Supplementary data Supplementary Table e-1. Flow cytometry ...
Transcript of Supplementary data Supplementary Table e-1. Flow cytometry ...
Supplementary data Supplementary Table e-1. Flow cytometry reagents and staining combinations
Reagents
Antibody Fluorochrome
conjugation
Clone Source
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-CF594 236A/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 N49-653 BD Biosciences
IL-4 PE-CF594 MP4-25D2 BD Biosciences
IL-22 PerCP-
eFluor710
22URTI eBioscience
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 BV421 100 Biolegend
LIVE/DEAD Fixable
Aqua Dead Cell Stain
N/A N/A Invitrogen
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
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.
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.
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-CSF-
expressing 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
random coefficient mixed effects model. Individual patient trajectories are shown
in grey.
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
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.
Supplementary Table e-2. T-cell expression of pro- and anti-apoptotic molecules pre- and post-DMF treatment
Molecules ΔMFI Pre-treatment
(95% CI)
ΔMFI Post-treatment
(95% CI)
p value (pre- vs. post-
treatment)
Pro-apoptotic
BIM 191 (148-234) 188 (130-247) ns
PUMA 2227 (501-3954) 2326 (1016-3635) ns
BAX 152 (-54-357) 168 (-14-351) ns
BAK 138 (115-161) 154 (121-187) ns
Anti-apoptotic
BCL-XL 311 (245-377) 315 (200-429) ns
BCL-2 2204 (704-3704) 2588 (971-4205) 0.0425
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-/post-
treatment samples; p values compare pre- and post-treatment mean MFIs using
paired t-tests). ns = not significant, MFI = mean fluorescence intensity.
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.