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Title: Forward genetic analysis of memory CD8+ T lymphocyte development and long-term maintenance by in vivo chemical germline mutagenesis
Author: Choi, Onjee
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2012
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The establishment and long-term maintenance of virus-specific memory CD8+ cytotoxic T lymphocytes (CTL) is genetically controlled. However, not all genes involved in this process have been identified in vivo to date and their identity and function have to be clarified. To identify novel genes which regulate the development of memory cells, we have undertaken an in vivo forward genetic approach based on germline mutagenesis screening. Random point mutations were introduced in the genome of wild-type mice by N-ethyl-N-nitrosourea (ENU) treatment. A library of third generation mutants was then screened for genetic defects affecting their immune responses to Lymphocytic Choriomeningitis virus (LCMV). Altered immune responses to LCMV in mutant mice were detected by measuring the percentage of antigen-specific CTL, their cell death and the composition of the LCMVspecific CD8+ T cell pool at the peak of expansion and at the beginning of the memory phase. Any individuals with outstanding cell population numbers or characteristics were isolated as phenodeviants. Once a mutation was confirmed to affect the germline, a stable strain was established and the mutation was mapped by positional cloning. Through this process, we have isolated 3 mutants: memi, binu and alois. Our first mutant, memi was isolated based on its very low CD8+ T cell memory population following LCMV infections. Using single nucleotide polymorphism linkage analysis, we identified memi as a loss-of-function mutation in the gene encoding deoxycytidine kinase (dCK), a crucial enzyme of the nucleoside salvage pathway. In addition to the immunological phenotype observed after viral infection, we have characterized significant changes in T and B lymphocyte development and their cell-intrinsic/extrinsic peripheral homeostasis in naive memi mice. This mutant is valuable especially to understand how a defect in nucleoside salvage impacts on lymphocyte development and behaviour. The other two mutants, binu and alois, which present increased and decreased CD8+ T memory precursors upon LCMV infection, respectively, are currently being mapped. To our knowledge, no mutant with exactly the same phenotypes as binu or alois has been described to date and we therefore believe that they have strong potential to be novel mutants. Overall, our ENU mutagenesis was successful both in terms of the number of mutants we have isolated and the types of mutations they represent, affecting known and potential new immune regulators.
Supervisor: Rutschmann, Sophie ; Ashton-Rickardt, Philip Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available