The human Philadelphia chromosome (Ph) arises from a translocation between chromosomes 9 and 22 [t(9;22)(q34;q11)]. The resulting chimeric BCR-ABLoncogene encodes a constitutively activated, oncogenic tyrosine kinase that induces chronic myeloid leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). The BCR-ABL tyrosine kinase inhibitor (TKI), imatinib mesylate, induces a complete hematologic and cytogenetic response in the majority of CML patients, but is unable to completely eradicate BCR-ABL–expressing leukemic cells, suggesting that leukemia stem cells are not eliminated. Over time, patients frequently become drug resistant and develop progressive disease despite continued treatment. Two major reasons cause the imatinib resistance. The first one is the BCR-ABL kinase domain mutations which inhibit the interaction of BCR-ABL kinase domain with imatinib; the second one is the residual leukemia stem cells (LSCs) in the patients who are administrated with imatinib. To overcome these two major obstacles in CML treatment, new strategies need further investigation. As detailed in Chapter II, we evaluated the therapeutic effect of Hsp90 inhibition by using a novel water-soluble Hsp90 inhibitor, IPI-504, in our BCR-ABL retroviral transplantation mouse model. We found that BCR-ABL mutants relied more on the HSP90 function than WT BCR-ABL in CML. More interestingly, inhibition of HSP90 in CML leukemia stem cells with IPI-504 significantly decreases the survival and proliferation of CML leukemia stem cells in vitro and in vivo. Consistent with these findings, IPI-504 treatment achieved significant prolonged survival of CML and B-ALL mice. IPI-504 represents a novel therapeutic approach whereby inhibition of Hsp90 in CML patients and Ph+ ALL may significantly advance efforts to develop a cure for these diseases. The rationale underlying the use of IPI-504 for kinase inhibitor–resistant CML has implications for other cancers that display oncogene addiction to kinases that are Hsp90 client proteins. Although we proved that inhibition of Hsp90 could restrain LSCs in vitro and in vivo, it is still unclear how to define specific targets in LSCs and eradicate LSCs. In Chapter III, we took advantage of our CML mouse model and compared the global gene expression signature between normal HSCs and LSCs to identify the downregulation of Pten in CML LSCs. CML develops faster when Pten is deleted in Ptenfl/fl mice. On the other hand, Pten overexpression significantly delays the CML development and impairs leukemia stem cell function. mTOR is a major downstream of Pten-Akt pathway and it is always activated or overepxressed when Pten is mutated or deleted in human cancers. In our study, we found that inhibition of mTOR by rapamycin inhibited proliferation and induced apoptosis of LSCs. Notably, our study also confirmed a recent clinical report that Pten has been downregulated in human CML patient LSCs. In summary, our results proved the tumor suppressor role of Pten in CML mouse model. Although the mechanisms of Pten in leukemia stem cells still need further study, Pten and its downstream, such as Akt and mTOR, should be more attractive in LSCs study.

Jak3, a member of the Janus family of tyrosine kinases, is essential for signaling via the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. These Jak3-dependent cytokines primarily activate STAT5 and are critical for lymphoid generation and differentiation. Using naïve CD4+ T cells from Jak3-deficient mice and wild type CD4+ T cells treated with a pharmacological inhibitor of Jak3, we report that Jak3-dependent cytokine signals are not required for the proliferation of naïve CD4+ T cells. This is illustrated by the similar percentage of divided cells, comparable cell divisions, intact cell cycle progression and unaffected regulation of cell cycle proteins in the absence of Jak3. In contrast to proliferation, differentiation of naïve CD4+ T cells into Th1 effector cells requires Jak3-dependent cytokine signals. In the absence of Jak3, naïve CD4+ T cells proliferate robustly, but produce little IFN-γ after Th1 polarization in vitro. This defect is not due to reduced activation of STAT1 or STAT4, nor to impaired up-regulation of the transcription factor T-bet. Instead, we find that T-bet binding to the Ifng promoter is greatly diminished in the absence of Jak3-dependent signals, correlating with a decrease in Ifng promoter accessibility and histone acetylation. These data indicate that while Jak3-dependent signals are dispensable for naïve CD4+ T cell proliferation, Jak3 regulates epigenetic modification and chromatin remodeling of the Ifng locus during Th1 differentiation.

The innate immune system is the first line of defense against infectious agents. It is essential for protection against pathogens and stimulation of long-term adaptive immune responses. Therefore, deciphering the mechanisms of the innate immune system is crucial for understanding the integrated systems of host defense against microbial infections, which is conserved from insects to humans. Despite lacking a conventional adaptive immune system, insects can mount a robust immune response against a wide array of microbial pathogens. These innate immune mechanisms have been widely studied in Drosophila melanogaster, because of the model system’s powerful genetic, genomic and molecular tools. The Drosophila immunity relies on cellular and humoral innate immune responses to fight pathogens. The hallmark of the Drosophilahumoral immune response is the rapid induction of antimicrobial peptide genes in the fat body, the homolog of the mammalian liver. Expression of these antimicrobial peptide genes is controlled by two distinct immune signaling pathways, the Toll pathway and the IMD (immune deficiency) pathway. The Toll pathway is activated by fungal and Gram-positive bacterial infections, whereas the IMD pathway responds to Gram-negative bacteria. Both pathways culminate in activation of the Rel/NF-кB transcription factors DIF (Dorsal-related immunity factor), Dorsal and Relish, which in turn translocate to the nucleus to induce the antimicrobial peptide genes. DIF and Dorsal are activated by the Toll pathway and control induction of antimicrobial peptide genes such as Drosomycin. The NF-кB precursor Relish, which is composed of an N-terminal Rel homology domain and a C-terminal IкB-like domain, is activated by the IMD pathway and initiates transcription of antimicrobial peptide genes such as Diptericin. Although many components of the Drosophila immune signaling pathways have been identified, the detailed mechanisms of signal trans

T lymphocytes are distinguished by the expression of αβ TCR or γδ TCR on their cell surface. The kinetic differences in the effector functions classifies γδ T cells as innate-like lymphocytes and αβ T cells as adaptive lymphocytes. Although distinct, αβ and γδ T cell lineages produce a common array of cytokines to mount an effective immune response against a pathogen. The production of cytokine IL-17 is a shared characteristic between the γδ T (Tγδ17) cells and the CD4 T (Th17) cells. γδ T cells develop into Tγδ17 cells in the thymus whereas CD4 T cells differentiate into Th17 cells in response to antigens in the peripheral lymphoid tissues. γδ T cells exported from the thymus, as pre-made effectors, are the early IL-17 producers compared with the late IL-17 producing Th17 cells. In this thesis we describe how TGFβ-SMAD2 dependent pathway selectively regulates Th17 cell differentiation but not Tγδ17 cells generation. We further illustrate the requirement of WNT-HMG box transcription factor (TF) signaling for the thymic programming of Tγδ17 cells. Cytokine TGFβ in co-operation with IL-6 induces the differentiation of Th17 cells. Conversely, TGFβ signaling also regulates the differentiation and maintenance of CD4+FOXP3+ regulatory T cells. The mechanism by which TGFβ signals synergize with IL-6 to generate inflammatory versus immunosuppressive T cell subsets is unclear. TGFβ signaling activates receptor SMADs, SMAD2 and SMAD3, which associate with a variety of nuclear factors to regulate gene transcription. Defining relative contributions of distinct SMAD molecules for CD4 T cell differentiation is critical for mapping the versatile intracellular TGFβ signaling pathways that tailor TGFβ activities to the state of host interaction with pathogens. We show here that SMAD2 is essential for Th17 cell differentiation and that it acts in part by modulating the expression of IL-6R on T cells. While mice lacking SMAD2 specifically in T cells do not develop spontaneous lymphoproliferative autoimmunity, Smad2-/- T cells are impaired in their response to TGFβ in vitro and in vivo and they are more pathogenic than controls when transferred into lymphopenic mice. These results demonstrate that SMAD2 is essential for TGFβ signaling in CD4+ T effector cell differentiation and that it possesses functional capabilities distinct from SMAD3. Although SMAD2 is essential for the differentiation of Th17 cells, TGFβ signaling via SMAD2 is not required for the thymic programming of innate Tγδ17 cells. Among different γδ T cells, Vγ2+ (V2) γδ T cells are the major IL-17 producing subsets. We demonstrate that Sry-high mobility group (HMG) box TFs regulate the development of V2 Tγδ17 cells. We show that the HMG box TF, SOX13 functions in a positive loop for the intrathymic generation of V2 Tγδ17 cells. SOX13 regulates the programming of Tγδ17 cells by controlling the expression of B-lymphoid kinase (BLK) in developing immature V2 γδ T cells. BLK is an Src-family kinase expressed by all Tγδ17 cells. Furthermore, we show another HMG box TF, TCF1, the nuclear effector of canonical WNT signaling, is the primary negative regulator of IL-17 production by all γδ T cells. We propose that the antagonism of SOX13 and TCF1 determines the generation of IL-17 producing γδ T cells. We also show that extrinsic cues from αβ T cells do not affect the generation of IL-17 producing γδ T cells. Using OP9-DL1 culture system, we demonstrate that the progenitors of V2 Tγδ17 cells are the c-Kit+ early thymic precursors.

Costimulation blockade protocols are promising alternatives to the use of chronic immunosuppression for promoting long-term allograft survival. However, the efficacy of costimulation blockade-based protocols is decreased by environmental insults such as viral infections. For example, lymphocytic choriomeningitis virus (LCMV) infection at the time of costimulation blockade treatment abrogates skin allograft survival in mice. In this dissertation, we test the hypothesis that viruses shorten allograft survival by activating the innate immune system through pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs). To investigate the role of innate immunity in shortening allograft survival, costimulation blockade-treated mice were co-injected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid, poly(I:C)), TLR4 (lipopolysaccharide, LPS), or TLR9 (CpG DNA) agonists, followed by transplantation with skin allografts 7 days later. Costimulation blockade prolonged skin allograft survival that was shortened in mice coinjected with TLR agonists. To investigate the underlying mechanisms of this observation, we used synchimeric mice, which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, co-administration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) upregulated CD44, and 3) failed to undergo apoptosis. We also demonstrate that costimulation blockade-treated CD8α-deficient mice exhibit prolonged allograft survival when co-injected with LPS. These data suggest that TLR agonists shorten allograft survival by impairing the apoptosis of alloreactive CD8+T cells. We further delineate the mechanism by which TLR agonists shorten allograft survival by demonstrating that LPS and poly(I:C) fail to shorten allograft survival in IFNRI- deficient mice. Interestingly, the ability of poly(I:C) to more potently induce IFN-α/β than LPS correlates with its superior abilities to shorten islet allograft survival and induce allo-specific CTL activity as measured by an in vivo cytotoxicity assay. The ability to shorten allograft survival and induce IFN-α/β is a TLR-dependent process for LPS, but is a TLR-independent process for poly(I:C). Strikingly, the injection of IFN-β impairs alloreactive CD8+T cell deletion and shortens allograft survival, similar to LPS and poly(I:C). These data suggest that LPS and poly(I:C) shorten allograft survival by inducing IFN-α/β through two different mechanisms. Finally, we present data showing that viruses (LCMV, Pichinde virus, murine cytomegalovirus and vaccinia virus) impair alloreactive CD8+T cell deletion and shorten allograft survival, in a manner comparable to LPS and poly(I:C). Similar to LPS, LCMV and MCMV exhibit an impaired ability to shorten allograft survival in MyD88-deficient mice. These data suggest that the MyD88 pathway is required for certain viruses and TLR-agonists to shorten allograft survival. In this dissertation, we present data supporting an important role for TLRs and IFN- α/β in shortening allograft induced by costimulation blockade. Our findings suggest that targeting these pathways during the peri-transplant period may enhance the efficacy of costimulation blockade protocols in the clinic.

Vascular Endothelial Growth Factor Receptor-3 (VEGFR3/Flt4) and its ligand Vegfc are necessary for development of both blood and lymphatic vasculature in vertebrates. In zebrafish, Vegfc/Flt4 signaling is essential for formation of arteries, veins, and lymphatic vessels. Interestingly, Flt4 appears to utilize distinct signaling pathways during the development of each of these vessels. To identify components of this pathway, we performed a transgenic haploid genetic screen in zebrafish that express EGFP under the control of a blood vessel specific promoter. As a result, we indentified a mutant allele of vascular endothelial growth factor c (vegfc), vegfcum18. vegfcum18 mutants display defects in vein and lymphatic vessel development but normal segmental artery (SeA) formation. Characterization of this allele led to the finding that the primary defect in vegfcum18 mutants was a general failure in vein and lymphatic vessel sprouting. Further genetic and biochemical analysis of this mutant revealed profound paracrine defects, which likely result in the observed loss of lymphatic and venous structures. Furthermore, double mutant analysis demonstrated that defects during SeA formation in vegfcum18 mutants were masked by inputs from the Vegfa signaling pathway. Endothelial cell autonomous expression of vegfcum18 induced angiogenic effects on blood vessels while endothelial cells lacking vegfc displayed defects in tip cell occupancy, suggesting a cell autonomous-autocrine role for Vegfc during developmental angiogenesis. Finally, we present genetic evidence that links processing of Vegfc by Furin during the formation of lymphatics in zebrafish. Together the data presented here suggest two discrete modes of signaling during blood and lymphatic vessel development, thus implying that regulation of Vegfc secretion and processing may play a pivotal role in the formation of these distinct vessel types in zebrafish.

Discovery of new antifungal chemo-therapeutics for humans is limited by the large degree of conservation among eukaryotic organisms. In recent years, the histone acetyl-transferase Rtt109 was identified as the sole enzyme responsible for an abundant and important histone modification, histone H3 lysine 56 (H3K56) acetylation. In the absence of Rtt109, the lack of acetylated H3K56 renders yeast cells extremely sensitive to genotoxic agents. Consequently, the ability to sustain genotoxic stress from the host immune system is crucial for pathogens to perpetuate an infection. Because Rtt109 is conserved only within the fungal kingdom, I reasoned that Rtt109 could be a novel drug target. My dissertation first establishes that genome stability provided by Rtt109 and H3K56 acetylation is required for Candida albicans pathogenesis. I demonstrate that mice infected with rtt109 -/- cells experience a significant reduction in organ pathology and mortality rate. I hypothesized that the avirulent phenotype of rtt109 -/- cells is due to their intrinsic hypersensitivity to the genotoxic effects of reactive oxygen species (ROS), which are utilized by phagocytic cells of the immune system to kill pathogens. Indeed, C. albicans rtt109 -/- cells are more efficiently killed by macrophages in vitro than are wild-type cells. However, inhibition of ROS generation in macrophages renders rtt109 -/- and wild-type yeast cells equally resilient to killing. These findings support the concept that ability to resist genotoxic stress conferred by Rtt109 and H3K56 acetylation is a virulence factor for fungal pathogens and establish Rtt109 as an opportune drug- target for novel antifungal therapeutics. Second, I report the discovery of a specific chemical inhibitor of Rtt109 catalysis as the initial step in the development of a novel antifungal agent. We established a collaboration with the Broad Institute (Cambridge, MA) to perform a high-throughput screen of 300,000 compounds. From these, I identified a single chemical, termed KB7, which specifically inhibits Rtt109 catalysis, with no effect on other HAT enzymes tested. KB7 has an IC50 value of approximately 60 nM and displays noncompetitive inhibition regarding both acetyl-coenzyme A and histone substrates. With the genotoxic agent camptothecin, KB7 causes a synergistic decrease in C. albicans growth rate. However, this effect is only observed in an efflux-pump mutant, suggesting that this compound would be more effective if it were better retained intracellularly. Further studies through structure-activity relationship (SAR) modifications will be conducted on KB7 to improve its effective cellular concentration.

Respiratory syncytial virus (RSV) causes a common infection that is associated with a range of respiratory illnesses from common cold-like symptoms to serious lower respiratory tract illnesses such as pneumonia and bronchiolitis. RSV is the single most important cause of serious lower respiratory tract illness in children < 1 year of age. Host innate and acquired immune responses activated following RSV infection have been suspected as contributing to RSV disease. Toll-like receptors (TLRs) activate innate and acquired immunity and are candidates for playing key roles in the host immune response to RSV. Leukocytes express TLRs including TLR2, TLR6, TLR3, TLR4, and TLR7 that can potentially interact with RSV and promote immune responses following infection. Using knockout mice, we have demonstrated that TLR2 and TLR6 signaling in leukocytes can activate innate immunity against RSV by promoting TNF-α, IL-6, CCL2 (MCP-1), and CCL5 (RANTES) production. As previously noted, TLR4 also contributed to cytokine activation (71, 90). Furthermore, we demonstrated that signals generated following TLR2 and TLR6 activation were important for controlling viral replication in vivo. Additionally, TLR2 interactions with RSV promoted neutrophil migration and dendritic cell activation within the lung. Collectively, these studies indicate that TLR2 is involved in RSV recognition and subsequent innate immune activation and may play a role in modulating acquired immune responses through DCs. Despite the fact that RSV is the single most important cause of infant upper respiratory tract disease, there are no licensed vaccines available to prevent RSV disease. We have developed a virus-like particle (VLP) vaccine candidate for RSV. The VLP is composed of the NP and M proteins of Newcastle disease virus (NDV) and a chimera protein containing the cytoplasmic and transmembrane domains of the NDV HN protein and the ectodomain of the human RSV G protein (H/G). BALB/c mice immunized with 10 or 40 μg total VLP-H/G protein by intraperitoneal or intramuscular inoculation stimulated antibody responses to G protein as good as or better than comparable amounts of UV-inactivated RSV. Furthermore, VLP-H/G induced robust CTL responses in vaccinated animals. Immunization with two or even a single dose of these particles resulted in the complete protection of BALB/c mice from RSV replication in the lungs. Upon RSV challenge of VLP-H/G immunized mice, no enhanced pathology in the lungs was observed, although lungs of mice immunized in parallel with formalin-inactivated RSV (FI-RSV) showed the significant pathology that has been previously observed with FI-RSV vaccination. Thus, the VLP-H/G candidate vaccine was immunogenic in BALB/c mice and prevented replication of RSV in murine lungs with no evidence of immunopathology. These data support further development of virus-like particle vaccine candidates for RSV.

The Rip2 kinase contains a caspase recruitment domain (CARD) and has been implicated in the activation of the transcriptional factor NF-кB downstream of Nod-like receptors. However, how Rip2 mediates innate immune responses is still largely unclear. We show that Rip2 and IKK-γ become stably polyubiquitinated upon treatment of cells with the Nod2 ligand, muramyl dipeptide. We demonstrate a requirement for the E2 conjugating enzyme Ubc13, the E3 ubiquitin ligase Traf6 and the ubiquitin activated kinase Tak1 in Nod2-mediated NF-кB activation. We also show that M. tuberculosisinfection stimulates Rip2 polyubiquitination. Collectively, this study revealed that the Nod2 pathway is ubiquitin regulated and that Rip2 employs a ubiquitin-dependent mechanism to achieve NF-кB activation. We also demonstrate that intraphagosomal M. tuberculosis stimulates the cytosolic Nod2 pathway. We show that upon Mtb infection, Nod2 recognition triggers the expression of type I interferons in a Tbk1- and Irf5-dependent manner. This response is only partially impaired by the loss of Irf3 and therefore, differs fundamentally from those stimulated by bacterial DNA, which depends entirely on this transcription factor. This difference appears to result from the unusual peptidoglycan produced by mycobacteria, which we show is a uniquely potent agonist of the Nod2/Rip2/Irf5 pathway. Thus, the Nod2 system is specialized to recognize bacteria that actively perturb host membranes and is remarkably sensitive to Mycobacteria, perhaps reflecting the strong evolutionary pressure exerted by these pathogens on the mammalian immune system.

Helicobacter pylori causes chronic gastritis, peptic ulceration and gastric cancer. This bacterium is one of the most prevalent in the world, but affects mostly the populations with a lower socioeconomical status. While it causes gastric and duodenal ulcers in only 20% of infected patients, less then 1% will develop gastric adenocarcinoma. In fact, H. pylori is the most important risk factor in developing gastric cancer. Epidemiological studies have shown that 80% of gastric cancer patients are H. pylori positive. The outcome of the infection with this bacterium depends on bacterial factors, diet, genetic background of the host, and coinfection with other microorganisms. The most important cofactor in H. pylori induced disease is the host immune response, even though the exact mechanism of how the bacterium is causing disease is unknown. The structural complexity of Helicobacter bacteria makes us believe that different bacterial factors interact with different components of the innate immunity. However, as a whole bacterium it may need mainly the TLR2 receptor to trigger an immune response. The type of adaptive immunity developed in response to Helicobacter is crucial in determining the consequences of infection. It is now known for decades that a susceptible host will follow the infection with a strong Th1 immune response. IFNγ, IL-12, IL-1β and TNF-α are the key components of a strong adaptive Th1 response. This is further supported by our work, where deficient T-bet (a master regulator for Th1 response) mice were protected against gastric cancer, despite maintaining an infection at similar levels to wild type mice. On the other hand, a host that is resistant to Helicobacter develops an infection that is followed by a Th2 response sparing the mucosa from severe inflammation. Human studies looking at single nucleotide polymorphism of cytokines, like IL-1β, IL-10 and TNF-α have clearly demonstrated how genotypes that result in high levels of IL-1β and TNF-α, but low IL-10 expression may confer a 50-fold higher risk in developing gastric cancer. The outcome of Helicobacter infection clearly relies on the immune response and genetic background, however the coinfection of the host with other pathogens should not be ignored as this may result in modulation of the adaptive immunity. In studying this, we took advantage of the Balb/C mice that are known to be protected against Helicobacter induced inflammation by mounting a strong Th2 polarization. We were able to switch their adaptive immunity to Th1 by coinfected them with a T. gondii infection (a well known Th1 infection in mice). The dual infected mice developed severe gastritis, parietal cell loss and metaplastic changes. These experiments have clearly shown how unrelated pathogens may interact and result in different clinical outcomes of the infected host. A strong immune response that results in severe inflammation will also cause a cascade of apoptotic changes in the mucosa. A strict balance between proliferation and apoptosis is needed, as its disruption may result in uncontrolled proliferation, transformation and metaplasia. The Fas Ag pathway is the leading cause of apoptosis in the Helicobacter-induced inflammation. One mechanism for escaping Fas mediating apoptosis is upregulation of MHCII receptor. Fas Ag and MHCII receptor interaction inhibits Fas mediated apoptosis by an impairment of the Fas Ag receptor aggregation when stimulated by Fas ligand. Because H. pylori infection is associated with an upregulation of the MHCII levels on gastric epithelial cells, this indeed may be one mechanism by which cells escape apoptosis. The link between chronic inflammation and cancer is well known since the past century. Helicobacter infection is a prime example how a chronic inflammatory state is causing uncontrolled cell proliferation that results in cancer. The cell biology of “cancer” is regarded not as an accumulation of cells that divide without any control, but rather as an organ formed of cancer stem cells, tumor stromal support cells, myofibroblasts and endothelial cells, which function as a group. The properties of the cancer stem cells are to self-renew and differentiate into tumor cells thus maintaining the tumor grow, emphasizing that a striking similarity exists between cancer stem cells and tissue stem cells. We looked into what role would BMDCs play in chronic inflammation that causes cancer. Using the mouse model of Helicobacter induced adenocarcinoma we discovered that gastric cancer originates from a mesenchymal stem cell coming from bone marrow. We believe that chronic inflammation, in our case of the stomach, sets up the perfect stage for bone marrow stem cells to migrate to the stomach where they are exposed to inflammatory stimuli and transform into cancer stem cells. One of the mechanism by which the MSC migrate to the inflammation site is the CXCR4/SDF-1 axis. Our work sheds new light on Helicobacter induced gastric cancer pathogenesis. I hope that our findings will promote the development of new therapies in the fight against this deadly disease.