Single-cell RNA sequencing highlights the role of distinct natural killer subsets in amyotrophic lateral sclerosis

Background Neuroinflammation plays a major role in amyotrophic lateral sclerosis (ALS), and cumulative evidence suggests that systemic inflammation and the infiltration of immune cells into the brain contribute to this process. However, no study has investigated the role of peripheral blood immune cells in ALS pathophysiology using single-cell RNA sequencing (scRNAseq). Methods We aimed to characterize immune cells from blood and identify ALS-related immune alterations at single-cell resolution. For this purpose, peripheral blood mononuclear cells (PBMC) were isolated from 14 ALS patients and 14 cognitively unimpaired healthy individuals (HC), matched by age and gender, and cryopreserved until library preparation and scRNAseq. We analyzed differences in the proportions of PBMC, gene expression, and cell-cell communication patterns in patients with ALS compared to HC, and their association with plasma neurofilament light (NfL) concentrations, a surrogate biomarker for neurodegeneration. Flow cytometry was used to validate alterations in cell type proportions. Results We identified the expansion of CD56 ^dim natural killer (NK) cells in ALS (fold change = 2; adj. p-value = 0.0051), which was mainly driven by the NK_2 subpopulation (fold change = 3.12; adj. p-value = 0.0001), a mature and cytotoxic CD56 ^dim NK subset. Our results revealed extensive gene expression alterations in NK_2 cells, pointing towards the activation of immune response (adj. p-value = 9.2x10 ^− 11 ) and the regulation of lymphocyte proliferation (adj. p-value = 6.46x10 ^− 6 ). We identified gene expression changes in other immune cells, such as classical monocytes, and distinct CD8 + effector memory T cells which suggested enhanced antigen presentation via major histocompatibility class-II (adj. p-value = 1.23x10 ^− 8 ) in ALS. The inference of cell-cell communication patterns demonstrated that the interaction between HLA-E and CD94:NKG2C from different lymphocytes to NK_2 cells is unique to ALS blood. Finally, regression analysis revealed that the proportion of CD56 ^bright NK cells along with the ALSFRS, disease duration, and gender, explained up to 76.4% of the variance in plasma NfL levels. Conclusion Our results reveal a signature of relevant changes occurring in peripheral blood immune cells in ALS and underscore alterations in the proportion, gene expression, and signaling patterns of a cytotoxic and terminally differentiated CD56 ^dim NK subpopulation (NK_2), as well as a direct role of CD56 ^bright NK cells in neurodegeneration.