Elisabetta Zucchi ^1* Federico Banchelli ² Cecilia Simonini ^2* Sara De Biasi ³ Ilaria Martinelli ² GIULIA GIANFERRARI ^4,5 Domenico Lo Tartaro ³ Andrea Cossarizza ³ Roberto D'Amico ⁶ Jessica Mandrioli ^7*

• ¹ Neuroscience PhD Program, University of Modena and Reggio Emilia, Modena, Italy
• ² Unità di Neurologia, Azienda Ospedaliera Universitaria di Modena, Modena, Lombardy, Italy
• ³ Department of Medical and Surgical, Maternal-Infantile and Adult Sciences, University of Modena and Reggio Emilia, Modena, Lombardy, Italy
• ⁴ University of Modena and Reggio Emilia, Modena, Emilia-Romagna, Italy
• ⁵ Neuroscience PhD program, University of Modena and Reggio Emilia, Modena, Italy
• ⁶ Unit of Statistical and Methodological Support to Clinical Research, Azienda Ospedaliero-Universitaria Modena, Modena, Italy
• ⁷ Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Emilia-Romagna, Italy

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. 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. Our results 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. In conclusion, Treg levels remained stable during the ALS observation period and we 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.