In this issue of Arthritis & Rheumatism, Dudek et al (1) report the identification of T cell epitopes on the La/SSB autoantigen that are recognized in humanized transgenic mice expressing the HLA class II molecule DR3 (DRA1/DRB1*0301) and its linked HLA–DQ counterpart (DQA1/DQB1*0201). This study represents a major step in advancing our understanding of the process involved in the loss of immunologic tolerance to self antigens and the pathogenetic mechanisms of disease induction in systemic autoimmunity, particularly as these relate to the autoimmune response to the La/SSB autoantigen in Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE).
Autoantibodies are widely recognized as a hallmark of the rheumatic diseases, and the presence of anti-La/SSB autoantibodies is a marker for SS (2–5). Less frequently, the presence of anti-La/SSB is a marker for SLE (1–5). Despite recognition of their association with SS and SLE, and despite previous studies of the evolution of autoantibody production against La/SSB (and the closely related autoantibody response to the Ro/SSA autoantigen), our understanding of the precise genesis of anti-La/SSB autoantibodies remains incomplete.
In addition to the production of autoantibodies, B cells can have additional roles in pathogenesis, including the ability to serve as antigen-presenting cells (APCs), which allows them to process and present antigens as well as provide costimulatory signals to T cells; the ability to secrete pathologic cytokines, such as interferon-γ, interleukin-6, and tumor necrosis factor α, that cause direct tissue damage as well as attract other cells to sites of inflammation through the release of proinflammatory cytokines; and the ability to mediate tissue injury through a variety of antibody-directed mechanisms, such as antibody-dependent cellular cytotoxicity. Studies of Ig isotype switching and gene rearrangement have suggested indirectly that T cell–derived cytokines have an important role in promoting the expansion of autoantibody-producing cells and in determining the fine specificity of anti-La/SSB autoantibodies; however, the precise nature of T cell–B cell interactions and T cell–derived B cell help for La/SSB autoantibody induction and maintenance has not been fully delineated.
Regarding HLA, it is instructive to recall that serologic association studies and, later, candidate gene studies of SS and SLE conducted in the 1980s (2–5) demonstrated that major histocompatibility complex (MHC)–encoded HLA–DR and HLA–DQ molecules or linked genes within the HLA–DR/DQ regions of the human MHC appeared to be important in determining susceptibility to the development of SS and the production of anti-La/SSB autoantibodies. Basic research, culminating in the Nobel Prize for Physiology or Medicine awarded to Benacerraf, Dausset, and Snell in 1980 and awarded to Doherty and Zinkernagel in 1996, demonstrated first that the HLA repertoire controls antigenic peptide selection, and second that HLA molecules are essential for the recognition of antigenic peptides by T cells (6, 7). This supported the concept that the association of SS with HLA polymorphisms could be due to their effect on HLA-dependent autoantigenic peptide selection and presentation to T cells.
Subsequent contributions by a number of investigators have demonstrated that CD4+ T cells are selected in the thymus based in part upon signaling of self peptides presented on class II MHC molecules, and that human CD4+ T cells recognize antigens in the context of peptides bound to class II MHC molecules, including the 3 human class II MHC molecules HLA–DR, HLA–DQ, and HLA–DP. X-ray crystallography of the MHC and T cell receptor (TCR) has further defined the details of molecular MHC–peptide–TCR interactions (8, 9). Molecular genetics studies have helped to define the complex organization of MHC genes encoded on the short arm of human chromosome 6 (2, 10).
Taken together, these studies and others have greatly refined our view of antigen recognition and immune tolerance. These studies emphasized the pivotal role of T cells in the induction and maintenance of the immune response and suggested that autoantigen-reactive T cells could be central to systemic autoimmune diseases, including diseases such as SLE and SS, which had previously been characterized by the presence of specific autoantibodies, including anti-La/SSB (2–5).
One major limitation of studies of MHC genetics in SS and other diseases is the strong linkage disequilibrium that exists between HLA–DR/DQ and the many other genes contained within the various HLA extended haplotypes (such as HLA–DR3/DQ2/DR52), which makes identification of the precise disease-associated genes/protein(s) difficult. The use of mice that express specific HLA transgenes helps to overcome this limitation of human genetic association studies.
Research defining the role of autoantigen-specific T cells in the pathogenesis of human autoimmune disease has progressed despite substantial technical challenges (11). Four groups of investigators have conducted studies examining patients and healthy controls for the presence of La/SSB-reactive human T cells, using a variety of approaches (12–15). Namekawa et al (14) observed oligoclonal TCR use by T cells isolated from the salivary glands of SS patients, supporting the possibility that T cell epitopes recognized on the autoantigen might be limited to select regions on the molecule. Davies et al (15) directly identified La/SSB-reactive T cells in the peripheral blood of SS patients and healthy controls, and identified 5 putative T cell epitopes on the La/SSB protein. Thus, while La/SSB-reactive T cells have been identified in the peripheral blood of patients and healthy controls, the precise importance of these T cells in the pathogenesis of SS has not been clearly defined (11–15).
More recently, however, the generation of mice that are transgenic for human MHC, resulting in the expression of specific HLA molecules, has provided an opportunity for the examination of characteristics of autoantigen-specific, HLA-restricted T cells in greater detail. Paisansinsup et al (16) were among the first to utilize transgenic mice as a model of SS by studying the response of the mice to an autoantigen in an analysis of the generation of antibodies to the Ro 60 antigen. This approach has proven successful in the analysis of T cell responses to a number of antigens, including other autoantigens (17, 18). One further extension of the approach has been the generation and analysis of mice expressing the product of >1 human MHC locus (i.e., HLA–DR and HLA–DQ), as were used by Dudek et al, whose article appears elsewhere in this issue (1).
Dudek et al immunized mice that were transgenic for both HLA–DR3 (DRB*0301) and HLA–DQ2 (DRB*0201) with recombinant antigen for either human La (hLa), a truncated mutant form of human La, or a 15–amino acid peptide containing the sequence for the mutant La plus adjuvant, and boosted them with hLa, mutant La, or peptide plus adjuvant. The investigators examined mice for anti-La/SSB autoantibodies using immunoblotting and enzyme-linked immunosorbent assay. They observed that the patterns of B cell responses found in transgenic mice recapitulated findings in patients, with a rapid, polyclonal, Ig class-switched autoantibody response to hLa and immune spreading to both Ro 52 and Ro 60, supporting this aspect of the model as a valid replication of human disease.
They then examined T cell responses using a classic approach for T cell epitope mapping, measuring T cell antigen–specific proliferation using 3H-thymidine incorporation following stimulation of transgenic murine T cells in vitro with a series of overlapping synthetic peptides spanning the La/SSB protein. From this they identified 7 peptides that stimulated T cells. Five of these 7 T cell epitopes were within the regions that Davies et al had previously identified in their studies using La/SSB fusion protein antigen–induced stimulation of peripheral blood mononuclear cells from patients with SS and controls (1, 15). Interestingly, 2 new T cell epitopes, hLa211–228 and hLa313–330, which were the most stimulatory, were identified in HLA-transgenic mice.
The authors determined, using anti–HLA–DR– and anti–HLA–DQ–specific blocking monoclonal antibodies, which MHC-encoded HLA molecules on APCs (DR3 and/or DQ2) were used in antigen presentation of La peptide to T cells. Somewhat unexpectedly, they found that La/SSB-reactive T cells recognized the antigen in the context of HLA–DR (DR3) but not HLA–DQ (DQ2). Consistent with this finding, they observed, using molecular modeling techniques, that the highly stimulatory peptides hLa211–228 and hLa313–330 had strong DR3 (DRB1*0301) binding motifs. Finally, they directly measured the binding of hLa211–228 and hLa313–330 peptides to DR3 and, as was predicted from the modeling studies, found that they bound with high avidity (1). Although those investigators observed that HLA–DQ2 was capable of binding the hLa peptides, they did not detect HLA–DQ2–restricted T cells in their experiments.
The study also examined T cell response to a 7–amino acid modified version of mutant La, which had first been identified as a frameshift mutation in a patient with both SS and SLE (1). Immunization of transgenic mice with mutant La revealed that the immunodominance hierarchy of the T cell response to La/SSB was substantially altered when mutant La was used as the immunogen, providing potential insight into how structural changes in an autoantigen during apoptosis, oxidative cleavage, or other modification may contribute to breaking T cell immune tolerance to self antigens (19).
The study by Dudek et al elegantly demonstrates how a transgenic murine model can provide insight into the cellular and molecular mechanisms of pathogenesis in a complex autoimmune disease such as SS (1). Their findings substantially advance our understanding of the role of autoantigen-specific T cells in a disease that has been characterized by the presence of a specific autoantibody. The study also provides an approach to dissecting complex genetic traits, such as the association of multiple linked MHC genes with susceptibility to SS, through the use of mice transgenic for multiple human immune response genes. Finally, the study evokes new questions regarding how structural modification of self antigens (in the case of mutant La) can lead to loss of immunologic tolerance. Further study of the model, focusing on these questions, will be of considerable interest.