The role and mechanism of exosomal aminopeptidase n (APN/CD13)-mediated CTL cell dysfunction in aplastic anemia

Abstract Introduction Severe aplastic anemia (SAA) is a life-threatening bone marrow failure disorder, currently recognized to arise primarily from cytotoxic T lymphocyte (CTL)-mediated attacks on hematopoietic stem cells. APN/CD13 is expressed in diverse cell types/tissues and can be shed as soluble CD13 (sCD13). It participates in multiple physiological processes, including cell proliferation, differentiation, migration, and immune cell regulation, with elevated expression observed in various autoimmune diseases and malignancies. This study investigates the impact of aberrant CD13 expression on CTL function and its association with AA, while preliminarily exploring the therapeutic potential of CD13 inhibitor. Methods Using exosomes as an entry point, we employed iTRAQ technology to analyze exosomal protein profiles in plasma from SAA patients versus healthy controls (HCs). ELISA validated elevated CD13 concentrations in SAA plasma. Flow cytometry was used to analyze CD13 expression on different immune cells from SAA patients, MDS patients and HCs. In vitro functional assays (CCK-8 and flow cytometry) evaluated the effects of CD13 and its inhibitor bestatin on CTL proliferation and inflammatory cytokine (TNF-α/IFN-γ) secretion. Western blotting elucidated molecular mechanisms underlying CD13-mediated T-cell regulation. Results Seven upregulated proteins (NT5E, FTH1, GPRC5C, SAA2, ANPEP, GGT1, CAVIN2) in SAA patients were obtained through iTRAQ proteomic analysis. We confirmed significant upregulation of CD13 in SAA plasma. Evidently, both MDS and SAA patients exhibit higher overall CD13 expression compared to healthy controls. However, unlike in MDS patients where sCD13 is primarily derived from granulocytes, the elevated plasma sCD13 levels in SAA patients likely originate from monocytes. In vitro, high CD13 levels enhanced CTL proliferation and pro-inflammatory cytokine (TNF-α and IFN-γ) release, and that increasing CD13 concentrations led to a gradual elevation in the proportion of CD8+ T cells and a corresponding decrease in the CD4/CD8 T lymphocyte ratio. This dose-dependent trend mirrors the disease progression of AA, suggesting that elevated CD13 levels may promote the pathogenesis of AA. Mechanistically, highly expressed CD13 promoted ERK1/2 phosphorylation, modulating T-cell function via the MAPK signaling pathway. These effects were reversible with bestatin treatment. Conclusions APN/CD13 is overexpressed in SAA patients and may drive disease progression by augmenting CTL proliferation and inflammatory cytokine production. The CD13 inhibitor can counteract this effect, potentially offering therapeutic benefits for the disease. Our findings may contribute to the identification of novel potential biomarkers for SAA, and could provide valuable insights for developing therapeutic or personalized treatment strategies for SAA.