Bishop Lab - Current Projects

 We are interested in the molecular mechanisms which underlie the processes of lymphocyte activation and tolerance. Our particular area of focus is antigen-specific, T cell-B cell interactions.

 The following is a sample of some projects ongoing in the lab –

A) The role of the cytoplasmic adapter protein TRAF3 in lymphocyte function.

Members of the tumor necrosis factor receptor (TNFR) superfamily participate in a large number of events that regulate cell activation and programmed cell death. A substantial majority of signals induced by receptors of this family are delivered via cytoplasmic (CY) adapter molecules belonging to the TNFR-associated factor (TRAF) family. Although numerous reports have focused upon how the widely used TRAF2 molecule contributes to positive signals by most TNFR family molecules, little has been known about the functions of TRAF3, which shares a highly overlapping receptor binding site with TRAF2. It is now clear from work performed by our group and others during the past several years that TRAF3 can play diverse, even sharply contrasting roles in signaling by different receptors. It has also been quite recently revealed that TRAF3 plays important roles in signaling by receptors of the innate immune system. To explore further the biological function of this understudied TRAF, we are making use of a conditional TRAF3 deficient mouse strain that we recently produced, together with TRAF3-deficient cell lines, to understand the varied roles played by TRAF3 in B and T cell activation.

B) How members of the TNFR superfamily interact to regulate lymphocytes in normal immunity and disease.

Receptors of the tumor necrosis factor receptor (TNFR) superfamily are expressed on a wide variety of cell types, and deliver many and diverse signals. Understanding how these receptors deliver cell signals, and how this process is regulated, is important to both basic knowledge about this large and multifunctional group of receptors, as well as how these signaling pathways can be manipulated to combat autoimmune disease, malignancy, and infection. This project builds upon prior progress that examined how the TNFR family member CD40 regulates signaling to B lymphocytes. The present workl extends our investigations to additional members of the TNFR superfamily, including CD120b (TNFR2), CD27, and the receptors for BAFF and APRIL. Because all cells, particularly immune cells, express many TNFR superfamily members simultaneously and/or sequentially, we also wish to examine the mechanisms and consequences of receptor interactions in determining biological outcomes of cell signaling.

C) How does the viral oncoprotein LMP1 utilize TRAF molecules in altering B lymphocyte function?

The tumor necrosis factor receptor (TNFR) superfamily member CD40 delivers multiple signals to B cells that play major roles in B cell survival, expansion, and differentiated functions. The EBV-encoded viral mimic of CD40, latent membrane protein 1 (LMP1) delivers strikingly similar signals to B cells, but does so in an amplified and sustained manner. This dysregulated signaling is consistent with the well-documented association of LMP1 function with human B cell lymphoma, and emerging information on the potential role of LMP1 in autoimmune disease. We recently discovered that CD40 and LMP1 use the same cytoplasmic adapter molecules (TNFR associated factors, TRAF) in unexpectedly distinct and even sharply contrasting ways. LMP1 signaling is independent of TRAF2, a major mediator of CD40 signals. Conversely, TRAF3 was revealed as a negative regulator of CD40 signaling, but a necessary positive mediator of key LMP1 signals. Both CD40 and LMP1 also employ TRAFs 5 and 6 in B cell signaling. However, CD40 directly binds TRAF6, while LMP1 uses TRAF6 indirectly. The opposite is true of TRAF5, which binds LMP1 directly, but contributes indirectly to CD40 signals. When expressed as transgenes in mice lacking endogenous CD40, the cytoplasmic tail of either CD40 or LMP1 can restore T-dependent humoral immunity. However, expression of molecules with the LMP1 CY domain result in an expanded B cell compartment, B cell hyperactivity and autoreactivity, and disordered lymphoid architecture. The major goals of the project are to build upon the information obtained on TRAF use and regulation to understand how LMP1 uses TRAFs 3, 5, and 6 in signaling to B cells, and to investigate the mechanistic basis by which LMP1 promotes B cell hyperactivity and autoreactivity, using both cell line and mouse models.

D) How do adaptive and innate receptor signals interact in B cells?

The global human population is developing an increasing need for new and better vaccines, to combat both infectious and malignant disease. A limiting factor in human vaccine development has been the narrow selection of safe adjuvants, to increase the effectiveness of vaccines, and stimulate effective responses with fewer immunizations. Scientists have made tremendous advances in understanding how the components of adjuvants, distinct pathogen-associated molecular patterns (PAMPs), trigger specific receptors of the innate immune system. Of particular interest are ligands for receptors that recognize special features of viral or bacterial nucleic acids, as these ligands can be readily produced synthetically, without the safety concerns associated with purifying substances from large quantities of infectious microbes. This basic immunology project focuses on gaining a more complete understanding of how receptors for microbial nucleic acids interact with receptors of the adaptive immune system in the activation of B lymphocytes, with the long-term goal of applying this knowledge to better strategies in vaccine development. All effective vaccines in use today elicit a robust antibody response; in the case of anti-viral vaccines, this response is capable of virus neutralization. B cells are also now appreciated to play important roles in cytokine production and antigen presentation. The proposed research will address questions considered key to understanding how innate and adaptive receptors stimulate B cell responses.

We hypothesize that distinct signaling pathways will influence ultimate outcome. This will be tested by measuring relevant cellular functions, then examining specific early molecular events that preliminary data indicate are key to these functions. Understanding gained should permit more precise design of antigen-adjuvant combinations for safe, effective vaccines in a variety of clinical and public health settings.

What are the effects of interactive signals between adaptive and innate receptors on the function of B cells as antigen-presenting cells (APC)? We hypothesize that interactions between these receptor types will enhance the APC ability of B lymphocytes. This information will allow design of vaccines that maximize the efficacy of B cell activation, not only to produce antibodies and cytokines, but also to activate cellular responses as APC.

How is function of innate receptors for microbial nucleic acids regulated by their structure, and how may sequential or concomitant engagement of distinct receptors for microbial nucleic acids influence immune reactivity? We will test the hypothesis that differences between structures of TLR7 and TLR8, as well as structural differences between mouse and human TLR8, will regulate key functional features of these receptors in immune stimulation. A second hypothesis to be tested is that prior signals through TLR7/8 may alter the subsequent cellular response to TLR9 signals, possibly contributing to the increased susceptibility to bacterial infection that often occurs during viral infections. The information gained in understanding of how these innate receptors work can inform design of better small molecule adjuvants that target these receptors.

E) Explore the potential of a natural plant product as a novel anti-inflammatory agent.

The natural product honokiol (HNK) is a small phenolic compound that has long been used in traditional Asian medicine to treat thrombotic, gastrointestinal and anxiety disorders . More recently, reports have shown that HNK inhibits the growth of cancer cell lines, and tumors in in vivo mouse models. To determine if HNK also has anti-inflammatory properties, we tested its effects in a mouse model of rheumatoid arthritis (RA), collagen-induced arthritis, or CIA. Our preliminary data show that both in vivo inflammation and in vitro cytokine production were markedly inhibited, without increased cell death. These preliminary findings suggest that HNK also has promise in the treatment of chronic inflammatory diseases such as RA, which affects a large number of people. HNK also inhibited signaling via CD40, an immune receptor expressed by B cells, macrophages, and dendritic cells. CD40 has been implicated as playing a role in RA. Because HNK has multiple anti-inflammatory actions, it could prove more effective than treatments targeting a single molecule. Its long prior use in traditional medicine, as well as the results of mouse studies suggest that it can be effective without dangerous side effects. We are currently testing the hypothesis that HNK will have effective anti-inflammatory properties in a mouse model of RA, and that it will exert important biological effects on B lymphocytes, and their specific pro-inflammatory signaling pathways. Understanding its mode of action should permit more effective therapeutic use of this natural plant product. We are currently characterizing in detail the ability of HNK to alter disease progression and inflammation in a mouse model of rheumatoid arthritis (RA), and determining the roles of TNF, IL-6, and GABA receptors in HNK’s anti-inflammatory properties.