Optimisation of immune effector function within the tumour microenvironment

Cancer represents a leading of cause of death in the developed world, inflicting tremendous suffering and plundering billions from health budgets. The traditional treatment approaches of surgery, radiotherapy and chemotherapy have achieved little in terms of cure for this deadly disease. Instead, life is prolonged for many, with dubious quality of life, only for disease to reappear with the inevitable fatal outcome. “Blue sky” thinking is required to tackle this disease and improve outcomes. The realisation and acceptance of the intrinsic role of the immune system in cancer pathogenesis, pathophysiology and treatment represented such a “blue sky” thought. Moreover, the embracement of immunotherapy, the concept of targeting immune cells rather than the tumour cells themselves, represents a paradigm shift in the approach to cancer therapy. The harnessing of immunotherapy demands radical and innovative therapeutic endeavours – endeavours such as gene and cell therapies and RNA interference, which two decades ago existed as mere concepts. This thesis straddles the frontiers of fundamental tumour immunobiology and novel therapeutic discovery, design and delivery. The work undertaken focused on two distinct immune cell populations known to undermine the immune response to cancer – suppressive T cells and macrophages. Novel RNAi mediators were designed, validated and incorporated into clinically relevant gene therapy vectors – involving a traditional lentiviral vector approach, and a novel bacterial vector strategy. Chapter 2 deals with the design of novel RNAi mediators against FOXP3 – a crucial regulator of the immunosuppressive regulatory T cell population. Two mediators were tested and validated. The superior mediator was taken forward as part of work in chapter 3. Chapter 3 deals with transposing the RNA sequence from chapter 2 into a DNA-based construct and subsequent incorporation into a lentiviral-based vector system. The lentiviral vector was shown to mediate gene delivery in vitro and functional RNAi was achieved against FOXP3. Proof of gene delivery was further confirmed in vivo in tumour-bearing animals. Chapter 4 focuses on a different immune cell population – tumour-associated macrophages. Non-invasive bacteria were explored as a specific means of delivering gene therapy to this phagocytic cell type. Proof of delivery was shown in vitro and in vivo. Moreover, in vivo delivery of a gene by this method achieved the desired immune response in terms of cytokine profile. Overall, the data presented here advance exploration within the field of cancer immunotherapy, introduce novel delivery and therapeutic strategies, and demonstrate pre-clinically the potential for such novel anti-cancer therapies

Extracellular vesicles from induced neurons trigger epigenetic silencing of a brain neurotransmitter.

Introduction: Antithrombin (AT) is a glycoprotein involved in the regulation of blood coagulation. It belongs to the family of serine-protease inhibitors and acts as the most important antagonist of different clot- ting factors. Two types of inherited AT deficiency can be distinguished: Type I (quantitative deficit), and Type II (qualitative deficit). The latter is characterized by an impaired inhibitory activity related to dysfunc- tional domains of the protein. Three Type II subtypes can be defined: Type IIa (reactive site defect), Type IIb (heparin binding site defect) and Type IIc (pleiotropic defect). This classification has clinical importance since these subtypes have a different thrombotic risk. No functional routine diagnostic assay, however, can be assumed to detect all forms of Type II deficiencies since false-negative results may hamper the diagnosis. Methods: We analysed the biochemical/biophysical association of ATT to EVs. We separated EVs from plasma of healthy or Type II affected patients or from cultured hepatocytes through differential ultracentrifu- gation followed by sucrose density gradient and/or immunoprecipitation. We next combined dot blot ana- lysis, WB, 2D electrophoresis and enzymatic assays to reveal the nature of ATT association to EVs. Results: We evidenced that ATT is associated to the external leaflet of EVs. We also found that specific ATT isoforms are enriched in EV preparations in respect to total plasma and that those isoforms are selectively associated to EVs when comparing healthy or ATT type II deficient patients. Summary/Conclusion: ATT selective association pat- tern to EVs might be related either to mutations in the primary sequence of the protein or alterations in the glycosylation process, hence experiments are ongoing to reveal the nature of this phenomenon. Our findings suggest that analysis of ATT enriched in EV prepara- tions might be useful to gain insights into the patho- genesis and be of support in the diagnostic algorithm of ATT deficiency. Funding: This work acknowledges FFABR (Fondo finanziamento attività Base di ricerca from MIUR, Ministry of Education, Universities and Research, Italy) for financial support

Dichotomic role of NAADP/two-pore channel 2/Ca2+ signaling in regulating neural differentiation of mouse embryonic stem cells

Poster Presentation - Stem Cells and Pluripotency: abstract no. 1866The mobilization of intracellular Ca2+stores is involved in diverse cellular functions, including cell proliferation and differentiation. At least three endogenous Ca2+mobilizing messengers have been identified, including inositol trisphosphate (IP3), cyclic adenosine diphosphoribose (cADPR), and nicotinic adenine acid dinucleotide phosphate (NAADP). Similar to IP3, NAADP can mobilize calcium release in a wide variety of cell types and species, from plants to animals. Moreover, it has been previously shown that NAADP but not IP3-mediated Ca2+increases can potently induce neuronal differentiation in PC12 cells. Recently, two pore channels (TPCs) have been identified as a novel family of NAADP-gated calcium release channels in endolysosome. Therefore, it is of great interest to examine the role of TPC2 in the neural differentiation of mouse ES cells. We found that the expression of TPC2 is markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebound during the late stages of neurogenesis. Correspondingly, perturbing the NAADP signaling by TPC2 knockdown accelerates mouse ES cell differentiation into neural progenitors but inhibits these neural progenitors from committing to the final neural lineage. Interestingly, TPC2 knockdown has no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Overexpression of TPC2, on the other hand, inhibits mouse ES cell from entering the neural lineage. Taken together, our data indicate that the NAADP/TPC2-mediated Ca2+signaling pathway plays a temporal and dichotomic role in modulating the neural lineage entry of ES cells; in that NAADP signaling antagonizes ES cell entry to early neural progenitors, but promotes late neural differentiation.postprin