*2.1. Patients*

In total, 34 patients who attended the Department of Neurology at the University Hospital Ulm between 2002 and 2004 before initiation of disease-modifying treatment (DMT) were included in the study. Initially, the MS diagnosis was made on the diagnostic criteria valid at time of study inclusion (McDonald 2001), but were adjusted for the most recent updates of the McDonald criteria (McDonald 2017). After study inclusion, 20 patients started treatment with glatiramer acetate, 12 patients were treated with interferon-beta (Avonex, Betaferon and Rebif) and 2 patients rejected DMT. All patients were then followed-up for 24 months with visits every 3 months in the first year and every 6 months in the second year. At all visits, clinical assessments including relapse evaluation, EDSS, Paced Auditory Serial Addition Test (PASAT), and serum sampling were performed. Relapses were defined as focal neurological disturbance lasting more than 24 h, without an alternate explanation. Furthermore, 17 patients received magnetic resonance imaging (MRI) scans at baseline, 12 months and 24 months. Detailed patients' characteristics are shown in Table 1 and the study schedule is shown in Table 2. Relapses were treated with high-dose corticosteroids (50–1000 mg) over 3–5 days after exclusion of contraindications. Age did not differ between patients with and without at least one relapse during follow-up and did not correlate with serum NfL levels at baseline.


**Table 1.** Patients' characteristics.

EDSS = Expanded Disability Status Scale; NfL = Neurofilament light chain; IQR = Interquartile range.


EDSS, Expanded Disability Status Scale; PASAT, Paced Auditory Serial Addition Test; NfL, neurofilament light chain; MRI, magnetic resonance imaging.

### *2.2. NfL Measurements*

Serum samples were stored in the local biobank according to recommended biobanking protocols at −80 ◦C [28]. Serum NfL was measured using the Simoa technology (Quanterix Corporation, Lexington, MA, USA). Samples were diluted, as recommended by the manufacturer, and concentrations were calculated using the corresponding standard curve.

### *2.3. Cytokines Measurements*

Cytokine profiles including IFN-γ, osteopontin (OPN), IL-2, IL-4 and IL-10 were determined in serum at study onset and at every visit during follow-up using the electrochemiluminescence detection multiplex technology of Meso Scale Discovery (MSD, Gaithersburg, MD, USA) according to the manufacturer's instructions as previously reported [29].

### *2.4. MRI Scans*

MRI scans of the brain and spinal cord were performed on a 1.5 Tesla clinical MRI scanner (Symphony Siemens, Erlangen, Germany) and the total number of hyperintense lesions in T2-weighted scans at the di fferent time points were visually quantified by an experienced rater.

### *2.5. Cognitive Functions*

Cognitive functions were assessed at every time point by the Paced Auditory Serial Addition Test (PASAT). Here, information processing speed and flexibility, as well as calculation ability are tested, which also means that this is not a global measure of cognitive dysfunction, but rather targets specific cognitive executive functions frequently a ffected in MS.

### *2.6. Statistical Methods*

All statistical tests were performed using the GraphPad Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA). Shapiro–Wilk test was used to examine the distribution of the data. Mann–Whitney U test was used to compare medians in skewed distributed parameters for unpaired samples and Wilcoxon matched-pairs signed-rank test for paired samples. Correlation analyses were performed with Spearman's rank correlation and corrected for multiple testing by the Bonferroni method. A *p*-value ≤ 0.05 was considered as statistically significant.

### *2.7. Ethical Statement*

The study was reviewed by the appropriate ethics committee of the University of Ulm (approval number 79/2001, approval date 14.11.2001) and was performed in accordance with the ethical standards of the current version of the Declaration of Helsinki. Written informed consent was obtained from all patients participating in this study.
