Indoleamine 2,3-dioxygenase 1 (IDO1) promotes immune effector cell-associated neurotoxicity syndrome (ICANS) after CAR-T therapy by disrupting brain endothelial cell integrity via KYNU–AA–Wnt/β-catenin pathway

Abstract

Abstract Background: Elevated serum tryptophan levels are observed in patients with immune effector cell-associated neurotoxicity syndrome (ICANS) following chimeric antigen receptor T-cell (CAR-T) therapy. Indoleamine 2,3-dioxygenase 1 (IDO1), the crucial rate-limiting enzyme in tryptophan catabolism, is implicated in neurodegeneration, inflammation, and tumorigenesis. Gene expression analysis of pre-treatment tumor biopsies showed that IDO1 levels were significantly higher in patients who developed severe ICANS (Grade >3) compared to those with mild toxicity (Grade 0–2) (p=0.002, ). However, the specific molecular mechanisms by which IDO1 contributes to ICANS pathogenesis remain unclear. Aims and Methods: This study aims to investigate the effect and underlying mechanisms of IDO1 in ICANS following CAR-T therapy. A syngeneic non-Hodgkin's lymphoma mouse model was established using CD19-targeted CAR-T cells. IDO1 expression and activity in brain tissue were assessed by liquid chromatography-mass spectrometry (LC-MS). Mice were treated with the IDO1 inhibitor 1-methyltryptophan (1-MT), and the integrity of the blood-brain barrier (BBB) and expression of tight junction proteins were subsequently analyzed. Cognitive and anxiety-like behaviors were assessed in mice during active phase. A monolayer blood-brain barrier (BBB) model was established using the human cerebral microvascular endothelial cell line (hCMEC/D3) in vitro. Cells were exposed to supernatant from co-cultures of CAR-T cells and Raji cells, and endothelial integrity was analyzed. The role of IDO1 was investigated using both the pharmacological inhibitor 1-MT and IDO1-specific shRNA. LC-MS was employed to profile IDO1-related metabolites. Results: IDO1 was significantly upregulated and activated in our ICANS mouse models, concomitant with increased BBB permeability and reduced tight junction protein expression. Behavioral studies revealed that mice receiving CD19 CAR-T cells displayed increased anxiety and cognitive deficits compared to mice receiving non-transduced T cells. The behavior deficits and BBB integration were improved after IDO1 inhibition. In vitro, co-culture supernatant significantly activated endothelial cell and upregulated IDO1 expression. Meanwhile, endothelial permeability increased accompanied by downregulation of tight junction proteins (ZO-1, occludin, claudin1), which correlated with suppression of Wnt/β-catenin signaling pathway. Both pharmacological and genetic IDO1 inhibition attenuated these effects, restoring barrier function and partially rescuing Wnt/β-catenin activity. Mechanistically, kynureninase (KYNU), a key downstream enzyme of IDO1, was upregulated in parallel with IDO1 activation and downregulated upon IDO1 inhibition, suggesting that its metabolite anthranilic acid (AA) may contribute to endothelial dysfunction. LC-MS analysis confirmed that anthranilic acid (AA) levels were elevated upon IDO1 activation and decreased following IDO1 inhibition. Furthermore, supplementation with exogenous AA not only attenuated the protective effects of IDO1 inhibition on endothelial barrier integrity, such as the restoration of tight junction protein expression, but also suppressed the reactivation of the Wnt/β-catenin signaling pathway, indicating a central role for the IDO1–KYNU–AA axis in modulating Wnt-dependent endothelial function. Conclusion: IDO1 promotes ICANS by impacting brain endothelial cell integrity through KYNU-AA-Wnt/β-catenin pathways. Targeting IDO1 may offer a therapeutic strategy to reduce neurotoxicity in CAR-T therapy.