Research Interests

The long-term interests of our lab include the regulation of autoreactive B lymphocytes and the development of high affinity B cell immune responses and memory cells. More recently, we have also developed an interest in the mechanisms by which T cells are activated in Graft vs. Host Disease (GVHD) and the subsets of T cells that are pathogenic. We have emphasized in vivo models in all the work.

To study the regulation of autoreactive B cells in normal mice and the loss of regulation in autoimmune-prone animals, we have used transgenic (Tg) mouse models to ask how B cells which express a disease-related autoantibody, Rheumatoid Factor (RF), are prevented from causing harm in normal mice. We first demonstrated that these cells develop, mature and are immunocompetent in non-autoimmune mice (Hannum et al., 1996). Recent results demonstrate that these Tg RF B cells are spontaneously activated in autoimmune-prone mice, but only when the autoantigen is present (Wang and Shlomchik, 1999). We have recently used this system to develop a method to visualize the initiating events of B cell autoimmunity (William et al., 2005a; William et al., 2005b), an elusive goal. We expect this to be a powerful new model system for understanding of the origins of autoimmunity. For example, we have recently found that the early and perhaps chronic events in the MRL.Faslpr lupus model are not taking place in the germinal center (GC) as originally thought, but instead involve continuous proliferation at the outer edge of the T cell zone, where T and B cells normally interact only transiently (William et al., 2002). Quite surprisingly, we have shown that very active somatic hypermutation of Ig V regions is taking place at this site, thus dissociating mutation from the GC environment for the first time and also raising the possibility that mutation outside of GCs can lead to loss of self-tolerance (William et al., 2002). Interestingly, we found that autoreactive B cells on a normal BALB/c background still entered GCs and began to mutate there, but never made autoantibodies, thus suggesting a new self-tolerance checkpoint. We are actively investigating how autoreactive B cells become activated at extrafollicular sites, using a new method that we discovered to induce it (rather than wait for it to happen spontaneously) as well as a cell transfer system. These techniques greatly improve our ability to dissect the mechanisms. We are currently investigating the roles of TLRs, DCs, T cells, BAFF/APRIL family members and other costimulatory molecules using a combination of knockout mice and inhibitors. We also recently developed a BCR knockin version of the AM14 mouse, which undergoes isotype switch. This facilitates our identification of activated cells, the study of switch regulation, and also the development of memory.

An emerging interest in this area is the role of Toll-like receptors in activating autoreactive B cells (Leadbetter et al., 2002, Vigliante et al., 2003, Christensen, et al., submitted, 2005). These studies, partly in collaboration with Dr. Marshak-Rothstein at Boston University, have provided significant new insights into how self-Ags stimulate the immune system in autoimmunity by stimulating TLRs. In two recent studies, we have shown how this explains the generation of the two major types of lupus-related autoantibodies, anti-DNA and anti-RNA. Using knockout mice, we showed that the former is controlled by TLR9 and the latter by TLR7. Surprisingly though, we found that while deletion of TLR7 ameliorated disease, deletion of TLR9 exacerbated it; thus, only TLR7 appears to be a good therapeutic target (Christensen et al., 2005; Christensen et al., 2006). We are currently investigating the mechanism by which TLR9 normally regulates disease, as well as the cell types that need to express both TLRs to promote disease.

An important related focus has been on how activated autoreactive B cells contribute to pathogenesis in systemic autoimmunity. Traditionally B cells have been thought of mainly as sources of pathogenic autoantibody. We challenged this view with the notion that a main function of B cells is to activate autoreactive, pathogenic T cells, in an antigen-specific way. First, we deleted B cells from autoreactive mice and showed that T cell activation and disease was ablated (Chan and Shlomchik, 1998; Shlomchik et al., 1994). Second, we created a novel mouse model which had B cells expressing only the membrane form of IgM and that could not secrete Ab (Chan et al., 1999). In the autoimmune-prone mouse, such B cells promoted both T cell activation and disease. We have recently developed a Tg mouse that expresses human CD20 on all B cells and can be used to deplete B cells with anti-CD20 at any time. Using this model, we recently found that depletion of B cells in adult autoimmune-prone mice can indeed ameliorate disease, but at the same time we discovered that depleting B cells in the midst of autoimmunity is markedly more difficult than in normal strains, thus revealing an unexpected therapeutic barrier (Ahuja, et al, submitted). We are now working on why this is the case.

The second major interest in B cell biology is the development of high affinity B cell immune responses and memory. We are using a Tg mouse model which has B cells that cannot secrete antibody to explore whether antigen retained as immune complexes on follicular dendritic cells (FDCs) is necessary for the development and maintenance of memory B cells. B cells in these mice make normal primary and secondary immune responses including germinal centers and somatic mutation (Hannum et al., 2000). We have also recently found that normal memory B cells form in these animals survive without further cell division for months after their initial development (Anderson et al., 2006b). Thus, immune complexes on FDCs are not necessary for these processes. We are now investigating the identity and functional capacity of the memory cells formed and have used microarray expression profiling to identify a host of genes differentially expressed between naïve and memory B cells. Many of these have been confirmed at the protein level, providing new insights into the functions of memory B cells. We are currently using knockout mice to test the roles of some of these molecules in memory development and function in vivo We also have a program to study the dynamics of germinal center B cells using both experimental and computer modeling approaches, as well as in vivo multiphoton microscopy, a program led by Associate Research Scientist Dr. Ann Haberman (Hauser, et al., submitted).

Over the past several years we have developed an interest in the immunopathology surrounding stem cell transplantation We have focused on chronic GVHD (cGVHD), applying modern genetics and molecular techniques to a murine model. We have already shown (surprisingly) that host-derived antigen-presenting cells are not required to induce cGVHD. Further, we have found that host-derived CD4+/CD25+ T cells play an important role in regulating cGVHD (Anderson et al., 2005; Anderson et al., 2004). Most recently, in collaboration with Dr. Warren Shlomchik, we found that donor memory T cells do not cause GVHD, although they engraft and function (Anderson et al., 2003). This could represent a major therapeutic approach to minimize GVHD while preserving GVL and immunocompetence in recipients. We are currently investigating the reasons why memory T cells do not cause GVHD.

If you are interested in working on or discussing any of these areas, feel free to contact Dr. Mark Shlomchik anytime.

Somatic hypermutation of autoreactive RF B cells proliferating at the T zone red pulp border. Small numbers (10-20) of RF B cells (dark red) were microdissected in two separate clusters (11f3 and 11f4) and the Vk genes amplified by PCR, cloned and sequenced. The recovered sequences could be assembled into a genealogic tree depicting the relationship of mutations that were shared or unique among the sequences. The pattern shows that although all the sequences shared a number of mutations (in the "trunk" of the tree), there was a hierarchical pattern of partially shared and unique mutations indicative of ongoing somatic hypermutation among the RF B cells in each cluster.