Chronic Lymphocytic Leukaemia (CLL) remains the most abundant leukaemia in those aged over 65 years. It is characterised by the expansion of malignant monoclonal B-lymphocytes that were originally described as being functionally incompetent. Identified by immunophenotypic expression of monoclonal light chain restriction, it falls into the classification of chronic B-cell lymphoproliferative disorders (B-LPD). This thesis aims to demonstrate that CD160, an activating NK cell receptor, is aberrantly expressed in B-LPD and can function as a tumour specific antigen, which has clear translation roles within the clinical environment, aiding in the diagnosis of CLL and monitoring of minimal residual disease (MRD). More so, this study aims to provide an insight into the potential biological roles of CD160 within chronic B-cell malignancies. CD160 is an activating NK cell receptor whose major form is a glycosylphosphatidylinositol (GPI)-anchored cell surface molecule with a single immunoglobulin domain. In-vitro studies on a large cohort of B-LPD patients demonstrated that CD160 was primarily restricted to cases of CLL (98%) and Hairy Cell Leukaemia (HCL, 100%) with only a minor population of other B-LPDs expressing the antigen. More so, within the B-cell lineage, CD160 can be considered a tumour specific antigen (TSA) in that when looking for both transcript and protein, they were absent throughout the normal B-cell hierarchy. Many clinical studies base their entry criteria on clinical and biological prognostication, as this provides insights into the biology of CLL and its response to therapy. Disease eradication has been shown to be prognostic. This study demonstrates the feasibility and clinical importance of MRD detection utilising CD160 as novel marker of residual disease. Subsequently, CD160 analysis by flow cytometry (CD160FCA) demonstrated to be as sensitive and specific as other methodologies, and independent of the type of therapy. Further to this the early detection of MRD was correlated with known biological prognostic risk groups. Patients in CR had significantly different EFS based on their MRD status following treatment using the CD160FCA. For those patients with adverse prognostic markers (including CD38, ZAP-70 and M), the time to detection of MRD or relapsing disease ß2using CD160FCA, was significantly shorter than those with a normal or good prognosis. Within normal NK and T lymphocytes, CD160 has a multifunctional role that upon triggering results in a unique profile of cytokine production via the recruitment of Phosphatidylinositol 3-kinase (PI3K). In CLL cells, CD160 stimulation resulted in the recapitulation of these observations including cell survival, an increase in Bcl-2 family antiapoptotic proteins, and cell cycle progression. This thesis has demonstrated that CD160 is aberrantly expressed in malignant B-cells, it has a clear clinical translation role in terms of diagnosis and MRD monitoring, and multiple biological functions which recapitulate those observed in NK-cells.

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Tumor immunoediting is a dynamic process in which the immune response attacks tumor cells by detecting danger signals and tumor antigens. In order to survive, tumor cells develop mechanisms to avoid detection or destruction by the immune system. To counteract this, several strategies are being developed to enhance the antitumor immune response, including the depletion of immunosuppressive cells, enhancing the activation of antitumor immune cells and increasing tumor cell immunogenicity. These therapies have seen limited success individually, however, and it is likely that combination therapy with novel targets will be necessary to see reproducible beneficial responses. Epigenetic modifications are attractive therapeutic targets because they are reversible and affect gene expression in cancer cells. Within this framework, this study aimed to elucidate the role of the chromatin remodeling complex nucleosome remodeling factor (NURF) in cancer immunoediting by silencing of bromodomain PHD-finger containing transcription factor (BPTF), the largest and essential subunit of NURF. Using two syngeneic mouse models of cancer, BPTF was found to suppress T cell antitumor activity in the tumor microenvironment. In vitro, enhanced cytolytic activity was observed for individual CD8 T cell clones only from mice bearing BPTF-silenced tumors, implicating the involvement of novel antigens. Mechanistic investigations revealed that NURF directly suppresses the expression of genes encoding immunoproteasome subunits Psmb8 and Psmb9 and the antigen transporter genes Tap1 and Tap2. PSMB8 inhibition reversed the effects of BPTF ablation, consistent with a critical role for the immunoproteasome in improving tumor immunogenicity. Thus, NURF normally suppresses tumor cell antigenicity and its depletion improves CD8 T cell antitumor immunity. In a concurrent study using different tumor lines, BPTF was also found to suppress natural killer (NK) cell antitumor immunity in vivo. Enhanced NK cell cytolytic activity toward BPTF-depleted targets in vitro was dependent on the natural cytotoxicity receptors (NCR). Molecular studies revealed that BPTF directly activates heparanase (Hpse) expression, resulting in reduced cell surface abundance of the NCR co-ligands: heparan sulfate proteoglycans. Thus, NURF represses NCR co-ligand abundance and its depletion enhances NK cell cytotoxicity. Therefore, NURF emerges as a candidate therapeutic target to enhance CD8 T or NK cell antitumor immunity.

Natural killer cells (NK cells) are part of the immune system in human and other mammals. The task of these cells, which belong to the non-specific immunity, is to induce apoptosis in other cells of the body that may represent a threat for the body (i.e., tumour or virally infected cells). NK cells have a variety of surface receptors to recognize their target cells. A number of receptors are well-known today and they may be divided into groups based, e.g., on their structural similarities or on the type of signal which these receptors present to NK cells. Accordingly, we distinguish activation and inhibitory receptors. Inhibitory receptors inhibit NK cell response, while activation receptors elicit this response. During NK cell contact with another cell, the resulting NK cell behaviour is always the result of a certain balance of activation and inhibitory receptor responses. The NKp44 receptor is an immunoglobulin-like receptor. This receptor is very unique among other receptors in many respects, for example because it is associated with both activation and inhibitory motif. The ligand of this receptor is a proliferating cell nuclear antigen (PCNA). PCNA is a clamp protein important, inter alia, during DNA replication, in which it anchors other replisome proteins. This work is focused on...

Monoclonal antibodies (mAbs) have become an important part of therapy for a number of cancers. The first mAb to be approved for clinical use is rituximab, which is currently used for the treatment of various B cell malignancies. Despite its clinical value, the mechanisms in which rituximab induces tumor regression are unclear. Growing evidence suggests that multiple mechanisms involving complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved. However, the direct interactions between CDC and ADCC have yet to be investigated. My studies examine the relationship between complement fixation and the activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model, and clinical samples from patients. Using these systems, I demonstrate that the initiation of the complement cascade inhibits NK cell activation and ADCC induced by rituximab in vitro. I also show that depletion of complement enhances the activation of NK cells and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that NK cell activation correlates with decreased complement activity in patients after rituximab treatment. The studies described in this dissertation have furthered the understanding of the mechanisms involved in antibody therapy. These results have described a novel inhibitory role for complement activity in the anti-tumor responses of mAbs. Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of complement may improve how current mAbs are used and the how mAbs are designed in the future.

The immune system is comprised of specific cell types that communicate and interact via a range of soluble and surface-bound molecules to defend the body against pathogens. Many gaps remain in our understanding of the subcellular trafficking pathways that regulate the diverse functions of the immune system. The central aim of this thesis was to investigate transport through the endocytic pathway, focussing in particular on the unique organelles and functions of this pathway in immune cells. Two subsets of immune cells were of particular interest in this thesis, macrophages and natural killer (NK) cells. These cell types both perform a range of functions that contribute to both innate and adaptive immunity. Another common thread between these cells is that they both perform functions involving specialised endocytic organelles and pathways. Macrophages utilise their endocytic pathways to perform several unique functions; phagocytosis, endocytosis and degradation of foreign proteins for presentation on MHC class II molecules, and signalling of Toll-like receptors from endosomes. Even secretion of cytokines such as tumour necrosis factor alpha (TNFα) by macrophages requires transport through an endosomal compartment, the recycling endosome, as recently discovered in this laboratory (Murray et al., 2005a). NK cells utilise specialised secretory lysosomes to deliver a lethal hit to carefully identified target cells, providing an alternative example of specialised endocytic trafficking in the immune system. Of the many protein families that regulate subcellular trafficking, the SNARE, Rab, Munc and exocyst proteins were focussed on during this thesis. The localisation and function of members of these families in the endocytic pathway were investigated. Novel results in macrophages concerned the role of Vti1b in endocytosis, a process with implications for MHC class II antigen presentation and TLR detection of endocytosed particles. Alteration of Vti1b protein levels in the cells significantly decreased uptake and degradation of endocytic cargo. A role for Rab11 and the recycling endosome in antigen presentation was also studied. MHC class II was detected in recycling endosomes, and overexpression of a mutant Rab11 protein altered the distribution of MHC class II, suggesting a role for Rab11 in subcellular trafficking during antigen presentation. Preliminary results also suggest a role for the exocyst protein Sec15 at the recycling endosome in macrophages, providing a new target for investigation of the regulation of TNFα secretion. The recycling endosome is emerging as a vital transport hub during cytokine secretion, phagocytosis and possibly other cellular functions in macrophages. This project also involved the unique opportunity to examine primary NK cells from patients with a number of genetic immunodeficiencies caused by mutations to trafficking proteins. The autosomal recessive immunodeficiencies Griscelli syndrome type 2 (GS2) and familial haemophagocytic lymphohistiocytosis types 3 (FHL3) and 4 (FHL4) are associated with loss-of-function of Rab27a, Munc13-4 and syntaxin 11 (Stx11), respectively. These diseases involve a loss of cytotoxic function by cytotoxic CD8+ T lymphocytes and NK cells, but the precise molecular role of these proteins in granule release is incompletely understood. In freshly isolated, resting NK cells from healthy subjects, PMA and ionomycin stimulation or conjugation to susceptible target cells induced colocalisation of endogenous Rab27a and Munc13-4 to perforin-containing granules. In Rab27a-deficient cells, which showed defective degranulation and cytotoxicity induced by signals for both natural and antibody-dependent cellular cytotoxicity, Munc13-4 failed to colocalise with perforin upon activation. Unexpectedly, Rab27a and Munc13-4 localisation to lytic granules was selectively induced by different receptor signals, demonstrating specificity for regulation of lytic granule maturation by target cell ligand expression. Recruitment of the SNARE protein Vti1b, which has not previously been associated with NK cell function or secretory lysosome release, to perforin granules was also discovered. Unexpectedly, Stx11 was not localised to perforin granules. These experiments have contributed to our understanding of the precise molecular roles of Munc13-4, Rab27a and Stx11 in NK cell granule release. Overall, this thesis presents novel and important results from studies of subcellular transport through the endocytic pathways of macrophages and NK cells. These results advance our understanding of several immune functions, and a number of human genetic immunodeficiencies. This new knowledge of the role of endocytic organelles and fusion machinery in these processes provides exciting targets for future research.

Natural killer (NK) cells are an essential part of immune system, providing self-surveillance of virally infected, stress transformed or cancerous cells. NKR-P1 receptors and their ligands from clec2 gene family represent an alternate missing-self recognition system of NK cells based on interaction of highly related C-type lectin-like receptors. Human NKR-P1 has been described more than twenty years ago but still remains the sole human orthologue of this receptor family, particularly numerous in rodents. On binding to its cognate ligand LLT1, NKR-P1 can relay inhibitory or co-stimulatory signals. Although being interesting targets for their potential role in tumor immune evasion and autoimmunity, nature of their interaction is still unclear. To elucidate the architecture of their interaction, we developed a generally applicable method for recombinant expression of human NKR-P1 and LLT1 and their homologues based on transfection of HEK293S GnTI- cells. Further, we described a stabilizing mutation His176Cys, that enables for expression of highly stable and soluble LLT1. Finally, we have crystallized LLT1 and human NKR-P1 in different glycosylation states both as individuals and in complex. While both structures of LLT1 and NKR-P1 follow the classical C-type lectin-like superfamily fold, contrary to...

The definition of Natural Killer (NK) cells has undergone dramatic modification with the advance of immunological research tools. NK cells can no longer be classified only as indiscriminate killer innate immune cells. NK cells are now known to form functional relationships with accessory cells to shape and promote the adaptive immune response. Recently, antigen specific and cytokine induced memory NK cells have been demonstrated to exhibit the characteristic phases of an adaptive immune cell, including extended persistence and robust function in response to reexposure. New findings in basic NK immunobiology indicate that the role of NK cells is underappreciated and these cells may potentially be manipulated for the development of anti-viral therapies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20072号 / 医博第4165号 / 新制||医||1018(附属図書館) / 33188 / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 小川 誠司, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

NK cell based anti-tumour therapies demonstrate high efficacy in targeting hematological malignancies, however, treatments for advanced solid tumours face challenges. The immunosuppressive environment produced by tumours prevents NK cells from maintaining cytotoxic activity and reducing tumour burden. Enhancing NK cell activation is essential to improve their function against solid tumours. Genetic manipulation of primary NK cells with viral and non-viral methods has seen a drastic improvement in recent years. Lentiviral vectors are being used to generate CAR-NK cells ex vivo, while refinement of electroporation protocols has allowed for the generation of stable gene knockouts in primary NK cells. To establish and validate the generation of a stable knockout in primary human NK cells we focused on targeting the NCAM-1 (CD56) surface adhesion molecule. The high surface expression of CD56 in NK cells makes it a suitable target to establish the knockout protocol. Furthermore, despite its levels of expression being correlated to different functional phenotypes, the role of CD56 in NK cell function is not understood. Here we have shown that current lentiviral transduction protocols are not viable methods to deliver the sgRNA/Cas9 system into primary NK cells. However, we found that nucleofection of the sgRNA/Cas9 complex into NK cells is an efficient method to generate gene knockouts. Using newly generated CD56KO NK cells we have shown that the expression of CD56 has no effect on NK cell cytotoxicity, cytokine production, proliferation, and in vivo tissue trafficking. In parallel, we have also identified an intracellular pathway that is active in the tumour microenvironment and could inhibit NK cell function. Recent studies on the intracellular signaling of the E3 ubiquitin-protein ligase Cbl-b have highlighted its role in inhibiting NK cell tumour lytic and anti-metastatic activity. Immunosuppressive factors produced by tumours activate the Cbl-b pathway, leading to the targeted degradation of signaling proteins required for NK cell activation. We have shown that Cbl-b is upregulated in ex vivo expanded NK cells cultured with GAS6 or ovarian cancer ascites. Therefore, the generation of human primary Cbl-bKO NK cells could be a beneficial asset to enhance NK cell cancer immunotherapy. / Thesis / Master of Science (MSc)