Although clinical trials have demonstrated both the efficacy and safety of dupilumab, its impact on dendritic cells (DCs) remains unclear. TIM-3 has emerged as crucial regulators of immune responses in various inflammatory diseases. Understanding the interplay between TIM-3 and the type 2 inflammatory response in atopic dermatitis (AD) could provide valuable insights into the mechanisms underlying the efficacy of dupilumab.To investigate whether TIM-3 induce immune modulation of DCs and determine its role in dupilumab therapy.Using single-cell technology to screen for the expression landscape of immune checkpoints. Employing αIL-4/IL-13 monoclonal antibody treatment in mice to simulate the therapeutic effects of dupilumab treatment. Investigating the function of the immune checkpoint Tim-3 with αTim-3 monoclonal antibody. Finally, peripheral blood mononuclear cells (PBMCs) were collected from AD patients 16 weeks before and after dupilumab treatment (n=24) to validate the findings observed in the mouse experiments. Additionally, AD skin lesions were collected before treatment (n=8) and after treatment (n=5) for further validation. To verify the therapeutic effects of TGF-β1 and Galectin-9, we also collected PBMCs from untreated AD patients (n=21) and conducted in vitro stimulation experiments.Dupilumab upregulates the immune checkpoint HAVCR2 (encoding TIM-3) by inducing the secretion of TGF-β1 in myeloid DCs. Interestingly, TIM-3 also promotes the secretion of TGF-β1, thus forming a positive feedback loop. This process was found to promote myeloid DC apoptosis, contributing to the observed decrease in myeloid DC numbers and potentially enhancing the therapeutic effects of dupilumab. The analyses of PBMCs and skin lesions from AD patients before and after dupilumab treatment showed dupilumab significantly elevated the levels of TIM-3, which correlated with a reduced proportion of myeloid DCs and suppressed myeloid DC function. In vitro stimulation of PBMCs with TGF-β1 or Galectin-9 similarly revealed their ability to inhibit myeloid DCs function in AD, suggesting potential therapeutic effects.Dupilumab improves the symptoms of AD by inducing the expression of TIM-3 to suppress myeloid DCs function, treatment with the TGF-β1 or Galectin-9 enhanced these effects, demonstrating the therapeutic potential of targeting the TGF-β1/ Galectin-9-TIM-3 axis in AD.Copyright © 2025. Published by Elsevier Inc.