Regulatory T Cells in Human Autoimmunity

Gerald T. Nepom M.D., Ph.D.

Benaroya Research Institute at Virginia Mason

Funded in December, 2004: $675000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY . HYPOTHESIS . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Determining the Regulatory Defect in Autoimmunity and Developing Therapies to Correct It

Researchers from several Virginia Mason departments will collaborate to identify the regulatory defect that enables immune T cells to attack the pancreas in autoimmune juvenile diabetes. Additionally, the investigators will devise two methods for treating this regulatory defect and test both methods in the animal model of this disease. Their research could lead to fundamental changes in the way this and perhaps other autoimmune diseases are prevented or curtailed.

While most people have some immune T cells that fail to learn to distinguish foreign invaders from the body's own cells, their immune systems eliminate or contain these errant ("self-reactive") T cells. Since it is so important to prevent self-reactive T cell attacks, the immune system has evolved three ways to regulate their actions. In healthy people compared to those with autoimmune disease, the immune system either kills off the self-reactive T cells, or it suppresses them by secreting hormones (called "cytokines") or by activating a highly specialized group of suppressor T cells, called "T regulatory" cells. Moreover, scientists recently have identified a specific gene that is critical to the functioning of these regulatory T cells.

Significance: This research could lead to fundamentally new ways to prevent or arrest juvenile diabetes and perhaps other autoimmune diseases in which a genetic defect impairs regulatory T cells from preventing self-reactive T cells from wreaking destruction on the body's own organs.

ABSTRACT

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Regulatory T Cells in Human Autoimmunity

Immunological tolerance to self-antigens is a tightly regulated process. Several lines of evidence demonstrate two fundamental mechanisms for controlling immunologic reactivity to self tissues: A primary regulatory set-point for self tolerance through deletion of self-reactive cells in the thymus, and generation of a variety of peripheral regulatory cells to control auto-reactive cells that escape the thymus. The hypothesis that is tested in this proposal states that human autoimmune disease likely represents a breakdown in one or both of these mechanisms. The experiments described in this proposal are designed to both determine the mechanism that underlies loss of tolerance and to develop methodologies to overcome this loss. We will:

1. Determine whether self-reactive T cells from patients with autoimmune disease are refractory to normal regulatory signal;

2. Determine whether the ability to generate self-reactive regulatory T cells is defective in patients with autoimmune disease; and

3. Determine whether otherwise pathogenic T cells can be converted to antigen-specific regulatory cells through ectopic expression of the forkhead transcription factor FoxP3.

To accomplish these aims we will utilize novel reagents and experimental systems developed by the individual investigators. The data generated by these studies will provide critical insights on the mechanisms involved in loss of self-tolerance and provide important new information on the efficacy of these novel methods of regenerating self-tolerance for the treatment of autoimmune disease.

INVESTIGATOR BIOGRAPHIES

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Gerald T. Nepom M.D., Ph.D.

Dr. Nepom received his bachelor's degree in Biochemistry from Harvard, magna cum laude 1972. He attended the University of Washington, receiving his Ph.D. in Biochemistry in 1977 and his M.D. in 1978. After post-doctoral work in immunogenetics in the Department of Pathology at Harvard Medical School, he returned to Seattle, joining the Fred Hutchinson Cancer Research Center and the University of Washington Medical School Faculty in 1982. Beginning in 1985, he founded the Immunology and Diabetes Research Programs at the Benaroya Research Institute at Virginia Mason (BRI). BRI is widely recognized as a research leader for Type 1 diabetes, housing the Pacific Northwest TrialNet center for diabetes clinical trials, the NIH coordinating center for the North American Type 1 diabetes genetics consortium, and the JDRF Center for Translational Research.

Ongoing projects in Dr. Nepom's laboratory are focused on identifying and understanding molecular and genetic mechanisms triggering of autoimmune disorders, particularly Type 1 diabetes, and on improving experimental therapies for diabetes prevention, intervention and transplantation. Dr. Nepom serves on numerous editorial boards and professional advisory boards relating to molecular immunology, autoimmunity, and immunotherapy; nationally, he recently chaired the Expert Panel for the Autoimmunity strategic plan for the National Institutes of Health and currently chairs the executive committees for the NIDDK diabetes consortia coordinating group and the NIAID autoimmunity diseases prevention centers.

Jane Hoyt Buckner, M.D. is an Assistant Member at the Benaroya Research Institute and Clinical Assistant Professor at the University of Washington in the Division of Rheumatology. Dr. Buckner received her medical degree from Johns Hopkins University School of Medicine, and completed her training in Internal Medicine at the University of Minnesota, and in Rheumatology at the University of Washington. Dr. Buckner completed research fellowships in molecular immunology and immunogenetics at the University of Washington and the Virginia Mason Research Center, and received two physician scientist awards from the American College of Rheumatology, the American College of Rheumatology Senior Fellow Award and the ACR Arthritis Investigator Award.

The central focus of Dr. Buckner's laboratory is to identify autoantigens that are the targets of autoimmunity in human disease and understand the role T cells specific for these autoantigens play in autoimmunity. Current projects in Dr. Buckner's laboratory are designed to understand how T cell responses are regulated in humans, how that regulation is lost in autoimmunity, and how to create regulatory T cells to suppress pathogenic autoreactive T cells for novel therapy of autoimmune diseases. Her work includes studies of Type 1 diabetes, rheumatoid arthritis, and relapsing polychondritis. In addition to her laboratory research, Dr Buckner heads the Translational Research Program at BRI. This program was established to facilitate the acquisition of clinical data and samples from individuals with autoimmunity, in order to extend our knowledge of immunology and genetics to human subjects in the context of clinical trials. Dr. Buckner continues to care for patients with rheumatic diseases and to teach.

The goal of her work is to identify the underlying defects that lead to autoimmunity and develop more directed methods to treat these diseases.

Steven F. Ziegler, Ph.D., is a Member at the Benaroya Research Institute, and Director of the Immunology Program. During his Ph.D. work at UCLA in Dr. Owen Witte's laboratory he developed new methods for the in vitro differentiation of B cells. Following post-doctoral training at the University of Washington, where he identified and cloned HCK, a novel gene involved in myeloid cell function, Dr. Ziegler spent five years as a staff scientist at Immunex, followed by three years as the director of Immunology/Molecular Biology at Darwin Molecular. He joined the Virginia Mason Research Center as an associate member, and the Immunology Department, as affiliate associate professor, in 1997.

The principal focus of his laboratory is the development and regulation of the immune system, using both mouse and human model systems. He recently identified FoxP3 as a gene critical for the development and function of a population of T cell involved in controlling autoimmunity.

HYPOTHESIS

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The Virginia Mason researchers hypothesize that people with autoimmune juvenile diabetes have a genetic defect that makes them unable to create enough, or strong enough, regulatory T cells. The investigators will first identify the failed regulatory pathway in the animal model of juvenile diabetes to see if this is the case. If they find that the genetic defect is present, they will create two types of experimental therapies and test them in the animal model. One method is to develop regulatory T cells in the laboratory. The other method is to develop a therapy aimed at the defective gene and its protein product. Both therapeutic approaches will be tested in the animal model. If successful, the researchers then would be in a position to apply to other funders to undertake human clinical trials in patients.

FINDINGS

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Lay Summary:
In patients with autoimmune disease, autoreactive T cells avoid regulation.  In this project, we examined why this system of regulation breaks down, in autoimmunity and how one could repair it.  During the three years of the grant, we have successfully addressed these questions by examining differences in the function of the immune system of individuals with Type 1 diabetes and healthy subjects.  We have been able to demonstrate that several of the mechanisms involved in regulating the immune system are intact in Type 1 diabetes, while other mechanisms have failed.  This new information will now allow us to design better therapeutics.

Scientific Summary:
This project addressed mechanisms by which the balance between regulatory T cells and autoreactive T cells is perturbed in individuals with autoimmune diseases.  The studies performed to examine this question focused on differences between healthy controls and subjects with type 1 diabetes, but the findings that have resulted from this work are applicable to other autoimmune diseases.  These studies have established: That adaptive Treg can be induced from the CD4+CD25-Foxp3- T cells of individuals with type 1 diabetes and are similar in function and number as those induced from healthy controls; that islet specific Tregs can be induced from the CD4+CD25- T cells of T1D and control subjects; that the effector T cells of individuals with T1D are resistant to suppression by Treg; that particular domains of Foxp3 differentially influence T cell function and differentiation; and that specific binding partners of Foxp3 may be crucial for controlling lineage commitment.  Translation of these findings into therapeutic trials involving Treg expansion are underway.

SELECTED PUBLICATIONS

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Carson B.D. and Ziegler S.F.  Impaired T cell receptor signaling in Foxp3+ CD4 T cells.  Ann N Y Acad Sci. 2007 Apr;1103:167-78.

Soper D.M., Kasprowicz D.J., and Ziegler S.F.  IL-2Rbeta links IL-2R signaling with Foxp3 expression. Eur J Immunol. 2007 Jul;37(7):1817-26.

Lopes J.E., Torgerson T.R., Schubert, L.A., Anover, S.D., Ochs, H.D., and Ziegler, S.F.  Analysis of FOXP3 reveals multiple domains required for its function as a transcriptional repressor.  J Immunol. 2006 Sep 1;177(5):3133-42.

Liu W., Putnam A.L., Xu-yu Z., Szot G.L., Lee M.R., Zhu S., Gottlieb P.A., Kapranov P., Gingeras T.R., Fazekas de St. Groth B., Clayberger C., Soper D.M., Ziegler S.F., and Bluestone J.A.  CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells.  J Exp Med. 2006 Jul 10;203(7):1701-11.

Bacchetta R., Passerini L., Gambineri E., Dai M., Allan S.E., Perroni L., Dagna-Bricarelli F., Sartirana C., Matthes-Martin S., Lawitschka A., Azzari C., Ziegler S.F., Levings M.K., and Roncarolo M.G.  Defective regulatory and effector T cell functions in patients with FOXP3 mutations.  J Clin Invest. 2006 Jun;116(6):1713-22.

Allan S.E., Passerini L., Bacchetta R., Crellin N., Dai M., Orban P.C., Ziegler S.F., Roncarolo M.G., and Levings M.K.  The role of 2 FOXP3 isoforms in the generation of human CD4+ Tregs.  J Clin Invest. 2005 Nov;115(11):3276-84.

Holzer U., Rieck M., and Buckner J.H.  Lineage and signal strength determine the inhibitory effect of transforming growth factor beta1 (TGF-beta1) on human antigen-specific Th1 and Th2 memory cells.  J Autoimmun. 2006 Jun;26(4):241-51.

Walker M.R., Carson B.D., Nepom G.T., Ziegler S.F., and Buckner J.H.  De novo generation of antigen-specific CD4+CD25+ regulatory T cells from human CD4+CD25- cells. Proc Natl Acad Sci U S A. 2005 Mar 15;102(11):4103-8.