Abstract
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons in the brain and spinal cord. Despite the crucial role of aberrant immune responses in ALS pathogenesis, studies investigating immunological profiles in the cerebrospinal fluid (CSF) of patients with ALS have reported inconsistent findings. Herein, we explored the intrathecal adaptive immune response and features of circulating T cells between CSF and blood of patients with ALS using single-cell RNA and T-cell receptor (TCR) sequencing. This study comprised a total of 11 patients with apparently sporadic ALS and three controls with non-inflammatory diseases. We collected CSF from all participants, and for three patients with ALS, we additionally obtained paired samples of peripheral blood mononuclear cells (PBMCs). Utilizing droplet-based single-cell RNA and TCR sequencing, we analysed immunological profiles, gene expression characteristics and clonality. Furthermore, we examined T-cell characteristics in both PBMC and CSF samples, evaluating the shared T-cell clones across these compartments. In the CSF, patients with ALS exhibited a lower proportion of CD4(+) T cells (45.2 versus 61.2%, P = 0.005) and a higher proportion of CD8(+) GZMK (hi) effector memory T cells (TEMs) than controls (21.7 versus 16.8%, P = 0.060). Higher clonality was observed in CD8(+) TEMs in patients with ALS compared with controls. In addition, CSF macrophages of patients with ALS exhibited a significant increase in chemokines recruiting CD8(+) TEMs. Immunohistochemical analysis showed slightly higher proportions of T cells in the perivascular and parenchymal spaces in patients with ALS than in controls, and CD8(+) TEMs co-localized with neurons or astrocytes in the motor cortices of patients with ALS. Clonally expanded CD8(+) GZMK (hi) TEMs primarily comprised shared T-cell clones between CSF and PBMCs. Moreover, the shared CD8(+) TEMs of PBMCs exhibited gene expression profiles similar to CSF T cells. Patients with ALS showed an increase in proportion and clonality of CD8(+) GZMK (hi) TEMs and activated features of macrophages in CSF. The shared T-cell clone between CSF and blood was mainly composed of expanded CD8(+) GZMK (hi) TEMs. In conclusion, single-cell immune profiling provided novel insights into the pathogenesis of ALS, characterized by activated macrophages and clonally expanded CD8(+) T cells potentially communicating with the central nervous system and peripheral circulation.