Browsing by Subject "Killer Cells, Natural"
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Item Antigen-Specific Natural Killer Cell Responses in Chronic Hepatitis C Virus Infection(2014-02-04) Bowman, Kathryn; Holz, Lauren; Rehermann, BarbaraChronic hepatitis C virus (HCV) infection results in an inflammatory liver disease leading to fibrosis and cirrhosis. The progression of liver disease is thought to be immune-mediated because HCV itself is non-cytopathic. Given that HCV-specific T cells are diminished in number and functionally exhausted in chronic HCV infection, it remains unclear which cell population drives disease pathogenesis. Here, we investigated the function of natural killer (NK) cells, the major innate immune cell population in the liver. The NK cell population increases further in the setting of chronic hepatitis C infection and have multiple mechanisms of cytotoxicity. We investigated whether NK cells could respond to HCV in an antigen-specific manner. PBMCs from 39 patients with chronic HCV infection (gt 1) not recently on medication (>2 years) were stimulated for 8 hours in a whole blood activation assay with pools of overlapping 18-mer peptides comprising HCV structural (E1, E2) and nonstructural (NS3) proteins. Cytokine production by NK cells and T cells was assessed by multicolor flow cytometry. The frequency of IFN-γ+ NK cells was 5 fold greater than the frequency of IFN-γ+ T cells. A minority of IFN-γ+ NK cells co-produced TNF-α. NK cell responses to HCV peptides varied between subjects, but did not correlate with T cell responses or viremia. This study demonstrates that NK cells are activated in an antigen-specific manner in chronic HCV infection and respond to both structural and nonstructural HCV proteins. Natural killer cell cytokine and cytotoxic responses were larger than corresponding T cell responses. The mechanism of antigen-specific NK cell activation is currently under investigation.Item Biochemical Characterization of IpaH E3 Ubiquitin Ligase Effector Proteins and Their Host Substrates(August 2021) Hansen, Justin Mark; Orth, Kim; Sperandio, Vanessa; Reese, Michael L.; Alto, NealShigella flexneri is a gram negative pathogen that utilizes its type 3 secretion system (T3SS) to inject effector proteins in the cytoplasm of host cells to manipulate host cells processes. T3SS effectors are able to post translationally modify host proteins to reprogram intracellular signaling pathways, actin dynamics, membrane trafficking, and innate immune pathways. This allows Shigella to modify the intracellular environment to be conducive to bacterial replication and dissemination to neighboring cells. Shigella flexneri and other bacteria including Salmonella and Yersinia secreted E3 ubiquitin ligases into the host cell cytoplasm via the Type III secretion system (T3SS) apparatus. The invasion plasmid antigen Hs (IpaHs) are a novel family of bacterial E3 ubiquitin ligases that are secreted by Shigella, Salmonella, and Yersinia. These bacterial enzymes highjack the host ubiquitin conjugation machinery by binding to ubiquitin-charged E2 conjugating enzymes and facilitating direct transfer of ubiquitin onto host substrates. IpaH effectors induce polyubiquitination and subsequent proteasomal degradation of their substrates during bacterial infection. The effector substrate interaction of IpaH1.4/2/5 and HOIP was previously characterized. I went on to identify that IpaH2.5 is able to inhibit the in vitro catalytic activity of HOIP via mono-ubiquitination of catalytic lysine residues in the HOIP ring-between-ring domain (RBR-C). Subsequent to this Ubiquitin activated interactive trapping (UBAIT) screening was then utilized to identify the host substrate of IpaH7.8, Gasdermin B (GSDMB). GSDMB belongs to a large family of pore forming cytolysins that execute inflammatory cell death programs. While genetic studies have linked GSDMB polymorphisms to inflammatory disease, its function in human physiology remains poorly understood. I investigated a previously unrecognized host-pathogen conflict between GSDMB and the IpaH7.8 effector protein encoded by Shigella flexneri. Through extensive biochemical and cellular characterization, I show that IpaH7.8 ubiquitinates and targets GSDMB for proteasome destruction. This virulence strategy protects Shigella from the bacteriocidic activity of Natural Killer cells by suppressing Granzyme-A mediated activation of GSDMB. In contrast to the canonical function of most Gasdermin-family members, GSDMB does not inhibit Shigella by lysing infected cells. Rather, GSDMB exhibits direct microbiocidal activity through recognition of phospholipids found on Gram-negative bacterial membranes. These findings place GSDMB as a central executioner of intracellular bacterial killing and reveals a mechanism employed by pathogens to counteract this host defense system.Item Natural Killer Cell Responses in Chronic Hepatitis C Virus Infection(2014-05-01) Bowman, Kathryn Anne; Rehermann, Barbara; Lee, William M.; Thiele, Dwain L.BACKGROUND: Hepatitis C virus (HCV) has infected 170 million people worldwide and is a leading cause of chronic viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. Liver injury and disease progression in hepatitis C virus infection is driven by the host immune response. Previous research has largely focused on the contribution of virus-specific T cells to this process. However, in chronic HCV infection, T cells are functionally exhausted and do not control viremia. Thus, it is likely that immune cells other than HCV-specific T cells contribute to disease pathogenesis. Natural killer (NK) cells constitute the major innate immune cell population in the liver. They are capable of producing cytokines and utilize multiple mechanisms of cytotoxicity. The high prevalence of NK cells in the liver, along with their potential for viral specificity, suggested we study NK cell function in the setting of chronic hepatitis C infection. An improved understanding of the endogenous mechanisms that regulate liver inflammation and disease pathogenesis in chronic hepatitis C infection may result in a new focus for therapeutic options to decrease the rate of disease progression in individuals who are unable to clear infection with antiviral agents. OBJECTIVE: Here we sought to characterize whether natural killer cells from individuals chronically-infected with hepatitis C virus respond in chronic HCV infection and to compare the frequencies of HCV-specific NK cell responses with corresponding virus-specific T cell responses. METHODS: We tested the NK cell function of chronically HCV-infected patients not currently on antiviral treatment using a whole blood activation assay. In this assay, heparin-anticoagulated whole blood from 39 anti-HCV positive HCV patients and 10 healthy blood donors was stimulated for 8 hours with pools of overlapping 18 amino acid peptides (OLPs) comprising the HCV envelope (E1, E2) and nonstructural (NS3) sequences. Specificity controls consisted of a pool of overlapping hepatitis D virus peptides and samples without peptide addition. As a readout for NK and T cell function, the frequency of IFNγ+ NK cells and T cells was assessed by multicolor flow cytometry. In addition, cytotoxicity was assessed by the frequency of NK or T cells expressing CD107a, a marker of cellular degranulation. Variance between responses in HCV-infected versus healthy donors and variance between NK versus T cell responses were analyzed by matched two-way ANOVA with multiple comparisons and Bonferroni post-test using Graphpad Prism software. RESULTS: This study demonstrates that NK cells from chronically HCV-infected individuals are activated in HCV infection in a whole blood peptide stimulation assay with both structural and nonstructural HCV proteins, including E1 and E2 envelope proteins and NS3 nonstructural protein. NK cells secrete cytokines assessed here by release of IFNγ, as well as NK cell degranulation, and degranulated, assessed by CD107a expression. As expected, there were minimal NK cell or T cell responses in healthy donor blood. NK cell cytokine responses were 4-6 times greater than HCV-specific T cell cytokine responses. CONCLUSION: This study demonstrates that NK cell cytokine production and degranulation is greater than that of corresponding T cells. Further research is needed to characterize the mechanism of these NK cell responses to understand antiviral mechanisms and factors influencing disease progression.Item NK Cell Function and Tumor Resistance in Mice Transgenic for Antibody to NK Inhibitory Receptors(2005-05-03) Gandhi, Namita Anikumar; Bennett, MichaelTumor surveillance has been proposed as a means whereby the immune system monitors and eliminates transformed cells before their growth. Transformed cells that survive the immune response are escape variants selected by nature as they have developed mutations in immune recognition components. To boost immune response to these tumors, several types of immunotherapies are being studied but so far have had minimal success when translated into patient studies. Among proposed immunotherapeutic approaches, monoclonal antibody treatments have shown the best efficacy in human clinical trials. NK cells, cytolytic effector cells of the innate immune system, are implicated in tumor surveillance. Inhibitory Ly49 receptors determine the specificity of murine NK cells by recognizing of MHC class I molecules expressed on the target cell. This allows the transmission of inhibitory signals through intracellular signals to block NK cytotoxicity. Many tumors express sufficient levels of self MHC class I and are able to escape lysis by NK cells. Our lab has been studying the inhibitory regulatory pathways in natural killer cells and has developed an approach for enhancing the ability of NK cells to kill tumor cells. We have focused on studying the inhibitory function of the murine Ly49 receptors and provided evidence that blocking of negative signals on two inhibitory receptors, Ly49C and I, with a monoclonal antibody (5E6), allow NK cells to kill syngeneic leukemia cells more efficiently providing an enhanced anti-tumor effect. To study further the effect of Ly49C/I receptor blockade and improve tumor rejection, we developed a transgenic model whereby the 5E6 Fab antibody fragments are constitutively secreted to allow the sustained blockade of the Ly49C/I receptor. These studies detail the generation of these Tg mice and their characterization in relation to NK and T cell receptor development, tolerance, autoimmunity and tumor surveillance. In addition, we demonstrated an effect of blocking inhibitory receptors on NK cells to delay tumor establishment in a nascent tumor model of murine chronic myelogenous leukemia.Item Role of Nlc Cells in Murine Models of T Cell-Dependent Responses(2007-12-17) Jennings, Paula Alessandra; Yuan, DorothyNK cells are part of the innate immune system, yet they can also modulate the acquired immune response. Activated NK cells, for instance, can increase antigen specific IgG2a production in response to T independent responses, mostly through IFN-gamma secretion. Previous experiments examining the effect of NK cells on T cell- dependent antigens in various laboratories have yielded inconsistent conclusions. Therefore attempts were made to further investigate this question. Whereas depletion of NK cells had no detectable effect on the response to a TD antigen in Ribi adjuvant injected intraperitoneally, the secondary IgG1 response can be significantly reduced. This result suggested a role for NK cells on the generation of memory T cells. Therefore experiments were initiated to investigate the effect of NK cells on T cell proliferation. The absence of NK cells during immunization was found to reduce primary T cell proliferation. Such effect was not observed when B cell antigen presentation was absent, which is known to be important for memory T cell generation. A direct effect of NK cells on B cell antigen presentation was assessed in vitro. These experiments showed that NK cells can upregulate B cell antigen presentation of ovalbumin to na?T cells expressing a transgene specific to the ovalbumin derived peptide, OVAp. This increase is contact dependent and can occur in the absence of IFN-gamma . Moreover, the NK cell enhancement of B cell presentation of intact protein was greater than the presentation of OVAp, which requires no processing. These experiments suggest that the upregulation involves both processing and presentation of antigen. These experiments show that NK cells have a direct effect on B cell antigen presentation and provide a mechanistic basis for the role of NK cells in modulating in vivo T cell- dependent antibody responses.Item The Role of Serine Protease Inhibitors in Regulating Hepatotoxicity During Viral Infection(2007-05-23) Stout-Delgado, Heather Winona; Thiele, Dwain L.Major hepatitis viruses, such as HBV and HCV, are not directly cytopathic; instead liver injury is due to a vigorous immune response. Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells kill virally infected and malignant cells by two major pathways, the perforin/granzyme pathway and the Fas and/or TNF death receptor pathways. In contrast to other target cells, virally infected hepatocytes are resistant to killing by perforin and granzyme-dependent cytotoxic effector pathways. This results in a more prominent role for Fas and TNFR-mediated killing of infected hepatocytes and clearance of hepatic viral infections. Human proteinase inhibitor 9 (PI-9/ serpinB9) and the murine orthologue, serine proteinase inhibitor 6 (SPI-6/ serpinb9) are members of a family of intracellular serine proteinase inhibitors (serpins). PI-9 and SPI-6 expression in immune-privileged cells, antigen-presenting cells, and cytotoxic T cells protects these cells against the actions of granzyme B and when expressed in tumor cells, confers resistance to killing by CTL and NK cells. Thus, one potential explanation for hepatocyte resistance to the perforin/granzyme pathway would be expression of PI-9/ SPI-6 within human or murine liver cells. The present studies were designed to assess whether factors present during hepatic viral infections as well as the associated antiviral immune responses, might induce the expression of SPI-6 in murine liver and thus, confer resistance of virally infected hepatocytes to perforin and granzyme B dependent hepatotoxicity. To this extent, we examined SPI-6 expression after IFN-alpha treatment and during in vivo adenoviral infection of the liver. To detect changes in SPI-6 gene expression, mRNA and cytosolic protein was isolated from liver and real time PCR and western blotting were performed. The results indicated that SPI-6 is the only PI-9/serpinB9 homologue that is significantly up-regulated in liver post IFN-alpha stimulation or during the course of viral infection. Increased SPI-6 gene expression during viral infection correlated with influxes of NK cells and CTL, as reflected by increased lymphocyte surface receptor mRNA levels within the liver. Additional experiments using virally infected, genetically altered mice unable to process or express granzyme B indicated that SPI-6 is a cytoprotective gene that is selectively up-regulated by hepatocytes in response to activated granzyme B. Knockdown of SPI-6 gene expression in vivo by hydrodynamic injection of siRNA specifically targeting SPI-6 resulted in a significant increase in serum alanine amino transferase (ALT) levels, a measure of hepatocellular injury, during early time points after viral infection. In addition, inhibition of SPI-6 resulted in accelerated clearance of AdCMV-LacZ encoded transgene products from the liver. However, expression during high dose AdCMV-LacZ infection resulted in early onset of lethal, acute liver failure. Given these results, we conclude that up-regulation of SPI-6 gene expression in hepatocytes protects against perforin/ granzyme B mediated killing during hepatic viral infection and thereby slows the rate of immune elimination of virally infected hepatocytes.Item The Roles of Major Histocompatibility Complex Class I and Foxk1 in Natural Killer Cell Development(2007-12-18) Moody, Leslie Ann; Bennett, MichaelPathways leading to the development of functionally mature Natural Killer cells from bone marrow progenitors are incompletely characterized. Several reports have indicated the necessity of class I Major Histocompatibility Complex-Ly49 interactions to generate functionally mature Natural Killer cells. Natural Killer cells from mice deficient in Major Histocompatibility Complex class I exhibit impaired lytic ability against class I+and class I- targets. It has been proposed that class I interactions with inhibitory Ly49s are required for generation of lytic Natural Killer cells; cells that do not receive these signals fail to become activated. To investigate further the role of class I-Natural Killer cell interactions during development, we produced chimeric mice using class I- mice, in which the hematopoietic system was derived from class I-expressing mice. We discovered that class I+ Natural Killer cells that are developed in a class I- environment are not functional, despite the presence of class I on hematopoietic cells. This indicates that the environment in which Natural Killer cells are developed determines their function and further supports the role of the bone marrow microenvironment in Natural Killer cell development. A complete understanding of Natural Killer cell development would involve determining which transcription factors drive development of Natural Killer cells from stem cells to mature, functional Natural Killer cells. Several transcription factors have been described to be necessary for Natural Killer cell development. Mice lacking these transcription factors often have a deficit in Natural Killer cells in vivo. Here we illustrate a role for the forkhead transcription factor, Foxk1, in Natural Killer cell development. Foxk1-/- mice have significantly fewer Natural Killer cells than do wild-type mice and their remaining Natural Killer cells have decreased cytotoxicity. An increase in the percentage of cells in a developmentally important expansion stage indicates that Foxk1 acts there. However, Foxk1 seems to play no role in the thymic development of Natural Killer cells; cells with phenotypic characteristics of thymus-derived Natural Killer cells are present in Foxk1-/- mice. Our studies show a clear role for Major Histocompatibility Complex class I and Foxk1 in the development of functionally mature Natural Killer cells in mice.Item [Southwestern News](2003-06-09) Maier, Scott