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1
Opel, Cary F. (Cary Francis). "T cell mediated combination immunotherapy." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/107075.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemical Engineering, February 2016.Cataloged from PDF version of thesis. "September 2015."
Includes bibliographical references (pages 128-131).
Immunotherapy is a broad treatment strategy that harnesses the immune system to fight off a particular condition or disease. Cancer immunotherapy is the specific application of agents designed to interact or stimulate the immune system to fight off tumors. Treatments as diverse as passive antibody therapy, cytokine support, and comprehensive adoptive T cell transfer make up the broad field of immunotherapeutics. Due to the naturally complex interactions inherent in the immune system, there are many options for therapeutic intervention, however, this same complexity makes it extremely difficult to optimize treatment strategies. Because of this, research into developing new immunotherapies, optimizing existing immunotherapies, and designing new combinations of immunotherapies is still critical in the fight against cancer. Although there have been ongoing successes of individual immunotherapies in the clinic, the complexity and interdependence of the immune system suggests that any single therapeutic intervention will be insufficient to reject established malignancies. Increased interest in applying combinations of immunotherapies in the clinic requires more thorough preclinical work to guide the designs of these studies. The work presented in this thesis focuses on developing combinations of immunotherapies to treat preclinical models of cancer, as well as studying the underlying mechanism of tumor control. T cells are potent mediators of cytotoxicity and when properly used in adoptive cell transfer (ACT) protocols, can be highly effective in the treatment of cancer. ACT consists of three steps: 1) harvesting and purifying T cells from the patient, 2) enriching or modifying the T cells to become tumor specific, and 3) reinfusing the T cells along with supporting therapies. Therapies given alongside ACT are often adjuvants designed to enhance T cell response. However, focusing therapies only on enhancing the activity of the transferred T cells may miss out on synergistic effects when other parts of the immune system are simultaneously engaged. To study the effect of adjuvant therapy on ACT, a preclinical murine model was analyzed. Large, established B16F10 tumors were controlled when pmel-1 T cells were given with a course of supportive MSA-IL2 cytokine therapy, however, no cures were observed. When a course of TA99 antibody therapy was added alongside ACT, a high rate of cures was observed. Flow cytometry of both circulating and tumor infiltrating pmel-1 cells showed massive expansion and activation. Additionally, tumor infiltration of neutrophils, NK cells, and DCs were greatly enhanced by adjuvant therapy. DCs in the tumor draining lymph nodes were largely unchanged by the therapies. Engagement of the humoral immune response was also observed in both treatment cases. Surprisingly, antibody therapy did not substantially alter any of the mechanistic observations made in this study, despite its critical role in achieving cures of tumors. While ACT is a highly effective therapy, its clinical applicability is hindered by the complexity of performing T cell transplants and manipulations. A more optimal solution would involve purely injectable treatments that could elicit the same level of tumor specific T cell response in conjunction with potent recruitment of the adaptive immune system against tumors. To achieve this, working in collaboration with the Irvine Lab, combinations of immunotherapy using up to four different components were tested to identify critical factors in the successful rejection of established tumors in preclinical models. The four components of tumor targeting antibody, cytokine support, checkpoint blockade, and cancer vaccine acted synergistically to reject tumors from B16F10, TC-1, and DD-Her2/neu cell lines. The cancer vaccine elicited large numbers of tumor-specific T cells, and acted as a replacement for ACT. By analyzing subset combinations of this full treatment, the roles of each therapeutic component were identified. CD8 T cells and cross-presenting DCs were critical to curing subcutaneous tumors. Cytokine therapy was indispensable for effective tumor control, promoted immune cell infiltration into the tumor, and led to an increase in DCs. In combination with the other therapies, vaccination against a tumor antigen elicited a strong immunological memory response that was able to reject subsequent tumor rechallenge, as well as promote antigen spreading to new epitopes. Successful combinations were demonstrated to be dependent on the recruitment of both the adaptive and innate branches of the immune system. Finally, the efficacy of this combination of treatments was demonstrated by controlling the growth of induced tumors in a BRaf/Pten model. Combination immunotherapy promises a future where synergistic treatments are specifically tailored to individual cancers leading to highly effective responses. However, determining the optimal combination of therapies, the complexity of dosing strategies, and the availability of targeted treatments are all barriers that must be overcome. The analysis presented here will make a significant contribution to the body of knowledge on immunotherapy as it has shown the importance of combining orthogonal immunotherapies in order to get durable cures to established tumors. These results will hopefully encourage combinations of orthogonally acting therapies based on T cells to achieve stronger clinical responses. By determining the necessary requirements for a strong, synergistic response to tumorous growths, more effective combination immunotherapy protocols may be designed in the future.
by Cary F. Opel.
Ph. D.
2
Goddard, Ruth Victoria. "Generation of in vitro B-cell chronic lymphocytic leukaemia-specific T cell responses using dendritic cells." Thesis, University of Plymouth, 2002. http://hdl.handle.net/10026.1/2695.
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Immunotherapy using dendritic cells has shown encouraging results in both haematological and non-haematological malignancies. In this study, monocyte-derived dendritic cells from patients with B-cell Chronic Lymphocytic Leukaemia were generated by culture in Interleukin-4 and Granulocyte Macrophage-Colony Stimulating Factor. Lysate-pulsed autologous dendritic cells were used as antigen presenting cells in co-culture with autologous B-cell Chronic Lymphocytic Leukaemia T-cells. B-cell Chronic Lymphocytic Leukaemia T-cells stimulated with B-cell Chronic Lymphocytic Leukaemia lysate-pulsed autologous dendritic cells showed a significant increase in cell surface expression of Interleukin-2 Receptor (CD25), Interferongamma secretion and cytotoxicity against autologous B-cell Chronic Lymphocytic Leukaemia B-cell targets hut not against targets from healthy volunteers. Responses were only stimulated by the B-cell Chronic Lymphocytic Leukaemia B cell lysate. Cytotoxicity was Major Histocompatibility Complex Class II restricted. The addition of maturation agents such as Lipopolysaccharide, Tumour Necrosis Factor-alpha and Polyriboinosinic Polyribocytidylic Acid to monocyte derived dendritic cells was unsuccessful at increasing anti-tumour responses. Pre-treatment of T cells with Interleukin-15 before stimulation by lysate pulsed autologous dendritic cells increased numbers of activated cells, cytokine secretion and specific cytotoxicity to B-cell Chronic Lymphocytic Leukaemia 8-cells. Fusion of monocyte derived dendritic cells and B-cell Chronic Lymphocytic Leukaemia B-cells generated both Major Histocompatibility Complex Class I and Class II restricted cytotoxicity to B-cell Chronic Lymphocytic Leukaemia B-cell targets. When B-cell lysates were analysed using reducing sodium dodecyl sulphate-polyacrylamide gel electrophoresis, a B-cell Chronic Lymphocytic Leukaemia specific hand at 42,000 Dalton and other patient specific bands were observed. Only the 65,000 Dalton and 42,000 Dalton hands were capable of stimulating comparable T cell responses as the whole lysate. The 65,000 Dalton band from normal healthy volunteers showed a dominant peptide that closely matched Human Serum Albumin. The 42,000 Dalton band from B-cell Chronic Lymphocytic Leukaemia patients showed a possible match with Human Actin.
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Wurzenberger, Cornelia. "Dendritic cell vaccines in tumor immunotherapy." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-95530.
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Harley, Eric. "Modeling Cancer Cell Response to Immunotherapy." Scholarship @ Claremont, 2004. https://scholarship.claremont.edu/hmc_theses/164.
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Significant work has been done modeling cancerous tumor growth and response to therapy under certain simplifying assumptions, specifically, the assumption of spatial homogeneity. We have chosen a spatially heterogenous model for cancer cell growth using a hybrid Lattice-Gas Cellular Automata method. Cell mitosis, apoptosis, and necrosis are explicitly modeled along with the diffusion of nutrients and a necrotic signal. The model implementation is verified qualitatively and is modified to execute on a parallel computer.
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Lute, Kenneth D. "Costimulation and tolerance in T cell immunotherapy." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1141850521.
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White, Matthew. "T-cell cancer immunotherapy : characterisation and manipulation of tumour antigen-specific T cell subsets for adoptive immunotherapy in mouse models." Thesis, Imperial College London, 2011. http://hdl.handle.net/10044/1/9148.
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Understanding the behaviour of T cells in context of tumour responses is crucial for designing adoptive immunotherapy strategies. The aim of the study is to characterise tumour-specific T cells, including conventional and regulatory T cells (Tregs), in tumour-bearing mice after adoptive transfer. Male-specific minor histocompatibility antigen HY was used as the tumour-associated antigen, expressed by MB49 murine bladder carcinoma and recognised by TCR transgenic CD4 (Marilyn) and CD8 (Matahari) T cells. Unlike male skin grafts, MB49 tumours express a panel of immune-suppressive molecules including PD-L1, FAS-L, TGF-β, IL-10 and IDO which contribute to the formation of a tolerogenic microenvironment. As a result, HY-specific CD8 T cells reject syngeneic male skin grafts but not MB49 tumours. In response to MB49 tumours, HY-specific Tregs underwent expansion. A fraction of proliferating Tregs also lost FoxP3 and became ex-Tregs, which upregulated IFNγ, and downregulated a panel of Treg-specific genes. In addition, it was observed that preferential ex-Treg differentiation took place in an IL-6-enriched microenvironment, such as in the mesenteric lymph nodes. The antigen-specific CD8 response to MB49 is insufficient for rejection. Retroviral modification of MB49 to express hIL-2 allows for induction of effective antigen-specific CD8 responses, providing a potent whole cell in vivo vaccine strategy for exploring factors mediating immune evasion. Finally, the role of anti-HY TCR transgenic T cells in GvHD is assessed. Lymphopenic male recipients lose weight after adoptive transfer of CD4 but not CD8 T cells. With radiation preconditioning, full-blown GvHD ensues, raising questions about this combination for clinical therapy. This study advances knowledge of antigen-specific T effector and Treg responses to HY in a range of environments, including MB49 tumours, male skin grafts and GvHD. It highlights the importance of understanding not only induction of effector responses, but potentially harmful side-effects of adoptive cell transfer.
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Cabezón, Cabello Raquel. "Tolerogenic dendritic cell-based immunotherapy in Crohn’s disease." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/310604.
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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.
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Vertuani, Simona. "Strategies to optimize T cell-based cancer immunotherapy /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-891-6/.
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Adikari, Sanjaya Bandara. "Cytokine-modulated dendritic cell immunotherapy in autoimmune diseases /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-149-0/.
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Jackson, Andrew Mark. "Cytokines, cell adhesion molecules and bladder cancer immunotherapy." Thesis, University of Edinburgh, 1993. http://hdl.handle.net/1842/19867.
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The intravesical administration of Bacillus Calmette Guerin for the treatment of transitional cell carcinoma of the bladder is the most effective immunotherapy for any solid human malignancy. Despite this awesome accolade relatively little is understood of its mechanism of action. This study details the in vitro interaction between IL-2 activated lymphocytes and tumour cells, the effect of cytokines produced as a result of immunotherapy on tumour cells and the relationship of these findings to the situation in vivo. Bladder cancer cells were not found to be susceptible to NK cell activity but were found to be differentially susceptible to IL-2 activated lymphocytes. No correlation was evident between the histopathological grade of the tumour. The interaction between these cells was observed to involve intimate contact and the tumour cells were found to constitutively express either ICAM-1 or ICAM-2. The expression of these cell adhesion molecules correlated significantly with the sensitivity of the tumour cells to LAK mediated cytolysis. Following BCG therapy a variety of cytokines including IFNγ and TNFα are detected in the urine. When bladder cancer cells were cultured in the presence of recombinant IFNγ and TNFα an increase in the levels of ICAM-1 expression was observed. The optimal stimulation was found after 24 hours culture with 100Uml-1 IFNγ, whilst TNFα stimulated to a lesser extent. Culture in the presence of both cytokines was observed to synergistically induce or augment ICAM-1 expression. Following culture with IFNγ, the tumour cells displayed increased susceptibility to LAK activity, this was significantly correlated with increased ICAM-1 expression. The levels of tumour cell response to IFNγ could not be correlated with either the abundance or affinity of specific receptors as determined by Scatchard analysis. Thus investigations were initiated into the events down-stream of the ligand-receptor interaction. Monoclonal antibodies to ICAM-1, decreased the sensitivity of tumour cells to LAK activity. However, monoclonal antibodies to LFA-1 (the ligand for ICAM-1) further blocked the action of LAK cells.
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Crompton, Joseph. "Targeting Akt in cell transfer immunotherapy for cancer." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709380.
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Escribà, Garcia Laura. "Cellular immunotherapy for B-cell lymphoma with B-cell lymphoma with NKT-cell agonists." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/386489.
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Las células NKT representan una pequeña población de linfocitos con especificidad única para antígenos glicolipídicos, presentados por el receptor CD1d presente, principalmente, en las células dendríticas. Estas células juegan un papel muy important en la respuesta inmune antitumoral ya que coordinan tanto el sistema inmune innato como adaptativo. Las células NKT pueden ser activadas por el lípido sintético α-galactosilceramida (α-GalCer), que estimula la secreción de citoquinas como el IFN-γ, IL-12, IL-4 e IL-17, contribuyendo a la activación de células dendríticas (CDs), NK, B y T. En este trabajo, se evaluó el efecto antitumoral de la combinación de CDs, células tumorales irradiadas y el agonista de células NKT α-GalCer en un modelo murino de linfoma B.
La vacuna terapéutica formada por CDs, células tumorales y α-GalCer eliminó el linfoma B en todos los ratones tratados, y su eficacia fue superior a la de otras combinaciones que se testaron como controles, incluyendo α-GalCer, células tumorales con CDs y CDs con α-GalCer. La terapia indujo la generación de una respuesta inmune de memoria, además de ser tumor-específica. El perfil de citoquinas inducido por la vacuna terapéutica resultó ser una combinación de respuesta Th1, Th2 e IL-17. Además, el tratamiento incrementó el número de células NKT y NK en los ratones tratados, así como de células NKT, NK y T secretoras de IFN-γ. Nuestro modelo de linfoma B resultó ser dependiente de células NK, además de presentar activación de células B.
La vacuna terapéutica compuesta por CDs, células tumorales y α-GalCer elimina eficazmente el linfoma B de forma terapéutica. El tratamiento induce una respuesta inmune de memoria y tumor-especifica, y está asociado a un incremento de células NKT y NK, así como de células NKT, NK y T secretoras de IFN-γ, y con la activación de células B.Natural killer T (NKT) cells are a small population of lymphocytes with unique specificity for glycolipid antigens presented by non-polymorphic CD1d receptor on APC, mainly presented on dendritic cells (DCs). NKT cells play a central role in tumor immunology since they coordinate innate and adaptive immune responses. These cells can be activated with the prototypic lipid α-galactosylceramide (α-GalCer), stimulating IFN-γ production and cytokine secretion (eg, IL-12, IL-4, IL-17) that contribute to the enhancement of DC function and the induction of NK, B and T-cell activation. We evaluated the antitumor effect of a combination of DCs and irradiated tumor cells with the NKT cell agonist α-GalCer in a mouse model of B-cell lymphoma. The therapeutic vaccine consisting of DCs, tumor cells and α-GalCer was able to eradicate B-cell lymphoma in all treated mice, and was superior to any vaccine combination, including α-GalCer alone, irradiated tumor cells with DCs, and DCs with α-GalCer. Importantly, treated mice with the vaccine were resistant to a tumor rechallenge, suggesting the development of a memory immune response. In addition, the immune response was tumor-specific since all the mice were unable to reject a syngeneic A20 B-cell lymphoma. In addition to in vivo evaluation of the therapeutic vaccine, the cytokine profile induced by the treatment was evaluated, showing a combination of Th1 and Th2 cytokines together with IL-17. When we looked at effector cells, we observed that the vaccine induces a high NKT and NK-cell expansion, as well as a high increase of IFN-γ secreting NKT, NK and tumor-specific T cells. NK cells played a critical role in the antitumor effect observed after the therapeutic treatment and there was also an activation of B cells since IgG antibodies against tumor cells were found in treated mice. The therapeutic vaccine consisting of dendritic cells, tumor cells and α-GalCer efficiently eradicates B-cell lymphoma in a therapeutic setting. This immune response is long-lasting, tumor-specific, and it is associated with an expansion of NK and NKT cells, an increase of IFN-γ secreting NK, NKT and T cells and B-cell activation.
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Raeiszadeh, Mohammad. "Reconstitution of CMV-specific T-cells following adoptive T-cell immunotherapy and haematopoietic stem cell transplantation." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6968/.
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This thesis investigated reconstitution of CMV-specific T-cells in two cohorts of HSCT patients and studied the potential role of Tumour Necrosis Factor Receptor 2 (TNFR2) in regulation of CMV-specific T-cell expansion post HSCT. The first cohort included patients of a randomized phase II trial of adoptive cellular therapy for CMV-specific CD8\(^+\) T-cells. Cellular therapy resulted in earlier and greater expansion of CMV-specific CD8\(^+\) T cells and also reconstitution of CMV-specific CD4\(^+\) and non-infused CMV-specific CD8\(^+\) T-cells. The number of infused therapeutic T-cells and circulating levels of Alemtuzumab were found to influence immunotherapy. Additionally, reconstitution of CMV-specific CD4\(^+\) T-cells was studied using HLA-class II tetramers. CMV-specific CD4\(^+\) T-cell count of >0.7x10\(^3\)/ml was found to protect from recurrent CMV reactivation. One third of specific CD4\(^+\) T-cells were perforin and granzyme-B positive indicating cytotoxic potential, whilst the majority expressed T-bet. Expression of CD57 molecule on CD4\(^+\) T-cells was demonstrated as a potential biomarker of immune response to CMV. Also, distinct cytokine receptor expression patterns in naïve versus memory T-cells were observed. The results showed rapid decrease in IL-6R and increase in expression of TNFR2 after T-cell differentiation from naïve to effector cells and engagement of TNFR2 led to the apoptosis of CMV-specific T-cells.
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Elders, Richard Christopher. "Development of targeted immunotherapy for canine mast cell tumours." Thesis, Royal Veterinary College (University of London), 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522197.
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Chua, I. C. "CD8 co-receptor modifications to enhance T cell immunotherapy." Thesis, University College London (University of London), 2013. http://discovery.ucl.ac.uk/1419099/.
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TCR gene transfer can generate tumour antigen-specific T cells for adoptive immunotherapy. Following TCR gene transfer, transduced T cells usually display the same functional avidity as the parental clone from which the TCR was isolated. However, tumour-antigen specific T cells typically recognize over-expressed self-antigen and are often of low/moderate avidity. It is known that optimal recognition of target cells by CTL requires binding of the cognate peptide MHC class I complex (MHCI) by both TCR and the CD8 co-receptor. Some CD8β chain mutations have been shown to increase CD8 binding affinity with peptide/MHCI and enhance T cell effector function. Murine CD8β chain mutants were generated affecting MHC binding sites (L58R, S53L, S54V and L58R/I25A) or glycosylation sites (T120A, T121A, T124A, and T120A/T121A/T124A). The mutated CD8β molecules were introduced into murine splenocytes using retroviral vectors together with tumour antigen-specific TCRs. The CD8β mutants or control CD8β wild type (WT) chains were first introduced into CD8aa T cells obtained from CD8β knockout mice. All T cells were co-transduced to express the murine F5-TCR which recognizes the model tumour antigen, influenza A nucleoprotein (NP366) presented by H2-Db. The L58R MHC binding CD8 co-receptor mutant (L58R) demonstrated better IFN-γ and IL-2 production in response to relevant peptide while the CD8 glycosylation mutant (T120A/T121A/T124A) mutant demonstrated the opposite effect. The in vitro function of CD4+ T cells transduced with F5-TCR showed that IL-2 and IFN-γ production was enhanced with CD8 co-receptor. In addition, introducing a L58R mutation in the CD8 co-receptor could further increase this effect. The effects of the human CD8 co-receptor with a homologous mutation (I59R) was also investigated in human CD4+ T-cells with a CMV-specific TCR. In vivo studies showed that introducing the F5-TCR alone did not endow CD4+ T cells with significant protection against injected lymphoma cells expressing NP366. However adding CD8 co-receptor to the CD4+ T cells enhanced tumour protection. The genetically modified CD4+ T cells persisted for greater than three months in surviving mice and when re-challenged with antigen the CD4+ T cells with both F5- TCR and CD8 co-receptor had greater proliferative capacity and had more central memory phenotype cells.
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Lim, Sean H. "Investigation of CD20-directed immunotherapy in B-cell malignancies." Thesis, University of Southampton, 2011. https://eprints.soton.ac.uk/376893/.
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Valia, Dhvani. "EMERGING NATURAL KILLER CELL IMMUNOTHERAPY FOR ACUTE MYELOID LEUKEMIA." Case Western Reserve University School of Graduate Studies / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=case1561938259242716.
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Chen, Hung-Chang. "Human γδ T cell-based immunotherapy for breast cancer." Thesis, Cardiff University, 2015. http://orca.cf.ac.uk/86751/.
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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.
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Alderson, Kory L. "Deleterious changes to the T cell compartment following immunotherapy." abstract and full text PDF (UNR users only), 2009. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3355571.
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Maxwell, Tammy Joy. "Dendritic cell mRNA delivery strategies for ovarian cancer immunotherapy." Thesis, Queensland University of Technology, 2007. https://eprints.qut.edu.au/16495/1/Tammy_Maxwell_Thesis.pdf.
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Ovarian cancer, with the highest mortality rate amongst gynaecological malignancies in Australia, is the eighth most common cancer and the fifth cause of cancer-related deaths in women. Currently, five-year survival for women diagnosed with ovarian cancer is only 40 % and despite many patients experiencing remission, approximately 80 % of them will relapse due to residual micrometastasis. The limited impact of standard therapies on the prognosis for recurrent chemotherapy-resistant disease and the need to identify less toxic alternatives has motivated the development of strategies to combat the aggressive and life-threatening burden of ovarian cancer. A novel therapy against cancer utilises dendritic cells (DC), potent antigen presenting cells, to deliver tumour antigens to the immune system for the stimulation of cytotoxic T-lymphocyte (CTL) responses.
DC immunotherapy has been used for the treatment of patients with ovarian cancer; however, clinical responses after the injection of antigen-loaded DC have been disappointing. Therefore, the identification of additional tumour associated antigens (TAA) is required. A TAA highly expressed in ovarian cancer cells, CA125, is a candidate target for DC-based immunotherapy. Initially, CTL responses to CA125 were studied in the context of HLA-A*0201. CD8+ T-cell responses specific for CA125 peptides (with high affinity for the MHC class I) were generated from cultures initiated with peptide-loaded monocyte-derived DC (Mo-DC).
To expand the evaluation of T-cell recognition of CA125 to non-HLA-A*0201 individuals, messenger RNA (mRNA) was investigated as an antigen-loading vehicle. RNA encodes for the repertoire of epitopes presented by the TAA, potentially inducing immune responses in the context of multiple MHC class I and II molecules to known/unknown antigens. One focus of this study was to investigate a novel mRNA transfection system utilising mannan for the delivery of mRNA into DC. Initially the immunomodulating effect of mannan was examined in terms of DC activation. Mannan induced the phenotypic and functional maturation of immature Mo-DC in vitro. Next, the ability of oxidised mannan (OxM) linked to mRNA was investigated for its capacity to deliver enhanced green fluorescent protein (EGFP) mRNA into DC. We observed high transfection efficiencies in the murine and in human DC systems using low mRNA concentrations, in the absence of significant cell viability impairment. Interestingly, upon mRNA delivery via the OxM-PEI complex, DC maturation was induced to considerably higher levels as compared with that achieved with electroporation and non-transfected controls, this was measured by phenotype (CD83) and IL-12 secretion. Within this study, OxM-PEI did not deliver TAA encoding mRNA into DC for the stimulation of CTL. In summary, mannan is a novel strategy to deliver mRNA and a strong maturation signal simultaneously to human Mo-DC. The functional capacity of this system requires further investigation before it can be considered for clinical use.
Electroporation has evolved as a superior method for mRNA delivery into DC as reported in the literature. Therefore, a comprehensive study was performed encompassing the critical issues associated with transfection efficiency, in order to standardise an electroporation protocol for use in DC immunotherapy schedules. EGFP was used as a model antigen to optimise mRNA uptake by Mo-DC by monitoring the expression of the reporter gene by FACS analysis. Influenza matrix protein 1 mRNA was, then, utilised as a model antigen for MHC class I restricted antigen presentation, for confirmation of the optimised loading parameters. The efficiency of this delivery system was assessed using CA125 mRNA in stimulating antigen-specific T-cell responses in PBMC of healthy individuals. CD4+ and CD8+ antigen-specific T-cell responses were generated recognising CA125 mRNA loaded Mo-DC and also ovarian cancer cell lines endogenously expressing CA125.
This study has identified CA125 specific T-cell responses in healthy donors, allowing further investigation into the potential for its use as a candidate TAA in ovarian cancer immunotherapy. Furthermore, the use of Mo-DC transfected with mRNA encoding TAA is a promising strategy for the delivery of TAA in the generation of antigen-specific T-cell responses. In summary, the results gained from this PhD thesis should be taken into consideration when designing future DC immunotherapy strategies to combat one of the leading causes of cancer mortality in women, ovarian cancer.
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Maxwell, Tammy Joy. "Dendritic cell mRNA delivery strategies for ovarian cancer immunotherapy." Queensland University of Technology, 2007. http://eprints.qut.edu.au/16495/.
Full textAbstract:
Ovarian cancer, with the highest mortality rate amongst gynaecological malignancies in Australia, is the eighth most common cancer and the fifth cause of cancer-related deaths in women. Currently, five-year survival for women diagnosed with ovarian cancer is only 40 % and despite many patients experiencing remission, approximately 80 % of them will relapse due to residual micrometastasis. The limited impact of standard therapies on the prognosis for recurrent chemotherapy-resistant disease and the need to identify less toxic alternatives has motivated the development of strategies to combat the aggressive and life-threatening burden of ovarian cancer. A novel therapy against cancer utilises dendritic cells (DC), potent antigen presenting cells, to deliver tumour antigens to the immune system for the stimulation of cytotoxic T-lymphocyte (CTL) responses. DC immunotherapy has been used for the treatment of patients with ovarian cancer; however, clinical responses after the injection of antigen-loaded DC have been disappointing. Therefore, the identification of additional tumour associated antigens (TAA) is required. A TAA highly expressed in ovarian cancer cells, CA125, is a candidate target for DC-based immunotherapy. Initially, CTL responses to CA125 were studied in the context of HLA-A*0201. CD8+ T-cell responses specific for CA125 peptides (with high affinity for the MHC class I) were generated from cultures initiated with peptide-loaded monocyte-derived DC (Mo-DC). To expand the evaluation of T-cell recognition of CA125 to non-HLA-A*0201 individuals, messenger RNA (mRNA) was investigated as an antigen-loading vehicle. RNA encodes for the repertoire of epitopes presented by the TAA, potentially inducing immune responses in the context of multiple MHC class I and II molecules to known/unknown antigens. One focus of this study was to investigate a novel mRNA transfection system utilising mannan for the delivery of mRNA into DC. Initially the immunomodulating effect of mannan was examined in terms of DC activation. Mannan induced the phenotypic and functional maturation of immature Mo-DC in vitro. Next, the ability of oxidised mannan (OxM) linked to mRNA was investigated for its capacity to deliver enhanced green fluorescent protein (EGFP) mRNA into DC. We observed high transfection efficiencies in the murine and in human DC systems using low mRNA concentrations, in the absence of significant cell viability impairment. Interestingly, upon mRNA delivery via the OxM-PEI complex, DC maturation was induced to considerably higher levels as compared with that achieved with electroporation and non-transfected controls, this was measured by phenotype (CD83) and IL-12 secretion. Within this study, OxM-PEI did not deliver TAA encoding mRNA into DC for the stimulation of CTL. In summary, mannan is a novel strategy to deliver mRNA and a strong maturation signal simultaneously to human Mo-DC. The functional capacity of this system requires further investigation before it can be considered for clinical use. Electroporation has evolved as a superior method for mRNA delivery into DC as reported in the literature. Therefore, a comprehensive study was performed encompassing the critical issues associated with transfection efficiency, in order to standardise an electroporation protocol for use in DC immunotherapy schedules. EGFP was used as a model antigen to optimise mRNA uptake by Mo-DC by monitoring the expression of the reporter gene by FACS analysis. Influenza matrix protein 1 mRNA was, then, utilised as a model antigen for MHC class I restricted antigen presentation, for confirmation of the optimised loading parameters. The efficiency of this delivery system was assessed using CA125 mRNA in stimulating antigen-specific T-cell responses in PBMC of healthy individuals. CD4+ and CD8+ antigen-specific T-cell responses were generated recognising CA125 mRNA loaded Mo-DC and also ovarian cancer cell lines endogenously expressing CA125. This study has identified CA125 specific T-cell responses in healthy donors, allowing further investigation into the potential for its use as a candidate TAA in ovarian cancer immunotherapy. Furthermore, the use of Mo-DC transfected with mRNA encoding TAA is a promising strategy for the delivery of TAA in the generation of antigen-specific T-cell responses. In summary, the results gained from this PhD thesis should be taken into consideration when designing future DC immunotherapy strategies to combat one of the leading causes of cancer mortality in women, ovarian cancer.
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Sachamitr, Supatra. "Exploiting the use of induced pluripotent stem cell derived immune cells for immunotherapy." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:89315b6b-a8cd-4a6f-8c43-3506d8dd1725.
Full textAbstract:
Immunotherapy traditionally made use of biological agents such as cytokines and monoclonal antibodies. Such first generation therapies lack antigen specificity and fail to induce immunological memory, suggesting that cell therapies may provide the next generation of treatments that are more discerning in their mode of action. Nevertheless, difficulties in obtaining sufficient immunologically-relevant cell types from patients has limited their success. Given that induced pluripotent stem cells (iPSC) may be generated from patients, we have investigated the feasibility of deriving two cell types whose availability is restricted in vivo: regulatory T cells (Tregs) and CD141+ cross-presenting dendritic cells (DCs). We describe the optimization of protocols for differentiation and purification of CD141+ DCs, focussing on their utility as a therapeutic vaccine for HIV-1. We investigate their phenotype, chemotactic capacity, phagocytic ability and propensity to harbour infectious virus. We also assess their immunostimulatory capacity and ability to cross-present exogenous antigen to MHC class I-restricted T cells. Our findings led us to speculate that iPSC-derived DCs (iPDCs) possess fetal phenotype, which is characterised by excessive secretion of IL-10 and failure to secrete IL-12, under all but the most stringent conditions. We hypothesised that constitutive secretion of IL-10 may be responsible for maintaining the fetal phenotype, a hypothesis we tested by developing an appropriate mouse model. iPSCs were derived from WT and IL-10-/- mice and were shown to differentiate into iPDCs which recapitulate the fetal phenotype observed among human cells. However, loss of the endogenous Il-10 gene failed to restore full immunogenicity and IL-12 secretion. Finally, we developed protocols for differentiation of FoxP3+ Tregs from iPSCs, a feat that has not previously been achieved. These findings pave the way for the differentiation of Tregs from iPSCs reprogrammed from antigen-specific pathogenic T cells, thereby creating a source of Tregs with matched specificity for therapeutic intervention.
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Antar, Ramy. "Assessing B cell subsets changes in HIV subjects receiving a dendritic cell immunotherapy." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=86938.
Full textAbstract:
Dendritic cells (DC) have a central role in cell-mediated immunity and they are today in the middle of many immunotherapy strategies. Recently, a clinical trial was initiated at the Montreal Chest Institute, to evaluate the effect of an immunotherapy (AGS-004) containing autologous DC to amplify the T cell immune responses of HIV-1-infected subjects. However, concerns have been raised that B cell activation following DC immunotherapy may lead to the development of autoimmune diseases. Here, we studied the safety, patient tolerance and changes in total B cells and B cell subsets following administration of AGS-004 in ten HIV-1 subjects receiving antiretroviral therapy (ART). Clinically, AGS-004 was safe, well tolerated and caused few mild side effects. Moreover, CD4 and CD8 cell counts and HIV-1 viral load were unchanged throughout the 48-weeks follow-up study period. In addition, total B cells and B cell subsets, which were measured as an indicator of the immune activation status, did not change over time, except that the proportion of B memory cells significantly increased after receiving the AGS-004 immunotherapy (P=0.005). Collectively, these data show that the AGS-004 is relatively well tolerable and induces an increase in B memory cells. Further investigations would need to be done to confirm the presence of an activated immune status including functional properties of these B memory cells and antibody measurements.Les cellules dendritiques (DC) ont un rôle central dans l'immunité à médiation cellulaire et elles sont aujourd'hui au centre de nombreuses stratégies d'immunothérapie. Récemment, un essai clinique à base des DC autologues, a été initié à l'Institut Thoracique de Montréal pour évaluer l'effet d'une immunothérapie (AGS-004) à amplifier les réponses immunitaires des lymphocytes T chez les sujets infectés par le VIH-1. Cependant, le risque potentiel de l'activation des lymphocytes B, menant au développement de maladies auto-immunes, a été soulevé suivant des immunothérapies à base de DC. Dans le présent travail, nous avons évalué l'innocuité, la tolérance et les changements des lymphocytes B et leurs sous-populations après DC immunothérapie chez dix sujets infectés par le VIH-1 et recevant une thérapie antirétrovirale. Cliniquement, l'AGS-004 était sans danger, bien tolérée avec quelques effets secondaires bénins. De plus, les valeurs de la charge virale et les décomptes lymphocytaires CD4 et CD8 n'ont pas été modifiées tout au long de la période de suivi de 48 semaines. En outre, les pourcentages de lymphocytes B totaux et leurs sous populations n'ont pas changé, à l'exception des lymphocytes B mémoires qui ont considérablement augmenté après l'immunothérapie (P=0.005). Collectivement, ces résultats montrent que l'AGS-004 est relativement bien tolérée et induit une augmentation des lymphocytes B mémoires. Il demeure donc intéressant de confirmer ces résultats et d'étudier la fonction de ces lymphocytes de manière plus poussée ainsi que de mesurer la production des anticorps. fr
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Krzywinska, Ewelina. "Study of tumor cell metabolism and its relationship with NK cell-mediated immunotherapy." Thesis, Montpellier 1, 2014. http://www.theses.fr/2014MON1T012/document.
Full textAbstract:
La formation et le développement d'une tumeur sont provoqués par une série de défauts qui se produisent à l'intérieur de la cellule cancéreuse et dans son microenvironnement. Ces anomalies permettent à la cellule de développer ses propres stratégies de croissance, de prolifération, de différenciation et de métabolisme. Toutes ces adaptations, ainsi que la création d'un micro-environnement unique favorisent la croissance de la tumeur et inhibent la réponse immunitaire anti-tumorale. Le métabolisme des cellules cancéreuses et l'évasion immunitaire sont des points très sensibles dans le développement des cancers et peuvent être utilisés en clinique. Les études récentes suggèrent que ces deux phénomènes sont liés, et que le métabolisme des cellules cancéreuses peut amener à l'échappement immunitaire par la tumeur. Le métabolisme des cellules tumorales a tendance à éviter l'activité mitochondriale et la phosphorylation oxydative, et est principalement basée sur la glycolyse pour la production d'énergie (effet Warburg). Mon travail de thèse est divisé en deux parties. Dans la première partie nous avons proposé un concept thérapeutique novateur avec une nouvelle thérapie combinatoire pour le traitement de cancers hématologiques. Cette thérapie est basée sur l'induction de changements métaboliques par le dichloroacétate (DCA), et elle est associée avec la chimiothérapie conventionnelle (doxorubicine, vincristine) pour réactiver les fonctions de p53. Les tumeurs avec p53 mutantes sont résistantes à cette combinaison. Dans ce cas, nous avons constaté que le DCA peut coopère avec 17-AAG (l'inhibiteur de Hsp90) pour éliminer spécifiquement les cellules cancéreuses. En conséquence, une meilleure compréhension des signaux et des mécanismes par lesquels le DCA sensibilise les cellules tumorales à la chimiothérapie est nécessaire pour en comprendre le mode d'action. En outre, l'identification de ce mécanisme permettra d'élucider les voies métaboliques impliquées dans la survie des cellules cancéreuses. La deuxième partie de ma thèse se concentre sur la biologie des cellules NK. Les cellules NK sont des lymphocytes du système immunitaire inné et possèdent une cytotoxicité naturelle contre les cibles, c'est à dire les cellules tumorales. L'utilisation optimale des cellules NK en clinique nécessite leur expansion et leur activation in vitro. Les cellules NK s'activent en présence de cytokines ou par le contact avec les cellules cibles. L'activation des cellules NK induit la prolifération, mais celle-ci dépend aussi de la présence d'autres cellules immunitaires. L'activation, par les cytokines et par les cellules cibles, induit un différent ARNm/microARN profil d'expression. L'analyse détaillée des isoformes de la protéine tyrosine phosphatase CD45 a permis de caractériser de nouvelles populations de cellules NK anti-tumorales humaines. L'identification de différentes populations de cellules NK est très importante pour la compréhension de leur physiologie et pour l'amélioration de leur utilisation en immunothérapie clinique. Cela peut également donner des informations précieuses sur l'état physiologique de l'hôte. En effet, l'augmentation des cellules CD45RAdim et CD45RO + dans le compartiment des cellules NK matures identifie clairement les patients avec des hémopathies malignes. Nous pensons que leur détection peut être utilisée comme un outil de diagnostic et également pour évaluer l'efficacité des traitements anti-tumoraux, car ces populations de cellules NK spécifiques devraient diminuer lors de l'élimination de cellules tumorales cibles. Dans l'avenir, nous voulons combiner le traitement du métabolisme de la tumeur avec la thérapie anti-tumorale basée sur les cellules NK. Sur la base de nos données préliminaires, nous pouvons proposer le traitement des cellules cancéreuses par des médicaments métaboliques pour augmenter la sensibilité et la reconnaissance par les cellules NK activéesTumor formation and development are caused by a range of defects that occur inside the cancer cell and in the external cellular microenvironment. These abnormalities allow developing tumors to establish their own strategies of growth, proliferation, differentiation and metabolism. All these adaptations, as well as the creation of a unique microenvironment, promote tumor growth and suppress the anti-cancer immune response. Tumor cell metabolism and immune evasion are sensitive points of cancer development that can be targeted in clinic. Recent studies suggest that these two phenomena are related and that cancer cell metabolism may propel tumor immune escape. Tumor cell metabolism tends to avoid mitochondrial activity and oxidative phosphorylation (OXPHOS), and largely relies on glycolysis to produce energy (Warburg effect). My thesis work is divided into two parts. The first one proposes an innovative therapeutic strategy, which is the use of different combinatorial therapy depending on the p53 status for the treatment of hematological cancers. This is based on the induction of metabolic changes by dichloroacetate (DCA), combined with conventional chemotherapy (doxorubicin, vincristine) to reactivate wild type p53 functions. Mutant p53 tumors are resistant to this combination approach. However, we found that DCA synergized with the Hsp90 inhibitor 17-AAG to specifically eliminate these cells. Therefore, a clearer understanding of the signals and mechanisms by which DCA sensitize cancer cells to chemotherapy was needed to understand its mode of action. We uncovered it in our work. In addition, identification of this mechanism will help to elucidate metabolic pathways involved in cancer cell survival.The second part of my thesis is focused on the study of NK cell biology. NK cell is an innate immune system lymphocyte lineage with natural cytotoxicity against targets, i.e. tumor cells. Its optimal use in the clinic requires in vitro expansion and activation. Cytokines and the encounter with target cells activate NK cells, induce their proliferation, and cause clearly different mRNA/miRNA expression profile. Detailed analysis of the leucocyte-specific phosphatase CD45 isoforms allowed us to characterize new human anti-tumor NK cell populations. The identification of the different NK cell populations is important for understanding their physiology and for improving their therapeutic use in the clinic. It can also give valuable information about the host physiological status. Indeed, the increase of CD45RAdim and CD45RO+ cells in the mature NK cell compartment clearly identifies patients with hematological malignancies. We thus hypothesize that their detection could be used as a diagnostic tool, and also to assess the efficacy of antitumor treatments, because these specific NK cell populations should decrease upon removal of the targeted tumor cells. Our future goal is to use a novel combinatorial therapy in hematological cancers that will combine metabolic drugs and NK cell-based therapy. Based on our preliminary data, we propose that the treatment of cancer cells with metabolic drugs could increase their sensitivity and recognition by activated NK cells
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Tsitoura, Daphne-Chryssoula. "Modulation of human TH cell functions by altered peptide ligands." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338647.
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Curnow, Stephen John. "Interactions of antibody derivatives with target and effector cell surfaces." Thesis, University of Southampton, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316375.
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Siesjö, Peter. "Immunotherapy of rat brain tumors with mutagen induced, cross-reactive tumor cell variants." Lund : Section of Tumor Immunology, Dept. of Cell and Molecular Biology, University of Lund, 1997. http://books.google.com/books?id=TXZrAAAAMAAJ.
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Anderson, John Edward. "Pancreatic cancer as a target for adoptive T-cell immunotherapy." Thesis, University of Manchester, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.490060.
Full textAbstract:
The aim of this thesis was to investigate pancreatic cancer as a target for chimeric immune (CIR) based adoptive T ceil therapy and to irlcntiif\! presence of carcinoembryonic (CEA) and 5T4 antigen targets pancreatic tumour tissue. order to confirm the effectiveness of any it is important to identify a suitable model system. The immunotherapy mechanism on this thesis is based requires CIR specific recognition of the target antigens CEA or 5T4. Therefore two pancreatic and one gastric cancer cell expressing these were identified by immunofluorescent staining and Western Blot analysis. The general is numbers of T cells are required when targeting cancer tissue with adoptive immunotherapy. It is therefore necessary to be able to generate the of cells required a laboratory setting. different methods of T cell activation, using either anti-CD3s OKT3 or antibiotin T cell activation beads, were assessed comparing the rates of cell expansion, viability and cytokine production. To enable T cells to express CIR requires the genetic modification of T lymphocytes, also termed transduction. this thesis a retrovirus based on a murine leukaemia was used. As part the genetic modification process a retroviral clone expressing the 5T4 trans gene was produced. Pancreatic cancer patients' T cells were transduced two alternative transgenes . expressing a CIR specific for either the 5T4 or CEA antigen. levels of transduction efficiency achieved were assessed by staining and flow cytometer analysis. The specific cytotoxic function of the transduced cells was compared against that of untransduced T cells same patients by co-culturing the cells with the three Cancer lines previously shown to express CEA and 5T4 surface antigens. Cancer survival was assessed using a WST-1 cell proliferation assay. results confirmed specific cancer cell cytolysis was mediated by CIR T cells. After showing the adoptive immunotherapy model used was effective at reducing pancreatic and gastric cancer cell numbers vitro, it was important to identify the target antigens' expression true solid pancreatic cancer tissue specimens. Tissue was collected from pancreaticoduodenectomy that had been resected because of the suspicion cancer. Immunohistochemical staining identified the presence of CEA 5T4 antigens the pancreatic tumour tissue. of the findings of the in vitro model used this thesis and the expression of the target antigens indicated by immunohistochemical an~ true pancreatic cancer tissue, adoptive T cell immunotherapy have the potential, further development, to play a role the over survival outcome of pancreatic cancer patients.
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Chong, Tsung Wen. "Targeting the hypoxic tumour phenotype with specific T-cell immunotherapy." Thesis, University of Oxford, 2004. http://ora.ox.ac.uk/objects/uuid:d22f1d74-44eb-4560-9249-f6127accd1b1.
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Vo, Dang Nghiem. "Natural Killer cell subsets in hematological diseases : learning for immunotherapy." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT013/document.
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Les cellules Natural Killer (NK) sont des lymphocytes cytotoxiques innés qui jouent un rôle important dans le contrôle immunitaire de la formation de cellules tumorales et de l'infection virale. Chez les personnes en bonne santé, les cellules NK représentent des populations hétérogènes définies par différents marqueurs phénotypiques et exécutant des fonctions spécifiques. Les cellules NK provenant de patients présentant des tumeurs malignes néoplasiques et une infection virale sont cependant typiquement distinctes des personnes en bonne santé par l'apparition de sous-ensembles de cellules NK, qui sont différenciées par leur profil d'isoformes CD45. CD45 est une tyrosine phosphatase leucocytaire commune abondamment exprimée sur toutes les cellules immunitaires hématopoïétiques nucléées. Un variant d'épissage alternatif a entraîné la génération de l'isoforme CD45RA longue et de l'isoforme courte CD45RO, qui s'expriment différemment sur les cellules T naïves et effectrices / mémoires. L'expression des isoformes CD45 sur les cellules NK est largement inconnue. Nous avons précédemment montré que l'expression différentielle des isoformes CD45RA et CD45RO a identifié des sous-ensembles de cellules NK spécifiques dans les maladies hématologiques. Une question reste floue: comment ces cellules CD45RARO + NK changent-elles lorsque leurs cellules cibles disparaissent? Nous avons utilisé des cellules NK de patients traités avec Lenalidomide et l'anticorps anti-CD20 Obinutuzumab pour étudier cela et montré une réduction des cellules CD45RARO / CD45RO + NK après la clairance des cellules tumorales (Chater 4). Nous avons observé la même chose chez les patients atteints de LMA après une chimiothérapie. Dans ce cas, le sous-ensemble de cellules CD45RARO + NK est fortement corrélé avec la trogocytose du marqueur monocyte / macrophage CD14 (Chapitre 5). L'immunophénotypage de cellules NK provenant de patients infectés par le VIH a révélé la présence de cellules CD45RAdim et CD45RO + avec une expression réduite de CD16 et une diminution de la modulation NKG2D totale. En résumé, les cellules NK des cancers hématologiques et l'infection par le VIH présentaient des caractéristiques dysfonctionnelles et l'analyse du profil isoforme CD45 dans ces conditions pathologiques dévoile ces caractéristiques.Enfin, afin de retrouver la réponse immunitaire anti-tumorale chez les patients cancéreux, nous présentons une méthode efficace pour l'expansion in vitro de cellules NK hautement activées à partir du sang du cordon ombilical (UCB). Ces cellules NK prouvent une cytotoxicité cellulaire dépendante des anticorps (ADCC) importante lorsqu'elles sont utilisées en combinaison avec des anticorps monoclonaux approuvés sur le plan clinique ciblant divers antigènes tumoraux. Ceci ouvre leur utilisation dans les immunothérapies à base de cellules NK allogéniquesNatural Killer (NK) cells are innate cytotoxic lymphocytes that play an important role in immune control of tumor cell formation and virus infection. In healthy people, NK cell represents heterogeneous populations defined by different phenotypical markers and performing specific functions. NK cells from patients with neoplastic malignancies and viral infection are however typically distinctive from healthy people by the appearance of NK cell subsets, which are differentiated by their CD45 isoform profile. CD45 is a common-leukocyte tyrosine phosphatase abundantly expressed on all nucleated hematopoietic immune cells. Alternative splicing variant resulted in generation of the long-isoform CD45RA and the short-isoform CD45RO, which express differently on naïve and effector/memory T cells. Expression of CD45 isoforms on NK cells is largely unknown. We have previously shown that differential expression of CD45RA and CD45RO isoforms identified specific NK cell subsets in hematological diseases. One question remained unclear: how do these CD45RARO+ NK cell changes when their target cells disappeared? We used NK cells from patients treated with Lenalidomide and the anti-CD20 antibody Obinutuzumab to investigate this and showed a reduction in CD45RARO/CD45RO+ NK cells upon clearance of tumor cells (Chater 4). We observed the same in AML patients after chemotherapy. In this case the CD45RARO+ NK cell subset strongly correlates with trogocytosis of the monocyte/macrophage marker CD14 (Chapter 5). Immunophenotyping of NK cells from HIV-infected patients revealed the presence of CD45RAdim and CD45RO+ cells with reduced CD16 expression and total NKG2D down-modulation. In summary, NK cell from hematological cancers and HIV infection displayed dysfunctional hallmarks and analyzing CD45 isoform profile in these pathological conditions unveils these hallmarks.Finally, in order to regain the anti-tumor immune response in cancer patients, we present an efficient method for expansion of highly activated NK cells from umbilical cord blood (UCB) in vitro. These NK cells prove substantial antibody-dependent cell cytotoxicity (ADCC) when used in combination with clinical-approved monoclonal antibodies targeting various tumor antigens. This paves their use in allogeneic NK cell-based immunotherapies
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CORBETTA, CRISTINA. "Refining strategies for dendritic cell (DC) –immunotherapy in glioblastoma patients." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2019. http://hdl.handle.net/10281/241211.
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Nello studio clinico DENDR1, attivo presso la Fondazione IRCCS Istituto Neurologico Carlo Besta, 24 pazienti affetti da glioblastoma primario sono stati sottoposti a sette vaccinazioni con cellule dendritiche (DC) in combinazione al chemioterapico temozolomide (TMZ) come adiuvante. 9 pazienti hanno beneficiato del trattamento e l’aumento della sopravvivenza è correlato ad una persistente risposta mediata da cellule natural killer, ma non da linfociti CD8. Nello specifico, il trattamento con TMZ ha avuto un impatto negativo sull’attivazione di una risposta mediata da linfociti CD8 compromettendo la formazione di una memoria immunologica, un requisito fondamentale per una risposta immunitaria anti-tumorale prolungata nel tempo. La resistenza alla TMZ è risultata essere legata all’espressione di ABCC3, una proteina coinvolta nella resistenza ai farmaci, nelle cellule natural killer ma non nei linfociti CD8.
Nello studio clinico DENDR2, 12 pazienti con GBM recidivo sono stati trattati con cinque vaccinazioni con cellule dendritiche in combinazione con TMZ. In questi pazienti non abbiamo trovato un’attivazione significativa dei una risposta immunitaria anti-tumorale o un beneficio in termini di sopravvivenza.
Dati recenti indicano che l’iniezione intradermica dell’anatossina tetanica/difterica nel sito di somministrazione delle cellule dendritiche determina l’insorgenza di un processo infiammatorio con aumento di fattori, quali la CCL3 che sembra favorisce la migrazione delle DC ai linfonodi e di conseguenza l’efficacia della vaccinazione. Questo tipo trattamento che viene definito preconditioning.
In uno studio pilota chiamato variant –(V) DENDR2, che include otto pazienti con GBM recidivo, le DC sono state somministrate a livello intradermico nella stessa area dopo 24 ore è stata iniettata la tossina tetanica al fine di predisporre un ambiente favorevole alla loro attivazione e migrazione. I pazienti arruolati in questo studio non hanno ricevuto TMZ come trattamento adiuvante. 5 pazienti, che hanno raggiunto una sopravvivenza di 9 mesi e sono stati pertanto definiti lungo-sopravviventi, hanno mostrato una forte attivazione di linfociti CD8 con formazione di memoria. Inoltre abbiamo osservato a un aumento di linfociti CD4 attivati dalla tossina tetanica parallelamente a un’attivazione di linfociti CD4 più propriamente associati all’azione delle DCs. Inoltre, il sito di vaccinazione è risultato essere molto infiltrato dal linfociti CD4 esprimenti CCL3, a supporto di una possibile maggior migrazione di cellule dendritiche ai linfonodi.
Complessivamente questi dati indicano che il protocollo di somministrazione della TMZ dovrebbe essere rivalutato in modo da potenziare la risposta immunitaria con il contributo dei linfociti CD8. Inoltre, il preconditiong del sito di vaccinazione e l’assenza di TMZ permetterebbero l’attivazione di linfociti CD8 e la formazione di una memoria immunologica, supportata da un aumento di linfociti CD4 che sostengono la persistenza di una risposta anti-tumorale durante il trattamento.In the DENDR1 clinical study, active at Fondazione IRCCS Istituto Neurologico Carlo Besta, 24 patients with first diagnosis of glioblastoma (GBM) were treated with seven vaccinations with dendritic cell (DC) combined to temozolomide (TMZ) as an adjuvant. 9 patients took advantage of this approach and the gain in survival was associated with a specific and long-lasting NK cell, and not CD8+ T cell, response. Specifically, TMZ administration limited the T cell response impairing on CD8+ T cell activation and memory formation, that is a critical requirement for a long-lasting anti-tumor immune-response. Resistance to TMZ was associated with the expression of the multidrug resistance protein ABCC3 in NK but not CD8+ T cells. In the DENDR2 clinical study, 12 patients with recurrent GBM were treated with five DC vaccinations combined with a dose-dense TMZ. These patients did not experience any significant immune response activation and survival advantage. Recent data suggested that pre-conditioning the vaccine site with the recall antigen tetanus/diphtheria toxoid induces a local inflammation with the involvement of CCL3, which improves lymph nodes homing of DC and consequently, the efficacy of DC vaccination. In a pilot study named variant (V)-DENDR2, including 8 patients with recurrent GBM, the vaccine site was pre-conditioned with tetanus toxoid (TT) 24 hours before each DC vaccination. TMZ was avoided. 5 patients, who reached OS9 and were indicated as long-term survivors, showed a strong CD8+ T cell activation and effector to memory transition. Moreover, a vaccine-specific CD4+ T cell activation was detected, accompanied by a TT- specific CD4+ memory T cell response. Moreover, the injection site was strongly infiltrated by CD4+ T cells expressing the chemokine CCL3, supporting a potential increase of DC - homing. Taken together these results suggest that the schedule of TMZ administration should be carefully reconsidered in order to improve and complete the immune response with the contribution of CD8+ T cells. Furthermore, pre-conditioning of the vaccine site and TMZ absence allowed CD8+ T cell activation and memory formation, supported by an increased frequency of CD4+ T helper cells sustaining the persistence of the anti-tumor response during the treatment.
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Almezel, Naseem A. "Generation of monocyte-derived dendritic cells for adoptive immunotherapy." Cincinnati, Ohio : University of Cincinnati, 2010. http://rave.ohiolink.edu/etdc/view.cgi?acc_num=ucin1267720999.
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Thesis (M.S.)--University of Cincinnati, 2010.Advisor: Thomas B. Leemhuis. Title from electronic thesis title page (viewed Apr. 20, 2010). Includes abstract. Keywords: Dendritic cell; Maturation; Generation. Includes bibliographical references.
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Williams, Katherine Spring. "The effects of cell-surface composition on natural killer cell activation: a modeling study." The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306866864.
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Hallett, William H. D. "Augmentation of natural killer cell-mediated anti-tumor effects by molecular targeting and regulatory T cell depletion." abstract and full text PDF (free order & download UNR users only), 2008. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3307716.
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Carlsten, Mattias. "Molecular specificities of NK cell-mediated recognition of human tumor cells." Stockholm, 2010. http://diss.kib.ki.se/2010/978-91-7409-686-6/.
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Mulati, Kumuluzi. "VISTA expressed in tumor cells regulates T cell function." Kyoto University, 2019. http://hdl.handle.net/2433/242370.
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Vikman, Sofia. "Towards Immunotherapy of Midgut Carcinoid Tumors." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8421.
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Minard-Colin, Véronique. "Immune effector mechanisms of malignant B cell depletion by CD20 Immunotherapy." Paris 11, 2010. http://www.theses.fr/2010PA11T073.
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Juffs, Helen Gwendolyn. "Immunogenicity of B-cell chronic lymphocytic leukemia and prospects for immunotherapy /." [St. Lucia, Qld.], 2001. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe16312.pdf.
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Alsaieedi, Ahdab Abdulazim. "T cell delivery of immune-stimulatory cytokines to enhance cancer immunotherapy." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/10040720/.
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Adoptive cell therapy using TCR-engineered T cells is an exciting area of research and has emerged as a promising strategy for treating cancer patients. However, the effector function of TCR-engineered T cells can be tuned down by local mechanisms of tumour-associated immunosuppression. The potential of cytokines to reverse local immune suppression and enhance tumour immunity has been described in the past. The main aim of this project was to engineer T cell specificity as well as effector cytokine production as a strategy to enhance cancer immunotherapy. This was achieved by combining TCR gene transfer with genetic engineering to achieve IL-12 and IL-27 production in therapeutic T cells. In vitro validation data demonstrated not only an enhanced production of IL-12 and IL-27 by the engineered T cells but also an enhanced effector function upon antigen-specific stimulation. In order to circumvent previously described toxic side effects observed with systemic IL-12 delivery, a tet-regulated gene expression system was utilised to regulate cytokine production by engineered T cells in vivo. Adoptive transfer of TCR-redirected T cells expressing regulated IL-12 in B16F10 melanoma-bearing mice resulted in an enhanced accumulation of transferred CD8+ T cells in the tumour and in a change of the innate immune cell composition in the tumour microenvironment. Importantly, regulated IL-12 delivery resulted in enhanced therapeutic efficacy of the transferred T cells without causing systemic toxicity. IL-27 delivery in engineered T cells also showed some effectiveness when combined with TCR gene therapy, although the therapeutic benefit of IL-27 was inferior to IL-12. The data in this study demonstrate the potency of additional genetic manipulation to tailor the TCR-redirected T cell effector function which can result in a substantial enhancement in their therapeutic efficacy, and thus, enhanced antitumor immune response.
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Matsubara, Hiroyuki. "Induction of Human Pluripotent Stem Cell-Derived Natural Killer Cells for Immunotherapy under chemically defined condition." Kyoto University, 2019. http://hdl.handle.net/2433/245316.
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付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラムKyoto University (京都大学)
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新制・課程博士
博士(医科学)
甲第22121号
医科博第106号
新制||医科||7(附属図書館)
京都大学大学院医学研究科医科学専攻
(主査)教授 濵﨑 洋子, 教授 河本 宏, 教授 生田 宏一
学位規則第4条第1項該当
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Lo, Jennifer Alys. "Regulation of the Inflamed Tumor Phenotype in Melanoma Immunotherapy." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493467.
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Immune checkpoint inhibitors targeting cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and programmed cell death-1 (PD-1) pathways can deliver durable anti-tumor effects. However, a major fraction of metastatic cancer patients fail to respond to checkpoint blockade.
Recent studies suggest that efficacy of checkpoint inhibitors is associated with inflammation in the tumor microenvironment. In this thesis, I demonstrate using genetically-defined murine models that sterile melanomas can be converted into inflamed tumors with improved responses to checkpoint blockade via two independent approaches: introduction of neoantigens and a novel combinatorial therapeutic strategy.
In addition to tumor inflammation, genomic studies have identified elevated numbers of neoantigens, mutated proteins that can serve as targets of immune responses, as potential predictors of clinical benefit. The preponderance of UVR-associated somatic mutations in melanoma has been proposed to play a role in mediating responses to immunotherapy, but model systems to study the contribution of such mutations to anti-melanoma immunity have been lacking. In chapter 2, I present a BrafV600E/Pten-/- syngeneic tumor graft murine model in which melanomas bearing numerous non-synonymous UVB-induced mutations were markedly more inflamed and responsive to PD-1 inhibition than matched parental melanomas.
For the treatment of neoantigen-deficient, poorly-inflamed tumors, in chapter 3 I tested the novel combination of imiquimod, ablative fractional photothermolysis (aFP), and checkpoint inhibitors. In anti-PD-1 resistant models of melanoma and pancreatic adenocarcinoma, addition of imiquimod and aFP produced abscopal tumor regressions with long-term survival in 50-60% of cases. Combination therapy stimulated autoimmunity against wildtype tumor-lineage antigens, suggesting that therapeutic strategies which enhance inflammation and responses against self-antigens may bypass a need for neoantigens and produce major regressions of cancers that are currently refractory to checkpoint blockade in the clinic.
In chapter 4 I show that PD-L1 expression is transcriptionally regulated by the melanocyte lineage factor and oncogene microphthalmia-associated transcription factor (MITF). PD-L1 expression is significantly correlated with MITF copy number in patient melanomas and is induced in skin as part of the MITF-dependent tanning response pathway. Our data suggest that loss of PD-L1 predisposes mice to apparent vitiligo after chronic UVR, suggesting that the UVR-MITF-PD-L1 axis represents a melanocyte lineage-specific mechanism of immune tolerance.Medical Sciences
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McBride, Harry Michael. "The recruitment of ribosomal inactivating protein or T cells by antibody derivatives in the treatment of B cell lymphoma." Thesis, University of Southampton, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.295851.
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Guven, Hayrettin. "The therapeutic potential of ex vivo expanded natural killer (NK) cells for immunotherapy of cancer /." Stockholm, 2005. http://diss.kib.ki.se/2005/91-7140-564-X/.
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Maciocia, P. M. "Targeting the T-cell receptor beta constant region for investigation and immunotherapy of T-cell malignancies." Thesis, University College London (University of London), 2017. http://discovery.ucl.ac.uk/1551063/.
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T-cell lymphomas and leukemias are aggressive, treatment-resistant cancers with poor prognosis. Immunotherapeutic approaches have been limited by a lack of target antigens discriminating malignant from healthy cells. While treatment of B-cell cancers has been enhanced by targeting pan B-cell antigens, an equivalent approach is not possible for T-cell malignancies since profound T-cell depletion, unlike B-cell depletion, would be prohibitively toxic. We propose an immunotherapeutic strategy for targeting a pan T-cell antigen without causing severe depletion of normal T-cells. The α/β T-cell receptor (TCR) is a pan T-cell antigen, expressed on >90% of T-cell lymphomas and all normal T-cells. An overlooked feature of the TCR is that the β-constant region comprises 2 functionally identical genes: TRBC1 and TRBC2. Each T-cell expresses only one of these. Hence, normal T-cells will be a mixture of individual cells expressing either TRBC1 or 2, while a clonal T-cell cancer will express TRBC1 or 2 in its entirety. Despite almost identical amino acid sequences, we identified an antibody with unique TRBC1 specificity. Flow cytometry (FACS) of T-cells in normal donors (n = 27) and patients with T-cell cancers (n = 18) revealed all subjects had TRBC1 and 2 cells in both CD4 and CD8 compartments, with median TRBC1 expression of 35% (range 25-47%). In addition, we examined viral-specific T-cells in healthy volunteers, by generation of Epstein Barr virus-specific primary cytotoxic T-cell lines (3 donors) or by identification of cytomegalovirus- (3 donors) or adenovirus- (5 donors) specific T-cells by peptide stimulation. We demonstrated similar TRBC1: 2 ratios in viral-specific cells, suggesting that depletion of either subset would not remove viral immunity. Next, using FACS and immunohistochemistry, we showed that TCR+ cell lines (n = 8) and primary T-cell lymphomas and leukemias (n = 55) across a wide range of histological subtypes were entirely restricted to one compartment (34% TRBC1). As proof of concept for TRBC-selective therapy, we developed anti-TRBC1 chimeric antigen receptor (CAR) T-cells. After retroviral transduction of healthy donor T-cells, comprising mixed TRBC1/2 populations, 90% of T-cells expressed CAR on the cell surface. No detectable TRBC1 T-cells remained in the culture, suggesting selective depletion of this population. Anti-TRBC1 CAR T-cells secreted interferon-γ in response to TRBC1-expressing target cell lines (p < 0.001) or autologous normal TRBC1+ cells (p < 0.001), and not TRBC2 cell lines or autologous normal TRBC2 cells. Anti-TRBC1 CAR killed multiple TRBC1 cell lines (p < 0.001) and autologous normal TRBC1cells (p < 0.001), and not TRBC2 cell lines or autologous normal TRBC2 cells. These cell-line based findings were confirmed using primary cells from two patients with TRBC1+ adult T-cell leukaemia/lymphoma. We demonstrated specific tumour kill by allogeneic or autologous T-cells in vitro, despite partial downregulation of surface TCR by tumour cells. We developed a xenograft murine model of disseminated T-cell leukemia by engrafting engineered firefly luciferase+ TRBC1+ Jurkat cells in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Bioluminescent imaging and FACS of marrow at 5 days following IV T-cell injection showed that mice treated with anti-TRBC1 CAR T-cells and not non-transduced (NT) T-cells had disease clearance (p < 0.0001). In a further model, mice were engrafted with equal proportions of TRBC1-Jurkat and TRBC2-Jurkat cells. FACS analysis of bone marrow at 5 days post T-cells demonstrated specific eradication of TRBC1 and not TRBC2 tumour by anti-TRBC1 CAR (p < 0.001). In summary, we have demonstrated a novel approach to investigation and targeting of T-cell malignancies by distinguishing between two possible TCR β-chain constant regions. Using CART-cells targeting TRBC1 we have demonstrated proof of concept. Unlike non-selective approaches targeting the entire T-cell population, TRBC targeting could eradicate a T-cell tumour while preserving sufficient normal T-cells to maintain cellular immunity.
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Edes, Inan. "Targeted transduction of T cell subsets for immunotherapy of cancer and infectious disease." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2016. http://dx.doi.org/10.18452/17669.
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Das Ziel der vorliegenden Arbeit bestand darin, ein Vektorsystem zu entwickeln, dass den simultanen Transfer verschiedener Transgene in CD8+ und CD4+ T-Zellen und dadurch die Herstellung eines immunotherapeutischen T-Zell-Produkts ermöglicht, welches aus zwei unterschiedlich modifizierten T-Zell-Subtypen besteht. Im ersten Teil der Arbeit wurde die Targeting-Technologie von lentiviralen auf γ-retrovirale Vektoren übertragen. Anschließend wird die Herstellung von Vektoren beschrieben, die spezifisch für murines CD4 oder CD8 sind. Deren Spezifität wurde zum einen durch die exklusive Expression von GFP in CD4+ oder CD8+ Zellen und zum anderen durch den Dosis-abhängigen Verlust des GFP-Signals nach Inkubation dieser Zellen mit CD4- und CD8-blockierenden Antikörpern nachgewiesen. Im dritten Teil der Arbeit wird gezeigt, dass MVm8 und MVm4 primäre T-Zellen spezifisch transduzieren. MVm8-vermittelter Transfer des Ovalbumin (OVA)-reaktiven TZRs OT-I führte zu T-Zellen, die OVA+ Tumor-Zelllinien erkannten und Interferon-γ sezernierten. Der vierte Teil dieser Arbeit beschäftigt sich mit der in vivo Transduktion primärer T-Zellen mithilfe von MVm8, welches den OT-I-TZR und eine Luciferase transferiert (MVm8/OT-I-luc). Durch systemische Applikation von MVm8/OT-I-luc wurden T-Zellen in vivo transduziert. Durch Immunisierungen konnten antigen-spezifisches Homing, Expansion und eine anschließende Kontraktion in vivo transduzierter T-Zellen gezeigt werden. Mäuse mit starker OT-I-luc-Expression waren gegenüber einer Infektion durch OVA-transgene listeria monocytogenes geschützt. Zusammenfassend lässt sich sagen, dass das in dieser Arbeit entwickelte Vektorsystem in der Lage ist zwischen Subtypen von T-Zellen zu unterscheiden und sie simultan mit unterschiedlichen Transgenen auszustatten. Für MVm8 konnte gezeigt werden, dass es T-Zellen direkt in vivo transduzieren kann.The aim of this thesis was to generate a vector system that allows the simultaneous transfer of different transgenes into CD8+ and CD4+ T cells, allowing the generation of a immunotherapeutic T cell product comprised of two differently engineered T cell subsets. The first part of the thesis describes the transfer of the measles virus (MV) envelope-based targeting technology from lentiviral (LV) to γ-retroviral (gRV) vectors. The second part reports the generation of two targeting vectors specific for murine CD4 or CD8. The exclusive specificity of MVm4 and MVm8 was proven by expression of GFP in CD4+ and CD8+ reporter cells, respectively, but not in CD4-CD8- cells after transduction, and by a dose-dependent loss of GFP signal after incubation of reporter cells with CD4 or CD8 blocking antibodies before transduction. The third part shows that MVm8 but not MVm4 transduced primary T cells. MVm8-mediated transfer of the ovalbumin (OVA)-reactive TCR OT-I resulted in T cells secreting interferon-γ (IFNγ) upon recognition of OVA+ tumor cell lines. The final part of this thesis describes the in vivo transduction of primary T cells using MVm8 transferring OT-I and a luciferase (MVm8/OT-I-luc). To this end, B6 mice deficient for Rag2 have been repopulated with either polyclonal (B6) or monoclonal T cells derived from P14-TCR transgenic mice (P14). One day later the transferred T cells were transduced in vivo by systemic application of MVm8/OT-I-luc. Upon immunization in vivo-transduced T cells homed, expanded and contracted repeatedly in an antigen-dependent manner. Finally, mice exhibiting strong luc-signals showed improved protection against infections by OVA-transgenic listeria monocytogenes (LM-OVA). In conclusion, the viral vector system developed within this thesis is able to discriminate between the two main T cell subsets and to equip them with distinct transgenes simultaneously.
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Woods, David Michael. "Histone Deacetylases as Targets for Melanoma Immunotherapy." Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4856.
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Cancer represents the second leading cause of death in the United States. For many malignancies, currently available treatment options offer little long-lasting survival benefits to patients. However, recent studies have shown immunotherapeutic approaches to be an attractive strategy to cancer treatment. While many current immunotherapeutic strategies convey durable responses, such responses are only seen in a minority of patients. An increased understanding of the mechanisms governing tumor immunogenicity and the biology of immune responses is crucial to improving upon the efficacy of current and future cancer immunotherapies. Histone deacetylases (HDACs), enzymes classically associated with regulation of gene expression, have been therapeutic targets in various cancers for several years due to their involvement in cell growth. However, it has become increasingly clear that HDACs are intimately involved in regulating both the immunogenicity of tumor cells and immune response of leukocytes and lymphocytes. In order to expand upon this growing knowledge, the therapeutic efficacy of the pan-HDAC inhibitor LBH589 in the treatment of melanoma was studied. The results presented here demonstrate that LBH589 is a potent inhibitor of growth in a wide variety of melanomas through induction of cell cycle arrest and apoptosis. Additionally, LBH589 increases the immune visibility of melanoma cells by increasing expression of several immune associated cell surface markers (e.g. MHC I, MHC II, CD80, CD86) in addition to upregulating expression of melanoma differentiation antigens. Furthermore, LBH589 treatment of immune cells results in an enhanced pro-inflammatory phenotype of both APCs and T-cells. These combined effects result in better activation of T-cells and ultimately prolonged survival in LBH589 treated, melanoma-baring mice. To further the understanding of the role of individual HDACs in the T-cell response, the biology of the newest HDAC, HDAC11, was further assessed. To this end, it is shown that HDAC11 is differentially expressed in T-cell populations, and expression is rapidly decreased following activation. Utilizing an HDAC11 knockout (HDAC11KO) mouse strain, it is found that both CD4+ and CD8+ T-cells lacking HDAC11 have an enhanced type 1 effector function characterized by increased proliferation and secretion of IL-2, TNF and IFN-γ. Additionally, HDAC11KO CD8+ T-cells have increased expression of both granzyme B and perforin. HDAC11KO T-cells also demonstrate enhanced resistance to inhibition by Tregs and anergy formation. As a possible mechanism for the observed phenotype, it is also demonstrated that HDAC11KO T-cells produce elevated levels of the transcription factors Eomes and T-bet, both at the basal state and post-activation. In vivo, T-cells lacking HDAC11 have a more potent and robust ability to cause GvHD and mediate an enhanced anti-tumor response. Collectively, these results demonstrate that targeting of HDACs is a viable approach to cancer immunotherapy, and that targeting of specific HDACs may be an attractive strategy for optimizing immunotherapy efficacy while minimizing side effects.
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McWilliams, Emily Mary. "Restoring Innate NK-cell Immunity with Antibody Therapeutics in CLL B-Cell Malignancy." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1479863842166353.
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Stark, Felicity. "The Role of CD8+ T Cell Phenotype and Cytotoxicity on Cancer Immunotherapy." Thesis, Université d'Ottawa / University of Ottawa, 2011. http://hdl.handle.net/10393/20278.
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Cancer vaccines can fail despite the induction of large numbers of CD8+ T cells. Two categories of memory CD8+ T cells have been defined; central memory (TCM, IL-7RαhighCD44highCD62Lhigh) and effector memory (TEM, IL-7RαhighCD44highCD62Llow). It is clear that the memory phenotype of CD8+ T cells can affect vaccine potential; however methods to augment a beneficial phenotype are not clear. I have compared three vaccine delivery systems: Listeria monocytogenes, Salmonella enterica serovar Typhimurium and the particulate liposomal adjuvant, archaeosomes, for their efficacy to protect against murine melanoma. My study revealed that the anti-tumour response is strongly influenced by the kinetics, phenotype, and lymph node homing potential of CD8+ T cells.
Listeria monocytogenes-ovalbumin (LM-OVA) induced TCM cells were adept at long lasting protection against B16-OVA melanoma due to their increased homeostatic and antigen-induced proliferation, interleukin-2 production, and ability to extravasate into tumour draining lymph nodes. Conversely, although Salmonella Typhimurium-ovalbumin (ST-OVA) induced TEM, produced IFN-γ, and killed target cells, this was insufficient for long-term tumour protection.
Selectin-ligand engagements of TCM cells influenced their homing potential and efficacy against murine melanoma. Fucosyltransferase deficient (FtDKO) mice, lacking functional selectin ligands, were vaccinated with LM-OVA; despite the activation of cytotoxic CD8+ T cells, there was a reduced protection against murine melanoma compared to wild-type. FtDKO CD8+ T cells exhibited reduced extravasation into FtDKO lymph nodes compared to wild-type. Additionally, fewer FtDKO CD8+ T cells compared to wild-type migrated into tumour sites.
Archaeosome vaccination was used to compare the influence of CD8+ T cell quantity versus phenotype. Single or multiple therapeutic vaccinations with archaeosome-OVA yielded transient melanoma tumour protection, despite an increased frequency of circulating and tumour infiltrating CD8+ T cells. This correlated with increased expression of Program death receptor-1 (PD-1) on CD8+ T cells and induction of regulatory T cells. Prophylactic archaeosome-OVA vaccination resulted in a maximal frequency of antigen-specific CD8+ T cells of ~50-60 % with just three injections, and ~50 % of the mice were of mice were afforded long-term tumour protection (> 90 days).
Overall, my study shows that the choice of vaccine adjuvant and/or vector can profoundly influence CD8+ T cell quality and cancer vaccine efficacy.
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Cheong, Siew Chiat. "Development of cancer immunotherapy based on parvoviral vectors and hybrid cell vaccination." Doctoral thesis, Universite Libre de Bruxelles, 2005. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211033.
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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