LAG-3 palmitoylation-inducing dysfunction of decidual CD4⁺T cells is associated with recurrent pregnancy loss

Recurrent pregnancy loss (RPL) profoundly impacts not only the physical health but also the psychological well-being of women. Despite its profound effects, the underlying pathophysiological mechanisms of RPL remain largely elusive, with few discernible warning signs. Lymphocyte activation gene-3 (LAG-3) is a crucial immune checkpoint that modulates immune responses during infection and tumor. In the present study, we examined the expression of LAG-3 on CD4⁺T cells during pregnancy via cytometry by time-of-flight and flow cytometry. Our findings revealed a higher frequency of LAG-3⁺ decidual CD4⁺T (dCD4⁺T) cells in response to trophoblasts during normal pregnancy. This specific LAG-3⁺ subset of dCD4⁺T cells was found to produce a greater number of anti-inflammatory cytokines. Notably, blocking LAG-3 was highly effective in inhibiting the production of anti-inflammatory cytokines, which is detrimental to the maintenance of pregnancy. A decrease in the number of LAG-3⁺dCD4⁺T cells was correlated with miscarriage. Interestingly, the RNA level of LAG-3 (data analyzed from the two published single-cell databases) remained stable in RPL. Palmitoylation might play a role in regulating LAG-3 expression during RPL, as the palmitoylation of LAG-3⁺dCD4⁺T cells was increased in RPL. Additionally, the general palmitoylation inhibitor 2-bromopalmitate was found to upregulate LAG-3 expression on dCD4⁺T cells both in vitro and in vivo. Collectively, these findings highlighted the significant roles of LAG-3 in regulating the function of dCD4⁺T cell and maintaining normal pregnancy. Furthermore, they suggested that lower LAG-3 expression on dCD4⁺T cells could serve as a potential biomarker for diagnosis of RPL.