A sub-network of signaling protein complexes in

Alopecia Areata may provide new molecular

candidates for pharmacologic targeting

Adam G. Schrum, PhD

Assistant Professor of Immunology

Mayo Clinic College of Medicine

CD28 a b

CD8

SLAP 130

TCR

a b CD3

e g

z z

CD4

CBP

CSK

CD45

LCK

FYN Zap70

Calcineurin NFATc

Calmodulin Ca2+

Bcl-10

CD3

de

IKKa

IKKb

IKKg TBK1

IKKe

TANK

IkB

NFkB

MALT1

GRB2

SOS1

RAS

RAF1

MEK1

ERK1/ERK2

DOK1

DOK2

SHIP1

GADS

SLP-76

Nck

PAK

WASP

WIP

F-actin

ARP2/3

ITK

CDC42 RAC1

MEKK1

JNKK1

JNK1

SHP1/2

CARMA1 LAT PLCg CpA

PKCq VAV

PI3K

AKT

PKCa

PKCb

Cbl-b

B7.1/2 MHC

1 2

APC MHC

1

T cell

THEMIS CD6

Do different signals through TCR affect

immunologic tolerance and AA?

Qualitative

Quantitative

Kinetic

What could be

“different” ?

Responders: Jurkat

APCs: Raji

+/- SEE, 5 min

PiSCES: Proteins in Shared Complexes detected by Exposed Surface epitopes:

a multiplex matrix analysis to assess PPI network signatures

Smith SEP, Neier SC, et al., Aug 2, 2016 Science Signaling (AAAS)

PiSCES network analysis &

visualization

Responders: Jurkat

APCs: Raji

+/- SEE, 5 min

SEE / null, 5 min SEE CD28+ / SEE CD28–, 5 min

Subject T cells

(millions) %CD4

SP %CD8

SP %CLA+CCR4+ Age Sex

aa1 2 20.9 56.9 12.9 29 m

aa2 3 65.4 28 74.2 50 f

aa3 8.4 14.7 76.3 17.4 50 f

aa4 3.87 88.9 8.7 44.5 81 f

aa5 1.32 21.6 59.3 21.7 39 f

aa6 1.58 85.1 7.7 71.5 41 f

aa7 1.58 69.3 25 16.6 59 m

cntl1 1.5 82.9 6.1 24.4 51 m

cntl2 1.6 78 17.9 50.5 77 m

cntl3 6.8 91.8 5.1 84.2 51 m

cntl4 6.2 98.1 0.3 79.1 76 m

cntl5 2.8 85.9 12.3 59 89 f

T cells from volunteer patient punch-biopsies

Overall similar response

pattern between Control

and AA patient T cells to

anti-CD3/anti-CD28

stimulation

PiSCES data distinguishes AA from Control patient T cells,

and suggests increased basal TCR signaling in AA

AA > Ctrl

Ctrl > AA

Difference in basal levels

ITK

Thy1

Fyb

TCR

a b

z z

LCK Zap70

CD3

de

GADS

Nck

SLP-76

LAT VAV

plasma membrane

GRB2

SOS1

LAT

PKCq

PLCg Fyn

Zap70

CD3

e g

SKAP55 Integrin activation

Cell adhesion

Ras-Raf-ERK Differentiation

Clonal expansion

AA > CtrlCtrl > AA

Upon exogenous stimulation, PiSCES network activity

suggests disease-associated signaling profile in

Alopecia Areata patients

Acknowledgements

Schrum Lab

• Stephen E.P. Smith, PhD

(currently Assistant

Professor, U. WA,

Seattle)

• Steven Neier

• Brendan Reed

• Tessa Davis

Diana Gil, PhD

• Alejandro Ferrer, PhD

• Robert Stiles

Collaborators

• Claudia Neuhauser, PhD (U. MN, Comput. Biol.)

• Jeanette Eckel-Passow, PhD (Mayo Biostatisitics)

• Jason P. Sinnwell (Mayo Biostatistics)

• Gabriel Sciallis, MD (Mayo Dermatology)

• Carrie Wieland, MD (Mayo Dermatology)

• Shelly Torgerson, MD/PhD (Mayo Dermatology)

Funding partners

• R01 NIGMS

• Mr. Bernie Fineman (Mayo Benefactor)