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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.

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Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 112-126).

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

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

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

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.