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

Abstract

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 between ALS patients and HC, as well as 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), mainly driven by a specific subpopulation, NK_2 cells (fold change = 3.12; adj. p-value = 0.0001), which represent 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 = 9.2 × 10^{- 11}) and the regulation of lymphocyte proliferation (adj. p-value = 6.46 × 10^{- 6}). We also 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 = 1.23 × 10^{- 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 compared to HC. Finally, regression analysis revealed that the proportion of CD56^bright NK cells along with the ALSFRS-r, 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 possible role of CD56^bright NK cells in neurodegeneration.

Keywords: ALS; Immune system; Natural killer cells; Neurodegeneration; scRNAseq.

Fig. 1

Alterations in the proportions of major peripheral blood mononuclear cell populations in ALS. (A) UMAP plot indicating the clusters of major PBMC populations in ALS and HC. Note that in this plot, CD56^dim NK cells are named as NK. (B) Box plot showing the significant expansion of CD56^dim NK cells, and the upregulation of classical monocytes and CD56^bright NK cells. Cell type proportions were calculated by propeller tool based on the total number of cells per sample that passed quality control after scRNAseq. PBMC: peripheral blood mononuclear cells; NK: CD56^dim NK cell; DC: dendritic cell; Mono: Monocyte; TCM: central memory T cell. TEM: effector memory T cell; gdT: gamma-delta T cell; HSPC: Hematopoietic stem and progenitor cell; MAIT: Mucosal-associated invariant T cell; dnT: double-negative T cell. ** adjusted p-value < 0.01

Fig. 2

Two NK subtypes drive the expansion of CD56dim NK cells in ALS. (A) UMAP plot showing the clusters of PBMC subpopulations in ALS and HC. (B) Box plot demonstrating that NK_2 cells and NK_4 cells are expanded in the blood of ALS patients. (C) Dot plot depicting the expression of the traditional FCGR3A (CD16) and NCAM1 (CD56), together with 7 other marker genes across subsets of human blood NK cells. Cell type proportions were calculated by propeller tool based on the total number of cells per sample that passed quality control after scRNAseq. PBMC: peripheral blood mononuclear cells; NK: CD56dim NK cell; DC: dendritic cell; Mono: Monocyte; TCM: central memory T cell. TEM: effector memory T cell; gdT: gamma-delta T cell; HSPC: Hematopoietic stem and progenitor cell; MAIT: Mucosal-associated invariant T cell; dnT: double-negative T cell. **adjusted p-value < 0.01, *** adjusted p-value < 0.001

Fig. 3

Most relevant gene expression alterations in peripheral blood mononuclear cells from ALS patients. Volcano plots displaying differentially expressed genes between the ALS and HC. The vertical axis (y-axis) corresponds to the − log10 adjusted p value, and the horizontal axis (x-axis) represents the log₂ fold change value obtained. Significantly differentially expressed genes are depicted with blue circles (adjusted p-value < 0.05), whereas gray circles display the nonsignificant genes. (A) Volcano plot displaying differentially expressed genes between the ALS and HC groups in NK_2 cells. (B) Volcano plot showing differentially expressed genes between the ALS and HC groups in classical monocytes. (C) Volcano plot displaying differentially expressed genes between the ALS and HC groups in CD8 TEM_5 cells. The most significant and interesting genes are depicted. PBMC: peripheral blood mononuclear cells. NK_2: CD56^dim NK cell subtype 2; TEM: effector memory T cell.

Fig. 4

Cell-cell communication changes. (A) Scatter plot showing dominant senders and receivers in a 2D space for ALS (left) and HC (right). (B) Scatter plot revealing the MHC-I signaling pathways alterations associated with NK_2 cells. (C) Dot plot displaying the expression of significant ligand–receptor pairs in the MHC-I pathway from all senders to NK_2 cells, splitted by ALS and HC. NK: natural killer cell; TCM: central memory T cell. TEM: effector memory T cell

Fig. 5

Validation of alterations in cell type proportions using flow cytometry. Box plot demonstrating that (A) CD56^dim NK cells, (B) CD56^dim FceR1G + NK cells, (C) CD56^bright NK cells and (D) classical monocytes are expanded in ALS blood. Cell type proportions are calculated based on the total number of gated peripheral blood mononuclear cells per sample. * p-value < 0.05, *** p-value < 0.001