ABSTRACTThe non-classical class Ib molecule human leukocyte antigen E (HLA-E) has limited polymorphism and can bind HLA class Ia leader sequence peptides (VL9). HLA-E-VL9 complexes interact with the natural killer (NK) cell inhibitory receptor NKG2A/CD94 and regulate NK cell-mediated cytotoxicity. Here we report a murine HLA-E-VL9-specific IgM monoclonal antibody 3H4 that enhanced killing of HLA-E-VL9-expressing target cells by a NKG2A+ NK cell line, most likely due to steric clashes between 3H4 and CD94/NKG2A when docked on the HLA-E-VL9 surface as determined by a 1.8 Å co-complex crystal structure. Key 3H4-mediated contacts with HLA-E-VL9 were generated by germline-encoded CDR-H3 residues. Human IgM HLA-E-VL9 reactive antibodies could also be isolated from CD10−/CD27- naïve B cells; these antibodies also recognized microbiome-derived peptides presented by HLA-E. Thus, a subset of natural antibodies that recognize VL9-bound HLA-E exist as part of the normal Ig repertoire with capacity to regulate NK cell function.