No description available.

Animals deficient in complement components C1q, C4, C3, and CR1/2 have severely impaired antibody responses. C1q is primarily activated by antibody-antigen complexes. Antigen-specific IgM in complex with an antigen is able to enhance the antibody response against that antigen. This is dependent on the ability of IgM to activate complement. Naïve mice have very low amounts of specific antibodies and therefore it is surprising that classical pathway activation plays a role for primary antibody responses. It was hypothesized that natural IgM, present in naïve mice, would bind an antigen with enough affinity to activate C1q. To test this, a knock-in mouse strain, Cm13, with a point mutation in m heavy chain, making its IgM unable to activate complement was constructed. Surprisingly, the antibody responses in Cm13 were normal. Puzzled by the finding that the ability of IgM to activate complement was required only for some effects, the immunization protocol was changed to mimic an infectious scenario. With this regime, Cm13 mice had an impaired antibody response compared to wildtype (WT) mice. The antibody response in WT mice to these repeated low-dose immunizations was also enhanced. These observations suggest that IgM-mediated enhancement indeed plays a physiological role in initiation of early antibody responses. IgM-mediated enhancement cannot however compensate for the dependecy of T-cell help. Although IgM from WT mice enhanced the antibody response, the T-cell response was not enhanced. The connection between classical pathway activation and CR1/2 is thought to be generation of ligands for CR1/2. In mice, CR1/2 are expressed on B cells and follicular dendritic cells (FDC). Although CR1/2 are crucial for a normal antibody response, the molecular mechanism(s) are not understood. To investigate whether CR1/2 must be expressed on B-cells or FDC to generate a normal antibody response, chimeric mice between WT and CR1/2-deficient mice were constructed. The results show that CR1/2+ FDC were crucial for the generation of antibody responses. In the presence of CR1/2+ FDC, both CR1/2+ and CR1/2- B cells were equally good antibody producers. However, for an optimally enhanced antibody response against IgM-antigen complexes, both B cells and FDC needed to express CR1/2.

Infections with the bacterial pathogen Chlamydia trachomatis are a critical public health problem. Chlamydia remains the number one cause of preventable blindness worldwide and the leading cause of bacterial sexually transmitted infections in the United States. In humans, repeat and persistent infections with Chlamydia result in severe inflammation. Inflammation in the conjunctiva can result in blindness, while inflammation in the genital tract can result in pelvic inflammatory disease, ectopic pregnancy or infertility. In order to curb the increasing incidence of Chlamydia infections worldwide it will be necessary to develop a protective vaccine that affords long-term protection and prevents pathologies. To better inform vaccine development we must understand the mechanisms that drive long-term immunity in the genital tract and elucidate critical interactions between Chlamydia and host cells to uncover potential mechanisms of immune evasion.

<p>Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE.</p><p>The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project.</p><p>The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.</p>

Vaccination with DNA encoding the encephalitogenic autoantigen myelin oligodendrocyte glycoprotein (MOG), pMOG91-108, induce a protective immunity against experimental autoimmune encephalomyelitis (EAE), an animal model of human multiple sclerosis. By injection of a DNA vaccine that contains a DNA region encoding short interfering RNA specific for IFNβ (pMOG-IFNβ) the protective effect of the DNA vaccination is totally inhibited. This demonstrates that IFN-β is directly involved in the protective mechanism against EAE. The objective of this project was to study how molecules involved in the inflammatory process in EAE are regulated by suppressive DNA vaccination. mRNA expression of IL-1β, TGF β, IL-23p40 and Axl receptor tyrosine kinas did not show any significant differences between the groups vaccinated with these DNA vaccines. IL-6 and IFNγ mRNA expression after MOG stimulation in rats treated with pCI, a control vaccine was significantly higher compared to the group vaccinated with vaccine containing pMOG-IFNβ. IL-17 m RNA expression after MOG stimulation in pCl-treated rats was significantly higher compared to the group vaccinated with vaccine containing pMOG-91-108. Of these results the mRNA expression of IL-17 and IL-6 were of interest for the project. The immune system normally protects the body against infections and T-cells have an important role in this defence system. In MS and EAE, the immune system attacks the myelin and this process is caused by a dysregulation of the T-cells. IL-17-producing Th17 cells mediate EAE. Naïve CD4 T-cells in the presence of IL-6 and TGFβ are differentiated to Th17 cells instead of differentiating into T-helper or regulatory T-cells. These IL-17-producing T-cells are highly pathogenic and essential for the development of EAE. The results showed that pMOG IFNβ vaccine had an effect at the immune response, which resulted in an inhibition of the IL-6 production and that vaccination with pMOG91-108 impairs differentiation of IL-17-producing T-cells.

Antibodies can enhance or suppress the immune responses against their specific antigens. This phenomenon is known as antibody-mediated feedback regulation. We have studied the mechanisms underlying IgE-, IgM-, and IgG3-mediated enhancement of immune responses in mouse models using intravenous immunization. We attempted to answer the following questions: 1) Which cell type presents IgE-complexed antigens to CD4+ T cells? 2) Is complement activation required for specific IgM to enhance antibody responses? 3) Does IgM enhance CD4+ T-cell responses? 4) How are IgG3-antigen complexes transported into B-cell follicles? We found that CD23+ B cells transporting IgE-antigen complexes into B-cell follicles were not required to prime the antigen-specific CD4+ T cells in vivo, whereas CD11c+ cells were indispensable. After examining the three most common subpopulations of CD11c+ cells in the spleen, we determined that it was CD8α- conventional dendritic cells migrating into the T-cell zone following immunization that presented IgE-complexed antigens to CD4+ T cells. Next, we showed that specific IgM from Cµ13 mice, which is unable to activate complement, failed to enhance either antibody or germinal center responses whereas wild-type IgM enhanced both responses. Therefore, specific IgM must activate complement to enhance humoral responses. In addition, wild-type IgM did not up-regulate CD4+ T-cell responses. Finally, we showed that IgG3-antigen complexes were transported by marginal zone B cells into B-cell follicles via binding to complement receptors 1 and 2 (CR1/2) on those cells. The immune complexes were captured by follicular dendritic cells as early as 2 h after immunization. Germinal center responses were also enhanced by IgG3. Using bone marrow chimeric mice, we found that CR1/2 expression was required on both marginal zone B cells and follicular dendritic cells to provide an optimal enhancement of antibody responses.

Polymorphism in the Human Leucocyte Antigen (HLA) region of chromosome is the major source of host genetic variability in outcome of HIV-1 infection. However, there is limited understanding of the mechanisms underlying the beneficial effect of protective class I alleles such as HLA-B57, B27 and B51. Taking advantage of a unique cohort (SM cohort) infected with clade B’ HIV-1 through contaminated blood, in which many variables, such as the length of infection, the infecting viral strain and host genetic background are controlled, we performed a comprehensive study in order to understand HLA-B51 associated HIV-1 control. We first focused on the T cell responses against three dominant HLA-B51 restricted epitopes: GagNI9 (NANPDCKTI), Pol TV8 (TAFTIPSV) and Pol LI9 (LPPVVAKEI), and HLA-B51 associated escape mutations in these three epitopes. A sequential selection of epitope mutations (i.e., epitope Pol LI9, Pol TV8 and Gag NI9) was observed. Good control of viral load and higher CD4+ counts were significantly associated with at least one detectable T cell response to un-mutated epitopes. HLA-B51 restricted CD8+ T-cell clones, generated from the patients, could effectively inhibit HIV-1 replication when wild type epitopes are properly processed and presented. We then assessed the evolution of escape mutations under the selecting pressure of HLA-B51 CTLs in vitro by co-culturing HLA-B51 CTL clones with HIV-1 infected target cells (Virus Evolution Assay). Our data showed that three dominant HLA-B51 restricted CTL responses have driven the sequential escape mutations within the epitopes, leading to the loss of viral control, which confirmed our in vivo findings. Furthermore, applying Virus Evolution Assay, we assessed the impact of antigen sensitivity and TCR usage as well as founder virus effect on HIV-1 evolution and control. Our data suggested that antigen sensitivity plays an important role in anti-viral efficacy of CTLs; the TCR usage of CTLs has stronger effect on virus evolution. More importantly, our study highlighted the major impact of the founder virus sequence on viral control. It has been shown that HIV-1 has adapted to the T-cell responses to epitope Pol TI8 in other HLA-B51+ patient cohorts. However, in our cohort, T-cell responses targeting this epitope, with Valine at position 8 (Pol TV8), provide the hosts with a long-term protection against HIV-1 infection, because of a fine balance of efficient viral control, lower level of immune pressure and the slower rate of development of escape mutations. In addition, we assessed the ex vivo phenotypic characteristics of HLA-B51 restricted dominant T cell responses and our preliminary data indicated that the early differentiated and less senescence phenotype of CD8+ T cell responses in HIV-1 chronic infection is likely to be a result of low viral antigen exposure due to T cell driven escape. In conclusion, immune-dominant T-cell responses targeting three HLA-B51 restricted epitopes (Pol LI9, Pol TV8 and Gag NI9) could be advantageous for the host. In particular, the responses against epitope Gag NI9 with slow development of escape mutations or epitope Pol TV8 with a fine balance of moderate immune pressure and delayed escape mutations, are beneficial for long-term control of HIV-1 infection.

Optimal CD4+ T cell activation is key to the generation of a productive immune response. Naïve circulating CD4+ T cells are quiescent under normal conditions and undergo activation only upon encounter of the T cell receptor (TCR) with Major Histocompatibility Complex (MHC)-encoded class II molecules on antigen presenting cells (APCs). Processed antigens (derived from pathogens, tumors or self tissue during autoimmunity) in complex with MHC class II are recognized by specific TCRs on CD4+ T cells. After this encounter, the highly complex and regulated process of CD4+ T cell activation results in the differentiation of naïve T cells into effectors and their clonal expansion. Apart from binding to its cognate peptide-MHC-II complex, several other factors define the extent and magnitude of T cell activation. This context is an important determinant of the nature of the subsequent T cell response. One of the factors involved is the strength of the signal (SOS) which is delivered to the cell upon ligation of the TCR to the MHC-peptide complex. The SOS, which can vary from weak to strong, is determined by the affinity/avidity of the TCR for the MHC-peptide complex, antigen concentrations, the duration of engagement, etc. Extreme weak or strong signals can lead to non-productive T cell responses with the former resulting mostly in anergy and the latter in cell death. Signals of optimal strength are the ones that translate into functional T cell responses. However, mechanisms by which signal strength information is translated into distinct T cell responses are still not very well understood. Binding of the TCR to the MHC-peptide complex triggers several signaling cascades and leads to generation of intracellular signaling intermediates, including Ca2+. Rise in intracellular Ca2+ levels is one of the first events to occur upon initiation of T cell activation. The initial increase is brought about due to release of Ca2+ from intracellular smooth endoplasmic reticulum stores. Once intracellular stores have been emptied, the increase is sustained by a process termed as capacitative Ca2+ entry, involving opening of Ca2+ channels in the plasma membrane known as Ca2+ release activated channels (CRACs). Consequently, Ca2+ flows from the extracellular milieu into the cell. A sustained Ca2+ increase is essential for activation of the transcription factor, NF-AT whose primary job is to initiate transcription of IL-2, a cytokine crucial for CD4+ T cell proliferation. The other intracellular signaling intermediates which are the focus of work presented in this study are reactive oxygen species (ROS). TCR signaling leads to generation of ROS, which may be either mitogenic or detrimental to T cell activation. Low levels of ROS, especially H2O2, inactivate phosphatases leading to activation of kinases and signaling pathways resulting in increased proliferation. However, high levels of ROS cause oxidative stress leading to reduced T cell activation, hyporesponsiveness and death. The experimental system used for this study consists of purified mouse lymph node CD4+ T cells. These cells were activated with varying strengths of the primary signal to better understand the roles of Ca2+ and ROS in modulating T cell activation and function. The signal strength was either varied by addition of different concentrations of ionomycin or thapsigargin, pharmacological agents that increase intracellular Ca2+ concentrations. Alternatively, signal through the surface TCR-CD3 complex was initiated using anti-CD3 in two modes: soluble (weak signal) or plate immobilized (strong signal). Increasing concentrations of ionomycin or thapsigargin or changing the mode of anti-CD3 from soluble to plate bound enhances IL-2 amounts, thereby converting a weak signal to a strong one. The work presented has been divided into three parts, each dealing with a distinct aspect of T cell activation. I. SOS and CTLA4-CD80/CD86 interactions: The binding of the TCR to its cognate MHC-peptide complex delivers the primary signal. However, this alone is not sufficient to drive T cell activation and an additional costimulatory signal emanating from the binding of CD28, a constitutively expressed receptor on T cells, to its ligands CD80 and CD86 is required. Another receptor that binds to CD80 and CD86 is CTLA-4 although it does so with a ~100 fold higher affinity. CTLA-4, unlike CD28, is expressed upon T cell activation and is considered to downregulate T cell activation. Its role as a negative regulator is highlighted by the phenotype of Ctla4 -/-mice which die of massive lymphoproliferation. However, there have also been reports of some plasticity in the effects mediated by CTLA-4. Previous work from our laboratory showed that CTLA-4-CD80/CD86 interactions could either inhibit or stimulate T cell activation depending on the SOS. To identify the molecular mediators of the differential effects of CTLA-4, the role of Ca2+ and ROS was evaluated. During activation with phorbol myristate acetate (PMA) and low amounts of ionomycin, intracellular amounts of Ca2+ were greatly reduced upon blockade of CTLA-4-CD80/CD86 interactions. Further experiments demonstrated that CTLA4-CD80/CD86 interactions reduced cell cycling upon activation with PMA and high amounts of ionomycin or thapsigargin (strong SOS) but the opposite occurred with PMA and low amounts of ionomycin or thapsigargin (weak SOS). These results were confirmed by activating cells with anti-CD3 either in the soluble or plate bound form. Considerably higher amounts of intracellular Ca2+ were present in cells activated with plate bound anti-CD3 compared to those activated with soluble anti-CD3. These amounts, further augmented by CTLA-4-CD80/CD86 interactions, probably became toxic to cells as increased proliferation was observed, using reagents that blocked these interactions. The opposite, however, was seen in cells activated with soluble anti-CD3. Also, CTLA4-CD80/CD86 interactions enhanced the generation of ROS. Studies with catalase revealed that H2O2 is required for IL-2 production and cell cycle progression during activation with a weak SOS. However, the high amounts of ROS produced during activation with a strong SOS reduced cell cycle progression. Together, this study identifies intracellular Ca2+ and ROS to play important roles in the modulation of T cell responses by CTLA4-CD80/CD86 interactions. II. SOS and CD4 downregulation: This study was initiated to identify early T cell functional responses that would help predict the strength of the primary signal. Using the in vitro culture system of varying signal strengths, it was found that CD4 surface expression was controlled by signal strength. CD4 is a surface glycoprotein expressed on the TH subset along with the TCR. It performs two main functions: First, binding to MHC class II and strengthening the TCR-MHC interaction, i.e. functioning as a coreceptor. Second, due to its association with p56lck a src family tyrosine kinase, the presence of CD4 along with the TCR enhances signal transduction. Also, CD4 acts as a receptor for entry for the AIDS virus. It is known that CD4 is downregulated from the surface and degraded upon T cell activation by a protein kinase-C dependent process in human and mouse T cells. Experiments presented in this study showed increased CD4 downregulation with a strong signal. The roles of intracellular mediators were assessed and high intracellular Ca2+ amounts, but not PMA activation, was required for sustained CD4 downregulation. Also, increased H2O2 amounts in cells activated with a strong SOS inhibited CD4 downregulation. Most interestingly, the pattern of CD4 downregulation was different between peripheral T cells and thymocytes, suggesting a correlation with CD4+ T cell development. III. Modulation of CD4+ T cell activation by small molecule plant growth regulators: An important area of investigation in T cell biology is the identification of molecules that modulate T cell activation. Towards this end, the mechanisms by which small molecule plant growth regulators, naphthalene acetic acid (NAA), 2,4 dichlorophenoxyacetic acid (2,4D) and indole acetic acid (IAA), influence CD4+ T cell activation was studied. It is useful to recall that IAA is the natural auxin present in plants, NAA is a synthetic auxin and 2,4D is a herbicide. These compounds, but not structurally similar control molecules, increased the activation and IL-2 production in CD4+ T cells activated with either soluble anti-CD3 or a combination of PMA and ionomycin. An investigation into the mechanisms of action by these compounds revealed increased early generation of intracellular ROS and Ca2+. Interestingly, the nature of their effects was found to rely on the strength of the primary signal: IL-2 and proliferation were increased in cells activated with a weak signal, but lowered proliferation was observed in cells activated with a strong signal. Cells activated with strong signal posses high amounts of ROS and Ca2+ and further increase in their amounts by IAA, NAA and 2,4D resulted in growth suppression. However, augmentation of Ca2+ and ROS amounts in cells activated with a weak signal was mitogenic. The role of these compounds during in vivo T cell responses needs to be addressed. Taken together, results presented in this study emphasize the importance of the role of SOS in determining T cell responses. In addition, the roles of Ca2+ and ROS downstream of the primary signal in modulating CD4+ T cell activation were demonstrated.

The development of a malaria vaccine is necessary for disease eradication. Successful vaccine candidates to date have targeted the asymptomatic, pre-erythrocytic stage of the disease, however even the most efficacious vaccines are only partially protective. Research undertaken in our laboratory has demonstrated that one such regimen, using an 8 week prime-boost viral vector approach of ChAd63 ME-TRAP and MVA ME-TRAP, induces sterile efficacy in 21% of vaccinees, with a key role identified for TRAP-specific CD8⁺ T cells. The work described in this thesis explores the most immunogenic regimen by which to administer these two pre-erythrocytic malaria vaccines. A shortening of the prime-boost interval from 8 to 4 weeks, and the addition of an extra ChAd63 ME-TRAP priming vaccination, both demonstrated improved T cell immunogenicity over the standard 8 week regimen. Further to this, novel assays were developed to aid the evaluation of vaccine-induced immune responses. Adaptations of the existing methodology for ELISpot analysis and to whole blood flow cytometry techniques, enabled more detailed analyses of paediatric vaccine-induced T cell responses in The Gambia. This work also permitted the comparison of vaccine immunogenicity in this paediatric population, with malaria-naïve and malaria-exposed adult vaccinees. The results suggest that vaccine-induced T cell responses in infants of 8 weeks and older are comparable to that of adults. A second approach involved the development of a novel functional assay. This assay quantitatively measured the in vitro inhibition of intrahepatic Plasmodium parasite development using T cells from ChAd63.MVA ME-TRAP vaccinated volunteers. The assay demonstrated the ability of CD8⁺ T cells to inhibit parasite development in a TRAP-specific manner, and provides a platform with which to further explore pre-erythrocytic immune responses.

While adenovirus (Ad) vectors have been shown to elicit potent antigen-specific T cell responses, the kinetics and nature of antigen-specific mucosa! and systemic T-cell responses has rarely been examined, especially following mucosal administration of Ad-based vectors. In the present studies, the phenotypic and functional characterization of antigen-specific CD8+ T cell responses following intrarectal (i.r.) vaccination with an Ad vector expressing Gallus gallus ovalbumin (OVA) was conducted. The frequencies of OVA-specific CD8+ T cells was maximal at 2 weeks post-vaccination in all tissues examined and then declined, demonstrating normal expansion and contraction kinetics. CD8+ T cells induced in the course of immunization exhibited phenotypic characteristics of effector memory T cells including up-regulation of the cell surface molecules CD43, CD44 and a low level of expression of CD127 at both local and systemic sites. While the discordance between the number of tetramer-reactive and cytokine-producing OVA-specific CD8+ T cells was observed, CD8+ T cells appeared to be fully functional in vivo. Upon secondary antigen exposure, the CD8+ T cell population expanded dramatically, particularly at the mucosa! surfaces. In addition, the CD8+ T cell response generated in the course of i.r. priming protected mice from intravaginal (i. vag.) vaccinia virus one month after immunization, thus underscoring the importance of inducing a tissue-resident effector memory T cell subset for protection against pathogens at mucosal surfaces. In developing future vaccines for mucosal diseases, the induction of a tissue-resident effector memory T cell subset should be one of the immunization objectives. Lentiviral vectors represent an attractive mode of genetic vaccination. Most commonly used, vesicular stomatitis virus glycoprotein (VSVG)-pseudotyped lentiviral vectors do not efficiently infect epithelial cells from the apical side, and, therefore, are not suitable as mucosa! vaccines. In the present studies, Ebola Zaïre strain glycoprotein (EboZ)-pseudotyped lentiviral vectors, which have been previously used to deliver transgene to the lung epithelium, were delivered i.r. and evaluated as a mucosal booster vaccine. Rectal delivery of EboZ-pseudotyped lentiviral vectors expressing β-galactosidase (β-gal) had resulted in low, but detectable levels of β-gal expression 2 weeks after administration. When delivered on its own, EboZ-pseudotyped lentivirus did not prime detectable antigen-specific immune response. However, when delivered i.r. 30 days after i.r. Adβ-gal immunization, a significant enlargement (boost) of β-gal-specific CD8+ T cell responses, especially in the colonic lamina propria (LP), was observed as compared to the delivery of EboZ-pseudotyped vector encoding different transgenes or VSVG-pseudotyped lentivirus expressing β-gal. When these animals were i. vag. challenged with vaccinia virus expressing β-gal, a dramatic expansion of β-gal-specific CD8+ T cells, especially in the vaginal tract, was observed. In addition, this prime and boost strategy protected the mice from i. vag. vaccinia virus challenge. Therefore, i.r. Ad-based priming followed by i.r. EboZ-pseudotyped lentiviral boosting was an effective strategy for eliciting protective mucosal CD8+ T cell responses. / Thesis / Doctor of Philosophy (PhD)