Humane Dendritische Zellen: Identifizierung und funktionelle Charakterisierung von neuen Oberflächenantigenen

DC sind APC, deren Hauptfunktion in der Antigenaufnahme und Induktion T-Zell-abhängiger Immunantworten besteht. In den letzten Jahren wurden viele neue Subpopulationen von DC beschrieben, was die hohe Heterogenität dieser Population deutlich macht. Die einzelnen Subpopulationen unterscheiden sich nicht nur in ihrem Phänotyp, sondern auch in ihrer Morphologie, Funktion und Gewebeverteilung. Die charakteristische Funktion einzelner Subpopulationen wird größtenteils durch die Oberflächenrezeptoren bestimmt, über die die Zellen mit ihrer Umgebung in Verbindung stehen. Um die Rolle der DC innerhalb des Immunsystems und die funktionellen Unterschiede zwischen den einzelnen DC-Subpopulationen besser verstehen zu können, ist es daher notwendig, diese Moleküle genau zu erforschen. In der vorliegenden Arbeit wurde mit Hilfe der kontralateralen Immunisierung eine Reihe von monoklonalen Antikörpern generiert, die drei BDC-spezifische Oberflächenantigene erkennen: BDCA-2, BDCA-3 und BDCA-4. BDCA-3 wird von CD11c+ CD123- myeloiden BDC ausgeprägt und ist identisch mit Thrombomodulin, einem Rezeptor des vaskulären Endothels, der mit Thrombin 1:1 Komplexe bilden kann und somit die Funktion eines natürlichen Antikoagulans besitzt. BDCA-2 und BDCA-4 werden im Gegensatz zu BDCA-3 von CD11c- CD123++ PDC ausgeprägt. Bei BDCA-4 handelt es sich um Neuropilin-1, einen neuronalen Rezeptor, der bei der Ausbildung von Axonen eine wichtige Rolle spielt. Zusätzlich wird Neuropilin-1 neben den PDC auch von endothelialen und Tumor-Zellen als Rezeptor für den vaskulären, endothelialen Wachstumsfaktor-A (VEGF-A) ausgeprägt und spielt eine Schlüsselrolle in der Angiogenese. Weiterhin wird die Ausprägung von BDCA-4 auf CD11c+ BDC, Monozyten und T-Zellen durch Stimulation bzw. in vitro-Kultivierung induziert. Die Funktion von BDCA-3 und BDCA-4 im Fall der BDC ist allerdings noch nicht bekannt. Ausprägungsklonierung von BDCA-2 ergab, dass es sich bei diesem Molekül um ein neues Typ II c-Typ-Lektin handelt, das eine 50,7%-ige Übereinstimmung der AS-Sequenz mit seinem murinen Ortholog, Dectin-2, aufweist. Das Ausprägungsmuster von BDCA-2 ist in allen bisher untersuchten Geweben ausschließlich auf die CD11c- CD123++ PDC beschränkt. Die Tatsache, dass anti-BDCA-2 mAk nach ihrer Bindung an BDCA-2 sofort internalisiert, prozessiert und über MHC Klasse II-Moleküle präsentiert werden, deutet daraufhin, dass BDCA-2 bei der Antigenaufnahme und -präsentation eine Rolle spielen könnte. Weiterhin induziert die Ligation von BDCA-2 mit dem spezifischen mAk AC144 eine nahezu vollständige Inhibition der Sekretion von IFN-a/b. Da IFN-a/b die Produktion von IL-12 inhibiert, verstärkt die Ligation von BDCA-2 gleichzeitig die CD40L-abhängige IL-12-Sekretion bei PDC. Aus diesem Grund wird durch die Ligation von BDCA-2 weniger die Polarisation der PDC-induzierten T-Zell-Antwort beeinflusst, sondern vielmehr ein Wechsel von IFN-a/b-kontrollierten zu IL-12-kontrollierten Immunität induziert. Da IFN-a/b und IL-12 sehr vielfältige und zum Teil ähnliche Funktionen ausüben, sind die in vivo-Folgen einer BDCA-2-Ligation nur sehr schwierig vorherzusagen. Gleichwohl macht die Tatsache, dass IFN-a/b eine wichtige Rolle bei einer Reihe von autoimmunologischen Erkrankungen spielt, BDCA-2 sehr interessant als Ziel zukünftiger Therapien

Organ-dependent in vivo priming of naive CD4+,but not CD8+,T cells by plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) play a pivotal role as cytokine-secreting accessory cells in the antimicrobial immune defense. In contrast, the capacity of PDCs to act as antigen-presenting cells in naive T cell priming remains unclear. By studying T cell responses in mice that lack conventional DCs (cDCs), and by the use of a PDC-specific antigen-targeting strategy, we show that PDCs can initiate productive naive CD4+ T cell responses in lymph nodes, but not in the spleen. PDC-triggered CD4+ T cell responses differed from cDC-driven responses in that they were not associated with concomitant CD8+ T cell priming. Our results establish PDCs as a bona fide DC subset that initiates unique CD4+ Th cell–dominated primary immune responses

Complement-opsonized nano-carriers are bound by dendritic cells (DC) via complement receptor (CR)3, and by B cell subpopulations via CR-1/2, and affect the activation of DC and B-1 cells

The development of nanocarriers (NC) for biomedical applications has gained large interest due to their potential to co-deliver drugs in a cell-type-targeting manner. However, depending on their surface characteristics, NC accumulate serum factors, termed protein corona, which may affect their cellular binding. We have previously shown that NC coated with carbohydrates to enable biocompatibility triggered the lectin-dependent complement pathway, resulting in enhanced binding to B cells via complement receptor (CR)1/2. Here we show that such NC also engaged all types of splenic leukocytes known to express CR3 at a high rate when NC were pre-incubated with native mouse serum resulting in complement opsonization. By focusing on dendritic cells (DC) as an important antigen-presenting cell type, we show that CR3 was essential for binding/uptake of complement-opsonized NC, whereas CR4, which in mouse is specifically expressed by DC, played no role. Further, a minor B cell subpopulation (B-1), which is important for first-line pathogen responses, and co-expressed CR1/2 and CR3, in general, engaged NC to a much higher extent than normal B cells. Here, we identified CR-1/2 as necessary for binding of complement-opsonized NC, whereas CR3 was dispensable. Interestingly, the binding of complement-opsonized NC to both DC and B-1 cells affected the expression of activation markers. Our findings may have important implications for the design of nano-vaccines against infectious diseases, which codeliver pathogen-specific protein antigen and adjuvant, aimed to induce a broad adaptive cellular and humoral immune response by inducing cytotoxic T lymphocytes that kill infected cells and pathogen-neutralizing antibodies, respectively. Decoration of nano-vaccines either with carbohydrates to trigger complement activation in vivo or with active complement may result in concomitant targeting of DC and B cells and thereby may strongly enhance the extent of dual cellular/humoral immune responses

Microbiota Sensing by Mincle-Syk Axis in Dendritic Cells Regulates Interleukin-17 and -22 Production and Promotes Intestinal Barrier Integrity

We are grateful to members of the D.S. laboratory and Dr. E. Fernández-Malavé for discussions and critical reading of the manuscript. We appreciate the support of A. Tomás-Loba, G. Sabio, P. Martín, A. Tsilingiri, A.R. Ramiro, C.L. Abram, C.A. Lowell, J.M. García-Lobo, M. Molina, and M.C. Rodríguez for providing reagents and support. We thank the staff at the Fundación Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC) facilities for technical support. M.M.-L. received a Formación de Personal Universitario (FPU) fellowship (AP2010-5935) from the Spanish Ministerio de Educación. S.I. is funded by grant SAF2015-74561-JIN from the Spanish Ministerio de Ciencia, Innovación, y Universidades (MCIU) and Fondos Europeos de Desarrollo Regional (FEDER). G.D.B and D.M.R. are supported by the Wellcome Trust and the MRC Centre for Medical Mycology at the University of Aberdeen. S.L.L. is supported by the Swiss National Science Foundation (PP00P3_150758). Work in the D.S. laboratory is funded by the CNIC and grant SAF2016-79040-R from MCIU, the Agencia Estatal de Investigación, and FEDER; B2017/BMD-3733 Immunothercan-CM from Comunidad de Madrid; RD16/0015/0018-REEM from FIS-Instituto de Salud Carlos III, MCIU, and FEDER; the Acteria Foundation; the Constantes y Vitales prize (Atresmedia); La Marató de TV3 Foundation (201723); the European Commission (635122-PROCROP H2020), and the European Research Council (ERC-2016-Consolidator Grant 725091). The CNIC is supported by the MCIU and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (SEV-2015-0505).Peer reviewedPublisher PD

The clinical application of cancer immunotherapy based on naturally circulating dendritic cells

Dendritic cells (DCs) can initiate and direct adaptive immune responses. This ability is exploitable in DC vaccination strategies, in which DCs are educated ex vivo to present tumor antigens and are administered into the patient with the aim to induce a tumor-specific immune response. DC vaccination remains a promising approach with the potential to further improve cancer immunotherapy with little or no evidence of treatment-limiting toxicity. However, evidence for objective clinical antitumor activity of DC vaccination is currently limited, hampering the clinical implementation. One possible explanation for this is that the most commonly used monocyte-derived DCs may not be the best source for DC-based immunotherapy. The novel approach to use naturally circulating DCs may be an attractive alternative. In contrast to monocyte-derived DCs, naturally circulating DCs are relatively scarce but do not require extensive culture periods. Thereby, their functional capabilities are preserved, the reproducibility of clinical applications is increased, and the cells are not dysfunctional before injection. In human blood, at least three DC subsets can be distinguished, plasmacytoid DCs, CD141+ and CD1c+ myeloid/conventional DCs, each with distinct functional characteristics. In completed clinical trials, either CD1c+ myeloid DCs or plasmacytoid DCs were administered and showed encouraging immunological and clinical outcomes. Currently, also the combination of CD1c+ myeloid and plasmacytoid DCs as well as the intratumoral use of CD1c+ myeloid DCs is under investigation in the clinic. Isolation and culture strategies for CD141+ myeloid DCs are being developed. Here, we summarize and discuss recent clinical developments and future prospects of natural DC-based immunotherapy

Efficient virus assembly, but not infectivity, determines the magnitude of hepatitis C virus-induced interferon alpha responses of plasmacytoid dendritic cells.

Worldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV

Plasmacytoid dendritic cells respond to Epstein-Barr virus infection with a distinct type I interferon subtype profile

Infectious mononucleosis, caused by infection with the human gamma-herpesvirus Epstein-Barr virus (EBV), manifests with one of the strongest CD8$^{+}$ T-cell responses described in humans. The resulting T-cell memory response controls EBV infection asymptomatically in the vast majority of persistently infected individuals. Whether and how dendritic cells (DCs) contribute to the priming of this near-perfect immune control remains unclear. Here we show that of all the human DC subsets, plasmacytoid DCs (pDCs) play a central role in the detection of EBV infection in vitro and in mice with reconstituted human immune system components. pDCs respond to EBV by producing the interferon (IFN) subtypes α1, α2, α5, α7, α14, and α17. However, the virus curtails this type I IFN production with its latent EBV gene products EBNA3A and EBNA3C. The induced type I IFNs inhibit EBV entry and the proliferation of latently EBV-transformed B cells but do not influence lytic reactivation of the virus in vitro. In vivo, exogenous IFN-α14 and IFN-α17, as well as pDC expansion, delay EBV infection and the resulting CD8$^{+}$ T-cell expansion, but pDC depletion does not significantly influence EBV infection. Thus, consistent with the observation that primary immunodeficiencies compromising type I IFN responses affect only alpha- and beta-herpesvirus infections, we found that EBV elicits pDC responses that transiently suppress viral replication and attenuate CD8$^{+}$ T-cell expansion but are not required to control primary infection