating that the virus is not able to replicate in the developing DCs.
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Fig. 3. Mixed lymphocyte reaction of HHV-8 treated and untreated DC before and after induction to maturation with LPS. The DC maturation was obtained by adding LPS at the 6th day of iDC culture, for an additional 48 h. Each bar represents 3 H-thymidine incorporation (cpm ± S.D.) of two representative experiments. Ratio DC:T = 1:10.
3.2. Allostimulatory capacity of iDCs treated with the active and the inactivated HHV-8
Fig. 1. Verification of the viral presence in HHV-8 treated iDCs: (a) by DNA PCR; Lane 1 marker, Lane 2 iDC non-treated (mock), 3: HHV-8 positive BC3, 4: HHV-8 treated iDC after 3 days, 5: HHV-8-treated iDCs after 6 days, 6: HHV8 negative PBLs. The amplified products were visualized on 2% agarose gels. B and C: IFA for LANA; a monoclonal antibody was used to detect LANA expression in HHV-8 treated iDCs (b) and untreated iDCs were used as negative control (c).
We next analyzed in a MLR assay the ability of cells treated or not treated with HHV-8 and cultured for 6 days with IL-4 and GM-CSF to induce T-cell proliferative response. A strong reduction of the allostimulatory activity of HHV-8 treated iDC was found. These experiments were carried out with iDCs derived from four healthy individuals and the mean of four experiments is represented in Fig. 2a. We tested if HHV-8 could inhibit the
Fig. 2. Mixed lymphocyte reaction (MLR): MLRs were performed with immature dendritic cells (iDC) treated with HHV-8 in the presence of IL-4 and GM-CSF (a), in the presence of ␣IFN and GM-CSF (b) or with UV-inactivated HHV-8 (c) in comparison to untreated iDC (mock) on day 6 of culture. The mean of four experiments is shown. P < 0.01 was obtained by comparing the MLR results of HHV-8 treated and untreated iDC. The statistical significance of the studies was calculated by the t-test analysis. (d and e) The efficiency of viral inactivation was evaluated by testing LANA expression in 293 cells infected with the active (d) and inactive (e) virus.
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iDC function in the presence of ␣-IFN and GM-CSF. Indeed, as seen in Fig. 2b, in the presence of this combination similarly resulted in reduced allostimulatory capacity. Since we did not find any viral replication in iDCs, we asked if the virus binding to monocytes was sufficient for the observed reduction of functional activity of the DC progeny. For this purpose, a replication incompetent HHV-8 was prepared by UV irradiation and by heat
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inactivation. For UV irradiation, the virus was exposed to the UV light (230 mW/sq cm for 20 min at a 10 cm distance) and for heat inactivation, the virus stocks were incubated in a waterbath at 56 ◦ C for 1 h. Again, a strong reduction of the allostimulatory activity of iDC was observed in cells exposed to UV-inactivated virus Fig. 2c. These data indicate that the inhibition of allostimulaory capacity is independent of virus replication and that this
Fig. 4. Surface protein expression of the HHV-8 treated and untreated iDC. Cells were cultured for 6 days and the surface expression of the indicated markers was analyzed by immunostaining and FACS analysis.
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effect could be mediated by the viral binding to the cell surface molecules. Here again, the experiments were performed from four healthy donors, and the mean is shown in Fig. 2c. The efficiency of the virus inactivation was measured by verifying LANA expression in 293 cells. Fig. 2d shows LANA expression when the cells are infected with the active virus and 2e instead shows 293 cells infected with the inactivated virus. 3.3. Allostimulatory capacity of HHV-8 treated DCs after maturation with LPS We further asked if the LPS treatment might restore allostimulatory function of HHV-8-treated DC. For DC maturation, LPS from Salmonella abortus equi (Sigma) at 1 mg/ml was added 6 days after culture onset and kept for an additional 48 h. Fig. 3 shows that the mature HHV-8 treated DCs did not recover
their allostimulatory capacity. The difference with the untreated mature DCs was even more significant when both were induced to full maturation with LPS (Fig. 3). Furthermore, we asked if this reduction was correlated to a difference in IL-12 (p70) production in the DC untreated or treated with HHV-8. A six-fold reduction in the DC exposed to the virus was observed. Similar results were obtained when ␣-IFN was used instead of IL-4 (data not shown). 3.4. Phenotypic analysis of immature and mature HHV-8 treated DCs We tested both the DC differentiation markers as well as molecules involved in antigen presentation in virus treated or untreated immature and mature DCs. To this end, CD1a, CD80, CD83, CD86 and HLA-DR expression was investigated by flow
Fig. 5. Surface protein expression of the HHV-8 treated and untreated mDC. After 6 days, the HHV-8 treated and untreated iDC were induced to maturation with LPS and surface expression of the indicated markers was analyzed by immunostaining and FACS analysis.
M. Cirone et al. / Immunology Letters 113 (2007) 40–46
cytometry. The experiments were performed in parallel to MLR. The results shown in Fig. 4 indicate that exposure of iDC to HHV-8 reduced CD1a and CD80 expression (both as mean fluorescence intensity and as percentage of positive cells) whereas only marginal difference in the expression of CD86, CD83 and HLA-DR was observed. After maturation with LPS, the expression of CD80 remained low in the cells exposed to HHV-8, and again a minimal difference in the expression of the other molecules was observed (Fig. 5). 4. Discussion The presence of HHV-8-specific CTLs in peripheral blood from KS patients has been reported [12], however these cells are unable to clear the virus-infected cells from circulation or tissues. An inhibition of DC function could account for this inefficiency of the T-cell mediated immune-response. Indeed the present study shows that HHV-8, independently of viral replication interferes with monocyte differentiation into iDC (as indicated by a reduced CD1a expression), impair their allostimulatory activity and is also responsible for the reduction of CD80 on their cell surface. We also report that monocyte-derived iDC previously exposed to HHV-8 did not recover their ability to stimulate alloproliferative T-cell response after maturation with LPS. Since we found no viral replication in these developing DCs, we sought to investigate if HHV-8 interferes with the monocyte differentiation into DC by interaction of viral particles with monocyte surface molecules. Indeed, results obtained with UV light-inactivated virus, which is unable to replicate but maintains the ability to bind to cellular surface molecules, is consistent with this notion. Our results provide a potential mechanistic insight as to how through inhibition of DC development, HHV-8 might contribute to the pathogenesis of classic KS (cKS) which arise in the absence of apparent immunosuppression. Interference with DC function by binding of the viral particles with surface molecules, has been previously reported for other herpesviruses such as EBV [6] and Human Herpesvirus 6 (HHV6) [13]. As for HHV-8, previous studies indicated that the virus is able to enter into CD34+ stem cells derived from cord blood and to enhance the allostimulatory activity of DC derived from these infected precursors [14] and more recently it was shown that dendritic cells can be directly infected with HHV-8 resulting in a reduced ability to process and present Ag to memory CD8+ T lymphocytes [15]. The major difference between the latter study and our findings is that while Rappociolo et al., used DCs, we have used monocyte precursors. Our study thus underlines that also the myeloid DC precursor differentiation can be negatively influenced by HHV-8. The inhibition of monocytic differentiation through interaction with their surface molecules resulting in reduction of their allostimulatory activity adds another aspect of immune modulation mediated by this virus. Studies are in progress in our laboratory to evaluate the presence of HHV-8 DNA in monocyte precursors of healthy HHV-8 seropositive individuals and in patients affected with cKS. It will be interesting to determine if the presence of viral DNA in these precursors could influence the allostimulatory function of their progeny.
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Recently, a quantitative and functional defect of dendritic cells in classic Kaposi’s sarcoma patients, in comparison to HHV-8 seronegative healthy individuals has been reported [16]. It will be important to evaluate if there are differences in DC function between patients with cKS and healthy HHV-8 seropositive individuals, and if there is also a correlation with viral load in both groups. The interference of DC function is a common strategy among viruses which can be accomplished either by the virus particles or by soluble factors induced by them [16]. In cKS, such studies might provide useful information about the strategies through which HHV-8 escapes from the host immune responses [17,18], and contribute to the development of HHV-8 associated diseases. Acknowledgments This work was supported by grants from the MIUR, Ministero della Sanit`a, Progetto AIDS, and from the Associazione Italiana per la Ricerca sul Cancro. We thank Dr. Sabrina Rotolo for statistical analysis and Mr. Sandro Valia for skillfull technical assistance. References [1] Boshoff C, Weiss R. AIDS-related malignancies. Nat Rev Cancer 2002;2:373–82. [2] Schulz TF. The pleiotropic effects of Kaposi’s sarcoma herpesvirus. J Pathol 2006;208:187–98. [3] Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells. Cell 2001;106:263–6. [4] Raftery MJ, Wieland D, Gronewald S, Kraus AA, Giese T, Schonrich G. Shaping phenotype, function, and survival of dendritic cells by cytomegalovirus-encoded IL-101 . J Immunol 2004;173:3383–91. [5] Grigoleit U, Riegler S, Einsele H, Laib Sampaio K, Jahn G, Hebart H, et al. Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cells. Br J Haematol 2002;119:189–98. [6] Li L, Liu D, Hutt-Fletcher L, Morgan A, Masucci MG, Levitsky V. Epstein-Barr virus inhibits the development of dendritic cells by promoting apoptosis of their monocyte precursors in the presence of granulocyte macrophage-colony-stimulating factor and interleukin-4. Blood 2002;99:3725–34. [7] Salio M, Cella M, Suter M, Lanzavecchia A. Inhibition of dendritic cell maturation by herpes simplex virus. Euro J Immunol 1999;29: 3245–53. [8] Zhu FX, Yuan Y. The ORF45 protein of Kaposi’s sarcoma-associated herpesvirus is associated with purified virions. J Virol 2003;77: 4221–30. [9] Lallemand F, Desire N, Rozenbaum W, Nicolas JC, Marechal V. Quantitative analysis of human herpesvirus 8 viral load using a real-time PCR assay. J Clin Microbiol 2000;38:1404–8. [10] Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles D, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 1994;266:1865–9. [11] Santarelli R, De Marco R, Masala MV, Angeloni A, Uccini S, Pacchiarotti R, et al. Direct correlation between human herpesvirus-8 seroprevalence and classic Kaposi’s sarcoma incidence in Northern Sardinia. J Med Virol 2001;65:368–72. [12] Osman M, Kubo T, Gill J, Neipel F, Becker M, Smith G, et al. Identification of human herpesvirus 8-specific cytotoxic T-cell responses. J Virol 1999;73:6136–40. [13] Smith AP, Paolucci C, Di Lullo G, Burastero SE, Santoro F, Lusso P. Viral replication-independent blockade of dendritic cell matura-
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tion and interleukin-12 production by human herpesvirus 6. J Virol 2005;79:2807–13. [14] Larcher C, Nguyen VA, Furhapter C, Ebner S, Solder E, Stossel H, et al. Human herpesvirus-8 infection of umbilical cord-blood-derived CD34+ stem cells enhances the immunostimulatory function of their dendritic cell progeny. Exp Dermatol 2005;14:41–9. [15] Rappocciolo G, Jenkins FJ, Hensler HR, Piazza P, Jais M, Borowski L, et al. DC-SIGN is a receptor for human herpesvirus 8 on dendritic cells and macrophages. J Immunol 2006;176:1741–9.
[16] Della Bella S, Nicola S, Brambilla L, Riva A, Ferrucci S, Presicce P, et al. Quantitative and functional defects of dendritic cells in classic Kaposi’s sarcoma. Clin Immunol 2006;119:317–29. [17] Rahim Rezaee SA, Cunningham C, Davison AJ, Blackbourn DJ. Kaposi’s sarcoma-associated herpesvirus immune modulation: an overview. J Gen Virol 2006;87:1781–804. [18] Means RE, Choi JK, Nakamura H, Chung YH, Ishido S, Jung JU. Immune evasion strategies of Kaposi’s sarcoma-associated herpesvirus. Curr Top Microbiol Immunol 2002;269:187–201.
Human herpesvirus 8 (HHV-8) inhibits monocyte differentiation into dendritic cells and impairs their immunostimulatory activity Mara Cirone ∗ , Giuseppe Lucania, Paola Bergamo, Pankaj Trivedi, Luigi Frati, Alberto Faggioni Istituto Pasteur-Fondazione Cenci Bolognetti, Department of Experimental Medicine University of Rome “La Sapienza”, Rome, Italy Received 12 April 2007; received in revised form 12 July 2007; accepted 21 July 2007 Available online 21 August 2007
Abstract Several viruses interfere with the host immune response by infecting dendritic cells and by altering their functional activity. Here, we report that exposure to Human herpesvirus 8 (HHV-8) of human dendritic cell (DC) monocyte precursors resulted in impaired immature DC (iDC) formation as indicated by a reduced CD1a expression. In accordance, the immunostimulatory ability of such iDC was significantly reduced, as indicated by mixed lymphocyte culture (MLR) assays. The immunostimulatory functions of DCs were similarly inhibited by the UV inactivated viral stocks, suggesting that the virus binding is sufficient to determine the observed effect. Furthermore, HHV8 mediated inhibition of the DC allostimulatory function was present in lipopolysaccharide (LPS) matured DCs. A strong reduction of the expression of the costimulatory molecule CD80 on the surface of the virus-exposed cells was observed as well. Impairment of dendritic cell development and function might represent an important strategy used by HHV-8 to escape from the host defense mechanisms. © 2007 Elsevier B.V. All rights reserved. Keywords: Human herpesvirus 8; Dendritic cell; Immunostimulatory activity
1. Introduction Human herpesvirus 8 (HHV-8), also named Kaposi’s sarcoma-associated herpesvirus (KSHV), is a lymphotropic herpesvirus linked to several clinical disorders [1,2]. HHV-8 has been detected in biopsies of all clinical and epidemiological forms of Kaposi’s sarcoma (KS). The virus is present in the spindle-shaped cells that are considered to be the tumor cells of KS, where it establishes a latent infection, and also in the perilesional mononuclear cells where the infection can also be lytic. In addition, HHV-8 has been detected in peripheral blood mononuclear cells of KS patients, including B-cells, T-cells and monocytes. Besides KS, HHV-8 has been also linked to lymphoproliferative disorders such as primary effusion lymphomas (PEL) and multicentric Castelman’s disease (MCD). PEL are latently infected in vivo but can be induced to lytic infection in vitro by treating the cells with phorbol esters or sodium ∗ Corresponding author at: Dipartimento di Medicina Sperimentale, Viale Regina Elena 324, 00161 Roma, Italy. Tel.: +39 06 4463542; fax: +39 06 4454820. E-mail address: [email protected] (M. Cirone).
0165-2478/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2007.07.013
butyrate and this treatment is currently used for viral stock preparation. Dendritic cells (DC) play a pivotal role in the initiation and maintenance of immune responses [3]. They are involved in T-lymphocyte activation and in the polarization of immune responses towards Th1 or Th2 cytokine secretion patterns. Triggering of Th1-type immune responses and development of strong cytotoxic activity usually require efficient DCs mainly derived from myeloid precursors, such as blood monocytes. Consistent with an essential role of DC in anti-virus immune response, several viruses have developed strategies to target these cells and impair their function in the attempt of evading and subverting the immune response. In particular viruses that establish persistent infection such as herpesvirus have been shown to inhibit the T-cell-stimulatoty function of DC by interacting with DC or their precursors. Examples are Cytomegalovirus (CMV) that inhibits DC function by directly infecting these cells and/or by secretion of an homolog of IL-10 that affects their maturation [4,5]. Epstein-Barr virus (EBV) which has been shown to inhibit DC function by promoting apoptosis of their monocyte precursors [6] and Herpes Simplex virus that inhibits DC maturation in the presence of LPS [7].
M. Cirone et al. / Immunology Letters 113 (2007) 40–46
We wanted to assess whether HHV-8 is able to interfere with the development of immature-DCs (iDC) from monocyte precursors in the presence of granulocyte macrophage-colonystimulating factor (GM-CSF) and interleukin 4 (IL-4) and to affect their allostimulatory activity. 2. Materials and methods 2.1. Virus preparation For the virus preparation, the HHV-8 positive but EBVnegative PEL cell line BCBL-1 was treated with 20 ng/ml tetradecanoyl phorbol acetate (TPA) for 3–5 days to induce HHV-8 replication. The virus-containing supernatant was centrifuged at 70,000 × g (2 h at 4 ◦ C). The resulting pellet was resuspended in cold PBS in 1/200 of the original volume of medium and filtered through a 0.45 m-pore filter. For HHV8 purification, the concentrated viral stocks were centrifuged through 30–60% sucrose step gradient at 17,000 rpm for 4 h. The band at the gradient junction was collected. The virions were then purified in a second round of gradient centrifugation [8]. Quantitation of viral yields was performed using polymerase chain reaction (PCR) amplification by comparison to the viral copy number of BCBL-1, which is known to have 70 viral copies/cell [9]. Approximately 1 × 108 –1 × 109 ml viral copies were present in the purified viral stocks. 2.2. Generation of monocyte-derived DC and HHV-8 infection To generate monocyte-derived DC, human peripheral blood mononuclear cells were isolated by Ficoll-Paque gradient centrifugation (Pharmacia, Uppsala, Sweden) from buffy coats of healthy volunteers after they had given informed consent to the procedure. CD14+ monocytes were then positively selected using anti-CD14 MAb-conjugated magnetic microbeads (Miltenyi Biotec, Auburn, CA). Purified monocytes (approximately 95% pure, as determined by flow cytometry using Mab directed against CD14) were cultured at a density of 1.5 × 106 cells/3 ml in 12-well plates for 6 days in the following medium: RPMI 1640 (Euroclone) containing 10% heat inactivated fetal calf serum (Gibco), 2 mM l-glutamine, 100 U/ml penicillin G, 100 g/ml streptomycin, 50 mM 2-mercaptoethanol (Sigma), and recombinant human granulocyte-macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) (20 ng/ml each, Biodesign International). In some experiments, instead of IL-4, recombinant ␣ IFN (5000 U/ml, Alfa Wassermann, Bologna, Italy) was used together with GM-CSF. Cytokines were replenished every other day by adding 20% fresh medium to each well. For DC maturation LPS from Salmonella abortus equi (Sigma) at 1 mg/ml was added 6 days after culture onset and kept for 48 h. HHV-8 infection was performed by incubating 1 × 106 monocytes for 2 h at 37 ◦ C with 100 l of viral stock containing approximately 1 × 108 –1 × 109 viral copies/ml. Cells were then washed in PBS and cultured with the above-mentioned supplements to induce differentiation into iDC as described above.
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For PCR analysis, total DNA was extracted with a DNA extraction kit (Invitrogen, San Diego, CA) from iDC exposed or not exposed to HHV-8 and cultured for 3 and 6 days. One hundred nanogram of DNA was then amplified for HHV-8 DNA sequences by PCR using the following primer set: forward 5 -GCCGAAAGGATTCCACCAT-3 and reverse 5 -TCCGTGTTGTCTACGTCCA-3 from KS330 BamH1 fragment [10]. BC-3 was used as positive control. For detection of HHV-8 antigens by indirect immunofluorescence, iDC exposed for 2 h or not exposed to HHV-8 were subsequently cultured for 6 days. These cells were then air-dried, fixed in cold acetone and methanol (1:1) and incubated with previously characterized human sera from KS patients [11] or with mAbs directed against latent (LANA) or lytic (ORF K8.1) antigens purchased from ABI, USA. The secondary antibodies to rat, mouse or human Ig were purchased from Cappel. 2.3. Mixed lymphocyte reaction MLR was carried out in 96-well flat-bottom microtiter plates by adding three different numbers of irradiated (3000 rad) iDC (previously treated or not treated with HHV-8) to 2 × 105 allogeneic PBMCs. After 6 days at 37 ◦ C, cell proliferation was assessed by uptake of 3 H thymidine (1 Ci/well for 16 h). The cells were harvested onto glass microfiber filters (Packard) and counted using a Canberra Packard scintillation counter. 2.4. Flow cytometry For cell surface staining, after 6 days of culture, iDC were washed in PBS and incubated for 30 min at 4 ◦ C with monoclonal antibodies directed against CD1a, CD86, CD83 (Becton-Dickinson), CD-80 (ImmuneKontakt) and HLADR (Cymbus Biotech Ltd.). The binding of antibodies was revealed by goat FITC-conjugated or PE-conjugated anti-mouse Ig (Cappel). Samples were analyzed using a FACScan cytometer. 3. Results 3.1. Detection of HHV-8 in developing iDCs After concentration and purification of HHV-8 from BCBL1, CD14+ purified monocytes were exposed to the virus. The cell viability was not affected by the virus exposure, as indicated by trypan blue exclusion. To verify if the virus was able to enter into developing DCs, the cells were harvested on days 3 and 6 of culture. After three washes with PBS, DNA was extracted for PCR analysis. Viral DNA was detected in HHV-8-infected iDC in one step PCR both at days 3 and 6 post-infection (Fig. 1a). The expression of HHV-8 latent and lytic antigens in the iDC was assessed in parallel by IFA at day 6. As shown Fig. 1b, HHV-8 treated iDC showed the intracellular speckles typical of LANA while Fig. 1c shows control uninfected DCs. At 6 days postinfection, no positive cells for lytic antigen K8.1 were observed (data not shown), indicating that the virus is not able to replicate in the developing DCs.
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Fig. 3. Mixed lymphocyte reaction of HHV-8 treated and untreated DC before and after induction to maturation with LPS. The DC maturation was obtained by adding LPS at the 6th day of iDC culture, for an additional 48 h. Each bar represents 3 H-thymidine incorporation (cpm ± S.D.) of two representative experiments. Ratio DC:T = 1:10.
3.2. Allostimulatory capacity of iDCs treated with the active and the inactivated HHV-8
Fig. 1. Verification of the viral presence in HHV-8 treated iDCs: (a) by DNA PCR; Lane 1 marker, Lane 2 iDC non-treated (mock), 3: HHV-8 positive BC3, 4: HHV-8 treated iDC after 3 days, 5: HHV-8-treated iDCs after 6 days, 6: HHV8 negative PBLs. The amplified products were visualized on 2% agarose gels. B and C: IFA for LANA; a monoclonal antibody was used to detect LANA expression in HHV-8 treated iDCs (b) and untreated iDCs were used as negative control (c).
We next analyzed in a MLR assay the ability of cells treated or not treated with HHV-8 and cultured for 6 days with IL-4 and GM-CSF to induce T-cell proliferative response. A strong reduction of the allostimulatory activity of HHV-8 treated iDC was found. These experiments were carried out with iDCs derived from four healthy individuals and the mean of four experiments is represented in Fig. 2a. We tested if HHV-8 could inhibit the
Fig. 2. Mixed lymphocyte reaction (MLR): MLRs were performed with immature dendritic cells (iDC) treated with HHV-8 in the presence of IL-4 and GM-CSF (a), in the presence of ␣IFN and GM-CSF (b) or with UV-inactivated HHV-8 (c) in comparison to untreated iDC (mock) on day 6 of culture. The mean of four experiments is shown. P < 0.01 was obtained by comparing the MLR results of HHV-8 treated and untreated iDC. The statistical significance of the studies was calculated by the t-test analysis. (d and e) The efficiency of viral inactivation was evaluated by testing LANA expression in 293 cells infected with the active (d) and inactive (e) virus.
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iDC function in the presence of ␣-IFN and GM-CSF. Indeed, as seen in Fig. 2b, in the presence of this combination similarly resulted in reduced allostimulatory capacity. Since we did not find any viral replication in iDCs, we asked if the virus binding to monocytes was sufficient for the observed reduction of functional activity of the DC progeny. For this purpose, a replication incompetent HHV-8 was prepared by UV irradiation and by heat
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inactivation. For UV irradiation, the virus was exposed to the UV light (230 mW/sq cm for 20 min at a 10 cm distance) and for heat inactivation, the virus stocks were incubated in a waterbath at 56 ◦ C for 1 h. Again, a strong reduction of the allostimulatory activity of iDC was observed in cells exposed to UV-inactivated virus Fig. 2c. These data indicate that the inhibition of allostimulaory capacity is independent of virus replication and that this
Fig. 4. Surface protein expression of the HHV-8 treated and untreated iDC. Cells were cultured for 6 days and the surface expression of the indicated markers was analyzed by immunostaining and FACS analysis.
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effect could be mediated by the viral binding to the cell surface molecules. Here again, the experiments were performed from four healthy donors, and the mean is shown in Fig. 2c. The efficiency of the virus inactivation was measured by verifying LANA expression in 293 cells. Fig. 2d shows LANA expression when the cells are infected with the active virus and 2e instead shows 293 cells infected with the inactivated virus. 3.3. Allostimulatory capacity of HHV-8 treated DCs after maturation with LPS We further asked if the LPS treatment might restore allostimulatory function of HHV-8-treated DC. For DC maturation, LPS from Salmonella abortus equi (Sigma) at 1 mg/ml was added 6 days after culture onset and kept for an additional 48 h. Fig. 3 shows that the mature HHV-8 treated DCs did not recover
their allostimulatory capacity. The difference with the untreated mature DCs was even more significant when both were induced to full maturation with LPS (Fig. 3). Furthermore, we asked if this reduction was correlated to a difference in IL-12 (p70) production in the DC untreated or treated with HHV-8. A six-fold reduction in the DC exposed to the virus was observed. Similar results were obtained when ␣-IFN was used instead of IL-4 (data not shown). 3.4. Phenotypic analysis of immature and mature HHV-8 treated DCs We tested both the DC differentiation markers as well as molecules involved in antigen presentation in virus treated or untreated immature and mature DCs. To this end, CD1a, CD80, CD83, CD86 and HLA-DR expression was investigated by flow
Fig. 5. Surface protein expression of the HHV-8 treated and untreated mDC. After 6 days, the HHV-8 treated and untreated iDC were induced to maturation with LPS and surface expression of the indicated markers was analyzed by immunostaining and FACS analysis.
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cytometry. The experiments were performed in parallel to MLR. The results shown in Fig. 4 indicate that exposure of iDC to HHV-8 reduced CD1a and CD80 expression (both as mean fluorescence intensity and as percentage of positive cells) whereas only marginal difference in the expression of CD86, CD83 and HLA-DR was observed. After maturation with LPS, the expression of CD80 remained low in the cells exposed to HHV-8, and again a minimal difference in the expression of the other molecules was observed (Fig. 5). 4. Discussion The presence of HHV-8-specific CTLs in peripheral blood from KS patients has been reported [12], however these cells are unable to clear the virus-infected cells from circulation or tissues. An inhibition of DC function could account for this inefficiency of the T-cell mediated immune-response. Indeed the present study shows that HHV-8, independently of viral replication interferes with monocyte differentiation into iDC (as indicated by a reduced CD1a expression), impair their allostimulatory activity and is also responsible for the reduction of CD80 on their cell surface. We also report that monocyte-derived iDC previously exposed to HHV-8 did not recover their ability to stimulate alloproliferative T-cell response after maturation with LPS. Since we found no viral replication in these developing DCs, we sought to investigate if HHV-8 interferes with the monocyte differentiation into DC by interaction of viral particles with monocyte surface molecules. Indeed, results obtained with UV light-inactivated virus, which is unable to replicate but maintains the ability to bind to cellular surface molecules, is consistent with this notion. Our results provide a potential mechanistic insight as to how through inhibition of DC development, HHV-8 might contribute to the pathogenesis of classic KS (cKS) which arise in the absence of apparent immunosuppression. Interference with DC function by binding of the viral particles with surface molecules, has been previously reported for other herpesviruses such as EBV [6] and Human Herpesvirus 6 (HHV6) [13]. As for HHV-8, previous studies indicated that the virus is able to enter into CD34+ stem cells derived from cord blood and to enhance the allostimulatory activity of DC derived from these infected precursors [14] and more recently it was shown that dendritic cells can be directly infected with HHV-8 resulting in a reduced ability to process and present Ag to memory CD8+ T lymphocytes [15]. The major difference between the latter study and our findings is that while Rappociolo et al., used DCs, we have used monocyte precursors. Our study thus underlines that also the myeloid DC precursor differentiation can be negatively influenced by HHV-8. The inhibition of monocytic differentiation through interaction with their surface molecules resulting in reduction of their allostimulatory activity adds another aspect of immune modulation mediated by this virus. Studies are in progress in our laboratory to evaluate the presence of HHV-8 DNA in monocyte precursors of healthy HHV-8 seropositive individuals and in patients affected with cKS. It will be interesting to determine if the presence of viral DNA in these precursors could influence the allostimulatory function of their progeny.
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Recently, a quantitative and functional defect of dendritic cells in classic Kaposi’s sarcoma patients, in comparison to HHV-8 seronegative healthy individuals has been reported [16]. It will be important to evaluate if there are differences in DC function between patients with cKS and healthy HHV-8 seropositive individuals, and if there is also a correlation with viral load in both groups. The interference of DC function is a common strategy among viruses which can be accomplished either by the virus particles or by soluble factors induced by them [16]. In cKS, such studies might provide useful information about the strategies through which HHV-8 escapes from the host immune responses [17,18], and contribute to the development of HHV-8 associated diseases. Acknowledgments This work was supported by grants from the MIUR, Ministero della Sanit`a, Progetto AIDS, and from the Associazione Italiana per la Ricerca sul Cancro. We thank Dr. Sabrina Rotolo for statistical analysis and Mr. Sandro Valia for skillfull technical assistance. References [1] Boshoff C, Weiss R. AIDS-related malignancies. Nat Rev Cancer 2002;2:373–82. [2] Schulz TF. The pleiotropic effects of Kaposi’s sarcoma herpesvirus. J Pathol 2006;208:187–98. [3] Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells. Cell 2001;106:263–6. [4] Raftery MJ, Wieland D, Gronewald S, Kraus AA, Giese T, Schonrich G. Shaping phenotype, function, and survival of dendritic cells by cytomegalovirus-encoded IL-101 . J Immunol 2004;173:3383–91. [5] Grigoleit U, Riegler S, Einsele H, Laib Sampaio K, Jahn G, Hebart H, et al. Human cytomegalovirus induces a direct inhibitory effect on antigen presentation by monocyte-derived immature dendritic cells. Br J Haematol 2002;119:189–98. [6] Li L, Liu D, Hutt-Fletcher L, Morgan A, Masucci MG, Levitsky V. Epstein-Barr virus inhibits the development of dendritic cells by promoting apoptosis of their monocyte precursors in the presence of granulocyte macrophage-colony-stimulating factor and interleukin-4. Blood 2002;99:3725–34. [7] Salio M, Cella M, Suter M, Lanzavecchia A. Inhibition of dendritic cell maturation by herpes simplex virus. Euro J Immunol 1999;29: 3245–53. [8] Zhu FX, Yuan Y. The ORF45 protein of Kaposi’s sarcoma-associated herpesvirus is associated with purified virions. J Virol 2003;77: 4221–30. [9] Lallemand F, Desire N, Rozenbaum W, Nicolas JC, Marechal V. Quantitative analysis of human herpesvirus 8 viral load using a real-time PCR assay. J Clin Microbiol 2000;38:1404–8. [10] Chang Y, Cesarman E, Pessin MS, Lee F, Culpepper J, Knowles D, et al. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma. Science 1994;266:1865–9. [11] Santarelli R, De Marco R, Masala MV, Angeloni A, Uccini S, Pacchiarotti R, et al. Direct correlation between human herpesvirus-8 seroprevalence and classic Kaposi’s sarcoma incidence in Northern Sardinia. J Med Virol 2001;65:368–72. [12] Osman M, Kubo T, Gill J, Neipel F, Becker M, Smith G, et al. Identification of human herpesvirus 8-specific cytotoxic T-cell responses. J Virol 1999;73:6136–40. [13] Smith AP, Paolucci C, Di Lullo G, Burastero SE, Santoro F, Lusso P. Viral replication-independent blockade of dendritic cell matura-
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