LAG3 Gene Therapy Attenuates Autoimmune Uveitis by Regulating the PI3K/Akt Signaling Pathway

Purpose: Lymphocyte-activation gene 3 (LAG3) is a critical immune checkpoint molecule, yet its role and mechanisms in autoimmune uveitis remain inadequately understood. This study sought to elucidate the expression dynamics, therapeutic potential, and molecular mechanisms of LAG3 in the context of experimental autoimmune uveitis (EAU). Methods: The EAU model was established in C57BL/6J mice through subcutaneous administration of interphotoreceptor retinoid binding protein (IRBP) peptide emulsified in complete Freund's adjuvant and intraperitoneal injection of pertussis toxin. Alterations in LAG3 expression throughout disease progression were monitored. Adeno-associated virus serotype 2 (AAV2)-LAG3 was administered via intravitreal injection. Disease severity and blood-retinal barrier (BRB) integrity were evaluated using clinical scoring, histopathological analysis, Evans blue leakage assay, and transmission electron microscopy. Quantitative reverse-transcription PCR, western blot analysis, and RNA sequencing (RNA-seq) were utilized to examine associated molecular expression and signaling pathways. Functional rescue experiments employing a phosphoinositide 3-kinase (PI3K) activator were conducted to further explore the underlying mechanism. Results: The expression of retinal LAG3 was downregulated during the peak inflammatory phase of EAU. Administration of AAV2-LAG3 significantly mitigated both clinical and pathological manifestations of EAU, re-established the Th17/Treg cell balance, and preserved the integrity of the BRB. Analyses via RNA-seq and western blotting suggested that these protective effects were associated with modulation of PI3K/Akt signaling pathway activation. Notably, the protective effects conferred by LAG3 were largely attenuated by application of the PI3K activator 740Y-P. Conclusions: This study elucidated the downregulation of LAG3 expression in EAU and suggests that AAV2-LAG3 attenuates disease, likely by regulating the PI3K/Akt pathway and restoring Th17/Treg cell balance and BRB integrity.