Molecular characterization of HLA class II binding to the LAG‐3 T cell co‐inhibitory receptor

Abstract

Immune checkpoint inhibitors (antibodies that block the T cell co-inhibitory receptors PD-1/PD-L1 or CTLA-4) have revolutionized the treatment of some forms of cancer. Importantly, combination approaches using drugs that target both pathways have been shown to boost the efficacy of such treatments. Subsequently, several other T cell inhibitory receptors have been identified for the development of novel immune checkpoint inhibitors. Included in this list is the co-inhibitory receptor lymphocyte activation gene-3 (LAG-3), which is upregulated on T cells extracted from tumor sites that have suppressive or exhausted phenotypes. However, the molecular rules that govern the function of LAG-3 are still not understood. Using surface plasmon resonance combined with a novel bead-based assay (AlphaScreen TM), we demonstrate that LAG-3 can directly and specifically interact with intact human leukocyte antigen class II (HLA-II) heterodimers. Unlike the homologue CD4, which has an immeasurably weak affinity using these biophysical approaches, LAG-3 binds with low micromolar affinity. We further validated the interaction at the cell surface by staining LAG-3 + cells with pHLA-II-multimers. These data provide new insights into the mechanism by which LAG-3 initiates T cell inhibition.

LAG3’s Enigmatic Mechanism of Action

“…demonstrated that strong affinity of antigenic peptide for MHCII and expression of MHCII accessory molecules substantially increase LAG3 binding to MHCII. As the authors note, this indicates that LAG3 function is dependent on APC properties, the presented peptides and MHC haplotypes, bestowing a target selectivity that is unique among immune checkpoints ( 35 , 40 ).…”

“…Some have suggested that LAG3 inhibitory function is a result of competitive inhibition of CD4 due to its shared evolutionary origin and the fact that it binds MHCII with far greater affinity ( 35 , 39 , 40 , 46 ). However, no evidence suggests LAG3 inhibits CD4-ligand interaction during T cell activation.…”

Immune Checkpoint LAG3 and Its Ligand FGL1 in Cancer

“…Some studies suggested that pHLA-II is a LAG3 receptor ( 50 – 52 ), and recently, Maclachlan et al. showed that LAG3 can directly and competitively bind with pHLA-IIto influence the bind of CD4 with pHLA-II, further inhibiting T cell activation ( 53 ).…”

“…From above all, the discrepancy of LAG3 exerts positively or negatively in immune functions can be attributed to the complexity of tumor immune microenvironment and tumor heterogeneity, besides, LAG3 may not create immunosuppression when acting alone, but only when acting in combination with other immune checkpoints such as PD1. Some studies suggested that pHLA-II is a LAG3 receptor (50-52), and recently, Maclachlan et al showed that LAG3 can directly and competitively bind with pHLA-IIto influence the bind of CD4 with pHLA-II, further inhibiting T cell activation (53).…”

Seven mysteries of LAG-3: a multi-faceted immune receptor of increasing complexity

“…Consequently, our lab has recently used surface plasmon resonance (SPR) (see Technical box ) to directly measure binding between HLA-DR1 and LAG-3:Fc. Using titrated amounts of immobilised proteins on the sensor chip, we estimated the monovalent interaction between HLA-DR1 bearing a high-affinity peptide (Influenza A Haemagglutinin 306–318 ) and LAG-3 to be in the low micromolar range (K D ≈ 13 µM) [ 35 ]. This affinity measurement is in a similar range as HLA-I binding co-inhibitory receptors, such as the ILT/LILRB family [ 36 ] but still significantly stronger affinity than that of HLA-II/CD4 which is estimated at K D = 2.5 mM [ 37 ].…”