Using Dendritic Cell Receptors to Enhance Immunity

Using Dendritic Cell Receptors To Enhance Immunity Jessica Li ORCID: 0000-0003-1951-8320 Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy May 2017 Department of Microbiology and Immunology The University of Melbourne Abstract Dendritic cells (DCs) are the most potent initiators of immune responses, being highly specialised for the uptake and presentation of antigens (Ag) to activate T cells. Their priming potential can be harnessed to generate stronger immune responses by targeting Ag to DCs via monoclonal antibodies (mAbs) specific for DC-expressed surface receptors. This thesis builds upon the concept of targeting DCs in two main ways: firstly, by investigating a novel method of targeting adjuvant to DCs, and secondly, by investigating how DC-targeting constructs can be used to prime and boost responses. It was considered whether not only Ag, but also adjuvants could be targeted to DCs to improve their efficacy. A recent finding that the DC receptor DEC-205 can bind to and mediate the immunostimulatory effects of CpG oligonucleotide (ODN) adjuvants led to the hypothesis that CpG ODNs could be targeted to DCs via DEC-205 in order to enhance their potency. The interaction between DEC-205 and CpG ODNs was further characterised to determine the molecular properties of ODNs required for binding. This information was then used to enhance the DEC-205 binding capacity of a particular CpG ODN that normally only weakly binds DEC-205. Enhanced DEC- 205 binding was found to significantly improve the stimulatory capacity of this ODN, demonstrating that targeting adjuvant to DCs could be a viable method to improve adjuvant potency. Another receptor, CD14, has also been reported to bind CpG ODNs, so the potential for CD14 to act in synergy with DEC-205 was investigated. However, CD14 was not observed to mediate the uptake or stimulatory effects of CpG ODNs. The identification of natural ligands of DEC-205 is critical for understanding its physiological function. Although ODNs are synthetic molecules, their binding to DEC-205 may signify that DEC-205 is capable of binding other types of DNA that structurally resemble ODNs. A panel of biological DNA samples was screened for DEC-205 binding. While none of the DNA samples were observed to bind DEC-205, some DNA samples were found to bind another receptor, RAGE, suggesting a role for RAGE as a detector of both pathogenic and self-DNA. i Most vaccines must be administered more than once, or “boosted”, to achieve optimal efficacy, and DC-targeted vaccines should be no exception. However, our data suggested that simply administering the same DC-targeting construct twice does not effectively boost the response. This was due to interference from the primary antibody response, which can cross-react with and neutralise a subsequently administered boosting construct. To overcome this issue, the efficacy of various heterologous prime-boost strategies designed to reduce the reactivity of the primary response against the boosting construct was assessed. Ultimately, a combination of anti-Clec9A and anti-XCR1 targeting constructs was found to induce the least cross-reactivity and strongest response after boosting. These findings contribute to the development of better adjuvants and immunisation strategies that optimise the efficacy of DC-targeted vaccines. More broadly, they also highlight the value of understanding the underlying biological mechanisms that drive immune responses, which can then be applied to the rational design of more effective vaccines. ii Declaration This is to certify that: (i) this thesis comprises only my original work towards the PhD except where indicated in the preface; (ii) due acknowledgment has been made in the text to all other material used; (iii) this thesis is fewer than 100,000 words in length exclusive of figures, tables, and references. Jessica Li iii iv Preface This work was conducted in the laboratories of Associate Professor Irina Caminschi at the Burnet Institute and Monash University and Professor William Heath at the University of Melbourne. This work was funded by grants from the National Health and Medical Research Council. My studies were supported by an Australian Postgraduate Award. Some of the work presented in this thesis was collaborative, and the contribution of others is duly acknowledged in the text. The approximate proportion of work that was my original contribution is as follows: Chapter 3: 95% Chapter 4: 70% Chapter 5: 100% Chapter 6: 100% v vi Acknowledgments This PhD was only made possible through the support of many people around me, to whom I would like to express my sincerest gratitude. First and foremost, thank you to my supervisors, Irene Caminschi and Bill Heath, who took me under their wing as an Honours student, and guided me through years of ups and downs with utmost patience and dedication. I could not have asked for more inspiring and supportive mentors to foster my development both as a scientist and as a person. To Ken Shortman, Mireille Lahoud, Meredith O’Keeffe, Jose Villadangos, Nicole La Gruta and Andrew Lew, thank you for providing valuable insight and advice on many occasions. Thank you to Mireille for providing many key reagents, and to Meredith for providing feedback on Chapter 3. To the members of the Caminschi, Lahoud and O’Keeffe and Heath labs, thank you for assisting with both experimental and moral support. Having such friendly and open colleagues certainly made my days in the lab more enjoyable. In particular, thank you to Fatma Ahmet, for teaching me almost every experimental technique I know, taking care of every little matter to ensure the lab runs smoothly, and always being such a cheerful presence in the lab. To my fellow students who shared the Honours and PhD journey with me, thank you for the good times and for your care and support during the tough times. I wish you all the best in your future endeavours. A special thank you to Ee Shan, Zen, Filipp and Ting, for being so good at distracting me from work, but mostly for being there for me when I needed it the most. Finally, thank you to my parents, who have done everything possible to help me in any way they can. Your unconditional love and support has been the biggest source of encouragement and inspiration for me throughout this journey. vii viii Abbreviations Ab antibody ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt AF488 Alexa Fluor 488 AF647 Alexa Fluor 647 AF700 Alexa Fluor 700 Ag antigen APC allophycocyanin APC antigen presenting cell Batf3 basic leucine zipper ATF-like transcription factor 3 BDCA blood dendritic cell antigen bp base pairs BSA bovine serum albumin BSS balanced salt solution CD cluster of differentiation cDC conventional dendritic cell CFSE carboxyfluorescein succinimidyl ester CHO Chinese hamster ovary Clec9A C-type lectin domain family 9 member A CLR C-type lectin-like receptor CRD carbohydrate recognition domain CTL cytotoxic T lymphocyte CTLD C-type lectin-like domain DAMP damage-associated molecular pattern DC dendritic cell DEC DEC-205 DMSO dimethyl sulfoxide DNA deoxyribose nucleic acid ds double-stranded EDTA ethylenediaminetetraacetic acid ELISA enzyme-linked immunosorbent assay FCS fetal calf serum ix FITC fluorescein isothiocyanate Foxp3 forkhead box P3 g gravitational acceleration constant g gram G gauge GM-CSF granulocyte-macrophage colony-stimulating factor H2O water hDEC human DEC-205 hi high HIV human immunodeficiency virus HMGB1 high mobility group box 1 hr hour HRP horseradish peroxidase IgG immunoglobulin G IL interleukin ITAM immunoreceptor tyrosine-based activation motif ITIM immunoreceptor tyrosine- based inhibitory motif i.v. intravenous ICS intracellular cytokine staining IFN interferon Irf interferon regulatory factor kb kilobase kDa kilodalton kg kilogram L litre lo low LPS lipopolysaccharide M molar mAb monoclonal antibody mDEC mouse DEC-205 MFI mean fluorescence intensity mg milligram MHC I major histocompatibility complex class I MHC II major histocompatibility complex class I x min minute ml millilitre mM millimolar MyD88 myeloid differentiation primary response 88 μF microfarad μg microgram μl microlitre μm micrometre μM micromolar NET neutrophil extracellular trap NF-κB nuclear factor kappa B NK cell natural killer cell ng nanogram nm nanometre nM nanomolar nmol nanomole O.D. optical density ODN oligodeoxynucleotide OVA ovalbumin PAMP pathogen-associated molecular pattern PBMC peripheral blood mononuclear cells PBS phosphate buffered saline pDC plasmacytoid dendritic cell PE phycoerythrin PI propidium iodide polyIC polyinosinic-polycytidylic acid PRR pattern recognition receptor RAGE receptor for advanced glycation end-products RNA ribonucleic acid rpm revolution per minute RT room temperature s.c. subcutaneous SEM standard error of the mean ss single-stranded xi TCR T cell receptor Th T helper TLR Toll-like receptor TNF tumor necrosis factor Treg regulatory T cell TRIF TIR-domain-containing adapter-inducing interferon-β V volt v/v volume per volume w/v weight per volume WEHI Walter and Eliza Hall Institute ºC degrees Celsius xii Table of Contents Abstract .................................................................................................................................. i Declaration .......................................................................................................................... iii Preface ...................................................................................................................................

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