Single-cell analysis reveals expanded CD8+ GZMKhigh T cells in CSF and shared peripheral clones in sporadic amyotrophic lateral sclerosis

Abstract

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 ALS patients have reported inconsistent findings. Herein, we explored the intrathecal adaptive immune response and features of circulating T cells between CSF and blood of ALS patients, 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 ALS patients, we additionally obtained paired samples of peripheral blood mononuclear cells (PBMCs). Utilizing droplet-based single-cell RNA and TCR sequencing, we analyzed 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, ALS patients exhibited a lower proportion of CD4+ T cells (45.2% vs. 61.2%, p = 0.005), and a higher proportion of CD8+ GZMKhi effector memory T cells (TEMs) than controls (21.7% vs. 16.8%, p = 0.060). Higher clonality was observed in CD8+ TEMs in ALS patients compared to controls. In addition, CSF macrophages of ALS patients 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 ALS patients than in controls, and CD8+ TEMs co-localized with neurons or astrocytes in the motor cortices of ALS patients. Clonally expanded CD8+ GZMKhi 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. ALS patients showed an increase in proportion and clonality of CD8+ GZMKhi TEMs and activated features of macrophages in CSF. The shared T cell clone between CSF and blood was mainly composed of expanded CD8+ GZMKhi 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 communicate between the central nervous system and peripheral circulation.