Investigating the molecular and cellular basis of antibody affinity maturation in germinal centres

Project Overview

Antibodies are essential components of adaptive immunity and their induction is critical for the effectiveness of most vaccines. However, efforts to develop effective vaccines against various important pathogens such as HIV and malaria have so far failed and so there is an urgent need to better understand how protective humoral responses develop.

The quality and affinity of antibodies improves over the course of immune responses through a remarkable process known as affinity maturation. Antibody affinity maturation occurs in transient structures called germinal centres that form in the B cell follicles of secondary lymphoid tissues in the days following infection or immunization and may last for a couple of weeks to many months depending upon the challenge. Here, germinal centre B cells engage in a greatly accelerated form of Darwinian evolution that involves them deliberately introducing random somatic point mutations into their immunoglobulin variable region (antibody encoding) genes at a rate of approximately 1-2 nucleotide changes per cell, per day (a million times higher than background rates). Newly mutated B cells then compete with each other, based upon their ability to gather antigen, for survival and for cues that drive their further clonal expansion. As such, “better” clones are preferentially “selected”. Affinity improvements are the product of iterative rounds of this mutation and selection process. However, the molecular and cellular events involved in selection are only partly understood (discussed in detail in Bannard and Cyster, Current Opinions Immunology 2017, PMID 28088708).

The Bannard lab seeks to develop and apply novel genetic in vivo tools to investigate the issues discussed above. DPhil opportunities exist in the lab to investigate a range of fundamental questions in germinal centre biology, including:

Projects may involve the development and use of sophisticated genetic modified host/viral models. There will be opportunities to learn and implement cutting edge imaging, sequencing, single B cell cloning and flow cytometry technologies. As such, students can expect to receive sound intellectual and practical science training.

Informal enquiries are welcomed and can be directed to oliver.bannard@ndm.ox.ac.uk.

Training Opportunities

The projects will suit someone with a strong interest in mechanistic aspects of immune responses and who enjoys thinking deeply about complex problems. The student will learn to conduct in vivo experiments in mice. Approaches are likely to include single cell genomics, next-generation sequencing, transgenic mouse generation and experimentation, bone marrow reconstitution (including retrovirally transduced bone marrow), confocal microscopy, live cell imaging and multi-color flow cytometry. There will also be opportunities to develop and manipulate viruses. Other immunology, imaging and molecular biology techniques will be employed as needed. This study will be supervised by Dr. Oliver Bannard and will be conducted in the Weatherall Institute of Molecular Medicine (WIMM) as part of the MRC Human Immunology Unit.

Students will be enrolled on the MRC WIMM DPhil Course, which takes place in the autumn of their first year. Running over several days, this course helps students to develop basic research and presentation skills, as well as introducing them to a wide-range of scientific techniques and principles, ensuring that students have the opportunity to build a broad-based understanding of differing research methodologies.

Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.

As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford.

Theme

Immunology & Infectious Disease

Admissions

Project reference number: 1014

Funding and admissions information

Supervisors

Name Department Institution Country Email
Dr Oliver Bannard Experimental Medicine Division Oxford University, Weatherall Institute of Molecular Medicine GBR oliver.bannard@ndm.ox.ac.uk
Professor Richard J Cornall FMedSci FRCP Centre for Cellular and Molecular Physiology Oxford University, Henry Wellcome Building for Molecular Physiology GBR richard.cornall@ndm.ox.ac.uk

Stewart I, Radtke D, Phillips B, McGowan SJ, Bannard O. 2018. Germinal Center B Cells Replace Their Antigen Receptors in Dark Zones and Fail Light Zone Entry when Immunoglobulin Gene Mutations are Damaging. Immunity, 49 (3), pp. 477-489.e7. Read abstract | Read more

Adaptive immunity involves the development of bespoke antibodies in germinal centers (GCs) through immunoglobulin somatic hypermutation (SHM) in GC dark zones (DZs) and clonal selection in light zones (LZs). Accurate selection requires that cells fully replace surface B cell receptors (BCRs) following SHM, but whether this happens before LZ entry is not clear. We found that most GC B cells degrade pre-SHM receptors before leaving the DZ, and that B cells acquiring crippling mutations during SHM rarely reached the LZ. Instead, apoptosis was triggered preferentially in late G1, a stage wherein cells with functional BCRs re-entered cell cycle or reduced surface expression of the chemokine receptor CXCR4 to enable LZ migration. Ectopic expression of the anti-apoptotic gene Bcl2 was not sufficient for cells with damaging mutations to reach the LZ, suggesting that BCR-dependent cues may actively facilitate the transition. Thus, BCR replacement and pre-screening in DZs prevents the accumulation of clones with non-functional receptors and facilitates selection in the LZ. Hide abstract

Bannard O, Cyster JG. 2017. Germinal centers: programmed for affinity maturation and antibody diversification. Curr. Opin. Immunol., 45 pp. 21-30. Read abstract | Read more

The seminal discovery by Eisen that antibodies undergo improvements in antigen-binding affinity over the course of an immune response led to a long running search for the underlying mechanism. Germinal centers in lymphoid organs are now recognized to be critically involved in this phenomenon, known as antibody affinity maturation. As well as improving in affinity for specific epitopes, some antibody responses maintain or even increase their breadth of antigen-recognition over time. This has led to another intense line of research aimed at understanding how broadly neutralizing anti-pathogen responses are generated. Recent work indicates that germinal centers also play an important role in the diversification process. We discuss current understanding of how germinal centers are programmed to support both affinity maturation and antibody diversification. Hide abstract

Bannard O, McGowan SJ, Ersching J, Ishido S, Victora GD, Shin JS, Cyster JG. 2016. Ubiquitin-mediated fluctuations in MHC class II facilitate efficient germinal center B cell responses. J. Exp. Med., 213 (6), pp. 993-1009. Read abstract | Read more

Antibody affinity maturation occurs in germinal centers (GCs) through iterative rounds of somatic hypermutation and selection. Selection involves B cells competing for T cell help based on the amount of antigen they capture and present on their MHC class II (MHCII) proteins. How GC B cells are able to rapidly and repeatedly transition between mutating their B cell receptor genes and then being selected shortly after is not known. We report that MHCII surface levels and degradation are dynamically regulated in GC B cells. Through ectopic expression of a photoconvertible MHCII-mKikGR chimeric gene, we found that individual GC B cells differed in the rates of MHCII protein turnover. Fluctuations in surface MHCII levels were dependent on ubiquitination and the E3 ligase March1. Increases in March1 expression in centroblasts correlated with decreases in surface MHCII levels, whereas CD83 expression in centrocytes helped to stabilize MHCII at that stage. Defects in MHCII ubiquitination caused GC B cells to accumulate greater amounts of a specific peptide-MHCII (pMHCII), suggesting that MHCII turnover facilitates the replacement of old complexes. We propose that pMHCII complexes are periodically targeted for degradation in centroblasts to favor the presentation of recently acquired antigens, thereby promoting the fidelity and efficiency of selection. Hide abstract

Bannard O, Horton RM, Allen CD, An J, Nagasawa T, Cyster JG. 2013. Germinal center centroblasts transition to a centrocyte phenotype according to a timed program and depend on the dark zone for effective selection. Immunity, 39 (5), pp. 912-24. Read abstract | Read more

Germinal center (GC) B cells cycle between the dark zone (DZ) and light zone (LZ) during antibody affinity maturation. Whether this movement is necessary for GC function has not been tested. Here we show that CXCR4-deficient GC B cells, which are restricted to the LZ, are gradually outcompeted by WT cells indicating an essential role for DZ access. Remarkably, the transition between DZ centroblast and LZ centrocyte phenotypes occurred independently of positioning. However, CXCR4-deficient cells carried fewer mutations and were overrepresented in the CD73(+) memory compartment. These findings are consistent with a model where GC B cells change from DZ to LZ phenotype according to a timed cellular program but suggest that spatial separation of DZ cells facilitates more effective rounds of mutation and selection. Finally, we identify a network of DZ CXCL12-expressing reticular cells that likely support DZ functions. Hide abstract