Anergy in self-directed B lymphocytes from a statistical mechanics perspective

The ability of the adaptive immune system to discriminate between self and non-self mainly stems from the ontogenic clonal-deletion of lymphocytes expressing strong binding affinity with self-peptides. However, some self-directed lymphocytes may evade selection and still be harmless due to a mechanism called clonal anergy. As for B lymphocytes, two major explanations for anergy developed over three decades: according to "Varela theory", it stems from a proper orchestration of the whole B-repertoire, in such a way that self-reactive clones, due to intensive interactions and feed-back from other clones, display more inertia to mount a response. On the other hand, according to the `two-signal model", which has prevailed nowadays, self-reacting cells are not stimulated by helper lymphocytes and the absence of such signaling yields anergy. The first result we present, achieved through disordered statistical mechanics, shows that helper cells do not prompt the activation and proliferation of a certain sub-group of B cells, which turn out to be just those broadly interacting, hence it merges the two approaches as a whole (in particular, Varela theory is then contained into the two-signal model). As a second result, we outline a minimal topological architecture for the B-world, where highly connected clones are self-directed as a natural consequence of an ontogenetic learning; this provides a mathematical framework to Varela perspective. As a consequence of these two achievements, clonal deletion and clonal anergy can be seen as two inter-playing aspects of the same phenomenon too

“Rogue” B cells: An investigation into B cells that break tolerance in autoimmune disease

A breakdown in B cell self-tolerance can lead to antibody-mediated autoimmune disease. This thesis aims to explore how B cell tolerance can be broken in two distinct, but complementary projects within the context of the Goodnow somatic mutation hypothesis. In both scenarios, B cells that escape self-tolerance and generate autoantibodies are referred to as “rogue” B cells. First, this thesis aimed to elucidate the precise steps undertaken by expanded rogue B cell clones in patients with chronic Hepatitis C virus (HCV)-associated cryoglobulinemic vasculitis, an autoimmune disease characterised by the production of a rheumatoid factor cryoglobulin autoantibody. The rogue B cell clones in the HCV cryoglobulinemic vasculitis patients were confirmed to be the source of the autoantibody. The rogue B cell clone precursor antibodies failed to bind the HCV envelope glycoprotein E2, yet bound multimerised self-antigen IgG relative to membrane IgM density. These findings disfavour a molecular mimicry hypothesis, and instead indicate IgG immune complexes may be sufficient to drive recruitment of the rogue B cell clone precursors. Finally, the rogue B cells clones were found to carry somatic lymphoma-associated, non-immunoglobulin gene mutations and chromosomal aberrations, predicted to cause hyperactivation of the NF-kB signalling pathway and escape of B cell tolerance. This finding provides additional evidence in support of the Goodnow somatic mutation hypothesis. Second, this thesis examined rogue germinal centre (GC) B cells that arise in the absence of the receptor FAS. Rogue GC B cells loose specificity for the foreign antigen and incidentally generate autoantibodies. However, the accumulation of rogue GC B cells cannot be explained by our current understanding of affinity-based selection in the GC. This work revealed rogue GC B cells, unlike “conventional” GC B cells undergoing affinity maturation to the foreign antigen, can be identified by low expression of CD21 and high expression of B220 (CD21loB220hi). Moreover, rogue GC B cells were found to be rapidly entering cell cycle, enriched for a dark zone phenotype and T-cell dependent, reminiscent of positively selected GC B cells. Thus, rogue GC B cells typically removed by FAS, likely persist in the competitive GC microenvironment despite their loss of BCR specificity to foreign antigen, because they retain the capacity to undergo T-cell dependent positive GC selection

Predicting the outcomes of HIV treatment interruptions using computational modelling

In the past 30 years, HIV infection made a transition from fatal to chronic disease due to the emergence of potent treatment largely suppressing viral replication. However, this medication must be administered life-long on a regular basis to maintain viral suppression and is not always well tolerated. Any interruption of treatment causes residual virus to be reactivated and infection to progress, where the underlying processes occurring and consequences for the immune system are still poorly understood. Nonetheless, treatment interruptions are common due to adherence issues or limited access to antiretroviral drugs. Early clinical studies, aiming at application of treatment interruptions in a structured way, gave contradictory results concerning patient safety, discouraging further trials. In-silico models potentially add to knowledge but a review of the Literature indicates most current models used for studying treatment interruptions (equation-based), neglect recent clinical findings of collagen formation in lymphatic tissue due to HIV and its crucial role in immune system stability and efficacy. The aim of this research is the construction and application of so-called ‘Bottom-Up’ models to allow improved assessment of these processes in relation to HIV treatment interruptions. In this regard, a novel computational model based on 2D Cellular Automata for lymphatic tissue depletion and associated damage to the immune system was developed. Hence, (i) using this model, the influence of spatial distribution of collagen formation on HIV infection progression speed was evaluated while discussing aspects of computational performance. Further, (ii) direct Monte Carlo simulations were employed to explore the accumulation of tissue impairment due to repeated treatment interruptions and consequences for long-term prognosis. Finally, (iii) an inverse Monte Carlo approach was used to reconstruct yet unknown characteristics of patient groups. This is based on sparse data from past clinical studies on treatment interruptions with the aim of explaining their contradictory results

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

Itk is a Dual Action Regulator of Immunoreceptor Signaling in the Innate and Adaptive Immune System: A Dissertation

The cells and molecules that comprise the immune system are essential for mounting an effective response against microbes. A successful immune response limits pathology within the host while simultaneously eliminating the pathogen. The key to this delicate balance is the correct recognition of the pathogen and the appropriate response of immune cells. Cellular activation originates through receptors that relay information about the state of the microenvironment to different compartments within the cell. The rapid relay of information is called signal transduction and employs a network of signaling mediators such as kinases, phosphatases, adaptor molecules, and transcription factors. IL-2 inducible T cell kinase (Itk) is a non-receptor tyrosine kinase that is an integral component of signal transduction downstream of many immunoreceptors. This dissertation describes two distinct pathways that utilize Itk in both phases of the immune response. T cells use the TCR to sense a multitude of peptide-based ligands and to transmit signals inside the cell to activate cellular function. In this regard, the diversity of ligands the T cells encounter can be portrayed as analog inputs. Once a critical threshold is met, signaling events transpire in close proximity to the plasma membrane to activate major downstream pathways in the cell. The majority of these pathways are digital in nature resulting in the on or off activation of T cells. We find, however, that altering the TCR signal strength that a T cell receives can result in an analog-based response. Here, the graded expression of a transcription factor, IRF4, is modulated through the activity of Itk. We link this graded response to an NFAT-mediated pathway in which the digital vs. analog nature has been previously uncharacterized. Finally, we demonstrate that the repercussions of an analog signaling pathway is the altered expression of a second transcription factor, Eomes, which is important in the differentiation and function of T cells. These results suggest that Itk is crucial in the modulation of TCR signal strength. Mast cells primarily rely on the IgE-bound FcεR1 for pathogen recognition. Crosslinking this receptor activates mast cells and results in degranulation and cytokine production via an expansive signaling cascade. Upon stimulation, Itk is recruited to the plasma membrane and phosphorylated. Little else is known about how Itk operates inside of mast cells. We find that mast cells lacking Itk are hyperresponsive to FcεR1-mediated activation. This is most apparent in the amount of IL-4 and IL-13 produced in comparison to wild-type mast cells. Increased cytokine production was accompanied by elevated and sustained signaling downstream of the FcεR1. Finally, biochemical evidence demonstrates that Itk is part of an inhibitory complex containing the phosphatase SHIP-1. These results indicate a novel function for Itk as a negative regulator in FcεR1- mediated mast cell activation

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

Immune cell tracking following hematopoietic cell and gene therapy

In this project we combined standard cellular and molecular assays with a custom PCR-based technology based on high throughput sequencing to track genetically engineered cells in treated patients by means of viral integration sites (IS) analysis. We leveraged this analytical pipeline to 1) assess whether naïve T cells can still be produced for many years even in absence of any supply by multipotent progenitors in the bone marrow and 2) to investigate the origin of CAR-T cells that mediate anti-leukaemic responses or long-term immune surveillance. In a clinical trial X-linked Severe Combined Immunodeficiency patients received an infusion of autologous hematopoietic stem/progenitor cells corrected via retroviral vector encoding the interleukin-2 common cytokine receptor gamma chain. In these patients, many years after gene therapy only vector-positive T and NK cells persist while no other genetically engineered blood cell populations are detectable. By a comprehensive long-term immunophenotypic, molecular and functional characterization we demonstrated that the thymus is actively producing a new and diverse repertoire of vector-positive naïve T cells (TN). This suggests that, even if gene corrected HSC are absent, a de novo production of genetically engineered T cell is maintained by a population of gene-corrected long term lymphoid progenitors (Lt-LP). Moreover, tracking IS clonal markers overtime, we inferred that Lt-LP can support both T and NK cells production. In a separate clinical trial of CD19-CAR-T cells for the treatment of haematological malignancies, we used IS analysis to investigate the origin of short- and long-term circulating CAR-T cells that mediate early anti-leukaemic responses or long-term immune surveillance. We compared IS between the product and CAR-T cells at early/late timepoints in vivo. This analysis suggested that T memory stem cells (TSCMs) contained in the infused cell product, contributed the most to the generation of CAR-T cell clones during the peak response phase as well as to the generation of long-term persisting CAR-T cells

CANCER IMMUNOTHERAPY: TARGETED CELLULAR VEHICLE-MEDIATED IMMUNOGENE THERAPY AND DENDRITIC CELL-BASED VACCINE

Ph.DDOCTOR OF PHILOSOPH