LAG-3 Negatively Regulates T Cell Functions

LAG-3 Interacts with Multiple Ligands and Inhibits T cell Responses

Lymphocyte Activation Gene 3 (LAG-3), also known as CD223, is a type I transmembrane protein that belongs to the immunoglobulin superfamily (IgSF). It is expressed as a dimer or as an oligomer on the surface of activated CD4+ and CD8+ T cells, regulatory T cells, Tr1 cells, natural killer cells, and plasmacytoid dendritic cells (pDCs).1, 2 Similar to PD-1 and CTLA-4, LAG-3 is also up-regulated on exhausted T cells in cancer.2,3 The LAG-3 protein is structurally homologous to CD4 in that it has four extracellular Ig-like domains, but it also contains an extra loop on the membrane-distal Ig-like domain that is not present in CD4. This extra loop allows the LAG-3 protein to bind to MHC class II molecules expressed on antigen-presenting cells (APCs) or tumor cells with significantly higher affinity than CD4, and negatively regulate T cell receptor (TCR) signaling.4, 5

In addition to MHC class II molecules, Galectin-3, LSECtin, and Fibrinogen-like protein 1 (FGL1) are also LAG-3 ligands.2, 6-8 Significantly, all three of these proteins are expressed on various tumor cell types and inhibit T cell responses, making it unclear which ligands are primarily responsible for the immunosuppressive functions of LAG-3.6, 8 Although the mechanisms by which LAG-3 signals are not well understood, LAG-3 has been shown to negatively regulate T cell activation, proliferation, and cytokine production, inhibit pDC activation, and enhance the suppressive activity of regulatory T cells.9-15 LAG-3 has also recently been suggested to inhibit cytokine secretion by mature natural killer cells.16 On regulatory T cells, LAG-3 was found to engage MHC class II on dendritic cells (DCs) and inhibit DC maturation and their immunostimulatory capacity .17 Conversely, a soluble variant of LAG- 3 was shown to bind to MHC class II molecules on immature DCs and induce their maturation, leading to enhanced CD8+ T cell antigen cross-presentation.18, 19

Similar to PD-1, LAG-3 is frequently up-regulated on tumor-infiltrating lymphocytes (TILs) and while blockade of LAG-3 alone was found to be only weakly effective in reducing tumor growth in some mouse tumor models, blockade of both LAG-3 and PD-1 synergistically enhanced anti-tumor immunity and survival, when compared with anti-PD-1 alone.20, 21 In many of these mouse tumor models, the improvement in the anti-tumor immune response was attributed to an increase in the number of tumor-infiltrating CD8+, or CD8+ and CD4+ T cells, and an accompanying increase in IFN-gamma production that was observed following anti-LAG-3/anti-PD-1 treatment. As a result, researchers are now evaluating an antagonistic anti-LAG-3 antibody either alone, or in combination with anti-PD-1, in clinical trials.22, 23 Additionally, due to the observation that soluble LAG-3 has immune-stimulatory effects, a soluble recombinant form of LAG-3, known as IMP321, is also being evaluated in phase I testing in combination with anti-PD-1.

Bio-Techne offers R&D SystemsTM bioactive recombinant proteins for LAG-3 and its ligands, along with fluorochrome-conjugated antibodies for detecting LAG-3, Galectin-3, and LSECtin to further our understanding of the immunomodulatory effects of LAG-3.

LAG-3 and LAG-3 Ligands - Products by Molecule

Galectin-3 Galectin-3C Hepassocin/FGL1 LAG-3 LSECtin/CLEC4G

LAG-3 Binds to MHC II, Galectin-3, FGL1, and LSECtin and Suppresses T Cell Activity

T cell-expressed LAG-3 binds to multiple ligands, inhibits T cell functions, and enhances the suppressive activity of Tregs.

LAG-3 is an immunosuppressive receptor that inhibits T cell activity and promotes the suppressive activity of regulatory T cells. LAG-3 binds to multiple ligands including MHC class II, Galectin-3, LSECtin, and Fibrinogen-like protein 1 (FGL1), which have all been reported to have T cell inhibitory effects. Similar to PD-1, TIM-3, TIGIT, and BTLA, LAG-3 is also up-regulated on exhausted T cells and natural killer cells in cancer and is thought to contribute to their dysfunction (left). On regulatory T cells, LAG-3 expression enhances their suppressive function and can inhibit dendritic cell maturation and immunostimulatory capacity through its interaction with MHC class II (right).

Analysis of the Binding Activity of R&D Systems Recombinant Human LAG-3 and Hepassocin/FGL1 Proteins

Analysis of the binding response between R&D Systems Recombinant Human LAG-3 and Recombinant Human FGL-1 proteins.

LAG-3 Binds to Hepassocin/FGL-1. Recombinant Human LAG-3 Fc Chimera (R&D Systems, Catalog # 2319-L3) was coated on a plate at 1 ug/mL and the indicated concentrations of Recombinant Human Hepassocin/FGL1 His tag (R&D Systems, Catalog # 10285-HE) were added. The concentration of Recombinant Human Hepassocin/FGL1 that produces 50% of the optimal binding response is 0.05-0.4 ug/mL.

Assessment of the Bioactivity of R&D Systems Recombinant Cynomolgus Monkey LAG-3 Protein

Analysis of the effect of R&D Systems Cynomolgus Monkey LAG-3 protein on TNF-alpha secretion by immature dendritic cells.

LAG-3 Induces TNF-alpha Secretion by Mouse Immature Dendritic Cells. JAWSII mouse immature dendritic cells were treated with the indicated concentrations of Recombinant Cynomolgus Monkey LAG-3 (R&D Systems, Catalog # 9992-L3). TNF-alpha secretion was measured in cell culture supernatants using the Mouse TNF-alpha Quantikine™ ELISA Kit (R&D Systems, Catalog # MTA00B). The ED50for this effect is 0.15-0.9 ug/mL in the presence of a cross-linking antibody, Mouse Anti-His Tag Monoclonal Antibody (R&D Systems, Catalog # MAB050R).

References

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  11. Baixeras, E. et al. (1992) Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J. Exp, Med. 176:327. PMID: 1380059.

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  16. Narayanan, S. et al. (2020) LAG-3 is a central regulator of NK cell cytokine production.

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