Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disorder associated with the Philadelphia chromosome (Ph) that arises from a reciprocal translocation between chromosomes 9 and 22, thereby resulting in the formation of the chimeric BCR-ABL oncogene encoding a constitutively activated tyrosine kinase. BCR-ABL tyrosine kinase inhibitors (TKIs) induce a complete hematologic and cytogenetic response in the majority of chronic phrase CML patients. However, TKIs cannot efficiently eradicate leukemia stem cells (LSCs) because of the insensitivity of LSCs to TKIs. Therefore, developing new strategies to target LSCs is necessary and critical for curing CML, and success of this approach depends on further understanding the molecular mechanisms by which LSCs survive and are maintained. In Chapter I, I briefly introduce CML disease, BCR-ABL oncoprotein, and TKIs. I also describe the identification and features of LSCs. Several key pathways in LSCs including Wnt/ß-catenin, hedgehog, FoxO, Bcl6 and HIF1, are discussed. I also propose our strategy to identify unique molecular pathways that are important for LSCs but not their normal stem cell counterparts. In Chapter II, I describe our finding about the function of the positive regulator, HIF1α, in CML development and LSC survival. I show that loss of HIF1α impairs the maintenance of CML through impairing cell cycle progression and inducing apoptosis of LSCs, and I also report that p16Ink4a and p19Arf mediate the effect of HIF1α on LSCs, as knockdown of p16Ink4a and p19Arf rescues the defective colony-forming ability of HIF1α-/- LSCs. As detailed in Chapter III and IV, through comparing the global gene expression profiles of LSCs and HSCs, I find two novel regulators, Blk and Scd1, which act as tumor suppressors in CML development. In Chapter III, I show that Blk is markedly down-regulated by BCR-ABL in LSCs, and that c-Myc and Pax5 mediate this down-regulation. Deletion of Blk accelerates CML development; conversely, Blk overexpression significantly delays the development of CML and impairs the function of LSCs. I also demonstrate that p27, as a downstream effector, is involved in the function of Blk in LSCs. Blk also functions as a tumor suppressor in human CML stem cells, and inhibits the colony-forming ability of human CML cells. In Chapter IV, I investigate the function of another negative regulator, Scd1, in CML LSCs, and find that expression of Scd1 is down-regulated in mouse LSCs and human CML cells. We report that Scd1 acts as a tumor suppressor in CML, as loss of Scd1 causes acceleration of CML development and overexpression of Scd1 delays CML development. Using a colony-forming assay, I demonstrate that Scd1 impairs the maintenance of LSCs due to the change of expression of Pten, p53 and Bcl2. Importantly, I find that both Blk and Scd1 do not affect normal hematopoietic stem cells (HSCs) or hematopoiesis. Taken together, our findings demonstrate that HIF1α is required for the maintenance of CML LSCs, and conversely that Blk and Scd1 suppress the function of LSCs, suggesting that combining TKI treatment with specific targeting of LSCs will be necessary for curing CML.

High frequencies of CTL recognizing laboratory strains of HIV-1 are present in HIV-1 infected adults as early as preseroconversion. The presence of HIV-1 specific CTL during primary infection has been correlated with better control of early viremia and a more delayed onset of CD4 lymphocyte loss. Previous experiments in our laboratory have demonstrated that, unlike HIV-1 infected adults, the majority of vertically infected infants lack CTL which recognize laboratory strains of HIV-1 within the first year of life. ADCC antibody responses against laboratory strains of HIV-1 env gene products are also delayed until at least two years of age. As a possible correlate, disease progression is also more rapid in vertically infected infants. We hypothesized that HIV-1-specific CTL are type-specific in early infancy and that the use of target cells expressing laboratory strain gene products might limit the detection of HIV-1-specific CTL. To address this hypothesis, HIV-1 env genes from early isolates of four infants were PCR amplified, cloned, and used to generate recombinant vaccinia vectors (vv). The frequencies of CTL precursors (CTLp) recognizing env gene products from autologous isolates and the IIIB strain of HIV-1 were measured at time points from early infancy to 19 months using limiting dilution analysis (LDA). ADCC titers were also measured against autologous and IIIB env gene products at 4 time points spanning 2 months to 2 years of age. CTL precursors from 3 of 4 of these patients were specific only for autologous HIV-1 env gene products during the first 6 to 12 months of age. A pattern of CTL responsiveness was observed in these 3 patients in which type-specific CTL precursors observed in early infancy were replaced by cross-reactive, group-specific CTL by 6 to 12 months of age. CTL precursors from a fourth patient at 12 months of age recognized IIIB env and 1 out of 2 envs derived from 2 autologous viral isolates. High titers titers of ADCC antibodies against autologous env were detected in two infants prior to the detection of ADCC antibodies to IIIB. In two other infants, group specific ADCC antibody responses were detected in late infancy. Our results demonstrate that young infants can mount HIV-1 specific CTL and ADCC responses. The ability of young infants to mount cellular immune responses to HIV-1 also provides support for the concept of perinatal vaccination to prevent HIV-1 transmission. Furthermore. the lack of broadly-reactive CTL in early infancy suggests that the use of vaccines based on laboratory strains of HIV-1 may not afford protection from vertical infection.

Hantaviruses are members of the virus family Bunyaviridaethat cause two potentially life-threatening diseases in humans: hemorrhagic fever with renal syndrome (HFRS) and hantavirus pulmonary syndrome (BPS). HFRS is caused by Old World hantaviruses that are endemic in many Asian and European countries. Infections with Old World hantaviruses can range in severity from asymptomatic to moderate or severe, depending primarily on the infecting serotype of virus. HPS is caused by New World hantaviruses in North and South America. New World hantaviruses are rarely asymptomatic and are severe in the majority of cases. These syndromes are distinct from one another in the primary target organ of virus infection (kidney vs. lung), but have important clinical features in common, including fever, thrombocytopenia, and a capillary leak syndrome. These common clinical manifestations suggest that the underlying mechanisms of disease may be similar in the two syndromes. The precise mechanisms of pathogenesis of HFRS and HPS are poorly characterized, but may be mediated in part by immunopathology. Hantaviruses are able to establish infections in many human cell types, including primary human endothelial cells, without having any cytopathic effect on these cells. Human infections with hantavirus result in a robust activation of the humoral and cellular immune response, and we hypothesize that these immune responses contribute to the pathology of disease. Evidence for the activation of T lymphocytes, and their potential involvement in immunopathology, includes increases in the number of circulating, activated CD8+ T cells during HFRS, the presence of lymphocytic infiltrates (predominantly CD8+T cells) in kidney biopsies from patients with acute HFRS, and associations between certain HLA haplotype and disease severity following hantavirus infection. This thesis is the first examination of human T lymphocyte responses that are generated during HFRS. Initially, we studied memory T cell responses in scientists who were sub-clinically infected with Hantaan virus (HTNV), the prototype hantavirus. We later investigated memory T cell responses in healthy Finnish adults who had HFRS caused by Puumala virus (PUUV), a hantavirus endemic primarily in Scandinavia. At the onset of these studies, there was no available information on human T lymphocyte responses to Old World hantaviruses. Virus-specific CD8+ and CD4+human T cell lines had been isolated from patients with acute HPS caused by Sin Nombre virus (SNV) infection. In that study, conducted in our laboratory, several human T cell epitopes on the nucleocapsid (N) protein and G2 envelope glycoprotein of SNV were identified and characterized. We decided to perform similar analyses on PBMC from donors who had been infected with HTNV and PUUV, in order to determine the specificity and diversity of the T cell response to Old World hantaviruses. The initial study of three donors who had sub-clinical infections with HTNV demonstrated that virus-specific T cell responses could be detected in all the donors following in vitro stimulation of PBMC with inactivated virus. In two of the donors, the virus-specific cytolytic T cells (CTL) recognized the HTNV N protein, and in the third donor the virus-specific CTLs recognized the HTNV G1 glycoprotein. Isolation and characterization of virus-specific T cells from two donors resulted in the identification of two CD8+ T cell epitopes on the HTNV N protein, which were restricted by either HLA A1 or B51. These CTL lines included both HTNV-specific (HLA B51-restricted) and serotype-cross reactive (HLA A1 restricted) lines. In one subject, these virus-specific T cell responses were detectable in IFN-γ ELISPOT assays following peptide stimulation, and in bulk cultures after short-term stimulation with inactivated HTNV. These results indicated that the CD8+CTL responses of humans after sub-clinical infection with HTNV were readily detectable and were directed against a limited number of viral proteins and epitopes. In addition, sub-clinical infection resulted in the generation of both virus-specific and cross-reactive CTL responses. We reasoned that hantavirus infections that lead to clinical illness may result in the generation of more robust and/or diverse virus-specific T cell responses than in sub-clinical infections. To address this question, we studied the memory CD8+ T cell responses in a group of healthy adults from Finland who had HFRS caused by PUUV infection between the years 1984 and 1995. We detected virus-specific CTL in the bulk cultures of seven of eleven immune individuals tested following stimulation with infectious virus. The PUUV proteins N, G1 and G2 were recognized by CTLs in six, five, and two donors respectively. Extensive cloning of T cells from two donors resulted in the isolation of sixty-three virus-specific CTL lines, the majority of which (61/63) were specific for the PUUV N protein. Six novel CD8+ CTL epitopes and one CD4+ CTL epitope were identified on the N protein, all of which clustered in the center of the protein between amino acids 173 and 251. The CTL lines specific for these epitopes were restricted by a variety of HLA alleles including A2, A28, B7 and B8, and were primarily serotype specific when tested against target cells expressing HTNV or SNV N protein. IFN-γ ELISPOT analysis using the defined epitopes to stimulated PBMC, revealed high frequencies of circulating N-specific CD8+ T cells in eight of thirteen individuals tested. Finally, T cell receptor (TCR) Vβ analysis of CTL clones specific for one epitope (N204-12) demonstrated that cells in this population expressed up to five different Vβ chains. These results demonstrated that the PUUV N protein may be the dominant target of the CTL response, that the N-specific CD8+ CTL responses are diverse, heterogeneous, and primarily serotype specific, and that virus-specific memory CD8+T cells can persist at high levels for up to 15 years after the primary infection. In order to understand the pathology of HFRS and HPS, we must be able to assess the contribution of various factors that could potentially contribute to disease. The virus burden in the infected individual is likely to be an important factor in the severity of the resulting disease. Quantitative RT-PCR analysis of plasma samples from acute HPS patients demonstrated that a higher virus burden (as reflected by viral RNA copy number) is associated with more severe HPS. In order to perform similar analyses in patients with HFRS caused by PUUV, we established a quantitative RT-PCR assay for the detection of PUUV S segment RNA in patient plasma. The design and optimization of the PUUV-specific RT-PCR is described in this report. This assay will allow us to measure the virus burden in patients and compare these data with levels of T cell activation and with parameters of disease severity. In this way, we hope to gain an understanding of the kinetics and magnitude of both the virus burden and virus-specific T cell response during the acute illness. This thesis provides the first description of human virus-specific T cell responses to HTNV and PUUV. These data shed light on the nature of the CD8+ T cell responses that are generated following natural infections with PUUV and sub-clinical infections with HTNV. The studies of memory CD8+ T cell responses to PUUV, and the development of a PUUV-specific quantitative RT-PCR assay, establish the framework for future studies of the immunopathology of acute HFRS. Quantitative analysis of both virus burden and T cell responses during acute illness will provide insight into their relative contributions to the pathology of disease.

The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface.

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKε and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was discovered and originally thought to mediate the MyD88-independent pathway. However, Mal-deficient mice were found to be defective in both TLR2 and 4 mediated NF-кB activation. We hypothesized that other TIR domain containing adapters could mediate this MyD88-independent pathway of TLR3 and 4 leading to the activation of IRF3. Two additional TIR adapters were discovered, TRIF and TRAM. TRIF was shown to mediate TLR3 signal transduction. In this study, we report that both TRIF and TRAM mediate the activation of the MyD88-independent pathway in response to LPS/TLR4 activation. Unlike any of the other known TIR domain containing adapters, TRAM appears to be restricted to the LPS/TLR4 activation pathway while TRIF plays a role in both TLR3 and TLR4 pathways leading to IRF3 target gene expression. Our studies revealed that TRAM could be acting upstream of TRIF in the LPS/TLR4 pathway. To this end, we sought to determine the localization of TRAM within the cell. We found that TRAM localizes to the plasma membrane. TRAM localization is the result of myristoylation since mutation of the predicted myristoylation site (G2A) resulted in the re-distribution of TRAM from the membrane into the cytoplasm. Reconstitution of TRAM-deficient macrophages with TRAM G2A is unable to rescue LPS/TLR4 signal transduction. Thus, myristoylation and membrane association of TRAM are critical for LPS/TLR4 signal transduction. The data generated in this dissertation extends our understanding of the signaling pathways of the innate immune system. Indeed, the molecules and pathways described herein could prove to be beneficial targets for ameliorating symptoms of disease, both autoimmune and pathogen-associated. Finally, the research described here will spur further insight into the complex signaling pathways of a once ignored arm of the immune system.

Profound lymphopenia has been observed during many acute viral infections, and our laboratory has previously documented a type 1 IFN-dependent loss of most memory (CD44hi) and some naïve (CD44lo) CD8 T cells immediately preceding the development of the antiviral T cell response at days 2-4 following lymphocytic choriomeningitis virus (LCMV) infection. In this thesis, I will examine additional mechanisms involved in the early attrition of CD8 T cells and evaluate whether antigen-specific and non-specific CD8 T cells are equally susceptible. Lastly, I will examine whether the early attrition of CD8 T cells contributes to the generation of an effective immune response. Poly(I:C), a potent inducer of type 1 IFN, was previously shown to cause the attrition and apoptosis of CD8α+CD44hi cells in normal mice, but not in type 1 IFN receptor–deficient mice (IFN1-R KO). I questioned whether additional molecule(s) might contribute to the type 1 IFN-induced apoptosis of CD8α+CD44hi cells. I used a PCR array to determine the expression of 84 apoptosis-related genes at 6 hours post-poly(I:C) treatment, relative to an untreated control. There was an 11-fold increase in CD40 RNA expression in CD8α+CD44hi cells isolated from poly(I:C)-treated mice. CD40 protein expression was also increased on CD8α+CD44hi cells, peaking between 9 and 12 hours following poly(I:C) treatment, before declining thereafter. This increase in CD40 protein expression directly correlated with an increase in Annexin V reactivity, an indicator of early apoptosis. Nevertheless, CD40 was not required for the loss of CD8α+CD44hi cells, as both wildtype and CD40-deficient mice were equally susceptible to the poly(I:C)-induced attrition. Upon further characterization, I found this population of CD40+CD8α+CD44hi cells to be CD11c+B220-Thy1.2- MHCIIhi, which is consistent with a “lymphoid” CD8α+ DC phenotype. Kinetic analysis revealed a type 1 IFN-dependent increase in this CD8α+ DC population at 12 hours post-poly(I:C) treatment. This increase was only observed in the spleen, as no increase in percentage was observed in the peritoneal cavity (PEC), lungs, inguinal lymph nodes (iLN), or peripheral blood. Collectively, these results suggest that the type 1 IFN-dependent increase in splenic CD8α+DCs accounts for the observed increase in Annexin V reactive cells following poly(I:C) treatment. These findings required a re-evaluation of the type 1 IFN-induced attrition of CD8+CD44hi T cells with an anti-CD8β antibody, which is a more exclusive marker for T cells than the anti-CD8α antibody. Kinetic analysis revealed a significant decrease in splenic CD8β+CD44hi T cells at 12 hours post-poly(I:C) treatment. This reduction in splenic CD8β+CD44hi T cells was not due to trafficking to other organs, as the PECs, lungs, iLN, lungs, and peripheral blood all exhibited significant, although varying, decreases in the percentage of CD8β+CD44hi T cells at 12 hour following poly(I:C) treatment. These data support the notion that the type 1 IFN-induced attrition of CD8β+CD44hiT cells was a “global” phenomenon and could not be completely due to migration out of the spleen. The attrition of CD8β+CD44hi T cells was also dependent upon type 1 IFN at 3 days post-LCMV infection, as there was no significant reduction of this population in IFN1-R KO mice. The loss of wildtype CD8β+CD44hi T cells correlated with an increased activation of caspases 3 and 8, which are enzymes that play essential roles in apoptosis and inflammation. A significant loss of CD4+CD44hi T cells, which also correlated with an increased activation of caspases 3 and 8, was observed at 3 days post-LCMV infection. Collectively, these results suggest that attrition of both CD4+CD44hi and CD8β+CD44hiT cell populations is type 1 IFN-dependent and associated with the activation of caspases following LCMV infection. At 3 days post-LCMV infection, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had a higher frequency of cells with fragmented DNA, a hallmark characteristic of the late stages of apoptosis, as revealed by terminal transferase dUTP nick end labeling (TUNEL), relative to uninfected controls. This suggests that the loss of both populations was due to apoptosis. Therefore, I questioned whether the LCMV-induced apoptosis of both CD4+CD44hi and CD8β+CD44hi T cell populations occurred through a mitochondrial-induced pathway involving the pro-apoptotic molecule Bim. The attrition of both CD4+CD44hi and CD8β+CD44hi T cells was significantly higher in wildtype mice compared to Bim KO mice at 3 days post-LCMV infection. Moreover, both wildtype CD8β+CD44hi and CD4+CD44hi T cell populations had higher frequency of TUNEL+ cells, relative to Bim KO populations. These results suggest that the apoptosis of CD8β+CD44hi and CD4+CD44hiT cells, following LCMV infection, might occur through a mitochondrial-induced pathway involving Bim. Studies have shown “lymphoid” CD8α+ DCs to be involved in the phagocytosis of apoptotic lymphocytes. Therefore, I evaluated whether host CD8α+ DCs are capable of phagocytosing apoptotic lymphocytes by adoptively transferring CFSE-labeled wildtype donor splenocytes (Ly5.1) into congenic wildtype hosts (Ly5.2), followed by inoculation with poly(I:C). There was an increased frequency of donor cells (Ly5.1, CFSE+) within the host CD8α+CD11c+ gate at 9 and 12 hours post-poly(I:C) treatment. The results suggest that type 1 IFN-activated CD8α+DCs might aid in the rapid clearance of apoptotic cells during the type 1 IFN-induced attrition associated with viral infections. I next questioned whether TCR engagement by antigen would render CD8 T cells resistant to attrition. I tested whether a high concentration of antigen (GP33 peptide) would protect LCMV-specific naïve TCR transgenic P14 cells specific for the GP33 epitope of LCMV and GP33-specific LCMV-immune cells from depletion. Both naïve P14 and memory GP33-specific donor CD8 T cells decreased substantially 16 hours after inoculation poly(I:C), regardless of whether a high concentration of GP33 peptide was administered to host mice beforehand. The increased activation status of naïve antigen-specific cells via peptide inoculation did not confer resistance to type 1 IFN-induced depletion. Donor naïve P14 and LCMV-specific memory cells were also depleted from day 2 LCMV-infected (Clone 13) hosts by 16 hours post-transfer. These results indicate that antigen engagement does not protect CD8 T cells from the type 1 IFN-induced attrition associated with viral infections. Computer models indicated that early depletion of memory T cells may allow for the generation for a more diverse T cell response to infection by reducing the immunodomination caused by cross-reactive T cells. To test this in a biological system, I questioned whether the reduced apoptosis of the crossreactive memory CD8 population (NP205), in aged LCMV-immune mice (18-22 months), following heterologous virus challenge (PV), would allow it to dominate the immune response. At day 8 post-PV infection, the cross-reactive memory CD8 T cell response (NP205) was more immunodominating in aged LCMV-immune mice relative to younger LCMV-immune mice. This was indicated by the increased ratio of the cross-reactive NP205 response to the newly arising noncross-reactive, PV-specific NP38 response in older LCMV-mice relative to younger LCMV immune-mice, at day 8 post-PV infection. These data suggest that the early attrition of T cells allows for the generation of a more diverse T cell response to infection by reducing the immunodomination caused by crossreactive T cells. Collectively, these findings offer further insight into the early attrition of T cells associated with viral infections.

Mature αβ and γδ T cells arise from a common precursor population in the thymus. Much debate has focused on the mechanism of T cell lineage choice made by these multi-potential precursor cells. It is widely believed that the decision of these precursor cells to commit to the γδ or αβ T cell lineages is regulated primarily by a specific instructive signal relayed through the appropriate T cell receptor. Contrary to this model, we present evidence for a TCR-independent lineage commitment process. Comparison of global gene expression profiles from immature αβ and γδ lineage thymocytes identified Sox13, an HMG-box transcription factor, as a γδ T cell-specific gene. Unlike other HMG-box transcription factors such as TCF1, LEF1 and SOX4, that are critical for proper αβ T cell development, Sox13 expression is restricted to early precursor subsets and γδ lineage cells. Importantly, SOX13 appears to influence the developmental fate of T cell precursors prior to T cell receptor expression on the cell surface. Transgenic over-expression of Sox13 in early T cell precursors strongly inhibits αβ lineage development, in part, by inhibiting precursor cell proliferation and concomitantly, leading to increased cell death among αβ lineage subsets. Steady-state γδ T cell numbers, however, appear unaffected. Strikingly, the DP αβ lineage cells that do develop in Sox13 transgenic mice are imprinted with a γδ- or precursor-like molecular profile, suggesting that SOX13 plays an active role in the lineage fate decision process or maintenance. Sox13-deficient mice, on the other hand, have selectively reduced numbers of γδ thymocytes, indicating that SOX13 is essential for proper development of γδ T cells. We present additional data demonstrating that SOX13 is a canonical WNT signaling antagonist modulating TCF1 activity, raising a strong possibility that WNT signals, and their modulators, are at the nexus of γδ versus αβ T cell lineage commitment.

DNA damage response pathways are a complicated network of proteins that function to remove and/or reverse DNA damage. Following genetic insult, a signal cascade is generated, which alerts the cell to the presence of damaged DNA. Once recognized, the damage is either removed or the damaged region is excised, and the original genetic sequence is restored. However, when these pathways are defective the cell is unable to effectively mediate the DNA damage response and the damage persists unrepaired. Thus, the proteins that maintain the DNA damage response pathway are critical in preserving genomic stability. One essential DNA repair protein is the Breast Cancer Associated gene, BRCA1. BRCA1 is essential for mediating the DNA damage response, facilitating DNA damage repair, and activating key cell cycle checkpoints. Moreover, mutations in BRCA1 lead to a higher incidence of breast and ovarian cancer, highlighting the importance of BRCA1 as a tumor suppressor. In an effort to better understand how BRCA1 carried out these functions, researchers sought to identify additional BRCA1 interacting proteins. This led to the identification of several proteins including the BRCA1 Associated C-terminal Helicase, BACH1. Due to the direct interaction of BACH1 with a region of BRCA1 essential for DNA repair and tumor suppression, it was speculated that BACH1 may help support these BRCA1 function(s). In fact, initial genetic screenings confirmed that mutations in BACH1 correlated not only with hereditary breast cancer, but also with defects in DNA damage repair processes. The initial correlation between BACH1 and cancer predisposition was further confirmed when mutations in BACH1 were identified in the cancer syndrome Fanconi anemia (FA) (complementation group FA-J), thus giving BACH1 its new name FANCJ. These findings supported a previously established link between the FA and BRCA pathways and between FA and DNA repair. In particular, we demonstrated that similar to other FA/BRCA proteins, suppression of FANCJ lead to a substantial decrease in homologous recombination and enhanced both the cellular sensitivity to DNA interstrand cross-linking agents and chromosomal instability. What remained unknown was specifically how FANCJ functioned and whether these functions were dependent on its interaction with BRCA1 or other associated partners. In fact, we identified that FANCJ interacted directly with the MMR protein MLH1. Moreover, we found that the FANCJ/BRCA1 interaction was not required to correct the cellular defects in FA-J cells, but rather that the FANCJ/MLH1 interaction was required. Although both the FA/BRCA and MMR pathways undoubtedly mediate the DNA damage response, there was no evidence to suggest that these pathways were linked, until recently. Our findings not only indicate a physical link between these pathways by protein-protein interaction, but also demonstrated a functional link.

Hepatitis C Virus (HCV) is the primary cause of liver transplantation due to its chronic nature in up to eighty percent of infected cases. Around 3 percent of the world’s population is infected with HCV. Treatment for HCV is a combined Ribavirin and interferon-α (IFN-α) therapy effective in only fifty to eighty percent of patients depending on HCV genotype. The growing health concern with this disease is the lack of a cure despite liver transplantation. HCV targets hepatocytes, liver cells, but is not cytolytic. HCV has been shown to induce end stage liver disease through sustained inflammation from the host’s immune system in the liver. One of the key dilemmas in HCV research and the search for fully effective treatments or vaccines is the lack of animal models. HCV infectivity and disease is limited to primates, most specifically to humans, which cannot be fully replicated in any other living being. The mechanisms for HCV evasion or activation of the immune system are complex, many and discoveries within this field are crucial to overcoming this destructive hepatic infection. Toll-like receptors (TLR) are cellular activators of the innate immune system that have been a target of HCV. Activated TLRs trigger both the inflammatory and anti-viral pathways to produce inflammatory cytokines and interferons. HCV proteins have been reported to activate a number of TLRs in a variety of cell types. In order to identify possible targets of HCV within the TLR family, we first characterized TLR presence and function in both human hepatic carcinoma cell lines and purified primary human hepatocytes. RNA from TLRs 1-10 was observed to varying degrees in both the hepatoma cell lines and the primary hepatocytes. We show the extracellular and/or intracellular presence of TLR2, TLR1, TLR3 and TLR7 proteins in hepatoma cell lines. TLR3 and TLR7 are located within the endosome and recognize viral RNA products. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with either TLR1 or TLR6. We show NF-κB activation in hepatoma cells by TLR2/1, TLR2/6 ligand and HCV protein stimulation. In primary hepatocytes, HCV proteins induced both IL-8 and IL-6 production. We also show that primary hepatocytes initiate a Type 1 IFN response in addition to IL-8 and IL-6 production upon stimulation with a TLR7/8 ligand. Human hepatoma and primary hepatocytes are responsive to TLR2, TLR1, TLR6, TLR7/8 ligands and HCV proteins. Activation of these TLRs may contribute to the inflammatory mediated destruction caused by HCV or could be targets of HCV contributing to its immune evasion. We found previously that hepatoma cells and primary hepatocytes are responsive to TLR2 ligands and HCV proteins. We also reported that TLR2 is activated by HCV proteins. Here we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing siRNAs targeted to TLR2, TLR1 and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in Human Embryonic Kidney cells (HEK/TLR2) and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-α and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1 and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1 or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. TLR2 may utilize both TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins contributing to inflammatory disease. Two critical factors in chronic HCV infection are inflammatory disease and immune evasion. We have demonstrated that TLR2 and its co-receptors play a role in inflammatory-mediated induction via HCV NS3 and core administration. It has recently been shown that HCV targets the TLR3 pathway to aid in immune evasion. TLR3 is only one of four viral recognition receptors located within the endosome and it is plausible that HCV may target others. We hypothesized that HCV infection may interfere with the expression and function of TLR7, a sensor of single stranded RNA. Investigating any effect on TLR7 by HCV may reveal a new mechanism for HCV immune evasion. Low levels of both TLR7 mRNA and protein were measured in HCV replicating cells compared to control cells while reducing HCV infection with either IFNα or restrictive culture conditions restored the decreased TLR7 expression. Downstream of the TLR7 pathway, an increased baseline IRF7 nuclear translocation was observed in HCV replicating cells compared to controls. Stimulation with a TLR7 ligand, R837, resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV replicating cells showed impaired IRF7 activation. Use of RNA polymerase inhibitors on hepatoma cells, control and HCV replicating, revealed a shorter TLR7 half life in HCV replicating cells compared to control cells which was not seen in TLR5 mRNA. These data suggest that reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and results in impaired TLR7-induced IRF7-mediated cell activation. In conclusion, Hepatitis C Virus manipulates specific Toll-like receptors’ expression and their signaling pathways to induce cytokine production. HCV utilizes surface receptors TLR2 and its co-receptors which once activated could contribute to inflammatory disease by production of inflammatory cytokines and possibly immune evasion. HCV down-regulates TLR7, a viral recognition receptor, by decreasing mRNA stability which could facilitate evasion of host immune surveillance.

Tolerance-based stem cell transplantation using sub-lethal conditioning is being considered for the treatment of human disease, but safety and efficacy remain to be established. In order to study these two issues, we first established that mouse bone marrow recipients treated with sub-lethal irradiation plus transient blockade of the CD40-CD154 costimulatory pathway develop permanent hematopoietic chimerism across allogeneic barriers. Our conditioning regimen of 6 Gy irradiation, a short course of anti-CD154 mAb and 25 million fully allogeneic BALB/c bone marrow cells consistently produced long-term, stable, and multilineage chimerism in C57BL/6 recipients. Furthermore, chimeric mice displayed donor-specific transplantation tolerance, as BALB/c skin allografts were permanently accepted while third-party CBA/JCr skin allografts were promptly rejected. We next determined both the safety and efficacy of this protocol by infecting chimeric mice with lymphocytic choriomeningitis virus (LCMV) either at the time of transplantation or at several time points afterwards. Infection with LCMV at the time of transplantation prevented engraftment of allogeneic, but not syngeneic, bone marrow in similarly treated mice. Surprisingly, infected allograft recipients also failed to clear the virus and died. Post-mortem study revealed hypoplastic bone marrow and spleens. Hypoplasia and death in these mice required the combination of 6 Gy irradiation, LCMV infection on the day of transplantation, and an allogeneic bone marrow transplant but did not require the presence of anti-CDl54 mAb. Allochimeric mice infected with LCMV 15 days after transplantation were able to survive and maintain their bone marrow graft, indicating that the deleterious effects of LCMV infection on host and graft survival are confined to a narrow window of time during the tolerization and transplantation process. The final section of this thesis studied the mechanisms of graft rejection and death in sublethally irradiated recipients of allogeneic bone marrow and infection with LCMV at the time of bone marrow transplantation. Infection of interferon-α/β receptor knockout mice at the time of transplantation prevented the engraftment of allogeneic bone marrow, but the mice survived. Therefore, IFN-αβ is involved in the development of marrow hypoplasia and death, whereas a second mechanism is involved in blocking the development of chimerism in these mice. Through the use of depleting mAb's and knockout mice we demonstrate that three types of recipients survived and became chimeric after being given sublethal irradiation, anti-CD154 mAb, an allogeneic bone marrow transplant and a day 0 LCMV infection: mice depleted of CD8+ T cells, CD8 knockout mice, and TCR-αβ knockout mice. Our data indicate that the mediator of bone marrow allograft destruction in LCMV-infected mice treated with costimulatory blockade is a radioresistant CD8+ NK1.1- TCRαβ+ T cell. We conclude that a non-cytopathic viral infection at the time of transplantation can prevent engraftment of allogeneic bone marrow and result in the death of sub-lethally irradiated mice treated with costimulation blockade. The abrogation of allogeneic bone marrow engraftment is mediated by a population of CD8+ NK1.1- TCRαβ+ T cells and the mediator of hypoplasia and death is viral induction of IFN-αβ.