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Immunity and Tolerance

Research Summary

Michel Nussenzweig's research is focused on understanding the development and function of antibody-producing B lymphocytes and dendritic cells.

The function of the immune system is to protect vertebrates from a multitude of different pathogens. Two types of immune responses have evolved to accomplish this task: innate responses and adaptive responses. Innate immune responses depend on a series of pattern recognition receptors that have evolved to recognize a restricted set of frequently encountered pathogens. In contrast, adaptive immune responses display a much broader range of diversity and show immunologic memory.

Lymphocytes are the primary effectors of adaptive immunity. These cells assemble diverse-repertoire immune receptors by a somatic gene recombination process known as V(D)J recombination. Immune receptor assembly by random variable (V), diversity (D), and joining (J) gene sequence rearrangement enables the production of a large number of unique receptors that are able to recognize almost any antigen. V(D)J recombination also produces self-reactive receptors, however, which must be silenced to prevent autoimmune diseases.

To determine how frequently B cells develop autoreactive receptors and how they are regulated, we developed methods for cloning and expression of antibodies from single human B cells. We found that random immunoglobulin (Ig) gene recombination frequently produces self-reactive antibodies—as many as ~75 percent of newly generated B cells in the bone marrow. A significant fraction of these autoantibodies are polyreactive, and many recognize nuclear self-antigens. However, these antibodies are tightly regulated at two distinct checkpoints in B cell development. Thus, few self-reactive naïve B cells persist in human circulation, and these show only low levels of reactivity. We are using this baseline information to examine how tolerance breaks down in autoimmune diseases.

A cardinal feature of systemic lupus erythematosis (SLE) is the development of autoantibodies. The appearance of self-reactive antibodies in SLE precedes clinical disease, but where in the B cell pathway tolerance is first broken has not been defined. We have examined B cell tolerance in patients with SLE by cloning antibodies from single cells. We find the normal B cell tolerance checkpoints to be defective in patients with SLE. In these patients, 25–50 percent of the mature naïve B cells produce self-reactive antibodies even before they participate in immune responses, potentially accounting for a predisposition to disease development. Our ongoing research focuses on understanding checkpoint regulation and the mechanisms that veto autoimmune antibody production.

Once a B cell passes the quality control checkpoints in the bone marrow, it can be selected by antigen for antibody production in either T cell–dependent or T cell–independent immune responses. In the first half of the 20th century, Burnett and Talmage postulated that antibody affinity governs B lymphocyte selection during immune responses. To examine the role of antibody affinity in immune responses, we have produced mice that carry pre-recombined antigen receptor genes with high or low binding affinities for the hapten 4-hydroxy-3-nitrophenyl acetyl (NP). Our experiments revealed that there is an early affinity filter that precedes the germinal center reaction. Once in germinal centers, B cells undergo a fixed mutation program that is followed by selection, regardless of initial receptor affinity.

In germinal centers, B cells undergo two types of antibody gene modifications, somatic mutation and class-switch recombination. Class-switch recombination is a region-specific DNA recombination reaction that replaces one heavy-chain constant region with another, enabling a single variable region to be used in conjunction with several different constant regions, each with a different biological function. The switch reaction is initiated by activation-induced deaminase (AID), which catalyzes the deamination of cytidine residues in single-stranded DNA, thereby producing U:G mismatches, which can be repaired by alternative DNA repair pathways to produce either somatic hypermutation or class-switch recombination. We have shown that Nijmegen breakage syndrome protein (Nbs1) and phosphorylated histone H2AX are recruited to the DNA lesions induced by AID. These foci form in the G1 phase of the cell cycle, and switching is impaired in H2AX-, ATM-, Nbs1-, and Ku-deficient B cells. These experiments suggest that AID induces a double-stranded DNA break (DSB) during the switch recombination reaction and that the break is recognized as DNA damage by the cell. Our ongoing studies are focused on understanding the molecular mechanisms that mediate the initiation and resolution of the switching reaction.

Although B and T lymphocytes are similar in many respects, including diversification of their antigen receptor genes by V(D)J recombination, 95 percent of all lymphomas diagnosed in the Western world are of B cell origin. Many of these are derived from mature B cells and display hallmark chromosome translocations involving Ig genes and a proto-oncogene partner whose expression becomes deregulated as a result of the translocation reaction. These translocations are essential to the etiology of B cell neoplasms, and we have shown that they are initiated by AID by a mechanism that requires DNA cytidine deamination.

Under physiologic conditions, AID rarely induces c-myc/IgH translocations, suggesting the existence of surveillance mechanisms that detect DNA damage and prevent DSBs from being channeled to translocations or that kill cells that overexpress translocated c-myc. ATM deficiency but not deficiency in H2AX or 53BP1 results in accumulation of translocation events. In addition, mutations in the tumor-suppressor genes p53 and p19(ARF) also increase the observed frequency of c-myc/IgHtranslocations. These results are consistent with two independent mechanisms converging on p53 and protecting B cells against chromosome translocations, namely, the DNA damage response checkpoint through ATM activation and the oncogenic stress pathway triggered by translocated c-myc and p19 activation. Our current focus is understanding how cells protect themselves against AID-induced DNA damage and the ensuing translocations.

A second area of interest for our laboratory is the physiologic function of dendritic cells (DCs). To examine the function of DCs in the steady state, we devised an in vivo antigen delivery system, using a monoclonal antibody to a DC-restricted endocytic receptor, DEC-205. This route of antigen delivery is several orders of magnitude more efficient in inducing T cell activation and cell division than free peptide in strong adjuvants. The activation response is not, however, sustained, and T cells become unresponsive to systemic rechallenge with antigen. Coinjection of the DC-targeted antigen and anti-CD40 agonistic antibody changes the outcome from tolerance to prolonged T cell activation and immunity. These experiments indicate that in the steady state, the primary function of DCs is to maintain peripheral tolerance.

Our experiments are consistent with the notion that self-antigens, such as serum components and apoptotic cells captured and presented to T cells by DCs under physiological conditions, induce tolerance. In contrast, antigens taken up by DCs in the context of activation stimuli such as those found during inflammation or tissue destruction induce prolonged T cell activation. These two functions of DCs, maintaining tolerance to self and inducing immunity, are not in conflict because they are elicited under distinct circumstances, the steady state versus inflammation and infection. Moreover, the steady-state tolerizing function of DCs may be essential for their subsequent role in eliciting immunity. During inflammation or infection, DCs present self-antigens simultaneously with nonself. By establishing tolerance to self before challenge with pathogens, DCs can focus the adaptive immune system entirely on the pathogen, thereby avoiding autoimmunity. The ability to target antigens to DCs and control their function in vivo has significant implications for development of vaccines and therapies for autoimmunity.

Work on signal transduction by the B cell receptor and autoimmunity is supported in part by grants from the National Institutes of Health.

As of February 10, 2009

Scientist Profile

Investigator
The Rockefeller University
Immunology, Molecular Biology