The engineered mHVEM mutants have binding selectivity. (A) The relative binding affinities of mHVEM mutants with mBTLA, mCD160, and mLIGHT as measured by cell–cell or cell–protein methods. Error bars represent results from at least triplicates. The gray dashed line marks the averaged normalized affinities of WT mHVEM with mBTLA, mCD160, and mLIGHT. Blue dashed line marks the average background noise resulting from the non-specific interaction of negative control mCherry with mLIGHT. (B) The locations of the mutated residues on mHVEM. mHVEM is shown as a surface, with each CRD colored differently; G72, V74, H86, and L90 are marked on the mHVEM surface. Ligands BTLA, CD160, and LIGHT are modeled based on the HVEM structures and are shown as labeled gray surfaces. (C) The binding affinities of mHVEM^WT (WT mHVEM), mHVEM^−BT/160 (mHVEM G72R-V74A double mutein), and mHVEM^−LIGHT (mHVEM H86D-L90A double mutein) with mBTLA, mCD160, and mLIGHT as measured by Octet biolayer interferometry technology.