GVHD is a major complication of bone marrow transplantation (BMT) and results from donor T cells becoming activated and reacting to host antigens. Recently, lymphocyte activation gene-3 (LAG-3) has emerged as an important molecule, negatively regulating T cell activation and has been proposed to play an important role in CD4+CD25+Foxp3+ regulatory T cell (Treg) function. We investigated the functional in vivo role of LAG-3 in Treg and conventional T cells in murine GVHD with the hypothesis that LAG-3 engagement diminishes alloreactive T cell responses after BMT. Using murine models of acute GVHD in which allogeneic bone marrow cells are transplanted into lethally irradiated hosts, we and others have shown previously that donor Treg are able to suppress GVHD induced by donor allogeneic conventional T cells (Tcon). The role of LAG-3 in Treg function was evaluated both in vitro and in vivo by directly comparing Treg isolated from LAG-3−/− donor mice to Treg isolated from wild type donors (WT Treg). In vitro, in a mixed lymphocyte reaction assay, LAG-3−/− Treg efficiently suppressed the proliferation of alloreactive T cells in a manner similar to WT Treg. In vivo, a bioluminescent imaging assay (BLI) was utilized that allows for quantitative assessment of Tcon proliferation in addition to traditional metrics of GVHD severity including weight loss, survival and GVHD score. Both LAG-3−/− Treg and WT Treg were equally potent at suppressing Tcon proliferation as illustrated by BLI of luc+ T cells and demonstrated a significant increase in median survival time (MST) as compared to mice receiving Tcon only (35 days for Tcon vs. 58 and 68 days for WT and LAG-3−/− Treg, respectively, P=0.03), but there was no significant difference in MST between the groups receiving WT and LAG-3−/− Treg. Interestingly, when LAG-3−/− Tcon were used to induce GVHD in the absence of Treg, GVHD lethality was accelerated. Thus, all mice receiving LAG-3−/− Tcon showed decreased survival and significantly lower body weights than mice receiving WT Tcon (P=0.017). GVHD scores of LAG-3−/− Tcon recipients were also significantly higher than WT Tcon recipients at Day 20 post BMT (6.0 vs. 2.2, P=<0.0001). The addition of WT Treg induced only a modest yet statistically significant increase in median survival in mice receiving both LAG-3−/− Tcon and WT Treg as compared to mice receiving LAG-3−/− Tcon alone (45 days vs. 14.5 days, P=0.0075). In contrast, WT Treg more efficiently suppressed the proliferation of WT Tcon, increasing the MST to 70 days versus a MST of 26 days for mice receiving WT Tcon (P=0.0002). Re-isolation experiments using CFSE-labeled Tcon did not show differences in proliferation between WT and LAG-3−/− Tcon at five days following BMT. Since LAG-3 is upregulated as early as 2 days after T cell activation and gradually decreases over the next few days, is it possible that a difference in proliferation could be detected at an earlier timepoint thus explaining the difference in potency between the WT and LAG-3−/− Tcon. Together our results indicate, contrary to previous published results, that the absence of the LAG-3 molecule on Treg does not impair Treg function in our mouse model of acute GVHD. However, the absence of LAG-3 on Tcon induces a more severe GVHD suggesting that LAG-3 engagement on donor T cells diminishes alloreactive T cell response after BMT.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.