It is well established that females are more immunocompetent than males as evidenced by higher humoral antibody titers, lowered susceptibility to infection, and more efficient graft rejection. Furthermore, females also exhibit a much higher incidence of autoimmune disease. These observations have led investigators to believe that the male hormonal environment may play a key role in the regulation of immune response. For this reason, this study is concerned with the expression of autoimmunity and of immune function in the mouse. This study included the New Zealand Black (NZB) mouse strain, as an animal model for human SLE, as well as normal DBA/2 and Balb/c strains. Animals were administered testosterone via subcutaneous implants in silastic tubing or by injection. Mice used were intact females, intact males and castrated males. Animals were otherwise untreated or had been exposed to a sublethal dose (400-550 rads) of irradiation. Target organ weight changes, immune capacity and peripheral blood picture changes were measured.

The nervous system and immune system contact one another through two-way communication in order to establish and preserve homeostasis. The sympathetic neurotransmitter norepinephrine has an impact on how the immune system responds by affecting regional blood flow and activation of adrenergic receptors on leukocytes. Former studies showed that immune cells are capable of releasing nerve growth factor allowing for the establishment and continuation of sympathetic nerves in targeted tissues. From this gathered information, it was hypothesized that sympathetic nerves would prove to be less frequent in spleens from the immunodeficient R2G2 mouse strain (Envigo) when compared to 129P3/J (129) and C57BL/6 (C57) strains. R2G2 mice are an immunodeficient strain that lacks functional T, B, and natural killer cells. Ten to eleven week aged-matched male mice were measured by body weight, spleen weight, and temperature. Spleens were cut and fixed for histological investigation. Sympathetic nerves were labeled by immunostaining tyrosine hydroxylase (TH). Hematoxylin & eosin (H&E) was used to stain spleen sections in order to evaluate cytoarchitecture. Von Willebrand factor (VWF) was used to immunostain for megakaryocytes. R2G2 mice showed slightly higher temperatures and body weights but yielded a significantly smaller spleen weight (R2G2, 38.20 ± 1.48; 129, 65.08 ± 11.71; C57, 81.33 ± 8.38; P< 0.0001, ANOVA). TH stain revealed sympathetic innervation in all strains but location and morphology differed in R2G2 mice compared to controls. Control spleens had nerves which entered white pulp regions of the spleen and were closely related to leukocytes. Fiber profiles in the controls were filamentous with small acute bends. R2G2 differed by having (TH+) nerve fibers more associated with arteries and less localized in the surrounding parenchyma. The fibers were abnormally swollen and held a more granular shape instead of a filamentous shape. The H&E stain showed clear red and white pulp zones in the control spleens with 129 showing more distinct germinal centers than C57. R2G2 H&E sections showed cytoarchitecture with indistinct pulp areas. VWF staining revealed R2G2 mice had an abundant amount of megakaryocytes versus control mice megakaryocyte counts (R2G2, 11.28 ± 3.87 per 20X field; 129, 1.73 ± 0.70; C57, 1.42 ± 0.13; P< 0.0001, ANOVA) and extramedullary hematopoiesis was highly prominent. This evidence supports that leukocytes secrete neurotrophic factors or are vital to establishing normal growth of TH+ nerves toward the white pulp. Leukocytes may not be required for sympathetic innervation of blood vessels in the spleen, however, lack of leukocytes shows TH+ nerve fibers with abnormal morphology in severely immune threatened mice.

Influenza is one of the most common diseases worldwide, yet the vaccines against influenza are only 35% effective at protecting against infection. Creating a more effective vaccine requires an understanding of the foundation and the factors that contribute to a strong and protective adaptive immune response. T-bet [TBX21] is a transcription factor that plays an instrumental role in the orchestration of the type 1 immune response, which is the specialized response used by the immune system for a cell-mediated response against intracellular pathogens, such as influenza. It has yet to be explored in an influenza setting on the role T-bet in the production of antibodies. The aim of this study is to understand T-bet's role in production of antibody isotypes and identify whether expression of T-bet is more important for antibody production in T cells or B cells. We expected T-bet knockout (KO) mice to have IgG2a and that T-bet expression would be more important in T cells for antibody production. An enzyme-linked immunosorbent assay (ELISA) was used to measure the amount of virus-specific antibody in T-bet KO versus wild type (WT) mice infected with influenza. The results show that the T-bet KO and WT mice have relatively the same amount of IgG and IgG1, but the T-bet KO have a significantly lower level of IgG2a, confirming T-bet's importance for its production. To distinguish the importance of T-bet expression while T-bet expression in T cells was constant, a model was developed to allow us to control expression of T-bet in B cells. The results however were inconclusive, and the experiment will have to be repeated to make a firm conclusion on the roles of lymphocytes in the control of IgG isotypes. Overall, these results indicate that the manipulation of T-bet expression can be used as a vector to control IgG antibody levels, which holds potential for the improvement of vaccines.

CD4+ T cells play a central role in immunity. During virus infections, CD4+ T cells provide the necessary help for B cells to secrete anti-viral antibody and may act as effector cells themselves through the secretion of anti-viral cytokines such as IFN-γ and TNF-α. Recent studies in the lymphocytic choriomeningitis virus (LCMV) system have shown that CD4+ T cells are required to maintain the clearance of persistent viral infections as well as maintain virus-specific memory CD8+ cytotoxic T lymphocytes (CTL). Despite these important functions, surprisingly little information exists concerning the longevity, magnitude, and stability of the CD4+ T cell response following a virus infection. This thesis takes advantage of the well-studied LCMV system to address the above issues as well as to examine the role CD4+ T cells play during heterologous virus infections and to determine the fate of CD4+T cells following a high-dose LCMV infection. The cell surface phenotype of the CD4+ T cells was first examined in C57BL/6 mice acutely infected with LCMV. FACS analysis revealed the modulation of several activation markers on CD4+ T cells during an acute infection with LCMV, consistent with an activated cell phenotype. In addition, 25% of the CD4+ T cells were blast-sized by day 7 post-infection (p.i.) even though the total number of CD4+ T cells did not increase in the spleen during the acute infection. Additional studies were performed using CZ-1, a novel monoclonal antibody (mAb) previously generated in our laboratory that defines a sialic acid-dependent CD45RB-associated epitope. Examination of the expression of the CZ-1 antigen on CD4+ T cells following LCMV infection revealed that the blast-sized CD4+ T cells at day 6 p.i. were CZ-1 +. Further cell surface phenotyping showed that those blast cells activated at day 6 p.i. were CD45RB1oCD44hiCD62L-. This contrasts with the CZ-1-CD45RBhiCD441oCD62L+ resting cell population prior to infection. To determine if memory CD4+ T cells continued to express the CZ-1 epitope long after resolution of the LCMV infection, CD4+CZ-1+ and CD4+CZ-1- populations were purified by cell sorting and placed into an in vitro proliferation assay with LCMV-infected antigen-presenting cells (APC). It was found that the CD4+CZ-1+ population contained virtually all of the virus-specific memory. Thus, these studies indicate that the CZ-1 epitope defines a novel activation and memory marker for murine CD4+T cells. Examination of virus-specific cytokine production using ELISPOT assays showed a significant increase in the number of IFN-γ-secreting cells in the spleen during an acute LCMV-infection. CD8+ T cells made up the majority of the IFN-γ-producing cells, but analysis of the cell culture supernatants by ELISA revealed that the CD4+T cells produced more IFN-γ on a per cell basis. No significant increase in IL-4 levels was detected under these experimental conditions. These data suggest that LCMV infection induces primarily a virus-specific Th1 response that is characterized by increased IFN-γ production. No quantitative information was known about the frequency and longevity of the LCMV-specific CD4+ T cell response. Using limiting dilution assays (LDA), I examined the CD4+ T cell precursor (Thp) frequency in C57BL/6 mice infected with LCMV. The virus-specific CD4+ Thp frequency increased from <1/100,000 in uninfected mice to a peak of approximately 1/600 in FACS-purified splenic CD4+ T cell populations by 10 days p.i. with LCMV. After the peak of the response, the CD4+ Thp frequency decreased only about 2-fold per CD4+ T cell to approximately 1/1200 and remained stable into long-term memory. The CD4+ Thp frequency to each of the two known LCMV major histocompatibility complex (MHC) class II-restricted peptides dropped only 2- to 7-fold from the peak of the acute LCMV response into long-term memory. Thus, the CD4+T cell frequencies remain elevated after the acute infection subsides and remain extremely stable throughout long-term immunity. The above results show that LDA can account for +T cells as being virus-specific following LCMV infection. However, using newer, more sensitive assays based on intracellular cytokine production, >20% of the CD4+ T cells secreted IFN-γ after stimulation with phorbol myristic acid and ionomycin during the peak of the acute CD4+ T cell response. In addition, >10% of the CD4+ T cells secreted IFN-γ after stimulation with the LCMV MHC class II-restricted CD4 peptides. Thus, these new sensitive assays reveal a heretofore unappreciated, yet profound antigen-specific CD4+T cell response during LCMV infection. Infection of mice with a series of unrelated viruses, termed heterologous viruses, causes the reduction of memory CD8+ T cells specific to earlier infections. In order to examine the fate of CD4+ T cells under these conditions, I examined cytokine production and followed the CD4+ Thp frequency following heterologous virus infections. Challenge of LCMV-immune mice with vaccinia virus (VV) resulted in a significant increase in both the amount of IFN-γ protein and the frequency of IFN-γ-producing cells in the peritoneal cavity 3 days after infection as compared to control non-immune mice acutely infected with VV or to LCMV-immune mice alone. Intracellular IFN-γ staining revealed that both CD4+ and CD8+ T cells contributed to this increased IFN-γ production. LDA analysis of the LCMV-specific CD4+ Thp frequency following multiple heterologous virus infections or protein antigen immunizations, revealed that the CD4+ Thp frequency remains stable even under conditions that reduce the LCMV-specific CD8+ CTLp frequency. Additional studies using high-dose LCMV Clone 13 demonstrated that, like CD8+ T cells, there is a decline in detectable LCMV-specific CD4+Thp during overwhelming virus infections. The data presented in this thesis help provide a better understanding of the CD4+ T cell response during virus infections. I make several novel observations, including the demonstration that mAb CZ-1 defines a novel activation and memory marker for CD4+ T cells, that the LCMV-specific memory CD4+ Thp frequency remains extremely stable into long-term immunity, and that heterologous virus infections do not disturb the stable memory CD4+ T cell pool following a virus infection. I also provide data using new sensitive assays based on intracellular cytokine production that there is a much more profound antigen-specific CD4+ T cell response during viral infections than has previously been realized. Finally, I provide evidence that the virus-specific CD4+ T cells become unresponsive following a high-dose LCMV Clone 13 infection. Thus, the data presented in this thesis highlight some important similarities and differences between the CD4+ and CD8+ T cell responses during acute viral infections.

Glia, whose name derives from the original Greek word, meaning “glue,” have long been understood to be cells that play an important functional role in the nutritive and structural support of the central nervous system, yet their full involvement has been historically undervalued. Despite the strong evidence that glial reactions to cellular debris govern the health of the nervous system, the specific properties of damaged axonal debris and the mechanisms by which glia sense them, morphologically adapt to their presence, and initiate phagocytosis for clearance, have remained poorly understood. The work presented in this thesis was aimed at addressing this fundamental gap in our understanding of the role for glia in neurodegenerative processes. I demonstrate that the cellular machinery responsible for the phagocytosis of apoptotic cell corpses is well conserved from worms to mammals. Draper is a key component of the glial response machinery and I am able to show here, for the first time, that it signals through Drosophila Shark, a non-receptor tyrosine kinase similar to mammalian Syk and Zap-70. Shark binds Draper through an immunoreceptor tyrosine-based activation motif (ITAM) in the Draper intracellular domain. I show that Shark activity is essential for Draper-mediated signaling events in vivo, including the recruitment of glial membranes to axons undergoing Wallerian degeneration. I further show that the Src family kinase (SFK) Src42A can markedly increase Draper phosphorylation and is essential for glial phagocytic activity. Therefore I propose that ligand-dependent Draper receptor activation initiates the Src42A-dependent tyrosine phosphorylation of Draper, the association of Shark and the subsequent downstream activation of the Draper pathway. I observed that these Draper-Src42A-Shark interactions are strikingly similar to mammalian immunoreceptor-SFK-Syk signaling events in myeloid and lymphoid cells. Thus, Draper appears to be an ancient immunoreceptor with an extracellular domain tuned to modified-self antigens and an intracellular domain that promotes phagocytosis through an ITAM domain-SFK-Syk-mediated signaling cascade. I have further identified the Drosophila guanine-nucleotide exchange factor (GEF) complex Crk/Mbc/dCed-12, and the small GTPase Rac1 as novel modulators of glial clearance of axonal debris. I am able to demonstrate that Crk/Mbc/dCed-12 and Rac1 function in a non-redundant fashion with the Draper pathway to promote a distinct step in the clearance of axonal debris. Whereas Draper signaling is required early during glial responses, promoting glial activation and extension of glial membranes to degenerating axons, the Crk/Mbc/dCed-12 complex functions at later stages of glial response, promoting the actual phagocytosis of axonal debris. Finally, many interesting mutants have been identified in primary screens for genes active in neurons that are required for axon fragmentation or clearance by glia, and genes potentially active in glia that orchestrate clearance of fragmented axons. The further characterization of these genes will likely unlock the mystery surrounding “eat me” and “find me” cues hypothesized to be released or exposed by neurons undergoing degeneration. Illuminating these important glial pathways could lead to a novel therapeutic approach to brain trauma or other neurodegenerative conditions by providing a druggable means of inducing early attenuation of the glial response to injury down to levels less damaging to the brain. Taken together, my combined work identifies new components of the glial engulfment machinery and shows that glial activation, phagocytosis of axonal debris, and the termination of glial responses to injury are genetically separable events mediated by distinct signaling pathways.

This thesis proposes a mechanism for the induction of peripheral tolerance to protein antigens. I have investigated the mechanism of tolerance induction to soluble protein antigens by targeting an antigen to small, resting B cells. For this purpose I have used a rabbit antibody directed at the IgD molecule found on the surface of most small, resting B cells but missing or lowered on activated B cells. Intravenous injection of normal mice with 100 μg of an ultracentrifuged Fab fragment of rabbit anti-mouse IgD (Fab anti-δ) makes these mice profoundly tolerant to challenge with nonimmune rabbit Fab (Fab NRG) fragments. This tolerance is antigen specific since treated mice make normal responses to an irrelevant antigen, chicken immunoglobulin (Ig). Fab fragments of rabbit Ig (rabbit Fab) not targeted to B cells do not induce tolerance as well as Fab anti-δ. Evidence suggests that the B cells must remain in a resting state for tolerance to be induced, since injection of F(ab)'2 anti-δ does not induce tolerance. Investigation of the mechanisms of the tolerance, by adoptive transfer, have shown that rabbit Fab specific B cell function has been impaired. The major effect however is in helper T cell function, as shown by adoptive transfer and lack of help for a hapten response. In vitro proliferation experiments show that the T cell response has not been shifted toward activation of different T cell subsets which do not help Ig production, nor is there any change in the Ig isotypes produced. Suppression does not appear to be the major cause of the helper T cell defect as shown by cell mixing experiments. This work shows that an antigen targeted to small B cells can induce tolerance to a soluble protein antigen, and suggests a role for small B cells in tolerance to self-proteins not presented in the thymus.

Neonatal mice have immature immune systems with defects in several components of inflammatory, innate, and specific immune responses and develop a preferential T helper type 2 (Th2) response following immunization with many vaccine antigens. Although maternal antibody is the major form of protection from disease in early life when the neonatal immune system is still immature, the presence of maternal antibody also interferes with active immunization, placing infants at risk for severe bacterial and viral infection. Recent studies have suggested that immunizing with DNA plasmids encoding the vaccine antigen of interest is highly efficacious in a variety of adult animal models. However, similar extensive studies have not been conducted in infants. In this dissertation, we examine both the quantitative and qualitative differences between neonatal and adult humoral and cell-mediated immune responses in the presence or absence of maternal antibody. First, we wished to determine if one-day-old neonatal mice immunized with plasmid DNA expressing influenza A/PR/8/34 hemagglutinin (HA) by either intramuscular (i.m.) or gene gun (g.g.) inoculation were capable of generating humoral responses comparable to those in mice immunized as adults. We found that newborn mice developed stable, long-lived, protective anti-HA-specific IgG responses similar in titer to those of adult DNA-immunized mice. However, unlike the adult i.m. and g.g. DNA immunizations, which develop polarized IgG2a and IgG1 responses, respectively, mice immunized as neonates developed a variety of IgG1-, IgG2a-, and mixed IgG1/IgG2a responses regardless of the inoculation method. Boosting increased, but did not change these antibody profiles. We also found that, in contrast to the DNA immunizations, inoculations of newborn mice with an A/PR/8/34 viral protein subunit preparation failed to elicit an antibody response. Further, temporal studies revealed that both responsiveness to protein vaccination and development of polarized patterns of T help following DNA immunization appeared by 2 weeks of age. To determine if the disparity of polarized IgG responses between neonatal and adult DNA vaccinated mice was due to deficiencies in Th1 promoting cytokines, we addressed the ability of DNA encoding Th1 cytokines to bias the isotype of antibody raised by neonatal DNA immunization. We found that neonatal mice coimmunized with HA and either IL-12 or IFNγ-expressing DNAs developed IgG2a-biased immune responses, regardless of inoculation method, whereas these DNAs had no effect on IgG subtype patterns in adult DNA immunized mice. Consistent with the Th1-promoting effects of these cytokines, we also observed that codelivery of IL-12 or IFNγ DNAs raised T helper responses toward Th1 in mice immunized both as neonates or adults. Thus, codelivery of cytokine DNAs may be effective at tailoring immune responses depending on the required correlates of protection for a given pathogen. Finally, we addressed the effect of maternal antibody on the elicitation of humoral and cell-mediated immune responses. We tested the ability of i.m. and g.g. immunization with DNA expressing influenza HA and/or nucleoprotein (NP) to raise protective humoral and cellular responses in the presence and absence of maternal antibody. We found that neonatal mice born to influenza-immune mothers raised full antibody responses to NP but failed to generate antibody responses to HA. In contrast, the presence of maternal antibody did not affect the generation of long-lived CD4+ and CD8+ T cell responses to both HA and NP. Thus, maternal antibody did not affect cell-mediated responses, but rather it limited humoral responses, with the ability to limit the antibody response correlating with whether the DNA-expressed immunogen was localized in the plasma membrane or within the cell. We further observed that protection from influenza virus challenge was dependent on the presence of anti-HA IgG and was independent of the presence T cell responses. Taken together with other published studies, the data presented in this dissertation help better characterize the responses elicited by DNA vaccines at birth. This dissertation presents several novel observations including the temporal development of polarized IgG subtype responses, the ability of codelivered Th1 cytokine DNA to affect both antibody and T cell responses in the neonate, and the ability to generate humoral responses to intracellular, but not plasma membrane proteins, in the presence of maternal antibody. Furthermore, the data provides rationale for further development of DNA vaccines in the neonate.

The overall aim of this thesis was to determine how natural killer (NK) cells regulate virus infections in vivo. Anti-viral mechanisms by which NK cells control murine cytomegalovirus (MCMV) infection in the spleens and livers of adult C57BL/6 mice were first studied, revealing different mechanisms of control in different organs. Three days post-infection, MCMV titers in the spleens of perforin-deficient (perforin 0/0) mice were higher than in wild type controls, but no elevation of liver titers was found in perforin 0/0 mice. NK cell depletion in MCMV-infected perforin 0/0 mice resulted only in an increase in liver viral titers but not in spleen titers. Depletion of IFN-γ in adult C57BL/6 mice by injections with mAbs to IFN-γ resulted in an increase in viral titers in the liver but not in the spleen. Analyses using IFN-γ-receptor-deficient (IFN-γR0/0) mice, rendered chimeric with C57BL/6 bone marrow cells, indicated that even though the donor spleen cells could respond to IFN-γ, the depletion of NK cells in a recipient environment where the host cells could not respond to IFN-γ caused an increase in MCMV titers in the spleens but had little effect in the liver. IFN-γ has the ability to induce a variety of cells to produce nitric oxide (NO), and administrating the nitric oxide synthase (NOS) inhibitor Nω-monomethyl-L-arginine (L-NMA) into MCMV-infected adult C57BL/6 mice resulted in MCMV titer increases in the liver but not in the spleen. These data indicate that in adult C57BL/6 mice, there is a dichotomy in the mechanisms utilized by NK cells in the regulation of MCMV in different organs. In the spleen NK cells exert their effects in a perforin-dependent manner, suggesting a cytotoxic mechanism, whereas in the liver the production of IFN-γ by NK cells may be a predominant mechanism in the regulation of MCMV synthesis. These results may explain why the Cmv-1r (Cmv-1-resistant) locus, which maps closely to genes regulating NK cell cytotoxic function, confers an NK cell-dependent resistance to MCMV infection in the spleen but not in the liver. The ability of adoptively transferred cells to protect suckling mice from MCMV was another model used to study the mechanisms utilized by NK cells in the regulation of MCMV. Adoptive transfers of 129, C57BL/6 and perforin 0/0 spleen cells or lymphokine-activated killer (LAK) cells into 4 - 6 day old MCMV-infected C57BL/6 suckling mice significantly lowered the splenic MCMV titers in these mice compared to the infected controls. Adoptive transfers of C57BL/6 spleen cells into MCMV-infected 129 suckling mice also decreased the amount of MCMV in the 129 suckling mice, but C57BL/6 spleen cells could not regulate MCMV synthesis when adoptively transferred into 129/IFN-γR0/0 suckling mice. These results suggest that, in the suckling mouse model, the regulation of MCMV by the adoptively transferred NK cells is via an IFN-γ-dependent, perforin-independent, Cmv-1-independent mechanism. The Cmv-1 gene locus resides within the NK gene complex, in close proximity to the Ly49 NK cell receptor family. Analyses were carried out to determine if any of the 4 known Ly49 NK cell receptors (Ly49A, C, D and G2) played a role in the control of MCMV synthesis by NK cells. Studies comparing the expression of the different Ly49 NK cell subsets in the spleen and the peritoneal cavity revealed that there were differences in the distribution of the Ly49 receptors on NK1.1+ cells. Three days post-MCMV infection, the percentage of NK1.1+- Ly49+ NK cells in the spleen and the peritoneal cavity were different than in naive controls. Within the splenic NK1.1+ population, increases in NK1.1+ -Ly49A+ and NK1.1+-Ly49G2+ cells but decreases in NK1.1+-Ly49C+ and NK1.1+-Ly49D+ cells were observed. These changes in the spleen were accompanied by a concomitant decrease in NK1.1+ - Ly49A+ cells and increases in NK1.1+-Ly49C+, NK1.1+-Ly49D+ and NK1.1+-Ly49G2+ cells within the NK1.1+ population in the peritoneal cavity. These data suggest that 3 days post-MCMV infection, there may be movement of NK cells between the different organs. The role of Ly49 NK cell receptors in the regulation of MCMV was tested using adult C57BL/6 mice depleted of single or multiple Ly49 NK cell subsets. These in vivo depletions did not affect the ability of the residual NK cells to regulate MCMV synthesis. LAK cells sorted into the different Ly49 NK cell subsets and adoptively transferred into C57BL/6 suckling mice lowered the splenic MCMV titers in these mice. Together, these results indicate that even though there is a redistribution of the Ly49 NK cell subsets during MCMV infection, the presence or absence of anyone of the 4 tested Ly49 NK cell receptors does not affect the regulation of MCMV by NK cells. However, there remain a possibility that one of the undefined Ly49 receptors or an untested NK cell receptor may be important in the control ofMCMV. Most of the cloned NK cell receptors have been shown to bind to MHC class I molecules, and MHC class I antigens have been implicated as modulators of target cell sensitivity to NK cell-mediated lysis. The regulation of virus infections and the fate of NK cells and their natural targets was examined in β2-microglobulin-deficient mice [β2m (-/-)], which have defective MHC class I expression. Infections with either the NK cell-sensitive MCMV or the NK cell-resistant lymphocytic choriomeningitis virus (LCMV) significantly augmented NK cell activity in either C57BL/6 or β2m (-/-) mice. Depletion of NK cells in vivo with antiserum to asialo GM1 markedly enhanced the synthesis of MCMV but had no effect on the synthesis of LCMV in either strain of mouse. Adoptively transferred β2m (-/-) spleen cells lowered splenic MCMV titers in C57BL/6 suckling mice, not unlike adoptively transferred C57BL/6 spleen cells. Analysis of naturally NK cell-sensitive thymocyte targets from these virus-infected β2m (-/-) mice revealed no cell surface expression of class I MHC detectable by conformation-dependent or -independent antibodies, but the virus infections enhanced class I expression on thymocytes from C57BL/6 mice. The sensitivity of C57BL/6 thymocytes to NK cell-mediated lysis was markedly reduced after in vivo poly inosinic:cytidylic (poly I:C) treatment or viral infection; in contrast, the sensitivity of the β2m (-/-) thymocytes was significantly less affected by poly I:C or viral infection. These data indicate that the normal expression of MHC class I antigens on NK cells or their targets is not required for the anti-viral functions of NK cells against an NK-sensitive virus (MCMV) nor do they protect an NK-resistant virus (LCMV) from the anti-viral activity of NK cells. Together, the data presented in this thesis help to further our understanding of the mechanisms utilized by NK cells in the control ofMCMV in both adult and suckling mice, and also help clarify the roles played by Ly49 NK cell receptors and MHC class I molecules in the regulation of MCMV.

RNA interference (RNAi) in the nematode Caenorhabditis elegans is a type of homology-dependent post-transcriptional gene silencing induced by dsRNA. This dissertation describes the genetic analysis of the RNA interference pathway and inheritance properties associated with this phenomenon. We demonstrate that the RNAi effect can be observed in the progeny of the injected animal for at least two generations. Transmission of the interference effect occurs through a dominant extragenic agent. The wild-type activities of the RNAi pathway genes rde-l and rde-4 are required for the formation of this interfering agent but are not needed for interference thereafter. In contrast, the rde-2 and mut-7 genes are required downstream for interference. These findings provide evidence for germline transmission of an extragenic sequence-specific silencing factor and implicate rde-l and rde-4in the formation of the inherited agent. Other forms of homology-dependent silencing in C. elegansinclude co-suppression and transcriptional silencing of transgenes in the germline. We demonstrate that silencing of a germline transgene can be initiated by injected dsRNA, via the RNAi pathway, and then maintained on a different level. This observation indicates that post-transcriptional and transcriptional silencing of homologous genes could be connected. This dissertation also describes the connection between RNAi and developmental pathways of gene regulation in C. elegans. We show that inactivation of genes related to RNAi pathway genes, a homolog of Drosophila Dicer (dcr-l), and two homologs of rde-1 (alg-l and alg-2) cause heterochronic phenotypes similar to lin-4 and let-7 mutations. Further we show that dcr-l, alg-l, and alg-2 are necessary for the maturation and activity of the lin-4 and let-7small temporal RNAs that regulate stage-specific development. Our findings suggest that a common processing machinery generates guide RNAs that mediate both RNAi and endogenous gene regulation. Finally, this study illustrates the detection of small interfering RNAs (siRNAs), intermediates in the RNAi process, and describes requirements for their accumulation. We show that, in the course of RNAi induced by feeding dsRNA, C. elegans accumulate only siRNAs complementary to the target gene. This accumulation depends on the presence of the target sequence and requires activities of several RNAi-pathway genes. We show that selective retention or amplification of RNAi-active molecules can create a reservoir of memory antisense siRNAs that prevent future expression of the genes with complementary sequence. This suggests a parallel at the molecular level with the clonal selection of antibody forming cells and in the vertebrate immune system.

Hebert and Carruthers (1992) showed that the human erythrocyte glucose transporter is an allosteric complex of four GLUT1 proteins whose structure and substrate binding properties are stabilized by reductant-sensitive noncovalent subunit interactions. The GLUT1 tetramer dissociates into dimers upon exposure to reductant but subunits are not associated via disulfide bridges. Each subunit of SDS-denatured tetrameric GLUT1 exposes only two thiols while reduced denatured GLUT1 exposes all six sulfhydryl groups. They hypothesized that glucose transporter oligomeric structure and cooperative catalytic function resulted from noncovalent subunit interactions promoted or stabilized by intramolecular disulfide bridges. These interactions give rise to an antiparallel arrangement of substrate binding sites within the transporter complex. In the present studies, we tested aspects of this model. Specifically, we wanted 1) to understand why the native, noncovalent, homotetrameric GLUT1 complex is sensitive to reductant, 2) to determine whether the tetramer is more catalytically efficient than the dimer in situ, and 3) to test the hypothesis that it is the antiparallel arrangement of substrate binding sites between subunits that provides the transporter with its catalytic advantage. We used biochemical and molecular biological approaches to isolate specific determinants of transporter oligomeric structure and/or transport function in purified isolated transporter preparations, in intact red cells and in CHO cells. We have also examined the hypothesis that net sugar transport in the human erythrocyte is rate limited by reduced cytosolic diffusion of sugars and/or by reversible sugar association with intracellular macromolecules. Our findings support the hypothesis that each subunit of the parental glucose transporter contains a single intramolecular disulfide bridge located between cysteine residues 347 and 421. This disulfide seems to be necessary for GLUT1 tetramerization. Our findings suggest that GLUT1 N-terminal residues 1 through 199 provide contact surfaces for subunit dimerization but are insufficient for subunit tetramerization. Our studies also show that in situ disulfide disruption by cell impermeant reductants results in the loss of cooperative subunit interactions and a 3 to 15-fold reduction in the transport efficiency of the transporter. We further find that in situ GLUT1 is susceptible to exofacial proteolysis. Exofacial trypsin cleavage eliminates cooperativity between subunits but does not affect transporter oligomeric structure or transport activity. Thus catalytic efficiency does not derive directly from cooperative interactions between substrate binding sites on adjacent subunits. We have confirmed that 30MG transport in human erythrocytes is a diffusion limited process. We find that steady-state sugar uptake in red cells and K562 cells measures two processes - sugar translocation and intracellular sugar binding. We propose a model for native GLUT1 structure and function.