1. Figure e-1A

2. Figure e-1B

3. Figure e-1. Peripheral blood leukocyte immunophenotyping gating strategies. (A) PBMCs were gated according to their forward-scatter (FSC) and side-scatter (SSC). B and T cells were identified by expression of CD19 and CD3, respectively. CD19+ cells were further analyzed for expression of IgD and CD27. CD3+ cells were divided according to CD4 and CD8 expression. CD4+ and CD8+ T cells were further analyzed for CD45RA and CD45RO expression. CD3-CD19- population were analyzed for CD16 and CD56 expression to identify natural killer (NK) cells (CD16+CD56+). Monocytes were gated as CD14+ cells and Dendritic cells (DCs) as HLA-DR+ cells within the CD3-CD19-population. CD4+ and CD8+ cells T-regulatory cells were gated as CD127dimCD25+ cells. (B) Panel 2 was designed to study various T cell subtypes. PBMCs were gated as above, CD3+ T cells were analyzed for TCRγδ or TCRαβ expression. Within each CD3+CD4+ or CD3+CD8+ populations, Naïve cells were gated as CD197+CD45RA+, effector as CD197-CD45RA+, effector memory as CD45RA-CD197- , and central memory as CD197+CD45RA-. CD4+ and CD8+ cells were also categorized according to CD45RA versus CD62L expression and CD27 versus CD197 expression. Helper T cells (Th) identified within CD4+ cells included Th1 (CD119+), Th2 (CD294+ or CRTH2+), Th22 (CCR10+), Th17 (CD196+), follicular helper T cells (Tfh) as CXCR5+ cells. Activated T cells were gated as CD122+ within CD3+.

4. Figure e-2 A B

5. Figure e-2. Lymphocyte count and lymphopenia grade in RRMS patients
Figure e-2. Lymphocyte count and lymphopenia grade in RRMS patients. (A) The graph illustrates lymphocyte count (X109/L) of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10). Lymphocyte counts higher than 0.9X10^9/L were considered non-lymphopenic (grade 0), ≤0.9X10^9/L were scored as grade 1, ≤0.8 X10^9/L as grade 2, and ≤0.5 X10^9/L were scored as grade 3, ≤0.2 were scored as grade 4. The horizontal lines indicate the average mean of each group and the bars are the standard error of mean. P values are stated in the inset, those reaching statistical significance are highlighted by red. (B) The graph indicates the number of patients in each group with the indicated lymphopenia grade (color-coded).

6. A
Figure e-3 B C

7. Figure e-3. The T cell and its subtypes’ abundance are different in RRMS under FTY or DMF treatment. Flow cytometric analysis of peripheral blood of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10). (A) shows CD3+ T cell amounts, (B) is the number of CD3+CD4+ cells and (C) is the number of CD3+CD8+ cells (calculated as described in Methods). The horizontal lines indicate the average mean of each group and the bars are the standard error of mean. P values are indicated as insets, those reaching statistical significance are highlighted by red.

8. Figure e-4

9. Figure e-4. The frequencies of CD3+ T cells expressing homing receptors are significantly affected by FTY and DMF treatment. Flow cytometric analysis of peripheral blood of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10), looking at the frequencies of naïve (CD45RA+CCR7+), effector memory (CD45RA-CCR7-), effector (CD45RA+CCR7-) and central memory (CD45RA-CCR7+) populations within CD4+ cells (A) or within CD8+ cells (C). (B) and (D) illustrate the fractions of CD4+ cells and CD8+ cells expressing CD62L and CD45RA, respectively. (I). P values are indicated as insets, those reaching statistical significance are highlighted by red.

10. A B
Figure e-5 C D E F

11. Figure e-5. The overall abundance of helper T cells (Th) and TCRγδ T cells. The proportion of Th within PBMC was determined by flow cytometric analysis of peripheral blood of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10) for identification of Th1 by CD119+ cells (A), Th2 by CD294+ cells (B), Th17 by CD196+ cells (C), Th22 by CCR10+ cells (D), and Tfh by CXCR5+ cells (E), and TCRγδ T cells (F). The data are normalized to PBMC number. The horizontal lines indicate the average mean of each group and the bars are the standard error of mean. P values are indicated as insets, those reaching statistical significance are highlighted by red.

12. Figure e-6

13. Figure e-6. The B cell count is differentially affected in RRMS under FTY or DMF treatment. Flow cytometric analysis of peripheral blood of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10) . The graph illustrates B cell counts (as described in Methods). The horizontal lines indicate the average mean of each group and the bars are the standard error of mean. P values are indicated as insets, those reaching statistical significance are highlighted by red.

14. A
Figure e-7 B C

15. Figure e-7. The abundance of non-lymphocyte populations within PBMC is differentially affected in RRMS under FTY or DMF treatment. Flow cytometric analysis of peripheral blood of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10) for detection of monocyte population (CD3-CD19-CD14+, A), NK cells (CD3-CD19-CD56highCD16hich, B) and CD3- CD19-CD14-HLA-Dr+ cells (DCs, C). The horizontal lines indicate the average mean of each group and the bars are the standard error of mean. P values are indicated as insets, those reaching statistical significance are highlighted by red.

16. Figure e-8A A
Plasma

17. Figure e-8B and C B C
Unactivated
Activated

18. Figure e-8. The level of measured cytokines
Figure e-8. The level of measured cytokines. Multiplex assay cytokine analysis of untreated (NT, n=3), DMF-treated patients with no lymphopenia (DMF-N, n=3), and DMF-treated who developed lymphopenia (DMF-L, n=3). The graphs illustrate levels of all cytokines measured in plasma (A), and unactivated (B), or activated (c) PBMC supernatants.

19. Figure e-9 A B

20. Figure e-9. The frequency of CD4+ and CD8+ CD62L+ cells within T cells
Figure e-9. The frequency of CD4+ and CD8+ CD62L+ cells within T cells. Flow cytometric analysis of peripheral blood of Healthy controls (HC=13 )untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10). The graphs illustrate frequencies of CD62L expressing cells that are CD4+ (A) or CD8+ (B). The horizontal lines indicate the average mean of each group and the bars are the standard error of mean. P values are indicated as insets

21. Figure e-10 NT
FTY
DMF-N
DMF-L

22. Figure e-10. Visualization of overall abundance and heterogeneity of major PBMC populations. Flow cytometric analysis of peripheral blood of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10) was done by t-SNE (t-stochastic neighbor embedding). Original PBMC flow cytometry data were concatenated for each subject within a group (pooled into one population) and then downsampled to 100,000 cells as a representative of each category. Each population (defined as figure 2-e) is color-coded and the count refers to average of cells within that population.

23. Figure e-11 NT
DMF-N
DMF-L
FTY
RRMS011
RRMS023 A B

24. Figure e-11. Principal component analysis (PCA) of RRMS patients
Figure e-11. Principal component analysis (PCA) of RRMS patients. PCA identifies clustering of patients treated with FTY or patients who were treated with DMF and developed lymphopenia. PCA was based on flow cytometric analysis of peripheral blood of untreated (NT, n=19), FTY-treated patients (FTY, n=11), DMF-treated patients with no lymphopenia (DMF-N, n=15), and DMF-treated who developed lymphopenia (DMF-L, n=10). The frequency of gated populations, mean fluorescence intensity, and median fluorescence intensity for each marker as described in Figure 2-e in panel 1 (A) or panel 2 (B) were used to generate PCA. RRMS023 and RRMS011 (highlighted with circle) both are treated with DMF and have lymphocyte count of 0.9. Studied groups are color and shape-coded.

25. Table e-1. List of antibodies used in this study
Panel 1
Panel 2

26. Table e-2. List of cytokines measured in this study