Browsing by Subject "Natural killer cells"
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Item For Better or for Worse: The Influence of NK Cells, IFN-y and IL-4 on the Development of Protective Adaptive Immunity in Mycoplasma Respiratory Disease(2008-08-01) Bodhankar, Sheetal; Porunelloor A. Mathew; Stephen R. Grant; Rance E. BergSheetal Bodhankar, For better or for worse: The influence of NK cells, IFN-y and IL-4 on the generation of protective adaptive immunity in mycoplasma respiratory disease, Doctor of Philosophy (Microbiology and Immunology) August 2008, 136 pp., 13 illustrations, 3 tables, bibliography, 176 titles. The purpose of these studies was to evaluate the contribution of NK cells and the polarizing cytokines, IFN-y and IL-4, in the generation of protective adaptive immunity against mycoplasma infection. Presence of NK cells during the generation of adaptive immunity resulted in detrimental immune responses. However, upon depletion of NK cells, prior to nasal-pulmonary immunizations, mice demonstrated better clearance of mycoplasma from the respiratory tracts. That the NK cells hindered with the beneficial development of adaptive immune responses via lymphoid cells was demonstrated, since no protection was demonstrated in SCID mice. Furthermore, purified pulmonary T and B lymphocytes primed in a NK cell depleted environment as opposed to one’s primed in a versus non-depleted environment could transfer protection to naïve mice. Interestingly, this is the first time that a favorable role of functional CD4 T cells in mediating protection in mycoplasma respiratory disease was demonstrated. The presence of NK cells at the time of nasal-pulmonary immunization also modulated mycoplasma-specific IFN-y and IL-4 responses in lungs and lower respiratory nodes. In evaluating the roles of IFN-y and IL-4, it was demonstrated that the absence of a single cytokine alters a vast array of chemokines and cytokines produced in response to mycoplasma infection. Corresponding to the higher numbers of mycoplasma and severity in disease due to the loss of IFN-y, altered cytokine and chemokine responses in the lungs to mycoplasma infection were demonstrated. Nasal-pulmonary immunization of IFN-y mice exacerbated, rather than reduced, mycoplasma disease and infection, whereas immunization of IL-4 mice significantly enhanced protection along the respiratory tract particularly in the lungs. Prominent Th-2 type immune responses in the lungs of IFN-y mice corresponded to the severe immunopathologic reactions developed after mycoplasma infection and immunization. These studies demonstrated diverse but crucial functions for NK cells, IFN-y and IL-4 vital towards the development of protective adaptive immune responses against mycoplasma respiratory infection that will have a significant impact on future studies on respiratory immunology.Item Molecular Basis of Cancer Cell Recognition and Killing by Human Natural Killer Cells(2001-05-01) Boles, Kent S.; Bennett, Michale; Leudtke, Robert; Goldfarb, RonaldNatural Killer (NK) cells are a population of lymphocytes vital for the innate immune response. These cells protect the host during the early phase of infection before the adaptive immune response is effective. NK cells are direct effectors via cytoxicity towards neoplastic and infected cells. Additionally, they modulate the immune response by the production of cytokines, most notably interferon y and tumor necrosis factor a. Furthermore, NK cell receptors do not undergo rearrangement. Repetoires of activating and inhibitory receptors regulate the function of NK cells via a balance of signaling. NK cell receptors can broadly be divided by their ligand specificity as well. Most of the known receptors recognize MHC class I molecules and transduce inhibitory signals. This is the basis for the missing self hypothesis espoused by Karre and colleagues. The LY49 molecules server this function in the mouse and are related to the C-type lectins. In primates, a family of killer inhibitory receptors (KIR) appear to play the same role and are in the immunoglobulin (Ig) superfamily of receptors. Whether humans expressed the Ly49 receptors was a fundamental question in NK cell biology. In my attempt to address this issue, I isolated two receptors related to the C-type lectin receptors and localized to the human NK gene complex on chromosome 12 in a region syntenic to where the murine Ly49 genes reside. Functional characterization of these receptors will facilitate our understanding of NK cell biology. Additional activating receptors include the members of the CD2 subset of the immunoglobulin superfamily molecules expressed on NK cells and other leukocytes, including murine 2B4. 2B4 is the high affinity ligand for CD48. Engagement of 2B4 on NK cell surfaces with specific antibodies or CD 48 can trigger cell mediated cytotoxicity, IFN-y secretion, phosphoinositol turnover and NK cell invasiveness. This work describes the isolation and characterization of the human homologue of the 2B4 receptor. The putative peptide has a type I structure with one transmembrane domain. The extracellular region is comprised of two immunoglobulin like domains with six putative N-linked glycosylation sites. The cytoplasmic domain of 2B4 contains unique tyrosine motifs (TxYxxV/I) that associate with src homology 2 domain containing protein (SH2DIA) or signaling lymphocyte activation molecule (SLAM)-associate protein (SAP), whose mutation is the underlying genetic defect in the X-linked lymphoproliferative disease (XLPD). Impaired signaling via 2B4 and SLAM is implicated in the immunopathogenesis of XLPD. CS1 is a novel member of the CD2 subset that contains two of the unique tyrosine motifs present in 2B4 and SLAM. Signaling through 2B4, CS1 and other members of the CD2 subset may play a major role in the regulation of NK cells and other leukocyte functions.Item Molecular Regulation of Interferon Gamma in 2B4-Activated Natural Killer Cells: Functional Role in Tumor Rejection(2001-11-01) Johnson, Lori Ann; Mathew, Porunellor A.; Goldfarb, Ronald H.; Dimitrijevich, S. DanNatural killer cells are a third population of lymphocytes, distinct from T and B cells. NK cells are non-MHC-restricted cytotoxic effector cells which are effective against intracellular pathogens, virally-infected cells and tumor cells. 2B4 is a natural killer cell receptor originally identified in the mouse as a surface molecule involved in non-MHC-restricted killing and enhancement of IFN-γ secretion. The human and rat homologues of 2B4 have recently been cloned in our laboratory. Interferon gamma (IFN-γ) is a cytokine with potent anti-viral and anti-proliferative effects. In addition, this cytokine acts as a global immune regulator by regulating gene expression and serving to attract other immune cells. In this work, we establish the function of human 2B4 in a NK cell line, YT. We have shown that human 2B4 activation induces cytolytic function and enhances IFN-γ release in YT cells. Additionally we show that 2B4’s regulation of IFN-γ occurs at the transcriptional level, both through mRNA stability and increased promoter activity. We also demonstrate that several regions in the IFN-γ promoter respond to 2B4 activation and IFN-γ both separately and together in the rejection of metastatictumor cells in C57B7/6 mice. Our results confirm that both 2B4 and IFN-γ are critical in the rejection of metastatic tumor cells. Through the use of activating monoclonal antibodies, our studies indicate that 2B4’s anti-tumor activity is through IFN-γ as well as through cytolytic function of NK cells.Item Signaling in Natural Killer Cells: NK Cell Activation by LLT1 Receptor(2008-05-01) Bambard, Nowland D.; Jerry Simecka; Richard Easom; Harlan JonesBambard, Nowland D., Signaling in Natural Killer Cells: NK Cell Activation by LLT1 Receptor. Doctor of Philosophy (Microbiology and Immunology), May 2008, 162 pp., 1 table, 30 illustrations, bibliography, 179 titles. Natural Killer (NK) cells are large granular lymphocytes of the innate immune system that constitute the first line of defense against viral pathogens and cancer. Unlink cells of the adaptive immune response, NK cells do not recognize specific antigens expressed on MHC receptors, rather they recognize tumorgenic and virally infected cells through a complex balance of activating and inhibiting receptors expressed on the surface of human NK cells. LLT1 is expressed on numerous immune cells and subsequent functional analysis indicates that LLT1 plays an activating role on NK cells by way of stimulating interferon-gamma (IFN-G) secretion. LLT1 has also been shown to have a role on non-immune cells, inhibiting the formation and function of osteoclasts. Additionally, the natural ligand of LLT1 has been identified as NKR-P1A (CD161), an NK cell inhibitory receptor known to play an important role in immune regulation. We hypothesize that LLT1 employs multiple signaling pathways to accomplish its activating functions on human NK cells, and may be associated with one of four known transmembrane accessory proteins associated with NK cell activating receptors. We activated LLT1 on NK92 cells with target cells expressing its natural ligand CD161 and analyzing IFN-G production in the presence of pharmacological inhibitors specific for various signaling mechanisms. These results indicate that LLT1 employs Src-PTK, p38 and ERK signaling pathways, but not PKC, P13K or calcineurin. These results were followed up with phosphorylation analysis, which confirmed that the ERK signaling pathway is associated with LLT1 IFN-G production. Finally, by analyzing IFN-G mRNA we found that LLT1 activation is not associated with any detectable change in IFN-G mRNA levels, suggesting that LLT1 stimulates NK IFN-G production by modulating post transcriptional or translational events. Identification of the signaling pathways associated with LLT1 is of great medical significance as this may provide us with novel insights into activating NK cells to counter infection and cancer.Item uPAR Interaction and Regulation of Natural Killer Cell Integrins: Implications for the Modulation of NK Cell Migration and Invasion(2003-05-01) Gellert, Ginelle C.; Goldfarb, Ronald H.; Roque, Rouel; Hart, MarkGellert, Ginelle C. uPAR Interaction and Regulation of Natural Killer Cell Integrins: Implications for the Modulation of NK Cell Migration and Invasion. Doctor of Philosophy (Biomedical Sciences), May 2003; pp. 118, 2 tables; 12 figures; bibliography 163. The urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored receptor, devoid of an intracellular domain, but nevertheless initiates signaling, possibly through lateral interactions with integrins. Since adoptively transferred interleuking-2 (IL-2) activated natural killer (A-NK) cells can accumulate within established cancer metastases, these A-NK cells may integrate components of adhesion and proteolysis to facilitate their infiltration into tumors. The work in this dissertation investigates the hypothesis that uPAR directly interacts with and regulates the expression of integrins on the surface of NK cells in the potential modulation of NK cell migration and invasion. Crosslinking studies have revealed a relationship between the integrins and uPAR on the surface of the human NK cell line, YT. Crosslinking uPAR, which mimics uPAR clustering at focal adhesion sites, caused an increase in the expression of the αM, αv and β2 integrins. Although uPAR is GPI-linked to the plasma membrane and has no direct means of initiating intracellular signaling, crosslinking uPAR activated the MEK/ERK signaling cascade, as phosphorylation of both MEK ½ and ERK ½ occurred following receptor clustering. The MEK-specific inhibitors PD98059 and U0126 blocked MAP kinase phosphorylation, and PD98059 inhibited the increase in integrin expression induced by uPAR crosslinking. Furthermore, the binding of urokinase plasminogen activator (uPA) to uPAR also activated the MEK/ERK signaling pathway. Fluorescence microscopy revealed the cocapping of uPAR with the αv integrin, a process inhibited N-acetyl-D-glucosamine, which abrogates the lectin-like interactions that have been suggested to exist between uPAR and integrins. The work presented herein indicated that signaling initiated either by uPAR crosslinking, leading to increased integrin surface expression, or by uPAR occupancy with uPA may depend on the physical association of uPAR with integrins. These studies will enhance our understanding of the mechanisms utilized by NK cells for their adhesion to tumor vasculature and accumulation within established cancer metastases, thereby potentially identifying targets for enhancing their effectiveness during adoptive immunotherapy.