Understanding the dynamics of the adaptive immune repertoire by next generation sequencingRachel Bashford-Rogers, Wellcome Trust Sanger Institute Cambridge, UK
The adaptive immune response selectively expands B and T-cell clones following antigen recognition by the B- or T-cell receptors (BCR and TCR) respectively. Currently the diversification and dynamics of adaptive immune responses during infection, malignancy and autoimmunity remain poorly understood. To address this, we present a novel method of sampling human B and T-cell populations at highresolution, using next-generation sequencing of BCRs and TCRs coupled with novel repertoire analyses. These methods allow us to dissect the different components of the adaptive immune response and enable detection of lymphocyte clones in chronic lymphocytic leukemia (CLL) to exquisite sensitivity. We show BCR sequences organise into networks based on sequence diversity, which clearly distinguish between the diverse repertoires of healthy individuals and clonally expanded repertoires from individuals with CLL. Network connectivity measures of BCR diversity provide a proxy to the B-cell clonality status of individuals with decreasing network Rényi entropy strongly correlating with the time from CLL diagnosis. We correlated the repertoire diversity of the heavy and light immunoglobulin chains with TCR diversities to better understand the relationships between these immune subsets. Associations between BCR network entropy and disease stage or other clinical parameters offer a novel diagnostic & prognostic dimension for clonal blood disorders. This approach can also be used to enhance our understanding of the dynamics of the immune response in infectious disease and to help deconvolute hostpathogen interactions. These studies provide important insights into the evolution of both dominant and rare clonal species of B and T-cell populations, and the dynamic and complex adaptive immune responses.
Identification of host and viral factors of alphavirus-induced arthritis and myositis: the role of mannose binding lectin and the viral N-linked glycans Bronwyn Gunn, Department of Microbiology and Immunology, University of North Carolina, USA
Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus are mosquito-borne viruses that cause epidemics of debilitating myositis and polyarthritis in humans in various areas around the world. Studies conducted in a mouse model of RRV-induced disease have demonstrated a critical role of the inflammatory response in the development of disease. In particular, the host complement system contributes significantly to damage within target tissues through activation of CR3-bearing inflammatory cells. We have recently shown that the mannose binding lectin (MBL) activation pathway of the host complement system is the primary pathway required for complement activation and disease following RRV infection. MBL recognizes and binds to terminal carbohydrates, such as mannose found on glycosylated viral proteins or on infected cells. The RRV E2 envelope glycoprotein contains two N-linked glycosylation sites that are glycosylated with a combination of high mannose and complex glycans when replicating in mammalian cells. We hypothesized that these Nlinked glycans act as ligands for MBL binding and subsequent complement activation contributing to the development of severe disease. Using a panel of RRV mutants lacking one or more envelope glycans, we have found that the RRV E2 N-linked glycans contribute to MBL binding to RRV infected cells. Viruses lacking either E2 N-linked glycosylation sites cause reduced disease in mice, while a virus lacking both sites causes very mild disease. In addition, the role of the E2 glycans is independent of replication within host tissues and recruitment of inflammatory cells. Rather, the E2 glycans were required for MBL deposition and complement activation within target tissues in vivo. These results suggest that interactions between the viral Nlinked glycans and the MBL pathway play a central role in development of severe alphavirus-induced arthritis and may be an effective target for therapeutic treatment in patients infected with RRV.