TRACKING PERIPHERAL IMMUNE CELL PROFILES ALONG ALS PROGRESSION AT SINGLE-CELL RESOLUTION

Neuroinflammation is a key contributor to amyotrophic lateral sclerosis (ALS), involving not only resident glial cells but also infiltrating and activated peripheral immune populations. However, peripheral immune dynamics during disease progression remain poorly characterized at single-cell resolution.

We performed longitudinal single-cell RNA sequencing of peripheral blood mononuclear cells from 14 ALS patients and 16 cognitively unimpaired healthy controls. Using linear mixed-effects models, we assessed immune cell alterations in relation to clinical progression (ALSFRS-R) and neurodegeneration (plasma NfL). Additional analyses included functional module scoring for senescence and infiltration, differential gene expression, cell–cell communication, and trajectory inference, adjusting for age, sex, and disease duration.

After quality control, >170,000 cells were classified into 55 immune subpopulations. Faster ALS progression was associated with reductions in Treg memory cells (β=−0.51,p=0.02) and classical monocytes (β=−0.49,p=0.009). Treg cells from fast progressors displayed increased senescence signature (p=0.0002), whereas classical monocytes exhibited reduced senescence (p=9.3×10⁻¹⁴), increased infiltration scores (p=1.06×10⁻⁶), and a trajectory shift towards a pro-inflammatory, dendritic cell-like phenotype. Elevated plasma NfL levels were associated with expansion of CD56bright NK cells (β=0.26,p=0.003), which showed altered intercellular communication consistent with enhanced tissue homing. We also identified significant cell type-specific transcriptional changes, alterations in cellular trajectories, and intercellular communication patterns associated with ALS (adj.p<0.05).

Our findings highlight a central role for peripheral immune cells in ALS, with Treg dysfunction, classical monocyte remodeling, and NK cell alterations contributing to disease progression and neurodegeneration. These results indicate that immune priming drives ALS progression and neuronal loss.