Single-Cell Profiling of PBMCs Reveals an Immune Signature of irAEs in Anti-PD-1-Treated Acral Melanoma Patients

Qi BaoJie PanZhefang Wang^*

• Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China

The clinical efficacy of anti-PD-1 immunotherapy in melanoma is often limited by immune-related adverse events (irAEs), whose underlying mechanisms remain poorly defined, especially in the acral subtype. To identify the peripheral immune signatures of irAEs, we performed single-cell RNA sequencing on PBMCs from acral melanoma patients, comparing those who developed irAEs on anti-PD-1 therapy (AE) to treated patients without irAEs (NAE) and untreated controls (UNT). Our analysis revealed a profound reconfiguration of the CD8+ T cell compartment in AE patients. This was characterized by an expansion of cytotoxic CD8+ T cells, defined by high expression of GZMB, GNLY, and NKG7, and a concurrent contraction of a transitional CD8+ T cell population marked by GZMK. Consequently, the ratio of transitional to cytotoxic CD8+ T cells was decreased in the AE group. Pseudotime trajectory analysis confirmed that GZMK+ cells constitute an intermediate differentiation state between naïve and terminal cytotoxic phenotypes. Furthermore, AE patients exhibited an elevated proportion of proliferating T cells, and their T cells were enriched for gene pathways related to cell killing. Our findings propose a model where an imbalance in CD8+ T cell differentiation, favoring aggressive cytotoxic effectors over a putative buffering transitional population, underpins irAE pathogenesis in acral melanoma patients receiving anti-PD-1 therapy. The transitional-to-cytotoxic CD8+ T cell ratio emerges as an exploratory candidate biomarker for irAE risk, warranting validation in larger cohorts.