Lepin, Eric J.M.; (2002) Functional characterisation of HLA-F. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The work presented in this thesis is the functional characterisation of HLA-F, a human non-classical MHC class I molecule. HLA-F is thought to have evolved a specific immune function, similar to HLA-E and HLA-G, two other human non-classical class I molecules. HLA-F was first expressed and characterised in mammalian cell lines and bacteria. A prokaryotic system of expression was found to be more useful. Recombinant HLA-F heavy chain and beta-2 microglobulin proteins formed a stable complex when refolded in vitro in the absence of synthetic peptide. Furthermore, high-pressure liquid chromatography did not detect any bound peptides following acid elution of the refolded complex. This complex was used as an immunogen to produce a highly specific, high affinity monoclonal antibody (FGl) that was used to study the cell biology and tissue distribution of HLA-F. HLA-F was shown to have a restricted pattern of tissue expression in tonsil, spleen, and thymus. HLA-F could be immunoprecipitated from B cell lines, and from HUT-78, a T cell line. In B cell lines HLA-F was shown to bind TAP, but cell surface expression was only detected when these cells were grown at 26² C. HLA-F tetramers were generated to identify potential receptors for HLA-F. HLA- F tetramer binding could be conferred on non-binding cells by transfection with the inhibitory receptors ILT2 (LIRl) and ILT4 (LIR2). Finally surface plasmon resonance studies demonstrated a direct molecular interaction of HLA-F with ILT2 and ILT4. At present, the function of HLA-F remains unclear however, we now have substantial insight into the biochemistry and intracellular interactions of HLA-F and the cell types, which express and may interact with it.

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