AG Prof. Dr. Schirmbeck

Many vaccines are designed to prime multi-specific CD8 T cell responses, because the cellular immune system mediates protective immunity to many intracellular pathogens and tumours. We have established novel DNA- protein- and peptide-based vaccination strategies to induce CD8 T cell responses against viral, tumor and ´self´ antigens in different mouse models. In particular, we designed antigens that target endogenous “helper” molecules (endogenous adjuvant) and thereby facilitate priming of cellular and humoral immune responses under difficult conditions.

Exploring immune modulating strategies to restore antiviral effector functions of intrahepatic CD8 T cells

Reconstitution of antiviral immunity by vaccination is an attractive option to specifically control chronic hepatitis B virus (HBV) infection. To characterize HBV-specific CD8 T cell responses in the liver that produces all HBV antigens, we established the 1.4HBV-Smut transgenic (tg) line that harbours a replicating HBV genome in the liver, produces HBV large/middle surface (S) and core/precore (C/E) antigens but secretes neither small S particles, nor HBV virions. DNA vaccines efficiently induce core- (but not S-) specific CD8 T cell responses that transiently suppress HBV replication in the liver of 1.4HBV-Smut tg mice. Intrahepatic, antiviral CD8 T cells in vaccinated 1.4HBV-Smut tg mice show reproducible changes in their phenotype and cytokine profile that coincide with their loss of antiviral activity (‘exhaustion’). The project aims to elucidate systemic and intrahepatic regulatory effects that set off ´exhaustion´ of specific CD8 T cells in vaccinated 1.4HBV-Smut tg mice. We will use 1.4HBV-Smut tg mice crossed to well-defined tg or KO lines (deficient for coinhibitory molecules, regulator cells or cytokines) and immunize them with HBcAg- or HBsAg-encoding vaccines. We will combine specific vaccination with immune modulating protocols in an attempt to maintain an antiviral phenotype in the specific, intrahepatic T cells. We are in particular interested in: (i) the characterization of the kinetics of primary and boosted CD8 T cell responses in wt versus tg animals; (ii) changes in phenotype and cytokine secretion of CD8 T cells and hepatocytes; and (iii) epitope specificities of CD8 T cells. To selectively analyze intrahepatic effector CD8 T cell responses, we will use adoptive transfer experiments (CD8 T cells from vaccinated wt or KO mice transferred into wt or KO 1.4HBV-Smut tg mice). These studies may help to design specific immune intervention protocols that attenuate the functional ‘exhaustion’ of CD8 T cells in chronic infection with hepatotropic viruses.

Endogenously expressed HBV core particles capture mammalian RNA that facilitates priming of antiviral CD8 T cells by DNA-based vaccination

The HBV core (HBV-C) protein self-assembles into particles and encapsidates immune-stimulatory bacterial or yeast RNA through a cationic COOH-terminal (C150-183) domain. To investigate whether this domain has an impact on the RNA-binding and immunogenicity of endogenously expressed HBV-C, we developed an expression/purification system for HBV-C/RNA particles in vector-transfected HEK-293 cells and evaluated induction of Kb/C93-specific CD8+ T-cell responses in B6 and 1.4HBV-Smut tg mice by DNA-based immunization. We showed that HBV-C but not HBV-C149 particles (lacking the cationic domain) capture mammalian RNA. Prevention of specific serine-phosphorylation at three repeated SPRRR motifs, either by exchanging the entire cationic domain with an unrelated HIV-tat48-57-like sequence (HBV-C149tat) or by exchanging the serine residues S155, S162 and S170 with alanines (HBV-CAAA), significantly enhanced the RNA-binding of particles. Particle-bound mammalian RNA functioned as TLR-7 ligand and induced a Th1-biased humoral immunity in B6 and TLR-3-/- but not in TLR-7-/- mice. Encapsidation of RNA into particles, but not binding of RNA to a non-particulate HBV-CAAAad antigen, was crucial to elicit Kb/C93-specific effector CD8+ T-cells and inhibited HBV replication in the liver of 1.4HBV-Smut tg mice by DNA-based vaccination. HBV-C particles thus capture an endogenous RNA-based ´adjuvant´ activity with a potential therapeutic value by DNA-based vaccination.

Differential presentation of ´endogenous´ and ´exogenous´ hepatitis B surface antigens influences priming of CD8 T-cells in an epitope specific manner

Little is known whether presentation of endogenous and exogenous HBV surface antigens differs in virus-harbouring hepatocytes and in APCs targeted by DNA- or protein-based vaccines. We used HBV transgenic mice, expressing exclusively endogenous (1.4HBV-Smut) or secreted (Alb/HBs) surface antigens in the liver, to evaluate the specific epitope display by ex vivo isolated hepatocytes. 1.4HBV-Smut-derived hepatocytes exclusively present the Kb/S190-197 epitope, whereas Alb/HBs-derived hepatocytes present two (Kb/S190-197 and Kb/S208-215) epitopes to CD8 T-cells. These mice thus differ in the expression of circulating surface antigen and the presentation of the Kb/S208 epitope. Interestingly, the induction of CD8 T-cells in B6 mice also depends on endogenous and exogenous antigen delivery: Antigen-expressing DNA-vaccines preferentially induced Kb/S190-specific T-cells, whereas a recombinant surface antigen vaccine preferentially induced Kb/S208-specific T-cells. This selective priming of T-cells critically depends on a Kb/S190-mediated ´immunodominance´ operating in DNA- but not protein-immunized mice. Of note, the local surface antigen expression and exclusive Kb/S190 epitope presentation in 1.4HBV-Smut hepatocytes allowed priming of Kb/S208- but not Kb/S190-specific T-cells in 1.4HBV-Smut mice by protein- and DNA-based vaccines. These T-cells accumulated in the liver of 1.4HBV-Smut tg mice but could not inhibit HBV replication. These results have practical implications for the design of T-cell-stimulating therapeutic vaccines.

DNA-based, virus-like particle expressing cancer vaccines.

Cancer stays one of the leading causes of mortality within the human population worldwide. Therefore, development of prophylactic and therapeutic treatments has a high priority. Progress in cancer immunotherapy provides important insights in this direction. The adaptive immune system and, in particular CD8+ T cells has been shown to play an important role in tumor rejection.
DNA-based vaccination is an attractive technique to express vector-encoded antigen(s) and elicit antigen-specific immune responses in the host. Injection of antigen-expressing plasmids efficiently stimulates CD8+ T-cell responses, because they allow direct antigen expression and MHC class I-restricted epitope presentation by in vivo transfected APCs. Furthermore, ´cross-presentation of antigenic material, released from non-professional antigen-expressing APCs (e.g., myocytes) to professional APCs (e.g. DCs) facilitates priming of CD8+ T-cell responses. In our group, we are studying the efficiency of DNA-based vaccines expressing chimeric virus-like particles (VLPs), which self-assemble spontaneously from their subunits in the cell. We hypothesize that tumor antigen-specific domains and/or epitopes fused to a VLP in a vector DNA combines the strong immunogenicity of VLPs with the flexibility of plasmid DNA-based vaccines. As carriers for tumor-specific epitopes we have chosen the hepatitis core antigen (HBcAg). As a model for tumor experiments, we have chosen the TRAMP-C2 (mouse prostate adenocarcinoma) cell line in which a single point mutation resulted in an amino acid exchange at a position 251 and generated the H-2Db-restricted Spas-1244-252 epitope (STHVNHLHC) (Fasso et al., 2008, PNAS).
In this project, we will optimize conditions that induce protective anti-tumor immune responses in therapeutic as well as in prophylactic settings. This goal could be achieved (i) by generation of recombinant VLPs expressing the TRAMP-C2-specific neo-epitope with several modifications (e.g., particle-, dimer- or monomer forming, RNA-binding) and (ii) by optimization of an immunization protocol, which includes combination of DNA vaccine, cytokines, co-stimulatory molecules and antagonists blocking immune check-point inhibitors.

Induktion und Hemmung autoreaktiver Präproinsulin-spezifischer CD8 T Zellantworten.

Unsere Arbeitsgruppe beschäftigt sich mit der Induktion von murinen CD8 T Zellantworten, sowie deren spezifischer Hemmung. Die gegenwärtig verfügbaren Technologien basieren auf synthetischen Peptiden, rekombinanten Proteinkonstrukten, DNA-basierten Expressionsvektoren und viralen Vektoren. Diese Strategien wurden eingesetzt, um protektive und therapeutische T Zellantworten in präklinischen Modellen der chronisch-persistierenden HBV-Infektion zu induzieren. Dabei ähneln die Ansätze in vielen Aspekten dem Durchbrechen von Selbsttoleranz in der Induktion experimenteller Autoimmunkrankheiten. Um die Aspekte der Infektions- und Autoimmunitätsforschung zu verknüpfen hat unsere Arbeitsgruppe in den letzten Jahren verschiedene Mausmodelle etabliert um die CD8 T Zell-vermittelte Induktion eines experimentellen Autoimmundiabetes (EAD) zu charakterisieren. Hierzu zählen unter anderem RIP-B7.1 transgene Mäuse, die das ko-stimulatorische B7.1 (CD80) Molekül in pankreatischen beta Zellen exprimieren sowie PD-L1-/- KO Mäuse denen selektiv das ko-inhibitorische PD-L1 Molekül fehlt. Durch Immunisierung mit spezifischen Insulinantigenen kann in RIP-B7.1 und PD-L1 Mäusen direkt ein EAD ausgelöst werden. Durch zusätzliche Ko-stimulation oder fehlende Ko-inhibition auf den beta Zellen können autoreaktive CD8 T Zellen mit dem Zielgewebe interagieren und die Insulin-exprimierenden beta Zellen (z.B. durch Freisetzung von Interferon-Gamma) spezifisch zerstören. Im Vergleich zum NOD System ist die Diabetesentwicklung in diesem System geschlechtsunabhängig. In unseren Immunisierungsstudien konnten wir neue Aspekte aufzeigen, wie Präproinsulin in nicht-pankreatischen Zellen für die Induktion von CD8 T Zellen prozessiert und präsentiert wird. Die wichtigsten Erkenntnisse sind: die Antigene müssen im Endoplasmatischen Retikulum (ER) exprimiert werden um Insulin-spezifische CD8 T Zellen zu stimulieren und rekombinante „exogene“ Antigene stimulieren keine diabetogenen CD8 T Zellen.

Im Zuge dieses Projekts sollen unsere induzierbaren Modelle und die bisherigen Erkenntnisse genutzt werden, um Strategien zur spezifischen Hemmung der induzierten diabetogenen CD8 T Zellen zu etablieren. Im Besonderen erscheint hierfür die antigenspezifische Induktion regulatorischer T Zellen als attraktiver Ansatz. Zu diesem Zweck wollen wir Antigene, Antigenformulierungen und/oder Verabreichungsformen identifizieren, welche in unseren Modellen die autoreaktiven diabetogenen CD8 T Zellen spezifisch hemmen. Durch den Einsatz vom Insulin abgewandelter Designer-Antigene, sollen die Antigenprozessierung und –präsentation in Antigenpräsentierenden Zellen (APZ) zu Gunsten regulatorischer anti-diabetogener T Zellantworten dirigiert werden. Um die Wirksamkeit verschiedener Designer-Antigene zu testen werden zunächst PD-L1 Mäuse mit diesen (in Form von DNA oder als Protein) vakziniert. Nach einer gewissen Karenzzeit wird dann versucht, durch Injektion von Präproinsulin-DNA, Diabetes in den Mäusen zu induzieren. Antigene die dabei eine Immuntoleranz gegen die Insulin-spezifischen diabetogenen CD8 T Zellantworten vermitteln, werden auf ihre Wirkweise hin untersucht. Insbesondere soll hierbei die Rolle natürlicher und induzierter regulatorischer Foxp3+ CD25+ CD4 T Zellen (nTreg und iTreg), sowie deren Marker- (z.B. CD62L, CTLA-4, GITR) und Zytokinprofil (z.B. TGF-beta, IL-10) untersucht werden. Die Aufklärung der Mechanismen der T Zell-Toleranzinduktion gegen autoreaktive CD8 T Zellen, sowie deren Kommunikation und Wechselwirkungen untereinander, soll durch adoptive Transferexperimente erfolgen. RAG1-defiziente Mäuse (welche keine B- und T Zellen besitzen) werden dabei als Empfänger eingesetzt. Durch Transfer einzelner Komponenten einer diabetogenen bzw. Toleranz-induzierenden Immunantwort können so die Wechselwirkungen verschiedener Zellpopulationen gezielt und unter definierten Bedingungen analysiert werden. Wir erwarten uns von diesem Projekt neue Erkenntnisse wie das Ungleichgewicht von autoreaktiven und protektiven T Zellantworten in autoimmunen Erkrankungen durch antigenspezifische Vakzinierung ausgeglichen werden kann.

Enhancing and restoring immune function by rejuvenating LT-HSCs

        - in collaboration with:
Prof. Dr. Hartmut Geiger (Institut für Molekulare Medizin)
Prof. Dr. Michael Denkinger  (AGAPLESION Bethesda Klinik Ulm)

Upon aging, immune function declines which is associated with higher susceptibility to autoimmune disease, an enhanced onset of cancer as well as an increased susceptibility to infections and a decreased responsiveness to vaccines. To restore adequate immune responses in older adults new approaches are indispensable. A hallmark of these aging associated changes, which are referred to as immunosenescence, is a decreased number of naïve T cells and overall changes in the numbers of lymphocyte populations including a reduction in the number of CD19+ B cells. As aging-associated changes in the T cell population has been thought to be primarily a consequence of thymic involution, more recently, also aging of long term hematopoietic stem cells (LT-HSCs) has been identified as an important contribution factor for immunosenescence. Upon aging LT-HSCS show an altered differentiation capacity, they are skewed towards the myeloid lineage which results in a reduction in the frequency of lymphoid progenitor. This finally results in decreased B and T cell lymphopoiesis upon aging. In previous experiments, it could be shown by the laboratory of Prof. Dr. Hartmut Geiger and some other research groups, that aging of HSCs is associated with an increased activity of the small RhoGTPase Cdc42. Furthermore, pharmacological inhibition of this protein results in a sustainable functional rejuvenation of aged HSCs. They regain their potential to differentiate towards the lymphoid lineage, suggesting that this could be a promising approach to attenuate immunosenescence. Within this project, we are analyzing the effect of rejuvenation of aged LT-HSCs on the immune system as well as on immune function. Thereby, our efforts concentrate on the adaptive immune system.

Characterization of Asialo-GM1+ CD8 T cells.

The cell surface expression of the glycosphingolipid Asialo-GM1 (ASGM1) was originally used to identify and deplete natural killer (NK) cells in laboratory animals. Often overlooked was the fact, that ASGM1 is also expressed on the surface of murine CD8+ cytotoxic T lymphocytes (CTL). As ASGM1 expression is upregulated on effector CTLs upon priming, e.g. after vaccination, it is assumed that ASGM1 expression represents a marker of CD8+ T-cell activation. Upon aging, a higher number of CTL express ASGM1, and aged animals also generate lower immune responses to vaccination. To further investigate the role of ASGM1 in this context, we are characterizing the expression of ASGM1 on CTLs of young and old mice. For this purpose, lymphocytes from spleen and liver of non-immunized mice and mice immunized with DNA encoding for well-defined antigens, are isolated and analyzed bymulticolor flow cytometry. Thereby, the surface expression of ASGM1 on the antigen specific CTLs and their subpopulations can be measured together with other activation or exhaustion markers. Furthermore, we are analyzing the effect of depletion of ASGM1+ cells on immuneresponses to DNA vaccines by injection of anti-ASGM1 antibody.





2005-2008  AIF-Proinno Projekt: Entwicklung von innovativen Impfstoffen und Evaluierung der generierten Antikörpern aus dem Eigelb von Tauben für Anwendungen in der Wissenschaft
2006-2008 DFG (SCH505/3-1) Priming specific, murine CD8+ T cell responses by complexes of cationic/antigenic fusion peptides with nucleic acids
2006-2009 DFG (SCH505/2-4) Immunogenicity of recombinant chaperone-complexed antigens
2007-2010 SFB518 Entzündung, Regeneration und Transformation im Pankreas: (Teilprojekt A01). Induktion und Kontrolle diabetogener, Präproinsulin-spezifischer CD8 T Zellen in transgenen Mausmodellen des Typ 1 Diabetes
2008-2010 ProInno-II Projekt: Fortentwicklung messenger RNA basierter Vakzine auf Grundlage immunologisch wichtiger Parameter
2008-2011 BMBF 01KI0772: Erkennung, Vermeidung und Behandlung von Infektionen des immun-kompromittierten Patienten. Teilprojekt D „Etablierung und Optimierung HCMV-spezifischer, CD8+ T- Zell-stimulierender Vakzinierungsstrategien im HLA-A2 transgenen Mausmodell“
2009-2014 GrK 1041 Molecular Diabetology and Endocrinology in Medicine. Immunoprofiling of HLA-A*0201-restricted, insulin-specific CD8 T cells in various stages of autoimmune diabetes induced in murine models of type 1 diabetes
2009-2011 DFG (SCH505/4-1) KFO142 (Teilprojekt 7) CD8 T Zellantworten und ihre Regulation im fortschreitenden Alter
2010-2013 DFG (SCH 505/5-1) Exploring immune modulating strategies to restore antiviral effector functions of intrahepatic CD8 T cells

Wissenschaftliche Publikationen