Senescent-like Blood Lymphocytes and Disease Progression in Amyotrophic Lateral Sclerosis

Abstract

Background and objectives: Aging is known to exacerbate neuroinflammation, and in the neurodegenerative disorder amyotrophic lateral sclerosis (ALS), an older age is associated with a worse prognosis. We have previously shown the activation of cell senescence pathways in the proteome of peripheral blood mononuclear cells and the increase of proinflammatory cytokines in blood from individuals living with ALS. In this single-center, retrospective study, we investigated the expression of senescent-like blood mononuclear cells in ALS.

Methods: We first applied multidimensional cytometry by time-of-flight (CyTOF) to study the senescent immunophenotype of blood mononuclear cells from 21 patients with ALS and 10 healthy controls (HCs). We then used targeted flow cytometry (FC) to investigate frequencies of senescent blood lymphocytes in 40 patients with ALS and 20 HCs. Longitudinal analysis included 2 additional time points in 17 patients with ALS. Frequencies of senescent-like lymphocytes were analyzed in relation to survival.

Results: Unsupervised clustering of CyTOF data showed higher frequencies of senescent CD4⁺CD27^-CD57⁺ T cells in patients with ALS compared with those in HCs (p = 0.0017, false discovery (FDR)-adjusted p = 0.029). Moderate to strong negative correlations were identified between CD4 T central memory-cell frequencies and survival (R = -061, p = 0.01; FDR-adjusted p < 0.1) and between CD95 CD8 cells and ALS functional rating scale revised at baseline (R = -0.72, p = 0.001; FDR-adjusted p < 0.1).Targeted FC analysis showed higher memory T regulatory cells (p = 0.0052) and memory CD8⁺ T cell (M-Tc; p = 0.0006) in bulbar ALS (A-B) compared with those in limb ALS (A-L), while late memory B cells (LM-B) were also elevated in A-B and fast-progressing ALS (p = 0.0059). Higher M-Tc levels separated A-B from A-L (AUC: 0.887; p < 0.0001). A linear regression model with prespecified clinical independent variables and neurofilament light chain plasma concentration showed that higher frequencies of LM-B predicted a shorter survival (hazard ratio: 1.094, CI: 1.026-1.167; p = 0.006).

Discussion: Our data suggest that a systemic elevation of senescent and late memory T and B lymphocytes is a feature of faster progressing ALS and of ALS individuals with bulbar involvement. Lymphocyte senescence and their memory state may be central to the immune dysregulation known to drive disease progression in ALS and a target for biomarkers and therapeutics discovery.

Figure 1. Unsupervised Clustering Analysis Reveals a Senescent Population of CD4 T Cells in Patients With ALS

(A) Heatmap showing phenograph clustering of 22 lineage markers identifying 19 clusters with a frequency above the 1% threshold, which were grouped into 17 meta-clusters according to their lineage marker expression. Clusters with a frequency of less than 1% were excluded and grouped into the “Excluded” cluster (NA, 6th row from the top). Cluster frequencies are shown together with their assigned cell type. (B) t-distributed stochastic neighbor embedding (tSNE) of 100,000 down sampled cells for patients with ALS and HCs (50,000 per HC and ALS group), colored by phenograph-determined cell types. (C) Frequency of CD4 senescent T cells (CD4 T SEN) as a percentage of live cells identified by unsupervised phenograph clustering in the HC and ALS groups. p values determined through quasi-likelihood negative binomial generalized linear models. ALS = amyotrophic lateral sclerosis; CD4 T SEN = CD4 cells expressing CD57; HC = healthy control.

Figure 2. CyTOF Analysis: Association Between Blood Mononuclear Cell Frequencies and Clinical Parameters

FDR-adjusted partial correlation analysis correcting for batch effect showed moderate to strong negative correlations between CD4 central memory (CM) cell frequencies and survival and between CD95 (Fas) CD8 cells and ALSFRSR at baseline. ALSFRS-R = amyotrophic lateral sclerosis functional rating scale revised; CyTOF = cytometry by time-of-flight.

Figure 3. FC Gating Strategy

Schematic representation of gating strategy for T cells (A), B cells (B) and T-cell senescence panels (shown for CD8⁺ T cells) (C) used in the FC experiments. FC = flow cytometry.

Figure 4. Analysis of T-Cell Subset Frequencies and Association With Site of Onset and Disease Progression in Individuals With ALS

(A) FoxP3⁺ Tregs expression is significantly higher in blood from patients with ALS compared with that in HCs. (B) M-Tregs are elevated in ALS and A-B. (C) M-Tc frequencies are significantly upregulated in A-B compared with that in A-L. (D) ROC analysis testing M-Tc ability to separate A-B from A-L. A-B = bulbar ALS; A-L = limb-onset ALS; ALS = amyotrophic lateral sclerosis; HCs = healthy controls; M-Tc = memory CD8⁺ T cells; M-Tregs = memory T regulatory cells; ROC = receiver operating characteristic.

Figure 5. Analysis of B-Cell Subset Frequencies and Their Correlation With Disease Progression in ALS

(A) Blood frequencies of LM-B are increased in A-F. The blood expression of LM-B separates ALS from HCs and A-S (ROC analyses). (B) Blood frequencies of M-B are higher in HCs compared with those in ALS. A-F = faster progressing ALS; ALS = amyotrophic lateral sclerosis; A-S = slow progressive ALS; HCs = healthy controls; LM-B = late memory B cells; M-B = memory B cells; N-B = naive B cells; ROC = receiver operating characteristic.

Figure 6. Changes of Lymphocyte Frequencies in Longitudinal Blood Samples

Box plot representation of ANOVA for repeated measures analysis (mixed model with missing values at random) of the changes from V1 to V3 showing a significant increase over time of CD3 (p = 0.0259) and the relative stability of CD4 and CD8. All remaining T-cell and B-cell subgroups under investigation, including those expressing markers of cell senescence, show variable patterns of expression but no significant changes across time points.