CD8+ T cells are a crucial component of the adaptive immune system and are required for optimal protection from most pathogens and cancer. They function by secreting pro-inflammatory cytokines and by directly eliminating infected and malignant cells. In order to be effective, CD8+ T cells must be activated through a complex sequence of steps involving engagement of the antigen-specific T cell receptor (TCR) and other receptors, which orchestrate transcriptional, epigenetic, and metabolic changes that direct the differentiation of the responding cells. Following optimal activation, naive CD8+ T cells rapidly undergo clonal expansion and effector differentiation that enables prompt resolution of infection. Following pathogen clearance, a fraction of effector CD8+ T cells differentiate into long-lived memory CD8+ T cells that provide robust protection from re-challenge with the same microbe. However, in the context of persistent abundance of antigen and inflammation, such as in chronic infections and in cancer, the T cells instead become gradually more dysfunctional – a state known as T cell exhaustion. The overarching goal of this thesis is to identify the cardinal features and molecular mechanisms associated with three main states in which CD8+ T cells exist: T cell memory, T cell exhaustion, and T cell effector differentiation. I used two complementary approaches to examine CD8+ T cells at the different states in vivo. First, I used classical immunology techniques including knockout mice and cellular phenotypic analyses to examine the role of cell surface molecules PD-1 and CD39 on CD8+ T cells in the context of memory and exhaustion, respectively. Secondly, I developed a novel experimental platform that enables gene perturbation in naive CD8+ T cells in vivo during their differentiation. I used this approach to systematically interrogate the transcriptional programming of activated CD8+ T cells and to identify novel regulators of effector differentiation. In a proof of concept study, I used this system to further define how the transcription factor BATF regulates CD8+ T cell activation. Additionally, I used this experimental platform to systematically interrogate the functional role of a set of ~80 transcription factors in CD8+ T cell differentiation, and identified TGIF1 as a novel regulator of this process. The role of the co-inhibitory receptor PD-1 on CD8+ T was examined in mice using an acute respiratory infection model. PD-1 is a co-inhibitory receptor that is up-regulated on T cells following activation and recruits SHP1/2 phosphatases to directly antagonize signals through the TCR and this way inhibit the activation of T cells. It is down-regulated following the resolution of an acute infection but remains persistently expressed on CD8+ T cells in chronic infections and cancer. As such, PD-1 has been exhaustively studied for its contribution to the functional exhaustion of T cells. However, its role in acute infections remains less defined. We found that this receptor prevents over-activation and over-expansion of CD8+ T cells following initial differentiation, and is crucial for optimal differentiation of effector CD8+ T cells into functional memory cells. Exhausted CD8+ T cells express several markers distinctive of the state. Some, like PD-1, Tim-3, and Lag-3 are well known. However, genome-wide transcriptional studies identified numerous additional genes that are differentially expressed in the exhausted state. Thus, we hypothesized that additional markers may provide characteristic features of the exhausted cell state and may function in chronic infections. We investigated one such gene – ENTPD1 – that encodes for CD39. This cell surface molecule is an ectonucleotidase that hydrolyzes extracellular ATP into ADP and AMP, which can be further broken down to immunosuppressive adenosine by CD73. In the context of the immune system, CD39 has largely been studied on CD4+ regulatory T cells, which use CD39 as a mechanism to suppress immune responses. However, surprisingly, we found that CD8+ T cells can also express CD39, but its expression is largely restricted to terminally exhausted CD8+ T cells. These cells are most dysfunctional as measured by the most limited proliferative capacity and ability to produce pro-inflammatory cytokines. We have observed this biology in both human and mouse chronic viral infections. Additional studies further demonstrated the importance of CD39 and the purinergic pathway in regulating lethal immunopathology associated with chronic LCMV infection in mice. In addition to interrogating memory and exhaustion fates of CD8+ T cells, we also examined the initial regulatory programs involved in CD8+ T cell differentiation in vivo through gene silencing. Gene perturbation in naive T cells without prior cellular stimulation has been a continuous challenge in the field. To circumvent this limitation, we engineered a novel experimental platform that enables inducible gene knock-down in any immune cell in mice in vivo without prior manipulation of these cells. Initially, I validated this system by knocking down BATF and confirmed its essential role in CD8+ T cell responses to acute LCMV infection. Additionally, leveraging the inducible nature of the platform, I showed that BATF functions in the early stages of T cell activation but becomes dispensable once its transcriptional program is initiated. Several other transcription factors such as T-bet, Eomes, Bcl6, and Blimp-1 have been described to regulate CD8+ T cell differentiation. However, numerous additional transcription factors may function in this process based on their rapid up-regulation following T cell activation. I used the novel platform to systematically test the functional relevance of ~80 additional transcription factors in a pooled setting. These experiments identified several novel candidate regulators of this process. We validated one such gene – Tgif1 – to confirm its role in the effector CD8+ T cell differentiation following acute LCMV infection and provide clues to the potential mechanism in which it may function. The above projects have benefited significantly from genome-wide transcriptional datasets of cells at various states or of different genotypes that we generated or that originate from published studies. One particularly powerful approach to examine differences between different groups is gene set enrichment analysis (GSEA) that examines coordinate up- or down-regulation of sets of genes rather than assessing differential expression of specific genes. This is particularly important because changes in biological processes are often guided by relative small changes of groups of genes that act in concert rather than by a robust expression change of a single gene. This approach, however, is only informative if a relevant gene-set collection is used to analyze the data. Existing collections are largely centered around cancer biology and general biological processes but no extensive gene-set collection existed that contained information describing immune processes. Thus, we created ImmuneSigDB – the largest collection of immunology-focused gene sets to date by identifying, annotating, and reanalyzing ~400 published immunology studies. To show its broad use, we used it to examine the cross-species conservation of transcriptional responses in the immune system. We focused on analyzing transcriptional data from systemic responses to sepsis using GSEA and a novel approach, called leading edge metagene analysis. Using these approaches, we uncovered shared and species-specific biology in mouse and human transcriptional responses to sepsis. Deciphering CD8+ T cell biology is key for conceptualizing new medical interventions that may boost their activation, memory development, and rejuvenation from functional exhaustion. We have determined that PD-1 is essential for optimal CD8+ T cell memory responses, and that BATF is a key transcription factor initiating effector T cell transcriptional programming. We also identified CD39 as a new marker of terminally exhausted CD8+ T cells and uncovered a key role for purinergic signaling in regulating lethal immunopathology in LCMV Clone 13 infection in mice. Furthermore, we developed a new experimental platform that enables systematic interrogation of gene function in any hematopoietic cell type by inducible knock-down of genes and identified TGIF1 as a novel negative regulator of CD8+ T cell responses. We have also developed a new computational resource to improve analyses of transcriptional profiles in the immune system. Together, the body of work presented in this thesis advances our knowledge of major states of CD8+ T cell biology, uncovering both novel mechanisms underlying CD8+ T cell function, as well as highlighting potential novel therapeutic targets that may be transformative in creating better vaccines, treating infections, or fighting cancer. / Medical Sciences

In order to counter pathogen infection while preventing autoimmune responses, the human innate immune system must be precisely regulated to distinguish “self” from “non-self”. Pattern recognition receptors detect “non-self” pathogen RNAs and initiate antiviral signaling. Accumulated evidence suggests that host “self” RNAs contain modified nucleotides that evade or suppress immune signaling; however, the precise mechanisms are not understood. Defining these mechanisms is relevant toward understanding the biology of immunity as well as the applied use of RNAs as therapeutic molecules, where reducing ligand immunogenicity is essential. Evidence from our lab and others’ suggests that the cytosolic RNA helicase RIG-I (retinoic acid inducible gene-I) detects not only the 5’ terminus and double-stranded nature of RNA, but also the presence/absence of modified nucleotides. In the present study, we use a model RNA ligand (polyU/UC), derived from the 3’ untranslated region of the hepatitis C virus RNA, to dissect the mechanisms by which RNAs containing nucleotide modifications suppress or evade RIG-I signaling. Five assays were developed to test our hypothesis that eight different nucleotide modifications, both natural and synthetic, share a common mechanism of innate immune evasion. In vitro transcribed 5’-triphosphate polyU/UC RNA containing canonical nucleotides potently activates the RIG-I signaling pathway in transfected cells, culminating in an antiviral state. When transcribed with any of eight modified nucleotides, the polyU/UC RNA suppressed the RIG-I antiviral response. Unexpectedly, the modified nucleotides had different effects on RIG-I:RNA binding affinity, as well as RIG-I conformational change. The data suggest that multiple RIG-I evasion/suppression mechanisms associated with different modified nucleotides may have evolved to effect a common result of interrupting innate immune signaling responses to “self” RNA. Our findings hold implications for understanding the co-evolution of the innate immune response and RNA modification pathways across domains of life, as well as for defining approaches for testing the multitude of naturally occurring and synthetic nucleotides that may have utility in the design of therapeutic RNAs. / Medical Sciences

Endocytosis, which is normally associated with cellular processes such as nutrient uptake and ligand uptake, is used by many different viruses to gain entry into a susceptible cell and to promote infection. In earlier work it was shown that CD55 is the major attachment molecule for enterovirus 70 (EV70) on HeLa cells. EV70 is a member of the enterovirus genus. Enteroviruses use a variety of receptors and enter cells by different mechanisms. For example, poliovirus binds to the poliovirus receptor (PVR), and has been shown to enter by a novel endocytic mechanism which is clathrin-, caveolin-, and dynamin-independent but does rely on cholesterol (Danthi and Chow, 2004; DeTulleo and Kirchhausen, 1998; Kronenberger et al., 1998). Coxsackievirus A9, which binds to coxsackievirus and adenovirus receptor (CAR) and the integrin alpha5beta3, and coxsackievirus B4, which binds to CAR and CD55, have been shown to enter via lipid rafts (Triantafilou and Triantafilou, 2004; Triantafilou and Triantafilou, 2003); coxsackievirus B3 which also binds to CAR and CD55 has been shown to enter via clathrin-coated pits (Chung et al., 2005); and echovirus I which binds to the alpha2beta1 integrin, enters via a caveolin-mediated and/or alternative route that is dynamin- and lipid raft-dependent (Marjomaki et al., 2002; Pietiainen et al., 2004). The major objectives of the research described in this thesis were to determine if EV70 entered HeLa cells by endocytosis, and, if so, which endocytic pathway was exploited by this virus. In studies using drug inhibitors of endocytosis, chlorpromazine, which blocks clathrin-mediated endocytosis, reduced EV70 infection of HeLa cells, but cholesterol sequestering drugs had little or no effect on virus entry. These results suggested that EV70 entry was dependent on clathrin, but not on caveolae or lipid rafts. EV70 conjugated with Alexa fluorochrome 555 appeared to co-localize with clathrin light chains tagged with enhanced yellow fluorescent protein (EYFP), consistent with a clathrin-mediated mechanism for EV70 entry into HeLa cells. Expression of the dominant-negative mutant of epidermal growth factor receptor pathway substrate clone 15 (eps15), a protein required for recruitment of adaptor protein 2 (AP-2) and epsin (both required for targeting clathrin to the cellular membrane), and assembly of clathrin-coated pits and vesicles, reduced EV70 infection of HeLa cells by 30 % when compared to the null mutant of eps15, D3Delta2. These results seem to rule in clathrin-mediated endocytosis. However, expression of the dominant-negative mutant of caveolin-1 had little or no effect on EV70 infection of cells. These results would seem to rule out caveolin-mediated endocytosis. A dominant-negative mutant of dynamin-2 strongly inhibited EV70 infection of cells, and must play an essential role in entry. Thus, it is proposed that EV70 infection may occur through an endocytic mechanism that is dependent on both dynamin and clathrin, but independent of caveolin.

Current adenovirus retargeting strategies are unable to target the virus to specific cell types. In this study, we investigated whether fusion of a single-chain antibody, MR1, to the capsid protein IX (pIX) could target the virus to cancer cells expressing EGFRvIII. We show that addition of an endoplasmic reticulum signal peptide to pIX-MR1 significantly increased the ability of the fusion protein to bind its ligand. Use of the human CMV promoter rather than the native pIX promoter permitted a greater accumulation of the protein within the cell. Finally, addition of the HIV-1 Tat NLS caused pIX-MR1 to relocalize to the nucleus, the site of capsid assembly. Taken together, these results provide a foundation to design Ad vectors targeted to specific cells through the use of single-chain antibodies. Ultimately, the development of a tropism modified Ad vector would result in a tailored treatment for a particular acquired genetic disease.

Viral treatment of cancer has been described since the beginning of the twentieth century. With the increasing need for effective targeted cancer therapies, the oncolytic virus platform has been revisited. Clinical trials have demonstrated that oncolytic viruses are well tolerated, but their ability to eliminate tumor burden or effectuate cures is poor. This thesis attempts to address enhancing the oncolytic virus efficacy. The strategy is to modulate characteristics of the rhabdovirus by adding or swapping envelope genes of interest in order to enhance oncolytic activity, specifically by addressing tumor spread, immune evasion and safety. The fusogenic protein from reovirus is used to generate a recombinant VSV to enhance viral spread within the tumor. Similarly, recombinant VSVs are generated with envelope proteins either from other vesiculovirus members or from a beta-retrovirus, and used to demonstrate tumor infectivity in the presence of neutralizing antibodies. The recombinant VSV expressing the beta-retrovirus envelope protein demonstrates altered viral tropism, and like the replication incompetent VSV/FAST recombinant is not neurotoxic in animal models.

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HIV and the malaria parasite have great disease burdens world-wide, and because their endemic regions overlap, the risk of co-infection is great. Little is known about the impact one infection has on the other's progression, but given the number of people at risk of being co-infected and the magnitude of the disease burden associated with each disease, any interaction could have a large impact on public health As more attention has been given to the potential risks of this kind of co-infection, a number of studies have been initiated to investigate the effects of co-infection. Recent studies involving Plasmodium falciparum and HIV-1 co-infection have reported increased risk of clinical malaria episodes in HIV-infected individuals with the risk rising with increased immunosuppression. Malaria parasite infection has also been reported to increase the risk of HIV transmission and a more rapid disease progression. Due to ethical concerns and other complicating factors, such as other infections and malnutrition, co-infection studies in humans have provided limited information about the impact of co-infection. As such, an animal model for HIV/malaria parasite co-infection would greatly enhance investigating the impacts of co-infection This project sought to and was able to successfully combine two well established rhesus macaque models for HIV and P. vivax infections in humans. As a result of co-infection, there was an increased risk of a more rapid SIV progression. This was reflected in (1) a decreased group survival time, (2) a twice as rapid CD4+ T cell decline, (3) an impaired CD8+ T cell response, (4) an impaired B cell response, (5) an altered IgG response to both SIV and the malaria parasite, and (6) a more rapid decline in naive CD4+ and CD8+ T cells / acase@tulane.edu

Approximately 3% of the global population is chronically infected by the Hepatitis C Virus (HCV). The only approved treatment for chronic HCV infection, pegylated interferon-alpha (IFN-alpha) plus ribavirin clears the virus in less than half of genotype 1 infected patients. The precise molecular mechanisms of HCV resistance to IFN-alpha are unclear, therefore we sought to uncover HCV resistance mechanisms utilizing a stable human hepatoma cell line containing sub-genomic HCV that is resistant to interferon. Characterization of this resistant cell line revealed that a truncated IFNAR1gives rise to the IFN-alpha resistant phenotype. These in vitro findings were then confirmed in human explant liver samples obtained from patients with and without HCV infection. Next we employed a novel strategy utilizing constructs containing the TAD of STAT1 or STAT2 fused to IRF9 in an attempt to circumvent Jak-STAT cellular defects in the resistant cell line. The STAT2 TAD fusion product showed antiviral and immunoregulatory activity in the resistant cell line and caused minimal toxicity. A second novel strategy utilized a STAT1-CC double cysteine substituted construct that showed potent antiviral and immunoregulatory properties in the resistant cell line. In conclusion, IFNAR1 may play a role in IFN resistance in vivo and Jak-STAT pathway components can be engineered to overcome HCV resistance to IFN / acase@tulane.edu