Principal Supervisor: Professor Kai-Michael Toellner, Institute of Immunology and Immunotherapy
Co-supervisor: Professor Adam Cunningham, School of Immunity and Infection
PhD project title: Using BASP1 as a reporter of affinity matured plasma cells
University of Registration: University of Birmingham
Over recent years, generation of monoclonal antibodies has become the major method to generate and produce intelligently designed drugs. To achieve this, it is important to generate antibodies of high affinity. Currently, antibodies are generated by immunising animals and then using a non-selected collection of B cells and plasma cells from these animals to generate monoclonal antibody producing hybridoma cell lines by fusion with immortal myeloma cells. The process is inefficient as there is no selection of high affinity variants. This is done at later stages when the newly generated monoclonal antibodies are characterized. High affinity is mainly achieved by repeatedly immunising animals, not by specifically selecting cells that have affinity matured.
Antibody affinity maturation happens in germinal centres (1). We plan to produce a gene manipulated mouse line that will allow specific identification of affinity matured plasma cells by inserting Cre-recombinase into the Basp1 gene. We have shown that in immune cells Brain Acid Soluble Protein 1 (BASP1) is expressed specifically within germinal centre cells (2). Basp1Cre mice will be crossed with mice expressing membrane tagged fluorescent proteins containing floxed Stop codons. This will allow fluorescent detection of germinal centre derived cells, e.g. affinity matured plasma cells. As Cre-recombinase leads to permanent activation of the fluorescent reporter, this system will permanently label all germinal centre derived cells, including affinity matured plasma cells and memory B cells.
After generation of the new mouse strain it will be used to test whether it can be used for monoclonal antibody generation with higher frequency output of high affinity antibody producing hybridomas. Additionally, the new mouse strain will be used as a tool for basic research on the immunobiology of germinal centre derived cells. We will immunise Basp1Cre x flStopflXFP mice with model antigens, and then quantify and isolate germinal centre cells and germinal centre derived memory B cells and plasma cells by flow cytometry. Isolated cells will be used for downstream gene expression analysis. The new mouse strain will be used to detect onset and timing of germinal centre responses, and track memory B cells and plasma cells generated in germinal centres using flow cytometry, fluorescence microscopy and intravital microscopy.
This project is developed in collaboration with Medimmune, and we anticipate that the project will include some periods of work in an industrial setting; during development of the gene targeting vectors and/or during testing of the practical application on potential drug targets.
- Zhang, Y., L. Garcia-Ibanez, and K.-M. Toellner. 2016. Regulation of germinal centre B cell differentiation Immunol. Rev. 270: 8-19.
- Yu, D., M. C. Cook, D. M. Shin, D. G. Silva, J. Marshall, K. M. Toellner, W. L. Havran, P. Caroni, M. P. Cooke, H. C. Morse, I. C. MacLennan, C. C. Goodnow, and C. G. Vinuesa. 2008. Axon growth and guidance genes identify T-dependent germinal centre B cells. Immunol Cell Biol 86: 3-14.
BBSRC Strategic Research Priority: Molecules, Cells and Systems
Techniques that will be undertaken during the project:
- Genetic manipulation of animals
- Immunisation of animals
- Multi-parameter flow cytometry
- Multi-parameter fluorescence immunohistology
- Gene expression analysis using real time RT-PCR
- Intravital microscopy
- Stereology and statistical analysis of histology and flow cytometry.
Contact: Professor Kai-Michael Toellner, Institute of Immunology and Immunotherapy