Engineered α-Synuclein-specific nanobody CAR iTregs restrain neuroinflammation and proteinopathy in Parkinson’s disease mice

Parkinson’s disease (PD) is characterized by progressive DAergic neurodegeneration and the accumulation of aggregated α-Synuclein (αSyn), which drives chronic neuroinflammation through sustained activation of innate and adaptive immune responses. Regulatory T cells (Tregs) exert potent immunosuppressive functions and have shown neuroprotective effects in preclinical PD models; however, clinical translation of polyclonal Treg therapies has been limited by poor tissue specificity and insufficient therapeutic efficacy. To overcome these limitations, we engineered induced human Tregs (iTregs) expressing chimeric antigen receptors (CARs) directed against pathological αSyn aggregates. Among the CAR designs tested, only a nanobody-based construct incorporating NbSyn87 displayed selective antigen-dependent activation in response to αSyn preformed fibrils (PFFs). Intriguingly, despite the ability of the parental NbSyn87 nanobody to bind both monomeric and aggregated αSyn, incorporation into the CAR architecture conferred functional selectivity for aggregated conformers. This feature enabled discrimination between pathological extracellular aggregates and physiological monomeric αSyn, providing an important safety advantage. To evaluate therapeutic activity in vivo , we established an immunodeficient mouse model of synucleinopathy permissive to human cell engraftment. iTregs preferentially accumulated within αSyn-rich brain regions and, in the presence of astrocyte-derived human IL-2 with antigen-independent mechanism. Conversely, only CAR iTregs directed against αSyn significantly reduced microglial and astrocytic activation, decreased pro-inflammatory cytokine expression, and attenuated αSyn pathology. Collectively, these findings demonstrate that αSyn-specific CAR iTregs can selectively exert potent local immunomodulatory effects, establishing a promising antigen-specific cellular immunotherapy platform for PD and other synucleinopathies.