Medical Biology - Theses
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ItemB cell responses during severe malaria: the impact of inflammation on T follicular helper cell and germinal centre responses( 2015)Despite many advances in malaria control and elimination, infection by Plasmodium remains a significantly widespread cause of morbidity and mortality worldwide. Naturally acquired immunity to the parasite plays an important role in protection against malaria infection and the development of symptomatic disease. However, no evidence exists of sterile immunity to the disease and the development of sustained clinically protective antibody responses has been shown to require repeated infections. While many studies have focused on the complex nature of these responses against the antigenically diverse parasite, few have addressed the effect of malaria infection on the generation of memory B cell responses. A study of children in areas of high seasonal malaria transmission revealed a delay in malaria-specific MBC generation despite continual exposure to the parasite. In contrast, in a low transmission setting, lasting memory B cell responses were detected in adults following a single exposure to the parasite. These data indicate clinical malaria infections may hinder the generation and maintenance of malaria-specific memory B cell populations. Long-lived populations of B cells, including memory B cells and long-lived plasma cells, are generated during the germinal centre (GC) reaction in secondary lymphoid organs, such as the spleen. In support of the notion that clinical malaria episodes hinder the induction of humoral memory, histological studies revealed that human fatal malaria infections are accompanied by dramatic changes in splenic architecture, including impaired GC formation. The bulk of studies examining the induction of GC responses following malaria infection have made use of self-resolving infection models in mice. To specifically address the impact of severe malaria infections on these processes, the development of GC responses was assessed using the P. berghei ANKA model of severe malaria in comparison to immunisation with an equivalent antigenic load of attenuated parasites. This model permitted the uncoupling of the effects of severe malaria infection and parasite exposure, and demonstrated that severe malaria infections profoundly impede the correct generation of GC structures. Further, compared to immunised control animals, infected animals had reduced numbers of GC B cells. Critically, the excessive inflammatory processes caused by severe malaria infection directly impaired T follicular helper cell differentiation and lead to the preferential accumulation of Tfh precursors. As a consequence of impaired GC induction, memory responses were not efficiently generated following severe malaria. Collectively, the data presented in this thesis demonstrate a novel role for inflammation in the control of Tfh and GC responses and provide valuable insight into the mechanisms underlying inefficient B cell responses following clinical malaria infections in humans.
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ItemDistinct requirements for T-bet and Nfil3 for the generation of innate lymphoid cell populations( 2014)Innate lymphoid cells (ILCs) are key effector cells found at mucosal surfaces. They are crucial for lymphoid tissue development and preserving the delicate balance of tolerance, immunity and inflammation. ILCs are grouped into three distinct lineages namely ILC1, ILC2 and ILC3 based on their cytokine and transcription factor expression profiles. The lineage specification, development and function of ILCs depend on the timely expression of a precise set of transcription factors that regulate distinct checkpoints during their development and maturation. The full array of molecular networks that are responsible for generating ILC diversity is, however, yet to be fully characterized and is the focus of this thesis. Firstly, the role of the T helper 1 lineage specifying transcription factor T-bet (encoded by Tbx21) in regulating the diversity of ILC3 populations was investigated. Here T-bet was found to be the essential regulator of the NKp46 expressing population of ILC3 termed NCR+ ILC3. T-bet was necessary for the transition from NCR- ILC3 precursors into NCR+ ILC3s. Furthermore, expression of T-bet was intricately regulated by the Notch signaling pathway. T-bet regulated the expression of a precise set of molecules unique to NCR+ ILC3. Loss of T-bet also resulted in reduced protection against the intestinal pathogen Citrobacter rodentium (C. rodentium) which correlated with reduced IL-22 produced from ILC3. This pathology seen in T-bet deficient mice, however, was not solely due to loss of NCR+ ILC3. Mice specifically deficient in NCR+ ILC3 were able to broadly control C. rodentium to infection, although mild pathologies were evident. Secondly, the transcription factor Nfil3 is implicated in a number of immune cell lineages including natural killer (NK) cell development. This thesis further demonstrates that all ILC populations display high Nfil3 expression and genetic ablation of Nfil3 results in a global reduction of all ILC lineages including ILC precursors. Furthermore, loss of Nfil3 selectively disrupted Peyer’s patch but not lymph node formation. Loss of Nfil3 consequently resulted in reduced protection to Citrobacter rodentium infection. Therefore, T-bet and Nfil3 exquisitely control the diversity of not only the adaptive but also the innate immune system to confer protection against multiple pathogenic organisms that infect through barrier surfaces.
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ItemPlasmodium chabaudi adami: vaccine antigens and antigenic variation( 2003)There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studi Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.
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ItemMechanisms regulating MHC II antigen presentation for improved vaccination strategies( 2013)Activation of the adaptive immune system is critical for successful vaccination outcomes. Antigen presentation plays an instrumental role in activation of the adaptive immune response and thus vaccination success. Professional antigen presenting cells (APC) are capable of constitutive major histocompatibility complex (MHC) I and MHC II antigen presentation. The most well characterised APC are B cells, dendritic cells (DC) and macrophages. Targeting antigen to the different professional APC can dictate the resulting immune response and thus the outcome of vaccination. This Thesis focuses on the role of antigen presentation in activation of the immune system, in particular the mechanisms different APC utilise to present antigens, including protein trafficking, antigen uptake and antigen processing. Virus-like particles (VLP) are naturally occurring non-infectious immunogenic particles of viral proteins. Hepatitis B virus (HBV) small envelope protein (HBsAgS) VLP are utilised in the current HBV vaccine. In Chapter Three we demonstrate that HBsAgS VLP can be utilised to generate adaptive immune responses with presentation of both MHC I and MHC II antigens observed following administration in vitro and in vivo. Despite the inhibition of interferon α (IFNα) secretion by plasmacytoid DC (pDC) in vitro, DC are capable of generating specific CTL responses to epitopes within HBsAgS VLP. In Chapter Four we investigate targeting of antigen to surface receptors in order to generate adaptive immunity. We investigate bone marrow stromal cell antigen 2 (BST-2) as a targeting receptor for delivery of antigen to the MHC I and MHC II antigen presentation pathways. The ability of cell surface BST-2 to be internalised and trafficked to lysosomal compartments, provided evidence for BST-2 to act as a targeting receptor. Comparison of the antigen targeting ability of BST-2 to known receptors, SiglecH and DEC205, showed BST-2 targeted antigen is delivered for presentation, with better or similar efficiency than the known receptors, respectively. Importantly, the most efficient cell type at cross presenting BST-2 targeted antigens was not the pDC, which received the highest antigen load, instead the activated CD8+ conventional DC (cDC). Despite success in eliciting MHC I and MHC II antigen presentation during vaccination, a number of the mechanisms controlling these pathways are poorly understood. Ubiquitination plays a key role in regulating multiple aspects of the MHC II antigen presentation pathway. In cDC membrane associated RING-CH (MARCH) 1 is critical for the surface turnover of MHC II during the steady state. However following activation, cDC shutdown gene expression of MARCH 1, leading to increased surface expression of MHC II. The role of MARCH 1 in regulating MHC II turnover in macrophages is less defined. In Chapter Five we show that the continued expression of MARCH 1 following macrophage activation is partially responsible for the continued ubiquitination and turnover of MHC II. The role of MARCH 1 in regulating trafficking and turnover of MHC II and CD86 in APC is well characterised. However, the ubiquitination targets of the other MARCH family members remain unknown. In Chapter Six we adapt a recently established protocol for plasma membrane proteomics analysis by mass spectrometry to identify known targets of MARCH 1, confirming that this protocol can be utilised to identify targets of the other MARCH family members. Additionally, the individual MARCH family members have unique gene expression profiles in an array of immune cells, highlighting the need to target specific cell types for proteomics analysis. Utilising techniques in molecular, cellular, biochemical and advanced proteomics analysis this Thesis has advanced the fields understanding of the activation of the adaptive immune response through antigen presentation. In particular this Thesis has outlined the mechanisms regulating MHC II antigen presentation for improved vaccination strategies.
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ItemCostimulatory requirements of helpindependent anti-influenza CTL( 2012)This thesis focused on the role of costimulatory molecules, CD28 and CD40L, on the induction and expansion of help-independent CD8+ T cell response during primary influenza virus infection. It has been conventionally believed that the help provided by CD4+ T cells is mediated by CD40-CD40L interactions, which results in the licensing of DCs. However, during some infections such as influenza virus infection, CD8+ T cell responses can be mounted without the requirements of CD4+ T cell help. Although there have been numerous studies on the role of costimulation in CD8+ T cell immunity, many were done in the presence of CD4+ T cells. Hence it is difficult to distinguish which T cell subset (CD4+ or CD8+ T cell), or both, the costimulation has had an impact on. This thesis has dissected the role of CD28 and CD40L costimulation on the activation and expansion of help-independent CD8+ T cells during primary influenza infection. This thesis demonstrated that, unlike CD4+ T cell help, the generation of influenza-specific CD8+ T cells was dependent on the direct costimulation of CD28 and CD40L. The impaired generation of influenza-specific CD8+ T cells in the absence of CD28 costimulation, and CD40L costimulation to a lesser extent, correlated with virus titre in the lungs. In addition, this thesis also demonstrated that CD28 costimulation was required in the early activation of “helpless” influenza-specific CD8+ T cells. In contrast, the CD40L costimulation was required at the late phase of the primary response, suggesting the role of CD40L costimulation in the expansion of “helpless” CD8+ T cells, after the initial activation by CD28 costimulation. In vitro studies revealed that CD80/86 and CD40L on DCs were important for the priming of “helpless” CD8+ T cells. Correlating with in vivo studies, CD28-CD80/86 costimulation was required for the early activation of CD8+ T cells, while CD40- CD40L costimulation was required at the late phase for the expansion of CD8+ T cells. CD28-CD80/86 costimulation had also been demonstrated to promote the survival of CD8+ T cells.
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ItemStudies on the cellular basis of the immunological defect following thymectomy in the mouse( 1969)Answers have been sought for two key questions relevant to the proposed mode of action of the thymus as a “central” lymphoid organ. Does the peripheral lymphocyte population contain a large number of thymus-derived cells and do these cells respond to antigens by producing a progeny of antibody-forming cells? The number of small lymphocytes emerging from a thoracic duct fistula in young adult neonatally-thymectomized CBA mice was approximately 2% of that in intact mice of the same age and body weight. Multiple injections of chromosomally-marked thymus cells increased the size of the lymphocyte pool and the majority of thoracic duct cells, stimulated into division, carried the chromosome marker of the thymus cell donor. Thymectomy in adult life was not followed by a significant decrease in the output of thoracic duct lymphocytes until many months after the operation. Whole body x-irradiation resulted in a dramatic reduction in the number of cells drained from a thoracic duct fistula and, in mice protected from the lethal effects of haemopoietic failure, the reestablishment of the lymphocyte population was dependent upon the presence of the thymus. The data supports an increasing bulk of indirect evidence which, when taken in toto, strongly suggests that a large proportion of recirculating lymphocytes are thymus -derived cells or their descendants. The number of sheep erythrocyte antigen-reactive cells (ARC) in the thoracic duct lymphocyte population of neonatally-thymectomized mice was markedly reduced when compared with the number in the population from normal mice. The bone marrow did not contain ARC but was a potent source of ARC precursors. Neonatally-thymectomized mice did not lack precursor cells in the bone marrow but apparently lacked the thymus influence necessary for the differentiation of these precursors into ARC. Further studies on the thymus-dependent development of ARC hinted at the possibility that the entity known as "an ARC" required the presence of both thymus- and bone marrow-derived cells to express itself in terms of haemolysin production. Neonatally-thymectomized CBA mice failed to respond in normal fashion to a primary injection of sheep erythrocytes (SRBC) and the peak number of haemolysin plaque-forming cells (PFC) in the spleen was reduced by a factor of 1 log 10. The PFC response was increased by injections of either thymus or thoracic duct lymphocytes from CBA, (CBA x C57BL)F 1 hybrid, and C57BL donor mice. In thymectomized mice reconstituted with semiallogeneic and allogeneic cellular inocula, the PFC carried the immunogenetic characteristics of the host and not those of the inoculated cells. Hence, thymus and thoracic duct cells were not reconstitutive simply by virtue of the ability to transform into PFC. Thymus and thoracic duct cell inocula contained "reactor cells" which responded to SRBC antigens in irradiated mice by undergoing a burst of mitosis. Thoracic duct lymphocytes, unlike thymus and bone marrow cells, were able to produce PFC when injected together with SRBC into irradiated mice. However, the PFC response in irradiated recipients of thoracic duct lymphocytes was increased substantially by a simultaneous injection of bone marrow cells. Combinations of cells from semiallogeneic mice did not interact upon transfer to irradiated recipients but clear evidence of synergism in PFC production was apparent in adult-thymectomized irradiated mice protected with CBA bone marrow cells and injected two weeks later with (CBA x C57BL)F 1 thoracic duct cells. In this case, the vast majority of PFC were derived from the bone marrow inoculum. The results suggest that the bone marrow contains only PFC precursors, the thymus only "reactor cells", but that the thoracic duct lymph contains both cell types. It seems that the normal 19S haemolysin response to SRBC in the CBA mouse requires the collaboration of bone marrow-derived PFC precursors and thymus-derived "reactor cells". The neonatally-thymectomized mouse contains adequate numbers of PFC precursors but, after challenge with SRBC, few are recruited into 19S haemolysin production because of the severe deficiency in thymus-derived ''reactor cells".
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ItemResolving causes and consequences in a model of autoimmune disease( 2010)Antibody is an essential component of immunity that triggers a series of mechanisms to neutralise and clear pathogens. In a situation where the targets of antibody cannot be cleared, for instance when they are a normal part of the host, self-directed antibody activates these same mechanisms to drive continued destruction of host tissues. The production of antibody is therefore a critical point of regulation in the prevention of autoimmune disease. A sequence of control points exists that prevents B cells expressing self-reactive antibody from differentiating to form plasma cells. Once this differentiation step has occurred, a plasma cell can persist for years unimpeded. No current therapies target plasma cells and their capacity for persistence precludes any potential benefit of B cell depletion strategies. The factors required for plasma cell maintenance are inadequately characterised, and even less is known of the means by which plasma cell homeostasis is regulated. In systemic lupus erythematosus (SLE), an antibody-mediated autoimmune disease, plasma cells can accumulate and secrete excess antibody into the serum. When this antibody reacts against self-components it may trigger autoimmune disease. With time, the recruitment of auxiliary cell types into the disease process can compound disease development and promote the maturation of self-reactive plasma cells into high-affinity, isotype switched antibody-producing cells whose capacity for inflicting tissue damage is considerably amplified. This cyclic pattern of disease development confounds the study of underlying cell-intrinsic defects, obscuring causes of disease from those aspects of the phenotype that are a consequence of inflammatory processes occurring as part of disease progression. Using the Lyn-deficient mouse model of SLE, this thesis demonstrates that certain aspects of this antibody-mediated disease require the participation of T cells to establish destructive IgG-driven inflammatory processes. When the contribution of T cells is diminished, an IgA-mediated pathology is revealed that is entirely reliant on the presence of IL-6. Pharmacological inhibitors of IL-6 and concurrent blockade of T cell help may therefore be an effective strategy for preventing the production of pathogenic IgG and IgA autoantibodies in SLE patients. Occurring in parallel and independently of these factors is an accumulation of unswitched plasma cells in peripheral lymphoid organs of Lyn-deficient mice. These plasma cells appear to accumulate by virtue of their enhanced capacity for survival, which is a direct result of the loss of Lyn in these cells. Lyn normally limits STAT3 signalling following IL-6 stimulation of plasma cells and in its absence loss of negative regulation of STAT3 signalling may reduce the threshold for plasma cell survival by STAT3-dependent factors such as IL-6. This could allow plasma cells to accrue beyond normal homeostatic limits. An additional means of regulating the plasma cell response to IL-6 is the induction of SOCS3, which curtails the duration of STAT3 signalling. The consequences of deletion of SOCS3 in the B cell lineage were investigated, revealing a subtle role for this negative regulator during the antibody response to T cell-dependent antigen.