CHARACTERIZATION OF THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS-II BINDING-SITE ON LAG-3 PROTEIN

The lymphocyte activation gene-3 (LAG-3), selectively transcribed in h uman activated T and NK cells, encodes a ligand for major histocompati bility complex (MHC) class II molecules, Like CD4, LAG-3 ectodomain is composed of four Ig-like domains (D1-D4). Nothing is known about the LAG-3 regions or residues required to form a stable MHC class II bindi ng site, In contrast to CD4, soluble LAG-3 molecules stably interact w ith MHC class II molecules expressed on the cell surface, In addition, the first two N-terminal domains of soluble LAG-3 (D1 and D2) molecul es, alone, are capable of binding MHC class II, From a LAG-3 model str ucture, we designed mutants and tested their ability to bind MHC class II molecules in an intercellular adhesion assay, We found residues on the membrane-distal, CDR1-2-containing top face of D1 that are essent ial for either binding or repulsing MHC class II proteins, Most of the se residues are clustered at the base of a large extra-loop structure that is a hallmark of the LAG-S D1 Ig-like domain, In addition, as for CD4, oligomerization of LAG-3 on the cell surface may be required to form a stable MHC binding site because mutation of three residues in t he ABED beta-strands containing side of D1 results in a dominant negat ive effect (i.e., binding inhibition of coexpressed wild-type LAG-3).