The LEF1-LAG3 axis regulates CD4⁺ T cell function during Plasmodium yoelii NSM infection

CD4⁺ T cells are pivotal in coordinating anti-malarial immunity, while co-inhibitory receptors such as LAG3 critically regulate their function. However, the phenotype of LAG3⁺CD4⁺ T cells during Plasmodium infection and the upstream molecular mechanisms regulating LAG3 expression remain incompletely elucidated.

We established a murine model using Plasmodium yoelii NSM (P. yoelii NSM). A multifaceted approach, incorporating single-cell RNA sequencing (scRNA-seq), flow cytometry, magnetic bead-based cell sorting, real-time quantitative polymerase chain reaction (RT-qPCR), dual-luciferase reporter assays, and in vitro cultures with the Wnt agonist CHIR99021, was employed. We characterized splenic CD4⁺ T cell dynamics, the phenotypic and functional profiles of LAG3⁺CD4⁺ T cells, and the transcriptional regulatory relationship between lymphoid enhancer-binding factor 1 (LEF1) and Lag3.

Plasmodium yoelii NSM infection induced significant splenomegaly and remodeling of the splenic CD4⁺ T cell compartment, with increased absolute numbers of CD4⁺ T cells, upregulated activation markers (ICOS, CD69), downregulated naïve marker CD62L, and enhanced secretion of IL-10 and IFN-γ. Both scRNA-seq and flow cytometry confirmed that infection markedly upregulated LAG3 on CD4⁺ T cells. These LAG3⁺CD4⁺ T cells exhibited an activated phenotype, characterized by increased proliferative capacity (Ki67⁺), an increased proportion of the effector phenotype (CD44ʰⁱCD62Lˡᵒ), and concurrent upregulation of multiple co-inhibitory receptors (PD-1, TIM-3, TIGIT). Mechanistically, LEF1 expression was significantly downregulated in CD4⁺ T cells post infection. Dual-luciferase reporter assay demonstrated that LEF1 directly binds to the Lag3 promoter, acting as a transcriptional repressor. Furthermore, treatment with the Wnt agonist CHIR99021, which stabilizes the upstream signaling of LEF1, dose-dependently reduced the frequency of LAG3⁺CD4⁺ T cells.

This study suggests that the LEF1-LAG3 axis is involved in modulating CD4⁺ T cells during P. yoelii NSM infection. LAG3⁺CD4⁺ T cells exhibit an activated phenotype with regulatory potential, which may contribute to balancing anti-parasitic immunity and immunopathology. These findings suggest that modulating LEF1-mediated transcriptional repression of Lag3 offers a promising avenue for fine-tuning anti-malarial immune responses.