Wallace, Graham Robert; (1990) T cell epitope analysis: Using insulin as a model antigen. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

T cells recognise antigen only on the surface of another cell, the antigen presenting cell, in association with a cell surface molecule encoded by the major histocompatability complex. The nature of these interactions is still not fully understood. For instance, it is not clear how many antigen binding sites are present on MHC molecules, or whether the T cell receptor recognises both antigen and MHC, or just the antigen. Those regions of an antigen which T cells recognise are known as epitopes and attempts to define them has been the focus of many studies. The objective of this thesis was to investigate T cell epitopes on the protein insulin. A hapten group TNP was used to modify the protein and the initial aim was to generate T cell clones against the hapten, while altering the insulin molecule in specific ways to determine the effect of those changes on response to the hapten. However it was noted at an early stage that in H-2b mice the addition of TNP to pork insulin, to which H-2b mice are non-responders, stimulated a response. This finding, and the investigation of the mechanisms involved, form the basis of this thesis. It will show that the TNP group was allowing the recognition of an epitope which in the unhaptenated form of the protein is functionally silent. The TNP was not directly involved in T cell receptor recognition, but may be able to increase the affinity of binding between pork insulin and H-2b MHC molecules. A study on the derivatisation of insulin is discussed. The site of haptenation on the insulin molecule was determined by high performance liquid chromatography. This showed clearly, contrary to previous reports, the the TNP group was binding to the glycine residue at the carboxy terminal of the A chain. In addition, in an attempt to produce hybrid insulin proteins, site-directed mutagenesis was performed on the human proinsulin gene. The use of molecular biology techniques for the production of these hybrids is discussed.

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