Johns Hopkins Medical Institutions
Autoimmune Disease Research Center

Autoimmunity Day Presentations: 2001

Irun Cohen

Dr. Cohen discussed the possible treatment of type 1 diabetes with a peptide derived from the heat shock protein 60 (HSP60). HSP60 is ubiquitous (present from bacteria to humans), and expressed in nearly every cell type, where it is involved in response to many kind of stresses. HSP60 is the target of the autoimmune attack in other autoimmune diseases in addition to type 1 diabetes, such as in multiple sclerosis, inflammatory bowel disease and arthritis, and each of these diseases is associated with a distinct HSP60 peptide.

The discovery of the connection between HSP60 and type 1 diabetes was accidental. Dana Elias was assessing in young NOD mice whether antibodies to insulin could predict which mouse develop diabetes, and chose as control antibodies to HSP60. To the investigators' surprise antibodies to HSP60 appeared earlier and were more abundant than antibodies to insulin. This observation was followed by the isolation of a T cell clone from NOD mice that recognized the HSP60 and was capable of transferring diabetes into non diabetic mice, thus indicating that HSP60 is a major autoantigen in diabetes that develop in NOD mice. The HSP60 was then analyzed systematically and finally the peptide recognized by the diabetogenic T cell clone was identified and designated p277.

The p277 peptide was then injected in incomplete Freund's adjuvant (so in a non inflammatory context) into NOD mice that were on the verge of becoming diabetic (between 12 and 17 weeks of age) to test whether this immunization could arrest the autoimmune process. The p277 peptide was indeed capable of preventing furhter destruction of the pancreatic beta cells, and freezing the hyperglycemia at the level observed before the immunization. This p277 immunization was associated with a shift from a Th1 to a Th2 response, as demonstrated by an upregulation of the splenocyte production of IL-4 and IL-10 and a concomitant downregulation of IL-2 and IFNg, by a shift from IgG2a to IgG1 and IgG2b in the isotype of antibodies to GAD, and by a downregulation of the production of IFNg by the lymphocytes infiltrating the pancreas.

Based on these promising findings in mice, the response to p277 was then assessed in humans, and indeed it was shown that about 90% of patients with type 1 diabetes respond to the p277 peptide. This set the stage to study whether the injection of p277 in humans could have the same beneficial effect on the progression toward autoimmune diabetes as shown in the NOD mice. In a phase II clinical trials 30 patients (between 16 and 45 years of age) with newly diagnosed diabetes (less than 6 months) received one injection of 1 mg of p277 mixed in vegetable oil (which, in contrast to incomplete Freund's adjuvant, is degradable). All patients were males to avoid the transplacental passage of antibodies to p277 that may have unknown effects. After 10 months of follow up the patients that received p277 treatment had higher serum C peptide levels (thus indicating a better insulin reserve), a lower requirement of the insulin dosage and a reduced IFNg production than the placebo group.

Report prepared by Patrizio Caturegli

Scheherazade Sadegh-Nasseri [ Bio ]

Dr. Sadegh-Nasseri spoke about the basic immunological research conducted in her laboratory that has implications for treatment of autoimmune diseases. The focus of her work has been the induction of antigen-specific tolerance in T cells. First observations came from the in vitro studies using a T cell clone specific for a hemagglutinin peptide (HA1.7). T cells exposed to low doses of HA1.7 became anergic, as determined by their inability to proliferate and produce cytokines upon a subsequent stimulatory dose of the peptide. It was estimated that approximately 1 to 10 agonist MHC/peptide complexes per antigen-presenting cell induce T cell anergy. This work was published in the Journal of Immunology 1999, 162:6401-6409.

To further explore whether the phenomenon of low dose tolerance is true in vivo, Saied Mirshahidi, a post-doctoral fellow in Dr. Sadegh-Nasseri's laboratory, immunized mice transgenic for a specific T cell receptor (6.5 TCR) with a corresponding peptide in complete Freund's adjuvant. Dr. Sadegh-Nasseri was especially interested in tolerance of memory T cells since these cells are more likely to be responsible for the flares of autoimmune diseases. It was found that memory T cells, characterized as cells with a size smaller than activated T cells and with a CD25lowCD69lowCD45RBlowCD44highCD62Llow phenotype, appear five weeks after the immunization. As a next step, mice were immunized with a peptide and five weeks later given different doses of the same peptide. Nine days after the peptide challenge, mice were sacrificed, and their lymph node T cells were examined in vitro for the responsiveness to the peptide. T cells from those mice that were given a low dose peptide challenge did not respond to the in vitro stimulation with the peptide. Thus, it was confirmed that low dose tolerance works not only in vitro but also in vivo. Furthermore, memory T cells, an attractive target of a therapeutic immunosuppression in autoimmune diseases, become tolerant in response to a low dose of antigen.

Next, Dr. Sadegh-Nasseri decided to determine the mechanism by which the tolerance is induced. There are four general types of tolerance: ignorance, suppression, deletion, and anergy. By methodically excluding ignorance, suppression, and deletion, the researchers concluded that anergy is the most likely mechanism of low dose tolerance. The researchers found that CTLA-4, a molecule on the surface of T cells, mediates the induction of anergy in response to low dose agonist treatment. It was found that the anergized memory T cells express high levels of CTLA-4 on their surface. Furthermore, anergy was prevented by blocking CTLA-4 with a monoclonal antibody.

Dr. Sadegh-Nasseri concluded that the avidity, rather that the affinity, of the interaction between T cell receptor and MHC/peptide complex is important in determining whether a T cells will follow an activation or a tolerance pathway. Treatment with low doses of a specific peptide leads to a low density of agonist/MHC expression of antigen-presenting cells and results in low avidity interaction with T cell receptors. In response, T cells upregulate their surface CTLA-4 and become anergic.

Report prepared by Marina Afanasyeva

Robert Brodsky [ Bio ]

In the late 60s bone marrow transplant began to make its appearance in the medical practice, but a major block to its success was the need to suppress the patient's own immune system to the point where it would accept the bone marrow from other patients (allogeneic transplant). To achieve this goal, George Santos, the founder of the Johns Hopkins bone marrow transplant program, introduced the use of high-dose cyclophosphamide (Cytoxan), a well-known drug used long term in low doses as immunosuppressant. Lyle Sensenbrenner, another Hopkins oncologist, noted that several of the patients Santos treated with high-dose cyclophosphamide were recovering their own bone marrow, instead of the donor's. He then theorized that cyclophosphamide could be used to rebuild the bone marrow in patients with severe aplastic anemia (SAA) for whom all other therapeutic options had run out.

SAA is a lethal disease (about 80% mortality one year after the diagnosis) characterized by a >75% reduction in the cellularity of at least two of the three marrow blood cell lineages (erythroid, myeloid, and lymphoid), and mediated by an autoimmune attack of T lymphocytes against hematopoietic stem cells. Dr. Sensenbrenner treated 10 young patients with SAA using the high-dose cyclophosphamide regimen, but then left Hopkins in 1987 and no one ever kept track of how his cyclophosphamide-treated patients fared long term --- until 1994 when Dr. Robert Brodsky came along. To his amazement, Dr. Brodsky found that 7 of the 10 original SAA patients were not only alive but completely disease-free, with no relapse and no secondary disease. He published these findings in Blood (87: 491-494, 1996), concluding the paper with a note of caution : "Although encouraging, these results remain preliminary. The study was small and the patients were relatively young. Hence, further studies with high-dose cyclophosphamide need to be performed".

Dr. Brodsky then expanded the treatment to 20 additional patients with SAA using the following high dose cyclophosphamide protocol: 50 mg/Kg/day of cyclophosphamide for four days, followed by G-CSF to begin 6 days after the last cyclophosphamide dose. The treatment was successful in 85% of the patients, thus confirming and expanding the original findings. It is now known how cyclophosphamide works, and why the bone marrow responds to this drug by bouncing back and recreating its own three blood cell lineages. Cyclophosphamide is activated in the liver to phosphoramide mustard, which kills most of the actively replicating cells, such as the bone marrow cells. Phosphoramide mustard is inactivated by the enzyme aldehyde dehydrogenase; thus, the cells that express this enzyme are resistant to phosphoramide attack. T and B lymphocytes, the cells that mediate the damage in autoimmune diseases, contain very little aldehyde dehydrogenase, and so are easily destroyed by cyclophosphamide. Hematopoietic stem cells represent less than 1% of the bone marrow cells, and have the capacity to generate cells of all the three blood lineages. Stem cells can be distinguished into two pools. The low quality pool is made by large, lin+ and DR+ stem cells which do not express the enzyme aldehyde dehydrogenase; the high quality pool is made by small, lin-, DR- cells which express aldehyde dehydrogenase. Thus, stem cells of the high quality pool are resistant to cyclophosphamide. This means that patients receiving cyclophosphamide will have destroyed the existing mature blood cells, but will retain the ability to create new blood cells, avoid therefore the need for bone marrow transplant. Simply put, cyclophosphamide ablates the bone marrow, but reboots its ability to form new hematopoietic cells, including new, nonautoimmune lymphocytes.

The high-dose cyclophosphamide treatment is effective, safe, represents 1/3 of the cost of a classical bone marrow transplant and avoid the reinfusion of the autoreactive lymphocytes that may contaminate the graft. The treatment has a mild toxicity: complete alopecia (that begins about 2 weeks after the first dose), nausea and vomiting for the first 4-5 days, and lowering of the blood cell counts. During this period, patients require transfusion of blood products (usually 1-2 units of red blood cells and 2-3 units of platelets), develop febrile neutropenia in about 70% of the cases, and may develop infections that require hospitalization .

With this background, the high-dose cyclophosphamide treatment is being tried in patients with other severe autoimmune diseases. For example, it has been tried in 6 patients with pemphigus and in 13 patients with severe and unresponsive lupus erythematosus systemic. All 6 patients with pemphigus have a good response, specifically 4 are in complete remission and 2 are in partial remission after the treatment. Of the lupus patients, 6 are in complete remission, 3 in partial remission and 4 have no response. In summary, high-dose cytoxan a) effectively ablates all the bone marrow cells, except the high quality stem cells, which are protected through the enzyme aldehyde dehydrogenase and can thus repopulate the marrow and "reboot" the immune system; b) cures severe aplastic anemia; c) can also cure other severe autoimmune diseases.

Report prepared by Patrizio Caturegli

Jerry Winkelstein [ Bio ]

Primary immunodeficiencies are inborn error of metabolism, that can affect every component of the immune system (lymphocytic, phagocytic, and complement). They are usually inherited as single gene defect, although the two most common immunodeficiencies (selective IgA deficiency and common variable immunodeficiency) are polygenic. They classically present with increased susceptibility to infections. Dr. Winkelstein described five examples of immunodeficiencies that are paradoxically associated with rheumatic diseases. 1) X-linked agammaglobulinemia. It was the first immunodeficiency to be identified. It is caused by a defect in btk, a tyrosine kinase specifically expressed in B cells. Patients present with increased susceptibility to infections by encapsulated bacteria and enteroviruses, and have profound hypogammaglobulinemia. Possibly as a consequence of the enteroviral infection, some patients present lesions that resemble dermatomyositis. They have violaceous rashes over the eyelids and extremities, weakness of proximal limb girdle muscles and increased serum creatinine kinase. This rheumatic complication can be ameliorated by the administration of intravenous immunoglobulins. 2) Selective IgA deficiency. It is very common, affecting between 1 in 300 and 1 in 500 individuals of European origin, and is associated with rheumatic diseases such as rheumatoid arthritis and systemic lupus erythematosus. 3) Common variable immunodeficiency is a defect of both B and T lymphocytes and is also associated with the rheumatic diseases seen in selective IgA deficiency. The two diseases likely have a common genetic background and in the same family some members may have selective IgA deficiency and others common variable immunodeficiency. 4) Chronic granulomatous disease is a disorder of phagocytic cells that show deficient killing of intracellular bacteria and fungi. Many patients develop a lupus-like syndrome. 5) Complement deficiencies are rare. They may affect C1q, C3, C4 or, more commonly, C2. Patients with complement deficiencies also develop a lupus-like syndrome.

Report prepared by Mehrdad Hejazi and Patrizio Caturegli

David Moller [ Bio ]

Sarcoidosis is a multi-systemic disorder characterized by non-caseating granulomas, with infiltrating CD4+ T cells that produce mainly IL-12 and IFNgamma. The tissue antigen that drives this immunologic response remains unknown. Dr. Moller described the efforts done in his laboratory to identify such candidate antigens. Using mass spectrometry on lung tissue homogenates from patients with sarcoidosis, they identified two candidate antigens. The first one, corresponding to the mycobacterial catalase-peroxidase gene, is recognized, when expressed in recombinant form, by immunoglobulins present in the sera from patients with sarcoidosis. The second one, called T complex protein 1, is a heat shock protein and is still under characterization.

Report prepared by Mehrdad Hejazi and Patrizio Caturegli

Bevra Hahn

Dr. Hahn discussed the mechanisms of autoimmunity in systemic lupus erythematosus and the potential ways of disrupting those mechanisms for therapeutic purposes. Currently, 25% of patients with lupus develop end-stage renal disease or die within 10 years of diagnosis despite treatment. Dr. Hahn identified three potential directions for intervention.

The first direction is to suppress hyperactivation of T and B cells in an antigen-specific manner. The second direction is the induction of regulatory T cells. The third direction involves manipulations of cytokines to favor protective immune responses. To explore these directions, Dr. Hahn uses an animal model of lupus, NZB/NZW F1 mouse that spontaneously develop disease. She found that these mice have autoreactive T cells that recognize Vh region of syngeneic immunoglobulin with specificity to DNA. She identified specific peptides from that region that are recognized by T cells.

Next, she tried to suppress disease in mice by inducing an antigen-specific tolerance of T cells. She achieved this goal by giving mice intravenous injections of high doses of a peptide derived from the Vh region.

Report prepared by Marina Afanasyeva

David Hellman [ Bio ]

Dr. Hellman described the Johns Hopkins Vasculitis Center. The center was established in June 1998 with the goals of assembling an unique data bank for patients with vasculitis, of bringing together expertise of diverse collaborators, and of initiating translational research. Since its creation, the center has recruited 650 patients with vasculitis (143 with Wegener's granulomatosis, 93 with giant cell arteritis, 87 with polyarteritis nodosa, and 35 with Takayasu arteritis). The center is also coordinating a multicenter trial on the use of anti-TNF for the treatment of Wegener's granulomatosis.

More information about the center can be found at the center web site:

Report prepared by Patrizio Caturegli

Kamal Moudgil [ Bio ]

Arthritis can be induced in Lewis rats upon immunization with M. tuberculosis. The disease develop 10-12 days after the immunization, and resolves after about 7 weeks. Studies have shown that the antigen responsible for arthritis induction is the heat shock protein 65 (Hsp65), and in particular the epitope p180-188. Moudgil's laboratory has shown that the immunologic response to the main epitope is maintained throughout the disease and that during the recovery phase, the immune system recognizes new epitopes localized at the C terminus of the heat shock protein 65. Epitope spreading has been implicated in the propagation of autoimmunity in animal models of multiple sclerosis, diabetes, and systemic lupus erythematosus. Moudgil's laboratory tested the hypothesis that epitope spreading results in a suppression, rather than propagation, of disease. They injected in the rats a mixture of the C terminal peptides of Hsp65 and indeed showed a downregulation of the arthritis. It is likely that after induction of arthritis with M. tuberculosis, there is an upregulation of antigen processing that leads to processing also of the endogenous Hsp65. T cells are then induced that are capable to recognize the bacterial and the endogenous Hsp65. This diversification of the response leads to the recruitment of downregulatory T cells. Kamal discussed next the effect of the environment on the development of arthritis. Fisher rats are susceptible to the induction of adjuvant arthritis when kept in a clean facility, whereas are resistant when kept in a conventional facility. Also, when the same rats are transferred from the clean to the conventional facility, they become resistant to arthritis induction. They tested whether the difference between "clean" rats and "dirty" rats may be due to a different response to Hsp65. They show that spleen cells prepared from "clean" rats do not respond to the C-terminal peptide of Hsp65, whereas those from "dirty" rats do. Thus it is possible that "dirty" rats possess in their guts regulatory T cells that respond to Hsp65 and protect them against adjuvant-induced arthritis. The difference is specific for Hsp65, because "dirty" rats spleen cells do not respond to HEL. Finally they showed that nave spleen cells from "dirty" rats, when transferred to "clean" rats, confer protection against the induction of arthritis. These findings contradict the general observation that conventional housing promotes, rather than suppresses, autoimmune diseases.

Report prepared by Mehrdad Hejazi and Patrizio Caturegli

Thomas Kickler [ Bio ]

Dr. Kickler's interest has been focused on hematologic disorders of autoimmune etiology. He talked about one of the examples of such disorders, chronic autoimmune thrombocytopenia (ATP). ATP is a consequence of the generation of platelet-specific autoantibodies. A number of platelet surface glycoproteins (GPs) have been identified as specific targets for autoantibodies, including GPIIb/IIIa (also known as a fibrinogen receptor), GPIb/IX (also known as von Willebrand factor receptor), and GPIa/IIa (also known as collagen receptor). These autoantibodies, mainly of IgG isotype, bind to platelets and lead to platelet activation and aggregation resulting in thrombosis and platelet depletion and subsequent hemorragic rash and bleeding. Identification of these targets of autoaggression is important in designing therapies based on antigen-specific tolerance.

Report prepared by Marina Afanasyeva

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