Abstract Body (Do not enter title and authors here): Background:
Heart failure (HF) is characterized by myocardial fibrosis and impaired cardiac function. The transverse aortic constriction (TAC) model in mice replicates pressure overload-induced fibrosis. Immune-mediated inflammation, particularly through T lymphocytes and macrophages, plays a pivotal role in fibrosis progression.A critical limitation of in vitro-induced Treg (iTreg) is their in vivo instability, often characterized by the loss of Foxp3 expression and reduced immunosuppressive function. To overcome this challenge, CD28-deprived iTreg have been developed, demonstrating epigenetic stability and sustained Foxp3 expression. This study aims to evaluate the therapeutic potential of intravenously administered CD28-deprived Treg in a murine TAC model of HF.

Method:
CD28-deprived Treg were generated from conventional T cell (Tconv) under CD28-blocking conditions. Male BALB/c mice underwent TAC surgery to induce HF and were divided into three groups: Control, Tconv, and Treg. One week after surgery, intravenous administration of iTreg or Tconv was performed. Cardiac function was assessed weekly using transthoracic echocardiography. Myocardial fibrosis was evaluated via histological analysis using Picrosirius Red staining. Additionally, RNA sequencing of left ventricular tissue was conducted to investigate gene expression changes related to fibrosis and inflammation.

Results:
The Treg group, which received intravenously administered CD28-deprived iTreg, exhibited significantly preserved cardiac function compared to the control group, as measured by serial echocardiography. Histological analysis demonstrated a marked reduction in myocardial fibrosis in the Treg group. RNA sequencing revealed that the Treg group significantly downregulated fibrosis-related pathways, including TNF-α/NF-κB and TGF-β signaling. Moreover, iTreg administration promoted the polarization of macrophages from a pro-inflammatory (M1) to an anti-inflammatory (M2) phenotype, contributing to fibrosis suppression.

Conclusion:
Systemic administration of CD28-deprived Treg effectively attenuates cardiac dysfunction and fibrosis in a pressure overload-induced HF model. This therapeutic effect is mediated through suppression of pro-inflammatory pathways and regulation of macrophage polarization. These findings support the potential of Treg therapy as a novel strategy for the treatment of HF.