B Cell Tolerance in Systemic Lupus Erythematosus

Hedda Wardemann, Ph.D.

Max-Planck-Institute for Infection Biology

Funded in March, 2004: $300000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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How Do B Cell Antibodies Cause Autoimmune Lupus, and What are the Effects of Treatment?

Lupus is an autoimmune disease affecting many organs.  It occurs when patients’ antibodies, produced by immune B cells, which are part of the body’s second line of immune defense, mistakenly attack the body’s own cells.  Determining how these B cell antibodies fail to learn to differentiate the body’s own cells from foreign antigens remains a major challenge that the Rockefeller University investigators will address.  Previous studies have indicated that a high percentage (50 to 75 percent) of developing B cells in bone marrow mistake the body’s own cells as foreign antigens (referred to as “self antigens”).  In most people, however, these B cells are eliminated either before they leave the bone marrow or when they first begin circulating in blood.  This weeding out of errant B cells can occur at one of two checkpoints, either within the bone marrow or when the B cells leave the bone marrow and enter the bloodstream.  The investigators hypothesize that in patients who develop lupus the checkpoint in the bone marrow fails; B cells evolving in bone marrow do not learn to recognize and become tolerant of the body’s own cells.  The researchers also hypothesize that treatment exerts its effect by inducing B cells to become tolerant of the body’s cells.

The investigators will explore these hypotheses in two ways.  First, in newly diagnosed and untreated lupus patients, the researchers will clone and express patients’ antibodies from single B cells that are isolated from immature and mature B cells, and from “memory” B cells that remember the identity of an antigen once they encounter it.  The investigators will test these antibodies to see if they react against the patients’ cells and compare these antibody actions to those previously observed in studies of healthy people.  This should help to determine whether the development of lupus is associated with abnormal B cell tolerance (intolerance to the body’s own cells), which results in the release of these abnormal self-reactive B cells into the blood.

If this hypothesis is confirmed, the investigators will assess the effects of therapy on B cells in the same patients.  Lupus patients undergo periods of remission and relapse following initial therapy.  The investigators will once again study patients’ immature, mature, and memory B cell antibodies following initial therapy and determine whether the clinical severity and course of the disease is associated with increased tolerance by the antibodies to the patients’ own cells.

Significance:  This project should help to determine whether patients with lupus develop autoimmunity as a result of a faulty “checkpoint” during immune B cell development in bone marrow, allowing survival of B cells that mistake the body’s own cells as foreign.  The project also may reveal whether lupus treatment makes these errant B cells more tolerant of the body’s cells.  If so, the findings could lead to new approaches to correct this faulty checkpoint to prevent lupus, and perhaps similar autoimmune diseases.  The study also might lead to improved therapies for inducing B cell tolerance to the patients' own cells.

ABSTRACT

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B Cell Tolerance in Systemic Lupus Erythematosus

During B cell development in the bone marrow (BM), random immunoglobulin (Ig) gene rearrangement generates a highly diverse antibody repertoire, including B cells that express autoreactive antibodies.  Three mechanisms—receptor editing, deletion and anergy—have been described to account for loss or silencing of self-reactive B cells to ensure self-tolerance and prevent the development of autoimmunity.  We have recently shown that 55-75% of antibodies expressed by early immature B cells isolated from BM of healthy human donors recognize self-antigens.  Long and positively charged Ig heavy (IgH) complementarity determining region 3 (CDR3) sequences correlated with self-reactivity, and antibodies with these features were counter-selected during B cell development.  However, these features could not be used to predict self-reactivity. 
Self-reactive antibodies are lost from the repertoire at two checkpoints.  The first checkpoint is at the immature B cell stage in the BM where the majority of polyreactive and anti-nuclear antibodies (ANAs) are lost.  The second checkpoint is in the periphery before maturation into naive immunocompetent B cells.

Little is understood about how tolerance is broken in autoimmune diseases in humans, but the finding that large numbers of autoantibodies are produced under physiologic circumstances suggests that even small changes in the efficiency of autoantibody regulation would lead to increased susceptibility to autoimmunity.  ANAs including anti-DNA antibodies are a hallmark of the systemic autoimmune disease lupus erythematosus (SLE).  These antibodies are predominantly of the IgG isotype and are generated by antigen-experienced B cells. Antibody-antigen immune complex formation results in tissue damage and often leads to fatal organ failure. In mice, injection of human anti-dsDNA IgG antibodies causes kidney Ig deposition and pathologic changes such as thrombi formation and proteinuria.

We have obtained preliminary data that early checkpoints in B cell tolerance are abnormal in SLE and that B cells emerging from the bone marrow of such patients are not adequately filtered to remove autoreactivity.  Our working hypothesis is that such patients might be predisposed to the development of autoantibodies because they fail to establish normal tolerance to self in the bone marrow.  In this model, increased numbers of circulating self-reactive B cells in the periphery would increase the probability that high affinity pathogenic anti-self antibodies would emerge during antigen-driven immune response. The long-range goal of the proposed research project is to determine where B cell tolerance is broken in SLE.

INVESTIGATOR BIOGRAPHIES

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Hedda Wardemann, Ph.D.

Hedda Wardemann, Ph.D., performed her graduate studies at the Max-Planck-Institute for Immunobiology in Freiburg, Germany, on the development of B cells in the mouse.  She received her degree from the Albert-Ludwig University in Freiburg, Germany, in 2000 and then joined the Laboratory of Molecular Immunology under the direction of Dr. Michel Nussenzweig at The Rockefeller University in New York as a postdoctoral fellow in 2001.  Pursuing her interest in B cell research, her studies at Rockefeller University focus on the establishment of B cell tolerance in human and the failure to establish B cell tolerance in systemic autoimmune diseases such as Systemic Lupus Erythematosus.  She became research assistant professor in 2003.

FINDINGS

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Scientific Results:
In the context of autoimmune disease, the formation of memory B cells expressing self-reactive antibodies can have devastating consequences if the cells are constantly triggered by self-antigens to secrete pathogenic serum autoantibodies. The frequency of self-reactive memory B cells in healthy humans and patients with SLE is unknown. We decided to address this question by cloning and production of antibodies from single IgG expressing memory B cells isolated from peripheral blood of 4 healthy human donors and 4 untreated patients with active SLE. Surprisingly, we found high numbers (20-40%) of self-reactive IgG+ memory B cells in healthy humans and the frequency of such cells was similar in SLE patients. It is unclear why large numbers of self-reactive IgG memory B cells are tolerated in healthy individuals but they do not seem to cause any harm under normal conditions. However, we found a high frequency (15%) of highly self-reactive memory B cells, which recognized Ro and La self-antigens, in one SLE patient with serum autoantibodies of the same specificity. Our finding that individual patients show high numbers of Ro and La reactive IgG memory B cells suggests that the formation of self-reactive memory B cells may play an important role in the pathogenesis of SLE. Furthermore, patients with high numbers of self-reactive memory B cells may respond to therapy only if the self-reactive memory B cells are permanently eliminated.

In summary, our work helped to understand the underlying B cell self-tolerance defects in SLE and showed that immunosuppressive treatment is unable to restore these defects. Our data suggest that effective therapies should aim at restoring the early filters for self-reactive B cells. Furthermore, memory B cells represent an important therapeutic target in particular in patients with high levels of self-reactivity in this compartment.

SELECTED PUBLICATIONS

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Tiller T., Meffre E., Yurasov S., Tsuiji M., Nussenzweig M.C., and Wardemann H.  Efficient generation of monoclonal antibodies from single human B cells by single cell RT-PCR and expression vector cloning.  J Immunol Methods. 2008 Jan 1;329(1-2):112-24. PMID: 17996249.

Wardemann H. and Nussenzweig M.C.  B cell self-tolerance in humans. Adv. Immunol. 95:83-110 (2007). PMID: 17869611

Tiller T., Tsuiji M., Yurasov S., Velinzon K., Nussenzweig M.C.*, and Wardemann, H.*  Autoreactivity in human IgG+ memory B cells.  Immunity. 2007 Feb;26(2):205-13. PMID: 17306569

Yurasov S., Tiller T., Tsuiji M., Velinzon K., Pascual V., Wardemann H.*, and Nussenzweig M.C.*  Persistent expression of autoantibodies in SLE patients in remission.  J Exp Med. 2006 Oct 2;203(10):2255-61.  PMID: 16966430

Tsuiji M., Yurasov S., Velinzon K., Thomas S., Nussenzweig M.C.*, and Wardemann H.*  A checkpoint for autoreactivity in human IgM+ memory B cell development.  J Exp. Med. 2006 Feb 20;203(2):393-400. PMID: 16446381

Yurasov S., Hammersen J., Tiller T., Tsuiji M., and Wardemann H.  B-cell tolerance checkpoints in healthy humans and patients with systemic lupus erythematosus.  Ann N Y Acad Sci. 2005 Dec;1062:165-74.  PMID: 16461799

Yurasov S.*, Wardemann H.*, Hammersen J., Tsuiji M., Meffre E., Pascual V., and Nussenzweig M.C.  Defective B cell tolerance checkpoints in systemic lupus erythematosus.   J. Exp. Med. 2005 Mar 7; 201(5): 703-711. PMID: 15738055

Wardemann H., Yurasov S, Schaefer A., Young J.W., Meffre E., and Nussenzweig M.  Predominant autoantibody production by early human B cell precursors.  Science. 2003 Sep 5;301(5638):1374-7. PMID: 12920303