Development of Antibodies Specific for NKG2 Family Members

INTRODUCTION

NKG2A, NKG2C and NKG2E/H are members of the NKG2 family of type II transmembrane receptors, which have closely-related extracellular C-type lectin domains. These proteins are expressed in NK cells and some T cell subsets. NKG2E and NKG2H are splice variants encoded by the same gene, whereas NKG2A and NKG2C are encoded by different genes. NKG2A, NKG2C and NKG2E/H all require association with CD94 for cell-surface expression and functional activity. Although members of the NKG2 family have similar extracellular domains, their cytoplasmic domains are functionally distinct. The NKG2A/CD94 complex delivers an inhibitory signal through association with ITIM motifs in the cytoplasm. The NKG2C/CD94 and NKG2E/CD94 complexes associate with the ITAM-bearing DAP12 adapter protein and deliver an activating signal upon ligand binding.

Due to these functional differences, an antibody that is specific for NKG2A can help to distinguish between the expression of the inhibitory NKG2A receptor and the activating NKG2E receptor. We have developed and characterized panels of monoclonal antibodies (mAbs) that recognize members of the NKG2 family. Immunization of Balb/c mice with Ba/F3 NKG2A + CD94 transfectant generated one mAb (Clone # 131411) that specifically recognizes NKG2A by flow cytometry. The same fusion yielded several mAbs that cross-react with NKG2C, as well as mAbs specific for CD94. We show that anti-human NKG2A (Clone # 131411) specifically recognizes NKG2A transfectant cells, and not NKG2C, NKG2E/H, or CD94. In contrast, another commercially available clone (Clone # Z199) stained both NKG2A and NKG2E/H-expressing Ba/F3 transfectants, but was not cross-reactive with NKG2C or CD94.

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