Reassessment of HLA-G isoform specificity of MEM-G/9 and 4H84 monoclonal antibodies

Human leukocyte antigen (HLA)-G is a non-classical HLA class I molecule thought to play a key role in maternal-fetal tolerance. Although initial studies suggested that HLA-G expression is restricted to extravillous cytotrophoblasts, expression was subsequently reported in a wide variety of other human tissues and tumor cells. However, consensus as to the validity of these collective findings has proven difficult because the antibodies used to define the temporal and spatial expression patterns of HLA-G remain incompletely characterized. The aim of our study was to reassess two of the most widely used HLA-G antibodies (MEM-G/9 and 4H84) in HLA-G-positive (JEG-3 and HLA-G transduced) and -negative (dermal fibroblast, mesenchymal stem cell, K562, and Jurkat) lines using flow cytometry, immunofluorescence, and western blotting. We found that MEM-G/9 recognized HLA-G3 by flow cytometry, indicating that its epitope is present on the α1 domain of HLA-G. Although 4H84 preferably recognized unfolded HLA-G-free chains, it showed strong non-specificity under certain methodological conditions.