Anti-inflammatory cell contact-How do regulatory B cells sense and respond to apoptotic cells?

Chronic inflammatory autoimmune diseases are a significant burden to health worldwide, causing chronic pain, disability and significantly shortening a person's lifespan. 1% of the world's population suffers from autoimmune rheumatic diseases such rheumatoid arthritis and systemic lupus erythematosus (SLE). In the latter condition a strong correlation exists between antibodies to antigens expressed on apoptotic cells and disease severity. These diseases and others including multiple sclerosis are amenable to treatment with B cell depletion therapy (BCDT), which is as efficacious as anti-TNF therapy. However the majority of patients fail to go into remission and refinement and improvement of BCDT is needed to improve patient outcomes.

We have discovered that in health, apoptotic cells impart a tolerogenic signal to self-reactive B cells and this protects mice from autoimmune diseases such as collagen induced arthritis and EAE (a model of multiple sclerosis). Apoptotic cells are cells that die naturally in the body and we generate millions of them every day. A loss of tolerance to apoptotic cells is at the heart of autoimmune diseases. These cells express neo-antigens on their cell surface which have come from inside the cell and it is thought that the apoptotic cells need to be taken up very quickly to prevent them inducing autoimmunity. Antibodies that are secreted in autoimmune diseases are generally to these neo- antigens that have come to the cell surface when the cell has died. However the self reactive B cells that recognise these apoptotic cells actually need to 'see' these nuclear complexes called nucleosomes in order for them to induce tolerance in health. Hence it seems that the very antigens on apoptotic cells that prevent autoimmunity in health are the same antigens that result in disease when tolerance breaks down.

We wish to understand exactly what antigens the B cell receptor on regulatory B cells is seeing and if these are the same B cells that lose tolerance to self when disease develops. By studying the B cell receptor specificity we will be able to study the cells at a single cell level, which will give greater insights than following whole B cell subsets. We also wish to understand the B cell receptor specificity of human regulatory B cells and to know exactly what subsets of B cells are regulatory and responsive to apoptotic cells in health. We will compare this to the same B cells from patients with rheumatoid arthritis and SLE, thus building up a picture of how these cells function in health and disease. As the apoptotic cell is so central to the breakdown of tolerance in autoimmunity we wish to study these cells in greater detail asking if accessory molecules such as complement proteins and natural antibody that bind to the apoptotic cell surface when the nucleosomes are expressed on them are important to inducing tolerance to self in these self reactive B cells. Patients that lack complement proteins especially the first complement component C1q are at a very high risk of developing SLE and we believe that this molecule may be very important for signalling in the regulatory B cells.

We believe that by gaining a better understanding of these self-reactive innate like B cells that respond to apoptotic cells in health by inducing a tolerogenic signal, we will be able to ask if they can be used to induce tolerance or to prevent transplant rejection. If we can identify antigens that induce these regulatory B cells we will be able to induce them in vivo, thus increasing their number. If these innate like B cells that are important for inducing tolerance to apoptotic self in health turn out to be the same cells that secrete pathogenic antibody and secrete pro-inflammatory cytokines in disease, then we will have a much better target for future therapies. We may then finally be able to offer our patients the hope of a long-term remission from sterile destructive inflammation.

We have previously reported that apoptotic cells protect in autoimmune models of arthritis by inducing regulatory B cells and our new data indicate that B cells secrete IL-10 in response to TLR9 signaling. Endogenous DNA complexes expressed on the apoptotic cell surface in nucleosomes are an important source of these signals. We wish to understand the B cell receptor specificity of these regulatory B cells both in murine and human B cells. We will examine B cells from healthy volunteers and in patients with rheumatoid arthritis and SLE. We have previously shown that B cells that respond to apoptotic cells make stable interactions with them prior to secreting IL-10 and FACs sorting will isolate these cells within the marginal zone and B1a B cell subsets. ELISPOT analysis will determine both the cytokine secretion profile and antigen specificity. Hybridomas will be generated following a standard fusion protocol and the B cell receptor specificity of the selected hybridoma antibodies will be determined by proteome array. A20 B cell lines will be generated using the heavy and light chain variable regions of selected hybridoma antibodies and tested for signaling focusing particularly on TLR7 and TLR9.

The second aim of the proposal is to ask if accessory molecules such as C1q, sIgM and TIM4, which are associated with autoimmunity are crucial in mediating the regulatory response of B cells and this will be studied in cells deficient in these molecules.

The goal of this research proposal is to clearly delineate the B cell receptor specificity of regulatory B cells. Current studies focus on defining regulatory B cells by their surface phenotype, which encompasses whole subsets of cells. Clearly within the same B cell subset will be B cells with both inflammatory and regulatory potential. This has been found for human CD27 positive cells which are clearly both pathogenic and regulatory.

We have discovered that regulatory B cells secrete IL-10 after recognizing endogenous DNA expressed by apoptotic cells. They exist in the CD27 positive human subset and also the marginal zone and B1a subset in mice. By clearly defining the regulatory B cell receptor, we will be able to study these cells at a single cell level gaining a deeper knowledge on their role in regulation. We will learn how to manipulate these cells in health to increase immune regulation (for example following transplantation) and also to ask if these same cells can become pathogenic in autoimmune disease. If so we will have a specific cell for deletion rather than the wholesale B cell depletion that is currently used for autoimmune conditions such as rheumatoid arthritis.

Hence the immediate impact will be on those scientists working to further understand autoimmunity. However we will generate cell lines and eventually knock in mice that will be useful for researchers and industry alike. They will be able to use these mice to ask if regulation protects from autoimmunity and damaging inflammation from other causes (for example ischemia reperfusion injury). In addition the murine and human monoclonal antibodies will be useful to interrogate the pathogenesis of autoimmunity and also the possibility of using particular antibody/B cell specificities to augment regulation or block inflammatory responses. Ultimately the clear beneficiary will be patients with sterile inflammation and all those that work to find more effective treatments.