京都大学 / 新制・課程博士 / 博士(医学) / 甲第23783号 / 医博第4829号 / 新制||医||1057(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 中山 健夫, 教授 上野 英樹, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24085号 / 医博第4861号 / 新制||医||1059(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 髙折 晃史, 教授 生田 宏一, 教授 金子 新 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

<p>Pulmonary infections caused by viruses, bacteria, mycobacteria and fungi, are a leading cause of death world-wide. Intracellular pathogens such as influenza virus and <em>M.tb</em> live inside host cells, making it difficult for the host to eliminate the pathogen. Adaptive T cell immune responses are required to clear intracellular pathogens. Rapid T cell priming and recruitment of effector T cells to the lung is critical to eliminating the pathogen and ultimately host survival, however, unchecked T cell responses can be detrimental. The research in this thesis examines T cell priming during pulmonary intracellular infections and examines the consequences of impaired/enhanced T cell responses at the lung mucosa.</p> <p><em>M.tb</em>, the bacterium that causes tuberculosis evades detection by the host and delays T cell priming. This delay is believed to allow <em>M.tb</em> to establish chronic infection. We show that DAP12 deficient mice prime T cell responses days sooner than wt controls, resulting in enhanced control of virulent <em>M.tb</em>. Enhanced T cell priming in DAP12 deficient mice is due to increased antigen presentation by professional antigen presenting cells in the local draining lymph nodes. These findings indicate that accelerating antigen presentation in the lymph node and T cell priming can dramatically improve host resistance to tuberculosis infection.</p> <p>Influenza infection in DAP12 deficient hosts also results in enhanced T cell activation however caused lethal lung pathology. We identified a previously unidentified role for CD4 T cells expressing FasL causing immunopathology. Furthermore, we show that DAP12 deficient antigen presenting cells are responsible for priming hyperactivated CD4 T cells and contribute to influenza mortality.</p> <p>Because T cell responses are so important in host defence against intracellular infection we examined the feasibility of using ex-vivo manipulated antigen presenting cells as a vaccine to generate T cell responses in the lung. Collectively these findings shed light on the factors which regulate host immune responses in the lung following intracellular infection and provide evidence that antigen presenting cells can be manipulated ex-vivo to elicit protective immune responses at the lung mucosa.</p> / Doctor of Philosophy (PhD)

<p>Pre-clinical and clinical data strongly support the use of immunotherapies for cancer treatment. Cancer vaccines offer a promising approach, however, the outcomes of clinical vaccine trials have been largely disappointing, prompting a need for further investigation. Using the B16F10 murine melanoma, we have investigated the local events within growing tumors following recombinant adenovirus immunization. In chapter 2, we investigated the ability of a pre-clinical vaccine to elicit only transient tumor growth suppression. We observed that tumors were initially infiltrated by a small number of highly functional tumor-specific CD8+ T cells following vaccination that instigated a rapid adaptive response in the tumor that suppressed local immune activity. In chapter 3, we questioned whether increasing the rate and magnitude of early immune attack would result in more robust tumor attack prior to tumor adaptation. Increasing the rate of tumor-specific CD8+ T cell expansion following vaccination resulted in tumor regression and durable cures in approximately 65% of treated mice. Further analysis revealed that tumor regression correlated with an early burst in immune attack that outpaced tumor adaptation. In chapter 4, we explored whether the same vaccine could be improved when combined with immunomodulatory antibodies. Vaccination combined with anti 4-1BB and anti PD-1 resulted in complete tumor regression and durable cure of >70% of treated animals and was associated with increased local immune activity. Gene expression profiling revealed a unique gene signature associated with the curative treatment, which was also associated with positive outcome in human melanoma patients. The described research sheds new light on mechanisms that limit the efficacy of therapeutic cancer vaccines. Namely, rapid tumor adaptation, triggered by early vaccine-induced CD8+ T cells, acts to suppress the local immune response prior to maximal immune attack. Strategies to overcome these adaptive processes should therefore be considered in future vaccine design.</p> / Doctor of Philosophy (Medical Science)

Chronic itch has a significant impact on quality of life for millions of patients worldwide, on a level comparable to that of chronic pain. Yet, although there are a host of effective drugs available for pain, there are no therapies that specifically target chronic itch. Current experimental approaches to investigate the pathogenesis of chronic pruritus and to test novel therapeutic agents are largely limited to rodent models. However, rodent models display significant dermatological, neurophysiological, and immunological differences from humans with chronic itch. The disadvantages of the current rodent paradigms call for the design of a valid primate model of chronic itch. For four years, we have monitored scratching behavior in a primate colony (n=35) of Cynomolgus macaques (Macaca fascicularis) suffering from idiopathic chronic itch. By comparing molecular and genetic analyses of the primates’ skin to their quantified scratching behavior, we attempted to characterize the underlying mechanisms of chronic itch in this model. Furthermore, the expression of itch-related proteins was examined in both the primate model and in humans with pruritic diseases. The first aim of the study was to characterize the underlying molecular and genetic basis of chronic itch in the primate model. We were able to distinguish specific peripheral targets related to pruritus by correlating the genetic and protein expression results to the primates’ scratching severity. In Aim 1a, RNA-sequencing was performed on skin biopsies from the primates to identify differentially expressed genes in pruritic, lichenified versus non-pruritic, non-lichenified skin. These results were then correlated to the quantified primate scratching behavior. This led to the identification of over 400 genes that were differentially expressed in the skin based on scratching intensity. Many of these differentially expressed transcripts were associated with sensory nerve fibers, keratinocytes, mast cells, or lymphocytes. Selected genes that were overexpressed and correlated to itch intensity were then targeted for immunohistochemical and proteomic analysis in Aim 1b. Immunohistochemical examination of the primate skin biopsies revealed that histamine levels were not elevated in primates that exhibited increased scratching behavior. However, mast cells containing tryptase were significantly increased in the skin of primates with severe scratching as compared to primates with mild scratching. The increased levels of gastrin-releasing peptide and substance P in lichenified skin were also found to be correlated to the primates’ scratching behavior. Of note, transient receptor potential channels V1, V3, and A1 were increased in the epidermis of primate skin, but the numbers of TRPV1+ and TRPA1+ nerve fibers were not significantly different between lichenified and non-lichenified skin. Transcriptome analysis of the opioid receptors and their ligands showed that primates with severe scratching behavior had a significant imbalance between the µ- and κ-opioid receptors and ligands. The µ-opioids had upregulated gene expression, while the κ-opioids were downregulated. In Aim 2, to further characterize this primate model of chronic itch, we compared immunohistochemical results from the primate studies to human findings. Lesional and non-lesional skin biopsies from patients with atopic dermatitis, psoriasis, and cutaneous T-cell lymphoma underwent immunohistochemical analysis in order to reveal the similarities and differences between the primate model and different types of chronic itch in humans. As in the primate model, substance P was found to be increased in the skin of lesional atopic and psoriasis skin. Additionally, similar to primate skin, human atopic and psoriatic skin had high levels of tryptase and its receptor in the epidermis. While IL-31 was only slightly elevated in primates, patients with cutaneous T-cell lymphoma or atopic dermatitis showed a significant correlation between itch severity and IL-31 levels. In conclusion, our primate model displayed expression patterns of many endogenous pruritogens and receptors that were similar to those of humans with atopic dermatitis or psoriasis. While the primate model did not completely mimic these specific pruritic diseases, the overlap of pruritic components suggests a commonality of signaling pathways across several different chronic itch states. The similarity of this primate model to human disease offers the combined advantages of experimental modeling and long-term behavioral follow-up. / Biomedical Sciences

M.tb, the causative agent of pulmonary tuberculosis (TB) remains one of the leading causes of infectious disease-based death worldwide. BCG, the only clinically approved TB vaccine, has been in use for almost a century to vaccinate against TB. Despite its success in protecting against disseminated forms of TB, it is unable to provide protection against pulmonary M.tb infection. Although there have been many recent efforts to enhance or replace BCG, our lack of understanding towards host immunity against M.tb has substantially hindered this goal. One aspect of pulmonary M.tb infection that remains poorly understood is the induction of Th1 immunity, which is substantially delayed in comparison to other pulmonary infections. This allows the bacteria to establish an infectious foothold within the host and impairs the ability of the host to clear the infection. Given the importance of the innate immune response in the induction of adaptive immunity, this delay in the establishment of Th1 immunity following pulmonary M.tb infection is likely due to a defect in the early innate immune response. However, the specific roles of this immune compartment in regards to T cell activation following pulmonary M.tb infection is still not well understood. As such, the scope of this thesis is to gain an increased understanding towards the role of the innate immune compartment in the generation of Th1 responses. Such insights will allow us to develop new strategies to improve upon future and existing TB vaccine design. / Thesis / Doctor of Philosophy (PhD)

A biomarker is an indicator of (ab)normal molecular or cellular processes found in tissues, blood, or other body fluids. It can also serve as an indicator of the response to an intervention, such as a drug response, toxic exposure, or disease. As a measurable and evaluable characteristic that indicative underlying biological processes, biomarkers can be used in immunology, toxicology, cancer research, and in a variety of application scenarios. These applications include the use of biomarkers as diagnostic tools, predictors of disease prognosis, monitoring clinical response to an intervention, or as tools for staging a disease. Here, we aimed to discover gene-based biomarkers using transcriptome data, including microarray and RNA-sequencing (RNA-Seq). This thesis consists of two thematically distinct parts, but with the common goal of identifying and confirming robust biomarkers through meta-analysis approaches. In the first part, we identified and verified biomarker signatures that robustly evaluate T-cell activation in a time-resolved manner. We applied a meta-analysis of anti-CD3/CD28 induced CD4+ T-cell activation kinetics of publicly available transcriptome-wide time series using a random effects model. We used non-negative matrix factorization, an unsupervised deconvolution method, to infer changes in biological patterns over time. For verification and to further map a wider variety of the T-cell landscape, we performed a time series of transcriptome-wide RNA sequencing on activated blood T-cells. We identified time-resolved gene expression profiles comprising 521 genes of up to 10 disjunct time points during activation and different polarization conditions. They cover sustained repressed, intermediate, and late response expression rates across multiple T-cell populations, thus defining consensus biomarker signatures for T-cell activation. These biomarker signatures are a valuable source for e.g., monitoring transcriptional changes during T-cell activation or assessing dysregulated functions of human T-cell immunity. In the second part, we developed a prognostic gene signature that predicts the clinical outcome (biochemical recurrence, BCR) of prostate cancer and is suitable for supporting clinical decision-making. We previously developed the prognostic ProstaTrend RNA signature based on transcriptome‐wide microarray and RNA-Seq analyses, primarily of prostatectomy specimens. An RNA-Seq study of formalin-fixed paraffin-embedded (FFPE) tumor biopsies have now allowed us to use this test as a basis for the development of a novel test that is applicable to FFPE biopsies as a tool for early routine PCa diagnostics. The prognostic relevance was evaluated using the Transcriptomic Risk Score (TRS). Validation of the TRS using the original ProstaTrend signature in the cohort of FFPE biopsies revealed a relevant impact of FFPE-associated degradation on gene expression and consequently no significant association with prognosis in FFPE tissue. However, the TRS based on the new version of the revised ProstaTrend signature, which included 204 genes (of originally 1396 genes), was significantly associated with BCR in the FFPE biopsy cohort and retained prognostic relevance when adjusted for Gleason Grade Groups. We confirmed a significant association with BCR in 9 independent cohorts including 1109 patients. Comparison of the prognostic performance of the TRS with 17 other prognostically relevant PCa panels revealed that the revised ProstaTrend signature was among the best-ranked panels.:Part 1 A time-resolved meta-analysis of consensus gene expression profiles during human T-cell activation 1 1 Introduction 3 1.1 Basic concepts of T-cells in the adaptive immunity 3 1.2 T-cell exit from quiescence 5 1.3 Hallmarks of T-cell activation 6 1.4 Modelling dynamic biological processes using time-series transcriptome data 9 1.5 Dimensionality reduction for signature inference 11 1.5.1 Introduction to NMF 13 1.5.2 Parts-based representation 14 1.5.3 Framework of NMF 16 1.5.4 Non-convexity 17 1.5.5 Objective function 18 1.5.6 Update rules 19 1.5.7 Interpretation of factorized matrices in transcriptome data 20 1.6 Motivation 22 2 Development of time-resolved consensus gene signatures 24 2.1 Methods 24 2.1.1 Data sources 24 2.1.2 Pre-processing 28 2.1.3 Statistical analysis 29 2.2 Towards time-resolved consensus gene signatures for T-cell activation 31 2.3 Time-resolved transcriptome-wide changes depict common trends across T-cell populations 36 2.3.1 Differential gene expression analysis . . . 36 2.3.2 Meta-analysis strategy 38 2.4 Discovery Set: NMF for each T-cell population 44 2.4.1 Determination of the factorization rank 44 2.4.2 Illustration of the NMF for time series data 46 2.4.3 Temporal categorization of metagenes 46 2.4.4 NMF revealed coherent metagenes across T-cell populations 47 2.4.5 Refining the variety of gene expression profiles 50 2.5 Discovery Set: Consensus gene expression profiles 53 2.6 Verification and mapping to a wider variety of T-cell transcriptional landscape 57 2.6.1 DGEA: activated vs unactivated Pan T-cells 57 2.6.2 Finalization of the consensus gene expression profiles 59 3 Consensus gene expression profiles in single-cell transcriptomics 61 3.1 Methods 61 3.2 Dataset 1: (un)activated CD4 T-cells for 4h 62 3.3 Dataset 2: Consensus gene expression profiles are enriched in CAR T-cell products from patients with low-grade ICANS 64 4 Discussion 69 4.1 Conclusion 71 Part 2 ProstaTrend, a prognostic gene-expression signature for prostate cancer 73 5 Introduction 75 5.0.1 Improved prognostic performance by combining genomic and clinicopathological features 76 5.0.2 ProstaTrend: a prognostic signature for long-term prognosis . . . . . . . 77 5.1 Motivation 79 5.1.1 From ProstaTrend to revised ProstaTrend 79 6 Development of the revised ProstaTrend signature 81 6.1 Methods 81 6.1.1 Data sources 81 6.1.2 Pre-processing 84 6.1.3 Statistical analysis 86 6.2 From ProstaTrend to revised ProstaTrend 88 6.2.1 Applying the Transcriptomic Risk Score to the FFPE biopsy cohort 89 6.2.2 Confounding factors and gene filtering . . 91 6.2.3 Filtering of genes based on prognostic impact in the TCGA cohort 93 6.3 Association of the TRS with BCR is confirmed in 9 validation cohorts 94 6.4 Meta-analysis for ProstaTrend genes across 13 cohorts 101 7 Integrated single-cell and spatial transcriptomics of human PCa 104 7.1 Methods (scRNA-Seq) 104 7.1.1 Analysis strategy 104 7.1.2 Differential expression analysis (DGEA) 106 7.1.3 Evaluation of cell type/lineage specific markers 107 7.1.4 Tumor cell identification using inferCNV 107 7.2 Methods (Spatial transcriptomics) 108 7.3 Toward the PCa single-cell atlas 109 7.3.1 First iteration: Post-clustering filtering 109 7.3.2 Second iteration: QC and cell cluster annotation 111 7.3.3 Final PCa atlas 116 7.4 ProstaTrend expression in the PCa single-cell atlas 119 7.4.1 Relating TRS of (revised) ProstaTrend to cell compartments 119 7.4.2 Association of ProstaTrend genes with cell lineages/types 119 7.5 ProstaTrend in the spatial context of prostate tissue 122 7.5.1 Annotation and carcinoma associated spot cluster 122 7.5.2 Relating TRS of (revised) ProstaTrend to spots 125 8 T-cell landscape of PCa 126 8.1 Elucidation of T-cell identities 126 8.1.1 T-cell cell cluster marker 126 8.1.2 Annotated T-cells from the re-analyzed datasets 128 8.1.3 Correlation between cell identities and immune cell populations 129 8.1.4 Enrichment of T-cell molecular mechanisms’ gene sets 131 8.1.5 Cell composition and summary of patient specimens 131 8.2 Consensus gene expression profiles for T-cell activation 132 8.2.1 Sample-level inferences 136 9 Discussion 138 9.1 The revised prognostic signature ProstaTrend (Chapter 6 & 7) 138 9.2 Activation states in the T-cell landscape of PCa (Chapter 8) 140 Summary (Part 1 & 2) 142 A Appendix 144 1 Part 1 145 1.1 DGEA and meta-analysis 145 1.2 Non-negative matrix factorization 152 1.3 CAR T-cell products 168 2 Part 2 170 2.1 Revised ProstaTrend and validation 170 2.2 PCa cell atlas 181 2.3 PCa T-cell atlas 191 List of Figures and Tables 198 Acronyms 203 Bibliography 204 Acknowledgements 218

Discovered in 1980, HTLV-1 (Human T-cell Leukemia Virus-1), was the first identified human retrovirus and is shown to be associated with a variety of diseases including: adult T-cell leukemia lymphoma (ATLL), tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM), chronic arthropathy, uveitis, infective dermatitis, and polymyositis. The mechanism by which the virus causes disease is still unknown. HTLV- 1 infection has been reported in many regions of the world but is most prevalent in Southern Japan, the Caribbean basin, Central and West Africa, the Southeastern United States, Melanesia, parts of South Africa, the Middle East and India. Approximately 30 million people are infected by HTLV-1 worldwide, although only 3-5% of the infected individuals evolve Adult T-cell Leukemia (ATL) during their life and the prognosis for those infected is still poor. The retroviral proteases (PRs) are essential for viral replication because they process viral Gag and Gag-(Pro)-Pol polyproteins during maturation, much like the PR from Human Immunodeficiency Virus-1 (HIV-1). Various antiviral inhibitors are in clinical use and one of the most significant classes is HIV-1 PR inhibitors, which have used for antiretroviral therapy in the treatment of AIDS. HTLV-1 PR and HIV-1 PR are homodimeric aspartic proteases with 125 and 99 residues, respectively. Even though substrate specificities of these two enzymes are different, HTLV-1 PR shares 28% similarity with HIV-1 PR overall and the substrate binding sites have 45% similarity. In addition to the 125-residue full length HTLV-1 PR, constructs with various C- terminal deletions (giving proteases with lengths of 116, 121, or 122 amino acids) were made in order to elucidate the effect of the residues in the C-terminal region. It was suggested that five amino acids in the C-terminal region are not necessary for the enzymatic activity in Hayakawa et al. 1992. In 2004 Herger et al. had suggested that 10 amino acids at the C-terminal region are not necessary for catalytic activity. A recent paper suggested that C-terminal residues are essential; and that catalytic activity lowers upon truncation, with even the last 5 amino acids necessary for full catalytic activity (1). The mutation L40I has been made to prevent autoproteolysis and the W98V mutation was made to make the active site of HTLV-1 PR similar to HIV-1 PR. We have characterized C-terminal amino acids of HTLV-1 PR as not being essential for full catalytic activity. We have discovered potential new inhibitors by in silico screening of 116-HTLV-1 PR. These small molecules were tested kinetically for various constructs including the 116, 121 and 122-amino acid forms of HTLV-1 PR. Inhibitors with the best inhibition constants were used in HTLV-1 infected cells and one of the inhibitors seems to inhibit gag processing.

Recombinant oncolytic virus (OV) vaccines that encode tumour-associated antigens are potent boosting agents for adoptive transfer of tumor-specific T cells (adoptive T cell therapy or ACT). Current strategies to exploit boosting vaccines for ACT rely on a priori knowledge of targetable tumour epitopes and isolation of matched epitope-specific T cells. Therefore, booster vaccines must be developed on a patient-by-patient basis, which severely limits clinical feasibility. To overcome the requirement for individualized pairing of vaccines and T cells, we propose a “universal” strategy for boosting tumor-specific T cells where the boost is provided through a synthetic receptor that can be engineered into any T cell and a matched vaccine. To this end, we are employing chimeric antigen receptors (CARs), which confer MHC-independent antigen specificity to engineered T cells, and a paired OV vaccine that encodes the CAR target. As proof-of-concept, we have developed and evaluated a model where murine TCR transgenic T cells are engineered with boosting CARs against a surrogate antigen for studies in immunocompetent hosts. In chapter 3, I optimized a murine CAR-T cell manufacturing protocol that allows for generation of highly-transduced T cells that maintain a predominantly central memory (Tcm) phenotype. This protocol leads to generation of highly functional CAR-T cell products that can be cryopreserved at the end of ex vivo culture for future use in adoptive transfer and vaccination studies. In chapter 4, I evaluated the in vivo boosting potential of our dual-specific CAR-T cells with paired OV vaccines. Adoptive transfer of these CAR-engineered tumor-specific T cells followed by vaccination with paired oncolytic vesicular stomatitis virus (VSV) vaccine leads to robust, but variable and transient, CAR-mediated expansion of tumour-specific CD8+ T-cells, resulting in delayed tumour progression in aggressive syngeneic tumour models. In chapter 5, I investigated the role of OV-induced type I interferon (IFN-I) responses on CAR-T cell boosting. I found that CAR-T cell expansion and anti-tumour function following OV vaccination is limited by the IFN-I response and can be further enhanced by blocking interferon alpha and beta receptor subunit 1 (IFNAR1). This IFN-I-mediated T cell suppression was found to be T cell-extrinsic and related to premature termination of OV infection and antigen expression in vivo. In chapter 6, I investigated the role of CD4+ T cell help in vaccine-mediated T cell boosting and evaluated different genetic engineering strategies to integrate pro-survival STAT5 signaling into the CAR-T cell product in an effort to improve persistence and long-term anti-tumour efficacy. The work presented herein describes a novel and clinically feasible approach to enhancing adoptive T cell therapies and contributes to the basic understanding of T cell biology in the context of CAR-engineering and cancer vaccination. / Thesis / Doctor of Philosophy (PhD) / Despite recent advances in cancer prevention, detection, and treatment, 2 in 5 Canadians are expected to be diagnosed with cancer in their lifetime and approximately 1 in 4 will succumb to their disease. New, more specific therapies are needed to improve responses to treatment and reduce therapy-related side effects. Cell therapy is a new way to treat cancer that uses the patient’s own immune cells as a living drug. The immune cells are taken from a patient’s blood or tumour, trained to attack cancer in the laboratory, and infused back into the patient where they will find and kill cancer cells. A major challenge with this strategy is that the trained immune cells do not always survive in the patient for long enough to get rid of the tumour. To “boost” the immune cells, we are developing a new strategy where the immune cells are genetically modified and combined with a vaccine to enhance their anti-tumor activity. Just like a vaccine against a bacteria or virus, this vaccine will tell the modified immune cells to turn on, make more of themselves, and to find and kill the cancer cells. By delivering this “go” signal through a vaccine, we think that the immune cells will be better able to survive and generate a stronger, longer-lasting immune response against the cancer. This thesis tests this approach in relevant mouse models of cancer and aims to understand how we can best design the immune cells and vaccine to work together in their tumour-killing activities.

Porcine circovirus associated disease (PCVAD) is currently one of the most economically important diseases facing the global swine industry. Porcine circovirus type 2 (PCV2) is the primary and essential causative agent of PCVAD, but development of clinical disease typically requires co-infection with other swine pathogens such as porcine reproductive and respiratory syndrome virus (PRRSV). The specific mechanisms of co-infection that lead to clinical disease are not fully understood, but immune modulation by the co-infecting viruses is thought to play a critical role. The ability of dendritic cells (DC) infected with PRRSV, PCV2 or both to induce regulatory T cells (Tregs) was evaluated in vitro. DCs infected with PCV2 significantly increased CD4+CD25+FoxP3+ Tregs (p<0.05) and DCs co-infected with PRRSV and PCV2 induced significantly higher numbers of Tregs than with PCV2 alone (p<0.05). This Treg induction was found to be dependent on TGF-β and not IL-10. Further investigation of the in vivo swine immune response to acute co-infection with PCV2 and PRRSV failed to detect activation of Tregs in peripheral blood mononuclear cells (PBMCs) or bronchoalveolar lavage samples. The Treg response to in vitro and in vivo PRRSV challenge in pigs persistently infected with PCV2 or vaccinated against PCV2 was evaluated. There was no significant difference in Tregs in PBMCs among chronically PCV2-infected, vaccinated PCV2 challenged or negative control pigs. However, following in vitro infection of monocyte-derived dendritic cells with PCV2, PRRSV, or both viruses, co-cultured lymphocytes from chronically infected and PCV2 vaccinated pigs had significantly (p<0.05) decreased Treg expression in the virus infected groups compared to the negative controls. In separate experiments, pigs vaccinated against PCV2 and subsequently challenged with an attenuated PRRSV strain and its pathogenic parental strain developed increased CD4+CD25+FoxP3+ Tregs (p<0.05) in PBMC samples compared to uninfected controls, and this correlated with increased suppressor activity and IL-10 expression. The findings from these studies indicate that the interaction of PCV2 and PRRSV in swine modulates the host immune response mediated in part through the activity of Tregs. However, the extent to which Tregs orchestrate a dysregulated immune response in the pathogenesis of PCVAD in vivo remains to be determined. / Ph. D.