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Supplemental Material for: Distinct T Cell Signatures Define Subsets of Multiple Sclerosis Patients Mark C. Johnson, Emily R. Pierson, Andrew J. Spieker, A. Scott Nielsen, Sylvia Posso, Mariko Kita, Jane H. Buckner, and Joan M. Goverman 1

Transcript of nn.neurology.org€¦ · Web view2016/08/23  · Comparable IFN-γ+ and IL-17+ responses to...

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Supplemental Material for:

Distinct T Cell Signatures Define Subsets of Multiple Sclerosis Patients

Mark C. Johnson, Emily R. Pierson, Andrew J. Spieker, A. Scott Nielsen, Sylvia Posso,

Mariko Kita, Jane H. Buckner, and Joan M. Goverman

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figure e-1. Comparable IFN-γ+ and IL-17+ responses to stimulation with tetanus

toxoid and anti-CD3/28 antibodies are observed for HCs, brain-predominant and

spinal cord-predominant MS patients. PBMCs from HCs (n = 45), brain-predominant

(n = 29), and spinal cord-predominant (n = 16) MS patients were stimulated with either

(A) tetanus toxoid or (B) anti-CD3/CD28 antibodies and analyzed by ELISPOT.

ELISPOT = Enzyme-linked immunosorbent spot; HC = Healthy control subject; IFN =

interferon; IL = interleukin; MS = multiple sclerosis; PBMCs = Peripheral blood

mononuclear cells; SC = spinal cord

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figure e-2. MHC class II-, but not MHC class I-, blocking antibodies significantly

reduce MBP-specific IFN-γ+ frequencies. PBMCs from a subset of subjects were

analyzed for MBP-specific IFN-γ responses in samples that were pre-incubated with (A)

a MHC class I–blocking antibody or (B) MHC class II-blocking antibodies. Responses in

wells containing blocking antibodies and MBP (white bars) were directly compared to

wells incubated with MBP alone (black bars). *p < 0.05, ***p < 0.001, Two-way

ANOVA with Tukey’s multiple comparison test. Data represent mean ± SEM.

IFN = interferon; MBP = myelin basic protein; MHC = Major histocompatibility

complex; PBMCs = peripheral blood mononuclear cells

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figure e-3. Myelin antigen responses are not significantly different between HCs and

non-stratified MS patients. (A) PBMCs stimulated with MBP from HCs (n = 45) and

MS patients not segregated by neuroinflammatory pattern (n = 45) were analyzed for

IFN-γ+ and IL-17+ responses to MBP by ELISPOT. (B) MOG-specific IFN-γ+ and IL-17+

responses were calculated in a sub-set of HCs (n = 16) and combined MS patients (n =

29). ELISPOT = Enzyme-linked immunosorbent spot; HC = Healthy control subject; IFN

= interferon; IL = interleukin; MBP = myelin basic protein; MOG = myelin

oligodendrocyte glycoprotein; PBMCs = peripheral blood mononuclear cells; MS =

multiple sclerosis

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table e-1. HC and MS Patient Demographics

Group Healthy Controls(n = 45)

Brain-predominant(n =29)

Spinal cord-predominant

(n = 16)Female 35 (77.8%) 23 (79.3%) 13 (81.25%)

Male 10 (22.2%) 6 (20.7%) 3 (18.75%)

Age at draw

38.6 ± 9.5 40.6 ± 9.8 40.9 ± 8.2

EDSS score NA 0.7 ± 0.9 2.3 ± 2.5

EDSS = Expanded disability status scale; HC = healthy control subject; MS = multiple

sclerosis

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Glossary

AUC = Area under the curve

EDSS = Expanded disability status scale

ELISPOT = Enzyme-linked immunosorbent spot

EAE = Experimental autoimmune encephalomyelitis:

FPR = False positive rates

HCs = Healthy control subjects

IFN = Interferon

IL = Interleukin

MBP = Myelin basic protein

MHC = Major histocompatibility complex

MOG = Myelin oligodendrocyte glycoprotein

MS = Multiple sclerosis

PBMCs = Peripheral blood mononuclear cells

ROC = Receiver operating characteristic

RRMS = Relapsing-remitting multiple sclerosis:

TPR = True positive rates

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