Medical Biology - Theses
Permanent URI for this collection
Search Results
1 - 10 of 10
-
ItemThe role of ZC3H12C in the posttranscriptional regulation of Tnf( 2021)Tumour Necrosis Factor (TNF) is one of the most potent pro-inflammatory cytokines and it is secreted in response to danger signals, such as those caused by pathogen infection. High levels of TNF have been associated with many chronic and inflammatory diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD) and psoriasis. To prevent high TNF levels and uncontrolled inflammation, Tnf mRNA is degraded when not required, making post-transcriptional regulation a central mechanism to control Tnf expression. Post-transcriptional control operates through cooperation between cis-elements present in the 3’UTR and trans-acting proteins such as RNA-binding proteins. Until recently, knowledge about post-transcriptional regulation of Tnf was limited to the role of the AU-Rich Element (ARE), and to a lesser extent to that of the Constitutive Decay Element (CDE). In 2015, our group identified a New Regulatory Element (NRE), which changed the view on the post-transcriptional regulation of Tnf. Importantly, we have discovered a cooperative mechanism between two or more elements to regulate Tnf mRNA stability in vitro. In this thesis, I have translated our previous in vitro observations, in vivo, using the CRISPR/Cas9 technology to generate mice with deletions of one or two regulatory elements in the Tnf 3’UTR. I showed that the variety of phenotypes of the mice changed greatly in severity, including a concomitant deletion of the ARE and the CDE causing embryonic death. This suggests that not only different cis-elements cooperate to destabilise Tnf mRNA efficiently, but the mechanisms involved also appear to operate in a cell- and tissue-specific manner. Furthermore, I aimed to characterise a new trans-acting RBP called ZC3H12C that was previously identified by our group as involved in the post-transcriptional regulation of Tnf in vitro. To study the physiological role of ZC3H12C and the consequences of its loss, and its expression in vivo, I engineered a mouse deficient in Zc3h12c, in which the green fluorescent protein GFP that can be used as a marker of expression replaces ZC3H12C. Zc3h12c-deficient mice are found as adults at the expected Mendelian frequency and look outwardly normal. In particular, they do not present with any phenotype related to an excess of TNF (like cachexia or arthritis), even at an advanced age. However, loss of Zc3h12c causes aberrations in the structure of secondary lymphoid tissues, and hypertrophic skin-draining lymph nodes with supernumerary B cells and inflammatory dendritic cells in ageing mice. Flow-cytometry analysis of our GFP-reporter mouse showed that dendritic cells (DCs) are the immune cell type expressing ZC3H12C the most. RNA-seq analysis on splenic DCs suggested that loss of Zc3h12c affected the anti-viral immune response. Accordingly, when challenged with chronic LCMV, Zc3h12c-deficient mice presented with an abnormally exaggerated immune response. I characterised the impact of the loss of Zc3h12c in the context of psoriasis to confirm previous Gene Wide Association Studies (GWAS) suggesting that Zc3h12c was one of the risk genes involved in psoriasis incidence in human. I found that loss of Zc3h12c did not impact psoriasis’ development, but this observation could be due to the limits of the psoriasis model used in my study. I further characterised the role of Zc3h12c in skin homeostasis by mimicking the Toxic-Epidermal Necrolysis disease using subcutaneous injection of SMAC-mimetics to induce TNF-dependent cell death in the skin. In this context, loss of Zc3h12c appeared to be beneficial and reduced the lesions and the cell death induced by the SMAC mimetic compound. Finally, to evaluate the potential role of TNF in this phenotype, I generated mice lacking both Tnf and Zc3h12c. While double-deficient (DKO) mice never developed lymphadenopathy, around 30% of the Tnf/Zc3h12c DKO mice developed severe multiorgan inflammation, including pancreatitis, myocarditis, otitis, myositis, pyelonephritis, anaemia, extramedullary haematopoiesis and bone marrow failure. Histopathological analysis suggested that concomitant loss of Tnf and Zc3h12c rendered the mice immunocompromised and potentially sensitive to the opportunistic pathogen Pasteurella pneumotropica, for which they tested positive. To evaluate the role of the TNF-TNFR2 signaling in the phenotype, and given the widely known role of TNFR2 in autoimmunity development, I generated Tnfr2 and Zc3h12c double-deficient mice. While I failed to observe a single Tnfr2/Zc3h12c DKO mouse falling sick, I also observed an absence of disease development in the Tnf/Zc3h12c DKO mice and this coincided with the clearance of P. pneumotropica from the animal facility. These observations raise new questions on the role of Tnf and Zc3h12c in the control of immune responses and inflammation, and further investigation will have to be conducted. Overall, my work suggests that Zc3h12c might be a risk factor in the context of anti-TNF treatment leading to autoimmunity in some patients.
-
ItemThe Transcription Factor T-bet in the Control of Germinal Centre Dynamics in Malaria( 2019)With reductions in the global malaria burden stalled, this preventable and curable infectious disease caused by the Plasmodium parasites, remains a public health challenge that affects the world’s most vulnerable populations. Naturally acquired immunity plays an important role in protection from disease; however, there is long-standing evidence that it requires years of repeated infections to develop. The reasons for this are largely elusive, but immuno-epidemiological studies support that protective antibodies and memory B cells are short-lived and inefficiently generated to infection. Moreover, recurrent infections are associated with an expansion of atypical memory B cells that may have impaired function. Histological analyses revealed significant disorganisation of the spleen in severe malaria patients, which led to the concept that acute infection may undermine the acquisition of B cell memory. T helper 1 pro-inflammatory responses induced by blood-stage infection were subsequently shown to compromise the induction of humoral immunity by inhibiting effective T follicular helper (Tfh) cell differentiation and germinal centre (GC) reactions. The relative contribution of the T helper 1 lineage-defining transcription factor, T-bet, in CD4+ T cells and B cells to GC development in malaria, was investigated using the P. berghei ANKA blood-stage infection model. T-bet expression in CD4+ T cells limited the differentiation of Tfh cells that supported GC development in the spleen. This led to an impaired generation of antibody-secreting cells and memory B cells following infection. In addition to its impact on CD4+ T cells, T-bet was highly up-regulated in GC B cells elicited by infection, and limited the magnitude of the GC response in a B cell-intrinsic manner. Strikingly, T-bet expression in the B cell compartment modulated the transcriptional landscape of GC B cells to promote the GC dark zone program but constrained light zone development. In particular, T-bet suppressed expression of the regulator of G-protein signaling 13, which down-regulates the responsiveness of B cells to migrate towards the chemokine CXCL12, for effective dark and light zone transition within the GC. T-bet-driven dark zone skewing of the GC reaction following malaria infection associated with enhanced somatic hypermutation of GC B cells, and improved the avidity of antibodies against the parasite. Therefore, this thesis supports a model in which malaria-elicited inflammation mediated by T-bet, exquisitely modulates the dynamics of the GC reaction, promoting GC B cell dark zone polarization that promotes the generation of B cells with increased affinity for antigen, consequently enhancing affinity maturation. This provides novel insight into the cellular mechanisms that underlie the development of humoral immune responses in malaria, and has implications for other chronic infections and autoimmune disease that are characterised by a similarly potent inflammatory milieu.
-
ItemTargeting regulators of natural killer cell homeostasis in cancer immunotherapy( 2018)The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory receptors and from cytokines such as IL-15. The ability to recognise tumour cells in the absence of antigen presentation has garnered significant interest in NK cells as novel targets for immunotherapy development. However, successful developments in this area have led to limited success. This is, in part, due to the lack of understanding of the underlying mechanisms governing inhibitory and stimulatory pathways in NK cells. In this work, we aimed to identify the key regulator of NK cell proliferation in order to further our understanding of NK cell activity both in the steady-state and in the setting of inflammation. Here, we have identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signalling in NK cells. IL-15 is the main driver of NK cell proliferation, survival, differentiation and function, and thus a highly relevant checkpoint in NK cell homeostasis. We found Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFNγ production and cytotoxicity toward tumours, in vitro. This was associated with increased JAK-STAT signalling in Cish-deficient NK cells. Cish-deficient mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity, uncovering CIS as a potent intracellular checkpoint in NK cell-mediated tumour immunity. Under homeostatic conditions, phenotypic changes in NK cells lacking Cish were observed in vivo. This included an increase in terminally differentiated NK cells as well as increased expression of cell cycle markers, suggesting that under steady-state conditions, CIS also plays a role in maintaining IL-15 driven regulation of NK cells in vivo. Additionally, the changes observed in steady state Cish-deficient NK cells manifested in a lower activation threshold, evidenced by the redundancy of exogenous IL-15 to induce augmented production of inflammatory cytokines and cytotoxicity when stimulated ex vivo. These data suggest that Cish not only regulates NK cell responsiveness to IL-15, but may also play a role in maintaining an activation threshold, consequently regulating effector functions in vivo. Furthermore, inhibition of CIS was found to be conserved between human and mouse NK cells, emphasising its potential role as a novel immunotherapy target for the treatment of human cancer.
-
ItemCharacterisation of mice deficient for the pro-survival BCL-2 family member A1/BFL-1( 2018)Apoptosis is important for the development and maintenance of a healthy immune system. The intrinsic apoptotic pathway is regulated by the BCL-2 family of proteins, which consists of both pro-survival and pro-apoptotic members. The balance between these two sub-groups determines whether a cell lives or dies. The levels of different BCL-2 family proteins is influenced by the signals that an immune cell receives – for example, cytokine signalling drives pro-survival protein expression, whilst cellular stress from cytokine deprivation upregulates pro-apoptotic proteins. Studies using knockout mouse models have highlighted the importance of some of the pro-survival proteins in different haematopoietic cell types, such as BCL-2 in mature lymphocytes and MCL-1 in haematopoietic stem cells. The pro-survival protein A1/BFL-1 is highly expressed in many different immune cell subsets and is upregulated after immune cell activation. However, little is known about the physiological importance of A1/BFL-1 in the immune system. This is because the presence of three murine A1 isoforms complicates the generation of a knockout mouse model. We have generated a completely A1-deficient mouse strain through sequential gene targeting in embryonic stem cells. This thesis presents an analysis of A1’s role in the haematopoietic system through the study of these A1-/- mice. A1/BFL-1 is reportedly expressed in B and T lymphocytes, neutrophils, mast cells, and conventional dendritic cells (cDCs). We characterised the cell numbers for these populations in the A1-/- mice in the steady state. There were no major differences found when compared to wild-type mice, although there was a small but significant decrease in cDC numbers in the spleen, and also small reductions in memory T cell populations. Given that A1/BFL-1 is upregulated by immune cell activation, we proceeded with in vitro activation assays with the various different immune cell subsets. Despite the reduction in memory T cells, in vitro survival of T cells after activation was unperturbed. A1-/- cDCs, however, had a marked survival disadvantage in tissue culture. We further characterised the A1-/- mice in response to immune challenge in vivo, with influenza infection, LCMV infection, and T cell-dependent immunization. The A1-/- mice responded normally to all of these models. We reasoned that A1/BFL-1 may have overlapping roles with other pro-survival BCL-2 family proteins. To this end, we generated compound mutant mice that are deficient for A1 and heterozygous for BCL-2, BCL-X or MCL-1. We focused on the lymphocytic compartment of these mice, based on the co-expression of these pro-survival proteins in B and T lymphocytes. Again, we found no overt differences in the compound mutant mice when compared to control mice. It is likely that complete deletion of BCL-2/BCL-X/MCL-1 in combination with A1 loss is required to decipher these overlapping roles. Overall, despite high expression of A1/BFL-1 in different immune cell types, loss of A1 is tolerable in mice and, therefore, A1 is a redundant pro-survival BCL-2 family member in the haematopoietic system.
-
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.
-
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.
-
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.
-
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.
-
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.
-
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.