Supplementary Table e-1. Flow cytometry reagents and staining combinations

Transcription

1 Supplementary data Supplementary Table e-1. Flow cytometry reagents and staining combinations Reagents Antibody Fluorochrome Clone Source conjugation CD3 FITC UCHT1 BD Biosciences CD3 PerCP-Cy5.5 SK7 Biolegend CD3 BUV395 SK7 BD Biosciences CD3 APC-R700 UCHT1 BD Biosciences CD4 BV786 SK3 BD Biosciences CD4 BUV661 SK3 BD Biosciences CD8 AlexaFluor700 RPA-T8 BD Biosciences CD8 BV786 RPA-T8 BD Biosciences CD45RA FITC HI100 BD Biosciences CD45RO PE UCHL1 BD Biosciences CD45RO APC-H7 UCHL1 BD Biosciences CCR7 APC 3D12 BD Biosciences CD31 PE WM59 BD Biosciences CD25 BV421 2A3 BD Biosciences CD127 PC7 R34.34 Beckman Coulter FoxP3 PE-CF A/E7 BD Biosciences IFNγ BV650 4S.B3 BD Biosciences GM-CSF PE BVD2-21C11 BD Biosciences IL-10 APC JES3-19F1 BD Biosciences IL-17 BV421 N BD Biosciences IL-4 PE-CF594 MP4-25D2 BD Biosciences IL-22 PerCPeFluor710 22URTI ebioscience

2 Annexin V BUV395 N/A BD Biosciences Propidium iodide N/A N/A BD Biosciences Integrin β7 FITC FIB504 Biolegend CCR9 PE-Cy7 L053E8 Biolegend CCR5 BV421 J418F1 Biolegend CCR2 PerCP-Cy5.5 K036C2 Biolegend CLA PE HECA-452 BD Biosciences CCR4 PE-CF594 1G1 BD Biosciences BIM AF488 C34C5 Cell Signaling technology PUMA PE EP512Y Abcam BAX AF488 2D2 Biolegend BAK PE D4E4 Cell Signaling technology BCL-XL AF488 54H6 Cell Signaling technology BCL-2 BV Biolegend LIVE/DEAD Fixable N/A N/A Invitrogen Aqua Dead Cell Stain Staining combinations CD3-PerCP; CD4-BV786; CD8-AlexaFluor700; CD45RA-FITC; CCR7-APC; Live/Dead CD3-APC-R700; CD4-BV786; CD45RO-APC-H7; CD31-PE; CD25-BV421; CD127- PC7; FoxP3-PE-CF594 CD3-FITC; CD4-BV786; CD8-AlexaFluor700; CD45RO-PE; CCR7-APC; CD25- BV421; CD127-PC7; Annexin V-BUV395; Propidium iodide CD3-BUV395; CD4-BV786; CD8-AlexaFluor700; IFNγ-BV650; GM-CSF-PE; IL-10- APC; IL-17-BV421; IL-4-PE-CF594; IL-22-PerCP CD3-BUV395; CD4-BUV661; CD8-BV786; Integrin β7-fitc; CCR9-PE-Cy7; CCR5- BV421; CCR2-PerCP-Cy5.5; CLA-PE; CCR4-PE-CF594 CD3-BUV395; CD4-BUV661; CD8-BV786; BIM-AF4888; PUMA-PE CD3-BUV395; CD4-BUV661; CD8-BV786; BCL-XL-AF488; BCL-2-BV421 CD3-BUV395; CD4-BUV661; CD8-BV786; BAX-AF488; BAK-PE

3 Supplementary Figure e-1. T-cell subset staining and gating strategy Supplementary Figure e-1. T-cell subset staining and gating strategy. Representative example of T-cell subset staining by flow cytometry and gating strategy used from a MS patient sample.

4 Supplementary Figure e-2. Annexin V/propidium iodide staining Supplementary Figure e-2. Annexin V/propidium iodide staining. Representative example of Annexin/PI staining of CD8+ (top) and CD4+ (bottoom) T-cell subsets from a healthy control subject under each exposure. UNTX = medium alone, VEH = vehicle (DMSO), DMF = dimethyl fumarate, MMF = monomethyl fumarate, PI = propidium iodide.

5 Supplementary Figure e-3. DMF treatment in vivo alters the regulatory to effector cell balance Supplementary Figure e-3. DMF treatment in vivo alters the regulatory to effector cell balance. Greater losses in putatively pro-inflammatory effector T-cell populations versus putatively regulatory T-cell subsets were seen with DMF treatment of MS patients, leading to changes in regulatory to effector cell ratios. Increases were seen in the ratios of regulatory T-cells (Treg) to Th1 (A), Tc1 (B) and GM-CSFexpressing CD4+ T-cells (C), as well as in the ratios of IL-10-expressing CD4+ T- cells to Th1 (D) and GM-CSF-expressing CD4+ T cells (E). Data shown is from multiple sclerosis patients (n=13) pre-treatment (Month 0) and up to 12 months following DMF treatment initiation. The p values displayed represent the statistical significance of the exponential decay trajectory (shown in red) in a

6 random coefficient mixed effects model. Individual patient trajectories are shown in grey.

7 Supplementary Figure e-4. Susceptibility of circulating T-cells to dexamethasone-induced apoptosis pre- and post-dmf treatment Supplementary Figure e-4. Susceptibility of circulating T-cells to dexamethasone-induced apoptosis pre- and post-dmf treatment. Annexin V/PI staining to capture T-cell apoptosis following exposure to different concentrations of dexamethasone. Representative example of dose-dependent increase in dexamethasone-induced T-cell apoptosis (top row) compared to vehicle control (bottom row) using purified T-cells from a DMF-treated patient and gating on all CD3+ T-cells (A). Summary graphs showing frequencies of Annexin

8 V+/PI+ apoptotic cells (corrected for degree of apoptosis with vehicle control: Δ% An+/PI+ = % An+/PI+ with dexamethasone minus % An+PI+ with corresponding vehicle concentration) do not change significantly pre- versus on-treatment (n=3 patients; highest 3 concentrations of dexamethasone shown) (B). An = Annexin V, PI = propidium iodide, PRE = pre-treatment, ON = on-treatment.

9 Supplementary Table e-2. T-cell expression of pro- and anti-apoptotic molecules pre- and post-dmf treatment Molecules p value (pre- vs. posttreatment) Proapoptotic Antiapoptotic ΔMFI Pre-treatment (95% CI) ΔMFI Post-treatment (95% CI) BIM 191 ( ) 188 ( ) ns PUMA 2227 ( ) 2326 ( ) ns BAX 152 ( ) 168 ( ) ns BAK 138 ( ) 154 ( ) ns BCL-XL 311 ( ) 315 ( ) ns BCL ( ) 2588 ( ) Supplementary Table e-2. T-cell expression of pro- and anti-apoptotic molecules pre- and post-dmf treatment. CD3+ T-cell expression of pro- and anti-apoptotic molecules pre- and post-in vivo treatment with DMF. Results are shown as mean ΔMFI (MFI of molecule minus MFI of isotype control) and 95% confidence intervals (n=3 matched pre-/posttreatment samples; p values compare pre- and post-treatment mean MFIs using paired t-tests). ns = not significant, MFI = mean fluorescence intensity.

10 Supplementary Figure e-5. Relative changes in tissue-homing T-cell populations with DMF treatment Supplementary Figure e-5. Relative changes in tissue-homing T-cell populations with DMF treatment. Representative example of staining for gut (CCR9+beta7integrin+), skin (CLA+CCR4+) and brain (CCR2+CCR5+) homing T-cell populations, gating on CD4+ cells (A). Summary graphs showing the change in relative frequency between matched pre- and 3 month post-treatment samples of each tissue homing population amongst CD4+ (B) and CD8+ (C) T-cells (n=3 patients; p values shown represent adjusted p values from repeated measures one-way ANOVA with Tukey s post test). DMF = dimethyl fumarate, ns = not significant.