Uterine Natural Killer cells are the major lymphocyte population in the pregnant uterus in early gestation; outnumbering both T and B cells. The numerical expansion of uterine Natural Killer cells is thought to result from the expansion of preexisting progenitor cells resident to the uterus, recruitment of Natural Killer cells from the circulation, or a combination of both pathways. Uterine Natural Killer cells are capable of cytotoxic killing and express receptors that can recognize foreign paternal antigens. Therefore, it has been argued that uterine Natural Killer cell activation can lead to killing of fetal cells and abortion. However, fetal rejection by uterine Natural Killer cells does not occur in normal pregnancies and other functions for uterine Natural Killer cells have been proposed. These include: the regulation of maternal blood supply responsible for providing oxygen to the fetus, regulation of maternal blood pressure and, in species with invasive placentation, regulation of decidualization, the process of endometrial cell expansion and transformation during the menstrual cycle and during pregnancy. These cell functions juxtapose the concept that uterine Natural Killer cell activation is harmful to the fetus and offer a new perspective that uterine Natural Killer cells regulate functions unrelated to traditional transplantation immunology. In this dissertation, work is presented showing that uterine Natural Killer cells express molecules which regulate blood pressure and decidualization. Also presented are data supporting the hypothesis that the numerical increase of uterine Natural Killer cells is due to the recruitment of Natural Killer cells from the blood. These results support roles for uterine Natural Killer cells other than cytotoxic killing and advance the understanding of uterine Natural Killer cells as dynamic players that support pregnancy-associated biological processes unrelated to traditional understandings of immune surveillance. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-12-15 11:33:37.11

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.

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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.

Although structurally unrelated, the human killer cell immunoglobulin-like (KIR) and the rodent lectin-like Ly49 receptors serve similar functional roles in natural killer (NK) cells. Moreover, both gene families display variegated and mostly mono-allelic expression patterns established at the transcriptional level. DNA methylation, but not histone modifications, has recently been shown to play an important role in maintenance of the expression patterns of KIR genes but the potential role of DNA methylation in the expression of Ly49 genes was unknown. My thesis focuses on the role of epigenetic modifications, especially DNA methylation, in the maintenance of mouse Ly49 gene expression. I show that hypomethylation of the region encompassing the main promoter of Ly49a and Ly49c in primary C57BL/6 (B6) mouse NK cells correlates with expression of these genes. Using B6 x BALB/c Fl hybrid mice, I demonstrate that the expressed allele of Ly49a is hypomethylated while the non-expressed allele is heavily methylated, indicating a role for epigenetics in maintaining mono-allelic Ly49 gene expression. Furthermore, the Ly49a promoter region is heavily methylated in fetal NK cells but variably methylated in non-lymphoid tissues. In apparent contrast to the KIR genes, I show that histone acetylation state of the promoter region strictly correlate with Ly49A and Ly49G expression status. Also, the instability of Ly49G expression on some lymphoid cell lines is at least in part due to changes in the level of histone acetylation of the promoter region. As for the activating Ly49 receptors, it seems that although DNA methylation levels of the promoter regions do correlate with the state of expression of these receptors, the pattern of DNA methylation is different from that of the inhibitory Ly49a and c genes. In conclusion, my results support a role for epigenetic mechanisms in the maintenance of Ly49 expression. Moreover, these epigenetic mechanisms appear to vary among the Ly49 genes and also differ from those governing KIR expression.