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Ulm University Department of Pediatrics and Adolescent Medicine Director: Prof. Dr. med. Klaus-Michael Debatin Regulation of human granzyme Bproducing plasmacytoid dendritic cells by viral stimuli Dissertation submitted in fulfillment of the requirements for the degree of Doctor of Medicine (Dr. med.) to the Faculty of Medicine of Ulm University by Verena Michaela Panitz born in Ulm, Germany 2015 Acting Dean: Prof. Dr. rer. nat. Thomas Wirth First Examiner: PD Dr. med. Dorit Fabricius Second Examiner: Prof. Dr. med. Marion Schneider Date of Graduation: November 18th, 2016 Parts of this dissertation have already been published in the following journal articles: The Journal of Immunology: Fabricius, D, Nussbaum, B, Busch, D, Panitz, V, Mandel, B, Vollmer, A, Westhoff, MA, Kaltenmeier, C, Lunov, O, Tron, K, Nienhaus, GU, Jahrsdorfer, B, and Debatin, KM: Antiviral vaccines license T cell responses by suppressing granzyme B levels in human plasmacytoid dendritic cells. J Immunol 191: 1144-1153 (2013) doi:10.4049/jimmunol.1203479 Journal of Vaccines & Vaccination: Jahrsdörfer, B, Panitz, V, and Fabricius, D: Human Immunodeficiency Virus Arrests Plasmacytoid Dendritic Cells in a Granzyme Bhigh Tolerogenic State. J Vaccines Vaccin 7: 337 (2016) doi:10.4172/2157-7560.1000337 For my family Table of contents List of abbreviations ................................................................................................... VII 1 Introduction ............................................................................................................. 1 1.1 Plasmacytoid dendritic cells- mediators between innate and adaptive immunity ............................................................................................................................. 1 1.2 Granzyme B- new roles and functions ........................................................................... 4 1.3 Plasmacytoid dendritic cell-derived granzyme B ......................................................... 5 1.4 Clinical importance of viruses used in this study ......................................................... 6 1.5 Hypothesis and aims ........................................................................................................ 9 2 Material and Methods............................................................................................ 10 2.1 Samples ............................................................................................................................ 10 2.2 Buffers ............................................................................................................................... 10 2.3 Viral stimuli ....................................................................................................................... 11 2.4 Isolation of specific cell subsets.................................................................................... 13 2.5 Cell culture ....................................................................................................................... 18 2.6 Enzyme-linked immunosorbent assay ......................................................................... 21 2.7 Flow cytometry................................................................................................................. 23 2.8 Data analysis and statistics ........................................................................................... 26 3 Results ................................................................................................................... 27 3.1 Herpesviruses modulate granzyme B production and secretion by plasmacytoid dendritic cells in a concentration-dependent manner ....................... 27 3.2 Human immunodeficiency virus 1 increases granzyme B secretion by plasmacytoid dendritic cells ........................................................................................... 31 3.3 Regulation of granzyme B production and secretion by plasmacytoid dendritic cells is virus-specific ....................................................................................... 33 3.4 Interferon-α secretion by plasmacytoid dendritic cells is differentially regulated by viruses ........................................................................................................ 34 V 3.5 Herpesviruses modulate the expression of distinct surface molecules on plasmacytoid dendritic cells ........................................................................................... 37 3.6 Herpesviruses and human immunodeficiency virus 1 in the concentrations or multiplicities of infection used do not relevantly alter viability of plasmacytoid dendritic cells ........................................................................................... 41 3.7 The viral components of the different herpesvirus sources are responsible for the effects on plasmacytoid dendritic cells ............................................................ 45 3.8 Herpesvirus- or human immunodeficiency virus 1-stimulated plasmacytoid dendritic cells modulate CD4+ T cell proliferation in mixed lymphocyte reactions ........................................................................................................................... 49 4 Discussion ............................................................................................................. 54 4.1 Virus-specific regulation of granzyme B in plasmacytoid dendritic cells ................ 54 4.2 Interaction of viruses with plasmacytoid dendritic cells ............................................ 56 4.3 Modulation of plasmacytoid dendritic cell interferon-α production and phenotype by viruses ...................................................................................................... 60 4.4 The role of plasmacytoid dendritic cells in anti-viral immunity and viral pathogenicity .................................................................................................................... 65 4.5 Granzyme B and tolerogenic plasmacytoid dendritic cells in anti-viral immunity ........................................................................................................................... 74 4.6 Outlook.............................................................................................................................. 77 5 Summary ................................................................................................................ 78 6 References ............................................................................................................. 80 VI List of abbreviations 7-AAD 7-amino-actinomycin D Ab Antibody ACK Ammonium chloride potassium AIDS Acquired immunodeficiency syndrom AIM-V Adoptive Immunotherapy Media APC Allophycocyanin B7-H1 B7 homolog 1 BDCA Blood dendritic cell antigen BID BH3 interacting domain death agonist BSA Bovine serum albumin CCL Chemokine (C-C motif) ligand CCR C-C chemokine receptor CD Cluster of differentiation CD40L Cluster of differentiation 40 ligand CD45RA Cluster of differentiation 45RA CFSE Carboxyfluorescein diacetate succinimidyl ester CMV Cytomegalovirus cmvIL-10 Cytomegalovirus IL-10 CO2 Carbon dioxide CpG Cytosine-phosphate-guanine CpG A Class A cytosine-phosphate-guanine oligodeoxynucleotide CpG B Class B cytosine-phosphate-guanine oligodeoxynucleotide CpG C Class C cytosine-phosphate-guanine oligodeoxynucleotide CTL Cytotoxic T lymphocyte CXCL Chemokine (C-X-C motif) ligand CXCR C-X-C chemokine receptor d Day DC Dendritic cell DNA Deoxyribonucleic acid VII dsDNA Double-stranded deoxyribonucleic acid EBV Epstein-Barr virus EDTA Ethylenediaminetetraacetic acid ELISA Enzyme-linked immunosorbent assay ER Endoplasmic reticulum FACS Fluorescence activated cell sorting FBS Fetal bovine serum FcR Fc receptor FCS Fetal calf serum FGFR1 Fibroblast growth factor receptor 1 FITC Fluorescein isothiocyanate Flt3-L Fms-like tyrosine kinase 3 ligand g Gravitational acceleration GluR Glutamate receptor GrB Granzyme B h Hour H2SO4 Sulphuric acid HAART Highly active anti-retroviral therapy HCl Hydrochloric acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HFF Human foreskin fibroblast hIL-10 Human IL-10 HIV Human immunodeficiency virus HIV-1 Human immunodeficiency virus 1 HLA Human leukocyte antigen HRP Horseradish peroxidase ICAM-1 Intercellular adhesion molecule 1 ICOS-L Inducible T cell co-stimulatory-ligand IDO Indoleamine 2,3-dioxygenase IE Immediate-early IFN Interferon Ig Immunoglobulin IL Interleukin IL3Rα Interleukin-3 receptor α-chain (CD123) VIII IRF7 Interferon regulatory factor 7 ISG Interferon-stimulated gene KHCO3 Potassium bicarbonate LFA-1 Leukocyte function-associated antigen 1 Lin 1 Lineage 1, cocktail containing antibodies against CD3, CD14, CD16, CD19, CD20, CD56 mAb Monoclonal antibody MACS Magnetic activated cell sorting MAPK Mitogen-activated protein kinase min Minute MLR Mixed lymphocyte reaction MOI Multiplicity of infection mRNA Messenger ribonucleic acid MyD88 Myeloid differentiation primary-response gene 88 NaOH Sodium hydroxide NF-B Nuclear factor-kappa B NH4Cl Ammonium chloride NK cell Natural killer cell ODN Oligodeoxynucleotide ODN 2006 Class B cytosine-phosphate-guanine oligodeoxynucleotide, sequence: 5’-tcg tcg ttt tgt cgt ttt gtc gtt-3’ on phosphorothioate backbone ODN 2336 Class A cytosine-phosphate-guanine, oligodeoxynucleotide, sequence: 5’-g*g*g gac gac gtc gtg g*g*g* g*g*g-3’ (*phosphorothioate bonds, rest are phosphodiester bonds) PAMP Pathogen-associated molecular pattern PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline (without calcium and magnesium) pDC Plasmacytoid dendritic cell PE Phycoerythrin PE-Cy5 Phycoerythrin tandem with cyanin dye 5 IX PerCP-Cy5.5 Peridinin chlorophyll protein tandem with cyanin dye 5.5 PFA Paraformaldehyde PHA Phytohemagglutinin, lectin used for mitogenic stimulation of lymphocytes PI Propidium iodide PKR Protein kinase R PRR Pattern recognition receptor RIG-I Retinoic acid-inducible gene 1 RLH RIG-I-like helicase RPMI Roswell Park Memorial Institute RT Room temperature SIV Simian immunodeficiency virus ssRNA Single-stranded ribonucleic acid SV40 Simian virus 40 TBEV Tick-borne encephalitis virus TCR T cell receptor TLR Toll-like receptor TMB Tetramethylbenzidine TNF-α Tumor necrosis factor-α Tr1 Regulatory T1 cell Treg Regulatory T cell UV Ultraviolet light UV-CMV Ultraviolet light-inactivated cytomegalovirus in the section discussion VEGF Vascular endothelial growth factor vIL-10 Viral IL-10 Vpr Viral protein R VSV Vesicular stomatitis Indiana virus VZV Varicella-zoster virus w/o Without X 1 Introduction 1.1 Plasmacytoid dendritic cells- mediators between innate and adaptive immunity Dendritic cells (DCs) were first discovered in 1973 by Ralph M. Steinman and Zanvil A. Cohn [162]. DCs are known as major players in innate and adaptive immunity that have a decisive influence on the reactions of the immune system. The diverse subsets exert different functions as professional antigen-presenting cells that produce either pro-inflammatory or immunoregulatory cytokines and interferons (IFNs) [158] and decide that way over immunity or tolerance. DCs can be roughly subdivided in classical/ myeloid DCs and plasmacytoid DCs [178]. Plasmacytoid dendritic cells (pDCs), formerly known as professional type 1 interferon-producing cells [159], are a very rare blood cell subset representing only 0.2-0.8% of peripheral blood mononuclear cells (PBMCs) [109]. They were first isolated in human tonsils in 1997 [64]. The corresponding mouse cell was identified in 2001 [6, 14, 130]. Their name originates from their plasma cell-like morphology in steady-state and their potential to develop into dendritic cells upon maturation [64]. Human pDCs are characterized by the expression of the following surface molecules: Cluster of differentiation (CD)4+, CD45RA+, human leukocyte antigen (HLA)-DR+, CD123 (Interleukin-3 receptor α-chain (IL3Rα)high, CD11c-, lineage (lin) 1-. Lin 1- means that pDCs do not express several surface markers that characterize other immune cells: PDCs are negative for the T cell antigen CD3, for CD14 (on monocytes), CD16, CD56 (on natural killer (NK) cells), CD19 and CD20 (on B cells). Human blood pDCs can be further distinguished by their exclusive expression of blood dendritic cell antigen (BDCA)-2 and -4 (neuropilin-1) [44]. PDCs are constantly produced from bone marrow [15], depending on the growth factor fms-like tyrosine kinase 3 ligand (Flt3-L) [178]. Whether pDCs originate from the lymphoid or myeloid pathway is to date not clear since pDCs can be derived from both common lymphoid and myeloid progenitors [36, 157]. After production, pDCs circulate in the blood and migrate via high endothelial venules into T cell rich areas of secondary lymphoid organs such as lymph nodes [26], mucosaassociated lymphatic tissues and the spleen [109]. The life span of the murine counterpart of human pDCs is about two weeks [137]. As human pDCs die rapidly 1 in cell culture, interleukin (IL)-3 is often supplemented, representing a crucial survival factor for pDCs and therefore prolonging pDC survival ex vivo [64]. Physiologically, IL-3 is produced by sources such as activated T cells [65] or mast cells [143]. The key features of pDCs are the expression of the intracellular pathogen recognition receptors (PRRs) Toll-like receptor (TLR)7 and TLR9 [89, 92]. The pathogen-associated molecular patterns (PAMPs) that are recognized by these receptors are single-stranded ribonucleic acid (ssRNA) from viruses [39, 72, 115] and synthetic small molecules such as imidazoquinoline compounds [74] or loxoribine [71] for TLR7 and double-stranded deoxyribonucleic acid (dsDNA) containing unmethylated 2’deoxyribo-cytosine-phosphate-guanine (CpG) DNA motifs [9, 67, 73] for TLR9. The latter are common in bacterial and viral DNA and are included in synthetically produced immunostimulatory CpG oligodeoxynucleotides (ODN) that also act via TLR9 [96]. The signaling cascade downstream of TLR7 and TLR9 through the adaptor molecule myeloid differentiation primary-response gene 88 (MyD88) [2] leads to the rapid secretion of high amounts of type I interferons (IFNs), namely IFN-α, by pDCs. PDCs are the main producers of type I interferons in the immune system as they secrete up to 1000 times more type I IFN than any other cell upon appropriate stimulation [159]. This is possible due to the constitutively high expression of interferon-regulatory factor (IRF)7 [86] and a potent endoplasmic reticulum (ER) machinery in pDCs. Moreover up to 60% of the transcriptional machinery of pDCs is involved in producing type I IFNs [84]. IFNs induce the transcription of interferon-stimulated genes (ISGs) in diverse cell types. The expression of these anti-viral molecules makes the cells more resistant to viral infection as the cells become more sensitive for detection and elimination of entering viruses or the antiviral molecules directly interact with viral replication or promote apoptosis of virally infected cells [3]. Type I IFNs derived from pDCs can also boost innate and adaptive immune responses as they induce the maturation of classical DCs [152] and activate natural killer (NK) cells and cytotoxic T lymphocytes (CTLs) [3]. TLR7 and TLR9 can furthermore signal through nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) inducing the expression of costimulatory molecules such as CD80 and CD86 on the cell surface of pDCs and 2 the secretion of IL-6, Tumor-necrosis factor (TNF)-α and chemokines that recruit immune cells to the sites of infection [66, 164]. For example, pDCs can attract NK cells and activated T cells via secretion of chemokine (C-C motif) ligand (CCL)4 and chemokine (C-X-C motif) ligand (CXCL)10 [121]. In addition pDCs are able to promote the differentiation of B cells to antibody-producing plasma cells [90]. Besides, maturated pDCs are able to present antigens on HLA-DR and HLA-ABC molecules and can thereby initiate an adaptive immune response via antigen presentation to CD4+ T cells [175] or via rapid cross-presentation of antigens to CD8+ T cells in a proteasome-independent pathway [38]. Altogether, these properties enable pDCs to activate and link both innate and adaptive immunity especially in the course of viral infections. PDCs are not only important in anti-viral immune responses, they also play a role in several other diseases such as auto-immune diseases where they are found in the peripheral organs. PDCs have been found to infiltrate the synovia of patients with rheumatoid arthritis [24], skin lesions of patients with psoriasis [134] and of systemic lupus erythematosus patients [16, 48], where pDCs play a pivotal role in the pathogenesis as the major producers of type I IFNs [149], but also in the central nervous system in a mouse model for multiple sclerosis where pDCs rather have a regulatory function [7]. PDCs also infiltrate several tumors such as melanoma [173], breast cancer where infiltration goes along with bad prognosis [169] and ovarian cancer [182] where pDCs possibly play a role in immune evasion and tolerance induction to tumor cells [176]. Lastly, pDCs play an important role in the maintenance of both peripheral as well as central tolerance [68, 120]. For example, pDCs stimulated with different classes of CpG ODNs induced regulatory T cells (Tregs) [128] via expression of indoleamine 2,3-dioxygenase (IDO) [30]. In this study we assess the role of pDCs in the anti-viral immune response. The real clinical relevance of human pDCs for viral infections has not been extensively studied in the human system as pDCs display a rare immune cell subset. Study results from mouse models cannot be transferred uncritically into the human system as the murine and human immune systems are distinct from one another. Our work group selectively examines human pDCs. Although human pDCs share many similarities with their murine counterpart, there exist relevant differences: 3 Murine pDCs express CD11c on their surface, in the human system a marker for classical DCs. Moreover murine pDCs are able to secrete IL-12 [6]. Besides, all DC subsets in the murine immune system can respond to CpG DNA whereas only B cells and pDCs respond to CpG molecules in the human system [76]. In conclusion, human pDCs represent a rare cell subset with diverse functions in the immune system and display immunogenic as well as tolerogenic features. 1.2 Granzyme B- new roles and functions The serine protease Granzyme B (GrB) is a granule enzyme that cleaves after aspartate residues [156]. GrB has long been known as the major constituent of the granules of NK cells and cytotoxic CD8+ T cells [31, 168]. The classical function of GrB is the induction of cell death in target cells, like virally infected or tumor cells, in a perforin-dependent manner via cleavage of caspase-3 [37, 123] or BH3 interacting domain death agonist (BID) [8, 70]. Besides, many different nonclassical sources of GrB have been discovered such as cytolytic CD4+ T cells [31, 150], B cells [69], hematopoietic progenitor cells [12], mast cells [163], basophils [170], macrophages [94] and pDCs [87, 147]. Apart from the classical cell-death inducing function, it has been shown that GrB can exert other immunogenic functions: Not only death effector molecules are substrates of human GrB, GrB can also cleave and inactivate a wide range of intracellular molecules that are important in cellular homeostasis which allows GrB to interfere with viral replication (reviewed in [148]). Furthermore GrB can also directly cleave essential viral proteins [148]. GrB also seems to exert diverse functions in an extracellular milieu. GrB is able to cleave receptors important for cell survival such as Notch1 and Fibroblast growth factor receptor 1 (FGFR1) [111] or receptors important for cell signaling and interaction like glutamate receptor (GluR)3 on T cell receptor (TCR)-activated T cells or the T cell receptor ζ-chain [177]. GrB might also modulate cell survival via modulation of the extracellular matrix, thus inducing anoikis, programmed cell death as a result of the cleavage of anchor-proteins of cells to the extracellular matrix, for example via cleaving of laminin, fibronectin, vitronectin [22]. GrB can also cleave the cartilage proteoglycan aggrecan [57]. In addition GrB was shown to increase vascular 4 permeability via release of vascular endothelial growth factor (VEGF) present in the extracellular matrix [75]. It has been shown that GrB is regularly present in the plasma of healthy individuals; in one study the median level of GrB was determined as 11.5 pg/ml [161], whereas relevantly elevated extracellular levels of GrB have been detected in several inflammatory states. In rheumatoid arthritis GrB is markedly elevated in the synovial fluid of patients and to a lower extent in the plasma [161, 165]. In the bronchoalveolar lavage of patients suffering from atopic asthma or chronic obstructive pulmonary disease elevated GrB concentrations were measured [19, 77]. Not only in autoimmune diseases but also in responses to pathogens, especially to viruses, extracellular GrB seems to play an important role. In blood samples from patients infected with human immunodeficiency virus 1 (HIV-1) in an asymptomatic phase and in serum of patients with acute infectious mononucleosis GrB was elevated [161]. Also in dengue fever [23] and cytomegalovirus infection after renal allograft transplantation [166], GrB was found to be increased. In contrast to these rather immunogenic functions of GrB, GrB can exert tolerogenic functions: Human adaptive regulatory T1 (Tr1) cells expressed GrB and killed target cells in a perforin-dependent manner indicating a role of GrB in immune system homeostasis [63]. In mice GrB could mediate a cell contactdependent tolerogenic mechanism of regulatory T cells (Tregs) in a perforinindependent way [61]. Previous work from our lab found that human pDC-derived GrB could inhibit T cell proliferation [87]. 1.3 Plasmacytoid dendritic cell-derived granzyme B GrB expression in pDCs seems to be post-transcriptionally regulated; upon activation pDCs produce fresh GrB [87, 147]. GrB secretion by pDCs requires endosomal acidification and an active transport from the ER to the Golgi apparatus. In addition GrB seems to be peri-nuclearly localized in granules of pDCs and can be transferred to T cells in a perforin-independent but seemingly cell contact-dependent manner. Astonishingly, pDCs secrete markedly higher amounts of GrB than NK cells or CTLs. In mixed lymphocyte reactions (MLRs) our lab has found that pDC-derived GrB could mediate suppression of both CD4+ and 5 CD8+ T cell proliferation in a perforin-independent manner [87]. The antiproliferative effect on CD4+ T cells could be mediated by cleavage of the T cell receptor ζ-chain by pDC-derived GrB [47]. GrB production in pDCs is enhanced when pDCs are incubated with IL-3. Yet, the combination of IL-3 and IL-10 was the most potent stimulus for GrB-induction in pDCs. But even freshly isolated pDCs produce a small amount of GrB. When pDCs are stimulated with a class B CpG ODN (CpG B), a stimulus that imitates viral infection as it is an agonist of TLR9, GrB production is diminished and T cell proliferation in MLRs is enhanced again [87]. Previous work tested the effect of anti-viral vaccines containing ssRNA viruses on pDCs [47]. These anti-viral ssRNA vaccines diminished GrB production by pDCs. Additionally, pDCs isolated from patients who were recently vaccinated against tick-borne encephalitis virus (TBEV), produced less GrB than pDCs from unvaccinated control patients. PDCs treated with a TBEV vaccine did no longer transfer GrB to CD4+ T cells and T cell proliferation was increased. 1.4 Clinical importance of viruses used in this study Which role pDCs actually play in the anti-viral immune response is not clear. Disease models in mice try to reveal the real importance of pDCs for anti-viral immune responses in vivo. The authors of one review hypothesize that pDCs might be essential in systemic viral infections and for infections with lymphotropic viruses. Furthermore they suppose that pDCs might be relevant for the clearance of infections that depend on strong CTL responses [164]. The human herpesviruses varicella-zoster virus, cytomegalovirus and Epstein-Barr virus, but also the retrovirus HIV-1 are all lymphotropic viruses and give rise to systemic infections. For the immune response against these viruses, CTLs are particularly important. Thus, in analogy to the supposed role of pDCs in murine anti-viral immunity, the effects of these viruses on human blood pDCs are interesting to evaluate as human pDCs are ascribed an important role in the immune response against these viruses. The human herpesviruses varicella-zoster virus, cytomegalovirus, and EpsteinBarr virus are dsDNA viruses that share relevant common features. A high percentage of the human population is symptomless carrier of these viruses. After 6 primary infection herpesviruses can establish lifelong latency in the human host. Reactivation can occur under certain circumstances such as inflammation and above all immune suppression, for example caused by immunosuppressive treatment after transplantation or HIV-1 infection. Of note, herpesviruses have developed many mechanisms to co-exist with the host. They express a variety of proteins to interact for example with antigen presentation or cell death [58, 83, 112, 113]. Importantly, herpesviruses, efficiently kept under control by a healthy immune system, can cause relevant morbidity in the immunocompromised host. The α-herpesvirus varicella-zoster virus (VZV) is a dsDNA virus. VZV is highly contagious and is transmitted via droplet infection and enters respiratory epithelia of the mouth from where virus is distributed in two phases of viremia to the reticulo-endothelial system and thereafter to the skin. After an incubation period of 10 up to 21 days primary infection with VZV causes varicella. Varicella or otherwise named chickenpox presents itself as a vesiculo-pustular ubiquitous cutaneous rash accompanied by symptoms such as fever or lethargy. VZV establishes latency in neurons of spinal ganglia and can therefore be reactivated particularly in the elderly or in immunocompromised people. The reactivation named herpes zoster or shingles usually presents itself as a localized painful cutaneous efflorescence [32]. The commercially available VZV vaccines, one is Varilrix®, introduced in 1984 [167], contain a live attenuated VZV strain derived from a clinical isolate that is able to induce potent and protective immune responses in vaccinated people [32]. Human cytomegalovirus (CMV) is a dsDNA virus that belongs to the subfamily of β-herpesvirinae. In immunocompetent people primary CMV infection transmitted by bodily secretions does seldom present clinical symptoms. Only in rare cases it causes a febrile mononucleosis-like disease. A high percentage of the population that increases with age is CMV positive without ever presenting symptoms [127]. Nevertheless, CMV infection is inducing a transient immunosuppression of a few weeks to months [131]. After primary infection CMV establishes latency in leukocytes. CMV infection usually causes problems in immunocompromised individuals and in the course of vertical infections of fetuses inducing congenital defects such as hearing loss, chorioretinitis and mental retardation among others. Infection with CMV or its reactivation can cause major problems after transplantations or during other situations of a compromised immune system like 7 in acquired immunodeficiency syndrome (AIDS) where CMV may arise as an opportunistic infection for example as retinitis or pneumonitis [127]. Epstein-Barr virus (EBV), a dsDNA γ-herpesvirus, is transmitted orally and causes mononucleosis, the kissing disease, mainly in young people which goes along with generalized lymphadenopathy, fatigue and hepatosplenomegalie. A high percentage of the population is asymptomatic carrier of EBV as EBV establishes latency in B cells. EBV causes highly relevant problems in immunocompromised patients such as transplant receivers or patients suffering from AIDS as posttransplant lymphoproliferative disease and B cell lymphomas can occur. EBV is oncogenic and consequently a potent promotor of diverse cancers such as Burkitt’s lymphoma or nasopharyngeal carcinoma [146]. The (+) ssRNA retrovirus HIV-1 was discovered in 1984 and causes AIDS. The HIV epidemic represents a major global health problem. HIV-1 transmission is possible in a parenteral, sexual or vertical way. Primary infection with HIV-1 usually presents like an acute viral infection but is often associated with lymphadenopathy and weight loss. An asymptomatic phase follows with ongoing CD4+ T cell loss ultimately leading to the establishment of AIDS after a varying time span with the onset of opportunistic infections or the manifestation of AIDSdefining diseases. PDCs are known to play an important role during HIV-1 infection and it still remains to be elucidated whether their role is rather beneficial or more detrimental for the spread and course of the disease [98]. 8 1.5 Hypothesis and aims As pDCs are important mediators between innate and adaptive immunity especially in the course of viral infections, they are decisive for the induction of a potent immune response against viruses and in the establishment of immunity upon immunization with anti-viral vaccines. Recently it was shown that anti-viral vaccines against ssRNA viruses modulate GrB expression by pDCs [47]. Therefore in this thesis, the effects of an anti-viral vaccine against a dsDNA virus, the varicella-zoster virus (VZV) vaccine Varilix® consisting of a live attenuated virus strain, were assessed. Then, to go beyond the model of anti-viral vaccines, we analyzed two other dsDNA herpesviruses: Ultraviolet light (UV)-inactivated human cytomegalovirus (CMV) and Epstein-Barr virus (EBV) against which no vaccines have been developed so far. Due to the high pathogenicity of human immunodeficiency virus 1 (HIV-1) and the interesting role pDCs seem to play in the course of the disease, we also tested the effect of the ssRNA retrovirus HIV-1 on pDCs. We hypothesized that GrB in pDCs is differentially and specifically regulated by these viruses. The inhibition of GrB may contribute to an efficient anti-viral immune response whereas increased GrB levels might dampen the initiation of a potent T cell effector response. Thus GrB might be an additional variable affecting the capacity of pDCs to either hamper or trigger adaptive immune responses in the course of viral infections. We presumed that the vaccine-derived virus and the other three viruses must also have a distinct effect on the immunophenotype of pDCs. In order to test this hypothesis, we asked the following questions: • Is GrB production and secretion by pDCs differentially regulated by the viral agents tested? • Is the secretion of IFN-α, the major cytokine produced by pDCs, induced in pDCs upon stimulation with the viruses tested? • Do the viruses tested influence the maturation status and the immunophenotype of pDCs assessed via surface molecule expression? • Do virus-stimulated GrB-producing pDCs have distinct effects on the proliferation of CD4+ T cells in mixed lymphocyte reactions (MLRs)? 9 2 Material and Methods 2.1 Samples The present study was approved by the Ethics Committee at Ulm University. In order to isolate specific cell subsets buffy coats were obtained from the German Red Cross in Ulm or 50 up to 250 ml of fresh peripheral blood was taken from healthy volunteers after obtaining informed consent from each individual. 2.2 Buffers 2.2.1 Ammonium chloride potassium lysing buffer For 1 l ammonium chloride potassium (ACK) lysing buffer 0.15 M ammonium chloride (NH4Cl) (Merck KGaA, Darmstadt, Germany), 10.0 mM potassium bicarbonate (KHCO3) (Sigma-Aldrich, St. Louis, MO, USA), 0.1 mM ethylenediaminetetraacetic acid (EDTA) (Carl Roth, Karlsruhe, Germany) and 800 ml Ampuwa® Spüllösung Plastipur® Aqua ad iniectabilia (Fresenius Kabi France, Sèvres, France) were mixed and adjusted to a pH between 7.2 and 7.4 with sodium hydroxide (NaOH) (Sigma-Aldrich). Then Ampuwa® Spüllösung Plastipur® Aqua ad iniectabilia was added to a final volume of 1 l and the buffer was filtered sterile with a Vacuum Filtration System (PES, 0.2 µm, 1 l) (VWR International, Radnor, PA, USA) and stored at room temperature (RT). 2.2.2 Magnetic activated cell sorting buffer To prepare magnetic activated cell sorting (MACS) buffer 0.5% Albumin bovine Fraction V, pH 7.0 (bovine serum albumin (BSA)) (SERVA Electrophoresis GmbH, Heidelberg, Germany) and 2 mM EDTA were added to 1 l of phosphate buffered saline without calcium and magnesium (PBS) (Biochrom AG, Berlin, Germany). After mixing ingredients, pH was adjusted to a value between 7.2 and 7.4 with NaOH. Then buffer was filtered sterile, degassed and stored at 4 °C. 10 2.2.3 Fixation buffer The paraformaldehyde (PFA) (Merck KGaA) stock was diluted in PBS to a final concentration of 4%, filtered sterile and stored at 4 °C. For HIV-1 and EBV experiments a 1:1 dilution of the fixation buffer with PBS was used to be able to use the double amount of volume per tube. 2.2.4 Permeabilization buffer For 100 ml permeabilization buffer 100 ml of PBS was mixed with 0.25% Saponin (Sigma-Aldrich), filtered sterile and stored at 4 °C. 2.2.5 Roswell Park Memorial Institute medium Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, Carlsbad, CA, USA) was supplemented with 10% foetal bovine serum (FBS) (Life Technologies), 2 mM L-glutamine (Life Technologies), 1 mM sodium pyruvate (Biochrom AG) and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (Biochrom AG), which will be referred to as completed RPMI. 2.3 Viral stimuli 2.3.1 Varicella-zoster virus As a source of varicella-zoster virus (VZV) we used the VZV vaccine Varilrix® (GlaxoSmithKline Biologicals s.a., Rixensart, Belgium) that contains the live attenuated varicella-zoster virus strain OKA, a clinical isolate that was propagated in cultures of human diploid cells, the cell line MRC-5 that consists of human fetal lung fibroblasts. Further ingredients of the lyophilized vaccine are amino acids, human albumin, lactose, mannitol, sorbitol, phenol red, traces of neomycin sulfate, traces of remaining stock from the cell culture and the culture media such as salts, vitamins, sugar and BSA. The lyophilized vaccine was reconstituted in 0.5 ml water for injection that was included in the commercially available vaccine. 0.5 ml of the reconstituted vaccine 11 contains at least 103.3 plaque forming units. The whole reconstituted vaccine was used in volume dilutions as follows: 1:2000, 1:200, 1:20 and 1:10. To test whether the viral component in the varicella-zoster virus vaccine alone and no other ingredients of the vaccine were responsible for the effects on pDCs, the reconstituted vaccine was filtrated through a virus filter that allowed the removal of viral particles. A PALL® Acrodisc® 32 mm Syringe Filter with a 0.1 µm Supor® Membrane (Pall Corporation, Port Washington, NY, USA) that allowed only particles smaller than 0.1 µm to pass through was used. The varicella-zoster virus virion itself has a diameter of 180-200 nm [32]. Consequently, the filtrated varicella-zoster virus vaccine should contain no more virions. We also tested the effect of ultraviolet light (UV)-irradiated VZV vaccine on pDCs. The reconstituted vaccine was inactivated via UV-irradiation in a UV CrossLinker (CL-1000; UVP, Upland, CA). 2.3.2 Cytomegalovirus Human cytomegalovirus (CMV) was kindly provided by Dr. Giada Frascaroli, Institute of Virology, head Prof. Dr. med. Thomas Mertens, University Medical Center Ulm. The endotheliotropic CMV strain TB40E was produced using human foreskin fibroblasts (HFFs). Virus was inactivated via UV-irradiation with a UV CrossLinker (CL-1000; UVP, Upland, CA) at a wavelength of 366 nm for 2 times 2 min what corresponded to an energy of 200 kJ. Virus aliquots were stored at -80 °C. We used only UV-inactivated CMV in our experiments. For easier labeling we use the abbreviation CMV in the results section. In the discussion UV-inactivated CMV is refered to as UV-CMV to distinguish the inactivated virus from active CMV. As a control we were provided with ultra-centrifuged supernatant from mockinfected HFFs. 2.3.3 Epstein–Barr virus Epstein-Barr virus (EBV) was propagated on B95-8 cells, an EBV-producing B cell line derived from a tamarin (Saguinus oedipus) [125, 126], in RPMI medium (PAA The Cell Culture Company, Pasching, Austria) supplemented with 10% FCS (PAA The Cell Culture Company) and 1% penicillin/streptomycin (PAA The Cell Culture 12 Company). After 21 days, cells were spun down and supernatant containing EBV in RPMI medium was stored at either -30 °C or -80 °C. To test whether the viral component in the EBV stock and no other ingredients in the supernatant of the B95-8 cells were responsible for the effects on pDCs, the EBV stock was filtrated through a virus filter that allowed the removal of viral particles: A PALL® Acrodisc® 32 mm Syringe Filter with a 0.1 µm Supor® Membrane (Pall Corporation, Port Washington, NY, USA) that allowed only particles smaller than 0.1 µm to pass through. The EBV virion itself has a diameter of approximately 115 nm [133]. Consequently, the filtrated EBV stock should contain no more virions. 2.3.4 Human immunodeficiency virus 1 The human immunodeficiency virus (HIV) strain HIV-1_M_NL_43_wt was kindly provided by Dr. Ali Gawanbacht, Institute of Molecular Virology, head Prof. Dr. Frank Kirchhoff, Ulm University. Virus stock was generated by transient transfection of HEK293T cells (ATCC, Manassas, VA, USA), human embryonic kidney cells that are transformed with Adenovirus Type 5 and express simian virus 40 (SV40) large T-antigen [62], with the calcium chloride precipitation method. Mock transfection of HEK293T cells was performed as a control. The only difference to the protocol for virus generation was that no DNA was added during the transfection of HEK293T cells. All other steps in mock generation were performed as for virus generation. 2.4 Isolation of specific cell subsets 2.4.1 Isolation of peripheral blood mononuclear cells In order to isolate peripheral blood mononuclear cells (PBMCs) from buffy coats or fresh blood, blood was diluted 1:2 with PBS (Biochrom AG). Up to 35 ml of diluted blood were layered on 15 ml of Biocoll separating solution (Biochrom AG) in a 50 ml polypropylene conical tube (Becton Dickinson, Franklin Lakes, NJ, USA) for density gradient centrifugation. 13 Tubes were filled up to 50 ml with PBS and were spun at 1000 g for 15 min at RT with brakes off for density gradient formation. Most of the erythrocytes and granulocytes are to be found at the bottom of the tube because of their higher density. Beneath the blood plasma on top, mononuclear blood cells or so called peripheral blood mononuclear cells (PBMCs) form a thin white ring that was harvested in 2 fresh 50 ml tubes. Then the tubes were filled up to 50 ml with PBS. After centrifuging the tubes at 300 g for 10 min at RT, supernatant was discarded; cell pellets were re-suspended and unified in one tube per donor. Cells were washed again with PBS in 50 ml volume (200 g, 15 min, RT). After centrifugation supernatant was discarded, the cell pellet re-suspended and 7 ml of ACK lysing buffer were added per tube and mixed with the cells to lyse erythrocytes. After 7 min of incubation at RT, tubes were filled up with PBS to 50 ml and cells were washed (300 g, 10 min, RT). Supernatant was discarded and the cell pellet re-suspended. After filling up the tube to exactly 50 ml about 150 µl of cell suspension per donor were taken to count the cells and to analyze the percentage of pDCs per donor via flow cytometry, if pDC isolation was the final aim. For cell count 10 µl of a mix of 5 µl cell suspension and 95 µl trypan blue (SigmaAldrich) were administered into a Neubauer counting chamber (Laboroptik, Friedrichsdorf, Germany). Cells were counted at a 10-fold magnification with a CK30 culture microscope (Olympus Corporation, Tokyo, Japan). Cell numbers were determined with the formula: Sum of 4 cell counts/4 x 20 (dilution factor with trypan blue) x 104 (0.1 µl volume per cell count field)/ml. The tubes containing the cell suspension were centrifuged (200 g, 15 min, RT), supernatant was discarded and the cell pellet was re-suspended. Then cells were centrifuged (300 g, 10 min, RT) in 20 ml of MACS buffer to adapt PBMCs to the new buffer milieu used for further cell subset isolation via MACS cell separation reagents (Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany). 2.4.2 Isolation of plasmacytoid dendritic cells To assess whether the isolated PBMCs contained enough pDCs per donor to continue isolation, a flow cytometry analysis was performed. 100 µl of cell suspension of PBMCs at the counting step were stained with 3 µl of a 14 phycoerythrin (PE)-conjugated BDCA-2 monoclonal antibody (mAb) (Miltenyi Biotec GmbH) and 1 µl of a fluorescein isothiocyanate (FITC)-conjugated lineage cocktail 1 (lin 1) (Becton Dickinson) per tube for 15 min in the dark. The lin 1 cocktail consists of mAbs against the following molecules: Cluster of differentiation (CD)3, CD14, CD19, CD20, CD56. After a washing step (2 ml PBS per tube, 350 g, 7 min, RT) cells were analyzed with a FACS Scan flow cytometer (Becton Dickinson) for viability and percentage of pDCs. PDCs were defined as BDCA-2+, lin 1– cells. 2.4.3 Positive selection of plasmacytoid dendritic cells PDCs were positively selected from PBMCs with a CD304 (BDCA-4/Neuropilin-1) MicroBead Kit human (Miltenyi Biotec GmbH) according to the manufacturer’s protocol. The PBMC pellet adapted to MACS buffer during the last washing step was resuspended in 150 µl MACS buffer per 108 cells. Then 50 µl of FcR Blocking reagent per 108 cells and 50 µl of CD304 MicroBeads per 108 cells were added and cells were incubated for 15 min at 4 °C in the dark. Cells were washed with 20 ml MACS buffer (300 g, 10 min, RT), supernatant was discarded and the pellet re-suspended in 500 µl MACS buffer per 108 cells. One LS column (Miltenyi Biotec GmbH) per donor was put into the quadroMACS™ Separation Unit (Miltenyi Biotec GmbH) and moistened with 3 ml MACS buffer. Then the cell suspension was applied to the columns. After letting the whole cell suspension run through the column, the columns were washed three times with 3 ml MACS buffer, adding the new volume only after the previous one had gone through the column. Then 5 ml MACS buffer were added to each column and magnetically labeled pDCs retained in the column were flushed out by firmly pushing the plunger into the upper part of the column. The eluted suspension, containing pDCs, was once again applied to a rinsed LS column to increase purity. This second column was again three times washed with 3 ml MACS buffer and pDCs retained in the column were flushed out into a 15 ml polypropylene conical tube (Becton Dickinson) by firmly squeezing 5 ml of MACS buffer through the column with a plunger. Cell count was performed with a Neubauer counting chamber (10 µl of a mix of 10 15 µl cell suspension and 10 µl trypan blue). Flow cytometry analysis of 100 µl cell suspension was performed as described above. Viability and purity of pDCs, defined as percentage of viable BDCA-2+, lin 1– cells, were assessed. Median purity after positive selection of pDCs was > 90% BDCA2+, lin 1- cells. Median viability was > 48%. The cell suspension of purified pDCs was spun down (300 g, 10 min, RT) and the cell pellet re-suspended in Adoptive Immunotherapy Media (AIM-V) (Life Technologies) or in completed RPMI in order to start cell culture. 2.4.4 Negative selection of plasmacytoid dendritic cells For negative selection of pDCs from PBMCs the Plasmacytoid Dendritic Cell Isolation Kit human (Miltenyi Biotec GmbH) was used according to the manufacturer’s protocol. The PBMC pellet, adapted to MACS buffer during the last slow washing step, was re-suspended in 200 µl MACS buffer per 108 cells. 50 µl per 108 cells of a BiotinAntibody Cocktail were added to and mixed with the cells. The Antibody cocktail contained Abs against molecules expressed on cells such as myeloid DCs, monocytes, T cells, B cells, NK cells, granulocytes and erythroid cells in order to label cells other than pDCs. After an incubation time of 10 min at 4 °C in the dark, cells were two times washed with 5-10 ml MACS buffer per 108 cells (300 g, 10 min, RT). Then the cell pellet was re-suspended in 200 µl MACS buffer per 108 cells and 50 µl Anti-Biotin MicroBeads per 108 cells were added and mixed with the cells. Cells were incubated for 15 min in the dark at 4 °C. After a washing step with 5-10 ml MACS buffer per 108 cells (300 g, 10 min, RT) the pellet was re-suspended in 500 µl MACS buffer per 108 cells. LD columns (Miltenyi Biotec GmbH) were put into the quadroMACS™ Separation Unit and moistened with 2 ml MACS buffer. 108 cells were applied per LD column and columns were washed twice with 2 ml MACS buffer. Flow-through containing pDCs was collected in 15 ml tubes. PDCs were counted in a Neubauer counting chamber (10 µl of a mixture of 10 µl cell suspension and 10 µl trypan blue) and a viability and purity flow cytometry 16 analysis was performed like described above. Median purity of BDCA2+, lin 1- cells after negative selection was > 89%, median viability was > 86%. The cell suspension was spun down (300 g, 10 min, RT) and cells were resuspended in AIM-V or completed RPMI medium. 2.4.5 Negative selection of CD4+ T cells In order to isolate CD4+ T cells from PBMCs the CD4+ T Cell Isolation Kit II human (Miltenyi Biotec GmbH) was applied according to manufacturer’s protocol. PBMCs adapted to MACS buffer were re-suspended in 40 µl MACS buffer per 107 cells. 10 µl per 107 cells of the Biotin-Antibody Cocktail containing Abs against CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRγ/δ and Glycophorin A were added and the mixture was incubated for 10 min at 4 °C in the dark. Then 30 µl of MACS buffer per 107 cells and 20 µl of Anti-Biotin MicroBeads per 107 cells were added and the mixture was re-incubated at 4 °C for 15 min in the dark. The cell suspension was then washed with 20 ml MACS buffer (300 g, 10 min, RT) and up to 108 cells were re-suspended in 500 µl MACS buffer. For magnetic separation of CD4+ T cells LS columns were put into a quadroMACS™ Separation Unit and moistened with 3 ml MACS buffer. The cell suspension was applied to the column and the column rinsed three times with MACS. Flow-through containing purified CD4+ T cells was collected in 15 ml tubes. Cells were counted in a Neubauer counting chamber and viability and purity of cells was assessed via flow cytometry after staining the cells like described above with a CD3-PE mAb (Becton Dickinson). Median purity of T cells after negative selection was > 95% CD3+ cells, median viability was > 95%. Cells were spun down (300 g, 10 min, RT) and re-suspended in AIM-V or completed RPMI medium or were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) (CellTrace™ CFSE Proliferation Kit; Life Technologies) as described in the section mixed lymphocyte reaction (MLR). 17 2.5 Cell culture 2.5.1 Short term cell culture For analysis of Granzyme B (GrB) production and GrB and IFN-α secretion pDCs were incubated in AIM-V directly after positive selection. PDCs were seeded at a density of 5 x 105 cells/ml, 200 µl/well in 96-well flat-bottom plates (Becton Dickinson). For most experiments, pDCs were incubated in medium supplemented with IL-3 at a final concentration of 10 ng/ml. For experiments with HIV-1 the cytokine IL-10 was applied at a final concentration of 25 ng/ml. PDCs were incubated for 16 h at 37 °C and 5% carbon dioxide (CO2) in a WTB Binder incubator (Binder GmbH, Tuttlingen, Germany). PDCs for experiments with EBV were incubated 1:1 in AIM-V and RPMI supplemented with 10% FCS and 1% penicillin/streptomycin, since the EBV stock was based on RPMI supplemented with 10% FCS and 1% penicillin/streptomycin. After 16 h of incubation, pDCs were stained for intracellular GrB and surface molecules and supernatant was analyzed for GrB and IFN-α secretion. 2.5.2 Mixed lymphocyte reactions PDCs for mixed lymphocyte reactions (MLRs) were negatively selected and then incubated for 24 or 48 h in IL-3-supplemented AIM-V medium (10 ng/ml) in the presence of different stimuli at a density of 5 x 105 cells/ml, 200 µl/well in a WTB Incubator at 37 °C and 5% CO2. To prepare pDCs for MLRs, first 150 µl of supernatant were harvested, 150 µl of fresh completed RPMI were added to the cells and then pDCs were harvested in 2 ml safe-lock tubes (Eppendorf AG, Hamburg, Germany). Wells were washed two times with 200 µl completed RPMI to harvest all cells. PDCs were washed at 270 g, RT, for 5 min using a 5417C centrifuge (Eppendorf AG). Supernatant was discarded, the cell pellet was re-suspended in 1 ml completed RPMI and cells were counted with a Neubauer chamber and trypan blue. Cells were washed again (270 g, RT, 5 min). Then pDCs were re-suspended in completed RPMI. CD4+ T cells were negatively selected on day one or two. After isolation CD4+ T cells were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE). 18 T cells were re-suspended in 380 µl PBS per 1 x 107 cells, CFSE at a final concentration of 2.5 µM was added, properly mixed and the whole tube content was transferred into a new one. Cells were incubated for 10 min at 37 °C and 5% CO2. Then 10 ml completed RPMI were added to block the staining reaction during another 10 min at RT. Afterwards tubes were filled up to 50 ml with PBS and washed twice (300 g, 10 min, RT). Washing included discarding of supernatant and re-suspension of cells in fresh PBS. Cells were counted in a Neubauer chamber with trypan blue staining. The third washing step was performed with completed RPMI medium. Then cells were re-suspended in completed RPMI. Proper and equal staining of CD4+ T cells with CFSE was evaluated via flow cytometry with a FACS Scan flow cytometer. PDCs and CD4+ T cells were seeded at different ratios in 96-well round-bottom wells (Becton Dickinson). 2 x 105 CD4+ T cells were seeded with pre-treated allogeneic pDCs at the ratios 1:50, 1:250 and 1:1250 in 200 µl completed RPMI. Cells were co-incubated for six or seven days and then harvested into FACS tubes for staining with a PE-conjugated CD3 mAb to label T cells and shortly before analysis with a LSR II (Becton Dickinson) cells were also stained with 7-aminoactinomycin D (7-AAD) (Merck KgaA, Darmstadt, Germany) to exclude dead cells. Staining for flow cytometry is described in detail in the section flow cytometry. Proliferated CD4+ T cells were defined as CD3+, 7-AAD-, CFSElow cells for experiments with VZV and CMV. For experiments with EBV and HIV-1 proliferated CD4+ T cells were defined as CD3+, CFSElow cells. PDCs die during a cell culture time of six or seven days. As controls 2 x 105 CD4+ T cells were incubated with 0.25 or 0.5 µl CD3/CD28 beads (Dynabeads® CD3/CD28 T cell Expander, Dynal Biotech ASA, Oslo, Norway) or in 200 µl completed RPMI alone. As a control for potent pDC stimulation for allogeneic induction of T cell proliferation we incubated pDCs with a class B cytosine-phosphate-guanine oligodeoxynucleotide (CpG B) with the sequence 5’-tcg tcg ttt tgt cgt ttt gtc gtt-3’ (ODN 2006; Invivogen, San Diego, CA, USA). PDCs incubated with 2.5 µg/ml CpG B produce less GrB than pDCs incubated in IL-3-supplemented medium alone and induce T cell proliferation in MLRs [87]. 19 2.5.3 Mixed lymphocyte reactions with HIV-1-infected CD4+ T cells CD4+ T cells were isolated as described above. 3 x 106 T cells were re-suspended in 1 ml supplemented RPMI medium (Life Technologies) (10% heat-inactivated FCS, 350 µg/ml L-glutamine, 120 µg/ml penicillin, 120 µg/ml streptomycin) and transferred into a 50 ml cell culture flask for suspension cells (Sarstedt AG & Co., Nümbrecht, Germany). T cells were stimulated with 25 µl CD3/28 beads (Dynabeads® Human T-Activator CD3/CD28, Life Technologies) per 1 x 106 T cells. Beads were prepared by washing them once with at least 1 ml PBS (PAA Laboratories GmbH, Pasching, Austria/Life Technologies). Volume of PBS should be at least the volume of beads used. A 5 ml polystyrene round-bottom tube (Becton Dickinson) containing beads in PBS was vortexed and then put for 1 min in a DynaMag™ magnet (Life Technologies). PBS was discarded and the tube was filled with supplemented RPMI and added to T cells in the flask. Finally, 2 x 106 T cells were re-suspended in 1 ml supplemented RPMI each and incubated for three days at 37 °C, 5% CO2 (STERI cult, Thermo Fisher Scientific, Waltham, MA, USA) in a 50 ml cell culture flask for suspension cells (Sarstedt AG & Co.). At day one (only in one experiment) or yet at d0 10 ng/ml IL-2 (Miltenyi Biotek GmbH) were added to the medium. After three days of stimulation, beads were removed from the cells with a DynaMag™ magnet. Therefore, content of the cell culture flask was transferred into a 15 ml tube (Sarstedt AG & Co.), the tube was vortexed and put into a DynaMag™ magnet for 1 min, fluid was transferred into a new tube whilst beads stayed attached to the walls of the tube. 1 x 106 T cells were put each into 5 ml tubes. After centrifugation (340 g, 3 min, RT) supernatant was discarded and 300 µl virus stock were added to 1 x 106 T cells or centrifuged supernatant of mock-transfected HEK293T cells was added as a control. Cells were transduced for 6 h at 37 °C, 5% CO2 (HERAcell® 240, Thermo Fisher Scientific). Cells were washed twice in PBS, then 3 ml supplemented RPMI plus 10 ng/ml IL-2 (Miltenyi Biotec GmbH) were added per tube. Tubes were vortexed, content was transferred into 6-well plates (Greiner Bio-One, Frickenhausen, Germany) and incubated at 37 °C, 5% CO2 for three days. 20 On day seven, pDCs were negatively selected as described above and stimulated with IL-3 (10 ng/ml), IL-10 (25 ng/ml) or a combination of IL-3 and IL-10 (IL-3 + IL10). PDCs were co-incubated with HIV-infected or mock-infected T cells in a ratio of 1 x 105 pDCs: 2 x 105 T cells in 200 µl supplemented RPMI in a 96-well flatbottom plate (Greiner Bio-One). At day eight, 150 µl supernatant were collected and stored at -20 °C for enzymelinked immunosorbent assay (ELISA) analysis. 2.6 Enzyme-linked immunosorbent assay 75 µl supernatant from pDCs incubated for 16 or 48 h were taken in order to measure GrB or IFN-α in the supernatant. Supernatant was usually stored at –80 °C for experiments with VZV and CMV, at -20 °C or -40 °C for EBV and at -20 °C for HIV experiments. 2.6.1 Granzyme B ELISA Plates were assembled using Nunc-Immuno™ Modules (Thermo Fisher Scientific, Waltham, MA, USA). For analyzing GrB in supernatant the Human Granzyme B ELISA kit (Mabtech AB, Nacka Strand, Sweden) was applied according to the manufacturer’s protocol. One day before ELISA the plate was covered with 100 µl/well of a GrB–Coating Antibody (mAb GB10) that was diluted to a final concentration of 2 µg/ml with PBS. The plate was covered with Parafilm “M” Laboratory Film (Pechiney Plastic Packaging, Menasha, WI, USA) and stored overnight in a humid chamber at 4 °C. The next day the Ab-covered plate was washed twice with 200 µl/well PBS and subsequently incubated for 1 h with 200 µl/well of incubation buffer to block unspecific binding (PBS with 0.05% Tween 20 (Sigma-Aldrich) and 0.1% BSA). GrB standards were prepared with incubation buffer ranging from 4000 pg/ml to 62.5 pg/ml in dilution steps of 1:2. Following five washing steps with 200 µl/well PBS, 100 µl/well of samples diluted with incubation buffer and GrB standards were added to the plate; incubation buffer was used as blank. The plate was covered with a plate sealer (Human IFN-α Multi-Subtype ELISA Kit; PBL Biomedical Laboratories, Piscataway, NJ, USA) and incubated for 2 h at RT in the dark. Plate 21 was washed five times. Then 100 µl/well of a Biotin tagged GrB-Detection Ab (mAb GB11-biotin) diluted in incubation buffer to a final concentration of 1 µg/ml were added. After 1 h of incubation and five washing steps 100 µl/well of Streptavidin-HRP 1:1000 diluted with incubation buffer were added and incubated for 1 h at RT. Finally a 1:1 mixture of colour reagent a (stabilized peroxide solution) and colour reagent b (stabilized chromogen solution) from a Substrate Reagent Pack (R&D Systems, Minneapolis, MN, USA) was added and incubated for 20 min in the dark without covering. Color reaction was stopped with 50 µl/well of 2N sulphuric acid (Sigma-Aldrich). ELISA was measured immediately at a wavelength of 450 nm with a Mithras LB 940 micro-titer plate reader (Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany) for VZV and most CMV samples, whereas EBV, HIV-1 and some CMV samples were measured with a Thermomax® microplate reader (Molecular Devices, LLC., Sunnyvale, CA, USA). 2.6.2 Interferon-α ELISA For measuring of IFN-α in the supernatant of pDCs the Human IFN-α MultiSubtype ELISA Kit (PBL Biomedical Laboratories, Piscataway, NJ, USA) was used according to the manufacturer’s protocol. Standards were prepared by diluting Human IFN Alpha standard (10,000 pg/ml) with dilution buffer. Extended range ELISA IFN-α standards covered a range between 5000 pg/ml and 78.125 pg/ml in steps of 1:2 dilutions. High sensitivity IFN-α ELISA standards ranged between 500 pg/ml and 6.25 pg/ml and were prepared by 1:2 dilutions from 200 pg/ml down to 6.25 pg/ml. 100 µl of samples diluted with dilution buffer, standards and dilution buffer as blank were added to the pre-coated IFN-α ELISA plate. Plate was covered with a plate sealer and incubated for 1 h in the dark. After washing the plate twice with 250 µl/well wash buffer (Wash solution concentrate 1:20 diluted in Ampuwa® Spüllösung Plastipur® Aqua ad iniectabilia), 100 µl/well of diluted antibody solution (Antibody concentrate diluted in dilution buffer according to the lot-specific certificate of analysis) was added and incubated for 1 h in the dark. Three washing steps (250 µl/well wash buffer) were followed by addition of 100 µl/well of diluted HRP solution (HRP conjugate concentrate diluted in HRP 22 conjugate diluent according to the lot-specific certificate of analysis). Plate was covered and incubated for 1 h. The plate was washed four times (250 µl/well wash buffer) and a Tetramethylbenzidine (TMB) substrate, warmed to room temperature, was added (100 µl/well) and incubated for 15 min in the dark without covering. After adding 50 µl/well of stop solution color reaction was measured immediately at a wavelength of 450 nm with a Mithras LB 940 micro-titer plate reader in the case of most VZV and some CMV samples or in case of EBV, HIV and some CMV and VZV samples with a Thermomax® microplate reader. As a control for potent IFN-α induction in pDCs we used 1 µg/ml of a class A cytosine-phosphate-guanine oligodeoxynucleotide (CpG A) with the sequence 5’g*g*g gac gac gtc gtg g*g*g * g*g*g-3’ (*phosphorothioate bonds, rest are phosphodiester bonds) (ODN 2336; Coley Pharmaceutical Group, Ottawa, ON, Canada). 2.7 Flow cytometry 2.7.1 Surface staining PDCs intended to be stained for intracellular GrB were incubated for 4 h with 1 µg/ml Brefeldin A (Epicentre Biotechnologies, Madison, WI, USA) prior to harvesting. Staining for surface molecules was performed before intracellular staining for GrB. Other pDCs and T cells were only stained for surface molecules. For surface staining, cells were harvested and 3 µl of the respective mAb were added, cells were vortexed and incubated for 15 min in the dark. Cells were washed (2 ml PBS, 350 g, RT, 7 min), supernatant aspirated except for 200 µl and cells were analyzed with a flow cytometer. MAbs against the following surface molecules were used: CD3, CD14, CD16, CD19, CD20, CD40, CD54, CD56, CD80, CD83, CD86, CD123, HLA-ABC and HLA-DR (all from Becton Dickinson). The mAbs were conjugated with different fluorescent dyes: Allophycocyanin (APC), FITC, PE, phycoerythrin tandem with cyanin dye 5 (PE-Cy5) and peridinin chlorophyll protein tandem with cyanin dye 5.5 (PerCP-Cy5.5). 23 2.7.2 Staining for intracellular granzyme B After 16 h of incubation of pDCs at 37 °C, 5% CO2, 150 µl of supernatant were taken for analysis with ELISAs. This volume was re-substituted. In case of experiments intended for the analysis of the effect of VZV on pDCs, 75% of the initial amount of the incubation reagents medium, IL-3 and VZV volume dilutions was re-substituted. In case of experiments with CMV, EBV and HIV-1 no virus or cytokines, only medium was re-substituted. After re-substitution of volume into the wells, cells were incubated for 4 h at 37 °C, 5% CO2 with 1 µg/ml Brefeldin A (Epicentre Biotechnologies, Madison, WI, USA). Samples for the analysis of the basic GrB-production level in freshly isolated pDCs, named d0 samples, were incubated with Brefeldin A for 4 h directly after isolation. Brefeldin A is a reagent that blocks the transport of proteins from the ER to the Golgi apparatus. Therefore intracellular GrB accumulated in the ER. Then pDCs were harvested in 5 ml polystyrene round-bottom tubes (Becton Dickinson) and were stained for surface molecules. 3 µl per mAb were used per tube, except for lin 1-FITC where 1 µl per tube was used. Cells were vortexed and incubated for 15 min in the dark. Afterwards cells were washed with 2 ml PBS at 350 g for 7 min at RT. Supernatant was aspirated and the cell pellet with about 100 µl of volume was left in the tube. Then cells were fixed for 15 min in 100 µl of fixation buffer. In case of incubations with HIV or EBV, 200 µl of a 2% PFA solution were used. After another washing step (2 ml PBS, 350 g, RT, 7 min), supernatant was aspirated and 100 µl of permeabilization buffer were added per tube. At once 2 µl of FcR Blocking reagent human (Miltenyi Biotec GmbH) were added, cells were vortexed and incubated for 10 min in the dark. Then 2 µl of a mAb for GrB (Sanquin Blood Supply, Amsterdam, The Netherlands) that was conjugated with PE or the isotype control Ab (Mouse immunoglobulin (Ig)G1 isotype control PE-conjugated; R&D Systems, Minneapolis, MN, USA) were added, tubes were vortexed and cells incubated for another 15 min in the dark. Cells were washed and 200 µl volume per tube was left after aspiration of supernatant to have enough volume left to analyze cells with a flow cytometer, either a FACS Scan, a FACS Calibur or a LSR II (Becton Dickinson). 24 2.7.3 Compensation controls For compensation, PBMCs left from the pDC isolations were incubated overnight and were stained for CD45 conjugated to the respective dyes according to the staining protocol for the pDCs intended for the same readout; if pDCs were fixed and permeabilized, PBMCs, used for compensation, were first stained for CD45 and then fixed and permeabilized as well. All CD45 Abs, conjugated with the fluorescent dyes APC, FITC, PE, PE-Cy5, PerCP-Cy5.5, used for staining of compensation controls were provided by Becton Dickinson. 2.7.4 Staining with 7-amino-actinomycin D To be able to exclude dead cells during flow cytometric analysis of MLRs, 3 µg/ml of 7-amino-actinomycin D (7-AAD) were added to the cells shortly before measurement. 2.7.5 Annexin V/Propidium iodide staining In order to analyze viability of cells incubated with VZV vaccine or UV-inactivated CMV, pDCs were stained for Annexin V and Propidium iodide (PI) after 16 or 24 h of incubation. Cells were harvested and washed twice with Annexin V binding buffer (Sterofundin with HEPES buffer 10 mM) (2 ml, 350 g, 7 min, RT) that contained the Ca2+ ions essential for Annexin V binding to phospholipids on the surface of dying cells. PBMCs used for compensation with PI, Annexin V and cells for positive controls were either incubated with 2 µl ethanol (100%) for 20 min or 3 or 5 µl hydrogen peroxide (H2O2) for 10 min and subsequently adapted to the Annexin V binding buffer. Then cells were stained with 1 µl lin 1-FITC and 5 µl Annexin V-APC or 10 µl of a self-mixed lin 1-APC and 5 µl Annexin V–FITC. All cells were incubated for 15 min in the dark and samples were washed once, supernatant was aspirated except for about 200 µl and cells were at once analyzed via flow cytometry. Shortly before analysis with the flow cytometer, PI (5 µg/ml) was added to each sample separately. 25 2.8 Data analysis and statistics Data of the experiments are demonstrated as averages ± standard error of mean (SEM). For the analysis of statistical differences the paired two-tailed student’s t test was applied. *p-values < 0.05 were considered as statistically significant. Data and statistics were analyzed with Microsoft Excel 2007 and 2010 and Microsoft Powerpoint 2007 and 2010 (both Microsoft Corporation, Redmond, WA, USA). 26 3 Results 3.1 Herpesviruses modulate granzyme B production and secretion by plasmacytoid dendritic cells in a concentration-dependent manner In order to test the hypothesis that viral stimuli with a dsDNA genome can modulate GrB production and secretion by pDCs we first tested a vaccine containing live attenuated varicella-zoster virus (VZV). The varicella-zoster virus vaccine Varilrix® was used in different volume dilutions of the reconstituted lyophilized vaccine. Additionally we analyzed the effects of UV-inactivated human cytomegalovirus (CMV) on pDCs in different multiplicities of infection (MOIs) and the effects of Epstein-Barr virus (EBV) in different volume dilutions of the virus stock. Intracellular GrB production was determined via flow cytometry as MFI of GrB in the viable, lin 1-, CD123+ cell gate. The gating strategy is shown in figure 1A. When purity of pDCs was very high and staining for additional surface molecules required the application of other mAb the MFI of GrB was measured in the viable cell gate alone. Freshly isolated pDCs (d0) already produced a small amount of GrB that was not markedly increased after 16 h of incubation in AIM-V medium alone (figure 1B). Moreover, pDCs incubated in AIM-V medium alone produced the same amounts of GrB as pDCs incubated in AIM-V medium with VZV in increasing concentrations (figure 1B). In contrast, IL-3 incubation led to a markedly higher GrB production than medium alone. This production could be modulated by VZV in a concentration-dependent manner. Therefore, all other experiments with pDCs and VZV, UV-inactivated CMV or EBV were only done with pDCs incubated in IL-3supplemented medium or only data with pDCs incubated in IL-3-supplemented medium are shown. Whereas the VZV vaccine dilutions 1:2000 and 1:200 had no marked effect on GrB production and secretion by pDCs incubated in IL-3-supplemented medium compared to that of pDCs incubated with IL-3 alone, the vaccine dilutions 1:20 and 1:10 significantly reduced GrB production in pDCs [47]. PDCs treated with VZV in a volume dilution of 1:10 produced 28.63 ± 3.14% less GrB than pDCs treated with IL-3-supplemented medium alone. This decrease of GrB production in pDCs 27 incubated with VZV in high concentrations was observed after 24 h of incubation as well (data not shown). To verify these results, GrB secretion by pDCs upon viral stimulation was measured with a GrB-specific ELISA after 16 h of incubation of pDCs. GrB secretion by pDCs was decreased in a concentration-dependent manner by VZV vaccine dilutions 1:20 and 1:10 [47]. PDCs incubated with vaccine dilution 1:10 secreted significantly less GrB; GrB secretion was reduced to 76.67 ± 10.57% (figure 1C). Thus the suppressive effect of VZV on GrB observed via flow cytometry could be reproduced using a GrB ELISA. As a second step we looked at the effects of human UV-inactivated cytomegalovirus (CMV). PDCs incubated with CMV produced less GrB in a dosedependent manner. PDCs incubated with MOI 1 and MOI 10 produced significantly less GrB than pDCs incubated with IL-3-supplemented medium alone. MOI 1 reduced GrB production to 76.32 ± 3.89% and MOI 10 to 41.69 ± 2.55% compared to IL-3-supplemented medium alone (figure 1D). Similar to GrB production, GrB secretion by pDCs was dose-dependently decreased by UVinactivated CMV. PDCs incubated with CMV in MOI 1 and 10 secreted significantly less GrB; GrB secretion was diminished to 69.13 ± 5.64% (CMV MOI 1) and to 55.23 ± 6.41% (CMV MOI 10) (figure 1E). Third, we analyzed another dsDNA virus: Epstein-Barr virus (EBV). In contrast to VZV and CMV, EBV increased GrB production by pDCs in a concentrationdependent manner. The volume dilution EBV 12.5 increased GrB production compared to pDCs incubated in IL-3-supplemented medium alone about 12.39 ± 4.73%, pDCs incubated with EBV 25 produced 26.51 ± 7.75% more GrB. EBV 50 did not further enhance GrB production by pDCs (figure 1F). In analogy to GrB production, GrB secretion was also dose-dependently higher when pDCs were stimulated with EBV. EBV 50 represented the most potent stimulus and increased GrB secretion by pDCs to 291.70 ± 50.12% (figure 1G). 28 29 Figure 1: Varicella-zoster virus and cytomegalovirus decrease whereas Epstein-Barr virus increases plasmacytoid dendritic cell-derived granzyme B. (A+B) Plasmacytoid dendritic cells (pDCs) were incubated for 16 h in AIM-V medium (M) without (w/o) interleukin (IL)-3 and with varicella-zoster virus (VZV) vaccine in the indicated volume dilutions or in IL-3-supplemented AIM-V medium with VZV. Granzyme B (GrB) production was determined via flow cytometry. (A) Representative dot plots of one donor and gating strategy are + shown. PDCs for isotype control (Iso) were treated with IL-3 alone. Dot plots show GrB cells in the - + viable, lin 1 , CD123 gate. (B) Bar graphs show average values of median fluorescence intensity (MFI) of intracellular GrB relative to pDCs incubated in M with IL-3 from at least three different donors. The d0 sample was stained directly after positive selection of pDCs. Error bars indicate standard error of mean (SEM), *p-values indicate significant differences compared to pDCs incubated in M with IL-3. (C) GrB secretion was measured by enzyme-linked immunosorbent assay (ELISA) in the supernatant of pDCs incubated for 16 h in IL-3-supplemented medium (M) with VZV in the indicated dilutions. Bar graphs represent average values relative to M of at least nine different donors. Error bars indicate SEM, *p-values indicate significant differences compared to M. (D+E+F+G) PDCs were incubated for 16 h in IL-3-supplemented medium (M) with UV-inactivated human cytomegalovirus (CMV) in the indicated multiplicities of infection (MOIs) or with Epstein-Barr virus (EBV) in the indicated volume dilutions. (D+F) GrB production was determined via flow cytometry after intracellular staining. Bar graphs show average values of MFI of intracellular GrB relative to M from at least 17 different donors for CMV and from at least nine different donors for EBV. Error bars indicate SEM, *p-values indicate significant differences compared to M. (E+G) GrB secretion was measured in supernatant by ELISA. Bar graphs represent average values relative to M of at least 12 different donors for CMV and of 10 different donors for EBV. Error bars indicate SEM, *p-values indicate significant differences compared to M. (B+C) See [47]. 30 3.2 Human immunodeficiency virus 1 increases granzyme B secretion by plasmacytoid dendritic cells In a separate set of experiments we investigated the effects of the ssRNA retrovirus human immunodeficiency virus 1 (HIV-1) on human pDCs. PDCs were incubated for 16 h with different cytokines and 5 ng/ml p24 antigen HIV-1. GrB production was analyzed with flow cytometry after staining for intracellular GrB. As expected pDCs incubated only in AIMV-medium or with different cytokines produced different amounts of GrB: PDCs incubated with medium alone or with IL-10 25 ng/ml for 16 h did not produce more GrB than freshly isolated pDCs. PDCs incubated with IL-3 or a combination of IL-3 and IL-10 (IL-3 + IL-10) produced high amounts of GrB at comparable levels. When pDCs were additionally stimulated with HIV-1 for 16 h, no difference of intracellular GrB to incubation without HIV-1 could be seen for all incubation conditions (figure 2A). As another readout we measured GrB secretion in the supernatant of pDCs incubated with different cytokines and HIV-1. In contrast to flow cytometry data measuring intracellular GrB production, the ELISA showed differences between the GrB secretion by pDCs incubated with or without HIV-1. HIV-1 increased GrB secretion in all the different incubation conditions (figure 2B and 2C) particularly when HIV-1 was combined with IL-3 or IL-3 + IL-10 [88]. PDCs incubated with IL-3 and HIV-1 secreted 39.65 ± 8.42% more GrB than pDCs incubated with IL-3 alone and IL-3 + IL-10 plus HIV-1 pDCs secreted 44.15 ± 7.46% more GrB than pDCs incubated with IL-3 + IL-10 alone (figure 2B). 31 Figure 2: Human immunodeficiency virus 1 increases granzyme B secretion by plasmacytoid dendritic cells. Plasmacytoid dendritic cells (pDCs) were incubated for 16 h in medium (M) supplemented with different cytokines and with or without (w/o) 5 ng/ml human immunodeficiency virus 1 (HIV) as indicated. (A) Granzyme B (GrB) production was determined via flow cytometry after intracellular staining. The d0 sample was stained directly after positive selection of pDCs. Bar graphs show average values of median fluorescence intensity (MFI) of intracellular GrB relative to M with interleukin (IL)-3 w/o HIV. Averages are derived from at least three different donors. Error bars indicate standard error of mean (SEM). (B+C) GrB secretion was measured in supernatant by enzyme-linked immunosorbent assay (ELISA). (B) Bar graphs represent average values relative to IL-3 w/o HIV. Averages are derived from at least five different donors. Error bars indicate SEM, *pvalues indicate significant differences compared to samples incubated w/o HIV. (C) Bar graphs represent average values relative to M w/o HIV. Averages are derived from at least five different donors. Error bars indicate SEM. (B) See [88]. 32 3.3 Regulation of granzyme B production and secretion by plasmacytoid dendritic cells is virus-specific As the two summary figures show, UV-inactivated CMV had a higher potential than vaccine-derived VZV to decrease GrB production and secretion by pDCs. EBV potently induced GrB secretion by pDCs and also to some extent GrB production. HIV-1 also had the potential to increase GrB secretion but it did not alter GrB production compared to pDCs incubated with IL-3 alone (figure 3A and 3B). Figure 3: Virus-induced regulation of granzyme B production and secretion by plasmacytoid dendritic cells varies between different viruses. The figures summarize the results from figure 1 and 2. Data are the same as in figure 1 and 2. Averages are derived from the same experiments. Plasmacytoid dendritic cells (pDCs) were incubated for 16 h in interleukin (IL)-3-supplemented medium (M) with varicella-zoster virus (VZV) vaccine or Epstein-Barr virus (EBV) in different volume dilutions, alternatively with UV-inactivated human cytomegalovirus (CMV) in different multiplicities of infection (MOIs) or human immunodeficiency virus 1 (HIV). (A) Bar graphs show average values of median fluorescence intensity (MFI) of intracellular granzyme B (GrB) production relative to M. (B) GrB secretion was measured in supernatant by enzyme-linked immunosorbent assay (ELISA). (A+B) Bar graphs represent average values relative to M. Error bars indicate standard error of mean, *p-values indicate significant differences compared to M. 33 3.4 Interferon-α secretion by plasmacytoid dendritic cells is differentially regulated by viruses Next, we tested the potential of the four different viral stimuli to induce IFN-α in pDCs, the typical type I interferon produced by pDCs upon viral stimulation. IFN-α secretion was measured with an IFN-α-specific ELISA in the supernatant after 16 h of incubation of pDCs in the presence or absence of different viruses. PDCs incubated with IL-3 alone do hardly produce any IFN-α. In contrast, pDCs incubated for 16 h with VZV in increasing concentrations secreted high amounts of IFN-α. PDCs incubated with VZV 1:10 secreted 9.71 ± 3.50 ng/ml IFN-α (figure 4A). As the high standard error of mean (SEM) shows, we observed in general a high variance in IFN-α secretion between the different donors. As a positive control for IFN-α secretion by pDCs, we used 1 µg/ml CpG A as a potent inductor of IFN-α production in pDCs. Data are not shown because IFN-α secretion by CpG A-stimulated pDCs was too high to measure within the area of our standard curve. UV-inactivated human CMV also induced potent IFN-α secretion in pDCs: PDCs stimulated with CMV MOI 10 produced 24.29 ± 1.62 ng/ml IFN-α (figure 4B). EBV induced only little IFN-α secretion by pDCs: EBV 25 induced 71.06 ± 29.89 pg/ml IFN-α and EBV 50 126.46 ± 6.6 pg/ml IFN-α. For EBV these are data from three different donors (figure 4C). In another experiment with another EBV stock we could not detect any IFN-α production by pDCs. In the experiments intended for the analysis of the effect of HIV-1 on IFN-α secretion by pDCs, pDCs incubated with AIMV-medium alone or only with the cytokines IL-3, IL-10 or IL-3 + IL-10 did hardly produce any IFN-α. When pDCs were additionally incubated with HIV-1 IFN-α secretion was slightly increased by medium-pDCs and by IL-10-pDCs (80.62 ± 23.77 pg/ml), more obviously in the presence of IL-3 (151.55 ± 34.51 pg/ml) and most with IL-3 + IL-10 (284.35 ± 136.35 pg/ml), even though the IFN-α amounts remained on a low level (figure 4D). We also tested IFN-α secretion in mixed lymphocyte reactions (MLRs) with pDCs and HIV-1- or mock-infected CD4+ T cells. After 16 h of co-culture pDCs coincubated with HIV-1-infected T cells produced markedly high amounts of IFN-α. The IFN-α amounts produced in these MLRs were - even in high dilutions - too high to measure correctly within our applied high sensitivity standard curve. As a consequence the results for IFN-α in these MLRs can only give hints of the actual 34 values. In contrast, pDCs co-incubated with mock-infected T cells produced only small amounts of IFN-α (figure 4E). 35 Figure 4: Regulation of interferon-α secretion by plasmacytoid dendritic cells is virusdependent. (A+B+C+D) Plasmacytoid dendritic cells (pDCs) were incubated for 16 h in interleukin (IL)-3supplemented medium (M) with varicella-zoster virus (VZV) vaccine or Epstein-Barr virus (EBV) in the indicated volume dilutions, UV-inactivated human cytomegalovirus (CMV) in the indicated multiplicities of infection (MOIs) or with different cytokines and with or without (w/o) 5 ng/ml human immunodeficiency virus 1 (HIV) as indicated. Supernatant was harvested after 16 h of incubation and interferon (IFN)-α concentration in supernatant was measured with an IFN-α-specific enzymelinked immunosorbent assay (ELISA). (A+B) Bar graphs represent average IFN-α concentrations in ng/ml in supernatants from four different donors for VZV and from at least four different donors for CMV. (C+D) Bar graphs represent average IFN-α concentrations in pg/ml in supernatant from three different donors for EBV and from at least five different donors for HIV-1. (A+B+C+D) Error bars represent standard error of mean. (A+B+C) *p-values indicate significant differences compared to M. (D) *p-values indicate significant differences compared to samples w/o HIV. (E) PDCs were either incubated alone with different cytokines or with different cytokines and HIV-1- or mock+ infected CD4 T cells. After 16 h of co-incubation supernatant was harvested and IFN-α concentrations were measured with an IFN-α-specific ELISA. Bar graphs represent average IFN-α concentrations in ng/ml in supernatants from at least 6 different pDC:T cell donor combinations. Error bars represent standard error of mean, *p-values are not shown due to measurement of MLR values out of range of the ELISA standard curve. 36 3.5 Herpesviruses modulate the expression of distinct surface molecules on plasmacytoid dendritic cells To test the expression of different surface molecules on pDCs stimulated with VZV, CMV or EBV, surface molecule expression was measured via flow cytometry after 16 h of incubation. Surface molecule expression was assessed most times as MFI in the viable, lin 1-, CD123+ cell gate but if purity of pDCs was high and staining for additional surface molecules required the application of other mAbs, surface molecule expression was determined after gating on viable cells. The antigen-presenting molecule HLA-ABC was expressed at higher levels on pDCs incubated with VZV than on pDCs incubated with IL-3 alone [47]. The concentrations 1:200, 1:20 and 1:10 had the same effect on the surface expression of HLA-ABC on pDCs. VZV 1:10 increased HLA-ABC expression by 83.41 ± 16.97% (figure 5A). The intercellular adhesion molecule CD54 was also slightly higher expressed on pDCs incubated with VZV [47] in increasing concentrations than on pDCs incubated with IL-3 alone: PDCs incubated with VZV 1:20 expressed 46.55 ± 10.69% more CD54, VZV 1:10 had a similar effect and led to 45.11 ± 9.77% more CD54 expression than on pDCs incubated with IL-3supplemented medium alone. In contrast, pDCs treated with VZV expressed the same levels of the pDC maturation marker CD83 compared to pDCs incubated with IL-3-supplemented medium alone [47]. Additionally, neither the high expression of HLA-DR on pDCs maturated with IL-3 [47], nor the expression levels of the co-stimulatory molecules CD40 [47], CD80 and CD86 [47] on pDCs incubated with IL-3 alone were further modulated by VZV. We also measured the effect of UV-inactivated human cytomegalovirus in different MOIs on the expression of different surface markers on pDCs after 16 h of incubation: The antigen-presenting molecule HLA-ABC was expressed at higher levels on pDCs incubated with CMV. The MOIs 0.1, 1 and 10 almost had the same effect on the surface expression of HLA-ABC on pDCs. CMV MOI 10 increased HLA-ABC expression by 138.13 ± 22.00% (figure 5B). The high expression of HLA-DR on pDCs maturated with IL-3 was not further modulated by CMV as was the expression of the co-stimulatory molecules CD80 and CD86 not altered by CMV compared to the incubation of pDCs with IL-3 alone. In contrast to the results 37 for VZV, the expression of the intercellular adhesion molecule CD54 was not modulated by CMV. On the contrary, EBV, the third herpesvirus analyzed, again slightly enhanced the surface expression of the intercellular adhesion molecule CD54 on pDCs. PDCs stimulated with EBV 25 expressed 9.82 ± 0.87% more CD54 than pDCs stimulated with IL-3 alone. But EBV did not modulate the expression of the antigenpresenting molecules HLA-ABC and HLA-DR on pDCs compared to the expression of these molecules on the surface of pDCs incubated in IL-3supplemented medium alone (figure 5C). 38 39 Figure 5: Herpesviruses modulate the expression of human leukocyte antigen (HLA)-ABC and Cluster of differentiation (CD)54 on the surface of plasmacytoid dendritic cells. PDCs were incubated for 16 h in interleukin (IL)-3-supplemented medium (M) with varicella-zoster virus (VZV) vaccine or Epstein-Barr virus (EBV) in the indicated volume dilutions or UV-inactivated human cytomegalovirus (CMV) in the indicated multiplicities of infection (MOIs). Surface molecule expression was measured via flow cytometry. Bar graphs represent average values of median fluorescence intensity (MFI) of human leukocyte antigen (HLA)-ABC, HLA-DR, Cluster of differentiation (CD)54, CD80, CD86, CD40 and CD83 relative to M from at least three different donors for VZV and EBV and from at least four different donors for CMV. Error bars indicate standard error of mean, *p-values indicate significant differences compared to M. (A) See [47]. 40 3.6 Herpesviruses and human immunodeficiency virus 1 in the concentrations or multiplicities of infection used do not relevantly alter viability of plasmacytoid dendritic cells To investigate the inhibitory effect of the VZV vaccine and human UV-inactivated CMV on GrB production and secretion by pDCs we analyzed the viability of pDCs incubated with increasing concentrations or MOIs of the vaccine or viruses used. Viability of pDCs incubated for 16 h with different viral stimuli in comparison to pDCs incubated with IL-3-supplemented medium alone was analyzed via flow cytometry. Viability was defined by gating on viable cells in the forward scatter (FSC)/side scatter (SSC) dot plot. Viability of pDCs incubated with IL-3 alone for 16 h was 71.66 ± 2.88% and 69.12 ± 3.24% for pDCs incubated with VZV 1:10. Overall this analysis showed no decrease in viability between pDCs incubated with IL-3 alone and pDCs incubated with VZV in increasing concentrations. The volume dilution 1:200 did even slightly increase viability of pDCs (figure 6A). CMV MOI 10 slightly reduced viability of pDCs after 16 h of incubation about 6.38 ± 2.59% compared to pDCs incubated with IL-3 alone (figure 6D). CMV MOI 0.1 and CMV MOI 1 did not affect viability of pDCs after 16 h. EBV 12.5 and 25 did not alter viability of pDCs after 16 h of incubation compared to pDCs incubated in IL-3-supplemented medium alone. EBV 50 slightly decreased viability of pDCs compared to IL-3-pDCs about 6.60 ± 2.46% (figure 6G). Incubation with HIV-1 for 16 h did not relevantly alter viability of pDCs if viability of pDCs incubated with different cytokines but without HIV-1 and the viability of pDCs incubated with the respective cytokines with HIV-1 are compared. HIV-1 only slightly enhanced viability of medium-pDCs. As expected, pDCs incubated with medium alone or only with IL-10 were in general less viable than pDCs incubated with IL-3 or IL-3 + IL-10 as IL-3 is an important survival factor for pDCs. Compared to IL-3-pDCs, pDCs incubated with IL-3 + IL-10 with HIV-1 were slightly more viable (figure 6H). To confirm that pDCs producing less GrB upon incubation with VZV or CMV were not just less viable, we performed Annexin V/Propidium iodide (PI) staining. PDCs were incubated for 16 or 24 h and as for analysis of GrB production, pDCs were additionally incubated for 4 h with Brefeldin A directly prior to staining for flow 41 cytometry. We first gated on viable, lin 1- cells, then we defined Annexin V-, PIcells in this gate. PDCs incubated for 16 or 24 h with VZV vaccine in increasing concentrations showed no relevant decrease in viability (figure 6B and 6C). Incubation of pDCs with UV-inactivated CMV in different MOIs for 16 or 24 h did also not affect viability of pDCs compared to pDCs incubated in IL-3-supplemented medium alone (figure 6E and 6F). Taken together, the viruses tested have had no significant influence on pDC survival. 42 43 Figure 6: Herpesviruses and human immunodeficiency virus 1 do not relevantly alter viability of plasmacytoid dendritic cells in vitro. (A+D+G) Plasmacytoid dendritic cells (pDCs) were incubated for 16 h in interleukin (IL)-3supplemented medium (M) with varicella-zoster virus (VZV) vaccine or Epstein-Barr virus (EBV) in the indicated volume dilutions or with UV-inactivated human cytomegalovirus (CMV) in the indicated multiplicities of infection (MOIs). (H) PDCs were incubated for 16 h in medium alone (M) supplemented with different cytokines and with or without (w/o) 5 ng/ml human immunodeficiency virus 1 (HIV) as indicated. (A+D+G+H) PDCs were subsequently stained for intracellular granzyme B (GrB) and analyzed via flow cytometry. Viable cells were gated in forward/side scatter dot plot. (A+D+G) Bar graphs represent average values of viable cells relative to M from at least six independent donors for VZV, from at least 17 independent donors for CMV and from 10 different donors for EBV. Error bars indicate standard error of mean (SEM), *p-values indicate significant differences compared to M. (H) Bar graphs represent average values of viable cells relative to IL-3 w/o HIV-1. Averages are derived from at least four different donors for HIV-1. Error bars indicate SEM, *p-values indicate significant differences compared to the indicated samples incubated without virus. (B+C+E+F) PDCs were incubated in IL-3-supplemented medium (M) with VZV vaccine in the dilutions indicated or with UV-inactivated CMV in the MOIs indicated. After 16 or 24 h, pDCs were incubated as for intracellular staining. Subsequently cells were stained with Annexin V and Propidium iodide (PI) and analyzed via flow cytometry. PDCs were gated as for analysis of - - - GrB production (viable, lineage cocktail 1 (lin 1) cells) and the percentage of Annexin V , PI cells was determined. (B+E) Dot plots from one representative donor are shown. Annexin V/PI staining is shown for M sample and pDCs incubated with VZV 1:10 or CMV MOI 10 for 16 and 24 h. (C+F) - - Bar graphs represent average values of Annexin V , PI cells from at least two donors for VZV. For CMV averages are derived from three different donors. Error bars indicate SEM. 44 3.7 The viral components of the different herpesvirus sources are responsible for the effects on plasmacytoid dendritic cells In order to test whether the viral particles alone and no other ingredients of the viral agents were responsible for the effects on pDCs we performed studies with filtrated viral vaccine or virus or culture supernatants of herpesviruses. In the case of VZV the whole vaccine was filtrated through a virus filter that allowed the removal of virions. Consequently, the filtrated VZV vaccine contained no more varicella-zoster virus virions. In contrast to the whole vaccine, the filtrated vaccine in the highest concentrations used, had no effect on GrB production (figure 7A). Additionally, the filtrated vaccine did no longer induce HLA-ABC expression on pDCs (figure 7B). PDCs incubated with the filtrated vaccine still produced some IFN-α but by far not the same amount as pDCs incubated with the whole vaccine (figure 7C). However, UV-irradiated non-filtrated VZV vaccine still decreased GrB production by pDCs and enhanced HLA-ABC expression on pDCs to a similar extent as the whole vaccine (data not shown). Therefore VZV particles themselves, not other vaccine components are primarily responsible for pDC activity modulation. As CMV was harvested in the supernatant of CMV-infected human foreskin fibroblasts (HFFs) we used supernatant of non-CMV-infected HFFs in respective amounts to test the specificity of the negative effect of UV-inactivated CMV on GrB production by pDCs. PDCs were incubated for 16 h with UV-inactivated CMV in increasing MOIs or supernatant of HFFs in the respective volume. GrB production was analyzed via flow cytometry after intracellular staining for GrB. Supernatant of HFFs did only mildly influence GrB production compared to medium; it did not decrease GrB production by pDCs but slightly induced GrB production by pDCs: Surprisingly, HFF supernatants increased GrB production up to 17.83 ± 4.51% (figure 7D). Hence CMV particles and not supernatant components were needed for the GrB suppressing effect. To compare the ability of UV-inactivated human CMV and HFF supernatant to induce IFN-α secretion by pDCs, pDCs were incubated for 16 h in IL-3-supplemented AIMV-medium with CMV in the indicated MOIs or with the same amounts of HFF supernatant as a control. The HFF supernatant control did not induce IFN-α secretion in pDCs (figure 7E). 45 Similar experiments were also performed for EBV. EBV stock was filtered through a virus filter to remove virions. Filtration abrogated the effect of the EBV stock on GrB production and secretion and no difference to pDCs incubated in IL-3supplemented medium alone was observed (figure 7F and 7G). As far as IFN-α secretion is concerned, EBV particles were only weakly capable of inducing IFN-α in pDCs. In this low range, filtrated EBV supernatant had a similar effect. EBV 50 and filtrated EBV 25 and EBV 25 and filtrated EBV 50 produced the same amounts of IFN-α in this experiment. These results that detect IFN-α at pg levels are difficult to compare with the IFN-α-data derived with other viruses that lie in the ng range. It has also to be pointed out that data are derived from only three donors and one experiment (figure 7H). In summary, the virus particles themselves but not the soluble components of the viral agents were responsible for the effects observed. 46 47 Figure 7: The viral components of the different herpesvirus sources are responsible for the effects on plasmacytoid dendritic cells. Plasmacytoid dendritic cells (pDCs) were incubated for 16 h in interleukin (IL)-3-supplemented medium (M) with the whole varicella-zoster virus (VZV) vaccine or with the filtrated varicella-zoster virus vaccine in the dilutions indicated, with UV-inactivated human cytomegalovirus (CMV) in the multiplicities of infection (MOIs) indicated or with supernatant from mock-infected human foreskin fibroblasts (HFFs) in respective amounts as a control or with Epstein-Barr virus (EBV) or the filtrated EBV stock in the dilutions indicated. (A+D+F) Granzyme B (GrB) production was determined via flow cytometry after intracellular staining for GrB. Bar graphs represent average median fluorescence intensity (MFI) relative to M from at least five different donors for VZV, four different donors for CMV and from three different donors for EBV. Error bars indicate standard error of mean (SEM), *p-values indicate significant differences to M. (B) Surface expression of human leukocyte (HLA)-ABC was determined via flow cytometry. Bar graphs represent average MFI relative to M from at least four donors. Error bars indicate SEM, *p-values indicate significant differences compared to M. (C+E+H) Interferon (IFN)-α concentration in supernatant was measured with an IFN-α-specific enzyme-linked immunosorbent assay (ELISA). (C+E) Bar graphs represent average IFN-α concentrations in ng/ml in supernatants from four different donors for VZV and from four different donors for CMV. (H) Bar graphs represent average IFN-α concentrations in pg/ml in supernatants from three different donors for EBV. (C+E+H) Error bars represent SEM, *pvalues indicate significant differences compared to M. (G) GrB secretion was measured by ELISA in supernatant of pDCs incubated for 16 h with EBV or the filtrated EBV stock in the dilutions indicated. Bar graphs represent average values relative to M of three different donors. Error bars indicate SEM, *p-values indicate significant differences compared to M. 48 3.8 Herpesvirus- or human immunodeficiency virus 1-stimulated plasmacytoid dendritic cells modulate CD4+ T cell proliferation in mixed lymphocyte reactions To analyze the immunostimulatory potential of pDCs primed with different viral stimuli on CD4+ T cells we performed mixed lymphocyte reactions (MLRs) of CD4+ T cells with allogeneic pDCs pre-incubated with different viruses in high concentrations for 24 or 48 h. Proliferated CD4+ T cells were defined as CFSElow cells in viable, CD3+, 7-AAD- gate after six (experiments with VZV and CMV) or seven days (experiments with EBV and HIV-1) of co-incubation. As a control for high T cell proliferation CD4+ T cells were stimulated with CD3/28 beads. Representative histograms of one donor combination show gating strategy and controls (figure 8A). Representative histograms of one donor combination are shown for VZV and CMV (figure 8B), EBV (figure 8C) and HIV-1 (figure 8D). CpG B-activated pDCs were used as a positive control for the potential of allogeneic pDCs to induce T cell proliferation. CpG B-pre-treated pDCs potently enhanced CD4+ T cell proliferation in all pDC:T cell ratios. In the pDC:T cell ratio 1:50 pDCs pre-incubated with VZV 1:20 had a significant stimulatory effect on T cell proliferation (43.3 ± 4.12%) that was as high as the effect of CpG B-pre-treated pDCs (44.66 ± 2.94%). VZV 1:10-activated pDCs were also able to significantly enhance T cell proliferation compared to IL-3-pDCs in the pDC:T cell ratio 1:50 (VZV 1:10: 30.83 ± 2.60%, IL-3: 20.75 ± 3.16%). In the ratio 1:250 pDCs activated with VZV 1:20 still significantly enhanced T cell proliferation (27.53 ± 3.87%) compared to IL-3-stimulated pDCs (14.48 ± 3.15%) whereas VZV 1:10 only showed a small additional effect to IL-3 alone with 21.64 ± 2.25% T cell proliferation [47]. In the pDC:T cell ratio of 1:1250 no difference in the proliferation of T cells that had been co-incubated with IL-3-stimulated pDCs to the proliferation of T cells that had been co-incubated with VZV–stimulated pDCs was observed (figure 8E). Next we investigated the effect of pDCs pre-treated with UV-inactivated CMV. PDCs pre-treated with CMV MOI 1 increased T cell proliferation in the pDC:T cell ratio 1:50 to a higher extent (27.25 ± 3.71% proliferation) than pDCs pre-treated with IL-3 alone (11.86 ± 2.00%); whereas the immunostimulatory potential of CMVpre-treated pDCs was not as high as the potential of CpG B-pre-treated pDCs 49 (41.85 ± 3.00%). In the pDC:T cell ratio 1:250 a slight positive effect on T cell proliferation by CMV-pDCs compared to IL-3-pDCs was still found (CMV: 15.58 ± 2.50%, IL-3: 10.66 ± 0.59%). But in the ratio of 1:1250 no difference to IL-3-pretreated pDCs was observed anymore (figure 8F). In a third step the effect of pDCs pre-treated with EBV on T cell proliferation was tested. In contrast to pDCs pre-treated with VZV or CMV, pDCs pre-incubated with EBV did not alter the level of T cell proliferation compared to pDCs incubated in IL3-supplemented medium alone. This result was observed for all pDC:T cell ratios (figure 8G). PDCs pre-incubated with CpG B, as expected, potently induced T cell proliferation in all pDC:T cell ratios. Importantly, when pDCs were pre-incubated with CpG B plus EBV, EBV did reduce the proliferation-inducing potential of CpG B-pDCs to a small extent (data not shown). Therefore, EBV-pDCs not only fail to induce T cell proliferation, there is even a trend towards suppression of CpG BpDC-induced T cell proliferation. In order to investigate the inhibitory mechanism of EBV-pDCs we analyzed GrB production in pDCs used for this MLR after 48 h of pre-incubation. Concordantly to our previous results, EBV enhanced GrB production in pDCs and CpG B diminished GrB production in pDCs in the presence of IL-3. But when pDCs were incubated in the presence of IL-3 plus CpG B the addition of EBV slightly elevated GrB production again and was able to counteract the suppressive effect of CpG B on pDC-derived GrB production to a certain extent (data not shown). Therefore GrB seems to mediate at least in part the suppressive effect of EBV-pDCs on T cell proliferation. Finally we analyzed the effect of HIV-1-pre-treated pDCs on T cell proliferation. The immunostimulatory capacity of pDCs primed for 48 h with 5 ng/ml HIV-1 or CpG B in the presence of IL-3 was tested in a MLR with allogeneic CD4+ T cells. PDCs pre-incubated with HIV-1 did not relevantly alter the level of T cell proliferation compared to pDCs incubated in IL-3-supplemented medium alone, but as expected T cell proliferation was higher when pDCs were pre-incubated with IL-3 plus CpG B (figure 8H). When pDCs were pre-incubated in the presence of IL-3 with CpG B in combination with HIV-1, HIV-1 could not influence the CpG B effect on pDCs as T cell proliferation remained on a similar level as with CpG BpDCs alone (data not shown). We also analyzed GrB production in pDCs used for this MLR after 48 h of pre-incubation with different stimuli. GrB production of pDCs additionally incubated with HIV-1 was similar to those incubated with IL-3 alone. 50 PDCs incubated with CpG B or CpG B + HIV-1 in the presence of IL-3 produced both markedly less but comparable amounts of GrB (data not shown). In summary, VZV- and CMV-pDCs markedly induced CD4+ T cell proliferation, whereas EBV- and HIV-1-pDCs were not capable of provoking such a T cell response. The inhibitory effect of EBV- and HIV-1-pre-incubated pDCs seems to be mediated, at least to some extent, by GrB. 51 52 Figure 8: Herpesvirus- or human immunodeficiency virus 1-stimulated plasmacytoid + dendritic cells modulate CD4 T cell proliferation. Plasmacytoid dendritic cells (pDCs) were pre-incubated for 24 or 48 h in interleukin (IL)-3supplemented AIM-V medium (M) with a class B cytosine-phosphate-guanine oligonucleotide (CpG B) (2.5 µg/ml), varicella-zoster virus (VZV) vaccine in the concentrations indicated, UV-inactivated human cytomegalovirus (CMV) in the multiplicity of infection (MOI) 1, Epstein-Barr virus (EBV) in the indicated volume dilutions or with 5 ng/ml human immunodeficiency virus 1 (HIV). PDCs were washed and co-incubated with isolated carboxyfluorescein diacetate succinimidyl ester (CFSE)+ + stained Cluster of differentiation (CD)4 T cells in a pDCs to CD4 T cell ratio of 1:50, 1:250, 1:1250 for six days (VZV-pDCs and CMV-pDCs) or for seven days (EBV-pDCs and HIV-1-pDCs). As + controls CD4 T cells were incubated alone or with CD3/28 beads. In order to identify viable 7- + amino-actinomycin D (7-AAD) , CD3 T cells, co-cultures were harvested and either stained for CD3 and 7-AAD or gating on viable cells in the forward/side scatter dot plot was performed. T cell low proliferation was measured via flow cytometry. Proliferating T cells were determined as CFSE cells. (A) Representative dot plots and histograms for gating strategy and for controls are shown. (B+C+D) For each virus representative histograms of one experiment are shown. Data represent low viable CFSE + low CD4 T cells. (E+F) Bar graphs represent average values of CFSE + CD4 T cells after co-incubation with VZV-pDCs or CMV-pDCs from at least four donor combinations of at least low one independent experiment. (G+H) Bar graphs represent average values of CFSE + CD4 T cells after co-incubation with EBV-pDCs or HIV-1-pDCs from one independent experiment each. (E+F+G+H) Error bars indicate standard error of mean, *p-values indicate significant differences to + CD4 T cells co-cultured with pDCs pre-incubated in M. Some error bars are not shown due to the fact that some bar graphs represent average values from less than 3 donors. (E) See [47]. 53 4 Discussion 4.1 Virus-specific regulation of granzyme B in plasmacytoid dendritic cells Plasmacytoid dendritic cells are essential for linking innate and adaptive immunity in viral infections. Our group recently described pDCs as potent sources of GrB [87] and that anti-viral vaccines against ssRNA viruses modulate GrB production by pDCs [47]. In the present study we investigated the effects of different dsDNA herpesviruses, VZV, CMV and EBV, and the (+) ssRNA retrovirus HIV-1 on pDCs. Especially the potential of these four viruses to regulate GrB production and secretion by pDCs was analyzed. PDCs were routinely incubated with IL-3 as IL-3 is a potent survival factor for pDCs in cell culture [64]. IL-3 induces substantial GrB production in pDCs. But, as has previously been shown, the combination of IL-3 and IL-10 has proven to be the most potent stimulus to induce GrB production in pDCs [87]. The α-herpesvirus VZV in form of a live attenuated virus strain derived from the commercially available vaccine Varilrix® reduced GrB production and secretion by pDCs [47]. UV-inactivated human β-herpesvirus cytomegalovirus (CMV), named in this section UV-CMV, reduced GrB production and secretion by pDCs as well. In contrast, the γ-herpesvirus EBV did enhance GrB production and notably GrB secretion by pDCs. The mechanisms why herpesviruses can have a contrasting impact on pDC-derived GrB are not understood. One could speculate that IL-10 plays a role in this system. IL-10 was previously shown as a potent stimulus for GrB production in pDCs when added to IL-3 [87]. It was observed that EBV produces a homologue of human IL-10 encoded by the gene BCRFI [174], a late transcript during viral replication [82]. It may be possible that the EBV stock used for our study contained a viral (v) IL-10. This vIL-10 might be an additional factor influencing the stimulatory potential of EBV on GrB expression in pDCs. But CMV can encode a homologue of human IL-10, cmvIL-10, as well [95]. We did not rule out that cmvIL-10 might be a component of the CMV virus stock we used and might have influenced our results. Nonetheless CMV inhibited GrB production by pDCs in contrast to our previous results derived from IL-10-stimulated pDCs. In consequence, it will be important for future experiments to measure IL-10 in viral 54 stocks to determine the role of IL-10 in the regulation of GrB production and secretion by different herpesviruses. In a separate set of experiments, we analyzed the regulation of pDC-derived GrB by the (+) ssRNA retrovirus HIV-1. Our data demonstrate that HIV-1 induced a higher GrB secretion in pDCs [88] but did not alter the GrB production levels in pDCs. The difference in the results obtained by intracellular staining for GrB versus measuring of GrB in the cell-free supernatant by ELISA might be due to the different readouts in the two methods: ELISAs measure the extracellular accumulation, intracellular staining detects intracellular accumulation of GrB in pDCs only in a very short phase. For intracellular staining it is therefore more difficult to determine the optimal time point to detect possible differences in GrB production. Hence, we conclude it may be more reliable and comparable to measure GrB secretion via ELISA. Another possibility could be that HIV-1 triggers higher secretion of GrB by pDCs. Despite potential higher production of GrB in pDCs after stimulation with HIV-1 compared to stimulation with different cytokines alone, equal levels of GrB are measured inside the cells of the two culture conditions, because more GrB leaves the cell in the HIV-1-treated pDC-cultures. In favor of our result that HIV-1 increases GrB secretion by pDCs one group measured slightly increased GrB levels in the plasma of asymptomatic HIVinfected individuals (n= 25). The median GrB level was 20 pg/ml and ranged from 1-52 pg/ml. The median GrB level in plasma of 54 healthy control subjects was 11.5 pg/ml [161]. Other authors were not able to detect elevated GrB levels in HIV infection. In their study, GrB levels in plasma of HIV+ patients receiving highly active anti-retroviral therapy (HAART) or not were at the detection limit of the ELISA with 8.78 pg/ml GrB [142]. In conclusion, our results show that GrB production and secretion is virusspecifically regulated in pDCs. The mechanisms underlying this differential regulation are not known to date. Additional virus-specific factors like for example IL-10 might play a regulatory role. The detection of differences in GrB production and secretion by pDCs upon viral stimulation seems to be readout- and timepointdependent and the assessement of GrB levels in vivo in infected individuals would be interesting for the future. 55 To rule out possible cytotoxic effects of the viral stimuli on pDCs, viability assays were performed. The four viruses examined did not have relevant effects on pDC viability. These results are in line with recent findings of our workgroup that antiviral vaccines containing ssRNA viruses do not relevantly alter viability of pDCs [135]. The viral stimulus VZV was derived from a commercially available vaccine. But as expected the vaccine compounds other than viral particles had no effect on pDC GrB production and secretion, pDC phenotype and IFN-α secretion as no known vaccine adjuvant is included in the VZV vaccine formulation. In addition, our workgroup could recently show that even the common potent vaccine adjuvant aluminium hydroxide does not relevantly affect GrB production in IL-3-pDCs and does not induce IFN-α production in pDCs [47]. 4.2 Interaction of viruses with plasmacytoid dendritic cells In order to further decipher the virus-specific regulation of GrB in pDCs, it is important to shed light on the interaction of the four viruses tested with pDCs and the mechanisms of recognition of the different viral agents by pDCs. First, it is important to analyze whether pDCs are permissive for the viruses tested, in other words, whether the viruses tested can replicate in pDCs and can thereby circumvent the anti-viral host defence. Concerning the interaction of VZV and pDCs, one study analyzed DCs in biopsies of varicella or herpes zoster lesions [81]. The VZV immediate-early (IE) antigens IE62 and ORF4 were detected in some infiltrating pDCs as a hint for productive infection of pDCs by VZV. In vitro experiments supported this observation as a relevant percentage of isolated pDCs were permissive for productive infection by VZV derived from VZV-infected HFFs: After co-culture of pDCs with VZV-infected HFFs early and late VZV antigens were detected in pDCs in vitro [81]. Therefore active VZV can infect pDCs. But data for the live-attenuated varicella-zoster virus vaccine that show productive infection of pDCs do not exist. Active CMV was shown to establish a non-permissive infection in peripheral blood pDCs as only IE-antigens were expressed in pDCs [171]. Another study compared two subsets of pDCs, peripheral blood pDCs and tonsillar pDCs [154]. PDCs from peripheral blood were again non-permissive for CMV with the expression of an IE protein only, whereas surprisingly tonsillar pDCs were permissive since an IE and 56 one late protein were detected. Nonetheless, infection rates were low at 1-10% for tonsillar pDCs [154]. Consequently, we suppose that CMV and especially UV-CMV are not able to productively infect peripheral blood pDCs. EBV was shown to enter pDCs upon interaction of its gp42 protein [144] with HLADR on pDCs [144, 155]. Whether EBV is afterwards able to establish a productive infection in pDCs is to date not clear as two authors did not observe infection of pDCs by EBV [50, 144] whereas others report that even latency genes were detected in pDCs indicating productive infection of pDCs by EBV [155]. For our experimental setting it is important to note that EBV can specifically enter pDCs. The mechanism by which HIV usually interacts with susceptible cells is well described: HIV first attaches to a CD4 receptor on the target cell and then to one of the co-receptors of CD4, either to the C-C chemokine receptor (CCR) type 5 (R5-tropic HIV strains) or to the C-X-C chemokine receptor (CXCR) type 4 (R4tropic HIV strains) [56]. PDCs are per se susceptible for HIV as they express CD4 and the co-receptors CCR5 and CXCR4 on their surface [140]. HIV enters pDCs via endocytosis upon interaction of gp120 with CD4 [10]. Some authors describe a productive infection of pDCs by HIV-1 [54, 139], others deny it [33]. One study found that HIV-1-infected thymic pDCs transmitted a R5-tropic HIV strain to thymocytes and blood pDCs transmitted a R5-tropic HIV strain to PBMCs [46]. Furthermore, blood pDCs were shown to transfer both R5- as well as X4-tropic strains to CD4+ T cells after being infected themselves [114]. In conclusion, the endocytosis of HIV-1 by pDCs is well described in the literature but whether pDCs are permissive for HIV-1 infection remains elusive to date. Some data point towards productive infection of pDCs as HIV can be transmitted to other immune cells by pDCs. To summarize all the aspects, it is not certain whether the live attenuated VZV strain was able to replicate in pDCs in our setting. But certainly, the vaccine strain had measurable effects on pDCs. Peripheral blood pDCs should not be infected by CMV, and especially not by UV-CMV. Again we could detect relevant impact on pDCs by UV-inactivated CMV. The literature about infection of pDCs by EBV is not unambiguous but it is certain that EBV can enter pDCs and is thereby able to influence pDCs. Concerning HIV-1, one has to presume that pDCs get infected by this retrovirus and the expression of the relevant receptors on the surface of pDCs ensures the entry of HIV particles into pDCs. We hypothesize that permissiveness 57 of pDCs towards the different viruses additionally influences the modulation pattern of the viral agents on pDCs. Next, the potential of recognition of attenuated or UV-inactivated viruses by pDCs will be discussed. As pDCs express both TLR7 and TLR9 [89, 92], they are per se well equipped to recognize ssRNA and dsDNA viruses. As the source of VZV, we applied a live attenuated virus strain of a vaccine while the human CMV strain was UV-inactivated (in this section named UV-CMV). Literature provides evidence that viral recognition by pDCs through TLR7 and TLR9 does not require active replication of viruses in pDCs: One model shows that pDCs internalize virions into the endosomal compartment where these virions are proteolytically degraded to unmask viral RNA or DNA for recognition by endosomal TLRs [35]. In this way, viruses might be recognized without replication in pDCs even in the case of non-permissiveness of pDCs for these viruses [141]. Additionally per se replication-defective viruses, such as UVinactivated or live attenuated viruses might be recognized. A study in mice showed that recognition of viruses by TLR7 was independent of the replication of the virus. In addition, TLR signaling and IFN feedback signaling prohibited viral replication. If one of these pathways was defective, virus replicated and induced IFN production in pDCs signaling via retinoic acid-inducible gene 1 (RIG-I)-like helicase (RLH) receptors [97]. Overall, according to one review [164], RLHs do not play an essential role for the activation of pDCs. The results of an additional study show that autophagy might be another possible way of delivery of viral antigens to endosomal TLRs. Vesicular stomatitis Indiana virus (VSV) RNA replication intermediates from the cytosol of pDCs were brought to endosomes via fusion with autophagosomes [102]. Using this pathway, endosomal TLRs might not only detect pathogens from the extracellular but also from the intracellular compartment, hypothesize other authors [141]. Normally, viral recognition by pDCs happens replication-independent but some viruses might be detected replicationdependent with the help of autophagy and TLR signaling or via cytosolic RLH signaling [97]. In consequence, the live attenuated varicella-zoster virus strain and UV-CMV should be detected by pDCs in a replication-independent pathway. Third, the known signaling pathways of the viruses used in this study in pDCs are of great interest. One study with PBMCs also used Varilrix® in a volume dilution of 1:20 as a source of VZV [179]. IFN-α secretion by PBMCs was partly dependent 58 on TLR9 signaling. As UV-inactivated VZV induced less IFN-α in PBMCs, the authors concluded that other pathways than signaling via TLR9 might contribute to the recognition of VZV replication and subsequent induction of IFN-α. They found that protein kinase R (PKR), known to recognize dsRNA, was partly responsible for IFN-α secretion by PBMCs upon stimulation with VZV as the inhibitor 2-aminopurine potently abrogated IFN-α secretion in VZV-stimulated PBMCs [179]. But as all experiments in this study were conducted with PBMCs, the exact pathway responsible for IFN-α production by pDCs upon interaction with VZV cannot be concluded from the results [179]. Another study investigated the signaling pathway of human CMV in pDCs [171]. CMV induced IFN-α in pDCs via TLR9 and/or TLR7. Which endosomal TLR receptor was responsible for signaling is not clear as an antagonist for both endosomal TLR receptors was used in this study to investigate the mechanistics behind IFN-α induction in pDCs by CMV [171]. Data concering EBV showed that EBV is able to signal through TLR9 in a replication-independent manner to induce IFN-α secretion by pDCs [50, 144] but TLR7 seemed to contribute to IFN-α secretion by pDCs upon EBV-stimulation as well [50, 155]. During latency in B cells, small RNAs, the so called EBERs containing ssRNA motifs, are synthesized [107]. These molecules alone were also able to stimulate pDCs to secrete IFN-α via TLR7 and are discussed to be also responsible for signaling of EBV in pDCs via TLR7 [107]. Other authors suppose as well that autophagy plays a role in the detection of EBV by pDCs. Both theories imply that replication of EBV in the cytosol is required for recognition of the virus by pDCs [155]. The recognition of HIV-1 by pDCs is mediated by the interaction of HIV RNA with TLR7 after receptor-mediated endocytosis of the virion [10]. Interestingly, pDCs are also able to sense HIV from HIV-infected cells. Lymphocytes infected with HIV, even with replication-deficient virus, seem to be detected through TLR7 [106]. The exact mechanism is to date unknown but depends on cell contact and endocytosis processes [153]. In conclusion, literature shows that pDCs are able to recognize VZV, CMV, EBV and HIV-1 through endosomal TLR pathways. According to the results of several authors, even replication-defective viruses such as the vaccine-derived VZV and UV-CMV should be sensed by TLRs in pDCs. Therefore we assume that some of 59 our results might be TLR-dependent. In future experiments the influence of specific antagonists for TLRs on the viral effects on pDCs should be analyzed. 4.3 Modulation of plasmacytoid dendritic cell interferon-α production and phenotype by viruses As pDCs are known to be the major producers of type I IFNs in viral infections [159] we analyzed the effect of viral stimuli on IFN-α secretion by pDCs. The live attenuated VZV strain did induce potent IFN-α secretion by pDCs. This is in line with a study which tested the effect of Varilrix® on PBMCs and identified pDCs as the main producers of IFN-α upon VZV stimulation. UV-inactivated VZV was markedly less efficient in inducing IFN-α production in PBMCs [179]. In contrast to these and our observations, other authors found that pDCs co-cultured with VZVinfected HFFs secreted hardly any IFN-α [81]. Even a TLR9 agonist could not overcome the IFN-α secretion-inhibiting VZV stimulus when added to the coculture. This observation was not due to impaired viability as Annexin V/PI staining could not detect different viability of pDCs co-cultured with VZV- or mock-infected HFFs [81]. When supernatant of co-cultures of pDCs with VZV-infected HFFs was added to naïve pDCs, these pDCs produced about 5700 pg/ml IFN-α, about half of the levels we measured. When the TLR9 agonist was added to pDCs stimulated with co-culture supernatant, the IFN-α amount was approximately as high as when naïve pDCs were cultured with the TLR9 agonist alone. Consequently the supernatant of infected pDCs alone could not block TLR9 agonist-induced IFN-α production by pDCs. Thus, the authors hypothesize that VZV-infected pDCs suppressed IFN-α production in uninfected bystander pDCs in a cell-contact dependent manner [81]. The differing results for IFN-α secretion upon VZV stimulation in this study might be a consequence of the use of cell-based HFFderived VZV. The authors assume that HFF-derived VZV might express proprietary regulatory proteins upon productive infection of pDCs that could influence the IFN response [81]. In contrast, the vaccine strain we used is attenuated and should not establish productive infection in pDCs, another factor that might explain the differing results. Unfortunately the authors did not check for IFN-α or free VZV particles in the supernatant of VZV-infected HFFs that might be responsible for the induction of IFN-α production in pDCs in the supernatant 60 experiments. A role of VZV-infected HFFs for inhibition of IFN-α production in cocultured pDCs is also possible. Overall, we assume that an experimental setting with cell-free virus to rule out side effects of feeder cells is more reliable to assess the direct effect of viruses on pDCs. In our hands, UV-inactivated human CMV markedly increased IFN-α in a dosedependent manner and it was the most potent stimulus of all four viruses tested for pDC-IFN-α secretion. PDCs also produced high amounts of IFN-α in response to active CMV in another study [99]. Interestingly, others showed that IFN-α production was on a similar level whether pDCs were stimulated with CMV or UVinactivated CMV. Moreover pDCs stimulated with CMV or UV-inactivated CMV secreted comparable amounts of IL-6, TNF-α, IL-10 and CCL3 [171]. In our experiments, EBV was only able to induce minimal amounts of IFN-α. Similarly, one paper showed that EBV induces some but not potential IFN-α secretion by pDCs [144]. In addition, one group found that EBV-stimulated pDCs were also able to secrete IL-6 and IL-8 in a TLR9-dependent manner [50]. Both studies found that it made no difference concerning IFN-α amounts whether pDCs were stimulated with active or UV-inactivated EBV [50, 144]. In contrast to the relatively low amounts of IFN-α detected by these two groups and us, another group found that active EBV induced high amounts of IFN-α secretion by pDCs. Contrary to this, UV-inactivated EBV induced much less IFN-α in their hands [155]. Unfortunately, the amounts of IFN-α presented in the figures of the different papers are not directly comparable as detailed information about the experimental settings is not provided. In one study using a comparable experimental set up, EBV-stimulated pDCs secreted as in our hands only IFN-α in pg levels [108]. Of note, in this publication, the EBV stock itself contained IL-10 and pDCs stimulated with EBV produced IL-10 additionally to IFN-α [108]. When an anti-IL-10 mAb was added to the culture, IFN-α levels increased to approximately 300 pg/ml giving rise to the suspicion that IL-10 was responsible here for lower IFN-α production by pDCs. As mentioned above, we did not check for IL-10 in the EBV stock or in the supernatant of pDCs incubated with EBV. This would be quite interesting to examine as our EBV stock was produced similarly to the method reported in this study and IL-10 might also play a role in our experimental setting [108]. In our work, HIV-1 5 ng/ml p24 antigen was a slightly better stimulus than EBV to induce IFN-α secretion by pDCs, but was still not able to induce potent IFN-α 61 responses by pDCs. But pDCs co-incubated for 16 h with HIV-1-infected CD4+ T cells did produce relevant amounts of IFN-α although the exact amounts of IFN-α production in these MLRs remain elusive, as the high IFN-α values measured were out of range in our ELISA standard curve. Recently it was shown that HIV-1 might delay IFN-α production in pDCs [110]. PDCs that were maturated over night with IL-3 and consequently stimulated for 4 or 8 h with HIV-1 secreted very small amounts of IFN-α. The maximum of IFN-α production took place 24 to 48 h after stimulation with HIV-1. The peak of IFN-α production for other viruses such as influenza virus was however after 4 h. Overall, even the peak IFN-α secretion by pDCs challenged with HIV-1 was markedly lower than upon stimulation with the other viruses tested, such as influenza or Sendai virus [110]. This goes along with our observation that pDCs stimulated with HIV-1 for 16 h do not produce substantial IFN-α. One further explanation for these results might be that binding of HIV gp120 to pDCs could inhibit TLR9 signaling [118]. HIV gp120 was actually able to bind to BDCA-2 on pDCs [118] ligation of which is known to inhibit IFN production by pDCs [45]. In another study, infectious as well as non-infectious HIV-1 induced IFN-α in pDCs via TLR7 signaling and consecutive involvement of autophagy processes. 500 ng/ml p24 antigen were used for stimulation of 1 x 105 pDCs for 16 h. About 6500 pg/ml IFN-α were measured in the supernatant of likewise stimulated pDCs in this publication [181]. We used 5 ng/ml p24 antigen, a much lower concentration that is closer to actual concentrations in primary infection, to stimulate pDCs [110]. To explain the high IFN-α amounts measured in our co-cultures of HIV-1-infected CD4+ T cells with pDCs, it is important to remark again that pDCs are able to recognize HIV-1-infected lymphocytes. This process depends on interaction with CD4 on pDCs and on endocytotic mechanisms [153] finally leading to signaling via TLR7 [106]. It is known that pDCs produce markedly increased and substantial amounts of IFN-α on encounter with these HIV-1+ CD4+ T cells [153]. Overall this shows that HIV-infected T cells are much more potent stimuli for pDCs than HIV virions alone [106]. In addition, others suppose that 300-1000 ng/ml p24 are needed to induce substantial IFN-α secretion by pDCs [52]. Consequently our results for IFN-α secretion by pDCs alone or after co-culture with HIV-1-infected CD4+ T cells are plausible and in line with published data. 62 As pDCs also interact with other immune cells via their surface molecules, surface molecule expression was analyzed on pDCs incubated with the three different herpesviruses. As a pre-requisite for this analysis, it has to be taken into account that IL-3 alone has the potential to maturate pDCs and therefore influences surface molecule expression. Maturation of pDCs with IL-3 leads to upregulation of co-stimulatory molecules CD80 and CD86 on pDCs as well as to upregulation of HLA-DR [91], the maturation marker CD83 [44] and the intercellular adhesion molecule CD54 [64]. This fits our data because when pDCs were incubated with VZV [47], UV-CMV or EBV in addition to IL-3 neither any modulation of the high expression of HLA-DR nor of maturation markers on IL-3-pDCs were seen. Previous research shows that pDCs incubated with CMV in IL-3-supplemented medium could upregulate HLA-DR [99], CD 40 and CD80 [154], but the basically low expression of CD83 was not affected [99]. Another study demonstrated for pDCs incubated without IL-3 that CMV upregulated HLA-DR and the maturation marker CD83 on the surface, but not CD80 and CD86 [171]. The differing results compared to ours may be due to the fact that we used UV-CMV instead of replicable CMV and that we used IL-3 as an important survival factor for pDCs in vitro [64]. In one additional study UV-CMV did not alter surface molecule expression on IL-3-pDCs [154], a result partly in line with our data. Data on surface molecule regulation on pDCs by EBV are ambiguous and have been tested with pDCs incubated without supplementation of IL-3 [50]. However one has to take into account that the amounts of EBV applied and other experimental settings are not mentioned and probably differed in these studies. Two authors reported that co-stimulatory molecules such as CD40, CD80 and CD86 as well as HLA-DR are rather upregulated on the surface of pDCs upon challenge with EBV [50, 108]. In contrast, others found that EBV was not able to maturate pDCs efficiently, as CD80 and CD86 were hardly increased on the cell surface and pDCs secreted also less TNF-α [155]. This is in line with our data that EBV did not enhance HLA-DR expression on IL-3-pDCs. In conclusion, we suppose that pDCs rather display an immature phenotype in EBV infection. In addition, we investigated two other important molecules for the interaction of pDCs with T cells: HLA-ABC and CD54. VZV proved itself as a potent inducer of HLA-ABC expression on pDCs [47]. Even high volume dilutions induced HLA-ABC expression on pDCs. UV-CMV was also a potent inducer of HLA-ABC on pDCs. 63 By contrast, EBV did not induce HLA-ABC. An upregulation of HLA-ABC might enhance interaction of VZV- or UV-CMV-stimulated pDCs with cytotoxic CD8+ T cells. Moreover, this interaction may be supported in a particularly efficient way as pDCs are equipped with intracellular pre-formed stores of HLA-DR and of HLAABC [38]. HLA-ABC is localized within the recycling endosomal compartment. Exogenous viral antigen is taken up and loaded on HLA-ABC in a proteasomeindependent manner and without a detour through the cytosol. Furthermore, HLAABC is upregulated on the surface of pDCs within 30 min upon stimulation with influenza virus. Antigen loaded HLA-ABC is then able to mediate crosspresentation of exogenous antigen to CD8+ T cells. Therefore cross-presentation enables pDCs to induce a very rapid adaptive immune response to viral agents besides the prompt anti-viral effects of type I IFNs [38]. VZV was also able to relevantly enhance CD54 expression on pDCs [47] whereas EBV only minimally induced CD54 expression. CD54, otherwise named intercellular adhesion molecule 1 (ICAM-1), interacts with leukocyte functionassociated antigen (LFA-1) that is expressed for example on lymphocytes. The interaction of CD54 with LFA-1 is important for T cell activation. Recently, it was found that CD54 can recruit HLA-ABC molecules to the cell surface, further facilitating the activation of CD8+ T cells [101]. In line with our results demonstrating enhanced expression of CD54 upon stimulation with VZV and EBV, one study in mice showed that pDCs upregulated CD54 expression upon stimulation with TLR7 or TLR9 ligands. CD54 on pDCs contributed thereafter to the proliferation and auto-Ab secretion of auto-reactive B cells by interacting with LFA-1 on B cells [41]. Enhanced expression of CD54 on the surface of pDCs might consequently contribute to the induction of an adaptive immune response via interaction with B cells and T cells. Overall in our study pDCs reacted with a more or less immunostimulatory phenotype to the herpesviruses tested: VZV induced a robust immunostimulatory phenotype with increased expression of both HLA-ABC and CD54 on IL-3-pDCs, whereas CMV only induced HLA-ABC and EBV only minimally induced CD54 on the surface of IL-3-pDCs. We suppose that pDCs might thereby stimulate a potent adaptive immune response in VZV infection and to a smaller extent in CMV infection. One should certainly not ignore the diverse mechanisms herpesviruses have developed to undermine the immune response, especially the antigen presentation machinery [58, 83]. Nevertheless 64 this point will not be discussed here as we performed only short term cell culture of 16 h and the herpesvirus stimuli except for EBV are probably not permissively infecting peripheral blood pDCs. The impact of soluble factors in the preparation of UV-CMV and EBV cannot be ruled out as we did not check for the influence of the supernatant controls on pDC surface molecule expression. In this study, we did not analyze surface molecule regulation by HIV-1. Others found that HIV-1 did not modulate the expression of co-stimulatory molecules and CD83 on pDCs incubated with IL-3 [153]. A more recent report found that HIV-1 was sensed in early endosomes, like CpG A, inducing a rather immature phenotype of pDCs [136]. But co-culture of pDCs with phytohemagglutinin (PHA)activated CD4+ T cells or with HIV-1-infected CD4+ T cells led to maturation of pDCs. Incubation with HIV-infected T cells additionally induced upregulation of CCR7 on pDCs mediating possible migration to lymph nodes [153]. Regarding IFN-α production as well, T cell-based HIV seems to be a more potent stimulus for the activation of pDCs finally leading to the induction of an immune response [153]. 4.4 The role of plasmacytoid dendritic cells in anti-viral immunity and viral pathogenicity The role of pDCs in viral infections in the human system is to date not entirely clear. In our study we investigated the herpesviruses VZV, CMV and EBV and the retrovirus HIV-1. Here we focus on discussing data concerning the role of pDCs during viral infections with VZV, EBV, CMV and HIV-1. To date, the role of pDCs in VZV infection has not been extensively investigated. In a single case report of a 23-year-old patient with acute varicella infection peripheral blood pDCs were reduced to 0.01% of PBMCs compared to the levels of four controls with 0.48% pDCs [59]. Additionally, influx of pDCs into the dermis was detected in infected skin biopsies. Three weeks later a percentage of 0.68% pDCs was measured in the patient’s blood [59]. Another study expanded these findings. In cutaneous biopsies of patients suffering from primary VZV infection or herpes zoster, pDCs were shown to immigrate into the infected dermis and to a lower extent into the epidermis in contrast to normal uninfected skin [81]. The loss of pDCs in peripheral blood might therefore result from relocation of pDCs into the 65 infected skin where pDCs may contribute to an efficient immune response by antigen uptake and other characteristic pDC functions. Whether VZV inhibits IFN-α secretion by pDCs [81] or induces some IFN-α, like in our hands, remains elusive. But the upregulation of CD54 and HLA-ABC on the cell surface of pDCs as described in our results rather implicates the induction of a protective immune response by VZV. The reduction of GrB production by pDCs upon VZV stimulation and the induction of T cell proliferation by VZV-pDCs in the MLRs underline this thesis. The VZV vaccine is clinically quite effective and was shown to induce equally potent T cell responses as the natural infection. When VZV+ patients got vaccinated, the numbers of T cells reacting to VZV even increased in these individuals [5], a fact in line with our results of GrB inhibition in pDCs by VZV licencing T cell proliferation. The role of pDCs in the immune response against CMV infections was investigated in several publications. The observations of one group that CMVstimulated pDCs activated B cells and additional T cell help or T cell-derived IL-2 allowed Ab production by B cells [171] are in line with the immunostimulatory function of UV-CMV-stimulated pDCs in our MLRs. But in contrast to our MLR results others found that IL-3-pDCs stimulated with UV-CMV did not alter CD4+ T cell proliferation in comparison to mock-infected pDCs [154]. In their hands, IL-3pDCs stimulated with active CMV even inhibited proliferation of CD4+ T cells in MLRs, possibly via a soluble factor in the supernatant of CMV-infected pDCs [154]. Of note, pDCs were stimulated with CMV MOI 50 what might account for the diverging results as well as the different pDC:T cell ratios. Another group showed as well that pDCs stimulated with active CMV or UV-CMV limited the proliferation of allogeneic CD4+ and CD8+ T cells [171] most likely due to diverging experimental conditions. One reason for the differences to our results might be the general pre-incubation of pDCs in IL-3-supplemented medium in our experiments. Lack of IL-3 may explain the failure of CMV-stimulated pDCs to upregulate costimulatory molecules observed by others [171]. Incubation with IL-3 upregulates per se CD80 and CD86 expression on pDCs and we did not observe a modulating effect of UV-CMV on the expression of these molecules compared to stimulation with IL-3 alone. Therefore the basic incubation of pDCs with IL-3 may have provided sufficient pDC surface molecule expression for pDC-T cell interaction so that other factors produced by pDCs and regulated by CMV like for example GrB 66 were able to further modulate the pDC proliferation-inducing capacity in our experiments; a fact that might be responsible for the diverging results. One has to take in account that IL-3 might play an important role for pDC survival not only in vitro [64], but also in vivo and might thereby influence the pDC response in vivo as well. On the other hand the difference in the MOI applied may have had diverging effects as well as the different pDC:T cell ratios. Overall, in contrast to our data, literature rather indicates a low allogeneic-stimulation potential of CMV-pDCs in MLRs. Moreover, the activation status of CMV seems to play an important role for pDC activation. Active CMV should be used in further experiments as relevant differences for the interaction of UV-CMV or active CMV with pDCs are present in literature. The interaction of human CMV with the immune system and pDCs is quite complex as the herpesvirus is well adapted to its human host and has developed a lot of strategies to undermine the host’s immune response with generally low pathogenicity [112, 113]. Mechanisms taking advantage of the host’s immune response have been described for CMV such as promotion of its own replication and reactivation by provoking an inflammatory immune status via TNF-α and IFN-γ induction [25]: CMV-infected pDCs induced CD69 on and IFN-γ and TNF-α in NK cells. On the other hand, CMV-infected pDCs did not stimulate cytotoxic activity of NK cells. The authors hypothesize that CMV might thereby evade an innate immune response as NK cells were impaired in their killing function [25]. Moreover, human CMV produces an IL-10 homologue (cmvIL-10) that diminishes IFN secretion upon TLR9 stimulation in pDCs up to 75% [28]. The CMV IL-10 homologue displayed the same inhibitory effects as recombinant human IL-10 (hIL-10) [28] and is a late antigen [27]. Still, the induction of maturation by TLR agonists was not hampered by addition of cmvIL-10 or hIL-10 [28]. The expression levels of HLA-DR and co-stimulatory molecules on pDC surface remained the same and as a consequence antigen presentation capacities of pDCs should not be affected [28]. To sum up, in line with our data showing enhanced expression of HLA-ABC on UV-CMV-pDCs, pDCs seem to represent potent antigen presenting cells in the course of CMV infection independent of potential interfering viral factors. But the authors hypothesize that cmvIL-10 might influence the interaction of pDCs with other immune cells via inhibition of a potent IFN response [28]. As IL10 also decreases the viability of pDCs activated with CpG ODNs [43] and 67 because IFN-α itself is a survival factor for pDCs, the hypothesis arose that CMV might try to decrease pDC viability by producing cmvIL-10 [28]. According to the authors, CMV seems to hinder an efficient early immune response and the above mentioned mechanisms might contribute to latency establishment of CMV [28]. This assumption is supported by the fact that pDC numbers in peripheral blood were diminished in immunocompetent patients suffering from a symptomatic primary mononucleosis-like CMV infection [172]. In conclusion, peripheral blood pDCs might normally have a protective role in CMV infection as their reduction was associated with symptomatic disease. PDCs could therefore be a factor influencing the wide range of pathogenicity of CMV infections. The reason why CMV infection is mostly asymptomatic in immunocompetent individuals remains elusive. One possible mechanism by which CMV promotes a sufficient anti-viral immune response is suppression of pDC-derived GrB as shown in our data. Moreover, certain individual factors such as biological age determine the outcome of an anti-CMV immune response. It was observed that IFN-α derived from pDCs stimulated with CMV could reduce telomerase activity in and induce loss of the costimulatory molecules CD27 and CD28 on CD4+ CMV-specific T memory cells [53]. The consequence might be a loss of these CMV-specific memory cells. The authors suggest that this process could take place in T cell areas of lymph nodes. CMV reactivation and consequent IFN-α production by pDCs could thereby affect longevity of other memory T cells as well. To sum up, these results show that the interaction of CMV with pDCs might influence the composition of adaptive immune cells and further underlines the relevance of the often asymptomatic CMV infection for carriers [53]. Overall the effects of CMV on the innate and adaptive immune system and especially on pDCs are diverse and need to be further elucidated as literature on CMV effects on immunity is quite controversial and the interactions are quite complex. We suppose that pDCs could play a protective role in the immune response against CMV. Based on our results we assume that suppressed GrB expression in peripheral blood pDCs might favor a protective T cell response against CMV infections and may keep CMV in check in CMV+ individuals at least in middle-aged humans. But as literature provides more evidence for an inhibiting effect of pDCs on T cell proliferation in CMV infection it would be interesting to 68 investigate the effects of active CMV on GrB production and the immunostimulatory potential of pDCs. Next, the role of pDCs in EBV infection shall be analyzed. It is still questioned whether EBV can infect pDCs or not. Some authors report that pDCs secreted moderate to low amounts of IFN-α in a replication-independent manner and upregulated co-stimulatory molecules on their surface upon EBV stimulation [50, 144]. Others showed permissiveness of pDCs to EBV and report that EBV induced high amounts of IFN-α whereas UV-inactivated EBV was only able to induce very little IFN-α in pDCs which was interpreted as a need for viral replication in the cytosol [155]. Autophagy seemed to be involved [155]. In addition, EBV induced only minor maturation in pDCs. Even the inducing effect of CpG C on pDC maturation was diminished by EBV [155]. These results are in line with our data indicating that CpG B-pDCs additionally incubated with EBV displayed a lower allogeneic stimulation potential in MLRs with T cells than pDCs incubated with CpG B alone. Moreover, pDCs stimulated with EBV expressed the regulatory molecules inducible T cell co-stimulatory-ligand (ICOS-L) and B7 homolog 1 (B7H1) on their surface leading to a reduced allogeneic CD4+ T cell answer [155]. In conclusion, these results imply that EBV is able to infect pDCs and to impair their potential to induce potent CD4+ T cell responses [155]. This effect might be mediated by the enhanced expression of inhibitory molecules such as ICOS-L [85] and B7-H1 [20] on the surface of pDCs at the expense of a sufficient expression of co-stimulatory molecules. This interaction with pDCs might favor the establishment of viral persistence [155]. Taken together, literature provides controversial data on EBV-incubated pDCs, either showing high or low IFN-α production, probably depending on the specific experimental settings. Most data point to a rather immunoregulatory phenotype of pDCs after EBV stimulation, including our own results showing a rather low allogeneic stimulation potential of EBV-pDCs. Nonetheless, it is well established that cellular immunity is decisive for the control of EBV infection. Consequently the induction of Ag-specific T cells is essential besides the activation of NK cells [129]. In contrast to the rather regulatory role of pDCs in the course of EBV infection described above, another group found that pDCs stimulated with EBV induced IFN-γ in CD3+ T cells in a TLR9-dependent 69 manner and additionally activated NK cells. Both processes were based on cell contact of pDCs with NK cells or T cells [108]. To summarize, contradictory data exist concerning the immunostimulatory potential of pDCs activated by EBV. Our results indicate a low immunostimulatory potential of EBV-activated pDCs going along with high GrB secretion levels. Interestingly, one group reported that GrB levels were elevated in the blood serum of patients suffering from EBV infection in different disease stages. GrB levels derived from 14 patients ranged from 1-4000 pg/ml with a median of 60 pg/ml. Despite these high inter-individual differences in GrB levels, the tendency was derivable, that high GrB levels were detected in patients suffering from acute mononucleosis and that these high GrB levels went down to normal levels after the acute phase of the infection. In this study the median GrB level of 54 healthy control subjects was measured with 11.5 pg/ml [161]. In conclusion, EBV can induce a rather immature phenotype in pDCs thereby hampering an efficient EBV immune response in particular after higher GrB induction. It remains to be determined to what extent high GrB secretion by pDCs stimulated with EBV is decisive for the clinical presentation of EBV infection. Factors produced by EBV upon infection such as vIL-10 may play a modulating role. Moreover, EBV could try to limit T cell responses not only by inducing high GrB levels in pDCs but also by enhancing the expression of other immunoregulatory molecules such as ICOS-L and B7-H1 on the surface of pDCs. Overall, it will be interesting to further elucidate the mechanisms responsible for the variable pathogenicity of EBV infection. One mechanism might be GrB regulation in pDCs as higher GrB levels may go along with higher pathogenicity of EBV infection. The most important features of chronic HIV infection and progression to AIDS are dysfunction, essentially the progressive loss of CD4+ T cells, but also hyperactivation of the host’s immune system. The role of pDCs in immunopathogenesis of HIV infection is complex and their potential to secrete high amounts of IFN-α is regarded as ambiguous concerning both a protective and a detrimental potential of this cytokine in the course of HIV infection. Lots of data are in favor of a rather protective role of pDCs in the course of HIV infection: 70 First, there is a loss of pDCs in the peripheral blood of patients suffering from advanced HIV-1 infection. Furthermore decreased pDC numbers were associated with elevated viral load and an increased appearance of opportunistic infections. These observations were seen as a hint for a protective role of pDCs in the course of HIV infection [138, 160]. Furthermore it was shown that pDC numbers were already decreased in the peripheral blood in early, primary HIV-1 infection [80, 151]. In some studies, the decline of pDCs in peripheral blood was accompanied by the accumulation of pDCs in lymphoid tissues, perhaps representing a redistribution of pDCs. CCL19 interacted with CCR7 that was upregulated on pDCs in HIV infection [55] and mediated migration of pDCs to secondary lymphoid tissues [51] where they might undergo apoptosis as described for pDCs in simian immunodeficiency virus (SIV) infection [21]. Accumulated pDCs in lymph nodes were immature [40] and secreted high amounts of IFN-α [105]. PDCs also accumulated in the spleen of viremic patients but there, surprisingly, they did not represent the main source of IFN-α [132]. Other reports found that classical DCs as well as pDCs were lost in lymph nodes of HIV patients in the chronic phase [13]. In another publication, pDCs were preserved in lymph nodes from patients in different disease stages and pDC numbers were comparable to those in lymph nodes of healthy controls [103], further contradicting the thesis of pDC loss due to redistribution. Another explanation for pDC loss might be that HIV-1 itself induced direct cell death in pDCs after fusion of the virion with pDCs [124]. The reason for the pDC loss in HIV infection consequently remains elusive. Besides, pDC function seems to be impaired in HIV-infected individuals: PBMCs from HIV-1-infected patients stimulated in vitro with TLR7 agonists secreted less IFN-α than PBMCs from healthy individuals. Intracellular staining revealed that less pDCs produced IFN-α [119]. HIV gp120 was shown to bind to BDCA-2 on pDCs and inhibited IFN-α, TNF-α and IL-6 secretion, NK cell stimulating capacity as well as CD83 expression by pDCs upon stimulation with a TLR9 agonist but not upon stimulation with a TLR7 agonist [118]. These results imply that HIV interferes also with the capacity of pDCs to respond to TLR9 signals such as other viral agents or bacteria [118] possibly facilitating opportunistic infections. Several authors showed that the crosstalk of pDCs with NK cells was affected [34, 145] and hence activation of NK cells by pDCs was impaired in HIV-1 infection [11]. One factor might be HIV viral protein R (Vpr) that was shown to inhibit type I IFN 71 production by pDCs [79]. What is more, HAART was not able to completely rescue pDC function and numbers, neither in adults [29, 49] nor in children [180]. The ability of HIV to hamper pDC functions is reflected in our data showing low IFN-α secretion by pDCs stimulated with HIV-1 and a low allogeneic stimulation potential of HIV-1-pDCs in MLRs with CD4+ T cells. On the contrary, a protective role of pDCs in HIV infection in some cases is underlined by the fact that HIV-1 maturated pDCs and enabled pDCs to activate and maturate bystander myeloid DCs and CD4+ T cells [55]. In addition, HIVantigen coupled to a lipopeptide or derived from HIV-infected apoptotic cells could be cross-presented by pDCs to CD8+ T cells [78]. Consequently pDCs seem to be able to induce adaptive immune responses against HIV under certain conditions. In elite controllers, representing HIV patients able to suppress viral replication under detectable levels for long time periods, a recent study found retained pDC numbers and IFN-α-producing capacity. Moreover pDCs were able to diminish viral replication and could bring about apoptosis of T cells [116]. PDCs from healthy individuals stimulated with HIV-1+ CD4+ T cells secreted IFN-α and were able to suppress viral replication in these T cells in an IFN-α-dependent manner [153]. CpG A-stimulated pDCs from HIV-1-infected patients limited viral replication in autologous CD4+ T cells in a partially IFN-α-dependent manner. Interestingly, even unstimulated pDCs from HIV-1-infected patients with low viral load were able to limit viral replication in autologous CD4+ T cells in a cell contact-dependent manner [124] further supporting a protective role of pDCs in HIV. Thus in some patients pDCs can help to control HIV infection. But there is also evidence that HIV abuses pDCs for its own advantage, manipulating pDC functions in a rather detrimental and pathogenic manner, especially in chronic disease stages. Currently immune hyperactivation is seen as one of the major causes of HIV progression and pathogenicity. Surprisingly enough, HIV infection in women develops faster into AIDS than in men relative to viral load [122]. Differences in the response of pDCs to TLR7 stimuli such as HIV-1 have been detected as female pDCs responded with higher IFN-α production [122]. Furthermore CD8+ T cells showed a more activated phenotype in women compared to men for the same viral load. Overall a higher immune activation seemed to contribute to faster disease progression in female HIV 72 patients [122], underlining the relevance of overactivation of the immune system in HIV pathogenesis. The exact role of IFN-α in HIV immunopathogenesis is quite controversial and diverging results are reported. In one study peripheral blood pDCs from patients in early stages of HIV infection were hyperresponsive to stimulation with a TLR7/8 agonist producing higher levels of diverse chemokines and cytokines, including IFN-α, in contrast to pDCs from uninfected controls [151]. Another study presented data that pDCs expressed less IFN-α per cell in early HIV infection [80]. The differences might be due to use of different methods. Another group showed that PBMCs from HIV-infected patients displayed a markedly higher expression profile of IFN-α compared to PBMCs from healthy individuals [103]. Especially the subtype IFN-α2 was increased in PBMCs from patients with HIV infection inversely correlating with CD4+ T cell counts [104]. On the other hand, lymphoid tissue mononuclear cells did not present higher IFN-α messenger (m) RNA levels in HIV patients [103]. Therefore, IFN-α levels in HIV infection show great variability, what may reflect different stages of infection. Our data support the assumption that poor IFN-α induction correlates with generally high viral pathogenicity as is the case in typical HIV infection. In conclusion the role of pDCs in HIV-1 infection still needs to be further elucidated and clarified. On one hand, pDCs might control viral replication in the early infection phase via IFN-α secretion and might induce a protective adaptive immune response against HIV-1. On the other hand, IFN-secreting pDCs might contribute to immune hyperactivation in lymph nodes leading to the loss of T cells in the chronic phase of infection. Another possibility is that pDCs represent a tolerogenic population limiting T cell proliferation that is lost in chronic infection and cannot limit the overactivation of the immune system anymore. As described in the following section, GrB might contribute to this regulatory function with limiting T cell expansion upon encounter with virus. Via inhibition of uncontrolled T cell proliferation GrB-secreting pDCs might potentially protect T cells from being killed. 73 4.5 Granzyme B and tolerogenic plasmacytoid dendritic cells in antiviral immunity PDC-derived GrB has been shown to limit T cell proliferation in a cell contactdependent but perforin-independent manner [87]. Anti-viral vaccines against ssRNA viruses inhibited GrB in pDCs and were therefore able to induce CD4+ T cell proliferation [47]. Concordantly to the results of our work that VZV and UVCMV inhibit GrB production and secretion by pDCs, proliferation of CD4+ T cells was enhanced when pDCs were pre-stimulated with VZV or CMV. However, EBV enhanced GrB production and secretion by pDCs. In MLR with CD4+ T cells and EBV-pre-treated pDCs, proliferation of allogeneic T cells was on the same low level whether pDCs were pre-incubated only with IL-3 or additionally with EBV in increasing volume dilutions. When pDCs were pre-incubated with CpG B and EBV, EBV even slightly decreased the proliferation-inducing potential of CpG BpDCs. This finding was accompanied by a slight increase in GrB production in pDCs pre-incubated with CpG B and EBV compared to CpG B stimulation alone (data not shown). Likewise HIV-1, another positive regulator for GrB secretion by pDCs, did not further modulate the effect of IL-3 alone-pre-incubated pDCs on allogeneic T cell proliferation. For UV-CMV, EBV and HIV-1 only one independent MLR was performed although with different donors. Consequently, it is difficult to derive a real statement from these results. But the different direction of GrB regulation in pDCs by viruses seems to inversely correlate with the effect of virusstimulated pDCs on CD4+ T cell proliferation in MLRs. Therefore one has to discuss which role pDC-derived GrB might play during viral infections. We hypothesize that pDC-derived GrB has a regulatory function. This is in line with reports that pDCs exert also other immunoregulatory functions. IL-3 + CD40L-stimulated pDCs could prime naïve CD4+ [85] and CD8+ T cells [60] to become regulatory T cells. PDCs incubated with different classes of CpG ODNs primed CD4+ Tregs [85, 128]. The mechanisms behind these tolerogenic functions might be additional immunoregulatory molecules. CpG ODN-stimulated pDCs expressed IDO and were able to induce inducible Tregs via this mechanism [30]. In another study pDCs expressed ICOS-L upon maturation and induced IL-10 expressing regulatory CD4+ T cells [85]. Interactions like that have been also described for natural viral ligands. For example, HIV enhanced IDO expression on 74 pDCs in a TLR7-dependent manner [117]. These pDCs were able to induce the generation of suppressive T cells from naïve allogeneic CD4+ T cells. These Tregs inhibited CD4+ T cell proliferation and classical DC maturation. The authors consequently attributed an immunoregulatory function to pDCs in the course of HIV infection [117]. Other authors support this finding. In their hands HIV-1 also induced IDO in pDCs that inhibited the proliferation of both CD4+ and CD8+ T cells [17, 18]. In allogeneic MLRs with pDCs derived from HIV-infected individuals pDCs showed equal stimulation capacity compared to pDCs from healthy subjects. But pDCs from highly viremic, acutely infected patients were not as efficient in stimulation of allogeneic T cell proliferation [151]. Another study supported this finding by showing a decreased potential of pDCs from HIV-infected individuals to promote proliferation of allogeneic HIV-, enriched T cells [42] maybe representing a protective mechanism against uncontrolled T cell activation mediated by pDCs. Summing up, our data showing elevated GrB secretion by pDCs upon challenge with HIV-1 and a limitation of CD4+ T cell proliferation in MLRs promote the hypothesis of pDCs being a tolerogenic population in HIV-1 infection that may hamper efficient T cell responses but can also limit hyperactivation of the immune system and concomitantly viral spread. Apart from pDCs incubated with HIV, pDCs stimulated with EBV can also express the immunoregulatory molecule ICOS-L on their cell surface [155]. Moreover, pDCs stimulated with HSV-1 induce regulatory IL-10 and IFN-γ-producing CD4+ T cells via IFN-α. Besides their suppressive function these T cells acquired cytotoxic functions by expressing GrB and perforin. By inducing regulatory T cells, virusstimulated pDCs might not only contribute to a contraction of the immune response but as a possible negative side effect also to the establishment of persistent viral infections [93]. PDCs stimulated with CMV induced IL-10 secretion in CD4+ memory T cells that were consecutively also able to produce IFN-γ. Such stimulated T cells were able to suppress allogeneic T cell proliferation and proliferated well themselves. These results implicate that pDCs are even able to control memory T cell responses. The theory of the authors is that pDCs allow Agspecific memory T cells to control the expansion of naïve T cells at sites of reinfection via IL-10 in favor of the expansion of Ag-specific memory T cells in early times of infection [100]. To sum up, pDCs seem to be able to orchestrate a lot of tolerogenic T cell responses especially in the course of viral infections. Overall 75 these tolerogenic properties might limit detrimental immune system overactivation and might guide specific T cell responses. The limitation of an immune response might on the other hand also facilitate the establishment of latent infections. We suppose a regulatory role of pDC-derived GrB that represents one possible effector molecule produced by pDCs to orchestrate immune responses. Of note, in contrast to our hypothesis of tolerogenic pDC-derived GrB, elevated GrB levels in the plasma are seen by some authors as a sign of immune activation. In their opinion GrB might promote inflammatory disease progression [4] and an elevation of GrB in the extracellular space might represent an overactivated immune system [1] which would be in line with the theory of detrimental immune system hyperactivation as shown for HIV infection. The exact role of pDCs in viral immunity needs further elucidation and especially their regulatory potential that might be in part mediated by GrB should be further analyzed. To sum up, our hypothesis that pDC-derived GrB is virus-specifically regulated was confirmed. T cell proliferation was modulated according to GrB levels produced by pDCs. The suppression of pDC-derived GrB by VZV and UV-CMV might facilitate the induction of a potent T cell response. EBV might profit from GrB elevation to hamper a rapid adaptive immune response. In the course of HIV-1 infection high GrB levels could represent a possible regulatory mechanism by which pDCs limit uncontrolled T cell proliferation. On the other hand, HIV-induced GrB induction may contribute to a failure of a HIV immune response which correlates with severe disease in most untreated individuals. In conclusion, pDCderived GrB seems to represent a tightly regulated molecule in anti-viral immunity. GrB produced by pDCs may inhibit uncontrolled proliferation of T cells, a favorable effect, but beyond that can lead to unfavorable immune suppression. 76 4.6 Outlook In the face of our data presented in this work and with regard to the discussed literature many questions remain to better understand the role of pDCs in anti-viral immunity. In the future it would be first of all interesting to further investigate the signaling pathways of different viral agents in pDCs leading to GrB production. This could be addressed with the help of specific TLR antagonists. The role of IL-10 that might be included in viral preparations but could also be produced by pDCs and infected bystander cells should be evaluated. One factor that might additionally influence our results in pDC cultures as well as in MLRs might be earlier viral infections reflected by the serostatus of the blood donors which was unknown. It would be interesting to analyze the serostatus of blood donors and to investigate whether this parameter has an impact on pDC responses or MLRs. Furthermore, the phenotype of CD4+ T cells co-cultured with virus-pre-treated pDCs should be specified. In particular the expression of regulatory molecules such as IDO and ICOS-L on pDCs should be measured. Besides, the effect of virus-stimulated pDCs on CD8+ T cells should be tested in MLRs. In addition, the exact mechanism by which GrB limits T cell proliferation needs to be elucidated beyond the finding of cleavage of the TCR ζ-chain [47]. 77 5 Summary Plasmacytoid dendritic cells (pDCs) are a rare but specialized immune cell subset that represents an important link between innate and adaptive immunity. The most known function of pDCs is their capability to respond with high amounts of type I interferons (IFNs) to viral stimuli. Through the expression of Toll-like receptor (TLR)7 and TLR9 pDCs are well equipped to respond to single-stranded ribonucleic acid (ssRNA) viruses such as human immunodeficiency virus 1 (HIV1) and double-stranded deoxyribonucleic acid (dsDNA) viruses such as the herpesviruses varicella-zoster virus (VZV), cytomegalovirus (CMV) and EpsteinBarr virus (EBV). Despite their immunogenic functions, especially in anti-viral immunity, pDCs are also known to exert tolerogenic functions. PDCs were found to express the serine protease granzyme B (GrB) in the past only known as the apoptosis-inducing effector protease of natural killer cells and cytotoxic T lymphocytes. Recent years of research have revealed new sources of GrB, such as pDCs, and have attributed many new functions to this molecule. PDC-derived GrB was shown to suppress T cell proliferation indicating that a regulatory mechanism is mediated by this molecule. In this study we evaluated the effects of diverse viral stimuli on human pDCderived GrB, the phenotype and function of human pDCs. The viruses investigated establish latency or chronic infection and adaptive immunity plays an important role for virus control. We measured GrB production and secretion as well as surface molecule expression and IFN-α secretion by pDCs upon stimulation with vaccine-derived live attenuated VZV, ultraviolet light (UV)-inactivated CMV, replicable EBV and HIV-1 with flow cytometry and enzyme-linked immunosorbent assays (ELISAs). Furthermore we analyzed the influence of virus-stimulated pDCs on proliferation of co-incubated carboxyfluorescein diacetate succinimidyl ester (CFSE)-labelled Cluster of differentiation (CD)4+ T cells in mixed lymphocyte reactions (MLRs). PDCs and T cells were isolated with magnetic beads from buffy coats or from fresh blood of healthy donors. GrB production and secretion were differentially regulated by the viral stimuli. VZV and CMV diminished GrB production in pDCs whereas EBV and HIV-1 increased GrB secretion indicating that GrB expression by pDCs is virus-specifically regulated. Robust IFN-α production was only observed for VZV and CMV. 78 In contrast, EBV and HIV-1 induced only very low levels of IFN-α. On the contrary, HIV-1-infected T cells led to relevant IFN-α secretion by pDCs in pDC-T cell cocultures. The herpesviruses VZV and CMV, but not EBV and HIV-1 induced a rather immunogenic phenotype in pDCs. CMV and VZV upregulated human leukocyte antigen (HLA)-ABC, VZV additionally CD54, on the cell surface of pDCs enabling better interaction with T cells and cross-presentation of viral antigens. CMV- and VZV-pre-stimulated pDCs were able to enhance T cell proliferation in MLRs whereas EBV and particularly HIV-1 failed to provoke significant T cell proliferation. We conclude that the effective adaptive immune response of healthy individuals to VZV might be supported by suppression of GrB in pDCs and the induction of an immunostimulatory phenotype on pDCs. Similarly, in CMV infection, peripheral blood pDCs could license CD4+ T cell responses by low GrB secretion levels. Of note, we examined UV-inactivated CMV. In fact, literature provides more evidence that pDCs stimulated with active CMV rather limit T cell proliferation. In our hands, EBV- and HIV-1-incubated pDCs did not enhance T cell proliferation, an observation that might be partly dependent on GrB induction in pDCs. Via elevation of pDC-derived GrB EBV might dampen a rapid protective immune response allowing viral spread and establishment of latency supported by the elsewhere published observation of relatively high GrB levels in active mononucleosis. In HIV infection, there is evidence that elevated GrB secretion by HIV-1-incubated pDCs might inhibit uncontrolled T cell activation and hyperinflammation limiting HIV disease progression but on the other hand might also represent one mechanism of progressive disease. Overall our MLR results inversely correlated with the regulation of GrB in pDCs and support the finding that pDC-derived GrB inhibits T cell proliferation. The data implicate that pDC-derived GrB plays a role in anti-viral immunity as it is virus-specifically regulated. Virusspecific lower pDC-GrB levels might rather facilitate, high levels might limit T cell responses. Further research is needed to elucidate the role of CD4+ T cell subsets primed by virus-stimulated pDCs and the effect on CD8+ T cells. Our findings contribute to further understand the interaction of viruses with pDCs. 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