Unravelling beta cell destruction in type 1 diabetes

Type 1 diabetes (T1D) results from the immune-mediated destruction of the insulin-producing beta cells. Genetic predisposition, impaired immune regulation, and beta cell (dys)function all contribute to disease initiation and progression. A critical gap in our knowledge is what causes the break in peripheral tolerance that eventually leads to beta cell destruction. We propose that neoepitopes generated by dysfunctional beta cells activate immune surveillance, causing beta cell autoimmunity. ER stress imposed both by intrinsic beta cell physiology and by external secondary triggers seems to be a crucial component in this process. Understanding the molecular mechanisms underlying beta cell dysfunction and neoantigen generation is critical to identify clinically relevant neoepitopes. This subsequently provides more insight in the disease dynamics as well as contribute to translational research in the development of biomarker assays and development of therapeutic strategies targeting autoreactive T-cells and beta cell function. Our task will be to restore the balance between immune reactivity and beta cell function, in order to prevent, treat, or cure type 1 diabetes.LUMC / Geneeskunde Repositoriu

Structural plasticity in I-Ag7 links autoreactivity to hybrid insulin peptides in type I diabetes

We recently provided evidence for promiscuous recognition of several different hybrid insulin peptides (HIPs) by the highly diabetogenic, I-Ag7-restricted 4.1-T cell receptor (TCR). To understand the structural determinants of this phenomenon, we solved the structure of an agonistic HIP/I-Ag7 complex, both in isolation as well as bound to the 4.1-TCR. We find that HIP promiscuity of the 4.1-TCR is dictated, on the one hand, by an amino acid sequence pattern that ensures I-Ag7 binding and, on the other hand, by the presence of three acidic residues at positions P5, P7 and P8 that favor an optimal engagement by the 4.1-TCR's complementary determining regions. Surprisingly, comparison of the TCR-bound and unbound HIP/I-Ag7 structures reveals that 4.1-TCR binding triggers several novel and unique structural motions in both the I-Ag7 molecule and the peptide that are essential for docking. This observation indicates that the type 1 diabetes-associated I-Ag7 molecule is structurally malleable and that this plasticity allows the recognition of multiple peptides by individual TCRs that would otherwise be unable to do so.Copyright © 2022 Erausquin, Serra, Parras, Santamaria and López-Sagaseta

Structural plasticity in I-Ag7 links autoreactivity to hybrid insulin peptides in type I diabetes

We recently provided evidence for promiscuous recognition of several different hybrid insulin peptides (HIPs) by the highly diabetogenic, I-Ag7-restricted 4.1-T cell receptor (TCR). To understand the structural determinants of this phenomenon, we solved the structure of an agonistic HIP/I-Ag7 complex, both in isolation as well as bound to the 4.1-TCR. We find that HIP promiscuity of the 4.1-TCR is dictated, on the one hand, by an amino acid sequence pattern that ensures I-Ag7 binding and, on the other hand, by the presence of three acidic residues at positions P5, P7 and P8 that favor an optimal engagement by the 4.1-TCR’s complementary determining regions. Surprisingly, comparison of the TCR-bound and unbound HIP/I-Ag7 structures reveals that 4.1-TCR binding triggers several novel and unique structural motions in both the I-Ag7 molecule and the peptide that are essential for docking. This observation indicates that the type 1 diabetes-associated I-Ag7 molecule is structurally malleable and that this plasticity allows the recognition of multiple peptides by individual TCRs that would otherwise be unable to do so

The genetics of experimental arthritis in rodents

Unravelling the genetic susceptibility to complex autoimmune diseases and understanding these pathologies on a mechanistic level are major obstacles to improve our possibilities for therapeutic intervention and an increase in the quality of life of affected patients. Studies in experimental rodent models, that can be run under stable environmental conditions, which itself can be subjected to experimental manipulation, and in cohorts of potentially unlimited size, hold significant promise for the understanding of genes and pathways involved in complex autoimmune diseases. In this thesis, which is based on five scientific manuscripts, we initially investigated the influence of the genetic background on the ability to detect three major genetic loci (Pia4/Cia12, Pia5/Cia3, Pia7/Cia13) for pristane induced arthritis (PIA) in the rat. We also investigated the effect of Pia1, which includes the RT1 region (major histocompatibility complex (MHC) in the rat). We could show that the major arthritis regulator NCF1 as well as the MHC are silent in certain genetic backgrounds, whereas their genetic effect on PIA susceptibility can be detected in other, distinct genetic setups, arguing for the importance of genetic interactions between MHC and non-MHC genes for PIA development. In the second and third paper, we used a unique approach with a heterogeneous stock (HS) derived inbred-outbred mouse cohort that had been backcrossed to the arthritis susceptible C57BL10/Q (BQ) mouse strain, in order to map clinical phenotypes and the autoantibody response during collagen induced arthritis (CIA) development. We defined numerous novel loci and fine mapped already described quantitative trait loci (QTL) associated with clinical disease and/or autoantibody production providing the to date most comprehensive mapping study in CIA. The papers 4 and 5 concern the positional identification of candidate genes for the CIA loci Cia21 and Cia22 in the mouse. We propose the costimulatory molecule CD2 as a female specific genetic risk factor for autommunity in the joint and the central nervous system (CNS). We also pinpoint the chitinase like gene Chi3l3, also denoted as Ym1, as an important immunomodulator in experimental murine arthritis models based on both active immunization with collagen (CII) and passive transfer of arthritogenic antibodies. Hopefully, the findings presented in this thesis will have clinical implications based on the novel genetic targets, we identified. In addition, our data demonstrate the difficulties and pitfalls that are associated with gene identification using a hypothesis free positional cloning approach in experimental rodent populations

Within and beyond immunomodulatory strategies against autoimmune diabetes : antigen-specific tolerance and endothelial regeneration

Type 1 diabetes (T1D) is a chronic disorder in which the cells of the immune system mediate selective destruction of the insulin-producing [beta]-cells in the islets of Langerhans in the pancreas. CD4+ effector T cells, including Th1 and Th17 cells, are crucial mediators during disease development. Therefore, therapeutic strategies against T1D should target both T cell subtypes. The mechanisms underlying the control of Th1 cells are well-defined, but those operating modulation of Th17 cells remain largely unknown due to the fact that Th17 cells are plastic and can drive the disease as convertible (Th17 to Th1) or stable T cells. To overcome these limitations, a tolerance induction model was developed to analyze the mechanisms underlying modulation of plastic Th17 cells. Indeed, upon induction of tolerance, convertible (Th17 to Th1) cells displayed downregulation of the chemokine receptor CXCR3 that was associated with diminished T-bet expression, leading to retention of the cells in the spleen and inhibition of trafficking to the pancreas. In contrast, stable Th17 cells downregulated RORγt but increased FasL expression and died by apoptosis under the same antigen-specific tolerance. Thus, the final signature transcription factor shapes the mechanism of tolerance in plastic Th17 cells. These findings suggest that effective strategies against T1D will require regimens that could drive both mechanisms of tolerance to overcome the disease. A core feature of autoimmune diabetes is the loss of the majority of insulin-producing [beta] cells. Therefore, reversal of overt T1D requires restoration of [beta]-cell mass in addition to effective control of islet inflammation. It has been established that [beta]-cell turnover relies on self-replication of pre-existing [beta]-cells; however, the diabetic state is tightly associated with a striking decrease of the islet endothelial cells, leading to poor [beta]-cell survival and function. Given that the endothelial progenitor cells (EPCs) reside in the bone marrow and th

Processing and presentation of the rheumatoid arthritis candidate autoantigen aggrecan, by antigen-specific B cells

The proteoglycan aggrecan, which is a major structural component of cartilage, has been identified as a candidate autoantigen in rheumatoid arthritis (RA). This is principally due to its degradation early in the disease pathology, its ability to induce an RA-like disease in mouse models and the presence of elevated numbers of reactive T and B cells in RA patients. Studies have also defined an essential requirement for autoantigen-specific B cells as antigen presenting cells (APC) in RA although the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by B cells remains unknown. To investigate the role of autoreactive B cells as APC in RA, I have used two complimentary approaches to generate B cells expressing an aggrecan-specific B cell receptor (BCR). The first was based on a modified monoclonal antibody production protocol and the second involved the transfection of B lymphoma cells with newly generated plasmids encoding an aggrecan-specific BCR. Using the second approach, I have successfully generated aggrecan-specific B cell lines (C71-4C5 and C71-5F10). I have shown that these B cells specifically bind aggrecan leading to efficient processing and the generation of the immunogenic T cell epitope 84-103 that is recognised by both aggrecan-specific T cell hybridomas and CD4+ T cells isolated from an aggrecan-specific TCR transgenic mouse. The aggrecan-specific B cells are able to present aggrecan at least 104 fold more efficiently than non-specific B cells, 102 fold more efficiently than macrophages and comparable to that seen by dendritic cells. By using a panel of inhibitors, I have also shown that the generation of the 84-103/MHC complex by aggrecan-specific B cells requires an acidic environment, proteolysis by aspartic, serine and metalloproteinases and the “classical” pathway of MHC class II biosynthesis. During this PhD, I have highlighted a novel role for aggrecan-specific B cells as important APC involved in aggrecan-presentation, as well as elucidating a role for metalloproteinases in aggrecan processing and presentation by APC.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Autoimmune Diseases

Autoimmune disease represents a group of more than 60 different chronic autoimmune diseases that affect approximately 6% of the population. Autoimmune diseases arise when ones immune system actively targets and destroys self tissue resulting in clinical disease with prime examples such as Lupus and Type 1 diabetes. The immune system is designed to protect us from foreign pathogens such as viruses and bacteria. However, during the process of generating immune cells for this purpose, as a negative consequence, self-reactive immune cells are also generated. This book aims to present the latest knowledge and insights regarding the different contributing factors and their interplay, discussions on several autoimmune diseases and their case studies, and therapeutic treatments, including stem cell, for autoimmune diseases

The evolution of CD4+ T cell clonality in a murine model of inflammatory arthritis

Immunological tolerance is an important aspect of immunity preventing responses being mounted against self-peptides and other innocuous foreign antigens. A breach in self-tolerance can lead to the development of autoimmune diseases such as Rheumatoid arthritis (RA). RA is a chronic inflammatory autoimmune disease that is characterised by synovial inflammation and joint erosion. CD4+ T cells have been shown to play a key role in disease progression, and their ability to infiltrate joints is associated with perpetuation of local and systemic inflammatory responses. A diverse range of T cell receptor (TCR) usage has been demonstrated in RA patients, however how such diversity arises and is shaped remains unclear. Understanding the development of CD4+ T cell antigen specific responses will therefore be important for the development and application of antigen-specific therapeutic tolerance regimes. To investigate these antigen specific responses, CD4+ T cell clonality was examined using the OVA induced breach of tolerance model of experimental arthritis, allowing the assessment of developing antigen specific responses in the early stages of arthritis. The initial articular CD4+ T cell response was found to be oligoclonal in nature, with enrichment of several TCRVβ families in the inflamed joint. Moreover, the enrichment for some families is associated with joint derived antigens. Next-generation sequencing analysis of CDR3β sequences of CD4+ T cell clones revealed the dynamics of clonal responses between the inflamed joint and its associated draining lymph node and how these responses change with the progression of the disease. Inflamed joints displayed similar CD4+ T cell repertoire diversity at early and late stages of the disease, while inflamed lymph nodes displayed increased repertoire diversity with disease progression. Moreover, the number of CD4+ T cell clones shared between the inflamed joint and lymph node decreased with time. However, correlation analyses of highly abundant clones between inflamed joints and lymph nodes suggested continued migration of CD4+ T cell clones from inflamed lymph nodes to the joints. The decreased diversity in inflamed lymph nodes at the later time point may be a reflection of epitope spreading to the initial inciting antigen as well as development of new responses to neo antigens released as a result of continued joint damage. The hypothesis is that the reduced CD4+ T cell diversity in the inflamed lymph nodes will eventually be mirrored in the joint if the disease continues untreated. This research provides insight on the dynamics of the antigen specific response between the inflamed tissue and its draining lymph node with disease progression, highlighting important site specific and temporal differences in clonal diversity with disease development and also highlights the role autoreactive CD4+ T cell responses play in disease progression. By understating the evolution of CD4+ T cell responses in RA, more informed decisions can be made on how antigen-specific therapeutics should be applied and will help develop more effective regimes to reinstate self-tolerance, with the ultimate goal of moving towards drug free remission and a cure