Quantitative detection of the HVEM-BTLA checkpoint receptor <i>cis</i>-complex in human lymphocytes

The herpesvirus entry mediator (HVEM) (TNFRSF14) engagement of the checkpoint inhibitory receptor B and T lymphocyte attenuator (BTLA) limits immune responses of T and B lymphocytes. HVEM and BTLA form signaling complexes in trans and when coexpressed, complexes in cis, creating a unique immune checkpoint. The function of the HVEM-BTLA cis-complex is not well understood primarily due to a lack of reagents that specifically measure the HVEM-BTLA cis-complex. We describe here a method to generate antibodies to receptor-ligand complexes using fusion immunogens, in this case, a BTLA-HVEM fusion protein. We identified 2 closely related antibodies that specifically recognize the HVEM-BTLA complex on the cell surface. In experiments utilizing the anti-HVEM-BTLA complex-specific antibody together with subunit-specific BTLA monoclonal antibodies, we were able to determine the precise ratio of free to cis-complexed BTLA on subpopulations of human lymphocytes. This is the first direct quantification of the HVEM-BTLA cis-complex. The method described here should apply to the detection of other receptor-ligand complexes.