MABF846 Sigma-AldrichAnti-LAG3 Antibody, clone C9B7W (Azide Free)

Inhibitory receptors on immune cells are pivotal regulators of immune escape in cancer. Among these inhibitory receptors, CTLA-4 (targeted clinically by ipilimumab) serves as a dominant off-switch while other receptors such as PD-1 and LAG-3 seem to serve more subtle rheostat functions. However, the extent of synergy and cooperative interactions between inhibitory pathways in cancer remain largely unexplored. Here, we reveal extensive coexpression of PD-1 and LAG-3 on tumor-infiltrating CD4(+) and CD8(+) T cells in three distinct transplantable tumors. Dual anti-LAG-3/anti-PD-1 antibody treatment cured most mice of established tumors that were largely resistant to single antibody treatment. Despite minimal immunopathologic sequelae in PD-1 and LAG-3 single knockout mice, dual knockout mice abrogated self-tolerance with resultant autoimmune infiltrates in multiple organs, leading to eventual lethality. However, Lag3(-/-)Pdcd1(-/-) mice showed markedly increased survival from and clearance of multiple transplantable tumors. Together, these results define a strong synergy between the PD-1 and LAG-3 inhibitory pathways in tolerance to both self and tumor antigens. In addition, they argue strongly that dual blockade of these molecules represents a promising combinatorial strategy for cancer.

Lymphocyte activation gene-3 (LAG-3) is a cell-surface molecule with diverse biologic effects on T cell function. We recently showed that LAG-3 signaling is important in CD4+ regulatory T cell suppression of autoimmune responses. Here, we demonstrate that LAG-3 maintains tolerance to self and tumor antigens via direct effects on CD8+ T cells using 2 murine systems. Naive CD8+ T cells express low levels of LAG-3, and expression increases upon antigen stimulation. Our data show increased levels of LAG-3 protein on antigen-specific CD8+ T cells within antigen-expressing organs or tumors. In vivo antibody blockade of LAG-3 or genetic ablation of the Lag-3 gene resulted in increased accumulation and effector function of antigen-specific CD8+ T cells within organs and tumors that express their cognate antigen. Most notably, combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8+ T cells in the tumor and disruption of the tumor parenchyma. A major component of this effect was CD4 independent and required LAG-3 expression by CD8+ T cells. Taken together, these data demonstrate a direct role for LAG-3 on CD8+ T cells and suggest that LAG-3 blockade may be a potential cancer treatment.

Regulatory T cells (Tregs) limit autoimmunity but also attenuate the magnitude of antipathogen and antitumor immunity. Understanding the mechanism of Treg function and therapeutic manipulation of Tregs in vivo requires identification of Treg-selective receptors. A comparative analysis of gene expression arrays from antigen-specific CD4(+) T cells differentiating to either an effector/memory or a regulatory phenotype revealed Treg-selective expression of LAG-3, a CD4-related molecule that binds MHC class II. Antibodies to LAG-3 inhibit suppression by induced Tregs both in vitro and in vivo. Natural CD4(+)CD25(+) Tregs express LAG-3 upon activation, which is significantly enhanced in the presence of effector cells, whereas CD4(+)CD25(+) Tregs from LAG-3(-/-) mice exhibit reduced regulatory activity. Lastly, ectopic expression of LAG-3 on CD4(+) T cells significantly reduces their proliferative capacity and confers on them suppressor activity toward effector T cells. We propose that LAG-3 marks regulatory T cell populations and contributes to their suppressor activity.

The lymphocyte activation gene-3 (LAG-3, CD223) is a CD4-related, activation-induced cell surface molecule that binds to MHC class II with high affinity. The function of murine LAG-3 on T cells is unclear. Here, we show that V beta 7/8(+)LAG-3(-/-) T cells expand poorly following staphylococcal enterotoxin B (SEB) stimulation in vitro. LAG-3(-/-) T cells proliferate at a normal rate, but exhibit increased cell death. Similar observations were made with LAG-3(-/-)CD4(+)OT-II TCR transgenic T cells following peptide stimulation. Despite reduced T cell expansion and increased cell death, LAG-3(-/-)OT-II(+) T cells secrete more IL-2 and IFN-gamma following stimulation. Antigen-driven expansion of LAG-3(-/-) T cells was restored by constitutive expression of LAG-3 via retroviral-mediated stem cell gene transfer. We further show that LAG-3 function is mediated via its cytoplasmic domain, for which a conserved 'KIEELE' motif is essential. Our data support a role for LAG-3 in regulating the expansion of activated T cells.

CD223 (LAG-3) is an activation-induced cell surface molecule, structurally similar to the T cell coreceptor CD4, that binds MHC class II molecules with high affinity. Little is known about the expression and function of murine CD223. Here, we show that mRNA expression is restricted to the thymic medulla, splenic red pulp and sparse cells in the adult brain cortex. In contrast, surprisingly high expression was seen in defined tracts at the base of the cerebellum and in the choroid plexus of day 7 postnatal brain. mCD223:Ig, but not CD4:Ig, fusion proteins stained cells expressing MHC class II molecules. Analysis of mCD223 cell surface expression was performed with a new monoclonal antibody (mAb) that recognizes an epitope in the D2 domain. Although it blocked mCD223 function in vitro, it did not block binding of mCD223 to MHC class II molecules. While very few TCRalpha beta T cells in the spleen and thymus of naive mice express surface mCD223 (<3 %), approximately 18 % TCR gamma delta T cells and approximately 10 % NK cells are positive. This small population of TCRalpha beta T cells are cycling memory T cells (BrdU(+), CD44(hi), CD62L(lo)). In contrast, all T cells express mCD223 2-3 days post activation. This study and the anti-CD223 mAb should greatly assist in the elucidation of CD223 function.