Computational analysis of total amino acid sequences indicate that select combinations that occur less frequently are correlated to increased immunogenicity in humans. Much evidence has been gathered in silico, but little is known about in vivo experimental validation. This concept can be applied to adjuvant research where increased immunogenicity is desirable and can aid in the potency and efficacy of vaccines. A rare peptide called 5mer4 was found to adjuvant influenza vaccines by increasing survival, humoral and cellular immune responses with a speculated NK cell mediated mechanism. Therefore we hypothesize that rare peptides are able to stimulate an increased immune response in comparison to common peptides through a NK-mediated fashion. The first aim of this study is to determine whether rare sequences are able to stimulate an increased immune response collectively in comparison to commonly occurring peptides. Mice vaccinated with rare, semi-common and common peptides indicate a trend of heightened cellular immune response from rare peptides. However, select rare peptide sequences based on high IFNγ responses do not always correlate directly to increased vaccine efficacy against H5N1-H05 influenza virus, indicating that additional immune parameters need to be taken into consideration. When compared against other adjuvants, 5mer4 performed better in both humoral and survival studies. Previous findings suggest NK cell involvement warranted the second aim of this thesis which is to further delineate the role of NK cells as rare peptide immune modulators. Macrophages were evaluated to determine the effect of peptide, but no increase in stimulation could be observed. NK cells incubated with rare peptides show increased levels of early activation marker CD69 in comparison to common peptides. Microscopy data indicates that rare, but not common peptides are able to bind to NK cells. Depletion of NK abrogated adjuvant activity of 5mer4 peptide, suggesting the necessary role of NK cells for adjuvant effect. Taken together, rare peptides have shown the ability to modulate the immune response through NK cell activation verifying our hypothesis. These findings can be extrapolated towards multiple fields such as anti-tumor therapies and can lead to the development of immunomodulators with high efficacy at a lower cost.

Computational analysis of total amino acid sequences indicate that select combinations that occur less frequently are correlated to increased immunogenicity in humans. Much evidence has been gathered in silico, but little is known about in vivo experimental validation. This concept can be applied to adjuvant research where increased immunogenicity is desirable and can aid in the potency and efficacy of vaccines. A rare peptide called 5mer4 was found to adjuvant influenza vaccines by increasing survival, humoral and cellular immune responses with a speculated NK cell mediated mechanism. Therefore we hypothesize that rare peptides are able to stimulate an increased immune response in comparison to common peptides through a NK-mediated fashion. The first aim of this study is to determine whether rare sequences are able to stimulate an increased immune response collectively in comparison to commonly occurring peptides. Mice vaccinated with rare, semi-common and common peptides indicate a trend of heightened cellular immune response from rare peptides. However, select rare peptide sequences based on high IFNγ responses do not always correlate directly to increased vaccine efficacy against H5N1-H05 influenza virus, indicating that additional immune parameters need to be taken into consideration. When compared against other adjuvants, 5mer4 performed better in both humoral and survival studies. Previous findings suggest NK cell involvement warranted the second aim of this thesis which is to further delineate the role of NK cells as rare peptide immune modulators. Macrophages were evaluated to determine the effect of peptide, but no increase in stimulation could be observed. NK cells incubated with rare peptides show increased levels of early activation marker CD69 in comparison to common peptides. Microscopy data indicates that rare, but not common peptides are able to bind to NK cells. Depletion of NK abrogated adjuvant activity of 5mer4 peptide, suggesting the necessary role of NK cells for adjuvant effect. Taken together, rare peptides have shown the ability to modulate the immune response through NK cell activation verifying our hypothesis. These findings can be extrapolated towards multiple fields such as anti-tumor therapies and can lead to the development of immunomodulators with high efficacy at a lower cost.

In the first phase of this study the natural cytotoxic activity of human extravascular lymphoid tissue was examined. In contrast to some previous investigations it is reported that the human palatine tonsil contains active natural killer (NK) cells. Like peripheral NK cells, tonsil cytotoxic lymphocytes possess a low buoyant density which allows their enrichment from non-cytotoxic cells. However, in contrast to blood borne natural killier cells which exhibit a large granular morphology, tonsil NK cells are agranular. Furthermore a functional distinction from classical NK cells is apparent, since although the cytotoxic activity of tonsil natural killer cells can be augmented with various immunopotentiating agents including interleukin 2, exposure to interferon (IFN)-a fails to influence reactivity. These data provide evidence for heterogeneity among human NK cells. The existence of NK cell resident within or recirculating through extravascular lymphoid tissue was confirmed by studies of axillary-and mesenteric lymph nodes. Cell populations isolated from these tissues were found to exhibit levels of NK activity equivalent to, or greater than, that observed with tonsil lymphocytes. In contrast to tonsil, NK cells, however, the cytolytic potential of some lymph node cell preparations could be significantly enhanced by IFN-a. It is suggested that natural killer cells within extravascular lymphoid tissues play an important role in providing systemic innate immunity.RThe second objective of this study was to examine the mechanisms of target cell resistance to natural cytotoxicity. Cloned variants of the human erythroleukaemic cell line K562 were isolated by limiting dilution and found to exhibit marked and stable differences in their resistance to NK lysis. Detailed examination of two such clones [E10/P2 and F9/P2] revealed that the observed variation in susceptibility to natural cytotoxicity was not attributable to differential expression of NK recognition determinants. In contrast to other studies in which isolated or induced target cell variants have been shown to exhibit a selective resistance to lysis by NK cells, the resistant clone [F9/P2] examined in this investigation was also less susceptible to antibody-dependent cellular cytotoxicity and complement-mediated lysis. These data indicate that mechanisms exist whereby a cell may exhibit reduced susceptibility to natural cytotoxicity through a generalised capacity to resist immunolytic processes. Cloned variants of K562 were also employed to examine the effect of differentiation-inducing agents, 12-0-tetradecanoylphorbol-13-acetate [TPA) and sodium n-butyrate [NaB] on resistance to NK lysis. It is reported that both TPA and NaB caused increased sensitivity of the resistant variant F9/P2. In contrast TPA, but not NaB, increased the resistance of the sensitive clone, E10/P2 to natural cytotoxicity. The data reveal that differentiation-induced alterations in sensitivity to NK lysis are variable among clones of the same cell line.

Natural killer (NK) cells are innate lymphocytes with inherent ability to eliminate infected cells and produce several cytokines/chemokines. They express surface receptors to sense environment and interact with other immune cells including the Dendritic cells (DC). Reciprocally, DCs are also shown to activate NK-cells. NK/DC cross-talk is well-documented, yet the molecular interactions and the diverse NK-cell activities regulated by DC remain unclear. Several target proteins such as MHC-1, Qa-1 mediate NK-cell target recognition. One such antigen, Ocil/Clr-b functions as a cognate ligand of NKR-P1B/D, NK-inhibitory receptor. In first aim of my study, I documented that deficiency of Ocil/Clr-b expression not only augmented the sensitivity of DC towards NK-cell cytotoxicity but also regulated the development of mature NK-cells. Thus suggesting NKR-P1B/D:Ocil to be another receptor:ligand system, besides Ly49:MHC-1, that regulates NK-cell responsiveness. Src homology region 2-containing protein tyrosine phosphatase-1 (SHP-1) transmits inhibitory signals of the specific NK-inhibitory receptors, including NKRP-1B/D. SHP-1 silenced NK-cells showed unaffected target recognition towards prototypic target cells in this study. In addition, these cells also displayed an unexpected phenotype of self-killing in-vitro, thus implicated SHP-1 as an important regulator of some other unappreciated NK-cell functions. The data from my third study suggest that DCs are directly implicated in the induction of NK-cell migration. In summary, using a novel live-cell imaging microfluidic platform and conventional transwell migration assay this project established a clear molecular link between DC-derived soluble factors such as IP-10 and NK cell-chemokine receptor such as CXCR3. Previously, GM-CSF was shown as an inflammatory cytokine, involved in the development of DC as well as in mediating Th-1 immune responses. In this study I found that GM-CSF regulates NK-cell migration negatively. Lastly, the fourth aim of my thesis highlighted the critical role of immature-DC in the induction of maturation receptors (NK1.1 & Ly49) on differentiating NK-cells. I successfully established a multi-stage in-vitro NK-cell differentiation model and found that differentiating NK-cells required an active engagement with DCs, in addition to the soluble factors. I believe my PhD project findings would impact the existing knowledge to harness DC-based NK cell therapies in clinical settings. / October 2014

Natural Killer (NK) cells are highly mobile, specialized sub-populations of lymphocytic cells that survey their host to identify and eliminate infected or tumor cells. They are one of the key players in innate immunity and do not need prior activation through antigen recognition to deliver cytotoxic packages and release messenger chemicals to recruit immune cells. Cytohesin associated scaffolding protein (CASP) is a lymphocyte specific adaptor protein that forms complexes with vesicles and sorting proteins including SNX27 and Cytohesin-1. In this study I show that by using stably integrated shRNA, CASP has a direct role in the secretion of messenger cytokines including IFN-γ, and impedes NK cell motility and ability to kill tumor cells. I also show that CASP is post-translationally modified by ubiquitination and cleavage by granzyme B. CASP is an essential and multi-faceted protein, which has a very diverse role in NK cell specific immune functions.

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.

Transient uterine Natural Killer (uNK) cells are the predominant leukocytes of early gestational human and murine uteri. Murine uNK cells promote changes in endometrial structure including initiation of perivascular smooth muscle reduction in spiral arteries. Less is known about human uNK cell functions due to sampling constraints. Xenogeneic engraftment of human lymphocyte progenitors to alymphoid mice has been useful in understanding human lymphocyte functions in vivo. Irradiation of recipients is required to create a niche for successful humanization of the mice but renders recipient mice sterile. The goal of my thesis was to develop a protocol enabling engraftment of human hematopoietic stem cells in alymphoid mice that would permit differentiation of functional human uNK cells. I then planned to evaluate human uNK cell functions and their regulation in vivo. Neonatal Rag2-/-Il2rg-/- mice, which lack T cells, B cells and NK cells were preconditioned with 5-fluorouracil and inoculated with syngeneic mouse bone marrow cells. As adults, inoculated female mice conceived and differentiated functional mouse uNK cells. In contrast, neonatally-preconditioned Rag2-/-Il2rg-/- mice inoculated with human cord blood hematopoietic stem cells conceived but differentiated non-lymphoid cells in sites normally occupied by uNK cells. Weekly injections of human IL-15, which is required for NK cell differentiation, proliferation and survival, did not promote uNK cell differentiation. Rather, treatment with IL-15 altered gestational uteri, even in mice receiving neither preconditioning nor hematopoietic stem cells. I was successful in developing a protocol that enables hematopoietic stem cell engraftment in neonatal mice without compromising mouse fertility. However, this model is apparently not suitable for in vivo studies of human uNK cell functions. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2010-08-30 16:27:07.522

In the present study, we characterized the ability of a novel oncolytic rhabdovirus - Maraba MG1 to boost Natural Killer (NK) cell activity. In tandem, we addressed the ability of this enhanced NK cell functionality to reduce the incidence of post-cancer surgery micrometastases. Due to the potential safety barriers associated with the use of a live virus immediately prior to surgery in cancer patients, we generated a single cycle replication virus (MG1-Gless) and UV-inactivated MG1 to stimulate NK cell function and reduce post-operative metastases. Our in vivo data demonstrate that significant NK cell activation and a similar level of reduction in postoperative tumor metastases was achieved with live MG1, MG1-Gless and UV-inactivated MG1, concluding that viral replication is important, but not necessary for NK cell activation. Mechanistically, we observed that dendritic cells (DCs) are necessary intermediates for MG1-induced NK cell activation. Finally, we characterized and compared a panel of UV-inactivated MG1 (2mins to 2hrs) to better understand the requirements for NK cell activation. Our results suggest that intact viral particle and cellular recognition and association are essential for NK cell mediated anti-tumor responses. These findings provide the preclinical rationale to develop safe and viable virotherapy-based interventional protocols that might reduce the risk of metastatic disease after cancer surgery.

Natural killer (NK) cells, lymphocytes of the innate immune system, can recognize a wide array of cells via several receptors families such as Ly49 and NKR-P1. The Nkrp1 gene family encode for C-type lectin-like receptors which can recognize their ligands, Clr, on target cells. Nkrp1 and Clr genes are intertwined in the NK gene complex and are thus inherited together. The Nkrp1-Clr genes in 129S6 and BALB/c mouse strains show significant sequence polymorphism compared to those of C57BL/6 mice while the overall gene organization and gene number are conserved. RT-PCR was utilized to study the expression of individual Nkrp1-Clr genes. In situ hybridization was performed to validate expression results from RT-PCR, as well as to verify the cell types in which Nkrp1-Clr genes are expressed. Surprisingly, our expression studies reveal an interesting pattern of expression of Nkrp1 and Clr genes not only in lymphoid tissues but also in the epithelial cells of the intestine, kidney, eye and lung, the myocytes of the heart and skeletal muscle, and possibly some endothelial cells, indicating novel functions of NK cells in these tissues.