Thesis (doctoral)--Lund University, 1998. / Added t.p. with thesis statement inserted.

Thesis (Ph.D.)--York University, 2007. Graduate Programme in Biology. / Typescript. Includes bibliographical references (leaves 224-257). Also available on the Internet. MODE OF ACCESS via web browser by entering the following URL: http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:NR32051

Despite progress through expanded use of bed nets and anti-malarial drugs, Plasmodium falciparum (Pf) malaria caused about 200 million cases and 500,000 deaths in 2015. An ideal vaccine would reduce the burden of disease and interrupt transmission. Despite decades of effort, there is no vaccine that can adequately address the global burden of malaria. This thesis focuses on two potential weaknesses in the parasite life-cycle. First, I investigate two vaccination strategies aimed at improving the antibody response to RH5, an essential and conserved protein for erythrocyte invasion. Due to instability of the resultant recombinant vaccine constructs, these efforts have required re-engineering of the vaccine platform, which remains an ongoing effort. Second, the immunogenicity and mechanism of protection of a live-attenuated whole sporozoite vaccine, PfSPZ Vaccine, was assessed. In a study that examined PfSPZ Vaccine at intravenous (IV) doses between 1.35 &tiles; 10⁵ to 4.5 &tiles; 10⁵ PfSPZ, I demonstrate that PfSPZ antibody responses correlated with durable sterile protection against controlled human malaria infection (CHMI). Surprisingly, the pre-vaccine frequency of Vγ9⁺Vδ2⁺ T cells, an innate T cell that recognizes conserved Plasmodium phosphoantigens, also correlated with durable sterile protection. Regarding the mechanism of protection, PfSPZ-specific antibodies as well as CD8 and CD4 T cells in the blood decreased substantially over time, yet sterile protection was maintained. In non-human primates, the CD8 T cell response in the liver at a memory time point was measured to be about 100-fold higher than found in the blood. Collectively, these data suggest that PfSPZ Vaccine confers durable protection in humans by long-lived, tissue-resident CD8 T cells. These findings were extended with a study using 9.0 x 10⁵ PfSPZ, wherein I demonstrate that T cell responses peaked immediately after the first vaccination with minimal T cell activation despite additional immunizations. This suggests that anti-PfSPZ immunity may be limiting the effectiveness of subsequent immunizations. Finally, I examined the T cell response to PfSPZ attenuated by chloroquine (termed PfSPZ-CVac). T cell responses were substantially higher than achieved with comparable PfSPZ Vaccine doses. Additionally, a significantly higher proportion of PfSPZ-specific CD4 T cells were polyfunctional, simultaneously expressing IFN-γ, IL-2, and TNF-α, in subjects that were protected from CHMI. In sum, these studies provide insight into the immunobiology of a protective immune response that may guide future malaria vaccine development efforts.

Les lymphomes T périphériques (PTCL) sont des néoplasmes rares et agressifs représentant environ 12% des lymphomes chez l’Homme. Nos travaux récents dans des souris p53-/- ont révélé une nouvelle entité de PTCL, émergeant de cellules Natural-Killer T-cell (NKT), un type particulier de lymphocyte T reconnaissant des antigènes lipidiques. Nous avons montré que ces lymphomes NKT (PTCL-NKT) présentent des caractéristiques de NKT stimulés chroniquement, et que la lymphomagenèse est initiée via l’activation chronique du TCR. Chez l’Homme, de nombreux PTCL sont suspectés pour être associés à des stimulations antigéniques chroniques, mais les mécanismes de transformation impliqués sont encore mal connus. Borrelia burgdorferi (Bb), l’agent responsable de la maladie de Lyme, provoque des infections chroniques dont l’implication dans certains lymphomes T cutanés (CTCL) a été suggérée. Cependant, cette observation manque de preuves cliniques et expérimentales. De manière intéressante, Bb est connue pour exprimer des glycolipides activateurs des NKT. Nous avons donc infecté des souris p53-/- avec des Bb vivantes, et montré que l’infection augmente significativement la fréquence des PTCL-NKT. Par traitement antibiotique précoce de souris infectées et par injections de Bb inactivées, nous avons également démontré que la chronicité de l’infection est nécessaire au développement de ces lymphomes. L’analyse phénotypique de ces PTCL-NKT a confirmé nos observations précédentes, montrant des caractéristiques de cellules NKT activées chroniquement, telles que l’expression de marqueurs d’activation et d’exhaustion (perte de NK1.1, surexpression de PD-1). Ces résultats suggèrent une implication de Borrelia dans la lymphomagenèse T. En se basant sur l’analyse de différents marqueurs phénotypiques et de leur production cytokinique, nous avons également montré que ces lymphomes présentent un profil dérégulé se rapprochant du sous-type NKT2. Une étude génomique par séquençage whole-exome sur 6 PTCL-NKT a révélé de larges pertes récurrentes du chromosome 13. Au sein de la zone minimale de délétion, nous avons identifié Jarid2, codant un facteur épigénétique impliqué dans le développement NKT par une activité histone-methytransférase. Ce gène est retrouvé altéré dans 20% des CTCL. De manière intéressante, les souris Jarid2-/- présentent une expansion périphérique de NKT au profil immature/NKT2, partageant donc des caractéristiques avec les PTCL-NKT. La perte de Jarid2 a été détectée dans presque tous les PTCL-NKT. Nous avons confirmé la perte de Jarid2 au niveau ARN et protéique. Nos résultats préliminaires montrent une hypométhylation de la lysine 9 de l’histone H3 (H3K9), la cible de Jarid2, soutenant un effet fonctionnel dans la physiopathologie des PTCL-NKT. Par conséquent, nous pensons que la perte de Jarid2 pourrait être un événement important de la lymphomagenèse NKT, puisque de plus en plus d’altérations de facteurs épigénétiques sont retrouvées dans les PTCL humains. Pour réponse à cette question, nous sommes notamment en train de générer des souris p53-/- x Jarid2-/-. En conclusion, nos données viennent renforcer le concept selon lequel certaines infections peuvent initier la transformation des cellules T par l’activation chronique du TCR. Nous avons également identifié un nouveau facteur épigénétique potentiellement impliqué dans la lymphomagenèse NKT / Peripheral T-cell lymphomas (PTCL) are aggressive and heterogeneous neoplasms that represent around 12% of Human lymphomas. Our recent work in p53-/- mice revealed a new PTCL entity, arising from Natural-Killer T-cell (NKT), a particular type of T cell recognizing lipidic antigens. We found that NKT lymphomas (NKTL) present features of chronically stimulated NKT-cells and that lymphomagenesis is driven through chronic TCR activation by microbial glycolipids. In human, many PTCL are suspected to be associated with chronic antigenic stimulation, but this transformation mechanism is still poorly understood.Borrelia burgdorferi (Bb), the causative agent of Lyme disease, induces chronic infection and has recently been suggested to be involved in cutaneous T-cell lymphomas (CTCL). However, this observation lacks clinical and experimental proofs. Interestingly, Bb is known to express NKT-activating glycolipids. We therefore infected p53-/- mice by live intradermal Bb injection and showed that Bb infection significantly increased NKTL rate. Phenotypic characterization of these NKTL confirmed our previously described features of chronically stimulated NKT-cells, with expression of activation and exhaustion markers (loss of NK1.1, upregulation of PD-1). Based on surface markers, transcription factors and cytokine production analysis, we also found that our lymphomas mostly present a NKT2 subtype profile, sometimes surprisingly mixed with NKT17 or NKT1. Genomic study by whole-exome sequencing on few of these lymphomas revealed recurrent large losses in the chromosome 13. Within the minimal deletion region, we identified Jarid2, a gene involved in NKT development by epigenetic regulation and which is found altered in 20% of CTCL. Jarid2 loss was detected in almost all NKTL. Interestingly, Jarid2-/- mice show increased NKT number in the periphery with an immature/NKT2 phenotype, sharing features with our NKTL.Thus, we believe that Jarid2 loss may be an important event in NKT lymphomagenesis, as more and more epigenetic factors are found mutated in several human PTCL. To answer this question we are currently breeding p53-/- x Jarid2-/- mice. In conclusion, our data reinforced the concept that chronic bacterial activation of T-cells through their TCR can effectively drive T-cell transformation. We also identified a new potential epigenetic factor that may be involved in lymphomagenesis

The importance of Cytotoxic T-Cell (CTL) reponses during the course of intracellular infections has received a lot of attention during the past few decades. CTLs respond to epitopes presented by the Major Histocompatibility Complex (MHC) originating from intracellular proteins for which they have an appropriate T-Cell Receptor (TCR) for. This response is crucial for the control of pathogens such as Influenza, Hepatitis, HIV and others by destroying the cell in which the pathogen replicates. Due to the extreme polymorphism of MHC molecules, Computational Immunology techniques have been developed to detect potential MHC ligands and as a consequence, potential CTL epitopes. The polymorphism factor needs to be taken into account especially when concerning the design of vaccines with a CTL response component to maximize population coverage. Tools have been constructed that combine the predictions tools concerning major steps in this pathway, that is, proteasomal cleavage, Transporter associated with Antigen Presentation (TAP) affinity, Major Histocompatibility Complex (MHC) affinity and Immunogenicity. In this study, a novel method is developed to combine the different steps in the pathway, which includes the development of a novel TAP predictor. Furthermore, by using a BLOSUM-based score in conjunction with the epitope prediction results, a novel CTL epitopebased clustering method was developed. Two pathogens with major CTL epitope components, but vastly different mutation rates were chosen to infer whether the aforementioned methods can be used to detect potential CTL epitopes and group sequences together based on shared immunogenicity. / Dissertation (MSc)--University of Pretoria, 2011. / Bioinformatics and Computational Biology Unit / unrestricted

The oncogene Ahi-1 was recently identified through provirus insertional mutagenesis in murine leukemias and lymphomas. Its involvement in human leukemogenesis is demonstrated by gross perturbations in its expression in several leukemic cells lines, particularly in cutaneous T-cell lymphoma (CTCL) cell lines (Hut 78 and Hut 102). Hut 78 is derived from a patient with Sezary syndrome, a common leukemic variant of the human CTCL mycosis fungoides. Aberrant expression of AHI-1 mRNA and protein has been found in CD4⁺CD7⁻ leukemic Sezary cells from patients with Sezary syndrome. Moreover, stable suppression of AHI-1 using retroviral-mediated RNA interference in Hut 78 cells inhibits their transforming activity in vitro and in vivo. In an effort to identify genes involved in AHI-1-mediated leukemic transformation in CTCL, microarray analysis was performed to compare six RNA samples from AHI-1 suppressed Hut 78/sh4 cells to five samples from Hut 78 control cells. Limma and dChip analyses identified 218 and 95 differentially expressed genes, respectively, using a fold change criteria of > or < 2 and a p-value threshold of ≤ 0.01. After evaluation of both analyses, 21 genes were selected based upon interesting structural and functional information, specificity to hematopoietic cells or T-cells, and previous connections to cancer. Expression patterns of these 21 genes were validated by qRT-PCR with p-values < 0.05 ranging from 1.97 x 10⁻¹⁰ to 6.55 x 10⁻³, with the exception of BRDG1 at 5.88 x 10⁻². The observed up-regulation of both BIN1 and HCK in AHI-1 suppressed Hut 78/sh4 cells as compared to control cells further confirmed at the protein level. The tumor suppressor BIN1 is known to physically interact with c-MYC, which also exhibits differential protein expression in these cells. Characterization of BIN1 identified 4 isoforms all of which contain exon 10 and demonstrate alternative splicing of exons 12A and 13. Additionally, qRT-PCR results from primary Sezary samples indicate there is clinical significance in the expression changes detected for BIN1, HCK, REPS2, BRDG1, NKG7 and SPIB. These findings identify several new differentially expressed genes that may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Chronic hepatitis C virus (HCV) infection has global effects on the immune system. CD8+ T-cells, responsible for viral clearance and control, are dysfunctional for as yet unknown reasons. It is hypothesized that IL-7 signaling pathway deficiencies contribute to this impairment. Blood-derived CD8+ T-cells in chronic HCV mono- and HIV-HCV co-infection had lower IL-7-induced activation of STAT5 and production of Bcl-2, and lower proliferation in co-infection, compared to controls. Lower Bcl-2 production was also associated with increased fibrosis. These changes were independent of the IL-7 receptor α expression and suppressor of cytokine signaling 1 or 3 expression. Intrahepatic CD8+ T-cells in HCV-infection did not activate STAT5 above basal levels with cytokine stimulation and had lower Bcl-2 expression than blood-derived cells. In conclusion, bulk CD8+ T-cells were impaired in response to IL-7 and the IL-7 signaling pathway may be one mechanism by which CD8+ T-cells are impaired in chronic HCV infection.

Angioimmunoblastic T-cell lymphoma (AITL) is a rare type of lymphoid malignancy with most affected patients presenting in their late 60’s with nonspecific symptoms, laboratory signs and advanced stage disease. 78 year old female with history of hypertension presented to the hospital with complaints of dyspnea, dry cough and fatigue of one week duration. She denied having fever, chills, night sweats or recent weight loss. Labs on admission were significant for leukopenia with lymphocytopenia, thrombocytopenia, hyponatremia and hyperglobulinemia. Computer tomography chest and abdomen showed bilateral multifocal lung infiltrates, large left side pleural effusion, diffuse lymphadenopathy and splenomegaly. She was started on empiric antibiotics for community acquired pneumonia. Sputum culture did not identify any offending organism and although thoracentesis was consistent with exudative fluid, microbiology and pathologic studies also did not offer a diagnosis. Fine needle aspiration of a suspicious lymph node was also negative. She was discharged home to complete treatment for pneumonia but over the next two months, she presented on three different occasions with the same respiratory symptoms. Serial thoracentesis thereafter showed chylous fluid that was fast re-accumulating. A repeat flow cytometry of the pleural fluid was concerning for a lymphoproliferative process and subsequent excisional biopsy with molecular studies performed on rearrangement of T-cell receptors resulted in eventual diagnosis of Angioimmunoblastic T-cell lymphoma stage IV due to associated lung involvement. She declined aggressive management and opted for palliative care. Lymphoma presenting with chylothorax is not common due to early diagnosis of most lymphoma but it can be the initial presentation of AITL because most patients present with nonspecific symptoms and lab findings that make diagnosis difficult and delayed. In our patient, eventual diagnosis was three months from initial presentation. This is not ideal because most patients who succumb to the disease tend to do so from progressive worsening nutritional status and immunosuppression that ensues as the disease advances. The recurrent chylothorax seen in our patient is likely a result of tumor burden that obstructed chyle drainage through the thoracic duct and may have resolved with adequate treatment of the disease. Multiple laboratory abnormalities and B-type symptoms in a patient with unclear primary process should prompt workup for a possible neoplastic disease, particularly lymphoma. Clinicians who suspect a lymphoproliferative process in patients with diffuse lymphadenopathy and pulmonary symptoms of unclear etiology should consider getting an excisional tissue biopsy for further diagnostic studies. PCR based studies that assess for cell locality is also helpful in particularly difficult cases.

archives@tulane.edu / Background: Human Immunodeficiency Virus-1 (HIV-1) has killed over 35 million and infects 1.8 million new people each year. Antiretroviral therapy (ART), although effective controlling plasma viremia and transmission, does not purge latent or persistent reservoirs necessary to eliminate infection, and must be maintained for life. It is thus imperative to discover therapeutics that provide both lifetime suppression of viral loads and depletion of viral reservoirs. Methods: To harness the immunosurveillance capacity of highly functional and persistent CMV-specific adaptive response, rhesus PBMCs were stimulated with rhCMV peptide pools (IE1, IE2, and pp65) to expand rhCMV-specific T cells. These cells were then genetically modified with retroviral vectors expressing a CD4 extracellular domain linked to T cell intracellular signaling domains that instruct CTL activity, converting them into HIV-specific effector cells. Vectors combine CD4 targeting with an maC46 fusion inhibitor to protect against viral entry. In a reversal of the critical step in the HIV viral lifecycle whereby virus targets new CD4+ host cells using its Env glycoprotein, these genetic modifications redirect host immune responses to target and kill Env expressing infected cells. We hypothesize that continuous stimulation of CD4-CAR T cells through their rhCMV-specific TCR will maintain activated T effector memory CTL capable of targeting HIV infected cells. Results: We find that autologous rhPBMCs can be expanded ex vivo with rhCMV peptides up to therapeutically relevant numbers for adoptive transfer. This rhCMV-specific T cell expansion enriches cells in a phenotype consistent with T effector memory differentiation. Following genetic modification and adoptive transfer, cells reach peak expansion at seven days post infusion into ART suppressed or unsuppressed SHIV infected Rhesus Macaques. We observe these cells capable of persisting in vivo for at least 2 years following reinfusion. Furthermore, these cells are maintained in vivo in an effector memory phenotype throughout the duration they were analyzed. Despite this, SHIV plasma viral loads remain unchanged. Conclusion: These studies establish use of rhCMV-specific T cells as an effective way to produce persistent genetically modified cells targeting SHIV. Future studies will need to further increase in vivo expansion, protection, and CTL activity as viral loads remain detectable. / 1 / Nathan Michel Johnson

Cyclosporine is used in veterinary medicine to treat a number of inflammatory and immune-mediated conditions, however firm oral dosing protocols have yet to be established in the dog. Traditionally a pharmacokinetic approach, through measurement of blood drug concentrations, has been the primary method of establishing if the given dose is effectively suppressing the immune system. However, there is some debate over how well blood drug concentrations correspond to immunosuppression, since individuals can vary in response to the same drug concentration. Our research group believes that a pharmacodynamic approach could alternatively be used to accurately determine cyclosporine dosages in individual patients since this will give a measurement of the immune system’s response to the drug, rather than simply how the body is processing it. This method will give a more accurate assessment of the patient’s immune system, and allow for better immunosuppressant therapy. The objective of this thesis was to develop a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay that could reliably predict patient outcome during cyclosporine treatment. This assay would essentially work as a diagnostic tool that clinicians can use to help determine if they were using an appropriate cyclosporine dose for their patients. The assay measures cytokine expression of activated T cells, which are the target cell for the active metabolite of cyclosporine. Our objectives were achieved, firstly, through validation of the assay. Since this assay will be used by clinicians throughout the nation, we first established if shipping conditions affected the sample, and therefore assay results. Once the effect of sample storage time and temperature were determined, optimal sample collection timing was established. Finally, cytokine levels were measured in samples from clinical cases and healthy control dogs to examine the difference in cytokine expression between these two groups. An effective and reliable treatment method for cyclosporine has yet to be established in the dog; therefore the results of this thesis will lead to better therapeutic monitoring and more efficient use of cyclosporine therapy in canine patients.