1,721,070 research outputs found
Modifying therapy in patients with advanced Hodgkin’s lymphoma by integrating early metabolic response by interim PET-CT
No full textDifferential organization of tonic and chronic B cell antigen receptors in the plasma membrane
Full text linkStimulation of the B cell antigen receptor (BCR) triggers signaling pathways that promote the differentiation of B cells into plasma cells. Despite the pivotal function of BCR in B cell activation, the organization of the BCR on the surface of resting and antigen-activated B cells remains unclear. Here we show, using STED super-resolution microscopy, that IgM-containing BCRs exist predominantly as monomers and dimers in the plasma membrane of resting B cells, but form higher oligomeric clusters upon stimulation. By contrast, a chronic lymphocytic leukemia-derived BCR forms dimers and oligomers in the absence of a stimulus, but a single amino acid exchange reverts its organization to monomers in unstimulated B cells. Our super-resolution microscopy approach for quantitatively analyzing cell surface proteins may thus help reveal the nanoscale organization of immunoreceptors in various cell types
Identification of T Cell Receptor Repertoire Signatures in Age, Autoimmune Conditions and Cancer using Next-Generation Sequencing
No full textThe immune system is an interactive network composed of cells, tissues and soluble factors which has evolved to protect the host from invading pathogens. T cells are specialized cells of the adaptive arm of the immune system and recognize antigens via their T cell receptor (TCR). The TCR is encoded by the random and irreversible recombination of V(D)J gene segments, a process resulting in a highly diverse repertoire of TCRs. The large diversity of the repertoire offers recognition of, and therefore immunological protection against, a wide range of antigens for the host. The clonal composition of the immune repertoire allows insights into the current state as well as the antigen exposure history of an individual and can be determined by next-generation sequencing (NGS). Increasing age and diseases like cancer can compromise this fine-tuned network thus leading to chronic inflammation, autoimmunity or insufficient responses towards harmful antigens.
The work presented here discusses age-, cancer and autoimmunity-associated alterations of the TCR repertoire and summarizes fundamental aspects of T cell immunity as well as applications and challenges of adaptive immune receptor repertoire sequencing (AIRR seq).
In the first publication, which arose from this thesis, we profiled more than 300 peripheral blood TCR repertoires from healthy individuals as well as patients with cancer of all age groups. These analyses revealed a continuous age-dependent decline of T cell diversity which was accompanied by a decrease in T cell richness beginning at the fourth decade of life. Moreover, we observed that patients with cancer who received chemotherapy reestablished their pre-treatment T cell diversity. Interestingly, the regeneration of the repertoire complexity after chemotherapy was best explained by rebound thymic activity rather than through recovery of T cell counts by peripheral clonal expansion only.
Taken together our data indicate that TCR metrics deteriorate gradually with increasing age, but age-specific repertoire metrics are restored after chemotherapy even in elderly patients with cancer.
In the second publication, we profiled the TCR repertoires of patients with autoimmune cytopenias (AIC) to understand if these patients present disease-specific immunological signatures that could reveal pathophysiological clues and eventually be exploited as bloodbased biomarker. We analyzed 25 newly diagnosed patients with primary or secondary (lymphoma-associated) AIC as well as three reference cohorts composed of age- and sexmatched healthy controls, patients with active autoimmune hepatitis (AIH; another primary autoimmune disease) as well as patients with chronic lymphocytic leukemia (CLL, without autoimmune complication). Global TCR repertoire metrics like diversity and clonality as well as VJ gene usage distribution showed uniform characteristics for patients with lymphoma, but no AIC-specific signature. However, clustering of T cells with overlapping antigen specificity using the GLIPH algorithm (grouping lymphocyte interaction by paratope hotspots) revealed a considerable lack of T cell clusters in patients with primary autoimmune disease (AIC as well as AIH) as compared to healthy donors. The functionality of these clusters will have to be elucidated in future studies. Nevertheless, the signature of T cell cluster loss may represent a readily accessible biomarker for autoimmune conditions.
Taken together, AIRR seq has deepened our understanding of the adaptive immune response and has helped thus far to answer pressing questions in fields of immunodiagnostics, vaccines, cancer immunotherapy, and antibody engineering. As technical and computational challenges are being solved, this technology will come to realize its true potential
Characterization of B-cell-immuno response after allogenic stem cell transplantation in multiple myeloma
No full textPatienten, die an einem Multiplen Myelom (MM) leiden und aus diesem Grund allogen knochenmarktransplantiert (allo-Tx) wurden, können daraufhin oligoklonale Proteinbanden (APB) in der Immunfixation entwickeln. Während die Ursache hierfür unbekannt ist, weiß man, dass das Auftreten von APB (als Ausdruck einer B-Zell-vermittelte Anti-Tumor-Immunantwort) mit einer Verbesserung der Prognose einhergeht. In dieser Arbeit wurden via Phagedisplay Mimotope selektiert, welche die post-Tx-Antikörper von sechs MM-Patienten spezifisch binden. Die so erhaltenen Phagen wurden daraufhin mittels ELISA auf ihre Bindungseigenschaften zu anderen post-Tx-Antikörpern evaluiert (Kreuzreaktivitätsanalyse). Es wurde ebenfalls mittels ELISA untersucht, wie sich die B-Zell-Immunantwort über die Zeit nach der allo-Tx quantitativ entwickelt. Für jeden post-Tx-AK konnte mindestens ein spezifisch bindendes Mimotop selektiert werden. Einige Mimotope zeigten weitreichende myelom-spezifische Kreuzreaktivitäten. Hierbei stellt sich eine signifikante Abhängigkeit der Polyreaktivität der Patientensera zu dem Ansprechen auf die allo-Tx (p < 0; 05) dar. In den Verlaufs-ELISAs konnte gezeigt werden, dass die Bindung zu den Mimotopen innerhalb eines Jahres eine
stärkste Ausprägung annimmt und dann mit größer werdendem Abstand zur allo-Tx sukzessive geringer wird.
In dieser Arbeit wurde die humorale Anti-Tumor-Immunantwort nach allo-Tx zum ersten mal auf Epitopebene untersucht. Eine Zuordnung eines gefundenen Mimotops zu einem dazugehörigen parentalem Antigen ist durch die durchgef¨uhrten Experimente nicht möglich, kann aber durch weitere Experimente erfolgen. Die Ergebnisse zeigen, dass die gefundenen Epitope spezifisch für das genannte Patientenkollektiv sind. Eine Polyepitopreaktivität stellt einen erstrebenswerten Zustand dar, der mit einer Besserung der Prognose einhergeht. Dies unterstützt die Theorie der Epitope als Ziele einer humoralen Anti-Tumor-Immunantwort. Diese Ergebnisse können weiter evaluiert werden und hieraus sowohl diagnostische als auch therapeutische Mittel entwickelt werden.Patients suffering from Multiple Myeloma who underwent allogenic stem cell transplantation allo-SCT may develop abnormal protein bands (APB) in immunofixation. It is known that the appearance of APB (which is presumably an expression of a B-cellmediated anti-tumor-immune response) leads to an improved prognosis.
In this thesis mimotopes binding specifically to the post-SCT antibodies of six patients were selected using phage display. The hereby obtained phages were subsequently examined via ELISA regarding their interaction towards other antibodies. Afterwards the chronological sequence of mimotope reactivity was evaluated. For each patient at least one mimotope could be found that was binding significantly. A significant amount of myeloma specific cross reactivities could be observed. The polyreactivity of a patient’s serum correlates significantly with the response of that patient to the allo-Tx. In the experiments examining the chronologiccal sequence of mimotope reactivity the strongest antibody mimotope binding could be observed approximately one year after allo-SCT and declines from there on.
In this thesis the b-cell mediated anti-tumor immunity following allo-SCT was examined on epitope level for the first time. A connection of the found mimotopes to their parental antigenes was not possible with the conducted experiments, but may be established using additional experiments. The results show a specificity of the found epitopes for MM patients with APB after allo-SCT. A polyepitope reactivity is a desirable situation that correlates with an improved prognosis. This supports the theory of these epitopes being targets of a humoral anti-tumor immunity. These results may be evaluated further in order to develop diagnostic as well as therapeutic tools
Evaluation of the humoral immune response against Cancer-testis-antigens after autologous and allogeneic stem cell transplantation in patients with multiple myeloma
No full textIdentification and characterisation of resistance mediating variants of the epidermal growth factor receptor (EGFR) and following signalling proteins under EGFR-targeted antibody therapy in patients with colorectal cancer and head and neck squamous cell carcinoma
No full textZu den drei elementaren Säulen der Krebstherapie (Chirurgie, Chemotherapie und Strahlentherapie) konnte in letzten Jahren ein weiterer Pfeiler hinzugefügt werden, die zielgerichtete Therapie, sog. targeted therapy. Zielgerichtete Therapeutika binden hoch spezifisch an auf Tumorzellen exprimierten Strukturen, sind somit lokal sehr effizient und schonen gesundes Gewebe (1,2). Eine solche Zielstruktur ist der epidermale Wachstumsfaktor Rezeptor (EGFR). In gesunden Zellen führt die Bindung des epidermalen Wachstumsfaktors (EGF) an den EGFR zu einer Signalgenerierung am Rezeptor, die über eine zellinterne Signalkaskade zu Zellwachstum und Proliferation führt, bevor das Signal über strikte Regulationsmechanismen abgeschaltet wird (3). In vielen soliden Tumoren, wie z.B. beim metastasierten kolorektalen Karzinom (mCRC) und in Kopf-Hals-Tumoren (head and neck squamous cell carcinoma = HNSCC), ist diese Signalkaskade jedoch fehlreguliert, was zu ständigem, unkontrollierten Zellwachstum führt (4,5). Dies macht den EGFR zum idealen Angriffspunkt zielgerichteter Therapien: Der therapeutische anti-EGFR Antikörper Cetuximab ist bei Patienten mit mCRC und HNSCC zugelassen, alternativ kann beim mCRC der anti-EGFR Antikörper Panitumumab eingesetzt werden. Beide Antikörper binden an die extrazelluläre EGFR-Domäne, blockieren so die EGF-Bindung und dadurch die Signalgenerierung am Rezeptor, was letztlich zur Hemmung des Tumorzellwachstums und der Proliferation führt (6-11).
Leider sind viele Patienten bereits vor Beginn einer Antikörpertherapie resistent oder entwickeln im Laufe der Behandlung sekundäre Resistenzen. Die zugrundeliegenden molekularen Mechanismen sind bisher nur teilweise geklärt (12-17). Ziel dieser Arbeit war die Wirkungsmechanismen therapeutischer Antikörper besser zu verstehen, molekulare Mechanismen, die zur Resistenz gegen anti-EGFR Antikörper führen aufzuklären und neue prognostische Biomarker zu etablieren.
Zunächst wurde das funktionelle Panitumumab-Epitop auf dem EGFR identifiziert und gegen das bereits bekannte Cetuximab-Epitop abgegrenzt. Anschließend wurde in Tumor-DNA und zirkulierender Tumor-DNA (ctDNA) von mCRC-Patienten unter Antikörpertherapie nach Mutationen in diesen Epitopbereichen gesucht. Als neue, resistenzvermittelnde EGFR-Mutation wurde in dieser Arbeit erstmalig die EGFR-Mutation G465R identifiziert welche zur Resistenz gegenüber beider EGFR-Antikörper Panitumumab und Cetuximab führt. Desweiteren konnte erstmalig gezeigt werden, dass es unter EGFR-Antikörpertherapie zu neu auftretenden aktivierenden rat sarcoma viral oncogene homolog (RAS)-Mutationen kommt, kommt welche neben den Mutationen im EGFR-Rezeptor die Entwicklung sekundärer Resistenzmechanismen im Tumor begründen.
Die Methode der liquid biopsy zeigt, dass resistenzvermittelnde minimale Subklone bereits detektiert werden können, noch bevor die Patienten eine klinische Resistenz gegen die therapeutischen Antikörper zeigen. Dadurch eignet sich das minimalinvasive Verfahren der liquid biopsy sehr gut für ein Monitoring möglicher resistenzvermittelnder Mutationen unter einer laufenden Therapie.
Die Ergebnisse dieser Arbeit sollten in weiteren klinischen Studien an größeren Patientenkollektiven evaluiert werden, da sie helfen können resistenzvermittelnde Subklone frühzeitig zu erkennen und die Therapieoptionen schnell und individuell anzupassen, um so unnötige Nebenwirkungen der Therapeutika in den Patienten zu vermeiden.In addition to surgery, chemo- and radiotherapy, a novel therapy option was developed in recent years, called targeted therapy. Targeted therapeutics bind with high specificity to tumor cells, which makes them very efficient in targeting tumor cells and sparing healthy tissue (1,2). A promising target for this purpose is the epidermal growth factor receptor (EGFR). In healthy cells, the binding of the epidermal growth factor (EGF) to the EGFR triggers an internal signal cascade, leading to cell growth and proliferation (3). In healthy tissues, the signal cascade is tightly regulated. In many solid tumors, like metastatic colorectal cancer (mCRC) or head and neck squamous cell carcinoma (HNSCC) these mechanisms are dysregulated, which leads to constant, uncontrolled cell growth (4,5). Since the EGFR is located in the cell membrane, the receptor is the ideal point of attack for targeted therapeutic antibodies: Cetuximab, a therapeutic EGFR-antibody is used in patients with mCRC and HNSCC. For mCRC-treatment, the EGFR-antibody Panitumumab may be used alternatively. Both antibodies bind to the extracellular EGFR-domain, blocking EGF binding and therefore the growth signal generation (6-11).
Unfortunately, many patients are already resistant to theses therapeutic antibodies or develop resistances in the course of treatment. The underlying molecular mechanisms are so far only partially understood (12-17). The aim of this study is to better understand these antibodies’ mechanisms of action and to discover novel molecular mechanisms leading to resistance against therapeutic EGFR-antibodies in patients with mCRC and HNSCC.
In the course of this study we first functionally dissected the exact epitopes of both therapeutic antibodies on the EGFR. This was followed by a screening for mutations within these epitopes in tumor and circulation tumor DNA (ctDNA) samples of antibody treated mCRC-patients. We found and functionally evaluated the EGFR-mutation G465R, which leads to resistance against Panitumumab and Cetuximab due to binding disruption. In ctDNA samples of HNSCC patients we did not find any EGFR-mutations. However, for the first time we could show that HNSCC-patients also developed activating RAS-mutations during Cetuximab therapy. A key feature of this work was a method called liquid biopsy, which enabled the detection of resistance mediating subclones, even before patients showed any clinical sign of resistance against the therapeutic antibodies. Therefore, liquid biopsy is suitable for monitoring resistance mediating mutations during therapy.
These results might help to detect resistance mediating tumor subclones even before clinical resistance occurs, which may help in tailoring EGFR-targeted therapies so unnecessary side effects can be avoided. A further validation of these results in large clinical studies of defined patient cohorts receiving EGFR-inhibiting antibodies is therefore highly desirable
RNA basierte Tiefensequenzierung des B-Zell Rezeptors zur Detektion und Überwachung von B-Zell Neoplasien
No full textWith our findings, we have established a functioning RNA-based B-cell receptor deep sequencing platform that allows:
• Detection of malignant clones in the peripheral blood of patients with multiple myeloma and potentially other B-cell neoplasms
• A potentially higher sensitivity yield compared to routine FACS analysis
• Tracking of the diversity of a circulating B-cell repertoire and of the presence of malignant clones throughout treatment
• Identification of the immunoglobulin class and subclass of a specific BCR
Nevertheless, regardless of the promising clinical applications, one must also be aware of the technological limitations of this technique.Wir konnten erfolgreich eine RNA-basierte Tiefensequenzierungsplattform des B-Zell Rezeptors in unserer Arbeitsgruppe etablieren. Diese Plattform ermöglicht uns:
• maligne Klone von B-Zell Neoplasien wie dem multiplen Myelom zu detektieren
• potentiell höhere Sensitivitäten bei B-Zell Detektion zu erzielen, als dies mit herkömmlichen Methoden wie der FACS Analyse möglich wäre
• den Verlauf von B-Zell Neoplasien unter Therapie näher zu verfolgen mit potentiellen Implikationen für die Therapie
• die Immunglobulin Klasse wie auch Subklasse aller B-Zell Rezeptoren in einer Blutprobe zu bestimmen
Neben den beschriebenen vielseitigen potentiellen zukünftigen Anwendungen dieser Technologie, dürfen die aufgeführten Limitationen nicht vernachlässigt werden
The Influence of Lenalidomide on the Immune Repertoire of Patients with Myelodysplastic Syndrome with the Chromosal Change del(5q)
No full textEs sind noch immer nicht alle Aspekte der Wirkungsweise von immunmodulatorischen Substanzen, allen voran Lenalidomid, bei der Behandlung von Erkrankungen des blutbildenden Systems verstanden. Ein Einfluss auf den funktionellen Status von Immunzellen wird in der Literatur seit längerem beschrieben. Ziel dieser Studie war es, die möglichen Effekte von Lenalidomid auf die Komposition des Immunrepertoires, folglich der Tund B-Zellen von Patienten mit myelodysplastischem Syndrom (MDS) mit einer Deletion des langen Armes von Chromosom 5 del(5q) im Knochenmark und peripheren Blut zu untersuchen. Hierfür wurden 15 Knochenmarkproben und 8 Proben peripheres Blut von MDS del(5q) Patienten vor und nach der Therapie und Proben von gesunden Spendern passenden Alters mittels modernster „Next Generation Sequencing“ (NGS) Technologie untersucht. Die Beobachtungen im B-Zell Kompartiment lassen auf einen proliferativen Effekt auf unreife noch nicht Antigen exponierte B-Zellen schließen. Dies wurde für Lenalidomid schon früher beschrieben. Mit Hilfe von Algorithmen konnten aus den Datensätzen der Proben T-Zellen mit höchstwahrscheinlich gleicher Antigenerkennung zu sogenannten Clustern zusammengefasst werden. Es ließ sich beobachten, dass eine große Zahl der zu beiden Probenzeitpunkten existenten Cluster eine hohe Entstehungswahrscheinlichkeit aufwies. Im Gegensatz dazu standen unter Therapie neu entstandene Cluster mit einer niedrigen bis mittleren Entstehungswahrscheinlichkeit, von denen einige bei mehreren Patienten zu finden waren. Dies spricht stark für eine Antigen gerichtete Generierung von neuen T-Zell Clustern gegen den MDS del(5q) Klon unter Lenalidomidtherapie. Unsere Ergebnisse sprechen dafür, dass Lenalidomid neben seinen bekannten Effekten auf den funktionellen Status von Immunzellen ebenfalls die Komposition des Immunrepertoires von Patienten mit MDS del(5q) beeinflusst.The effects of immune modulatory drugs such as Lenalidomide for the treatment of diseases of the haemapoietic system is currently a topic of research. However, medical literature describes an influence of Lenalidomide on the functional status of immune cells. The main focus of this study was to investigate the various effects of the drug on B and T-cells found in bone marrow und peripheral blood of patients with myelodysplastic syndromes (MDS) with a partial deletion of the long arm of chromosome 5 (del(5q)). Blood and bonemarrow was taken from 8 and 15 Patients with MDS del(5q) respectively, before and after treatment with Lenalidomide as well as donated blood and bonemarrow from healthy individuals of a similar age. All samples were examined with state of the art next generation sequencing technology.Our observations validate a proliferatve effect on young, non-antigen experienced B-cells in accordance with previously publications. An algorithm was then used to identify T-cells with a presumed identical antigen recognition which were assigned to specific ‘clusters‘. It was observed that the T-cell clusters that were found before and after the treatment with Lenalidomide had a high generation probability. A small number of new T-cell clusters emerged after the treatment with the drug. These clusters had a low to medium generation probability which indicates new T cell cluster generation in response to Lenalidomide. Our results show that Lenalidomide not only has an effect on the functional status of immune cells but influences the immune cell composition of patients with MDS del(5q)
Hospital population screening reveals overrepresentation of CD5− monoclonal B-cell lymphocytosis and monoclonal gammopathy of undetermined significance of IgM type
No full textMonoclonal B-cell lymphocytosis (MBL) and monoclonal gammopathy of undetermined significance (MGUS) result from clonal expansions of mature B or plasma cells. Here, we set out to determine the immunophenotypic/monoclonal immunoglobulin (M protein) features and co-prevalence of MBL and MGUS in a hospital-based cohort of 1909 non-hematooncological patients. Of the evaluable cases, 3.8 % showed evidence for MBL by immunophenotyping, while 9.8 % were screened positive for M protein by immunofixation. With six concomitant cases (0.4 %), MBL and MGUS were not statistically associated. At least in two of these coincident cases, MBL and MGUS were of different clonal origin since both clones had divergent light chain restriction. CD5(-) MBL (57.1 %) and IgM+ MGUS (24.7 %) were strikingly overrepresented compared to population-based screenings and did not progress to overt lymphoma or myeloma during the observation period (mean follow-up of 117 weeks or 110 weeks, respectively). Prevalence and phenotypes suggest that a substantial proportion of incidental MBL and MGUS in hospitalized patients may be attributed to transiently expanded B-cell clones in the context of disease-related immune stimulation rather than reflecting veritable precursors of clonal B-cell malignancies
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