Tesis sobre el tema "Cell-based immunotherapy"

The quality of life of a significant proportion of IBD patients is poor as a result of persistent disease activity and repeated surgery, among others. Current treatments for Crohn’s disease are not able to neither prevent this serious impact nor improve the long term prognosis of a significant proportion of patients. Therefore, new therapeutic approaches are needed in order to modify the immune response of these patients. We hypothesize that administration of ex-vivo generated autologous tol-DCs to Crohn’s disease patients may arrest Th1 lymphocyte proliferation and therefore may restore specific tolerance against non-pathogenic antigens in the gut. The overall objective of this thesis was to generate and characterize tol-DCs for the purpose of implementing an autologous immunotherapy treatment for Crohn’s disease patients. In the first study, we described the generation of tol-DCs from healthy donors and Crohn’s disease patients by use of clinical-grade reagents in combination with dexamethasone as immunosuppressive agent and characterized their functional properties. Our main findings demonstrated that the combination of dexamethasone with a specific cytokine cocktail yields clinical-grade DCs with the following characteristics: a semi-mature phenotype, a pronounced shift towards anti-inflammatory versus inflammatory cytokine production and low T-cell stimulatory properties. This characteristic tolerogenic profile is maintained when tol-DCs are activated using heat-inactivated Gram-negative bacteria as maturative stimulus. Whole microorganisms contain multiple PAMPs capable of stimulating DCs by different pathways. Our results clearly showed a strong inhibitory effect on DC phenotype, a robust inhibition of pro-inflammatory cytokines, increased IL-10 secretion, and inhibition of T-cell proliferation and Th1 induction. Interestingly, we showed that tol-DCs have reduced immunogenic capacity in autologous, allogeneic and antigen-specific T-cell responses. We further evaluated the ability of tol-DCs to induce CD4+ T-cell hypo-responsiveness. Our results demonstrated that T-cells or antigen-specific T-cells previously cultured with tol-DCs are anergic exhibiting a reduced capacity to proliferate as well as reduced IFN-gamma secretion when rechallenged with fully competent mDCs. With regard to tol-DCs clinical application, we importantly found that their tolerogenic properties remain stable after washing out dexamethasone and subsequent restimulation with LPS, CD40L or different Gram-negative enterobacteria strains. All these properties led us to conclude that this cell product is suitable to be tested in clinical trials of immune-based diseases such as Crohn’s disease. We further identified a positive biomarker for tol-DCs, MERTK receptor is highly expressed on clinical grade dexamethasone-induced human tol-DCs and contributes in their tolerogenic properties. Our results demonstrated that MERTK expression in human DCs is regulated by glucocorticoids and described a new function of this receptor in directly regulating T-cell response. Interestingly, our findings showed that neutralization of MERTK with monoclonal antibodies in allogeneic MLR cultures leads to increased T-cell proliferation and IFN-gamma production. The direct regulation of T-cell response was confirmed by the use of recombinant MERTK-Fc protein, used to mimic MERTK on DCs. Our results remarkably showed that MERTK-Fc suppresses naïve and antigen-specific memory Tcell proliferation and activation. These findings identified a new non-cell autonomous regulatory function of MERTK expressed on DCs. Additionally, we described that this regulation is mediated by the neutralization of MERTK soluble ligand PROS1. We also found that MERTK is expressed on T-cell surface and that PROS1 drives an autocrine pro-proliferative effect on these cells. In summary, the results of this work demonstrated that MERTK on DCs regulates T-cell activation and expansion through the competition for PROS1 interaction with MERTK in the T-cells. We showed that MERTK expression in human DCs has a key role in instructing adaptive immunity and identified MERTK as a potent suppressor of T-cell response. Therefore targeting MERTK may provide an interesting approach to effectively increase or suppress tolerance for the purpose of immunotherapy.
Esta tesis doctoral estudia el proceso de generación de células dendríticas tolerogénicas en grado clínico, con el objetivo de establecer un protocolo destinado al tratamiento de la enfermedad de Crohn. El estudio realizado ha permitido la caracterización de dichas células y sus propiedades tolerogénicas, incluyendo la descripción novedosa de un marcador de células tolerogénicas y el estudio de sus propiedades funcionales relacionadas con la inducción de tolerancia.

Scientific background. The inherent resistance of breast cancer stem cells (CSCs) to existing therapies has largely hampered effective treatments for advanced breast cancer. My research aimed at establishing novel immunotherapy approaches efficiently targeting CSCs by harnessing human γδ T cells as non-MHC-restricted killer cells and simultaneously as APCs to induce tumour-specific CD8+ T cell responses. Approach. An experimental model allowing reliable distinction of CSCs and non-CSCs was set up to study their interaction with γδ T cells and CD8+ T cells. FluM1 and CMVpp65 viral epitopes were used as surrogates for yet-to-be-discovered CSC-associated antigens. Results. Stable sublines with characteristics of CSCs and non-CSCs were generated from ras-transformed human mammary epithelial (HMLER) cells as confirmed by their (i) distinct expression profiles of CD24, CD44 and GD2, (ii) mesenchymal- and epitheliallike characteristics, (iii) differential growth patterns in mammosphere culture and (iv) distinct tumourigenicity, self-renewal and differentiation in NSG mice. The resistance of both CSCs and non-CSCs to γδ T cells could be overcome by inhibition of FPPS through pretreatment with zoledronate or FPPS-targeting shRNA, resulting in increased cytotoxicity and APC function of γδ T cells. CSCs presenting FluM1 or CMVpp65 exhibited stronger resistance to antigen-specific CD8+ T cells as compared to their non-CSC counterparts. Of note, pretreatment of Flu M1- or CMVpp65-presenting CSCs with γδ T cell conditioned supernatant significantly increased surface expression of MHC class I and ICAM-1 by both CSCs and non-CSCs as well as their susceptibility to CD8+ T cellmediated killing. Moreover, using the humanised anti-GD2 monoclonal antibody,Hu14.18K322A, a specific direction of γδ T cell responses against CSCs could be achieved. In addition to their direct cytotoxicity and ability to modulate the susceptibility of CSCs and non-CSCs to CD8+ T cell-mediated killing, γδ T cells concomitantly functioned as APCs to initiate de novo tumour-specific cytotoxic CD8+ T cell responses. Conclusions. My findings identify a powerful synergism between MHC-restricted and non-MHC-restricted T cells in the eradication of both CSCs and non-CSCs, thus establishing a powerful positive feedback loop for the eradication of residual cancer cells survived from killing by γδ T cells. My research suggests that novel immunotherapies may benefit from a two-pronged approach combining γδ T cell and CD8+ T cell targeting strategies that triggers effective innate-like and tumour-specific adaptive responses.

Cancer is a worldwide health problem and despite advances in traditional treatments i.e. surgery, chemotherapy and radiotherapy, the cure rate remains disappointing for some cancers. Different novel therapeutic strategies are being developed. In this thesis two nontraditional cancer therapy approaches are studied: gene therapy using viral vectors and antitumour vaccination with dendritic cell - tumour cell (DC/TC) hybrids.

We have developed a novel ELISPOT titration method for viral vectors that is based on the actual expression of the transgene in target cells. This method was developed with recombinant parvovirus MVM-IL2, but it should be adaptable for other vectors carrying expression cassettes for secreted transgene products for which antibodies are available. The ELISPOT titration method allows for faster and better quantification of transducing units present in vector stocks as opposed to titration by in situ hybridisation (annexe I). The MVMIL2 vector has shown an anti-tumour effect against melanoma in an immunocompetent mouse model (annexe IV). Previous work concerns photodynamic inactivation of adenoviral vectors for biosafety and an in vivo study in which a synergistic effect of antiangiogenesis gene therapy combined with radiotherapy could be shown (annexes V and VI).

DC/TC hybrids have been proposed as cancer vaccines for their simultaneous expression of antigen presentation machinery and tumour associated antigens. Hybrids are classically generated by polyethylene glycol (PEG) or electrofusion. These methods however require special skills and equipment and cause rather high cell lethality. Fusion via the expression of viral fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus-G (VSV-G) (annexe III) or the Gibbon ape Leukemia Virus (GaLV) FMG, have recently been described. We have mainly focussed on the latter. Transduction of cells with GaLV-FMG proved to be a limiting step for an efficient generation of hybrids. On the other hand, constitutive expression of GaLV-FMG leads to lethal syncytia formation in human cells. Therefore we developed a novel fusion strategy for the generation of DC/TC cell hybrids that involves the use of a non-human fusogenic cell line that constitutively expresses the GaLV-FMG. With this method we were able to generate reproducible yields of DC/TC triparental hybrids. The formation of tri-parental hybrids via the fusogenic cell line is an interesting alternative to existing DC/TC fusion methods because of its simplicity and its flexibility in the choice of fusion partners, i.e. autologous or allogeneic DCs and tumour cells.

Moreover, the tri-parent hybrid system offers the possibility to further enhance the immune response by the addition of transgenes that code for immuno-modulating factors to the fusogenic cell line (annexe II).
Doctorat en sciences biomédicales
info:eu-repo/semantics/nonPublished

22 patients with de novo or relapsed Acute Myeloid Leukaemia (AML) were recruited into a Phase I/II clinical trial aimed at vaccinating with autologous dendritic like leukaemia cells once in complete remission. At trial entry leukaemia cells were harvested and tested for their permissiveness to cytokine-induced dendritic cell differentiation. Study patients were then treated with induction chemotherapy. Five patients achieved both complete remission and had leukaemia cells that were permissive to differentiation, and were therefore eligible to proceed to vaccination. Four escalating doses of dendritic like leukaemia cells were administered weekly by subcutaneous injection. An increase in anti-leukaemic T cell responses was demonstrated in four patients. Vaccination was generally well tolerated. Two patients relapsed during or shortly after the vaccination schedule. In the remaining three patients, one relapsed at 12 months with two in continued remission more than 12 months post vaccination. In a parallel investigation, the potential of Tumour Cell / Dendritic Cell Fusion Hybrids to generate in vitro anti-leukaemic T-cell responses following co-culture with autologous remission lymphocytes was assessed in six patients with AML. Comparison was made to anti-leukaemic responses induced by mature dendritic cells (mDC) co-cultured with autologous, irradiated myeloid blasts. Fusion Hybrids induced anti-leukaemic T-cell responses in three out of six patients. Tumour pulsed mature dendritic cells induced T-cellular responses in two other patients. Only one of six patient’s remission lymphocytes failed to develop leukaemia directed immune responses following stimulation with either construct. Anti-proliferative properties of Fusion Hybrids against allergenic lymphocytes were observed in mixed lymphocyte-leukaemia reactions (MLLR) and were found not to be specific to the cell fusion partners and do not prevent the ability of AML-mDC heterokaryons to induce autologous anti-leukaemic cytotoxicity. In conclusion, Tumour Cell / Dendritic Cell Fusion Hybrids hold promise as a cellular vaccine for Acute Myeloid Leukaemia.

Herpes simplex virus 1 (HSV-1) naturally infects dendritic cells but this prevents the cell from undergoing maturation. Removal of the virus host shutoff protein (vhs) has been shown to improve the ability of both human dendritic cells and mouse bone marrow dendritic cells to mature following virus infection. An HSV-1 vhs" backbone was further modified by deletion of the ICP47 gene and mutation of the VP 16 gene resulting in a vector that does not interfere at all with the ability of mouse dendritic cells to mature. This virus therefore, provided a good candidate for use as vector for antigen delivery in immunotherapy. A recombinant vhs"ICP47"VP16" virus expressing the full length influenza nucleoprotein (NP) was constructed and used to determine the ability of this virus to induce immune responses upon immunisation with virus alone or upon immunisation with infected DCs. Both cellular and humoral responses were investigated and a CD8+ CTL response to both the NP gene and to the virus was obtained after different immunisation regimes confirming the potential of the virus to be used for immunotherapy. In the attempt to further improve the vector, mGMCSF was added to the viral constructs and viruses expressing both mGMCSF and NP or full length Plasmodium yoelii sporozoite surface protein 2 (PySSP2) or circumsporozoite protein (PyCS) were constructed. The effect of co-expressing mGMCSF from the virus was investigated. A convincing CD8+ CTL response was obtained against - the antigen upon direct virus injection though co-expression of mGMCSF did not seem to confer a significant advantage. Overall an HSV-1 vector has been developed and optimised for the infection of dendritic cells and then successfully used for induction of specific CTL and antibody response to full length NP, PySSP or PyCS.

T cells are one of the most pivotal cell types in the human adaptive immune system. They have the capacity to eradicate primary, metastatic, relapsed tumours and can ameliorate otherwise fatal viral infections. Not surprisingly therefore, the activation and expansion of T cells has become one of the main focuses for immunotherapy and immune gene therapy. However one of the problems of T cell mediated immunotherapy in terms of delivering significant clinical impact to patients, is the expansion of high numbers of functional antigen specific effector T cells. The current approaches for expanding T cells have a number of drawbacks in terms of timing, reproducibility and reliability. Many if not all the currently available systems rely on ex vivo cell manipulation, which concordantly leads to short T cell survival in vivo after infusion. In vivo artificial expansion systems would clearly circumvent this problem. Nevertheless active immunotherapy is not always the solution since sometimes in some patients, the T cells that could be potentially in- vivo expanded no longer exist because they have been deleted, killed or anergised. Therefore a flexible system should be constructed in order to performed both adoptive and/or active immunotherapy depending on the patients requirements. Currently there is no comprehensive artificial Antigen Presenting Cell system (aAPC) for both effective ex vivo and in vivo antigen specific T cell expansion. Therefore in order to address this we have constructed a novel artificial nano-sized super para magnetic antigen presenting cell system (aAPC) capable of priming and expanding antigen specific T cells ex vivo and in vivo. As defined by the NIH, nanotechnology uses nanoscale injectable, targeted and traceable devices capable of important immunological/clinical functions. This nano-system was constructed using the latest generation of immuno liposomes, approved for in vivo human use since they are non-toxic, biodegradable, avoid rapid recognition by the reticulo endothelial system, are safe in terms of size being 50 times smaller than average cells at lOOnm, have good stability and favourable pharmacokinetic behaviour for effective in vivo trafficking. We have coated these liposomes with an optimised number of MHC Class I / peptide complexes and a specific selected range of adhesion (anti LFA-1), early activation (anti CD28 and anti CD27), late activation (4-IBB) and survival receptors (anti CD40L) in the form of Fab antibody regions or monoclonal antibodies. We have made these immuno liposomes traceable since they carry fluorescent lipids and iron oxide super para magnetic nano particles or spios of 13nm size, which make them traceable in vivo using fluorescence and/or by Magnetic Resonance Imaging (MRI). The super para magnetic liposomes are also able to facilitate their own focusing to specific organs, tumour sites or body areas by applying external magnetic attraction. Production of this nano immune liposome system in a ready to use form is achievable in less than 48 hrs and viable for at least 7 days. After ex vivo stimulation with this artificial nano system using CMV pp65 as a model antigen, we have established successful expansions with high T cell numbers (55 to 200 fold) in CMV positive individuals, which are superior when compared with other systems such as peptide pulsed DCs, which are one of the standard methods currently used, coated Daudi cells, magnetic commercial beads and modified tetramers. The T cells are fully functional in terms of degranulation and production of cytokines when specifically challenged. They express predominantly effector-memory and memory phenotypes. We have demonstrated by double fluorescent staining that these liposomes activate T cells directly in an antigen specific fashion and also semi-directly by being incorporated on the surface of the natural APCs in a similar manner as exosomes. When tested in naive individuals, this nano system was also capable of accomplishing initial low levels of T cell priming without help of any adjuvants. In conclusion, we have generated an efficient artificial APC, which embodies a powerful, controllable and superior approach with enormous potential for cancer nanotechnology and T cell immunotherapy for use either in vivo or in vitro.

ABSTRACT Previous studies have shown that tumor-endothelial markers (TEMs) are upregulated in immunosuppressive, pro-angiogenic dendritic cells (DCs) found in tumor microenvironments. We reported that pro-angiogenic monocyte-derived DCs (Mo-DCs), utilized for therapeutic vaccination of cancer patients upon maturation, markedly differ in their ability to up-regulate tumor-endothelial marker 8 (TEM8) gene expression. A DC vaccination trial of 17 advanced cancer patients (13 melanoma and 4 renal cell carcinoma), carried out at the Cancer Institute of Romagna (I.R.S.T.) in Meldola, highlighted a significant correlation between delayed-type hypersensitivity test (DTH) and overall survival (OS). In the study, relative TEM8 mRNA and protein expression levels (mature (m) vs. immature (i) DCs), in DCs obtained for therapeutic vaccines were evaluated by quantitative real-time RT-PCR and cytofluorimetric analysis, respectively. mDCs from six healthy donors were included for comparison purposes. Eight non-progressing patients, all DTH-positive, had a mean fold increase (mfi) of 1.97 in TEM8 expression. Similarly, a TEM8 mRNA mfi = 2.7 was found in healthy donor mDCs. Conversely, mDCs from nine progressing patients, all but one with negative DTH, had a TEM8 mRNA mfi of 12.88. Thus, mDC TEM8 expression levels would seem to identify (p = 0.0018) patients who could benefit from DC therapeutic vaccination.

Despite the progress in the treatment of acute myeloid leukemia (AML) achieved in the last decades, a significant number of patients are still refractory to or relapse after standard cures. Hence, to improve cure rates of AML, it is crucial to develop novel therapeutic strategies. Immunotherapy with T cells genetically modified to express chimeric antigen receptors (CARs) represent a valid option in this sense. CARs are artificial molecules constituted by an extracellular-antigen-binding domain derived from a monoclonal antibody and an intracellular-signalling region that is immediately triggered after antigen recognition. Therefore, CARs combine the antigen binding properties of mononoclonal antibodies to T cell mediated effector functions, including the killing mechanism -that might be active against antibody resistant targets-, cytokine secretion- that might boost the anti-tumoral immune response- and capacity to efficiently home and infiltrate tumor sites. Different CARs have been generated so far, against a wide range of surface molecules expressed by many tumors and, currently, several phase I clinical trials are undergoing and the results obtained so far are very encouraging. The CARs approach can be employed to selectively target AML cells due to the overexpression of myeloid antigens, like CD33 and CD123. We recently demonstrated that expression of CD33-specific CARs in a population of ex-vivo activated T cells, called “cytokine induced killer” (CIK) cells, confers them potent in vitro anti-leukemic functions. However, since CD33 antigen is also expressed on normal haematopoietic stem/progenitors cells (HSPCs) resulting in a potential severe impairment of normal myelopoiesis, CD123 has recently been proposed as a new potential attractive molecule based on its differential expression pattern, being widely overexpressed by AML population and at the same time less expressed on HSPCs. In order to improve the safety profile against these cells we develop and test a novel CAR specific for the CD123 antigens. Here we describe the in vitro and the in vivo efficacy and the safety of this approach based on CIK cells genetically modified to express CAR molecules specific for the CD33 or CD123 antigen. The development and the optimization of the proposed strategy could be a good potential therapeutic tool in the context of minimal residual disease in high-risk transplanted AML patients. Moreover, CAR approach could be potentially used to treat patients resistant to conventional chemotherapeutic approaches or for whom high dose chemotherapy treatment could not be proposed.

Ovarian cancer commonly relapses after remission and activating tumour-specific T cells with dendritic cells loaded with tumour cells is a promising approach to target residual microscopic tumours. SK-OV-3 cells expressing HER-2/neu and MUC1 ovarian antigens were killed and their immunogenicity enhanced by hypochlorous acid (HOC1) oxidation. Treatment with 60 uM HOC1 for 1 h induced necrosis in all the cells. Oxidised, but not live SK-OV-3 were engulfed by monocyte-derived DCs. Autologous T cells primed with DCs of HLA-A2+ healthy volunteers loaded with oxidised SK-OV-3 (HLA-A2") recognised oxidised SK-OV-3 and HLA-A2-restricted epitopes of MUC1 and HER-2/neu. Responses were absent with heat- or hydrochloric acid (HCl)-killed SK-OV-3, thus HOC1 oxidation and not cell death/necrosis enhanced the immunogenicity of SK-OV-3. Oxidised SK-OV-3 primed T cells did not respond to oxidised melanoma, or vice versa. Next, T cells of ovarian cancer patients in remission, whose tumours overexpressed MUC1 and/or HER-2/neu, were tested using the same model. DCs of HLA-A2+ and A2" patients pulsed with oxidised SK- OV-3 stimulated T cells specific for oxidised SK-OV-3, and HER-2/neu and MUC1 epitopes. Oxidised SK-OV-3 loaded DCs further matured with CD40 agonistic antibody or monophosphoryl lipid A, primed stronger CD4+ and CD8+ responses than immature DCs. T cells stimulated this way also recognised autologous tumour cells from ascites, demonstrating that SK-OV-3 shared significant antigenic repertoire with tumours in patients. Finally, this DC-based vaccination was tested in vivo using the B16.F10 melanoma model. B16.F10 highly expressed tyrosinase-related protein (TRP)-2, and all the cells were necrotic after 1 h treatment with 60uM HOC1. Mice immunised intravenously and not intraperitoneally or subcutaneously with bone- marrow derived DCs loaded with oxidised B16.F10, responded to TRP-2 and oxidised B16.F10. Oxidised and not heat- or HCl-killed B16.F10, primed specific T cells in vivo. DCs were required for efficient delivery for processing and presentation of oxidised B16.F10 in vivo.

Notre étude aborde la question cruciale du rejet de greffe dans la transplantation rénale, où les médicaments immunosuppresseurs actuels présentent des risques significatifs. Nous proposons une approche novatrice de thérapie cellulaire utilisant des cellules dendritiques tolérogènes autologues (ATDC) pour promouvoir l'acceptation de la greffe. Sur la base de nos résultats encourageants chez les rongeurs, notre équipe a développé un processus de fabrication d'ATDC conforme aux BPF, qui a été évalué dans un essai clinique de phase I/II. La caractérisation in vitro des ATDC a mis en évidence une activité glycolytique accrue et le lactate produit a été lié à la suppression de la prolifération et de l'activation des cellules T CD4+. Dans notre essai, une activation réduite des cellules T CD8+ et une expression accrue de FoxP3+ ont été observées dans les cellules immunitaires circulantes des receveurs de greffe de rein traités avec des ATDC. En se concentrant sur la régulation des cellules T CD8+ par les ATDC, nos expériences in vitro ont démontré que les métabolites dérivés des ATDC suppriment la prolifération des cellules T CD8+ naïves et mémoires-effectrices (EM). Contrairement aux cellules T CD4+, le lactate n'est pas impliqué dans cette suppression. Cependant, le rôle critique de l'indoleamine-2,3-dioxygénase (IDO) produite par les ATDC pour supprimer les cellules T CD8+ a été mis en évidence à l'aide d'un inhibiteur spécifique. Les analyses métaboliques et transcriptomiques ont montré que l'environnement des ATDC favorise la phosphorylation oxydative dans les cellules T CD8+ naïves, associée à leur activation réduite et à une diminution de la phosphorylation oxydative dans les cellules T CD8+ EM. De plus, les ATDC diminuent leur capacité migratoire via un mécanisme dépendant du contact. En conclusion, nos résultats soutiennent l'efficacité des ATDC comme stratégie thérapeutique dans la transplantation rénale, en particulier pour supprimer le rejet de greffe médié par les cellules T CD8+
Our study addresses the critical issue of allograft rejection in kidney transplantation, where current immunosuppressive drugs pose significant risks. We propose a novel approach of cell therapy using autologous tolerogenic dendritic cells (ATDC) to promote graft acceptance. Based on our encouraging results in rodents, our team developed a GMP-compliant ATDC manufacturing process, which was evaluated in a phase I/II clinical trial. In vitro characterization of ATDC highlighted enhanced glycolytic activity and the produced lactate was linked to the suppression of CD4+ T cell proliferation and activation. In our trial, a reduced CD8+ T cell activation and an increased FoxP3+ expression were observed in the circulating immune cells of kidney transplant recipients treated with ATDC. Focusing on the regulation of CD8+ T cells by ATDC, our in vitro experiments demonstrated that ATDC- derived metabolites suppress naïve and effector memory (EM) CD8+ T cell proliferation. Unlike CD4+ T cells, lactate was not involved in this suppression. However, the critical role of indoleamine-2,3- dioxygenase (IDO) produced by ATDC to suppress CD8+ T cells was evidenced using a specific inhibitor. Metabolic and transcriptomic analyses highlighted that ATDC environment promotes oxidative phosphorylation in naive CD8+ T cells, associated with their reduced activation and decreased oxidative phosphorylation in EM CD8+ T cells. Moreover, ATDC decrease their migratory capacity via a contact- dependent mechanism. In conclusion, our findings support the efficacy of ATDC as a therapeutic strategy in kidney transplantation, particularly in suppressing CD8+ T cell-mediated allograft rejection

Les tumeurs sont composées de cellules malignes et d'une grande variété de cellules non-tumorales, en particulier des cellules immunitaires qui forment le micro-environnement tumoral (MET). Il a été démontré que la composition du MET était associée au devenir clinique des patients, en termes de survie et de réponses thérapeutiques. Avec le développement récent des immunothérapies qui ciblent des éléments spécifiques du MET, l'immunité anti-tumorale a soulevé un intérêt majeur. Plusieurs méthodologies ont été mises au point afin d'étudier la composition du MET, avec une précision toujours plus grande. En particulier, des méthodes comme MCP-counter permettent d'exploiter les données transcriptomiques de la tumeur entière afin de quantifier les différentes populations qui composent le MET. Le volet méthodologique de ce travail de thèse a ainsi consisté à proposer une amélioration de MCP-counter, en particulier pour l'analyse de données RNA-Seq. Une adaptation de la méthode pour des données issues de modèles murins (mMCP-counter) est également proposée. MCP-counter permet d'analyser rapidement le MET de larges séries de tumeurs. Un second volet de cette thèse consiste en l'application de cette méthode pour établir une classification immunitaire des sarcomes des tissus mous, un type de cancer rare, hétérogène et agressif. Cette classification immunitaire a permis de mettre en évidence des groupes de tumeurs faiblement ou fortement infiltrés, ainsi qu'un groupe marqué par une forte vascularisation. De manière intéressante, la classification immunitaire permet de prédire la réponse des patients aux immunothérapies. Ce travail a aussi démontré un rôle important des structures lymphoïdes tertiaires (SLT). Les SLT sont des structures de type noeud lymphatique composées de lymphocytes B et T qui se forment dans la tumeur ou à proximité de celle-ci. Au sein des SLT, une réponse immunitaire anti-tumorale peut se former et maturer. L'intérêt porté aux SLT est de plus en plus important pour de nombreux types de cancers. Dans la plupart des types de cancer, une forte infiltration de la tumeur par des lymphocytes T, en particulier CD8+, est associée à une meilleure survie des patients. Cependant, le carcinome rénal à cellules claires et le cancer de la prostate sont des exceptions à cette règle. En effet, dans ces deux cancers urologiques, la présence dans la tumeur de lymphocytes T est associée à une survie plus courte des patients, ainsi qu'à une rechute et une progression plus précoce. Ces exceptions sont détaillées dans une troisième partie de cette thèse, par une description minutieuse du MET, ainsi que par l'analyse de l'implication du système du complément. Dans leur ensemble, les résultats présentés dans cette thèse démontrent qu'en combinant différentes méthodes d'analyse, in silico, in situ et in vivo, il est possible d'obtenir une vision extrêmement complète du MET. La connaissance des types cellulaires présents dans la tumeur ainsi que leur orientation fonctionnelle permet de guider le soin apporté aux patients et d'améliorer leur devenir clinique. La description complète du MET ouvre la voie à une médecine personnalisée pour les patients atteints de cancer
Tumors are composed not only of malignant cells but also contain a vast variety of non-malignant cells, notably immune cells forming the tumor microenvironment (TME). The composition of the TME was shown to be associated with clinical outcome for cancer patients, in terms of survival and therapeutic responses. With the relatively recent development of immunotherapies targeting specific elements of the TME, tumor immunology has risen a strong interest and holds a strong therapeutic potential. Several methodologies have been developed to study the composition of the TME with an increased precision. Notably, some methods such as MCP-counter enable the use of the tumor bulk transcriptome to quantify cell populations composing the TME. The methodological aspect of this PhD project consisted in setting up an enhanced version of MCP-counter that can be readily applied to RNA-Seq data, as well as propose an adaptation of the method for mouse models. Using MCP-counter, the TME of large series of tumors can be easily analyzed. The application part of this PhD work consisted of applying MCP-counter to establish an immune-based classification of soft-tissue sarcoma, a rare, aggressive and heterogeneous cancer type. The immune classification notably allowed to identify immune low and high groups, and a group characterized by a strong vasculature. Interestingly, the classification was notably found to be predictive of the patients' response to immunotherapies. It also highlighted an important role of tertiary lymphoid structures (TLS). TLS are lymph-node-like structures composed of T and B cells that form within the tumor or in close proximity. They are a site of formation and maturation of antitumoral immune responses. TLS are raising a growing interest in many malignancies. In most cancer types, a strong infiltration by T cells, in particular CD8+ T cells, is associated with a favorable clinical outcome. However, clear-cell renal cell carcinoma and prostate cancer are exceptions to this general rule. Indeed, in these urological cancers, an increased infiltration by T cells is associated with a decreased patient survival and with earlier relapse and disease progression. In a third part of this thesis, these exceptions are investigated with more details by scrutinizing the TME, and questioning the implication of the complement system. Overall, this thesis presents how the combination of several analysis methods, in silico, in situ and in vivo, can help achieve an extremely precise description of the TME. Knowing accurately what cell populations and what their functional orientation can help guide patients care and improve clinical outcome. Complete description of the TME opens the way towards personalized medicine for cancer patients

Les "Antibody Recruiting Molecules" (ARMs) font partie des pistes prometteuses en immunothérapie contre le cancer et les pathogènes. Dans le contexte tumoral, un ARM a la capacité de relier la cellule cible et des composants du système immunitaire pour conduire à une réponse cytotoxique. Dans ce travail de thèse, nous avons décrit la conception et la synthèse par chimie click de plusieurs ARMs capables de cibler des protéines surexprimées à la surface des cellules cancéreuses par de "tumoral binding modules" (TBMs) basés sur des peptides et d’engager des anticorps endogènes naturels par l’intermédiaire de "antibody binding modules" basés sur des glucides. Nous avons ensuite démontré la formation d’un complexe ternaire cellule-ARM-anticorps et la destruction spécifique des cellules cancéreuses par nos agents uniquement en présence de sérum humain, comme seule source de d’effecteurs immunitaires, sans pré-immunisation. Nous avons également établi que la flexibilité de la molécule, la longueur de la liaison entre ABM et TBM, la nature des haptens (glucides et peptides) et leur valence jouent un rôle important. Compte tenu de la grande efficacité du processus de synthèse, une large gamme de ARM pourrait être facilement créée, ouvrant de nouvelles perspectives dans différents domaines thérapeutiques
The Antibody Recruiting Molecules (ARMs) are belongs to the promising alternative in immunotherapy against cancer and pathogens. In the tumoral contest, an ARM has the singular ability to interface between the target cell and components of the immune system present on the environment leading to an immune response. In this Ph.D. work, we reported the design and the synthesis by click chemistry of several ARMs able to target over-expressed proteins on cancer cell surface through peptides-based tumoral binding modules (TBMs) and to engage natural endogenous antibodies through carbohydrate-based antibody binding modules (ABMs). Next, we demonstrated the formation of a ternary complex between cell-ARM-antibodies and the specific killing of cancer cells by our agents only in the presence of human serum as unique source of immune effectors, without pre-immunization. In addition, we established that the molecule flexibility, the length of the linker between ABM and TBM, the nature of the haptens (carbohydrates and peptides) and their valency play a significant role. Due to the efficiency of the synthetic process, a large diversity of ARMs could be easily created, opening new outlooks in diverse therapeutic fields

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2014
O cancro actualmente é combatido com recurso à quimioterapia, cirurgia, e ainda, à radioterapia. Recentemente, juntou-se a imunoterapia no combate ao mesmo, que é concretizada em diversas formas desde vacinas de lisados virais a anticorpos monoclonais. A imunoterapia com recurso a entidades celulares é a mais promissora devido à intrínseca relação entre o cancro e estados inflamatórios, relacionados com o sistema imunitário. O sistema imunitário, pela sua complexidade e inter-relação de processos e vias metabólicas é uma área ainda por compreender de forma a se poder revolucionar as terapêuticas actualmente disponíveis. As principais células imunológicas envolvidas são linfócitos T, céulas Natural Killer (NK) e células dendríticas. É possível distinguir as células dendríticas como as entidades celulares com maior possibilidades de aplicabilidade na clínica. Estas podem ser utilizadas de formas distintas, tanto in vivo como ex vivo. Deste modo, diferentes abordagens tecnológicas têm vindo a ser estudadas tendo em vista a produção de vacinas oncológicas com recurso a células dendríticas. Os métodos de cultura, formas de colheita e isolamento destas células, métodos de formulação, factores intervenientes na eficácia terapêutica, constituem actualmente importantes desafios. Além disso, importa ainda referir que estão por definir a grande maioria das bases regulamentares necessárias a esta área actualmente reconhecida como extremamente promissora.

Adoptive T cell immunotherapy is a promising anti-cancer therapy that has the potential to become the ultimate therapeutic agents to treat a variety of diseases. Recently, chimeric antigen receptor (CAR) expressing T cells has demonstrated to be a very effective approach to treat B cell cancer patients. Despite optimistic results, there are several improvements that need to be made to enhance the safety and efficacy of current CAR T cell therapy. Fortunately, different synthetic biology tools can be implemented to overcome many of the current deficiencies of CAR T cell therapy. Here, we develop anti-Axl CAR and synNotch receptors to target Axl which is a tyrosine kinase receptor that is commonly overexpressed in many cancers and considered as one of important cancer therapeutic targets. Next, we develop a split, universal, and programmable (SUPRA) CAR system that can be used to switch targets without re-engineering T cells, fine-tune T cell activation level, and sense and logically respond to multiple antigens. These multiple features are useful in mitigating tumor relapse, limiting CAR-T induced toxicities, and enhancing tumor specificity. Orthogonal SUPRA CARs are also used to control different cell types and signaling domains, enabling diverse immune responses from SUPRA CAR T cells. Lastly, we demonstrate that SUPRA CAR can redirect the activity of both innate and adaptive immune cell types. We also expand the logic capabilities of SUPRA CAR T cells by integrating three inputs in a single immune cell. We also show intercellular logic gate by engineering immune cell-cell interaction. We further demonstrate controlling endogenous immune cell polarization using SUPRA CAR T cells. These wide-ranges of SUPRA CAR applications imply its versatility as a platform for engineering cell-cell interactions with advanced logic functions to enhance efficacy and safety of cell-based cancer immunotherapy.

Dissertação de Mestrado em Investigação Biomédica apresentada à Faculdade de Medicina
As vacinas de células dendríticas (CD) representam uma terapia segura que, apesar da sua comprovada atividade anti-tumoral em modelos pré-clínicos e clínicos, apresenta resultados terapêuticos limitados. Estudos anteriores sempre se focaram na geração de respostas de células T CD8+; no entanto, desvendou-se recentemente a relevância da ativação de outras células efetoras, especialmente as células Natural Killer (NK), na eficácia destas abordagens. Como resultado de uma extensa interação, as células NK interferem na maturação e capacidade secretora das CDs, enquanto que estas modulam a ativação e a atividade secretora e citotóxica das NK. Atendendo ao impacto na imunidade anti-tumoral, é crucial que esta interação seja explorada no desenho de novas vacinas de CD.Assim, na sequência do desenvolvimento de uma vacina de CDs para o tratamento de cancro, o principal objetivo deste trabalho é avaliar o impacto de novos fenótipos de CDs e o efeito do carregamento com lisados tumorais e da morte celular imunogénica (MCI) de células tumorais na interação CD-NK.Para tal, as interações CD-NK foram analisadas após 24h de co-cultura, recorrendo a citometria de fluxo para avaliar a expressão de moléculas de maturação nas CDs e marcadores de ativação nas células NK, bem como testar a sua desgranulação. A capacidade secretora das CDs e NK foi avaliada através de ELISA. Além disso, a morte tumoral mediada por células NK foi avaliada com o ensaio de libertação de LDH, e a proliferação de NK, bem como a das células T, foi analisada através de citometria de fluxo após marcação com CFSE.Após otimizar a cultura de células NK em meio com 50 IU/mL de IL-2 como o melhor método para preservar a viabilidade e atividade destas células, foram realizados ensaios funcionais para avaliar a interação CD-NK. Os resultados mostraram que o carregamento de CDs com lisados tumorais não afeta esta interação. Ao analisar a interação de três novos fenótipos de CDs (propriedade do grupo TECNIMEDE) com células NK, verificámos que o fenótipo F3 apresentou o maior aumento de moléculas de MHC, as CDs F1 evidenciaram-se na produção de IL-12, induzindo a secreção de IFN-γ por NK, enquanto que as CDs F2 demonstraram uma maior capacidade de promover a proliferação de NK quando na presença de células T. Por fim, verificámos que células tumorais submetidas a MTI, assim como os seus sobrenadantes, aumentam a maturação das CDs e a sua produção de IL-12 quando comparadas a células que sofreram o processo apoptótico tradicional. Isto refletiu-se na capacidade aumentada das CDs maturadas por MCI para promoverem a ativação de NK e a secreção de IFN-γ.Em suma, este trabalho destaca a relevância da interação CD-NK no contexto de vacinas de CDs, realçando o papel das células NK na resposta anti-tumoral. Revela também que o uso de novos fenótipos de CDs, bem como o carregamento de CDs com antigénios imunogénicos, poderá representar o próximo passo para o sucesso desta abordagem.
Dendritic cell (DC)-based vaccines represent a safe therapy that, although has proven anti-tumour activity in pre-clinical and clinical models, only presents modest therapeutic results. Previous research focused exclusively at generating CD8+ T cell responses; however, it is well-known that the stimulation of other effector cells, especially natural killer (NK) cells, plays a major role in the efficacy of these approaches. As a result from an extensive crosstalk, NK cells interfere with DC maturation and secretory capacity, while DCs modulate the activation, secretory and cytotoxic activity of NK cells. Having a direct impact on anti-tumour immunity, it is crucial for this interaction to be considered in the design of new DC-based vaccines.Therefore, in parallel with the ongoing development of a DC-based vaccine for cancer treatment, the main goal of this master thesis is to analyse the process of DC-NK crosstalk, exploiting the impact of novel DC phenotypes and the effect of tumour lysate loading and immunogenic cell death (ICD) of tumour cells in this process.To accomplish this, DC-NK interactions were analysed after 24 h of co-culture, performing flow cytometry to evaluate the expression of maturation molecules on DCs and the levels of activation markers on NK cells, as well as to test their degranulation ability. The secretory ability of both DCs and NK cells was evaluated by ELISA. Furthermore, the NK cell-mediated tumour cell killing was assessed using the LDH cytotoxicity assay, and the NK cell as well as T cell proliferation were analysed though flow cytometry using CFSE staining.After optimizing the culture conditions of NK cells with a supplement of 50 IU/mL of IL-2 as the best method to preserve NK cell’s viability and activity, functional assays were performed to evaluate the DC-NK crosstalk. Our results show that the loading of DCs with tumour antigens did not affect this interplay. After analysing the interaction of three novel DC phenotypes (property of TECNIMEDE group) with NK cells, we found that the F3 phenotype showed the greatest increase of MHC molecules, F1 DCs seemed to produced more IL-12, eliciting IFN-γ secretion by NK cells, whereas F2 DCs demonstrated a higher ability to promote the proliferation of NK cells when in the presence of T cells. Lastly, we found that tumour cells undergoing ICD, as well as their supernatants, increase the DC maturation and their production of IL-12 when compared to cells that have undergone the traditional apoptotic process. This was reflected by an enhanced capacity of ICD-matured DCs to promote NK cell activation and IFN-γ secretion.In sum, this work highlights the relevance of the DC-NK crosstalk in the context of DC-based immunotherapies, underscoring the substantial role for both the cytotoxic and the regulatory functions of NK cells in the development of DC-mediated antitumor immunity. It also reveals that the use of new DC phenotypes, as well as loading DCs with immunogenic antigens, could represent the next step for the success of this approach.
Outro - Financiamento proveniente de Fundos Nacionais através da FCT (Fundação para a Ciência e Tecnologia) pelos Projetos Estratégicos UIDB/04539/2020 e UIDP/04539/2020 (CIBB).
Outro - Projeto em co-promoção financiado pelo Programa Interface da ANI (Agência Nacional de Inovação)

碩士
國立陽明大學
微生物暨免疫學研究所
89
Dendritic cells (DCs) are potent antigen-presenting cells (APC) distributed among various tissues and could present antigens to T cells after migration to the lymphoid organs. DCs have the unique ability to uptake exogenous antigens and present antigens in the context of MHC class I to activate CD8+ T cells (cytotoxic lymphocytes; CTL), i.e. cross-priming. Recent years, heat shock protein 70 (HSP70) was reported to induce antibody and cytokine production as well as lymphocyte proliferation. The function of HSP70 was also suggested to be used as a vehicle for intracytoplasmic and intranuclear delivery of proteins or DNA to modulate gene expression and thereby control immune responses. In addition, HSP70 enhanced the ability of immature DCs to capture and present antigen to T cells. Based on the unique property of DCs and HSP70, we proposed to develop DC-based immunotherapy for the treatment of cancer patient. We utilized HSP70 to enhance the antigen capture ability of DCs in our experiments. According the report from the Administration of Health in Taiwan, Nasopharyngeal carcinoma (NPC) accounts for the tenth highest mortality rate in Taiwanese people, and is the seventh highest mortality rate in male Taiwanese. Therefore, how to control the incidence rate and to provide better strategy to treat NPC is an urgent need in current medicare. Our approach is to inoculate LMP1 transfected Balb/c 3T3 cells on both sides of thigh area, then use g-irradiation to induce tumor cells necrosis and apoptosis on one site, followed by injection of DCs and heat shock protein 70 (HSP70) to the irradiated area. Result from our study demonstrated that the growth of LMP1 overexpressing tumor cells on both the irradiated and non-irradiated sides was completely inhibited by this approach, indicating the success to elicite host immune response against LMP1 overexpressing tumor cells. This might provide a hope to develop DCs-based immunotherapy against human cancer in the future.

Neuroblastoma is an aggressive solid tumor that develops from immature cells of the nervous system and is almost exclusively diagnosed in infants and young children. Over the past decade a multitude of immune based therapies have been explored as therapeutic candidates for patients with neuroblastoma. The anti-GD2 monoclonal antibody, 3F8, and more recently, natural kill (NK) cell based therapies have been accepted as hopeful therapeutic options for patients with Neuroblastoma. These options however have many drawbacks including dose limiting pain, the development of tolerance, reliance on MHC mismatch and possible reliance on the invariant NK (iNK) cells population. Gamma Delta T cells, a subpopulation of T cells composed of a T cell receptor (TCR) with a gamma and a delta chain instead of an alpha and a beta; chain, have been shown to recruit a more robust immune response then both 3F8 and NK cells through their activation of antigen presenting cells (APCs) and non-reliance on MHC mismatch. Gamma Delta T cells are also able to recruit NK cells as well as other cytotoxic lymphocytes. For these reasons, it is believed that Gamma Delta T cell based treatment alone or in combination with an anti-GD2 monoclonal antibody may have a greater efficacy than either NK cells or an anti-GD2 monoclonal antibody alone. The intent of this thesis is to explore and evaluate the current state of Gamma Delta T cell based immunotherapy against the backdrop of NK cell based immunotherapy for neuroblastoma.

Tese de doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de Coimbra
O carcinoma da bexiga (CB) é a neoplasia maligna mais comum do trato urinário, sendo o 4º tipo de carcinoma mais comum nos homens e o 9º nas mulheres, no mundo ocidental. Na maioria dos casos os tumores são classificados como não músculo-invasivos, e são tratados por resseção transuretral e terapia intravesical. No entanto, cerca de 80% dos doentes apresenta propensão para recidivas e progressão para formas músculo-invasivas, geralmente refratárias à terapêutica. Apesar de já identificados alguns fatores de risco, ainda não estão bem estabelecidos os fatores preditivos da recidiva e progressão da doença. Estudos recentes associam a elevada taxa de recidivas e a baixa sobrevida à presença de células indiferenciadas com caraterísticas estaminais, denominadas de células estaminais cancerígenas (CSCs), com capacidade de autorrenovação e potencial tumorigénico. Uma eliminação efetiva das CSCs em fases iniciais da doença pode reduzir o risco de recidivas e progressão tumoral para estadios mais invasivos. Atualmente, as terapêuticas em vigor não conseguem eliminar as CSCs podendo até favorecer a seleção de clones resistentes e a sua expansão por autorrenovação. Esta tese teve como principal objetivo avaliar o potencial da aplicação de uma nova abordagem terapêutica baseada na imunoterapia adotiva com células Natural Killer (NK) na eliminação das CSCs no CB e o seu impacto na progressão tumoral. Numa primeira fase, demonstrámos que o CB do tipo músculo-invasivo contém uma população heterogénea de CSCs caraterizada pela expressão de fatores de pluripotência, de marcadores de células estaminais da camada basal do urotélio e de sistemas de desintoxicação de fármacos. Estas células apresentam ainda elevada resistência à cisplatina e ao metotrexato, e formam tumores heterogéneos com uma histologia semelhante à do carcinoma músculo-invasivo quando inoculadas por via ortotópica em ratinhos imunodeprimidos. Identificámos ainda uma potencial assinatura molecular definida pela expressão do SOX2 e da ALDH2, validada a nível proteico em secções de tecido tumoral, e que poderá ser útil na identificação de doentes com tumores músculo-invasivos mais propensos a evoluir para formas mais agressivas. A expressão preferencial de pelo menos dois marcadores de estaminalidade nos tumores músculo-invasivos reforça o papel das CSCs como a força motriz da sua patogénese e recidivas, destacando a necessidade de delinear novas abordagens terapêuticas tendo como alvo as CSCs. De seguida, avaliámos o potencial terapêutico da imunoterapia adotiva com células NK na eliminação das CSCs resistentes à quimioterapia. Verificámos que células NK isoladas de dadores saudáveis e ativadas com IL-2/IL-15 são eficazes na indução de uma resposta antitumoral contra as células do CB através da produção de TNF-α, IFN-γ e granzima B. Contrariamente à quimioterapia, as células NK ativadas demonstraram um elevado potencial citotóxico tanto para as células parentais como para as CSCs. Este efeito é sobretudo mediado pela sobreexpressão de ligandos à superfície das células alvo para os recetores ativadores, NKG2D e DNAM-1 nas NK. Adicionalmente, as células NK induziram uma alteração no fenótipo das CSCs para um estado mais diferenciado, acompanhada de uma diminuição da expressão dos marcadores de estaminalidade. Este efeito aumentou a sensibilidade das CSCs ao efeito da cisplatina, realçando os benefícios de uma possível terapêutica combinada. Pelo contrário, as células NK de doentes com CB exibiram uma expressão diminuída de recetores de citotoxicidade, de moléculas de adesão e apresentaram um fenótipo mais imaturo, sendo ineficazes na eliminação direta das CSCs bem como na indução da sua diferenciação. Os estudos in vivo comprovaram a elevada eficácia terapêutica das células NK isoladas de dadores saudáveis, em dois modelos animais de CB. A injeção intratumoral de células NK, no modelo subcutâneo, conduziu a uma remissão completa do tumor sem evidência de recidiva. Isto é consistente com a capacidade das células NK em eliminar tanto as CSCs como as células mais diferenciadas que constituem maioritariamente o tumor. No modelo ortotópico, a administração intravesical de células NK resultou numa diminuição massiva do tumor, entre 80 a 100% (remissão total), o que indica que as células NK mantêm a sua capacidade lítica no microambiente tumoral. Em conclusão, os nossos resultados demonstram a existência de subpopulações heterogéneas de CSCs no CB com elevado potencial tumorigénico e resistentes à quimioterapia convencional, mas extremamente suscetíveis à atividade lítica de células NK alogeneicas ativadas. Uma vez que as células NK são igualmente eficazes na eliminação das CSCs e das células mais diferenciadas, a sua administração intravesical poderá ser considerada como uma promissora estratégia para o tratamento dos doentes com CB, per se ou de forma complementar às opções terapêuticas atuais.
Bladder cancer (BC) is the most common malignancy of the urinary tract. It is the 4th most common cancer among men and the 9th in women in Western world. The majority of newly diagnosed cases are non-muscle-invasive tumors and can be effectively treated by transurethral resection and intravesical therapy. However, approximately 80% of patients have high propensity for tumor recurrence and to progress into muscle-invasive forms, being largely refractory to conventional therapy. Although some risk factors have been associated with disease appearance and progression, no concrete indicators are firmly established that can reliably predict its evolution and prognosis. Striking evidence associates the high recurrence rates and poor survival of BC patients to the presence of a distinct undifferentiated stem-like cell population, the so-called cancer stem cells (CSCs) endowed with extensive self-renewal and tumorigenic potential. An effective eradication of CSC in an early-stage is likely to reduce the risk of recurrences and the progression to a more invasive disease. Presently, current therapies mostly fail to eradicate CSC clones and instead may even favor expansion of the CSC pool and/or select resistant self-renewing clones. The main goal of this thesis was to evaluate the potential application of a novel therapeutic approach using Natural Killer (NK)-based adoptive immunotherapy in the eradication of competent CSCs and its impact on tumor progression. In the first part, we demonstrated that muscle-invasive BC harbor distinct cell subsets reflecting molecular features of stem-like cells, namely a gene expression pattern strongly related with stem cell pluripotency, basal urothelial stem cells and drug-detoxifying systems. Moreover, these cells displayed enhanced chemoresistance to cisplatin and methotrexate and formed a phenotypically heterogeneous tumor resembling the histological features of primary invasive BC when engrafted orthotopically in the bladder of immunocompromised mice. Based on a real time PCR analysis we identified a potential molecular signature defined by SOX2 and ALDH2, validated at protein level in tumor tissue sections, which could be useful on the recognition of patients with muscle-invasive tumors that are more prone to experience disease progression. The preferential expression of at least two stemness-related markers in muscle-invasive tumors strongly reinforces the role of CSCs as a driving force in the pathogenesis and relapse of invasive BC, highlighting the need to delineate novel therapeutic approaches considering CSCs as a target population. In the second part, we evaluated the therapeutic potential of NK cells based-immunotherapy against the chemoresistant bladder CSCs. We found out that NK cells isolated from healthy donors and activated with IL-2/IL-15 were highly effective in mediating an anti-tumor response by production of TNF-α, IFN-γ and granzyme B towards BC cells. Importantly, unlike chemotherapy, activated NK cells demonstrated enhanced cytolytic potential against both parental and bladder CSCs through an increased expression of ligands for NKG2D and DNAM-1 NK-activating receptors on the surface of BC cells. Besides the cytolytic/cytotoxic potential, NK cells were able to induce a phenotypic switching of the CSCs towards a more differentiated state and a concomitant down regulation of stemness-related markers. This NK-induced differentiation effect sensitized CSCs to cisplatin, highlighting the benefits of a possible combined therapy. In opposite, NK cells from BC patients displayed low density of NK cytotoxicity receptors, adhesion molecules and a more immature phenotype, and were ineffective in killing and inducing differentiation of CSCs. Conveying to an in vivo context, our results showed a remarkable and noteworthy anti-tumor effect of healthy activated-NK cells in BC xenografted models. The intratumoral delivery of activated-NK cells in subcutaneous tumors leads to a complete abolishment of tumor growth with no evidence of recurrence, consistent with the ability of NK cells to kill CSCs and non-CSCs populations. In the orthotopic model, the intravesical administration of NK cells resulted in a massive decrease in the tumor burden, ranging from 80 to 100% (complete remission), indicating that NK cells maintain their intrinsic killing ability in an in vivo tumor microenvironment. In conclusion, our results provide compelling evidence that BC harbors distinct CSCs populations that are highly tumorigenic and resistant to conventional chemotherapy but extremely susceptible to allogeneic activated-NK cells. Moreover, since NK cells targets both stem and non-stem cell populations and promotes CSCs differentiation, NK cells intravesical immunotherapy might be considered as a promising alternative or complementary therapeutic strategy for BC patients.

Kidney transplantation represents the best treatment for end-stage kidney disease, and in comparison to dialysis treatment has been shown to improve survival, quality of life, and reduce health-care costs over time. However, in order to prevent transplant failure from allograft rejection, immunosuppressive drug therapy is required. Immunosuppression is associated with significant systemic toxicities, and continues to impair optimal patient and graft outcomes. The avoidance or minimisation of immunosuppression via the promotion of tolerance of the allograft, or the use of targeted therapeutic strategies, in clinical transplantation is therefore an important goal that could have many benefits for patients. Dendritic cells (DC) are potent antigen-presenting cells that play a pivotal role in the initiation and maintenance of immune responses, and therapies utilising or targeting DC offer the potential to manipulate immune responses towards tolerance. This thesis seeks to develop the potential of DC based immunotherapies in a small and clinically relevant non-human primate (NHP) transplant model, the common marmoset monkey, and thereby facilitate translation of these therapies towards human clinical trials. Chapter 1 establishes the context for this thesis by outlining the background and providing a comprehensive review of relevant literature. Chapter 2 describes the materials and methods utilised in this thesis. Additional details of methods are contained in relevant chapters. Chapter 3 presents a comprehensive study of renal pathology in a colony of laboratory marmosets, including histology, immunofluorescence and electron microscopy, and correlates this for the first time with serum and urine biochemistry. This work demonstrates that the spontaneously observed glomerular pathology in marmosets represents a benign occurrence that would not impact on the assessment of renal function or histology in a marmoset kidney transplant model. Chapter 4 examines the trafficking behaviour in vivo of intravenously and subcutaneously administered allogeneic marmoset DC propagated in vitro from genetically disparate marmoset donors. The findings indicate that allogeneic marmoset DC do not necessarily exhibit normal trafficking behaviour in vivo, as they are not found in secondary lymphoid tissues at 48 hours, in contrast to similarly administered autologous DC. This finding lends weight to other recent studies of donor DC cellular therapy that indicate that the tolerogenic effects of this therapy are not mediated through cell to cell interactions with recipient T-cells, but rather through providing a source of donor antigen for acquisition and processing by recipient DC. Chapter 5 describes studies to develop a monoclonal antibody to marmoset DC-specific ICAM 3-grabbing non-integrin (DC-SIGN), which is a DC-specific marker. Ultimately, a marmoset cross-reactive commercially available anti-human DC-SIGN antibody (DCN46) was identified, and found to be suitable to utilise in the development of DC-SIGN targeted cell-specific therapy. Using this antibody, marmoset DC-SIGN positive cells were identified in the Lineage⁻ CD11c⁺ Class II⁺ fraction of marmoset spleen; in contrast in vitro propagated marmoset monocyte-derived DC have been confirmed to lack DC-SIGN expression. Chapter 6 describes the successful development of a novel nanocarrier targeted to DC: PLGA nanoparticles that target DC using the human and marmoset DC-SIGN cross-reactive antibody identified in Chapter 5. A series of preliminary studies have demonstrated that DC-SIGN targeted PLGA nanoparticles are taken up by Class II⁺ CD11c⁺ marmoset spleen cells, and that loading of the nanoparticles with the immunomodulatory drug curcumin shows evidence of in vitro immunosuppressive capacity, as shown in mixed leucocyte reaction; however the specificity for DC of immunosuppressive targeted PLGA nanoparticles remains to be demonstrated. Chapter 7 summarises the overall findings from this thesis, and proposes a series of necessary studies to exploit the identified potentials from this work further. Overall, the work in this thesis significantly advances the marmoset NHP model as a means to translate the potential of DC based immunotherapies towards clinical transplantation. The feasibility of DC-targeted therapy using nanoparticles has been established, and represents an opportunity to specifically target DC with immunosuppressive drugs in vivo, and thereby manipulate the immune response towards tolerance, while reducing the burden of non-targeted immunosuppression.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2013

Research Doctorate - Doctor of Philosophy (PhD)
Asthma is a chronic inflammatory disease of the airways that affects over 300 million people worldwide. The disease is characterised by episodes of breathlessness, coughing, wheezing and airway hyperresponsiveness (AHR). Asthma results from a dysregulation in immunity that is underpinned by a cohort of effector T cell populations including T helper (Th)1, Th2, Th17 and natural killer T (NKT) cells. These effector T cells produce numerous inflammatory cytokines and chemokines that induce eosinophil influx, mucus hypersecretion and AHR. Antigen presenting cells play key roles in priming these responses. Regulatory T cells (Tregs) are essential for suppression of aberrant immune responses and maintenance of immune homeostasis. Both the number and function of Tregs is impaired in asthmatics, compared to healthy individuals. This reinforces the importance of Tregs in regulating a balanced immune response. Microbial agents have been associated with increased or decreased risk of asthma. Microbial agents that have been associated with decreased asthma risk are under intense investigation for their potential utilisation in therapeutic strategies for asthma. Streptococcus pneumoniae vaccination has been associated with decreased asthma-related hospitalisations in children and the elderly. Furthermore, early mouse studies observed that S. pneumoniae infection attenuated blood eosinophilia during parasitic infection. More recent studies have shown that both live and ethanol killed S. pneumoniae suppress the development of allergic airways disease (AAD) in mice, including eosinophil recruitment to the lungs, Th2 cytokine release, mucus hypersecretion and AHR. Therefore S. pneumoniae has the potential for development into a novel immunotherapy for asthma. To examine this concept we first investigated the capacity of human S. pneumoniae vaccines, which were developed to prevent S. pneumoniae infection, to suppress AAD in mouse models (Chapter 2). In the next study, and in order to determine which components were required for S. pneumoniae-mediated suppression of AAD, S. pneumoniae components were tested for their capacity to suppress AAD (Chapter 3). Two potential S. pneumoniae-based immunotherapies were identified: the conjugate vaccine and the combination of type 3 capsular polysaccharide and pneumolysin (T3P+Ply). These S. pneumoniae immunotherapies suppressed the development of AAD when administered before, during and after sensitisation. Importantly, S. pneumoniae immunotherapy also attenuated established AAD. This demonstrated that S. pneumoniae immunotherapy has potential for therapeutic use in the prevention and/or treatment of asthma. To determine the mechanisms involved in S. pneumoniae-mediated suppression of AAD a number of investigations were performed. Tregs were shown to be induced by S. pneumoniae immunotherapy. Furthermore, anti-CD25 antibody-mediated depletion of Tregs reversed the effect of immunotherapy. Hence, Tregs were required for immunotherapy-mediated suppression of AAD. In the third study, Tregs were shown to be induced in a biphasic manner to suppress immune responses and AAD through a broad range of mechanisms (Chapter 4). Together, these studies have identified potential and novel S. pneumoniae immunotherapies for asthma and determined the mechanism of action that underpins suppression of AAD.