Natural autoantibodies are defined as self-reactive antibodies present in healthy individuals in the absence of purposeful immunization or any known antigenic stimulation. While most NAAbs are of the IgM isotype, T cell dependent isotypes such as IgG and IgA NAAbs have been reported. Natural antibodies are conserved through evolution; they have been detected in sharks and humans, the phylogenetic limits of vertebrates capable of producing antibodies. The phylogenetic conservation of NAAbs suggests that they play an important, but overlooked, role in the maintenance of physiological homeostasis of the immune system.My project involved the characterization of two NAAbs. The first part of my dissertation elucidates the production and functional characterization of an anti-T Cell Receptor NAAb, OR3. OR3 is an IgM heterohybridoma made from the peripheral blood B cells of a patient with Rheumatoid Arthritis. The results show that OR3 specifically binds to the Complementarity Determining Region 1 segment of TCRV b8 regions, is specific for a subset of T cells and blocks the antigen activation of these T cells. Importantly, OR3 does not induce apoptosis or necrosis of T cells. My results support our hypothesis that anti-TCR NAAbs are immunomodulatory and indicate that autoantibodies present in humans may have significant functional activity in the regulation of T cell responses.The second part of the dissertation describes the phenomenon of NAAbs to human delta opioid receptor. I affinity purified IgG antibodies to human DOR from intravenous immunoglobulin, a therapeutic blood product that contains purified IgG isolated from the plasma of thousands of healthy donors. The anti-DOR NAAbs bind to DOR protein, and initiate an activating signaling cascade, in a manner similar to a receptor specific agonist. Interestingly, there is a significant difference in the agonistic activity of the autoantibodies compared to the synthetic DOR agonist DPDPE. Unlike DPDPE, these autoantibodies display significant immunomodulatory activity as evinced by changes in CCR5 and CXCR4 chemokine receptor expression. The presence of functional autoantibodies in IVIG shows that they are a part of the normal healthy immune repertoire in humans. This work also presents data supporting the interconnecting network between the neuroendocrine and immune systems.

The Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disorder of impaired neuromuscular transmission. It is associated with small cell lung carcinoma (SCLC) in 60% of patients. There is considerable evidence that the defect in LEMS is caused by autoantibodies to voltage-gated calcium channels (VGCCs) on the nerve terminal. Two VGCC subtypes were demonstrated in cultured neuronal cell lines by the technique of K⁺-induced ⁴⁵Ca²⁺ influx: one subtype was inhibited by dihydropyridines (DHPs), the other by ω-conotoxin (ωCgTx). These may correspond to L and N channels previously described for neuronal tissue. The presence of these VGCC subtypes was confirmed by radioligand binding studies using [3 Hj-PN200-11 0 and 125 I-d)ωCgTx respectively. Pooled LEMS IgG inhibited K⁺-induced Ca²⁺ flux by 40% in SKNSH (human neuroblastoma) cells, while control IgG had no effect. The same LEMS pool reduced the density of ¹²⁵I-ωCgTx binding sites in SKNSH and MAR5 (human SCLC) cells by 57% and 43% respectively. These results provide further evidence that LEMS antibodies cause a loss of functional VGCCs. 78 LEMS and 88 control sera were tested for anti-VGCC antibodies by the precipitation of ¹²⁵I-ωCgTx-labelled VGCCs extracted from SKNSH cells. 42% of LEMS sera had significant levels of antibody (30-1466pM) compared to the healthy controls (<31pM). There was a high correlation between these results and those obtained using antigen extracted from MAR5 cells. Raised antibody titres (30-82pM) were also found among SCLC patients (47%) and patients with rheumatoid arthritis or systemic lupus erythematosus (56%). The incidence of positive sera was not significant among patients with other neurological disorders, including myasthenia gravis. Antibody titre did not correlate with disease severity across individuals. However, longitudinal studies in two LEMS patients showed an inverse relation between antibody titre and an electromyographic index of disease severity. Some of the antibodies detected may, therefore, be implicated in the neurological symptoms of LEMS. The assay may be a useful aid for the diagnosis of LEMS in some patients.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

Osteoporosis is a common complication of many autoimmune diseases that is typically attributed to disease specific factors rather than a direct autoimmune process. This thesis arises from the investigation of a patient with severe high bone turnover osteoporosis who was identified as having autoimmune disease but whose osteoporosis deteriorated despite appropriate treatment. This presentation led to the hypothesis that neutralising autoantibodies to the bone protective cytokine osteoprotegerin (OPG) may have developed. Serum from the index patient, but not healthy controls, was able to immunoprecipitate recombinant OPG protein, demonstrating that OPG had become the target of an autoimmune response. Purified immunoglobulins from the index case were able to inhibit the function of OPG in vitro, by suppressing OPG-mediated inhibition of a luciferase reporter cell line. This represents the first description of disease associated with neutralising antibodies to OPG. Whilst the immunoprecipitation assay did identify OPG antibodies in further patients these results were difficult to quantify. A more robust enzyme linked immunosorbent assay for OPG antibodies was developed using OPG as a capture antigen, which allowed the screening of patient cohorts. Presence of OPG antibodies was defined as a titre greater than the mean plus three standard deviations of 101 healthy volunteers. A low prevalence of 14/864 (1.6%) was seen in a general population cohort and no association with bone density or turnover was seen. An association with higher vascular calcification score in this cohort requires replication. A prevalence of 37/315 (11.7%) was seen in an osteoporosis cohort though no association was seen with bone density or response to treatment. In a coeliac cohort OPG antibodies were identified in 14/282 (5.0%) patients and presence of antibody was independently associated with reduced spine bone density. Functional inhibition of OPG was shown in vitro in 3/14 (21.4%) of the positive cases. Case finding of osteoporosis in the coeliac cohort was not improved by identification of OPG antibodies. These results are consistent with OPG antibodies being pathological in a small number of patients with osteoporosis but a clinical utility of measuring OPG antibodies has not been established.

Immune thrombocytopenia (ITP) is a heterogeneous immune-mediated blood disorder with multiple pathologies that cause thrombocytopenia. The primary source of this thrombocytopenia is platelet destruction by antiplatelet autoantibodies. Although several treatment options are available for ITP, they are often transient, and responses can be difficult to predict. Different studies show ITP plasma and autoantibodies can also inhibit platelet production, but the mechanism and its impact in causing thrombocytopenia remains unknown. By identifying the different mechanisms causing ITP thrombocytopenia, it may be possible to identify more effective and patient specific treatment options, as well as identify patients who could be at an increased risk of bleeding. To study the antibody mediated inhibition of platelet production, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own hematopoietic stem and progenitor cells (HSPC) as a starting cell source to grow megakaryocytes. I demonstrate this assay can use a small amount of peripheral blood to grow mature megakaryocytes that are capable of thrombopoiesis. Using this assay, I investigated the effect patient plasma had on platelet production. As such, this study is the first autologous investigation of the effect ITP plasma has on platelet production. I found no inhibition of megakaryopoiesis, but did find an effect on thrombopoiesis, indicating that the plasma is affecting the end stages of platelet production. Secondary observations also show that some ITP HSPC have an enhanced megakaryopoiesis potential, generating more mature megakaryocytes than what was observed with healthy donors. While screening monoclonal antiplatelet antibodies, I discovered an anti-GPIb antibody that inhibited megakaryocyte maturation and found this affect was also present with the Fab antibody fragment. From my research I have developed several tools that can be used to investigate impaired platelet production in ITP and further our understanding of this pathology. / Thesis / Doctor of Philosophy (PhD) / Immune thrombocytopenia (ITP) is an immune mediated blood disorder where autoantibodies target and destroy platelets, cells that are crucial for preventing blood loss. Evidence from different studies show that ITP autoantibodies are also affecting the cells producing platelets (megakaryocytes), but the mechanism of this effect remains unknown. To study antibody mediated inhibition of megakaryopoiesis, I developed a peripheral blood based megakaryopoiesis assay that used the patient’s own cells as a starting source to grow megakaryocytes. With this assay, I investigated the effect patient plasma had on platelet production. I found no inhibition of megakaryocyte growth but did find an effect on their ability to produce platelets. I also found a model antibody that affected the maturity of the megakaryocytes during their development. These tools can now be used to further investigate impaired platelet production in ITP patients and determine the impact this inhibition has on ITP pathology.

Autoantibodies are antibodies made abnormally to self-antigens that are expressed on the surface, in the cytoplasm, or in the nucleus of the cell. These antibodies are produced in certain people whose immune system has failed to work properly. It is a puzzle, however, how the B cells that produce autoantibodies which target the vital intracellular autoantigens and with potential deleterious effects to the cell, are able to exist in nature and not succumb to suicide. We have found a suitable model to answer this question. This is a newly-constructed mouse hybridoma (#476), which produces an IgG1 monoclonal antibody (mAb 476) to an epitope in the catalytic subunit of telomerase (TERT). This mAb stains TERT very well in both mouse and human cells. The unusual thing about hybridoma 476 is that, although it can grow and produce the antibody normally in culture, however, it fails repeatedly to grow in the peritoneum of normal BALB/c mice primed with an adjuvant, unlike other hybridomas of similar backgrounds that we have used. Control cell lines used in the study include a hybridoma (Mab2) which makes an IgG1 antibody to phosphorylcholine, and the myeloma fusion partner (Sp2/0) used to construct hybridoma 476. / When the antibody specificity was abrogated by genetic manipulation or by natural selection, the 476 cells were able to grow and produce ascites in vivo normally. This shows that the problem was specific, due to binding of the mAb with its antigen, TERT. Telomerase is a housekeeping enzyme in cells essential for maintaining the stability of the telomere, the DNA repeats found at the ends of chromosomes, failing which the cell undergoes senescence and eventually dies. It appeared that the peritoneum, unlike the culture medium, contained factors that presumably up-regulated the antibody production in the cells, which consequently increased the complex formation between TERT and mAb 476 in these cells, and led to cell apoptosis. Indeed, examination of the culture supernatant ("soup") of the peritoneal cells harvested from the adjuvant-primed mice, revealed no cytotoxic cytokines or chemokines present, but an abundant amount of IL-6. In parental 476 cells in culture, both soup and purified recombinant IL-6 were found to up-regulate the production of the intracellular antibodies, but not the secreted antibodies. The effect of these reagents on the Mab2 cells was different; both the intracellular and secreted antibodies were increased. (Abstract shortened by UMI.) / Niu Haitao. / "April 2005." / Adviser: Pak-Leong Lim. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0169. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 199-228). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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