Transkriptom-Analyse und Optimierung der Immuntherapie im Merkelzellkarzinom

Merkel cell carcinoma (MCC) is a rare, but highly aggressive neuroendocrine skin cancer, mainly caused by the Merkel cell polyomavirus. This virus can lead to MCC via integration into the host genome and expression of a truncated version of one of its proteins, called Large T antigen (truncLT). MCC is an immunogenic tumor that constantly needs to escape immune surveillance for example by aberant expression of the programmed cell death-protein 1/-ligand 1 (PD-1/PD-L1) or of the human leukocyte antigen (HLA). In context of cellular immunity, IFNɣ is a key cytokine mainly produced by activated lymphocytes. Although producing a variety of anti-tumoral effects, it paradoxically also exerts immunomodulatory functions which are misused by the tumor. Hence, a comprehensive understanding of the transcriptomic response of MCC cells to IFNɣ is of high interest. For this, three MCC cell lines were incubated with IFNɣ and the transcriptomic alterations were analyzed by single-molecule RNA sequencing using the Oxford Nanopore platform. Numerous significantly differentially expressed genes could be detected across all three cell lines as well as a clear upregulation of genes involved in tumor immune escape, suggesting these genes as potential targets in MCC immunotherapy. Furthermore, the transcriptome of a primary MCC and the corresponding cell culture were compared to each other and numerous alterations regarding tumor infiltrating lymphocytes, biomarker and immunosuppressive molecules were detected. In metastatic MCC, the treatment with checkpoint inhibitors (CPIs) has shown high clinical response rates of up to 60 %, but as nearly half of the patients do not respond to this treatment additional therapeutic options are urgently needed. For improvement of immunotherapy in MCC it was hypothesized that the combination of dendritic cell (DC) vaccination against the truncLT with checkpoint blockade could be an effective way to induce a strong and better immune response than each treatment alone. Therefore, the immunogenic potential of DCs loaded with the truncLT in presence or absence of three different CPIs (Ipilimumab, Pembrolizumab, Avelumab) was evaluated using the well-established human ex vivo cell culture system and the fraction of T cells, specific for the truncLT was determined by IFNɣ ELISPOT assays. Checkpoint inhibition had substantial effects on quantity and quality of antigen-specific T cells in healthy donors. Ipilimumab (anti-CTLA-4) strongly decreased the T-cell responses, Pembrolizumab (anti-PD-1) either drastically increased, or decreased the number of specific T cells and in presence of Avelumab (anti-PD-L1) a lower number of truncLT-specific T cells was detected. Furthermore, it was demonstrated that Avelumab results in antibody-dependent cellular cytotoxicity (ADCC) by binding on DCs and activating the NK cells, which then killed the DCs in the co-culture system finally leading to ineffective T-cell priming. Based on these data Avelumab should not be considered for combination with DC-based immunotherapy neither in MCC nor in other malignancies. In conclusion, the immunological characterization of MCC cell lines demonstrated an upregulation of immune escape genes after IFNɣ treatment and regarding the combination of CPIs and DC vaccines an Avelumab-mediated ADCC towards DCs via NK-cell activation was revealed. These data contribute to better understanding of T-cell responses under CPI influence and immune evasion in MCC what can lastly improve immunotherapy of MCC patients.Das Merkelzellkarzinom (MZK) ist ein seltender aber hochagressiver neuroendokriner Hautkrebs, welcher größtenteils durch das Merkelzellpolyomavirus ausgelöst wird. Dieses Virus kann durch Integration in das Wirtsgenom und durch Expression einer trunkierten Version eines seiner Proteine, des Large T Antigen‘s (trunkLT), zum MZK führen. Das MZK ist ein immunogener Tumor, welcher durchgehend der Kontrolle des Immunsystems entfliehen muss, z.B. durch abnormale Expression bestimmter Proteine, wie des programmed cell death-protein 1/-ligand 1 (PD-1/PD-L1) oder des humanen Leukozyten-Antigens (HLA). Wenn man die zelluläre Immunität betrachtet, ist Interferon-gamma (IFNɣ) ein Schlüssel-Zytokin, welches überwiegend von aktivierten Lymphozyten sekretiert wird. Obwohl es eine Vielzahl an anti-tumoralen Effekten bewirken kann, gehören paradoxerweise auch immun-modulatorische Funktionen zu seinem Repertoire, welche vom Tumor zweckentfremdet werden. Deswegen ist ein umfassendes Veständnis der transkriptionellen Antwort von MZK-Zelllinien auf IFNɣ von großem Interesse. Hierfür wurden drei MZK-Zelllinien mit IFNɣ inkubiert und Veränderungen in ihrem Transkriptom durch Einzelmolekül-RNA-Sequenzierung mit Hilfe der Oxford Nanopore Platform analysiert. Zahlreiche Gene, welche signfikant differentiell exprimiert waren, konnten in allen drei Zelllinien nachgewiesen werden, außerdem eine starke Hochregulation von Genen, die eine Rolle spielen, wie der Tumor der Kontrolle des Immunsystems entkommt. Diese Gene stellen potentielle neue Ziele für die Immuntherapie im MZK dar. Zudem wurden das Transkriptom eines Primärtumors sowie der daraus generierten Zellkultur miteinander verglichen, wobei zahlreiche Veränderungen festgestellt werden konnten, insbesondere bezüglich Tumor-infiltrierender Lymphozyten, Biomarkern und immunsuppressiv-wirkender Molekülen. Im metastasierenden MZK zeigte die Behandlung mit Checkpoint Inhibitoren (CPIs) sehr gute klinische Ansprechraten bis zu 60 %. Da jedoch umgekehrt fast die Hälfte der Patienten nicht auf diese Behandlungsmethode anspricht, werden weitere Therapieoptionen dringend benötigt. Um die Immuntherapie im MZK zu verbessern, wurde die Hypothese aufgestellt, dass die Kombination aus Vakzinierung mit dendritischen Zellen (DZs) gegen das trunkLT zusammen mit CPI Behandlung eine stärkere und bessere Immunantwort induzieren könnte, als DZ-Vakzine oder CPIs alleine. Hierfür wurde das immunogene Potential von DZs, die mit dem trunkLT beladen worden waren, in An- oder Abwesenheit von CPIs (Ipilimumab, Pembrolizumab, Avelumab) evaluiert, wobei ein gut etabliertes humanes ex-vivo Zellkultursystem eingesetzt wurde. Der Anteil an T-Zellen, die spezifisch für das trunkLT waren, wurde mittels IFNɣ ELISPOT-Assay bestimmt. Die Anwesenheit von CPIs hatte einen wesentlichen Effekt auf die Qualität und Quantität von Antigen-spezifischen T-Zellen in gesunden Spendern. Ipilimumab (anti-CTLA-4) führte zu einem starken Abfall an T-Zellantworten, Pembrolizumab (anti-PD-1) zeige zwei drastische Effekte, indem die Anzahl an spezifischen T-Zellen entweder stark erhöht oder stark erniedrigt war, und Avelumab (anti-PD-L1) führte zu einer deutlich niedrigeren Anzahl an trunkLT-spezifischen T-Zellen. Weiterhin konnte gezeigt werden, dass Avelumab an die DZs bindet und die natürlichen Killerzellen stark aktiviert, welche durch antikörperabhängige zellvermittelte Zytotoxizität (AZZ) die DZs in unserem Kokultursystem eliminierten, was letztlich zum ineffektiven T-Zell-Priming führte. Basierend auf diesen Daten, sollte Avelumab nicht in Kombination mit DZ-basierter Immuntherapie in Betracht gezogen werden, weder für das MZK noch für andere Tumorarten. Abschließend lässt sich sagen, dass die immunologische Charakterisierung von MZK-Zelllinien eine Hochregulation von Immunevasions-Genen nach Inkubation mit IFNɣ aufdeckte und außerdem im Rahmen der Kombination von CPIs und DZ-Vakzinen die Avelumab-vermittelte AZZ gegen DZs durch NK-Zell-Aktivierung aufgezeigt werden konnte. Diese Daten tragen zu einem besseren Verständnis von T-Zellantworten unter CPI-Einfluss sowie der Immunevasion im MZK bei, was letztlich die Immuntherapie von MZK Patienten verbessern kann

Relapse of acute myeloid leukemia after allogeneic stem cell transplantation: immune escape mechanisms and current implications for therapy

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the expansion of immature myeloid cells in the bone marrow (BM) and peripheral blood (PB) resulting in failure of normal hematopoiesis and life-threating cytopenia. Allogeneic hematopoietic stem cell transplantation (allo-HCT) is an established therapy with curative potential. Nevertheless, post-transplant relapse is common and associated with poor prognosis, representing the major cause of death after allo-HCT. The occurrence of relapse after initially successful allo-HCT indicates that the donor immune system is first able to control the leukemia, which at a later stage develops evasion strategies to escape from immune surveillance. In this review we first provide a comprehensive overview of current knowledge regarding immune escape in AML after allo-HCT, including dysregulated HLA, alterations in immune checkpoints and changes leading to an immunosuppressive tumor microenvironment. In the second part, we draw the line from bench to bedside and elucidate to what extend immune escape mechanisms of relapsed AML are yet exploited in treatment strategies. Finally, we give an outlook how new emerging technologies could help to improve the therapy for these patients, and elucidate potential new treatment options

T-Cell Responses in Merkel Cell Carcinoma: Implications for Improved Immune Checkpoint Blockade and Other Therapeutic Options

Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer with rising incidence and high mortality. Approximately 80% of the cases are caused by the human Merkel cell polyomavirus, while the remaining 20% are induced by UV light leading to mutations. The standard treatment of metastatic MCC is the use of anti-PD-1/-PD-L1-immune checkpoint inhibitors (ICI) such as Pembrolizumab or Avelumab, which in comparison with conventional chemotherapy show better overall response rates and longer duration of responses in patients. Nevertheless, 50% of the patients do not respond or develop ICI-induced, immune-related adverse events (irAEs), due to diverse mechanisms, such as down-regulation of MHC complexes or the induction of anti-inflammatory cytokines. Other immunotherapeutic options such as cytokines and pro-inflammatory agents or the use of therapeutic vaccination offer great ameliorations to ICI. Cytotoxic T-cells play a major role in the effectiveness of ICI, and tumour-infiltrating CD8+ T-cells and their phenotype contribute to the clinical outcome. This literature review presents a summary of current and future checkpoint inhibitor therapies in MCC and demonstrates alternative therapeutic options. Moreover, the importance of T-cell responses and their beneficial role in MCC treatment is discussed

Genetic interaction screen for severe neurodevelopmental disorders reveals a functional link between Ube3a and Mef2 in Drosophila melanogaster

Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene (Da, Mef2, Ube3a, Zfh1, XNP) and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities

Beyond Cancer: Regulation and Function of PD-L1 in Health and Immune-Related Diseases

Programmed Cell Death 1 Ligand 1 (PD-L1, CD274, B7-H1) is a transmembrane protein which is strongly involved in immune modulation, serving as checkpoint regulator. Interaction with its receptor, Programmed Cell Death Protein 1 (PD-1), induces an immune-suppressive signal, which modulates the activity of T cells and other effector cells. This mediates peripheral tolerance and contributes to tumor immune escape. PD-L1 became famous due to its deployment in cancer therapy, where blockage of PD-L1 with the help of therapeutic antagonistic antibodies achieved impressive clinical responses by reactivating effector cell functions against tumor cells. Therefore, in the past, the focus has been placed on PD-L1 expression and its function in various malignant cells, whereas its role in healthy tissue and diseases apart from cancer remained largely neglected. In this review, we summarize the function of PD-L1 in non-cancerous cells, outlining its discovery and origin, as well as its involvement in different cellular and immune-related processes. We provide an overview of transcriptional and translational regulation, and expression patterns of PD-L1 in different cells and organs, and illuminate the involvement of PD-L1 in different autoimmune diseases as well as in the context of transplantation and pregnancy

Breaking Entry-and Species Barriers: LentiBOOST^® Plus Polybrene Enhances Transduction Efficacy of Dendritic Cells and Monocytes by Adenovirus 5

Due to their ability to trigger strong immune responses, adenoviruses (HAdVs) in general and the serotype5 (HAdV-5) in particular are amongst the most popular viral vectors in research and clinical application. However, efficient transduction using HAdV-5 is predominantly achieved in coxsackie and adenovirus receptor (CAR)-positive cells. In the present study, we used the transduction enhancer LentiBOOST® comprising the polycationic Polybrene to overcome these limitations. Using LentiBOOST®/Polybrene, we yielded transduction rates higher than 50% in murine bone marrow-derived dendritic cells (BMDCs), while maintaining their cytokine expression profile and their capability to induce T-cell proliferation. In human dendritic cells (DCs), we increased the transduction rate from 22% in immature (i)DCs or 43% in mature (m)DCs to more than 80%, without inducing cytotoxicity. While expression of specific maturation markers was slightly upregulated using LentiBOOST®/Polybrene on iDCs, no effect on mDC phenotype or function was observed. Moreover, we achieved efficient HAdV5 transduction also in human monocytes and were able to subsequently differentiate them into proper iDCs and functional mDCs. In summary, we introduce LentiBOOST® comprising Polybrene as a highly potent adenoviral transduction agent for new in-vitro applications in a set of different immune cells in both mice and humans