Introduction: Liver transplantation is the most effective treatment for patients with end-stage liver disease. However, long-term dependence on immunosuppressive agents significantly increases the risk of infections, metabolic disorders, and malignancies, thereby compromising long-term outcomes. In recent years, clinical attempts have been made to minimize or even withdraw immunosuppressive therapy to induce immune tolerance. Nonetheless, the underlying mechanisms of immune tolerance remain poorly understood, hindering its clinical translation and individualized evaluation.

The establishment of immune tolerance is closely associated with the dynamic regulation of the immune microenvironment, particularly involving changes in T cell subsets and innate immune cells. Previous studies have suggested that immune-tolerant recipients may exhibit distinct patterns of immune cell remodeling. However, comprehensive profiling using high-dimensional technologies such as mass cytometry is still lacking.

In this study, we employed mass cytometry to analyze peripheral blood immune cell profiles from healthy individuals, immunosuppressed liver transplant recipients, and recipients who spontaneously discontinued immunosuppressants. Our aim is to identify the peripheral immune signatures associated with post-transplant immune tolerance and to uncover potential regulatory mechanisms. These findings may provide theoretical insights and candidate biomarkers to support the clinical implementation of immunosuppressant minimization or withdrawal strategies.

Methods: Peripheral blood samples were collected from six healthy controls, six immunosuppressed liver transplant recipients, and four recipients who had spontaneously discontinued immunosuppressants. PBMCs were isolated and analyzed using mass cytometry (CyTOF) with a panel of metal-conjugated antibodies targeting key immune cell markers.

Results: Mass cytometry revealed distinct immune profiles among the three groups. Compared with immunosuppressed recipients, drug-free tolerant recipients showed a significant increase in non-exhausted CD4⁺+FOXP3⁻ and CD8⁺+FOXP3⁻ T cells, along with elevated levels of activated macrophages and neutrophils. Despite an overall activated immune status, tolerant recipients also exhibited an increased proportion of CD4⁺+FOXP3⁺ and CD8⁺+FOXP3⁺ regulatory T cells (Tregs), indicating a potential compensatory immune-regulatory balance.

Conclusion: Our study demonstrates that spontaneous immune tolerance after liver transplantation is associated with a distinct peripheral immune signature characterized by both immune activation and enhanced regulatory responses. The coexistence of effector and regulatory immune components suggests a balanced immune remodeling process in tolerant recipients. These findings provide insights into the mechanisms of transplant tolerance and may facilitate the identification of biomarkers for safe immunosuppressant withdrawal.