MHC-E (HLA-E in human, Mamu-E in rhesus macaques) is a nonpolymorphic MHC class Ib molecule, with a species conserved evolutionary function that relates to NK-mediated monitoring of cellular homeostasis. This function relies on MHC-E presenting classical MHC class I leader sequence peptides VL9 (VMAPRT[L/V][V/L/I/F]L) in humans to NK cells, with recognition mediated by inhibitory NKG2A-CD94 and activating NKG2C-CD94 receptor engagement. The inhibitory signal mediated by NKG2A-CD94 engagement with HLA-E VL9 is the dominant effect on NK cells and when VL9 peptide supply is limited, such as in viral infection, loss of this inhibitory signal releases NK cells, causing cell lysis. In infection and cancer, MHC-E is often upregulated to engage the inhibitory NKG2A-CD94 receptor, to prevent NK cell-mediated killing of such cells.In a seminal study, Hansen et al. demonstrated that a RhCMV-vectored SIV vaccine elicited protective Mamu-E (rhesus HLA-E ortholog)-restricted CD8+ T cell responses in rhesus macaques against SIV challenge, revealing broad and diverse peptide epitopes. However, many of these non-VL9 peptides bind weakly to MHC-E, resulting in unstable complexes. HLA-E-restricted pathogen-derived peptides from viruses such as HCMV, HIV-1, HBV, HCV, EBV, SARS-CoV-2, and bacteria including Mycobacterium tuberculosis, and Salmonella enterica, have also been described in humans.Replicating these protective MHC-E-restricted CD8+ T cell responses in humans against HIV and other diseases through vaccination would be a major achievement for global health. Targeting these immunogenic HLA-E peptide complexes also presents a worthwhile venture as these could be universal across the human population given the near monomorphic nature of HLA-E globally. Two alleles, HLA-E*01:01 and HLA-E*01:03 dominate globally, that differ by a single amino acid in a region that does not affect peptide binding. However, challenges for such approaches are noteworthy given that many peptides bind weakly to HLA-E.This thesis centre on MHC-E, with the goal to improve reagents and methods that could aid the development of potential HLA-E-targeting therapeutics. The specific aims centred on the development of improved peptide binding assays to increase peptide binding to MHC-E and to explore the binding of antibodies that recognise peptides bound to HLA-E (so called T cell receptor mimics). The development of two MHC-E peptide binding assays – a thermal stability-based nano-differential fluorimetry (nDSF) and a fluorescent polarisation competition assay – is described. These assays were employed to compare HLA-E*01:03 and Mamu-E*02:04 peptide binding repertoires, with results depicting similar peptide binding characteristics but with subtle binding hierarchy differences. Subsequent crystallisation of Mamu-E*02:04 with VL9, aided by nanobody, VHH-AD01, enabled the first structural comparison to HLA-E VL9 and suggested that subtle differences between HLA-E and Mamu-E may subtly affect the positioning of the peptide mainchain in the peptide binding groove.To improve peptide binding to MHC-E, select mutations were introduced into the heavy chain within the peptide binding groove region. Successful refolding and stabilisation of HLA-E with mutants H99Y, F116Y, and S147W produced improved tetramer reagents, with no major structural changes observed for a HIV Gag-derived peptide RL9HIV with certain mutants. Finally, TCR mimics targeting HLA-E bound to a Wilm’s tumour antigen (WT1)-derived peptide were characterised for specificity and structurally (the latter aided by VHH-AD01, supporting potential immunotherapeutic development against HLA-E WT1 peptide expressing cancers. Overall, this work advances our understanding of MHC-E peptide binding and structure, and lays the foundation for future studies relating to the targeting of MHC-E therapeutically.