Tregs levels and phenotype modifications during Amyotrophic Lateral Sclerosis course

Abstract

Introduction: T regulatory cells (Tregs) inversely correlate with disease progression in Amyotrophic Lateral Sclerosis (ALS) and fast-progressing ALS patients have been reported to exhibit dysfunctional, as well as reduced, levels of Tregs. This study aimed to evaluate the longitudinal changes in Tregs among ALS patients, considering potential clinical and biological modifiers of their percentages and concentrations. Additionally, we explored whether measures of ALS progression, such as the decline over time in the revised ALS Functional Rating Scale (ALSFRS-r) or forced vital capacity (FVC) correlated Treg levels and whether Treg phenotype varied during the course of ALS.

Methods: Total Tregs (detected by CD3, CD4, FoxP3, CD25, and CD127) were quantified at five time points over 54 weeks in 21 patients in the placebo arm of the RAP-ALS trial; next they were characterized for the expression of surface markers including CD38, CD39, CXCR3, and PD1. Repeated measures mixed models were used to analyze the longitudinal course of Tregs, considering potential associations with other clinical and laboratory characteristics. Correlations between ALSFRS-r or FVC and Tregs over time were similarly investigated.

Results: Our study showed that Treg levels did not change significantly on average during the observation period in our ALS cohort. However, PD1+Tregs decreased and CD39+Tregs increased over time. Male sex and cholesterol levels were associated with increasing Tregs (%) over time, while monocytes positively affected Treg concentrations. Treg concentrations showed a modesty association with FVC decline but were not associated with ALSFRS-r decline.

Discussion: Treg levels remained stable during the ALS observation period and were not significantly associated with ALSFRS-r variations, suggesting that Treg numbers alone may have limited utility as a pharmaco-dynamic biomarker for ALS trials. However the observed changes in Treg phenotypes, such as the decrease in PD1+Tregs, indicate that phenotypic variations may warrant further investigation for their potential role in ALS progression and therapeutic targeting.

Keywords: ALS; ALSFRS-r; FVC (forced vital capacity); T regulatory cell; cholesterol; monocyte.

Figure 1

Gating strategy used to identify Tregs and their subpopulations. (A) Lymphocytes were identified by physical parameters, doublets were removed and alive CD3+ T cells were selected accordingly. In this population, CD4+ T were selected and within this, those who were CD25++, FoxP3+ and CD127- were defined as Tregs. In this population, by using two different panels (which shared the same backbone, such as Live Dead, CD3, CD4, CD8, CD127, CD25,FoxP3; all reported in (B), Tregs expressing HLA-DR, CD38, CXCR3, PD-1 and CD39 were identified.

Figure 2

Unadjusted linear trends over time of Tregs percentages over total CD4 T cells (A) and Tregs concentrations (B). Continuous lines represent observed trajectories for each single patient with ALS. Female patients’ Tregs trajectories are depicted in red color, while males’ ones in blue. Dotted orange line represents the average linear variation in Tregs percentage (A) and concentration (B) over time, expressed in months since onset of disease, with 95% confidence interval depicted by the yellow-shaded area. Tregs concentrations are expressed as cell count per microliter.

Figure 3

Unadjusted linear trends over time of ALSFRS-r (A) and FVC percentages (B). Continuous lines represent observed trajectories for each single patient with ALS, while dotted lines depict predicted trajectories. Female patients’ Tregs trajectories are depicted in red color, while males’ ones in blue. Dotted orange line represents the average linear variation in ALSFRS-r (A) and FVC (%) (B) over time, expressed in months since onset of disease, with 95% confidence interval depicted by the yellow-shaded area. Predicted values were calculated from an additive effect multivariable linear mixed model which considers clinical and laboratory characteristics as the independent variables.

Figure 4

Unadjusted linear trends over time of CD38+ (A), CXCR3+ (B), CD39+ (C), and PD1+ (D) Tregs subpopulations expressed in percentages over total Tregs. Continuous lines represent observed trajectories for each single patient with ALS. Female patients’ Tregs trajectories are depicted in red color, while males’ ones in blue. Dotted orange line represents the average linear variation in CD38+ Tregs (A), CXCR3+ Tregs (B), CD39+ Tregs (C), and PD1+ Tregs (D) over time, expressed in months since onset of disease, with 95% confidence interval depicted by the yellow-shaded area. Tregs subpopulations are expressed as the percentage over total Tregs.