**3.2 Signaling pathway of LAG-3**

LAG-3 expression is induced by activation of either TCR or various cytokines particularly interleukin-12 (IL-12). On the other hand, its transcription is regulated by interplay of inducing and regulating elements including transcription factor binding sites. There have been several transcription factors, reported to have inductive effects to LAG-3 expression, such as thymocyte selection-associated high mobility group box protein (TOX), nuclear factor of activated T cells (NFAT) [33], and nuclear receptor subfamily 4 group A (NR4A) [34]. In contrast, T-box transcription factor (T-bet) is inversely correlated with LAG-3 and leads to cytotoxic T cell differentiation. Thereby, it is a critical transcription factor in regulating immune exhaustion. However, the correlation between T-bet and LAG-3 is bidirectional. Deletion of either T-bet or LAG-3 enhances the expression of the other which suggests that LAG either promotes T cell exhaustion or vice versa [34]. LAG-3 interferes TCR:CD3 complex on the T cell surface; hence, TCR signal transduction is perturbed. As a result, cell proliferation as well as CD3-induced cytokine production are terminated. LAG-3 and CD3 engagement in the immunological synapse down-modulates TCR signal transduction which results in inhibition of TCR:CD3 dependent intracellular calcium fluxes to ultimately halt T cell responses [35].
