Table 1.

Unknowns of LAG3 Biology.

	Description of knowledge gap
Expression	• What are LAG3 expression levels on innate T cells, B cells and pDCs during autoimmunity, cancer or infectious disease? • What regulates LAG3 endocytosis? • How does LAG3 RNA level correspond to surface expression?  o Intracellular stores, endocytosis, and cleavage from cell surface complicate inferences of surface expression from RNA expression. • Does endocytosed LAG3 maintain function? • What % of intracellular LAG3 is de novo vs endocytosed?
Function	• What is the role of LAG3 on NK cells, B cells, pDCs and neurons? • Can LAG3 inhibit signaling through BCR, cytokine receptors, TLR?  o Can blockade of LAG3 reverse these effects? • What is the impact of LAG3 blockade on innate T cells? • How does interaction with each of the five LAG3 ligands impact LAG3 activity?  o What is the relative strength of each receptor?  o Does this differ based on context? • Is sLAG3 non-functional? • Do LSECtin and Gal-3 bind to glycosylated regions of LAG3? • Does endocytosed LAG3 still function if bound to ligand in endosome?
Mechanism	• Is dimerization necessary to bind to all ligands? • Is dimerization necessary for LAG3 function? • Is LAG3 colocalization with the TCR necessary for LAG3 function?  o If so, how do non-MHCII ligands facilitate this? • How does LAG3 inhibit activation following other stimuli (e.g., cytokines)?  o Does it act through a similar pathway as for TCR stimulation? • What protein(s) bind to LAG3 intracellular domain to facilitate its function?  o LAP is currently the only known interacting protein, but it cannot explain LAG3 function. Since ligand binding seems necessary for LAG3 function, it may be needed during screening for proteins that participate in LAG3 function through direct interaction. • What cytoplasmic motif(s) are involved in LAG3 function and what is their role?  o Are different motifs involved in different aspects of LAG3 function? • If LAG3 inhibits activity of some kinase, as is currently suspected, what kinase does it inhibit and how?  o Does LAG3 inhibit general activation by acting early in the pathway or does it act stronger on a particular pathway?