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  • Considering that loss of LAG expression


    Considering that loss of LAG-3 43 7 results in increased CD4+ T cell homeostatic expansion, and that the ability of a cell to proliferate is tightly coupled to its metabolic profile, we hypothesized that LAG-3 regulates naive CD4+ T cell metabolism. Our results indicate that, indeed, Lag3-deficient naive T cells exhibited enhanced oxidative and glycolytic metabolisms, with this being attributed to increased mitochondrial content. Also, this heightened metabolic profile supported greater homeostatic proliferation in vivo. Furthermore, Lag3-deficient T cells were more resistant to IL-7 deprivation and exhibited greater activation potential, in part due to increased signaling via STAT5. These findings substantiate that LAG-3 serves as a major metabolic regulator, charged with protecting against hyperproliferation and maintaining naive T cell quiescence, thereby ensuring controlled CD4+ T cell activation.
    Discussion Our results here indicate that expression of LAG-3 on naive CD4+ T cells negatively regulates mitochondrial biogenesis and metabolism as a means of controlling homeostatic expansion and quiescence. Further, we have demonstrated that LAG-3 is a key checkpoint in regulating downstream signaling of both the STAT5 and Akt pathways. Although LAG-3 expression on naive CD4+ T cells is known to be rather low as compared with activated or exhausted T cells, it is clear that the low expression levels are sufficient for mediating phenotypic alterations in these T cells (Durham et al., 2014, Workman and Vignali, 2005). The enhancement of mitochondrial metabolism and mass due to the lack of LAG-3 expression or LAG-3 blockade is consistent with the recent study published by Scharping et al. (2016). Their results indicated that increased expression of inhibitory receptors on exhausted CD8+ T cells correlated with reduced mitochondrial mass. Together, our work further supports a link between these receptors and mitochondrial maintenance. To delineate a direct link between LAG-3 signaling and T cell metabolism, we utilized a blocking antibody in vivo that is known to specifically interrupt LAG-3 downstream signaling (Workman et al., 2002). The fact that antibody blockade of LAG-3 on WT CD4+ T cells during adoptive transfer experiments yielded similar proliferation and metabolic responses as Lag3.OT-II cells (Figure 3) suggests that regulation is due to LAG-3 ligation and signaling, most likely through MHC class II interactions. Previous studies have confirmed LAG-3-mediated T cell inhibition (Huard et al., 1996, Workman et al., 2002, Workman and Vignali, 2003), yet a defined signaling pathway via LAG-3’s cytoplasmic tail remains elusive. Our data indicated that Lag3-deficient OT-II T cells were less dependent upon IL-7 due to elevated STAT5 and Akt activation (Figures 4 and 5). Another inhibitory receptor, Programmed Cell Death-1 (PD-1), regulates T cell metabolism via stabilization of the phosphatase PTEN (Patsoukis et al., 2013), thereby inhibiting Akt activation. Because some signaling redundancy exists between inhibitory receptors, it was logical that Lag3-deficient OT-II T cells exhibited a reduction in PTEN expression compared with WT OT-II T cells (Figure 4J). Lag3-deficient OT-II T cells also demonstrated a hyperproliferative phenotype (Figures 3B and 3C) and bypassed the requirement for IL-7 to promote proliferation and survival (Figures 4 and 5), analogous to what has been reported in Pten-deficient T cells (Newton and Turka, 2012, Suzuki et al., 2001). Together, this would suggest a potential link between LAG-3 and PTEN, and that Pten T cells might also display undisrupted mitochondrial oxidation during IL-7 deprivation, yet definitive studies are required. Naive Lag3 CD4+ T cells demonstrated greater glycolytic capacity and effector function upon activation, as compared with their WT counterparts (Figure 6). Likewise, it has been reported that memory T cells exhibit enhanced activation upon secondary challenge compared with naive T cells. This characteristic of memory T cells is supported by their increased mitochondrial mass and SRC (van der Windt et al., 2012, van der Windt et al., 2013). Therefore, we propose that the metabolic advantage exhibited by Lag3 CD4+ T cells contributes to their enhanced activation and glycolytic potential similarly. Here we have also shown that naive CD4+ T cells utilize their SRC to support homeostatic expansion in vivo (Figures 3G and 5D), thereby revealing an additional role for SRC in T cells.