Intratumorale T-Zellen in einem Spontanlymphommodell der Maus

Es gibt zahlreiche Bemühungen, neue Immuntherapien zur Behandlung von malignen Tumoren zu entwickeln, da die Prognose vieler Krebserkrankungen immer noch schlecht ist. Dazu muss das Verständnis, wie die verschiedenen Immunzellen innerhalb des Tumormilieus miteinander interagieren, verbessert werden. Mausmodelle für Spontantumoren spiegeln die klinische Situation besser wider als transplantierbare Tumoren und stellen somit eine gute Möglichkeit dar, die während der Tumorentwicklung stattfindenden Immunreaktionen zu analysieren. In der vorliegenden Dissertation wurde mit Mäusen gearbeitet, in deren B-Zellen das Onkogen c-MYC konstitutiv exprimiert wird und die nach einigen Wochen spontan B-Zell-Lymphome entwickeln. Frühere Arbeiten haben bereits gezeigt, dass natürliche Killerzellen (NK-Zellen) aus c-MYC-Tumoren im Vergleich zu Organen aus Wildtyp (wt)-Mäusen kaum Interferon-gamma (IFN-) produzierten und die Fähigkeit, Zielzellen zu töten, stark reduziert war. Dementsprechend sezernierten Tumor-infiltrierende dendritische Zellen (TIDZ) geringere Mengen Interleukin-12 (IL-12), das für die Induktion einer Th1 (T-Helferzelle 1) / ZTL (zytotoxischer T-Lymphozyt)-Antwort essenziell ist, aber vermehrt das immuninhibierende Zytokin IL-10. Für die Aktivierung von naiven T-Zellen ist die Hilfe von NK-Zellen und DZ notwendig, die in c-MYC-Mäusen offenbar nicht gegeben war. Es wurde daher erwartet, dass intratumorale T-Zellen nicht bzw. nur ungenügend aktiviert werden. Überraschenderweise wiesen Tumor-infiltrierende T-Zellen jedoch einen aktivierten Phänotyp auf, zeigten zum Teil sogar eine erhöhte IFN--Produktion, konnten degranulieren und proliferierten in vivo. Trotzdem übernahmen intratumorale T-Zellen im Gegensatz zu NK-Zellen keine immunüberwachende Funktion über die Lymphomentstehung, da c-MYC-Mäuse nach Depletion der T-Zell-Population nicht früher erkrankten als unbehandelte Tiere. Offenbar wurden T-Zellen in c-MYC-Tumoren Antigen-spezifisch aktiviert, was dazu führte, dass sie aufgrund der langanhaltenden Stimulation in einen Erschöpfungszustand geraten sind. Dies spiegelte sich in einer hohen Expression des Erschöpfungsmarkers PD-1 (Programmed Cell Death 1) wider. Die Interaktion des koinhibitorischen Rezeptors PD-1 mit dem entsprechenden Liganden PD-L1, der vor allem auf DZ in c-MYC-Tumoren detektiert wurde, führt in T-Effektorzellen zur Inhibierung des aktivierenden T-Zell-Rezeptor (TZR)-Signals. So konnten T-Zellen aus c-MYC-Tumoren im Vergleich zu T-Zellen aus normalen Mäusen in vitro über den TZR tatsächlich nicht mehr stimuliert werden, was ebenfalls für eine Erschöpfung sprach. Zudem exprimierten intratumorale T-Zellen neben PD-1 die koinhibitorischen Moleküle CTLA-4 (Cytotoxic T-Lymphocyte-Associated Antigen 4) und LAG-3 (Lymphocyte Activation Gene 3). Die Stimulierbarkeit der T-Zellen in vitro konnte durch Zugabe blockierender Antikörper (AK) gegen PD-1 und CTLA-4 verbessert werden. Aufgrund dieser Beobachtung wurden junge, noch gesunde c-MYC-Mäuse mit diesen AK behandelt. Durch die PD-1/CTLA-4-Blockade in vivo zeigten die Tiere ein signifikant verlängertes Überleben. Neben CD4+ T-Helferzellen und CD8+ zytotoxischen T-Lymphozyten wurde in c-MYC-Tumoren innerhalb der CD4+ T-Zellpopulation ein sehr hoher Anteil Foxp3+ regulatorischer T-Zellen (Treg) detektiert. Diese waren aktiviert, proliferierten in vivo und produzierten IL-10. Die koinhibitorischen Moleküle, welche auch auf erschöpften T-Zellen zu finden waren (PD-1, LAG-3 und CTLA-4), wurden ebenfalls von Treg exprimiert, was bekanntlich zur Verstärkung ihrer suppressiven Aktivität führt. Es wurden vor allem Helios-exprimierende natürliche Treg (nTreg) detektiert. Doch auch IL-10-produzierende, regulatorische Foxp3- Tr1-Zellen (T Regulatory Type 1 Zelle), die zu den induzierten Treg (iTreg) zählen, waren in c-MYC-Tumoren zu finden. Schließlich fanden sich auch T-Zellen, die sowohl IFN- als auch IL-10 produzierten. Diese könnten aus Th1-Zellen entstanden sein, welche die Zytokinexpression umgestellt haben. Treg unterdrückten in c-MYC-Tieren anscheinend eine effektive Antitumor-Immunantwort. Daher wurden Foxp3+ Treg in DEREG/c-MYC-Mäusen, die einen transgenen Diphtherietoxin (DT)-Rezeptor tragen, spezifisch durch DT-Injektionen in vivo depletiert. Erstmals wurde der DEREG-Mechanismus zur Treg-Depletion in einem endogenen Tumormodell angewandt. Die Depletion der Foxp3+ Treg verschaffte den DEREG/c-MYC-Mäusen einen leichten Überlebensvorteil. Diese Ergebnisse sind relevant für die Entwicklung neuer immuntherapeutischer Verfahren zur Behandlung maligner Erkrankungen in der Klinik.The prognosis for many cancers is still poor. Therefore, much effort is made to develop new strategies for immunotherapy targeting malignant tumours. For this purpose, the understanding of how immune cells in tumour microenvironments interact with each other has to be improved. Mouse models of spontaneously arising tumours reflect the clinical situation better than transplantable tumours. So, these models provide a good tool for analysing the occurring immune reactions. In the present work mice that constitutively express the oncogene c-MYC in their B cells and that develop B-cell lymphomas after a few weeks served as a tumour model. Former work has already demonstrated that natural killer (NK) cells derived from c-MYC tumours rarely produced interferon-gamma (IFN-) compared to NK cells from wildtype (wt) mice and showed strongly diminished killing of target cells. Accordingly, tumour-infiltrating dendritic cells (TIDC) only secreted low levels of interleukin-12 (IL-12) required for the induction of a Th1 (T-helper cell 1) / CTL (cytotoxic T lymphocyte)-response. Instead, TIDC increased the production of the immunosuppressing cytokine IL-10. A proper NK-cell and DC help that was obviously missing in c-MYC mice is required for activating naïve T cells. Hence, it was expected that intratumoural T cells are not or only insufficiently activated. Surprisingly, tumour infiltrating T cells exhibited an activated phenotype, partly even showed an increased IFN- production, could degranulate and proliferated in vivo. However, in contrast to NK cells, intratumoural T cells did not exert a tumour-protective function during lymphoma formation, since c-MYC mice did not develop lymphomas earlier after T-cell depletion. T cells in c-MYC tumours were activated through their specific antigen. However, due to prolonged stimulation, T cells got into an exhausted state. This was reflected by high expression of the exhaustion marker PD-1. The interaction of the coinhibitory receptor PD-1 (Programmed Cell Death 1) with the corresponding ligand PD-L1, which was detected mainly on DC in c-MYC tumours, leads to the inhibition of the activating T-cell receptor (TCR) signal in T-effector cells. Thus, in contrast to wt-T cells, T cells derived from c-MYC tumours could not be stimulated in vitro through the TCR. This also argued for T-cell exhaustion. In addition to PD-1, intratumoural T cells also expressed the coinhibitory molecules CTLA-4 (Cytotoxic T-Lymphocyte-Associated Antigen 4) and LAG-3 (Lymphocyte Activation Gene 3). T-cell stimulation in vitro could be improved through the addition of blocking antibodies (Ab) targeting PD-1 and CTLA-4. Based on this observation c-MYC mice that did not yet show clinical signs of tumour growth were treated with these Abs. As a result of the PD-1/CTLA-4 blockade, animals showed a significantly longer survival. Besides CD4+ T-helper cells and CD8+ cytotoxic T lymphocytes an augmented fraction of Foxp3+ regulatory T cells (Treg) within the CD4+ T-cell population was detected in tumour-bearing c-MYC animals. These cells were activated, proliferated in vivo and produced IL-10. The coinhibitory molecules that were found on exhausted T cells (PD-1, LAG-3 and CTLA-4) were also expressed on Treg. It is known that this increases the suppressive activity of Treg. Mainly Helios expressing natural Treg (nTreg) were detected. However, IL-10 producing regulatory Foxp3- Tr1 (T Regulatory Type 1) cells that belong to the induced Treg population (iTreg) were also found in c-MYC tumours. Finally, T cells producing IFN- as well as IL-10 were detected. These could originate from Th1 cells that switched their cytokine expression. Treg in c-MYC tumours apparently suppress an effective anti-tumour immune response. Therefore, Foxp3+ Treg were specifically depleted in vivo in DEREG/c-MYC mice carrying a transgenic diphtheria-toxin (DT) receptor by the injection of DT. For the first time, this DEREG mechanism is utilised in an endogenous tumour model. The depletion of Foxp3+ Treg provided a survival benefit for the DEREG/c-MYC mice. These results are relevant for the development of new immunotherapeutic approaches for treating malignant disease in the clinic

Methodology for site-specific determination of slope inclination using digital height information in the Plant Protection Application Manager (PAM)

In der Landwirtschaft werden viele Pflanzenschutzmittel im Zuge des Zulassungsverfahrens mit Hangneigungsauflagen belegt. Ziel ist es, den durch Oberflächenabfluss und Bodenerosion bedingten Eintrag von Pflanzenschutzmitteln in angrenzende Gewässer zu vermeiden. Aktuell existieren keine praktikablen, deutschlandweit verfügbaren Instrumente, die den Landwirt in einer zuverlässigen und nachvollziehbaren Weise bei der objektiven Ermittlung der Hangneigung unterstützten. Mit dem entwickelten PAM3D-Dienst wird dem Nutzer ein internetbasiertes Entscheidungshilfesystem zur Einhaltung von Hangneigungsauflagen zur Verfügung stehen. Gleichzeit wird diese Methode für die zuständigen Kon­troll­behörden der Länder zur Diskussion gestellt. Die Ermittlung der Hangneigung eines Feldes erfolgt automatisiert auf Grundlage von digitalen Höheninformationen mittels eines neuartigen Algorithmus, der Randpixelmethode. Im Gegensatz zu anderen Services gibt diese nicht nur einen Durchschnittswert pro Feld aus, sondern erlaubt eine teilflächenspezifische Beurteilung der Hangneigung. Die Methode ist gegenüber gängigen rasterbasierten Verfahren flexibel genug, Höheninformation aus verschiedenen Quellen (Laserscan-, Traktor,- Drohnen- und Satellitendaten) zu verarbeiten und ist zudem robust gegen­über unterschiedlichen räumlichen Auflösungen dieser Datensätze.In agriculture, many plant protection products (PPP) are subject to slope restrictions as part of their approval process. The aim is to prevent the discharge of pesticides into adjacent water bodies caused by surface runoff and soil erosion. There are currently no practicable instruments available throughout Germany, supporting farmers to deter­mine the slope inclination in a reliable and verifiable manner. The PAM3D-service will provide an internet-based decision support system for complying with slope requirements in the context of PPP risk management. The slope inclination of a field is determined automatically based on digital elevation information using a novel algorithm. Contrary to other services providing an unreliable average field value, this new methodology allows for a site-specific slope assessment. The service supports various sources of elevation information including official laser, tractor, satellite, and drone data, and it is robust to the spatial resolution of input elevation data

Rab3D is critical for secretory granule maturation in PC12 cells.

Neuropeptide- and hormone-containing secretory granules (SGs) are synthesized at the trans-Golgi network (TGN) as immature secretory granules (ISGs) and complete their maturation in the F-actin-rich cell cortex. This maturation process is characterized by acidification-dependent processing of cargo proteins, condensation of the SG matrix and removal of membrane and proteins not destined to mature secretory granules (MSGs). Here we addressed a potential role of Rab3 isoforms in these maturation steps by expressing their nucleotide-binding deficient mutants in PC12 cells. Our data show that the presence of Rab3D(N135I) decreases the restriction of maturing SGs to the F-actin-rich cell cortex, blocks the removal of the endoprotease furin from SGs and impedes the processing of the luminal SG protein secretogranin II. This strongly suggests that Rab3D is implicated in the subcellular localization and maturation of ISGs

Microenvironment‐induced restoration of cohesive growth associated with focal activation of P ‐cadherin expression in lobular breast carcinoma metastatic to the colon

Invasive lobular carcinoma (ILC) is a special breast cancer type characterized by noncohesive growth and E‐cadherin loss. Focal activation of P‐cadherin expression in tumor cells that are deficient for E‐cadherin occurs in a subset of ILCs. Switching from an E‐cadherin deficient to P‐cadherin proficient status (EPS) partially restores cell–cell adhesion leading to the formation of cohesive tubular elements. It is unknown what conditions control EPS. Here, we report on EPS in ILC metastases in the large bowel. We reviewed endoscopic colon biopsies and colectomy specimens from a 52‐year‐old female (index patient) and of 18 additional patients (reference series) diagnosed with metastatic ILC in the colon. EPS was assessed by immunohistochemistry for E‐cadherin and P‐cadherin. CDH1 /E‐cadherin mutations were determined by next‐generation sequencing. The index patient's colectomy showed transmural metastatic ILC harboring a CDH1 /E‐cadherin p.Q610* mutation. ILC cells displayed different growth patterns in different anatomic layers of the colon wall. In the tunica muscularis propria and the tela submucosa, ILC cells featured noncohesive growth and were E‐cadherin‐negative and P‐cadherin‐negative. However, ILC cells invading the mucosa formed cohesive tubular elements in the intercryptal stroma of the lamina propria mucosae. Inter‐cryptal ILC cells switched to a P‐cadherin‐positive phenotype in this microenvironmental niche. In the reference series, colon mucosa infiltration was evident in 13 of 18 patients, one of which showed intercryptal EPS and conversion to cohesive growth as described in the index patient. The large bowel is a common metastatic site in ILC. In endoscopic colon biopsies, the typical noncohesive growth of ILC may be concealed by microenvironment‐induced EPS and conversion to cohesive growth

Roles of Myosin Va and Rab3D in Membrane Remodeling of Immature Secretory Granules

Neuroendocrine secretory granules (SGs) are formed at the trans-Golgi network (TGN) as immature intermediates. In PC12 cells, these immature SGs (ISGs) are transported within seconds to the cell cortex, where they move along actin filaments and complete maturation. This maturation process comprises acidification-dependent processing of cargo proteins, condensation of the SG matrix, and removal of membrane and proteins not destined to mature SGs (MSGs) into ISG-derived vesicles (IDVs). We investigated the roles of myosin Va and Rab3 isoforms in the maturation of ISGs in neuroendocrine PC12 cells. The expression of dominant-negative mutants of myosin Va or Rab3D blocked the removal of the endoprotease furin from ISGs. Furthermore, expression of mutant Rab3D, but not of mutant myosin Va, impaired cargo processing of SGs. In conclusion, our data suggest an implication of myosin Va and Rab3D in the maturation of SGs where they participate in overlapping but not identical tasks

Impact of infection on proteome-wide glycosylation revealed by distinct signatures for bacterial and viral pathogens

Mechanisms of infection and pathogenesis have predominantly been studied based on differential gene or protein expression. Less is known about posttranslational modifications, which are essential for protein functional diversity. We applied an innovative glycoproteomics method to study the systemic proteome-wide glycosylation in response to infection. The protein site-specific glycosylation was characterized in plasma derived from well-defined controls and patients. We found 3862 unique features, of which we identified 463 distinct intact glycopeptides, that could be mapped to more than 30 different proteins. Statistical analyses were used to derive a glycopeptide signature that enabled significant differentiation between patients with a bacterial or viral infection. Furthermore, supported by a machine learning algorithm, we demonstrated the ability to identify the causative pathogens based on the distinctive host blood plasma glycopeptide signatures. These results illustrate that glycoproteomics holds enormous potential as an innovative approach to improve the interpretation of relevant biological changes in response to infection