Characterizing LAG3 binding to the TCR/CD3 complex and LAG3 ligands that modulate immune response

The National Institute of Allergy and Infectious Diseases, part of the Department of Health and Human Services, awarded the University of Pittsburgh $106,228 on February 10, 2027, under the Allergy and Infectious Diseases Research program (CFDA 93.855) to characterize lymphocyte activation gene 3 (LAG3) binding interactions with the T cell receptor/CD3 complex and LAG3 ligands that modulate immune response in the tumor microenvironment.

The research will elucidate the mechanism by which LAG3 interacts with the TCR/CD3 complex, evaluate the hierarchy of LAG3 ligand engagement in the tumor microenvironment, and assess the roles of multiple LAG3 ligands—including major histocompatibility complex class II, fibrinogen-like protein 1, L-selectin, and galectin-3—in modulating T cell function. The work addresses LAG3's role as an inhibitory receptor critical to T cell regulation and as a target for cancer immunotherapy. Performance of the work occurs in Pittsburgh, Pennsylvania, with an ultimate completion date of August 31, 2028. The assistance type is a project grant.

Abstract

T cells are crucial for defending against chronic infections and cancer, with dysregulation leading to autoimmunity. Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor (IR) that plays a critical role in regulating t cell tolerance, autoimmunity, and is a key target for cancer immunotherapy. LAG3 is upregulated upon t cell activation, inhibiting downstream t cell receptor (TCR) signaling and modulating effector functions. Several ligands bind LAG3 to mediate its inhibitory function. The canonical ligand, major histocompatibility complex class II (mhcii), binds to LAG3 with higher affinity than the coreceptor CD4. While peptide-mhcii (pmhcii) stabilizes LAG3 binding, its role in CD8+ t cells, which are restricted to mhci, is unclear. Additionally, fibrinogen-like protein 1 (FGL1), which is secreted by hepatocytes and expressed in various cancers, also binds LAG3, and its blockade can enhance anti-tumor immunity. Our laboratory has identified the TCR/CD3 complex as a ligand for LAG3. At the immune synapse, LAG3 associates with the TCR/CD3 complex independently of mhcii to inhibit TCR signaling. Other identified ligands, including lsectin and galectin-3, have unresolved mechanisms of LAG3 binding and functional implications. Although the binding sites for mhcii and FGL1 on LAG3 have been identified, the mechanism by which LAG3 interacts with the TCR/CD3 complex remains unknown. Additionally, the role of these ligands in the tumor microenvironment (TME) is not well understood. The goal of this research is to elucidate the interactions between LAG3 and the TCR/CD3 complex and to evaluate the hierarchy of LAG3 ligand engagement in the TME. The central hypothesis of this proposal is that the TCR/CD3 complex is the major ligand for LAG3 and plays a significant role in modulating LAG3 activity within the TME. To achieve the goals of this proposal, we will identify the binding site on LAG3 for the TCR/CD3 complex (aim 1) by evaluating whether the residues mediating binding to mhcii and FGL1 are distinct in TCR interaction. Additionally, we will investigate whether evolutionary conserved residues and solvent-accessible residues contribute to the LAG3-TCR interaction. To test this, we will mutate specific amino acid residues on LAG3 and assess its association to the TCR/CD3 complex using super-resolution microscopy and förster resonance energy transfer (fret). In aim 2, we will determine which ligands within the TME are essential for LAG3 function. This will be accomplished with in vivo tumor models using LAG3 mutants that are deficient in mhcii, FGL1, and TCR/CD3 binding. We will assess changes in anti-tumor immunity and competition within a murine melanoma model. These findings will define critical residues on LAG3 responsible for binding the TCR/CD3 and establish the functional hierarchy of LAG3 ligands within the TME. Additionally, this work may uncover previously unidentified LAG3 ligands. Ultimately, this work will provide mechanistic insights into LAG3 that could enhance immunotherapies or lead to novel treatments for inflammation and autoimmunity.