Characterization of the major histocompatibility complex class II binding site on LAG-3 protein

Abstract

The lymphocyte activation gene-3 (LAG-3), selectively transcribed in human activated T and NK cells, encodes a ligand for major histocompatibility 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 binding site. In contrast to CD4, soluble LAG-3 molecules stably interact with MHC class II molecules expressed on the cell surface. In addition, the first two Nterminal domains of soluble LAG-3 (D1 and D2) molecules, alone, are capable of binding MHC class II. From a LAG-3 model structure, 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 essential for either binding or repulsing MHC class II proteins. Most of these residues are clustered at the base of a large extra-loop structure that is a hallmark of the LAG-3 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 the ABED ␤-strands containing side of D1 results in a dominant negative effect (i.e., binding inhibition of coexpressed wild-type LAG-3).

MHC Class II Engagement by Its Ligand LAG-3 (CD223) Contributes to Melanoma Resistance to Apoptosis

“…Discrepancies between pathways activated by L243 mAb and sLAG-3 have also been reported in dendritic cells (42). The L243 mAb is known to recognize HLA-DR in a manner mimicking that of TCR recognition, whereas the interaction of LAG-3 with MHC II may rather mimic that of CD4 (15,27). Therefore, different epitopes recognized by each MHC II ligand might underlie the observed discrepancies between the effects of LAG-3 and anti-HLA-DR. LAG-3 and L243 also activate MAPK/Erk with different kinetics; engagement of MHC II in melanoma cells by L243 induces a sustained MAPK/ Erk activation for up to 24 h (10), whereas in this study we show that LAG-3 rather induces a transient activation from 2 to 4 h.…”

“…Recently, LAG-3 expression also defined an active CD4 + CD25 high FOXP3 + Treg subset, which is endowed with potent suppressor activity that requires cell-to-cell contact and is found at enhanced frequency in PBMCs of patients with melanoma and is expanded at tumor sites (26). As a soluble fusion protein, LAG-3 has also been shown to bind MHC II with a much higher avidity than CD4 (27), to increase the capacity of MHC II-positive macrophages and immature dendritic cells to induce T cell responses in vitro (28), and to enhance the in vitro induction of viral and tumor-specific cytotoxic T cells (29). Accordingly, the LAG-3 fusion protein has been suggested as a potential adjuvant for cancer vaccines (29), including those for melanoma, for which most attempts at immunotherapy have met with little clinical success.…”

A Soluble Lymphocyte Activation Gene-3 Molecule Used as a Vaccine Adjuvant Elicits Greater Humoral and Cellular Immune Responses to Both Particulate and Soluble Antigens

“…LAG-3 is expressed in activated CD4 ϩ and CD8 ϩ T lymphocytes where it is associated with the CD3/TCR complex at the cell surface (3,4). LAG-3, like CD4 (5), may oligomerize at the cell surface to interact more efficiently with MHC class II molecules, as shown by the finding of three dominant negative mutations in the LAG-3 domain 1 that were able to inhibit the binding of wild-type LAG-3 molecules to class II molecules in a cell-cell adhesion assay (6).…”

The immune microenvironment in Hodgkin lymphoma: T cells, B cells, and immune checkpoints

“…87,88 Subsequently it was determined that LAG-3, like PD-1, is upregulated on T cells during chronic antigen stimulation. 89 LAG-3 suppresses CD4+ T cell expansion in response to antigen, 90 and LAG-3 was found to be synergistic with CTLA-4 and PD-1 in mediating T cell suppression during chronic antigenic stimulation.…”