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Background <p>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 <i>Plasmodium</i> infection and the upstream molecular mechanisms regulating LAG3 expression remain incompletely elucidated.</p> Methods <p>We established a murine model using <i>Plasmodium yoelii</i> NSM (<i>P. yoelii</i> 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 <i>Lag3</i>.</p> Results <p><i>Plasmodium yoelii</i> 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 <i>Lag3</i> 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.</p> Conclusions <p>This study suggests that the LEF1–LAG3 axis is involved in modulating CD4⁺ T cells during <i>P. yoelii</i> 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 <i>Lag3</i> offers a promising avenue for fine-tuning anti-malarial immune responses.</p> Graphical Abstract <p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年5月26日

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  • Wenbo Peng,
  • Guikuan Liang,
  • Keyu Lu,
  • Feng Mo,
  • Xiongyu Xie,
  • Mingjie Chen,
  • Haiwen Yuan,
  • Lixin Luo,
  • Xingyue Wang,
  • Long Xu,
  • Haixia Wei,
  • Lu Li,
  • Shan Zhao,
  • Hongyan Xie,
  • Xingfei Pan,
  • Jun Huang

摘要

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Graphical Abstract