LAG3-MHC-II binding recruits LAG3 to the immune synapse, suppressing T-cell activation through induced formation of condensates between the LAG3 intracellular domain and that of the CD3ε subunit. The BiTS molecule designed by Du et al. mimics the effect of MHC-II binding by tethering LAG3 to the TCR. Over the past decade, immune checkpoint antagonists have gained prominence for their remarkable efficacy in the treatment of cancer. More recently, induced activation of inhibitory checkpoint receptors has emerged as a promising strategy for combating autoimmunity. A new study from Du et al. describes the engineering of Lymphocyte activation gene-3 (LAG3) agonists that effectively treat multiple autoimmune diseases in mice.1 LAG3 is an immune checkpoint protein that binds MHC class II (MHC-II) and suppresses effector T cells. The authors showed that MHC-II engagement functions by localizing LAG3 to the immune synapse, where it inhibits T-cell receptor (TCR) activation. This mechanism inspired the design of a bispecific T-cell "silencer" (BiTS) molecule that bypasses the need for MHC-II binding by physically tethering LAG3 to the TCR (Figure 1). T-cell activation is a complex process that involves TCR recognition of peptide–MHC complexes, as well as coreceptor (e.g., CD4, CD28) interactions between T cells and antigen-presenting cells. Thus far, the requirement for MHC-II in LAG3 function has been controversial since LAG3 has many potential ligands, including the secreted protein Fibrinogen-like 1 (FGL1) and lectins, Galectin-3 and LSECtin. The present study clearly demonstrates that MHC-II binding drives colocalization of LAG3 with the TCR/CD3 complex. This proximity facilitates condensate formation between the intracellular domains of LAG3 and CD3ε, which disrupts CD3ε interactions with the activating kinase Lck (Figure 1). LAG3 suppression was also shown to be independent of CD4, suggesting that competition for CD4-MHC-II binding is not the primary mechanism for LAG3-mediated immunosuppression. This CD4-independent effect was surprising in light of recent crystal structures showing that LAG3 occludes the CD4 binding site on MHC-II.2, 3 Collectively, these data indicate that LAG3 is capable of suppressing T-cell activation in the absence of CD4 despite being an effective CD4 competitor. One perplexing aspect of LAG3 biology is that LAG3 suppresses both CD4+ and CD8+ T cells, despite the latter not typically engaging MHC-II. This was previously attributed to a direct physical association between LAG3 and the TCR.4 However, the authors' data seem to refute these claims by demonstrating that LAG3 must be recruited to the immune synapse, either naturally via LAG3 engagement of TCR-bound peptide–MHC-II, or artificially through the administration of BiTS molecules. This discrepancy is partially explained by another recent report showing that MHC-II binding promotes ubiquitination of a lysine in the LAG3 intracellular domain, which dissociates the intracellular domain from the membrane to initiate suppressive signaling.5 The ubiquitination study proposed an integrated model in which LAG3 has a baseline suppressive effect (they refer to this as "tonic signaling") in the absence of ligand, and that MHC-II binding fully unleashes its inhibitory activity. In light of these findings, it will be interesting to see whether BiTS treatment promotes LAG3 ubiquitination similarly to MHC-II binding. The BiTS molecule consists of tandem single-chain antibody variable fragments (scFvs) targeting the TCR beta chain and the ligand-binding domain 1 (D1) of LAG3 (Figure 1). Both scFvs are linked to an IgG1 Fc domain that was mutated to attenuate Fc effector function. Through this design, the authors were able to convert formerly inhibitory LAG3 antibodies into "agonists" that potentiate LAG3-mediated T-cell suppression. The agonist effect was first demonstrated in vitro, where the addition of BiTS potently decreased IL-2 production by T cells recognizing model antigens. The efficacy was next demonstrated in the rat insulin promoter-ovalbumin (RIP-OVA) mouse model of autoimmune diabetes. In this context, administration of BiTS, but not a LAG3-binding-deficient mutant BiTS variant, conferred significant protection against diabetes development. BiTS treatment also reduced liver damage in a mouse model of hepatitis driven by the infiltration of inflammatory CD8+ memory T cells. As the prior two models are driven primarily by CD8+ T cells, the authors tested whether BiTS could treat experimental autoimmune encephalitis (EAE), a multiple sclerosis model driven by CD4+ T cells. Administration of BiTS reduced the clinical score in the EAE model, suggesting that LAG3 agonism is effective for suppressing autoimmune disease mediated by both CD4+ and CD8+ T cells. Although the authors' bispecific BiTS design is novel, another monoclonal antibody, IMP-761, has been shown to exhibit agonist function by targeting only D1 of LAG3.6 This antibody enhances LAG3 suppressive activity in vitro and cured 50% of macaques of delayed-type hypersensitivity (DTH)-associated erythema in a primate model of psoriasis inflammation.7 Comparing the molecular mechanisms of BiTS versus IMP-761 may provide new insights into LAG3 function, as it is currently unclear how IMP-761 activates LAG3 while nearly all other antibodies targeting D1 are inhibitory. More important than comparing agonist mechanisms will be to determine how LAG3 agonism compares to that of other inhibitory checkpoints. PD-1 agonist antibodies, for example, have been shown to have strong anti-inflammatory effects in mouse models of graft-versus-host disease and colitis, and agonists of additional checkpoint receptors are under development for the treatment of various autoimmune diseases.8, 9 Recent literature has also shown that LAG3 and PD1 inhibitors have different modes of action in vivo, with the former enhancing cytokine release and cytotoxic function, and the latter promoting T-cell expansion and proliferation.10 In the future, it will be critical to test whether agonism of different checkpoints has similarly distinct outcomes, and whether these outcomes can be leveraged for maximum efficacy in the treatment of autoimmune disease. V.C.L. and Q.M. are supported by NIH grant (National Cancer Institute) R01CA299899. Qianqian Ming: Writing – original draft. Vincent C Luca: Writing – original draft. V.C.L. is a consultant for Curie.Bio, Remunix, and Regeneron. Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
Ming et al. (Sun,) studied this question.