Medicine & Health

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  • (2023) Green, Nicole
    Thesis
    The onset of psychiatric disorders, such as autism spectrum disorder, schizophrenia, anxiety, and major depressive disorder, can be associated with both genetic and environmental factors. This thesis examines transcriptional changes caused by either genetic or environmental factors linked to psychiatric disorders. We investigated regulatory regions which harbour mutations associated with psychiatric disorders, and the effects of early life stress on the developing brain and its consequent adverse mental health outcomes. Here, we assessed the function of active enhancers in the brain to understand their regulatory roles, and their link to mutations associated with psychiatric disorders. This was achieved using a CRISPR interference (CRISPRi) screen to repress almost one thousand enhancers in primary human astrocyte cells. The effect of enhancer repression on transcription was measured using single cell mRNA sequencing (scRNA-seq), a which measures gene expression from individual cells. This thesis describes the first CRISPRi screen to be performed in brain cells, and the first to be performed in primary cells. Active enhancers targeted in this screen were characterised based on their chromatin state and through analysing enhancer marks from relevant published data. Regions of open chromatin are a marker for enhancers and were determined in primary human astrocytes using ATAC-seq, a method which transposes DNA in an open chromatin state, before using sequencing to determine regions of open chromatin. Candidate enhancers were filtered based on the ability to detect potential target genes of the enhancer by scRNA-seq, to which gene expression was required to be above an expression threshold. The expression of potential target genes was determined by mRNA-seq, and candidate enhancers were required to be in the same topologically associated domain as a detectable target gene expressed above the threshold. The CRISPRi screen was optimised with a pilot screen to determine the degree of silencing due to CRISPRi and the detectability of the transcriptional effect of silencing enhancers by CRISPRi using scRNA-seq. A large-scale CRISPRi screen was then performed to investigate and validate candidate enhancers in primary human astrocytes. This identified almost one hundred enhancers active in the human brain that have disease-associated variants, and 140 enhancer target genes associated with disease. We also investigated the transcriptional effect of early life stress using murine models. A limited nesting (LN) model was used, as this emulates human maternal neglect and has been previously shown to increase measures of anxiety like-behaviours in rats. We performed RNA sequencing on the prefrontal cortex (PFC) of rats that underwent an LN paradigm as a model for early life stress. Our results indicated that this primarily led to transcriptional repression in the PFC. We further identified a molecular signature of LN in rat PFC that, when compared with other studies, is observed across ELS protocols and replicable in other murine models.

  • (2023) Tesema, Azeb Gebresilassie
    Thesis
    Mortality from non-communicable diseases (NCDs) in Africa will overtake combined mortality from communicable, maternal, neonatal and nutritional diseases by 2030. Despite global declarations on the need for comprehensive primary health care (PHC) to address NCDs, the policy responses of African nations have been limited. In this thesis, I conduct a mixed methods study to assess the capacity of PHC systems to improve the prevention and management of NCDs in Africa, with a focus on Ethiopia. Section 1 summarises the NCD burden in Africa and provides an overview of the Ethiopian health system. Section 2 comprises Chapters 1 and 2. Chapter 1 maps progress against World Health Organization (WHO) targets for NCD care in the 47 countries of the WHO African Region. No country has met all recommended targets, and seven have met no targets. In Chapter 2, I propose priority areas to address these gaps, including (i) enhanced political commitment; (ii) service redesign; (iii) workforce capacity strengthening; (iv) improved treatment protocols; (v) comprehensive resource gap analysis; (vi) large-scale implementation; and (vii) support from multilateral institutions. In Section 3, which comprises Chapters 3–5, I focus on Ethiopia. Chapter 3 presents an in-depth qualitative study that corroborates the Chapter 1 findings, highlighting health system governance challenges to strengthening PHC (limited political commitment, inter-sectoral coordination and funding) combined with operational constraints (an insufficiently skilled and supported PHC workforce, fragmented information systems, and inadequate equipment and medicines). Chapter 4 presents a multi-level analysis of the National Ethiopia Health Extension Program (HEP) Survey. The analysis showed that provision of essential non-NCD HEP services was both positively and negatively associated with NCD service provision. In Chapter 5, I assess NCD training, supervision and performance appraisal for health extension workers (HEWs). I found variable quality in NCD training for HEWs and supervisors; inconsistent and somewhat punitive supervision practices; and non-transparent performance appraisal, which has led to HEW de-motivation. In Section 4, I synthesise all study findings and discuss the policy implications for Ethiopia and Africa more broadly. I also appraise the opportunities to strengthen NCD service delivery in the context of the COVID-19 coronavirus disease pandemic and the ongoing civil war in Ethiopia. I conclude with recommendations for future research and policy to increase Ethiopia’s capacity to provide comprehensive PHC that includes NCD care.

  • (2023) Afzal, Hafiz
    Thesis
    Sensory signals informing about frictional properties of a surface are used both for perception to experience material properties and for motor control to be able to handle objects using adequate manipulative forces. There are fundamental differences between these two purposes and scenarios, how sensory information typically is obtained. This thesis aims to explore the mechanisms involved in the perception of frictional properties of the touched surfaces under conditions relevant for object manipulation. Firstly, I show that in the passive touch condition, when the surface is brought in contact with immobilised finger, humans are unable to use existing friction-related mechanical cues and perceptually associate them with frictional properties. However, a submillimeter range lateral movement significantly improved the subject's ability to evaluate the frictional properties of two otherwise identical surfaces. It is demonstrated that partial slips within the contact area and fingertip tissue deformation create very potent sensory stimuli, enabling tactile afferents to signal friction-dependent mechanical effects translating into slipperiness (friction) perception. Further, I demonstrate that natural movement kinematics facilitate the development of such small skin displacements within the contact area and may play a central role in enabling the perception of surface slipperiness and adjusting grip force to friction when manipulating objects. This demonstrates intimate interdependence between the motor and sensory systems. This work significantly extends our understanding of fundamental tactile sensory processes involved in friction signaling in the context of motor control and dexterous object manipulation tasks. This knowledge and discovered friction sensing principles may assist in designing haptic rendering devices and artificial tactile sensors as well as associated control algorithms to be used in robotic grippers and hand prostheses.

  • (2023) Rosewarne, Emalie
    Thesis
    Non-communicable diseases (NCDs) are the leading cause of death and disability globally. NCDs are largely preventable, with the majority attributable to four behavioural risk factors: physical inactivity, harmful use of alcohol, tobacco use and exposure to tobacco smoke, and unhealthy diets. Dietary risks are a leading mortality risk factor: eight million deaths and 188 million disability-adjusted life years each year can be attributed to dietary risks worldwide. Intervention strategies to reduce dietary risks can be broadly grouped into environmental, behavioural, or multi-component strategies. Food environment policies focus on changing the physical, social, political, or economic environment that individuals and groups live in. Policies that directly impact the food environment include food reformulation targets and nutrition standards for publicly-funded institutions. Such interventions alter food access, availability, and affordability, and can create environments that support individuals to make healthier choices. The aim of this thesis is to evaluate the design, implementation and impact of nutrition policies and advocacy strategies to improve the food environment in Australia and globally. Formative research included two secondary analyses of a global review of salt reduction programs to understand the extent and scope of national reformulation strategies and nutrition standards for publicly funded institutions, including whether these policies are designed in line with current evidence. This was followed by comprehensive policy analyses of the Australian Government’s Healthy Food Partnership reformulation strategy and state- and territory-based nutrition standards for publicly funded institutions to evaluate policy design, potential impact, and scope for strengthening policies. Finally, a five-study, mixed methods evaluation of the Victorian Salt Reduction Partnership’s (VSRP) advocacy program was undertaken to evaluate the program’s design and implementation, and the impact on the food environment. This work found that one-third of countries globally had sodium reformulation programs and a similar number had institutional nutrition standards. Most policies were in high-income countries, and Europe was the region with the highest number of policies. Policy design was highly variable and substantial opportunity to better align policies with best practice evidence and global guidance was highlighted. These opportunities include applying sodium reduction targets to a wider range of foods using the World Health Organization’s global sodium benchmarks and applying school nutrition standards to all types of publicly funded institutions. There is ample opportunity to increase the public health impact of Australian food environment policies through alignment with evidence of best practice globally. The majority of government-led reformulation targets were determined to be too conservative, and therefore likely to have limited impact on the food supply. For most food categories, more than 43% of products were already meeting the reformulation targets prior to implementation, a greater than 10% difference from the government-proposed criteria of one-third. There were also no plans for implementation, monitoring, or evaluation. The design of institutional nutrition policies may be a barrier to implementation and prevent the policies from having their intended impact: policy designs were complex, many lacked key components such as accountability mechanisms, and there were differences within and between institution types and jurisdictions. The five-study evaluation of the VSRP advocacy strategy demonstrated the multi-faceted sodium reduction program had no impact on reducing sodium levels in the food supply or government policy; however, short-term objectives (e.g., project outputs) were achieved and important lessons were generated that will be useful for future public health partnerships and interventions. The establishment of a Partnership or guiding coalition with diverse skills and experience facilitated collaborative action, capacity building and execution of the intervention. Continual monitoring and evaluation of implementation informed strategy adaptations that allowed optimisation of the Partnership strategy. The political advocacy strategy, which involved advocating for stringent government-led sodium reformulation targets, enabled the dissemination of VSRP resources to food manufacturers, although had little impact on strengthening the draft targets. The media advocacy strategy, which involved the dissemination of sodium-monitoring data through media releases, was a useful tool to gain access to the media and reach consumers with salt reduction messages, and to engage food manufacturers in discussions about salt reduction. However, in the absence of an established government reformulation policy, this food industry engagement did not translate into meaningful reductions in sodium in the packaged food supply. More effective communication between strategic and implementation partners could have improved program implementation and outcomes. Greater emphasis on developing and maintaining relationships with policymakers could have increased influence on government policy and public health impact. Existing institutional nutrition policies in Victoria also require strengthening through improvements to policy design, governance, and support services, and overcoming barriers within and external to implementing organisations, to propel state-wide progress. Overall, this thesis identified substantial scope to accelerate the local and global impact of food environment policies. In addition to improving the design of food environment policies in line with best practice evidence and global guidance, implementation of food environment policies could be strengthened through the provision of support services, step-by-step guidance, and additional tools/resources. Further, performance measurement methods need to be integrated into policy monitoring systems to enhance evaluation approaches and facilitate policy enforcement. Strong government leadership and regular monitoring of measurable goals are essential elements of successful policies. Three step-wise frameworks were developed from the evidence generated and lessons learnt in this thesis to support i) governments to design impactful food environment policies and monitor them, ii) end-users to effectively implement food environment policies and iii) public health professionals to persuasively advocate for food environment policies. Thus, this thesis will be useful for informing future public health partnerships, interventions, and government policies to reduce the global burden of diet-related NCDs.

  • (2023) Samir, Jerome
    Thesis
    The adaptive immune response is responsible for recognising, containing and eliminating viral infection, and protecting from further reinfection. This antigen-specific response is driven by T and B cells, which recognise antigenic epitopes via highly specific heterodimeric surface receptors, termed T-cell receptors (TCRs) and B cell receptors (BCRs). The theoretical diversity of the receptor repertoire that can be generated via homologous recombination of V, D and J genes is large enough (>1015 unique sequences) that virtually any antigen can be recognised. However, only a subset of these are generated within the human body, and how they succeed in specifically recognising any pathogen(s) and distinguishing these from self-proteins remains largely unresolved. The recent advances in applying single-cell genomics technologies to simultaneously measure the clonality, surface phenotype and transcriptomic signature of pathogen- specific immune cells have significantly improved understanding of these questions. Single-cell multi-omics permits the accurate identification of clonally expanded populations, their differentiation trajectories, the level of immune receptor repertoire diversity involved in the response and the phenotypic and molecular heterogeneity. This thesis aims to develop a bioinformatic workflow utilising single-cell multi-omics data to explore, quantify and predict the clonal and transcriptomic signatures of the human T-cell response during and following viral infection. In the first aim, a web application, VDJView, was developed to facilitate the simultaneous analysis and visualisation of clonal, transcriptomic and clinical metadata of T and B cell multi-omics data. The application permits non-bioinformaticians to perform quality control and common analyses of single-cell genomics data integrated with other metadata, thus permitting the identification of biologically and clinically relevant parameters. The second aim pertains to analysing the functional, molecular and immune receptor profiles of CD8+ T cells in the acute phase of primary hepatitis C virus (HCV) infection. This analysis identified a novel population of progenitors of exhausted T cells, and lineage tracing revealed distinct trajectories with multiple fates and evolutionary plasticity. Furthermore, it was observed that high-magnitude IFN-γ CD8+ T-cell response is associated with the increased probability of viral escape and chronic infection. Finally, in the third aim, a novel analysis is presented based on the topological characteristics of a network generated on pathogen-specific, paired-chain, CD8+ TCRs. This analysis revealed how some cross-reactivity between TCRs can be explained via the sequence similarity between TCRs and that this property is not uniformly distributed across all pathogen-specific TCR repertoires. Strong correlations between the topological properties of the network and the biological properties of the TCR sequences were identified and highlighted. The suite of workflows and methods presented in this thesis are designed to be adaptable to various T and B cell multi-omic datasets. The associated analyses contribute to understanding the role of T and B cells in the adaptive immune response to viral-infection and cancer.

  • (2023) Ajisegiri, Whenayon
    Thesis
    Non-communicable diseases (NCDs) account for 71% of deaths globally, and 77% of NCD-related deaths occur in low-income and middle-income countries. In Nigeria, 29% of deaths in 2016 were attributed to NCDs. However this proportion is expected to substantially increase in the next decade. In response to the growing NCD burden, Nigeria has launched health reform policies with a focus on integrating NCD management with primary health care (PHC) services. This includes enhanced workforce capacity, particularly through training non-physician health workers. Despite the emphasis on PHC strengthening, there is a lack of timely and relevant evidence to inform policy and service delivery. This poses major challenges for the development of effective NCD prevention and control programs. In this thesis, I appraise policy, health service and workforce factors contributing to NCD prevention and control programs in Nigeria. Chapter 1 describes the NCD burden and provides an overview of the country’s health system structure. Chapters 2 and 3 include a document analysis of NCD prevention and control policies, clinical guidelines and subnational policies implemented at regional levels. Key findings include a lack of consideration for decentralisation and ineffective coordination mechanisms for the implementation of NCD programs. Chapter 4 includes mixed methods research to understand the organisational context of NCD service delivery in PHC facilities. Several systemic enablers and barriers to implementing NCD interventions were identified: physicians working at PHC services played a critical enabling role, and negative perceptions of PHC as inferior care acted as a major barrier. Chapter 5 includes further mixed methods research to understand the practices of community health workers (CHWs) in relation to NCD care. It was observed that many CHWs are conducting informal task-shifting, performing the work of physicians for NCD care in PHC facilities. Chapter 6 includes a discrete choice experiment with 300 CHWs to understand CHW motivational preferences for service delivery. Policy scenario modelling predicted that an employment package comprising educational opportunities, career progression opportunities and an additional 10% of salary as incentives would be most appealing to CHWs. In the final chapter (Chapter 7), I synthesise findings from all studies to identify the priority policy issues for improving NCD integration with PHC services in Nigeria. Recommendations focus on physical, workforce and organisational resource mobilisation via several ‘motive forces’, including improved goal alignment, harnessing of team action, enhanced organisational support, local and national policy responsiveness, and continuous learning.

  • (2023) Fichter, Christina
    Thesis
    Gene and cell therapies hold enormous promise for many presently incurable diseases. Although the efficiency and safety of site-specific genome editing systems have improved tremendously over the past decade, the delivery of these novel technologies to specific target cells remains a key technical bottleneck for the clinical translation. Since long-term expression of DNA-modifying enzymes can be associated with cytotoxicity, transient delivery is desirable to reduce the risk of mutagenesis from off-target activity. A main target for gene and cell therapies are T cells. In order to achieve gene transfer into human T cells, current gene delivery techniques involve significant levels of T cell activation and ex vivo expansion prior to lentiviral or retroviral transduction. However, ex vivo stimulation and expansion have been shown to significantly alter the natural physiology of T cells. To address these challenges, we have developed a non-integrative lentiviral gene therapy platform to focus on genetic modifications of resting CD4+ T cells. To overcome barriers, present in this notoriously hard to transduce cell population, we have re-engineered lentiviral particles from the inside-out. Firstly, we have identified key viral glycoproteins that can mediate cytosolic delivery of transgenes across resting CD4+ T cell plasma membranes. Secondly, by replacing HIV nucleocapsid with the coat protein of the MS2 bacteriophage and substituting lentiviral RNA packaging motifs (LTR and packaging signal Ψ) with a series of MS2 stem loops that facilitate RNA binding to the MS2 coat protein, we have enabled the packaging of CRISPR/Cas9 components into these particles. In this setting, we observed expression of biologically active mRNA in up to 65% of resting CD4+ T cells. Furthermore, with our CRISPR/Cas9 delivery platform we have achieved genome editing efficiencies of >85% in human cell lines, and >20% in primary CD4+ T cells. With the emergence of SARS-CoV-2, we have shifted our focus on the development of a novel viral vector for gene delivery. Using a SARS-CoV-2 VLP scaffold, we have enabled the packaging and transient delivery of mRNA into target cells by tethering a reporter gene to the recently identified SARS-CoV-2 packaging signal. Equipping these VLPs with Spike glycoproteins from emerging SARS-CoV-2 variants, we observed an increase in transient mRNA delivery to >50% of target cells. This platform may give us a novel set of viral vector solutions to use for many respiratory diseases, including coronavirus disease (COVID-19) itself. Taken together, this thesis describes the development of robust delivery platforms for the transient delivery of biologically active transgenes to multiple cell types.

  • (2023) Seyedzadeh, Hadi
    Thesis
    Cytotoxic T lymphocytes (CTLs) can target and induce apoptosis in cancer cells during the anti-tumour immune response. However, the cytotoxicity (or killing) function of CTLs can be perturbed directly by cancer cells or via the tumour microenvironment (TME). Among the various factors in TME that can influence T cell function, the effect that mechanical properties of the extracellular matrix (ECM) have on CTL responses is unclear. Research into CTL-mediated cytotoxicity is typically performed in either two-dimensional (2D) matrix-free culture or in complex in vivo animal models. In vitro, 2D studies are limited in recapitulating the CTL response in vivo, whereas it is very difficult to manipulate the TME and perform high-throughput experiments using in vivo models. Recently 3D culture models have been introduced to fill the gap between 2D and in vivo studies. In this study, we used an automated 3D bioprinter to incorporate OT-I T cells and cognate and non-cognate target cells in a polyethylene glycol (PEG)-based hydrogel and studied the killing efficiency in comparison with 2D culture and manually-prepared gels. Here, we showed that the 3D bioprinter embeds both CTLs and target cells in the hydrogel and enables control over the dimensions of the embedding matrix as well as the number and spatial organisation of cells. Moreover, the ability to digest the gel and release the cells allowed us to perform killing efficiency comparisons and downstream high-throughput CTL functional analyses using flow cytometry. This novel 3D cell culture system allowed us to investigate the effects of tunable ECM mechanical properties in a reproducible cytotoxicity model of matrix-embedded CTL and target cells. Our results demonstrate that in matrices with higher density, CTL killing efficacy was compromised. This demonstrates that matrix stiffness, independent of matrix porosity or other variable characteristics, has a large impact on CTL function. From another perspective, cancer cells can directly induce dysfunctional programming in CTLs. Repeated stimulation of the T cell receptor (TCR) on CTLs with the tumour-associated antigen leads to overexpression of inhibitory receptors such as programmed cell death (PD)-1 on the surface of T cells, leading to aberrant response and eventually tumour escape. TCR signalling machinery can be affected by the expression of inhibitory receptors, but it is not clear whether inhibitory receptors alone are responsible for the dysfunction of exhausted T cells or to what degree other mechanisms contribute. To address this, we used a mouse model of T cell dysfunction, finding that T cells could exhibit a dysfunctional phenotype with minimal upregulation of inhibitory receptors and without downregulation of TCR. Instead, we found a decrease in the proximal signalling kinases Lck and ZAP70, specifically in dysfunctional cells. To confirm these results, we developed a human primary in vitro CD8+ T cell dysfunction model, which allowed us to study the effect of repeated antigen stimulation on the inhibitory receptors expression and expression of Lck and ZAP70 in human T cells. In this model, we again found that dysfunctional T cells had lower expression of Lck and ZAP70, confirming the results from the mouse model. Future experiments could be performed in which Lck and/or ZAP70 expression is enhanced in dysfunctional T cells. If this restores the functional phenotype, it may confirm that a low level of Lck and ZAP70 protein expression is a cause of T cell dysfunction. This research sheds light on how the external (matrix stiffness) and internal factors (TCR stimulation) affect the CTL response.

  • (2023) Balachandran, Harikrishnan
    Thesis
    The protection offered by B cell responses after viral infection or vaccination features neutralising antibodies produced by plasmablasts and memory B cells (MBCs) which can provide long lasting immunity against reinfection. Hence understanding the factors associated with MBC persistence is crucial for vaccine design. Murine and human studies have implicated initial antigen affinity, bystander activation, activation-induced cytidine deaminase (AID) expression, and chemokine levels as being associated with persistence of antigen-specific MBCs, but detailed understanding of the genesis of long-lived MBCs in humans is lacking. During the initial phase of COVID-19 pandemic there were multiple studies characterising natural immune responses against SARS-CoV-2 infection, but then the interest shifted towards understanding vaccine-induced immunity. With the spread of variants of concern and breakthrough infections in vaccinees, understanding the longevity of natural immunity is essential to design improved vaccines. The primary objective of this thesis was to develop a highly specific tetramer for flow cytometry-based isolation of SARS-CoV-2 specific MBCs, evaluate their longevity, and identify biomarkers and transcriptomic signatures present early after infection that associate with durable MBCs. The samples used in this thesis were selected from the COVID-19 Outbreak Samples in NSW (COSIN) cohort, an ongoing prospective cohort evaluating the natural history of the SARS-CoV-2 infection in NSW. This thesis observed that at one year post infection, despite declining antibody titers, maintenance of neutralisation breadth and a variant specific protection (45% - 76%) against symptomatic disease. Encouragingly 80% of participants who had SARS-CoV-2 specific MBCs during early convalescence had detectable MBCs at one year. Based on MBC persistence, the cohort was divided into two groups – “Maintained” and “Dropped”. Early antigen specific CD4+ T cell responses was associated with maintenance of antibody neutralisation breadth and MBC durability. Smart-seq2 analysis of the MBCs identified ongoing maturation of the B cell receptor in both grand transcriptomic differences between the two groups. The transcriptomic analysis revealed that an enhanced activation profile of mature, proliferating MBCs was associated with persistence at one year. These findings provide initial identification of biomarkers that can be used to predict the durability of vaccine induced immunity.

  • (2023) Fernando, Bentotage Samitha Madushan
    Thesis
    Indoleamine 2,3-dioxygenase 1 (IDO1) is a critical immunoregulatory heme enzyme expressed in antigen-presenting cells (APCs). While IDO1’s immunoregulatory actions have been documented to alter a diverse range of diseases, less is understood about the biochemical pathways controlling the enzyme in APCs. Chapter 3 studies established that heme-iron metabolism regulates IDO1 in primary human APCs. Thus, iron chelators inhibited IDO1 expression independent of STAT1 or NF-κB, which represent important signals for IDO1 transcription. The heme precursor aminolevulinic acid (ALA) or heme biosynthesis inhibitor succinylacetone, stimulated or inhibited IDO1, respectively, at the posttranslational level. Prior upregulation of the heme catabolic enzyme heme oxygenase-1 (HO-1) inhibited IDO1 at the post-translational or transcriptional levels in IFNγ- or LPS-stimulated APCs, respectively. Mechanistic studies showed that HO-1 post-translationally controls IDO1 by reducing heme bioavailability for insertion into IDO1 and generating CO that directly inhibits IDO1 activity or suppressed IDO1 expression by inhibiting STAT1 and NF-κB. Chapter 4 studies showed that IFNγ and LPS differentially modulate glycolytic and mitochondrial parameters in human MDMs and that IDO1 is subject to transcriptional and/or post-translational control by different metabolic pathways including glycolysis, Krebs cycle metabolites itaconate and succinate, mitochondrial respiration and generation of reactive oxygen species, and mitochondrial fatty acid oxidation. These studies revealed complex regulatory crosstalk between cellular energy metabolism and IDO1. Chapter 5 examined the IDO1-dependent changes in the proteomic profile of MDMs stimulated with IFNγ/LPS or infected with Dengue or West Nile virus. Proteomic analysis indicated that IDO1 activity modulates various immune and metabolic pathways in MDMs, which have the potential to impact on their function. As cell immune responses play complex roles in the immunopathology accompanying virus infection, Chapter 6 established multiparametric flow cytometry workflows to comprehensively detail the changes in immune cell sub-set phenotypes in the lungs and lymph nodes of influenza-infected mice. These workflows are available to study the immunoregulatory actions of IDO1 in influenza. This work provides important new insights into the roles and regulation of IDO1 in APCs and established methods for studying IDO1’s immune function in vivo.