Saweirs, Walaa Wilson Matta.

2019-02-15T14:20:09Z

2019-02-15T14:20:09Z

2005

A quantitative and qualitative analysis of the various sub-types of antigenspecific CD4⁺ T cells in autoimmunity is important because they have a central role in an immune response. Although it has been possible to study the humoral response in detail in many autoimmune diseases, the analysis of antigen-specific T cells lags far behind. A major impediment has been the innate low affinity of the T cell receptor for its bipartite ligand - MHC plus peptide. The arrival of MHC class I tetramers shed new light on the CD8⁺ T cell response, and provided one solution to these challenges. Although monomeric HLA binding is of too low an affinity to be useful in T cell labelling, fluorochrome labelled tetrameric HLA-peptide complexes bind stably to antigen-specific CD8⁺ T cells due to the higher avidity gained by multimerisation. These MHC-peptide TCR interactions are, therefore, able to detect specific cellular immune responses in a similar way to the way antigen-antibody interactions have been used to examine the humoral immune response. There is currently no evidence that this technique overestimates the number of antigenspecific T cells. The need to explore the role of CD4⁺ T cells in autoimmunity and infection has lead several groups to try to develop MHC class II tetramers. All have met with varying technical difficulties, primarily because successful MHC class II multimer formation requires the conformationally correct interaction of three components - a and P chain, and peptide - rather than the two in MHC class I. However, the successes that have been reported suggested that MHC class II tetramer analysis could be undertaken and applied to examine autoimmune T cell populations.

I chose Goodpasture's disease as a pilot autoimmune disease to explore the potential of MHC class II tetramers. Although Goodpasture's disease is an uncommon autoimmune disorder that causes rapidly progressive glomerulonephritis and lung haemorrhage, it provides an amenable model for tetramer analysis of autoreactive T cells for a number of reasons: • The 'Goodpasture' antigen is known • Both predisposing and protective HLA class II molecules have been identified • Three nested sets of naturally processed peptides from the antigen that are presented bound to HLA-DR15 have been identified biochemically.

Here I describe the formation of functional recombinant MHC class II proteins that are capable of being multimerised to form fluorochrome labelled multimeric complexes. Two different approaches have been used. One approach utilised the Drosophila S2 expression system and adapted the C-terminals of the MHC class II alpha and beta chains through the addition of an acid-base leucine zipper motif in order to maintain the stability of their heterodimeric association in solution. Protein tags were also added to the C-terminals together with the BirA sitespecific biotinylation sequence. Both HLA-DR15 and HLA-DR7 were adapted in this way. The other, more novel approach, utilised a bacterial expression system and aimed to improve the heterodimeric chain pairing of the MHC class II peptidebinding domain through the construction of a two-domain single chain MHC class II peptide, linking the (31 domain directly to the al domain, similar to the rat RTlb construct of Burrows et al (Burrows et al., 1999). HLA-DR15, HLA-DR7 and I-Ed were adapted in this way. The conformationally correct nature of both of these approaches and their peptide-specific binding are demonstrated. Abstract

http://hdl.handle.net/1842/33830

The University of Edinburgh

Annexe Thesis Digitisation Project 2019 Block 22

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Formation of MHC class II - peptide multimers

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PhD Doctor of Philosophy