Mechanism and manipulation of antibody class-switching in human B cells

It is our immune system that recognises and destroys alien intruders in our body, such as bacteria, viruses and parasites. Malfunctions of the immune system always have serious consequences, and are often fatal (e.g. in cancer, anaphylactic shock and asthma, and autoimmune and infectious diseases). It is also the immune system which rejects an incompatible transplanted kidney, for instance, and causes mayhem when a patient is transfused with blood of the wrong group. Yet it can also be harnessed to mount an attack on cancer cells - a discovery that has led to one of the most momentous recent advances in treatment, the use of monoclonal antibodies - and this is now a subject at the forefront of medical research. The primary agents of the immune system are proteins, called antibodies. Each antibody recognises a particular substance (its antigen), as for example a protein on the surface of a bacterium. There are five classes of antibody in man, each of which acts in a different way or at a different location in the body, but all are made up of similar building blocks. To manipulate the immune system in our favour - to prevent rejection of an organ, or cause it to generate an antibody that will attack a tumour - we need to understand the highly complex programme of steps that controls the process of antibody construction. This mechanism involves rearranging the DNA sequences that encode the antibodies in a process called class switch recombination. What are the signals that instruct the cell to make an antibody of one class and not another? How does that signal set the machinery in operation? Why does it not initiate the activation of genes that will make the wrong protein? Most work in this area has been done on mice, but we have found important differences between the mouse and the human immune systems. We therefore work entirely on human antibody-producing cells. Our aim now is to disentangle the steps in the sequence of events that culminates in the production of an antibody of the required type and no other. We will employ novel methods that we have developed in our previous research to answer these questions. We will analyse the DNA sequences and the proteins that participate in class switch recombination using unique DNA probes and 'next generation' DNA sequencing technology to identify millions of protein and DNA complexes. We will also use ultra-high resolution microscopy to determine when and where in the cell that different steps in the process occur. We hope and expect that what we learn about this system will help in stimulating the formation of antibodies with the properties needed for an attack on different types of disease or suppressing the formation of harmful antibodies.

This work is directed at one of the major unresolved questions in immunology; namely, how antibody class switch recombination (CSR), the process by which the cell selects the antibody isotype to be synthesised, is regulated. The majority of past work in this area has relied on the mouse model. We have uncovered several important respects in which CSR in the mouse differs from that in man, and we shall therefore confine our studies to human B cells. We shall first seek to define the links between CSR and transcription of the germline genes, the modifications of histones in the critical 'switch regions' of chromatin, and the entry into the immunoglobulin gene locus of the enzyme AID responsible for initiating DNA rearrangement. We will determine whether a distinct pattern of histone modifications and/or the rate of transcription is responsible for AID binding to the DNA. We now know that AID is not solely confined to the immunoglobulin locus. If, then, we can determine its genomic distribution, and the positions at which it generates DNA breaks we will be on the way to discovering how its activity is induced or suppressed, according to where it is bound. An issue of especial interest is the nuclear locations at which CSR occurs. It is known that the bulk of transcriptional activity is restricted to as yet ill-defined nuclear regions, termed 'transcription factories'. AID appears to accrue in these centres, and since the migration of immunoglobulin genes inside the nucleus is known to exercise control over V(D)J recombination (the process that creates antibody specificity), we surmise that the transcription factories, or, more likely, a subset of these entities, which we term 'recombination factories', may be the site of CSR. We shall test this inference by FISH with novel precision probes that we have developed and immunohistochemistry using conventional and ultra-high-resolution (4pi) confocal microscopy.

This work is directed at one of the major unresolved questions in cellular immunology; namely, how antibody class switch recombination (CSR) is regulated, and how this process is restricted to the immunoglobulin genes. Inappropriate expression of immunoglobulin isotypes is an important element in many immunological disorders such as the over expression of IgE in asthma and allergy, and IgG in autoimmune diseases. If we can understand and learn to manipulate this process, many possibilities present themselves of developing new therapies to mitigate these diseases. In view of their proportions in society in this country and beyond, the social impact would then be considerable. The results generated by this project will be of intrinsic relevance to the progress of the field, but our long term aim is to use the knowledge that will be gained to develop new therapeutic modalities through intervention in CSR. The majority of previous work in this area has relied on the mouse model. However, we have discovered important differences between the regulation of CSR in man compared to the mouse, and we shall therefore confine our studies to human B cells. As well as publication in peer reviewed journals we give regular communications of our studies at internationally recognised meetings. However, we also share preliminary data with other members of the MRC and Asthma UK Centre in Allergic Mechanisms in Asthma (MRC-AUK), thus enhancing the immediate utility, as well as long-term value of our findings. Our work is brought to the attention of the general public through the efforts of the King's College public relations department, through lectures and symposia addressed to the lay public by the MRC-AUK Centre members, and press releases by the AUK. Through our activities in taking informed consent for the donation of samples we are brought into contact with all the donor patients and their relatives, allowing us to explain the nature and purpose of our research. The Division of Asthma, Allergy and Lung Biology maintains contacts with the local community through annual Guy's hospital Open Days, at which staff from the department are available to explain our work. Centre Heads recently participated in a meeting at the Houses of Parliament hosted by the Associated Medical Research Charities and attended by the cross-party parliamentary body responsible for funding UK science. We have fostered extensive links with industrial laboratories, especially UCB-Celltech and Novartis, and HG has supervised four BBSRC and MRC CASE students. We are proud of our record of translational success. HG's work has led to two Phase l clinical trials on IgE at Guy's hospital, one on cancer therapy and the other on asthma. KCL has a business development team whose activities include the identification of new opportunities for partnership, marketing, intellectual property protection, licensing, collaboration, consultancy and mentoring of spin-off companies. KCL Business Ltd. has been very successful in helping researchers protect and exploit their intellectual property and will be important in ensuring that our work makes the necessary impact. DF and HG have worked together for 10 years before DF moved to an independent position as an RCUK Fellow in the Division of Asthma, Allergy and Lung Biology in 2007. DF and HG have maintained a close association and have a long standing collaboration with Dr Felsenfeld (N.I.H., U.S.A.), with regular exchange visits by all parties to exchange information, plan future experiments and prepare publications. Our collaborations with Dr Felsenfeld and with other colleagues in the United States and Europe have proved especially fruitful, resulting in many joint publications and providing added value to all the associated research projects.