HLA Blockers for potentially treating Rheumatoid arthritis

Autoimmune diseases occurs when the immune system recognize self antigens in the body as foreign invaders leads to dysfunction of tissue. RA is an autoimmune disease, caused by improper recognition of self-peptides, particularly human cartilage glycoprotein and type II collagen, by specific human leukocyte antigen (HLA) receptors. Normally T-cell specific for these peptides are destroyed in the thymus before they are released, preventing autoimmunity. However, certain post-translational modifications, especially citrullination, can lead to “self-peptide” recognition by non-self T cells: in the case of RA, one HLA protein (DRB*0401), out of about 1700 possible ones, is responsible for 65% of RA cases. If this protein could be blocked, drugs could be developed that interrupt the disease at its root cause without affecting the rest of the immune system; this is the focus of research in the Trant Lab. This thesis will briefly overview the approach, including the drug design, and will focus on the molecular biology work accomplished to date. The main finding of our thesis are as follows. Human leukocyte antigen is a protein needed to advance the research and drug development in autoimmune diseases and cancers. In these early stages of this project, we showed that choice of HEK293 cell as a host to express HLA protein and IMAC chromatography as a purification system gives us a reasonable amount of α/β heterodimer glycosylated membrane HLA protein

Identification of Conserved and HLA Promiscuous DENV3 T-Cell Epitopes

Anti-dengue T-cell responses have been implicated in both protection and immunopathology. However, most of the T-cell studies for dengue include few epitopes, with limited knowledge of their inter-serotype variation and the breadth of their human leukocyte antigen (HLA) affinity. In order to expand our knowledge of HLA-restricted dengue epitopes, we screened T-cell responses against 477 overlapping peptides derived from structural and non-structural proteins of the dengue virus serotype 3 (DENV3) by use of HLA class I and II transgenic mice (TgM): A2, A24, B7, DR2, DR3 and DR4. TgM were inoculated with peptides pools and the T-cell immunogenic peptides were identified by ELISPOT. Nine HLA class I and 97 HLA class II novel DENV3 epitopes were identified based on immunogenicity in TgM and their HLA affinity was further confirmed by binding assays analysis. A subset of these epitopes activated memory T-cells from DENV3 immune volunteers and was also capable of priming naïve T-cells, ex vivo, from dengue IgG negative individuals. Analysis of inter- and intra-serotype variation of such an epitope (A02-restricted) allowed us to identify altered peptide ligands not only in DENV3 but also in other DENV serotypes. These studies also characterized the HLA promiscuity of 23 HLA class II epitopes bearing highly conserved sequences, six of which could bind to more than 10 different HLA molecules representing a large percentage of the global population. These epitope data are invaluable to investigate the role of T-cells in dengue immunity/pathogenesis and vaccine design. © 2013 Nascimento et al

Generation of a transplantable murine tumor model expressing the human breast cancer associated tumor antigen NY-BR-1 in HLA-DRB1*0401-transgenic mice

Prevalence of mutations and deregulated gene expression has resulted in a wide variety of tumor antigens which can be targeted for immunotherapy. NY-BR-1 is a breast cancer associated differentiation antigen which is overexpressed in more than 60% of breast cancers. In this study we established the first NY-BR-1 expressing transplantable tumor model using the murine mammary adenocarcinoma cell line EO771 as parental cells for the generation of NY-BR-1-expressing transfectants (EONY cells) which are compatible for transplantation into HLA-DRB1*0401tg mice. Since NY-BR-1 is not endogenously expressed in these mice, transplantation of NY-BR-1 expressing tumors led to the induction of CD4+ and CD8+ T cells specific for HLA-DR*0401- and H2-Db-restcried NY-BR-1 epitopes, respectively, previously identified in our lab. We also generated MHC I knockout cell lines with the aim of using them to study NK cell responses or as parental lines for transection of human MHC I molecules. Since several HLA-restricted NY-BR-1-specific T cell epitopes have been described, these cells could serve as an important tool to study the efficacy of using the epitopes as peptide vaccines and studying the synergistic effect of induced CD8+ and CD4+ T cells in HLA-transgenic mice co-expressing human MHC I and MHC II molecules. Additionally, we also confirmed that previously identified H2-Db-restrcited epitope was indeed naturally processed based on detection of CD8+ T cells which specifically recognized this epitope in mice following transplantation of NY-BR-1 expressing tumors or upon immunization with global NY-BR-1 antigen. We also observed that immunization with Ad.NY-BR-1 was more suitable for generating a NY-BR-1 specific CD8+ T cell line compared to immunization with nonameric peptide. We also obtained evidence suggesting that the H2-Db-restricted CTL epitope might actually be identical to the core sequence of one of the previously identified HLA-DRB1*0301-restricted CD4+ T cell epitopes. Thus binding of the CTL epitope to HLA-DRB1*0301 molecules on feeder cells might have resulted in predominant restimulation of HLA-DR3B1*0401-restricted CD4+ T cells in vitro. Cancer immunotherapy involving T cells have focused primarily on cytotoxic CD8+ T cells. However, a body of evidence has emerged in the recent past that argues for including CD4+ T cells because they not only eliminate tumor cells directly but also greatly enhance the outcome of cancer immunotherapy by various ways. One of the mechanisms by which CD4+ T cells can contribute to successful immunotherapy is by repolarizing tumor associated macrophages (TAMs) into a less immunosuppressive phenotype in an antigen dependent fashion. In this study we could demonstrate that NY-BR-1 specific CD4+ T cells could repolarize M2-like macrophages towards an M1-like phenotype upon antigen specific interaction. However, the majority of the CD4+ T cells infiltrating the EONY tumors appeared to have tumor promoting functions as depletion of CD4+ T cells resulted in delayed tumor growth and was accompanied by switch in the polarization status of TAMs towards an M-1like phenotype. Though Ad.NY-BR-1 immunization resulted in induction of NY-BR-1 specific CD4+ T cells and slight changes in polarization of intra-tumoral macrophages; it did not seem to drastically change the proportion of tumor-promoting CD4+ T cells in the tumor since the protective effect and switch in TAM phenotype was observed in both control virus and Ad.NY-BR-1 immunized mice. Therefore, antigen-specific instruction of TAMs by NY-BR-1-specific CD4+ T cells could not be demonstrated in vivo, possibly due to the prevalence of tumor infiltrating CD4+ T cells with a tumor promoting phenotype occurring in the EO771/NY-BR-1 model established within this thesis

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease

Rapid Regulatory and Effector Immune Responses in Toxic Shock Syndrome

Toxic shock syndrome (TSS) is an acute, potentially fatal condition characterized by high-grade fever, hypotensive shock and systemic inflammation. It is caused by exposure to staphylococcal and streptococcal superantigens (SAgs), which can activate up to 50% of T cells resulting in a hyperinflammatory ‘cytokine storm’ within hours. This inflammatory cascade progresses to a life-threatening illness with alarming rapidity, and SAg-exposed individuals can develop multi-organ failure within hours of onset of symptoms. However, there are currently no available treatments that efficiently mitigate the cytokine storm, which drives TSS immunopathology. Therefore, identifying and understanding the critical components underlying this process should hold the key to designing effective therapeutics to reduce TSS severity. In this thesis, I have utilized a clinically relevant humanized HLA-DR4 transgenic (DR4tg) mouse model of TSS to reveal the previously unrecognized roles of three rapid host responses in the initiation or control of the cytokine storm. First, genetic and antibody-mediated depletion of invariant natural killer T (iNKT) cells in DR4tg mice show that iNKT cells are pathogenic in TSS and contribute to the cytokine storm. Targeting iNKT cell responses with the T helper type-2 (Th2)-polarizing glycolipid agonist OCH also reduces TSS morbidity and mortality. Second, I found that granulocytic myeloid-derived suppressor cells (MDSCs) are rapidly recruited to the liver of DR4tg mice during TSS. These hepatic MDSCs potently suppress SAg-induced T cell responses and may therefore mitigate tissue injury in TSS. Lastly, I define the rapid production of interleukin-17A (IL-17A) by effector memory T cells as a novel mechanism promoting immunopathology in TSS. Blockade of IL-17A signaling in human blood mononuclear cells reduces the expression of multiple inflammatory mediators of TSS, suggesting that IL-17A contributes to the cytokine storm. Importantly, the treatment of DR4tg mice with an IL-17A-neutralizing antibody attenuates TSS-induced tissue damage, morbidity and mortality. Collectively, the results presented in this thesis delineate the novel contributions of iNKT cells, MDSCs and IL-17A to the early phase of TSS pathogenesis. Furthermore, my findings suggest that therapeutic approaches targeting iNKT cells or IL-17A responses may be effective in reducing TSS mortality

Cellular Responses of the Retina to West Nile Virus Infection

Age-related macular degeneration (AMD) is the leading cause of blindness in the developing world in people aged over 60 years, manifested as a loss of central vision in one or both eyes, with significant morbidity including loss of mobility and depression. This condition involves the degeneration of the macula, and although the exact aetiology of this disease is unknown, various epidemiological studies have shown it to be multifactorial. Current research points towards the involvement of a dysregulated immune system in the pathogenesis and progression of the disease: as the body ages, the immune system increasingly adopts a more inflammatory basal state. However, not all of the aged population develops AMD and it is highly likely that an additional stimulus or stimuli is/are needed to exploit this dysregulated immune environment to initiate this disease. Given the range of pathogens that can infect the retina, we hypothesize that this breaking point could manifest as a chronic inflammation as a result of a low-level infection. West Nile Virus (WNV) is a flavivirus that has come into international prominence ever since its spread into previously WNV-free regions following the 1999 New York outbreak. As several case reports have shown that WNV is capable of infecting the retina, and given its immunopathogenic properties, we believe the virus is a useful tool to model key immune pathways and responses that may be involved in the development and progression of AMD. Of significant interest are the processes involved in the breakdown of the outer blood-retinal barrier (BRB), which is an important step in the progression of AMD from an early stage to a more severe one. Additionally, deciphering and understanding the profile and populations of leukocytes that are recruited during an immunopathic infection in an organ regarded as being immunoprivileged is of great appeal. With this in mind, we set out to investigate the effects of WNV infection on the retinal pigment epithelium (RPE), which comprises the outer BRB. Previously, our laboratory established the WNV BRB model by quantitating various parameters, such as level of infectivity, viral output by WNV-infected RPE and effects of WNV infection on RPE proliferation/migration. The effect of WNV on the extracellular matrix (ECM) production by RPE was also investigated and increases in collagen I, IV and fibronectin were noted. Global ECM production induced a lowered rate of proliferation of RPE seeded on WNV-infected RPE ECM as opposed to mock-infected ECM. A full genome microarray was also undertaken on WNV-infected RPE to analyse differentially regulated gene mRNA production, and increases in several immune genes, as well as genes involved in the stress-response pathway and the TGFβ pathway were found. This current investigation expanded upon these results, and found that WNV infection produces a predominantly CCL5 chemokine response rather than a CCL2 response. Additionally, a lack of TNF production was noted, despite a high initial upregulation of the TNF gene in WNV-infected cells. WNV attenuation was found to be predominantly IFNβ-1-driven, while induction of indoleamine 2,3 dioxygenase activity was induced in part by IFNλ-1 and -2. The effects of WNV infection on RPE barrier integrity was investigated, and an initial increase in infected cells of barrier integrity was observed. Several investigations resulted in a conclusion of a soluble-mediator as the likely mechanism behind this initial increase, and while none of the chemokines tested appeared to contribute to this change, the results suggest that it may be TLR3/RIG-I independent. Finally, establishment of a murine WNV intravitreal model was also undertaken, and several key parameters were determined, including confirmation of WNV-infection of the murine retina, effect of WNV titre on mortality, and histological analysis of the effects of WNV infection on the murine retina. Quantification of the leukocyte profile recruited into the WNV-infected murine retina and choroid revealed significant increases in inflammatory Ly6Chi monocytes, as well as significant differences between immune mice and naïve mice intravitreally infected with WNV, and differences between 2 month old and 5 month old mice. Collectively, these results highlight the importance of the interferon response in both direct and indirect anti-WNV activities and immunomodulation, the changes in outer BRB integrity and possible contributors to its degradation, and the establishment of the murine intravitreal WNV model along with identification of several key leukocytes that are recruited at the peak of infection. These results will help guide further research and highlight possible immune pathways that may contribute to dysregulated inflammatory processes that may occur during the pathogenesis of AMD

Beyond conventional care : developing novel therapeutic approaches to combat arthritis

Rheumatoid arthritis (RA) is an autoimmune disorder without a definitive cure. Although RA is driven by systemic autoimmunity, its most pronounced manifestation is organspecific inflammation, particularly synovitis in joints. Persistent synovitis results in progressive joint damage and deformity, ultimately compromising joint function. The etiology of RA is multifaceted, intricately intertwining genetic, environmental, and immunological elements. While autoreactive agents have traditionally been viewed as pathogenic contributors to the development of arthritis, our research, utilizing multiple experimental arthritis models, has pinpointed several pivotal autoreactive mediators, which are surprisingly regulatory. In study I, we established a cartilage antibody induced arthritis (CAIA) model. The deficiency of Fc gamma receptor (FCGR) 2B enables swift onset of CAIA within a 12-hour time frame, and overrides the resistance arising from complement C5 deficiency. Notably, our results highlight that FCGR3 is essential and sufficient for CAIA development. The role of FCGR4 remains to be further elucidated. In Study II, we engineered a range of recombinant antibodies targeting the F4 epitope on type-II collagen (COL2). One of these antibodies, denoted R69-4, not only prevented the onset of CAIA, but also effectively suppressed the established disease. Further screening revealed that R69-4 binds to numerous targets in the synovial fluid (SF), including the complement C1q. As a result, R69-4 markedly dampens FCGR3 signaling in SF neutrophils, thereby interrupting neutrophil self-orchestrated recruitment. Given this efficacy, R69-4 emerges as a promising therapeutic candidate for RA, particularly during its acute stage. In study III, we introduced mutations to the immunodominant T cell epitope of COL2. A mutation resulting in higher affinity to major histocompatibility complex class II (MHC II) confers resistance to collagen-induced arthritis (CIA). However, the absence of either FCGR2B or neutrophil cytosolic factor 1 (NCF1) disrupts this tolerance. In particular, the deficiency of NCF1 leads to a reduction of regulatory T cells (Tregs), and a decrease of autoimmune regulator (AIRE) expression in medullary thymic epithelial cells (mTECs). In Study IV, we identified a subset of autoreactive B cells that are ubiquitously present across species. These B cells target the C1 epitope on COL2. Transferring these C1 B cells effectively suppressed arthritis of recipient mice in an antigen-specific manner. We further discerned that the suppressive efficacy of C1 B cells stems from the activation of Tregs and the functional integrity of CD72. In RA patients, we noted a reduced frequency of C1 B cells, possibly attributed to their differentiation into plasma cells. Interventions that can reverse this transition may contribute to preventing the onset of RA

Superantigen responsive T cells are required for nasopharyngeal infection by Streptococcus pyogenes

Streptococcus pyogenes is a human-specific pathogen that is responsible for serious morbidity and mortality worldwide despite being susceptible to common antibiotics. Furthermore, there is currently no licensed vaccine available against this organism. Previous research from our laboratory implicated a critical role for SAgs in a transgenic mouse model of acute nasopharyngeal infection by S. pyogenes. Herein, we are able to detect SAg production in vivo and establish that anti-SAg antibodies generated by either passive immunization or active vaccination with a MHC II-binding interface SAg toxoid reduces S. pyogenes nasopharyngeal burden. We were also able to demonstrate that this organism requires responsive Vβ-specific T cells in order to efficiently infect the upper respiratory tract. These experiments remarkably reveal that S. pyogenes manipulates T cells to promote infection and also supports targeting SAgs as vaccine candidates to prevent nasopharyngeal carrier and subsequent disease caused by this globally important pathogen

Das MHC II Ligandom : Massenspektrometrische Anwendungen in der Immunologie

The aim of this thesis was to establish a better knowledge of the human MHC class II peptide repertoire, the HLA ligandome, in general, and to outline a procedure which helps in the identification of class II-presented peptides from tumor associated antigens, in particular. To achieve these goals, biochemical and biomolecular methods as well as state-of-the-art mass spectrometric devices were used. To characterize the class II ligandome, HLA-DR peptides from the tumor-like cell line Awells, a human EBV transformed B lymphoblastoid cell line homozygous for HLA-DR4 – the HLA of interest –, were isolated and analyzed by MS using the rules of proteome analysis. 404 peptides with 173 different core sequences could be identified – the highest number of HLA ligands identified in a single experiment so far. Peptides from source proteins localized in virtually every cell compartment and participating in general cellular processes were presented under normal conditions on HLA class II molecules on the cell surface. In further experiments it could be shown that autophagy, a process involved in endosomal/lysosomal degradation and playing a role in tumor development, had a substantial impact on the class II ligandome. Cells undergoing autophagy over-presented class II peptides from intracellular source proteins by up to 131% in average as quantified by LC-MS. Thus, intracellular source proteins reach via autophagy the endosomal/lysosomal system and are there processed, corresponding peptides are loaded on class II molecules and presented on the cell surface. Posttranslationally modified naturally presented class II ligands could also be identified. Deamidated, cysteinylated and glycosylated HLA-DR peptides were characterized showing for the first time that naturally presented class II peptides can carry complex N-linked glycans. Finally, a strategy for the identification of naturally presented class II ligands from tumor associated antigens was set up. Fusion proteins targeting antigens of interest into the class II processing compartment were expressed in cells and the corresponding HLA-DR peptides isolated. By a differential mass spectrometric approach an HLA-DR4 ligand from cyclin D1 containing a CD4+ T cell epitope could be identified.Ziel dieser Arbeit war es ein besseres Verständnis des HLA Klasse II Peptid-repertoires, dem so genannten Klasse II Ligandom, herzustellen. Insbesondere sollte eine Methode entwickelt werden, die die Charakterisierung von HLA Klasse II-Liganden Tumor-assoziierter Antigene ermöglicht. Um diese Vorgaben zu erfüllen, wurden neben molekularbiologischen und biochemischen Methoden moderne massenspektrometrische Technologien eingesetzt. Zur Charakterisierung des Klasse II-Ligandoms wurden HLA-DR-Liganden von der Tumor-Zelllinie Awells, einer EBV-transformierten humanen B-lymphoblastoiden Zelllinie, die homozygot für HLA-DR4 ist, isoliert. Es konnten 404 unterschiedliche Peptide mit 173 Kernsequenzen, die bisher höchste Anzahl an identifizierten HLA-Liganden in einem einzigen Experiment, beschrieben werden. Eine Proteom-Analyse ergab, dass Peptide von Quellproteinen aus nahezu allen subzellulären Kompartimenten auf HLA-DR präsentiert werden. Des Weiteren nehmen die Quellproteine an generellen zellulären Mechanismen teil. In weiteren Experimenten konnte gezeigt werden, dass Autophagie, eine spezielle Form des endosomalen/lysosomalen Abbauweges, unter anderem involviert in der Tumorentwicklung, einen großen Einfluss auf das Klasse II-Peptidrepertoire hat. Autophagische Zellen überpräsentierten Peptide aus intrazellulären Quellproteinen durchschnittlich um 131%. Dies konnte mittels LC-MS gezeigt werden. Über Autophagie werden intrazelluläre Quellproteine in das endosomale/lysosomale System geschleust und dort abgebaut. Entsprechende Peptide werden dann auf Klasse II-Molekülen auf der Zelloberfläche präsentiert. Zusätzlich konnten auch posttranslational modifizierte Peptide identifiziert werden. So wurden deamidierte, cysteinylierte und glycosylierte Peptide charakterisiert. Unter anderem gelang es zum ersten Mal die Struktur eines natürlich präsentierten Klasse II-Peptids, modifiziert mit einem N-gebundenen Hexasaccharid, aufzuklären. Zur Identifizierung Klasse II-präsentierter Peptide aus Tumor-assoziierten Antigenen wurden in Zellen Fusionsproteine exprimiert, die Tumor-assoziierte Antigene in den Klasse II-Prozessierungsweg leiten, um anschließend HLA-DR Liganden der entsprechenden Antigene zu isolieren. Mit Hilfe einer differenziellen massenspektrometrischen Analyse konnte so ein HLA-DR4-Ligand aus Cyclin D1, der ein T-Helferepitop enthält, identifiziert werden

Autoimmune Disorders

The present edition entitled "Autoimmune disorders - Pathogenetic aspects" aims to present the current available evidence of etiopathogenetic insights of both systemic and organ specific autoimmune disorders, the crossover interactions among autoimmunity, cardiovascular morbidity and malignancy as well as novel findings in the exciting fields of osteoimmunology and immunology of pregnancy