Bei den konventionellen Krebstherapien kommt es häufig zu einer Wiederkehr des Tumors, da meist einzelne Tumorzellen und abgesiedelte Metastasen im Körper verbleiben. Vor diesem Hintergrund hat die Entwicklung neuer Behandlungsmethoden, die spezifisch die Tumorzellen erkennen und eliminieren und zudem gesunde Körperzellen schonen, eine große Bedeutung in der heutigen Krebsforschung. Eine erfolgsversprechende Strategie ist die Generierung von tumorspezifischen, zytotoxischen Immuneffektorzellen, zum Beispiel T-Lymphozyten und Natürlichen Killerzellen, durch die genetische Modifikation mit einem chimären Antigenrezeptor (CAR). Dabei gibt es bereits weitreichende Studien mit T-Lymphozyten, so dass sich nun das Forschungsinteresse immer mehr auf die NK-Zellen richtet. Im Gegensatz zu CAR-armierten T-Lymphozyten sind sie in der Lage ihr antitumorales Potenzial nicht nur gegen Antigen-positive sondern auch MHC-Klasse I-negative Tumorzellen zu richten. Mögliche Zielstrukturen der CAR sind tumorassoziierte Antigene, wie das Prostata-spezifische Stammzellantigen (PSCA). Es wird auf über 94 % der humanen primären Prostatakarzinome und deren Knochenmetastasen verstärkt exprimiert, jedoch kaum auf Normalgewebe. PSCA ist somit ideal für eine Immuntherapie geeignet. Die bisher in Studien verwendeten CAR-armierten NK-Zellen wiesen eine feststehende Spezifität gegenüber einem bestimmten Tumorantigen auf. Allerdings ist die Expression von Tumorantigenen innerhalb des Tumors sehr heterogen oder wird durch Tumorevasionsmechanismen herunterreguliert. Dies begrenzt die Reaktivität CAR-armierter NK-Zellen. Durch die Generierung eines CAR, dessen Spezifität gegenüber einem Tumorantigen ausgetauscht werden kann, wäre der universelle Einsatz CAR-armierter NK-Zellen in der adjuvanten Immuntherapie von Tumorerkrankungen möglich. Im Hauptteil dieser Arbeit wurden die permanente NK-Zelllinie YTS und primäre humane NK-Zellen mittels lentiviralen Gentransfers mit einem PSCA-spezifischen CAR, bestehend aus dem gegen PSCA gerichteten Einzelkettenantikörper αPSCA und dem aktivierenden NK-Zellrezeptor NKp46, armiert. Die generierten NK-Zellen wiesen eine über längere Zeiträume stabile Oberflächenexpression des CAR αPSCA-NKp46 auf. Die Kreuzvernetzung des CAR mit seinem Antigen führte zunächst zu keiner selektiven Immunantwort der CAR-armierten YTS und primären NK-Zellen gegenüber histogenetisch verschiedenen, PSCA-exprimierenden Tumorzelllinien. Erst nach gleichzeitiger Überexpression des mit NKp46 assoziierten Signaladaptermoleküls CD3-ζ wurde eine Aktivierung der Effektorfunktionen der YTS NK-Zellen induziert. Dies zeigte sich zum einen in der Expression von CD107a als Degranulationsmarker sowie der Freisetzung des inflammatorischen Zytokins IFN-γ. Zum anderen wiesen die CAR-armierten und CD3-ζ-exprimierenden YTS NK-Zellen eine spezifische Zytotoxizität gegenüber MHC-Klasse I- und PSCA-exprimierenden Tumorzellen auf. Im anschließenden Teil der Arbeit wurde ein modular aufgebauter CAR generiert, bei dem der Einzelkettenantikörper und folglich die Spezifität gegenüber Tumorantigenen austauschbar ist. Dazu wurden YTS NK-Zellen durch lentiviralen Gentransfer mit dem biotinylierbaren NKp46-NK-Zellrezeptor NKp46-KiBAP modifiziert, der über mehrere Monate stabil auf der Oberfläche exprimiert wurde. Die exogene als auch endogene Biotinylierung des Rezeptors wurde mittels einer Biotinproteinligase demonstriert. Unter Ausnutzung der sehr starken Biotin-Avidin-Bindung wurde die Assoziation mit einem Einzelkettenantikörper nachgewiesen. Dafür wurde exemplarisch der gegen PSCA gerichtete, biotinylierbare Einzelkettenantikörper αPSCA-BAP verwendet. Diese Arbeit zeigt, dass eine spezifische Erkennung und effiziente Lyse von PSCA-exprimierenden Tumorzellen durch die generierten CAR-armierten NK-Zellen erfolgte, wobei zum ersten Mal NKp46 als Bestandteil eines CAR verwendet wurde. Zudem wurde ein modular aufgebauter CAR generiert, dessen Spezifität gegenüber Tumorantigenen austauschbar ist. Diese neuartige Strategie ermöglicht erstmalig eine flexible Armierung von NK-Zellen und stellt damit einen wesentlichen Vorteil bei der Behandlung verschiedener Krebserkrankungen dar.

BACKGROUND: Natural killer (NK) cells are traditionally considered part of the innate immune system but have recently been shown to possess adaptive qualities similar to T cells in response to an infection with a pathogen. In addition to possessing adaptive features, NK cells have also been found to reside in different organs such as the liver, spleen, and lymph nodes and differ based on phenotypic markers and their responses to different cytokines. Utilizing these findings, several groups have isolated and identified CD49a as a marker for tissue-resident NK cells. In the liver, CD49a has also been shown to be a positive indicator for NK cell memory-like responses in murine models. Building off work that demonstrated antigen-specific responses in rhesus macaques, this project focuses on characterizing the phenotypic markers and functional profile of CD49a+ NK cells in non-human primates. To better understand the role of CD49a in memory-like NK cells outside of the liver, this project utilized spleen samples from rhesus macaques infected with SIV/SHIV. This work aims to help us better understand the dysfunction NK cells experience as a result of HIV-1 infection in humans and also to demonstrate the changes NK cells experience as the disease progresses. A thorough understanding of the adaptive capabilities of NK cells can pave the way for targeted therapies to increase NK cell antiviral activity in HIV and other infections. METHODS: To characterize the functional and phenotypic profiles of CD49a+ NK cells by multiparameter flow cytometry, thirteen samples of spleen from rhesus macaques were thawed and then stained with two different protocols. A phenotyping protocol involved staining with antibodies against surface markers as well as intracellular markers T-Bet and Eomes. For the functional characterization protocol, the same thirteen samples were stained intracellularly after being stimulated with a cocktail of PMA and ionomyocin. The antibodies used in this were for functional markers. Of the thirteen samples used, six were infected with SHIVSF162P3, two were infected with SIVmac239X, and the remaining five were uninfected. After staining, these samples were analyzed on an BD LSRII from BD Biosciences. The data obtained were then analyzed using FlowJo software to study NK cells, which were characterized as CD45+CD14-CD20-CD3-CD159+. RESULTS: The analysis compared NK cells with T cells, B cells, and NKB cells. Some increases were seen among CD49a+ NK cells in the frequency of CD336+ (NCR2/NKp44), CD337+ (NKp30), and CD366+ (Tim-3) after infection. Although there were some mild increases in CD107a and TNF- in infected samples compared to uninfected, a significant increase was observed in the frequency of IFN-ɣ among infected CD49a+ NK cells compared to uninfected. CONCLUSION: When comparing samples that were infected vs uninfected, it appears there were some mild decreases after infection in the ratio of NK cells to other lymphocytes. In addition, there did not appear to be a significant increase in the frequency of CD49a+ among these NK cells as a result of the infection. However, among the CD49a+ subpopulation, there were some observed non-significant decreases in CD56-CD16+ cells. Furthermore, there was found to be an almost significant increase in TNF- (p = 0.06) among CD49a+ cells after infection. These findings demonstrate an increase in cytotoxic activity in splenic NK cells associated with an adaptation to the virus. Although there does not appear to be significant changes in the ratio of NK cell populations in the spleen, the changes observed in phenotypic and functional markers associated with CD49a+ demonstrate an increase in the cytotoxic activity of NK cells as a result of infection with SIV/SHIV. However, it remains to be seen if CD49a is a direct indicator of this type of infection. Future work geared toward memory-like NK cells in non-human primate splenic tissue could look at the contrast in CD49a+ NK cells from different states of infection with HIV-1 and/or SIV (acute vs chronic) to better understand the integrin’s role in adaptation.

Natural killer (NK) cells belong to the family of innate immune cells with the capacity to recognize and kill tumor cells. Different phenotypes and functional properties of NK cells have been described in tumor patients, which could be shaped by the tumor microenvironment. The discovery of HLA class I-specific inhibitory receptors controlling NK cell activity paved the way to the fundamental concept of modulating immune responses that are regulated by an array of inhibitory receptors, and emphasized the importance to explore the potential of NK cells in cancer therapy. Although a whole range of NK cell-based approaches are currently being developed, there are still major challenges that need to be overcome for improved efficacy of these therapies. These include escape of tumor cells from NK cell recognition due to their expression of inhibitory molecules, immune suppressive signals of NK cells, reduced NK cell infiltration of tumors, an immune suppressive micromilieu and limited in vivo persistence of NK cells. Therefore, this review provides an overview about the NK cell biology, alterations of NK cell activities, changes in tumor cells and the tumor microenvironment contributing to immune escape or immune surveillance by NK cells and their underlyingmolecular mechanisms as well as the current status and novel aspects of NK cell-based therapeutic strategies including their genetic engineering and their combination with conventional treatment options to overcome tumor-mediated evasion strategies and improve therapy efficacy.

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<p>NK cells play a pivotal role in tumor rejection; however, once present in the tumor microenvironment, they are characterized by decreased cytotoxicity and reduced expression of activating receptors. The mechanisms governing the inactivation of NK cells within tumors remain poorly understood. Since tumor associated macrophages (TAMs) are a highly abundant and suppressive cell type within tumors, we hypothesized that they are capable of altering the function of NK cells. Following the co-culture of alternatively activated macrophages (M2) or TAMs with NK cells we observed that the expression of the cytotoxic marker CD27 on NK cells was down-regulated as well as the ability of these cells to kill YAC-1 cells in a killing assay. We have demonstrated that the mechanism by which M2 cells inhibit NK cells is TGF-β dependent. Notably, the developmental stage of NK cells after interaction with TAMs was altered and the NK cells became phenoytpically mature and potentially exhausted (CD27^lowCD11b^high). This prompted our interest in examining the developmental stage of NK cells from polyoma MT antigen (pyMT) transgenic mouse (MMTV-pMT) breast tumors. Interestingly, in contrast to the <em>in vitro</em> results, we have shown that NK cells isolated from pyMT tumors are developmentally immature; however maintain their maturity within the spleen. Their immature phenotype correlates well with their decreased expression of perforin, granzyme, and NKp46. Both our <em>in vitro</em> studies with TAMs and our <em>in vivo</em> developmental studies using the pyMT model demonstrate that NK cells are altered by their surroundings. A better understanding of how NK cells are modified by the tumor microenvironment will help to develop strategies aimed at bolstering immune responses against tumors.</p> / Master of Science (MSc)

<p>IL-15 is a cytokine that has effects on both innate and adaptive immune cells, including NK and CD8 T cells. The involvement of these cell types in tumor immunosurveillance and eradication has led to interest in IL-15 as an immunotherapy. Its role in spontaneous solid cancers has not been studied thoroughly. Here, we have shown for the first time that IL-15 overexpression in a spontaneous breast cancer model, MMTV-polyoma Middle T antigen (MT), leads to increased survival, tumor destruction, and decreased metastasis (IL-15 TG/MT). In contrast, lack of IL-15 led to decreased survival (IL-15 KO/MT) and increased metastasis. Protection in IL-15 TG/MT mice was dependent upon the presence of highly activated NK1.1+ cells, but not dependent upon CD8 T cells. The cytokine environment in IL-15 TG/MT tumors was capable of activating human NK cells to kill human triple negative breast cancer cells. In a model of metastasis, we found that overexpression of IL-15 protected from metastasis in a NK cell dependent manner. Lack of IL-15 promoted the polarization of CD4 T cells to a Th2 phenotype and they influenced polarization of macrophages to an M2 phenotype. M2 macrophages help establish tumors at the metastatic site. Here we found that M1 polarized macrophages could prevent engrafted breast tumor formation, whereas M2 macrophages promoted it. Overall, IL-15 is an extremely promising immunotherapy that has more anti-tumor effects on the immune system than were previously appreciated. Additionally, our data argues that it could generate immune responses against both primary breast cancer and metastasis.</p> / Doctor of Philosophy (Medical Science)

In last decades, with expansion of immunological and biological methods are developed new diagnostical and treatment processes, which enable stratification of patients into sanative groups and trend to individual therapy. Absolutely transparent are effects relevant to leukemia. Present treatment procedures enable not only longer survivance of patients, but often their stable sanation. In present time is in progress intesive research imunotherapy NK cells, which could be able to finish minimal residual disease after chemotherapeutical treatment, which is evoke by persistant malignant cells. Next advantage of this treatment procedure is elimination of system disease in cosequence of exactly pointed cure. In this work he attended in vitro testing to possibility of utilization imunotherapeutic treatment by NK cells acute and chronic myeloid leukemia and chronic lymfoblastic leukemia. Using flow cytometry methods we detected activation and inhibitory ligands which are recognized by NK cells on the cell surface of leukemia blasts. These are members of MHC complex HLA-E, molecules derived from MHC class I (MICA, MICB), UL16-binding proteins (ULBP-1, ULBP -2, ULBP -3, ULBP -4) and also Hsp70 protein according to the newest observation. We also atended to detection of expression inducible heat shock...

Since being identified as the cause of AIDS in 1983, HIV-1 infection has reached pandemic proportions. Despite public awareness about prevention, the growing incidence of HIV-1 infection and the limitations of current antiretroviral therapy underscore the imperative need for a vaccine. Understanding the basis of an immune response that controls infection or provides sterilizing immunity remains a major goal in the search for effective vaccines or immunotherapies. Research into correlates of immunity to HIV-1 have largely focused on CD8⁺ T cells or neutralising antibodies (NAbs) but to date these responses have not proved effective in containing viral replication in vaccinees who become infected. Natural killer cells (NKs), monocytes (MCs), and neutrophils (PMNs) are cells of the innate immune system with intrinsic cytotoxic function that can be enhanced by antibodies (Abs) in what is termed antibody-dependent cellular cytotoxicity (ADCC). In my studies I investigated the production of PMNs from human stem cells, the elimination of HIV-1 infected cells by these effector cells, the modulation of cellular cytotoxicity by Ab, and characterized how Abs facilitate a potent ADCC response. I developed a novel flow cytometry assay to measure cytotoxic activity against HIV-1 infected CD4⁺ T cells. Using this, effector cells were shown to have different cytotoxic capacities which were enhanced by Ab. Comparing ADCC mediated by patient serum revealed that higher levels correlated with IgG binding to infected cells. I observed no correlation between serum-mediated ADCC and markers of disease progression including patient status, viral RNA load, CD4⁺ T cell count, or NAb titers. The data presented here have implications for acquisition and control of early HIV-1 infection by NKs, MCs, and PMNs prior to activation of an adaptive immune response, at later stages in the presence of HIV-1-specific Abs, and are relevant to vaccine-induced anti- HIV-1 Ab-based effector mechanisms.