A structural investigation of human class Ib major histocompatibility complex molecules in innate and adaptive immunity

While the Human Leukocyte Antigen (HLA) locus is the most polymorphic region in the human genome, Major Histocompatibility Complex Class Ib (MHC-Ib) molecules display far less polymorphism and variation than the structurally similar MHC Class Ia (MHC-Ia) molecules. From an evolutionary perspective, the polymorphism of MHC-Ia stems from the advantage conferred by a heterozygotic MHC-Ia genotype, due to the recognised role of antigen presentation to clonotypic αβT-cell receptors (TcRs) in the adaptive immune system. In contrast, the primary roles of the various MHC-Ib molecules are in the regulation of the innate immune system, roles which are generally independent of allele variation. Indeed, the two most extensively studied Class Ib molecules in humans, HLA-G and HLA-E, are essentially monomorphic at the amino acid level, though a limited number of polymorphisms have been found in healthy individuals. The role of HLA-G in regulating the innate immune response to the semi-allogeneic foetus in pregnancy has been a recent focus of research in reproductive biology. Little is known about the role of the peptide presented by HLA-G in this context, though the crystal structure of HLA-G presenting the endogenous peptide RIIPRHLQL, has been determined in both monomeric (Clements et al., 2005) and dimeric (Shiroishi et al., 2006a) forms. It has been proposed that the nature of the bound peptide may influence binding to Natural Killer (NK) cell receptors of the Leukocyte Immunoglobulin-like Receptor (LILR/LIR/ILT) family, as well as KIR2DL4, a member of the Killer Immunoglobulin-like Receptor (KIR) family of NK receptors. Therefore, the structure of HLA-G was determined with two further endogenous peptides, KLPAQFYIL and KGPPAALTL, in order to investigate the effect of the bound peptide on the conformation of HLA-G. KIR2DL4, which also displays significantly less polymorphism relative to other members of the KIR family, was expressed recombinantly and studied using biochemical techniques including circular dichroism (CD) analysis and small angle x-ray scattering (SAXS) as part of the wider aim of solving the crystal structure of this NK receptor. HLA-E performs a distinct role in regulating the innate immune response, mediated by the presentation of related peptides, derived from the leader sequence of MHC-I molecules, to NK receptors of the CD94/NKG2 family. This role forms part of the ‘missing self’ reaction, with downregulation of pMHC-I (peptide/MHC-I) production (for example, in infected or damaged cells) resulting in downregulation of pHLA-E presentation and knockout of signals inhibiting NK cell-mediated lysis. Constitutive, activatory signals present in the NK cell are then able to mediate lysis of the infected or damaged cell. While the structure of HLA-E has been solved with several peptides and also in complex with CD94/NKG2A, a role in the adaptive immune system has recently been proposed based on the discovery of HLA-E-restricted T-cell populations in several individuals with latent or resolved Cytomegalovirus (CMV) infections. It has been proposed that the product of the open reading frame UL40 (a Type 1 membrane protein) of some CMV strains has evolved to contain mimotopes of the MHC-I leader sequences, and that in infected individuals whose MHC-I haplotypes do not match the viral mimotopes, a T-cell response may be generated (Ulbrecht et al., 2000). Based on the haplotypes of individuals, a range of non-self pHLA-E-reactive TcRs may be generated. To date, two groups of HLA-E restricted TcR have been characterised, based on peptide recognition patterns (Pietra et al., 2003). The TcRs GF4 (Group 1) and KK50.4 (Group 2) have been isolated as representative of each of these groups. To investigate the mechanism of this TcR:MHC-Ib interaction, the crystal structure of GF4 has been determined in complex with HLA-E presenting the two related peptides VMAPRTLVL and VMAPRTLIL. These structures have enabled comparison with the previously determined KK50.4:HLA-EVMAPRTLIL complex structure (Hoare et al., 2006) as well as those of MHC-Ia:TcR complexes. Collectively, the work presented in this thesis has provided insight into the basis of peptide presentation and subsequent TcR recognition by MHC-Ib molecules in humans.