Thesis (doctoral)--Lund University, 1994. / Added t.p. with thesis statement inserted.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

Thesis (M. Phil.)--University of Hong Kong, 2005. / Title proper from title frame. Also available in printed format.

The skin is an important barrier against a range of different environmental challenges. The skin associated immune system is able to detect breaks in the barrier to initiate a protective immune response. Langerhans cells express a high density of CD1a, which presents lipid antigens to T-cells. However little is known about CD1a-restricted lipid antigens and the role of CD1a-restricted T-cells in inflammatory skin disease. In order to investigate the role of T-cells that react to CD1a presenting lipid in skin disease, wasp and bee venom were studied as an antigen source. Venoms are able to cause allergic hypersensitivity reactions, associated with skin T-cell infiltration, a venom protein-specific T-cell response in allergic individuals and are independent of filaggrin. Using primary antigen presenting cells and target cells lacking surface MHC expression (K562 cells) transfected with CD1a, allowed investigation of polyclonal T-cells responses from unrelated donors. Bee and wasp venoms were shown to induce CD1a-restricted T-cell responses in both peripheral blood and skin. Surprisingly this activity was not contained within the lipid fraction of the venoms, but instead was mediated through the generation of a lipid ligand by venom phospholipase. Furthermore, wasp venom delivery results in the production of phospholipase products in the skin of humans. A significantly increased frequency of IFNγ-, GM-CSF- and IL-13-producing venom specific CD1a-restricted T-cells was observed in allergic individuals compared to healthy controls. During subcutaneous immunotherapy, frequencies of CD1a-reactive T cells were initially induced, peaking by weeks 5, but then reduced despite escalation of antigen dose. CD1a-reactive T cells were further investigated to characterise their physiological role by generating T cell lines which produced a range of different cytokines, including IL-22 on stimulation with phospholipase and other phospholipase containing allergens. In summary, we identified a novel pathway of skin inflammation where lipids generated by allergen-derived phospholipase can be recognised by CD1a-restricted T cells which produce type 1 and type 2 cytokines and associate with allergic reactivity. These findings have implications for novel therapeutic strategies for allergic disease.

HIV establishes a reservoir comprising long lived, latently infected CD4+ T cells and monocytic cells early during primary infection. This population represents a major barrier to an HIV cure. This thesis aimed to investigate the role of the immunological synapse in the failure of cytotoxic T lymphocytes (CTLs) to eliminate the HIV reservoir and the potential for engineered bispecific <b>I</b>mmune-<b>m</b>obilising <b>m</b>onoclonal <b>T</b> cell receptors <b>A</b>gainst <b>V</b>iruses (ImmTAV) to overcome this by redirecting fully functional CD8+ T cells against viral targets. A primary cell model of latency was used to investigate the expression of HIV Gag on latently infected cells and their susceptibility to ImmTAV-mediated elimination. A subset of cells expressed low levels of Gag without spreading infection and ImmTAV-redirected healthy donor CD8+ T cells were able to eliminate up to 40% of infected cells without latency reversal. CD8+ T cells from chronic HIV infected (CHI) donors showed impaired antiviral activity even with ImmTAV redirection. To investigate this further, confocal microscopy was used to study immunological synapse formation using primary CD8+ T cells from HIV-negative and CHI donors. CD8+ T cells from CHI donors were able to form conjugates with virus-infected cells but exhibited impaired synapse maturation, indicated by reduced Zap70 localisation, delayed microtubule-organising centre polarisation and impaired perforin recruitment to the synapse. ImmTAV redirection partially overcame these defects. Finally, the impact of antiretroviral agents on T cell mitochondrial function was explored. Exposure to zidovudine increased mitochondrial reactive oxygen species production and susceptibility to apoptosis. However, there was no evidence of impaired mitophagy. These data show that defects in CD4+/CD8+ T cell synapse maturation contribute to HIV persistence but nevertheless suggest that a subset of HIV reservoir cells may be susceptible to ImmTAV-mediated elimination. The therapeutic potential of ImmTAVs may depend in part on correction of CD8+ T cell exhaustion.

Many cancers, including lymphoma, are associated with increased numbers of T cells with suppressive properties, and it has been suggested that immune subversion is important in cancer pathogenesis. The ability of lymphoma cells to induce conventional CD25- T cells to adopt a regulatory phenotype was evaluated, with the aim of elucidating the factors and pathways governing this process and determining the clinical relevance. Regulatory T cell phenotype in both peripheral blood and nodal material from patients with lymphoma and healthy controls was also assessed. Preferential representation of Tregs in nodal tissue was noted with higher percentages seen than in peripheral blood. Contrary to previous studies, minimal evidence to suggest that lymphoma induces a regulatory phenotype from CD4+CD25- T cells was found. Furthermore, nodal Tregs displayed high expression of Helios and FOXP3, indicating a thymically-derived rather than induced origin. PD-1 expressing T cells were present in greater numbers in PBMCs from patients compared with healthy controls, suggesting an alternative reason for the immunosuppression that may be exhibited in these patients. These data support recruitment and amplification as the mechanism for the high proportion of Tregs seen in lymphoma. Hodgkin lymphoma is typified by a prominent reactive infiltrate and is the archetype of immune subversion in lymphoma. Inherited predisposition is demonstrated through familial and twin studies. Susceptibility loci have been identified in a number of genes that affect immune response, with the strongest association seen with the HLA region. 5 individuals with classical Hodgkin lymphoma from a family originating in North East Scotland were evaluated by linkage analysis and exome sequencing. Novel, shared variants predicted to disrupt protein function were identified in 2 genes on chromosome 3. Proximity to a previously described gene in familial Hodgkin lymphoma implicates this region as an important susceptibility locus.

archives@tulane.edu / Effective adaptive immune responses depend on the presentation to CD4+ T cells antigen peptides bound to major histocompatibility complex class II proteins. The structure of an antigen strongly influences its processing within the endolysosome and potentially controls the identity and abundance of peptides that are presented to T cells. The dissertation presented here sought to expand our understanding of how antigen structure and stability influence adaptive immune responses for two model antigens. Pseudomonas exotoxin A domain III (PE-III) functions as an ADP-ribosyltransferase with significant cellular toxicity and has been incorporated into a recombinant immunotoxin for the treatment of cancer. The bacterial component of the PE-III immunotoxin is highly immunogenic and generates neutralizing antibodies that render subsequent treatments ineffective. A group of six single-amino-acid substitutions in PE-III that were predicted to disrupt CD4+ T-cell epitopes have been shown to reduce antibody responses in mice. Here we demonstrate that only one of the substitutions, R494A, exhibits reduced folding stability and proteolytic resistance through the removal of a hydrogen bond. This destabilization significantly reduces its antibody immunogenicity while generating CD4+ T-cell epitopes that are indistinguishable from those of wildtype PE-III. PE-III specific B cells isolated from R494A-immunized animals contained fewer somatic mutations, which are associated with affinity maturation, and exhibited a weaker germinal-center gene signature, compared to B cells from wildtype-immunized animals. Chicken ovalbumin (cOVA) has been studied for decades primarily due to the robust genetic and molecular resources that are available for experimental investigations. cOVA is a member of the serpin superfamily of proteins that function as protease inhibitors, although cOVA does not exhibit this activity. As a serpin, cOVA possess a protease-sensitive reactive center loop that lies adjacent to the OT-II epitope. We took advantage of the previously described single-substitution-variant, OVA R339T, which can undergo the dramatic structural transition observed in serpins to study how changes in loop size and protein stability influences CD4+ T-cell priming in vivo. We observed that OVA R339T loop-insertion increases overall stability and protease resistance and significantly shortens the reactive center loop. This results in reduced CD4+ T-cell priming of the OT-II epitope in SJL mice. These findings have implications for the design of more effective vaccines for the treatment of infectious diseases and cancer as well as the development of more robust CD4+ T-cell epitope prediction tools. / 1 / Daniel Moss

Thymic negative selection has been identified as a crucial checkpoint in thymocyte development that purges the T-cell repertoire of autoreactive T cells through apoptosis of the cells after strong T cell receptor (TCR) stimulation. It has been well established that efficient thymic negative selection is required to prevent severe monogenic autoimmune diseases, such as Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy. The involvement of negative selection in other T cell-mediated autoimmune diseases remains unclear. This is largely due to the lack of fully-humanized physiological models for the study of human thymic negative selection. In the work presented here, I aim to study human thymic negative selection in healthy control (HC) immune systems and to determine whether negative selection is impaired in immune systems from individuals with Type 1 diabetes (T1D), a T cell-mediated autoimmune disease. To facilitate these studies, I developed novel humanized mouse and organ culture models. These models built on the previously described Personalized Immune (PI) mouse model [1]. The PI mouse model allows for the rederivation of a fully humanized immune system from a human donor by transplanting hematopoietic stem cells (HSC) and progenitors from an individual adult donor and a human fetal thymus fragment to immunodeficient mice. The HSCs then reconstitute all immune cell lineages, including T cells which develop in the human thymus fragment. This model is extremely powerful because it allows for the study of a human’s immune system in a model that is conducive to experimental replicates and interventions, unlike studies done directly on human patients. To optimize this model for the study of thymic negative selection, I developed two other PI models. The first is the TCR-transgenic PI thymic organ culture (TOC) model. This model allows for the study of the selection of a specific TCR in a culture system combining human HSCs and thymus fragments. The second model is the TCR-transgenic PI mouse model. This model allows for the study of the thymic selection of a specific TCR in a fully humanized in vivo model. The work presented here utilized these three powerful PI models to interrogate the thymic negative selection process in human health and disease at a depth not previously possible. Using these models, we demonstrated the first evidence for thymic negative selection of an insulin-reactive TCR that recognizes a naturally expressed antigen in healthy human immune systems. These studies also demonstrated that robust negative selection requires HSCs expressing the HLA-restriction element of the TCR, and without the expression of that HLA on HSCs, negative selection is reduced and performed in later stages of thymic development. When comparing the phenotypic and functional characteristics of thymocytes undergoing negative selection in HC and T1D immune systems, T1D thymocytes in some immune systems had differential expression of TCR-signaling and negative selection markers and resistance to apoptosis and cell death after strong TCR stimulation. Studies on the negative selection of a specific insulin-reactive TCR in healthy and T1D immune systems demonstrated that in healthy immune systems central tolerance to this TCR involved a combination of negative selection and T regulatory cell conversion. This is the first demonstration of combined tolerogenic induction in the human immune system. In contrast, some T1D immune systems demonstrated impaired negative selection of this insulin-reactive TCR and impaired conversion of these autoreactive T cells to T regulatory cells. Further, when comparing the gene expression profile of HC and T1D thymocytes undergoing negative selection, there are multiple genes important in thymic selection and apoptosis that are differentially regulated. Overall, this data provides unique insights into the process of thymic negative selection in healthy immune systems. It also provides the first evidence that thymic negative selection is impaired in some T1D immune systems and this impairment is possibly driven by differential gene expression. The models developed will allow for further study into human thymic selection in health and disease. These findings answer the important question of whether thymic negative selection is impaired in autoimmune disease, which has been debated in the field of T1D research and the wider immunology field. More importantly, it opens the door to targeting of the thymic negative selection pathway with therapeutics for T1D and other autoimmune diseases.

T lymphocytes (T cells) represent one of the major cell populations of the immune system. Named by the place of their development, the thymus, several types can be distinguished as the αβ T cells, the γδ T cells, the mucosa-associated invariant T cells (MAIT), and the natural killer T (NKT) cells. The αβ lineages of CD4+ THelper and the CD8+ T cytotoxic cells with the T cell receptor (TCR) composed of α- and β-chain are major players of the adaptive immune system. In the thymus, CD4+ and CD8+ single positive (SP) αβ cells represent the ultimate result of positive and negative selection of CD4+CD8+ double positive (DP) thymocytes. The DP population derives from the double negative (DN) thymocytes that develop from bone marrow-derived progenitors through different stages (DN1-DN4) that are characterized by CD25 and CD44 surface expression. NFATc1, a member of the Nuclear Factor of Activated T cells (NFAT) transcription factors family, is critically involved in the differentiation and function of T cells. During thymocyte development, the nuclear expression of NFATc1 reaches the highest level at the DN3 (CD44-CD25+) stage. The hematopoietic cell-specific ablation of NFATc1 activity results in an arrest of thymocyte differentiation at the DN1 (CD44+CD25-) stage. On the other hand, over-expression of a constitutively active version of NFATc1 results in an impaired transition of DN3 cells to the DN4 (CD44-CD25-) stage, suggesting that a certain threshold level of NFATc1 activity is critical at this point. ChIP-seq and RNA-seq analysis allowed us the identification of NFATc1/A target genes involved in lineage development as the Tcra and Tcrb gene loci. Furthermore, we identified multiple NFATc1-regulated genes that are involved in γδ T cell development. In the mouse models, Rag1Cre-Nfatc1fl/fl and Rag1Cre-E2fl/fl, in which the activity of NFATc1 or inducible NFATc1 in the latter is impaired during the early stages of thymocyte development, we observed increased numbers of γδ T cells. These γδ T cells showed an unusual overexpression of CD4, a lack of CD24 expression, and overexpression of the anti-apoptotic gene Bcl2a1a. We hypothesize that during the DN stages NFATc1 plays an important role in regulating crucial steps of αβ thymocyte development and when NFATc1 activity is missing this may disturb αβ development resulting in alternative cell fates like γδ T cells. / T-Lymphozyten (T-Zellen) sind eine wichtige Säule des Immunsystems. Benannt nach dem Ort ihrer Entstehung, dem Thymus, werden sie verschiedenen Linien zugeordnet, den αβ-T-Zellen, den γδ-T-Zellen, den Mukosa assoziierten invarianten T-Zellen (MAIT) und den natürlichen Killer T-Zellen (NKT). Bei den αβ-T-Zellen mit einem T-Zell-Rezeptor (TZR) aus α- und β-Kette unterscheidet man die CD4+ Helfer T- von den CD8+ zytotoxischen T-Zellen. Beide sind zentrale Bestandteile des adaptiven Immunsystems. Einfach positive (SP) CD4+ und CD8+ αβ-T-Zellen gehen aus der positiven und negativen Selektion doppelt-positiver (DP) CD4+CD8+ Zellen hervor. Zuvor durchlaufen sie als doppelt-negative (DN) Thymozyten, die von Vorläufern aus dem Knochenmark abstammen, verschiedene Stadien (DN1-DN4), die durch unterschiedliche Expression der Adhäsionsmoleküle CD25 und CD44 gekennzeichnet sind. Der Transkriptionsfaktor NFATc1, ein Mitglied der Familie Nukleärer Faktoren Aktivierter T-Zellen (NFAT), ist maßgeblich an der Differenzierung und der Funktion von T-Zellen beteiligt. Während der Thymozytenenreifung kann eine zunehmende nukleäre Expression von NFATc1 beobachtet werden, die im DN3-Stadium (CD44-CD25+) den höchsten Wert erreicht. Eine Deletion der NFATc1-Aktivität in frühen Vorläuferzellen führte zu einem Arrest der Thymozytendifferenzierung im DN1-Stadium (CD44+CD25-). Andererseits hemmte die Überexpression einer konstitutiv aktiven Form von NFATc1 den Übergang von DN3-Zellen in das DN4-Stadium (CD44-CD25-), was vermuten lässt, dass ein bestimmter Schwellenwert der NFATc1-Aktivität zu diesem Zeitpunkt von entscheidender Bedeutung ist. Mittels ChIP- und RNA-Sequenzierung gelang es, NFATc1-Zielgene im Thymus zu identifizieren. Hierzu zählten unter anderen die Tcra und Tcrb Gene. Transkriptions-analysen an isolierten DN-Thymozyten offenbarten daneben mehrere NFATc1-regulierte Gene, die an der Entwicklung von γδ-T-Zellen beteiligt sind. In den Mausmodellen, Rag1Cre-Nfatc1fl/fl und Rag1Cre-E2fl/fl, bei denen die Aktivität von NFATc1 bzw. der induzierbaren NFATc1-Aktivität zu frühen Stadien der Thymozytenentwicklung beeinträchtigt ist, stieg die Zahl von γδ-T-Zellen an. Diese γδ-T-Zellen zeigen eine unübliche Überexpression des CD4 Markers, eine fehlende Expression des Oberflächenmarkers CD24 und eine Überexpression des antiapoptotischen Gens Bcl2a1a. Wir vermuten daher, dass NFATc1 an der erfolgreichen Reifung von αβ-T-Zellen direkt beteiligt ist und fehlende NFATc1-Aktivität die Entwicklung alternativer Linien wie von γδ-T-Zellen befördert.

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