Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes a life-long latent infection of B cells. It is usually asymptomatic in healthy individuals; however, individuals with compromised immunity often develop EBV-induced lymphoma. EBV also encodes potential oncogenes that can contribute to tumorigenesis. Therefore, vaccine and immunotherapeutic strategies targeting EBV are desirable. Recent studies have shown that infusion of EBV-specific CD8+T cells can elicit remission of lymphomas arising after administration of immunosuppressive drugs during transplantation, suggesting an important role for T cells in the prevention of EBV-induced malignancy. A better understanding of the cellular immune components involved in the control of EBV will aid in the development of methods to prevent infection and/or treat EBV-associated disease. While EBV infection is usually acquired asymptomatically during childhood, primary infection of adolescents and young adults can result in an illness termed acute infectious mononucleosis (AIM). Because of the characteristic symptoms of the illness, individuals with AIM can be readily identified and diagnosed with acute EBV infection. Thus, primary CD4+ and CD8+ T cell responses against the virus can be evaluated. It has been previously found that there is a marked expansion of lytic EBV protein-specific CD8+ T cells early during AIM, with delayed detection of lower frequencies of latent EBV protein-specific CD8+ T cells. The magnitude and specificity of CD4+T cell responses during AIM has been less well characterized. This thesis dissertation presents data from both functional assays and direct staining experiments documenting the timing, magnitude, and antigen-specificity of CD4+ T cells over the course of primary EBV infection. Lytic and latent protein-specific CD4+ T cells were readily detected by intracellular IFN-γ production at presentation with AIM and declined rapidly thereafter. Blood EBV load was also quantitated and found to decrease over time following AIM. By contrast, CD8+T cell IFN-y responses remained high for several weeks following presentation with AIM. Direct staining of lytic epitope-specific CD4+ T cells during AIM revealed high frequencies of virus-specific cells with low proliferative and IFN-γ-producing potential. Blood EBV load in these patients was persistently high through 6 wk following AIM. These data suggest a relationship between high EBV load during acute infection and impaired EBV-specific CD4+ T cell responses, which are compatible with impaired CD4+ T cell responses reported during high viremia associated with other viral infections. This may represent a mechanism by which persistent viruses, such as EBV, are able to establish a life-long infection in their hosts.

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.

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.

There are four serotypes of dengue virus designated dengue 1, 2, 3 and 4 (D1, D2, D3 and D4) and epidemiological studies indicate that a severe complication of dengue virus infection - dengue hemorrhagic fever (DHF) is more likely to occur following a secondary infection. DHF is hypothesized to be immunologically mediated and may be triggered by virus-specific T cells. It is also likely that dengue virus-specific cytotoxic T lymphocytes (CTLs) are important for recovery from dengue virus infections. An analysis of the immune response during acute illness and when the patient has recovered from the infection (immune state) is therefore important as it will provide insights into the immunopathological nature of the disease. This thesis initially examines the CD8+CTL responses in volunteers who have received live attenuated dengue vaccines and then investigates acute and immune T cell responses in children following natural infection with dengue. When this project was initiated, there was little available information on the human CD8+ T cell responses to dengue viruses. PBMC from one donor had generated memory CD8+CTL to the nonstructural protein NS3 of dengue virus. Memory CD8+CTL responses were therefore analyzed to determine the diversity of the T cell response to dengue virus and to identify immunodominant proteins using PBMC from eight healthy adult American volunteers who had received monovalent live-attenuated candidate vaccines of the 4 dengue serotypes. All the donors had specific T cell proliferation to dengue viruses and to other flaviviruses that we tested. CTLs were generated from the stimulated PBMC of all donors and in the seven donors tested, dengue virus-specific CD8+CTL activity was demonstrated. The nonstructural proteins NS3 and NS1.2a and the structural protein E were recognized by CD8+CTLs from six, five and three donors respectively. All donors recognized either NS3 or NS 1.2a. In a donor who received a dengue 4 vaccine, CTL killing was seen in bulk culture against the premembrane protein (prM). This is the first demonstration of a CTL response against the prM protein. The CTL responses using PBMC of two donors were serotype-specific whereas all other donors had serotype-cross reactive responses. For one donor, CTLs specific for E, NSl.2a and NS3 proteins were all HLA-B44 restricted. For the three other donors tested the potential restricting alleles for recognition of NS3 were HLA-B38, A24 and/or B62 and B35. These results indicate that the CD8+CTL responses of humans after immunization with a single serotype of dengue virus are diverse and directed against a variety of proteins. The nonstructural proteins NS3 and NSl.2a appear to be immunodominant and should be considered when designing subunit vaccines for dengue. Previously T cell responses had not been examined in people who have had natural infections with dengue. The HLA diversity between North American Caucasians and populations where dengue is a serious health problem, calls for the analysis of immune responses in people who have been infected with natural circulating strains of the virus. We examined the memory cytotoxic T lymphocytic (CTL) responses of peripheral blood mononuclear cells (PBMC) obtained from patients in Thailand 12 months after natural symptomatic secondary dengue infections. In all four patients analyzed, CTLs were detected in bulk culture PBMC against nonstructural dengue proteins. Numerous CD4+ and CD8+ CTL lines were generated from the bulk cultures of two patients, KPP94-037 and KPP94-024, which were specific for the NSl.2a and NS3 proteins respectively. All CTL lines derived from both patients were crossreactive with other serotypes of dengue virus. The CD8+ NS1.2a specific lines from patient KPP94-037 were HLA-B57 restricted and the CD8+ NS3 specific lines from patient KPP94-024 were HLA-B7 restricted. The CD4+ CTL lines from patient KPP94-037 were HLA-DR7 restricted. A majority of the CD8+CTLs isolated from patient KPP94-024 were found to recognize a.a. 221-232 on NS3. These results demonstrate that after symptomatic secondary natural dengue infections in Thai patients, CTLs are mainly directed against nonstructural proteins and are broadly crossreactive. The data correlate with our observations that nonstructural proteins are immunodominant proteins in volunteers who received dengue vaccines. We were interested in examining CTL responses in children during their acute illness and comparing them to memory CTLs obtained from the same children a year or more after the infection. A detailed analysis on samples from nine patients during their acute illness failed to generate any dengue virus-specific CTL responses. We therefore decided to determine if cell mediated responses are altered during acute dengue infection. Decreased proliferative responses to mitogens and recall antigens have been observed in PBMC obtained during several acute human viral infections. All responses of PBMC during acute illness were compared to the same patients PBMC obtained at least 6 months after their infection. Proliferative responses to PHA, anti-CD3, tetanus toxoid and dengue antigens were significantly decreased in PBMC obtained during the acute infection. The proliferative responses to PHA were restored by the addition of gamma-irradiated autologous immune or allogeneic PBMC. Cell contact with the irradiated PBMC was necessary to restore proliferation. Non-T cells from the acute PBMC of dengue patients did not support proliferation of T cells from control donors in response to PHA, but T cells from the PBMC of patients with acute dengue proliferated if accessory cells from a control donor were present. Addition of anti-CD28 antibodies restored anti-CD3-induced proliferation of the PBMC of some patients. The percentage of monocytes was reduced in the acute sample of PBMC of the dengue patients. Addition of IL-2 or IL-7, but not IL-4 or IL-12 also restored proliferation of acute PBMC stimulated with anti-CD3. The results demonstrate that both quantitative and qualitative defects in the accessory cell population during acute dengue illness result in a depression of in vitro T cell proliferation. The data generated from this project shed light on the nature of the immune responses during acute natural dengue infections. It strengthens the existing data on the human memory CD8+CTL responses to dengue viruses and validates the observations by examining memory CTL responses after natural dengue infection in patients from Thailand. In addition, we demonstrate a profound defect in lymphoproliferative responses during dengue illness.

The immunological mechanisms involved in virus-induced hepatitis were examined by measuring the cytotoxic capabilities and the morphological and antigenic phenotypes of leukocytes isolated from the livers of virus-infected mice. Large granular lymphocytes (LGL) of both natural killer (NK) cell and cytotoxic T lymphocyte (CTL) phenoytpes [phenotypes] accumulated in livers of mice infected with lymphocytic choriomeningitis virus (LCMV) of either the nonhepatotropic Armstrong strain (LCMV-ARM) or the hepatotropic WE strain (LCMV-WE). NK cell activity and LGL number increased 3- to 4-fold between days 1 and 5 postinfection (p.i.). These LGL were characterized as NK cells on the basis of cell surface antigens, kinetics of appearance, target cell range, and morphology. By day 7 p.i., virus-specific, H-2-restricted, Thy-1+, Lyt-2+CTL activity was present in the liver, and its appearance correlated with a second wave of LGL accumulation. Total CTL activity, leukocyte numbers, and CTL/LGL numbers were at least 5-fold higher in the livers of LCMV-WE-infected mice than in the livers of LCMV-ARM-infected mice. Mice infected with the cytopathic viruses, mouse hepatitis virus and murine cytomegalovirus, experienced greater increases in NK/LGL by day 3 p.i. than did mice either infected with LCMV or injected with poly I:C. The early and late accumulations of LGL in the virus-infected liver were associated with the appearance of two waves of LGL with blast cell morphology expressing the phenotypes of NK cells and CTL, respectively. Thus, the organ-associated accumulation, blastogenesis, and in situ proliferation of cytotoxic LGL provide a means for the localization and site-specific augmentation of a host's cell-mediated antiviral defenses. The mechanism of inhibition of virus synthesis in vivo by immune splenocytes containing virus-specific CTL was examined in mice dually infected with two different viruses and then adoptively immunized with spleen cells immune to one of the two viruses. Only the titer of the virus to which the splenocytes were immune was reduced in titer, and no nonspecific antiviral effect was seen on the titer of the 'bystander' heterologous virus. These data are consistent with an in vivo mechanism of CTL-mediated antiviral resistance involving direct cytotoxicity rather than release and dissemination of antigen-nonspecific antiviral factors, such as interferon, following recognition of appropriate viral antigen.

Background Monoclonal Gammopathy of Undetermined Significance (MGUS) is an understudied precursor of multiple myeloma (MM), the second most prevalent hematologic malignancy in the United States. This dissertation was designed to: (1) Describe the trajectories of serum biomarkers over time in patients with an MGUS diagnosis, (2) Determine if an MGUS diagnosis is associated with changes in healthcare service utilization, and (3) explore the patient- and provider-level drivers of healthcare utilization in patients with MGUS. Methods Data sources include health claims and electronic health records from a community-based population of patients seeking care in central Massachusetts and primary qualitative data collected from providers and patients’ interviews. The analyses included descriptive statistics, group-based trajectory modeling, conditional Poisson regression, and qualitative data analyses. Results (1) Three distinct multi-trajectory groups of creatinine and hemoglobin were identified. (2) The rates of emergency room, hospital, and outpatient visits were higher for patients with MGUS than patients without MGUS. (3) Patients have a basic understanding of MGUS; however, some patients feel anxiety, which may affect other aspects of their lives. Patients primarily see hematologists for follow-up care; other providers have less knowledge about MGUS. Conclusions Biomarker trajectories characterize specific subpopulations of patients with MGUS over time. We found that an MGUS diagnosis is associated with higher healthcare utilization, especially during the months surrounding the diagnosis date. Finally, our study suggests that some patients with MGUS may need psychosocial support services and identifies a gap in knowledge around caring for MGUS patients among primary care providers.

The immune system generates discrete lineages of cells that are designed to respond optimally to environmental cues and infectious agents. Two distinct lineages of T cells, distinguished by expression of either an αβ or γδ T cell receptor (TCR), arise from a common progenitor in the thymus. The type of pathogen and the cytokine milieu directs effector differentiation of αβ T cells in the periphery through the induction of specific transcriptional networks. γδ T cell development is distinct from that of αβ T cells in its ordered rearrangement of TCR genes and the pairing of Vγ and Vδ chains to generate γδ T cell subsets that home to specific tissues. Unlike conventional αβ T cells, γδ T cells express a preactivated or memory phenotype prior to pathogen encounter, and recent evidence indicates that effector functions may be programmed during thymic development. To better understand the development and function of γδ T cells, we analyzed the gene expression profiles of subsets of γδ T cells segregated by TCR repertoire and maturation state in the thymus. We also determined the impact of TCR signaling and trans-conditioning on γδ T cell subset-specific gene signatures by analysis of Itk-/- and Tcrb-/- γδ T cell subsets. Our analysis has defined three stages of γδ T cell subset-specific differentiation, and indicates that γδ T cells may consist of at least two separate lineages, distinguished by the expression of a Vγ2 or Vγ1.1 TCR, that arise from different precursors during thymic development. Key transcriptional networks are established in immature γδ T cells during the first phase of development, independent of TCR signaling and trans-conditioning, with Vγ2+ cells expressing modulators of WNT signaling, and Vγ1.1+ cells expressing high levels of inhibitor of DNA binding 3 (ID3), which regulates E2A/HEB proteins. The second stage involves the further specification of the Vγ2+ subset specific gene signature, which is dependent upon ITK-mediated signals. In the third stage, terminal maturation of γδ T cell subsets occurs, dependent on both TCR and trans-conditioning signals. The expression patterns of Vγ1.1+ subsets that differ in Vδ usage diverge, and all subsets further elaborate and reinforce their effector programming by the distinct expression of chemokine and cytokine receptors. Alteration of WNT signaling or E2A/HEB activity results in subset specific defects in effector programming, indicating that the transcriptional networks established at the immature stage are crucial for the functional maturation of γδ T cells. These data provide a new picture of γδ T cell development, regulated by multiple checkpoints that shape the acquisition of subset-specific molecular signatures and effector functions.