Revisiting the role of immune privilege (IP)

in alopecia areata (AA) pathobiology

Ralf PausUniversity of Manchester Manchester UK

No relevant conflicts of interest

Alopecia areata (AA)

= Hair follicle (HF) cycling disorder

Perifollicular inflammatory infiltrates

1 Attack only anagen hair bulb

(anagen III-VI)

2 Catapult anagen HFs

prematurely into catagen

3 Central role of MHC class I-based

HF immune privilege (IP)

collapse CD8+ T cells amp IFNg

bdquoNo IP collapse no AAldquo

HF damage (dystrophy)

hair shaft breakage amp shedding

Hair loss

V Price

WCHR Miami Nov 2015 AA pathobiology

bull Several different pathobiology pathways lead to AA which all coalesce in a stereotypic HF damage response the AA phenotype

bull This AA response pattern also occurs in healthy HFs in individuals not genetically predisposed to AA

Most essential prerequisitebull Immune privilege (IP) collapse of anagen HFs

bull AA = bdquoterritorial diseaseldquo pathobiology can only be decipheredby intracutaneous analyses focused on IP collapse amp restoration

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991

Paus et al Yale J Biol Med 1993

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Christoph et al BJD 2000

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)Ito T et al JID 2008

Petukhova et al Nature 2010

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991Paus et al Yale J Biol Med 1993

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)

Immunoinhibitory

function of peri-

follicular mast cells

in HF physiology

Bertolini et al PLoS ONE 2014

Kinori etal EXD 2012 Breitkopf et al JID 2013 Wang et al JID 2014 Bertolini et al BJD 2016

Role of mast cells (MCs) in AA

Lesional AA skin

bull much more amp hyperproliferating MCs

bull more direct interatcionsbetween MCs amp CD8+ TCs

Compared to non-lesional AA skin ampto healthy human scalp skin

Pink cells= MCs Brown cells= CD8+ T-cells

Bertolini et al PLoS ONE 2014Collab K McElwee A Gilhar et al

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Alopecia areata (AA)

= Hair follicle (HF) cycling disorder

Perifollicular inflammatory infiltrates

1 Attack only anagen hair bulb

(anagen III-VI)

2 Catapult anagen HFs

prematurely into catagen

3 Central role of MHC class I-based

HF immune privilege (IP)

collapse CD8+ T cells amp IFNg

bdquoNo IP collapse no AAldquo

HF damage (dystrophy)

hair shaft breakage amp shedding

Hair loss

V Price

WCHR Miami Nov 2015 AA pathobiology

bull Several different pathobiology pathways lead to AA which all coalesce in a stereotypic HF damage response the AA phenotype

bull This AA response pattern also occurs in healthy HFs in individuals not genetically predisposed to AA

Most essential prerequisitebull Immune privilege (IP) collapse of anagen HFs

bull AA = bdquoterritorial diseaseldquo pathobiology can only be decipheredby intracutaneous analyses focused on IP collapse amp restoration

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991

Paus et al Yale J Biol Med 1993

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Christoph et al BJD 2000

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)Ito T et al JID 2008

Petukhova et al Nature 2010

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991Paus et al Yale J Biol Med 1993

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)

Immunoinhibitory

function of peri-

follicular mast cells

in HF physiology

Bertolini et al PLoS ONE 2014

Kinori etal EXD 2012 Breitkopf et al JID 2013 Wang et al JID 2014 Bertolini et al BJD 2016

Role of mast cells (MCs) in AA

Lesional AA skin

bull much more amp hyperproliferating MCs

bull more direct interatcionsbetween MCs amp CD8+ TCs

Compared to non-lesional AA skin ampto healthy human scalp skin

Pink cells= MCs Brown cells= CD8+ T-cells

Bertolini et al PLoS ONE 2014Collab K McElwee A Gilhar et al

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

WCHR Miami Nov 2015 AA pathobiology

bull Several different pathobiology pathways lead to AA which all coalesce in a stereotypic HF damage response the AA phenotype

bull This AA response pattern also occurs in healthy HFs in individuals not genetically predisposed to AA

Most essential prerequisitebull Immune privilege (IP) collapse of anagen HFs

bull AA = bdquoterritorial diseaseldquo pathobiology can only be decipheredby intracutaneous analyses focused on IP collapse amp restoration

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991

Paus et al Yale J Biol Med 1993

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Christoph et al BJD 2000

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)Ito T et al JID 2008

Petukhova et al Nature 2010

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991Paus et al Yale J Biol Med 1993

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)

Immunoinhibitory

function of peri-

follicular mast cells

in HF physiology

Bertolini et al PLoS ONE 2014

Kinori etal EXD 2012 Breitkopf et al JID 2013 Wang et al JID 2014 Bertolini et al BJD 2016

Role of mast cells (MCs) in AA

Lesional AA skin

bull much more amp hyperproliferating MCs

bull more direct interatcionsbetween MCs amp CD8+ TCs

Compared to non-lesional AA skin ampto healthy human scalp skin

Pink cells= MCs Brown cells= CD8+ T-cells

Bertolini et al PLoS ONE 2014Collab K McElwee A Gilhar et al

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991

Paus et al Yale J Biol Med 1993

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Christoph et al BJD 2000

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)Ito T et al JID 2008

Petukhova et al Nature 2010

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991Paus et al Yale J Biol Med 1993

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)

Immunoinhibitory

function of peri-

follicular mast cells

in HF physiology

Bertolini et al PLoS ONE 2014

Kinori etal EXD 2012 Breitkopf et al JID 2013 Wang et al JID 2014 Bertolini et al BJD 2016

Role of mast cells (MCs) in AA

Lesional AA skin

bull much more amp hyperproliferating MCs

bull more direct interatcionsbetween MCs amp CD8+ TCs

Compared to non-lesional AA skin ampto healthy human scalp skin

Pink cells= MCs Brown cells= CD8+ T-cells

Bertolini et al PLoS ONE 2014Collab K McElwee A Gilhar et al

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Physiological status

Immune privilege (IP) of anagen HF

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH

cortisol MIF

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Christoph et al BJD 2000

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)Ito T et al JID 2008

Petukhova et al Nature 2010

Main IP pillars

Ito T et al AJP 2004

Billingham amp Silvers JID 1971Westgate et al JID 1991Paus et al Yale J Biol Med 1993

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)

Immunoinhibitory

function of peri-

follicular mast cells

in HF physiology

Bertolini et al PLoS ONE 2014

Kinori etal EXD 2012 Breitkopf et al JID 2013 Wang et al JID 2014 Bertolini et al BJD 2016

Role of mast cells (MCs) in AA

Lesional AA skin

bull much more amp hyperproliferating MCs

bull more direct interatcionsbetween MCs amp CD8+ TCs

Compared to non-lesional AA skin ampto healthy human scalp skin

Pink cells= MCs Brown cells= CD8+ T-cells

Bertolini et al PLoS ONE 2014Collab K McElwee A Gilhar et al

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8)

Immunoinhibitory

function of peri-

follicular mast cells

in HF physiology

Bertolini et al PLoS ONE 2014

Kinori etal EXD 2012 Breitkopf et al JID 2013 Wang et al JID 2014 Bertolini et al BJD 2016

Role of mast cells (MCs) in AA

Lesional AA skin

bull much more amp hyperproliferating MCs

bull more direct interatcionsbetween MCs amp CD8+ TCs

Compared to non-lesional AA skin ampto healthy human scalp skin

Pink cells= MCs Brown cells= CD8+ T-cells

Bertolini et al PLoS ONE 2014Collab K McElwee A Gilhar et al

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Role of mast cells (MCs) in AA

Lesional AA skin

bull much more amp hyperproliferating MCs

bull more direct interatcionsbetween MCs amp CD8+ TCs

Compared to non-lesional AA skin ampto healthy human scalp skin

Pink cells= MCs Brown cells= CD8+ T-cells

Bertolini et al PLoS ONE 2014Collab K McElwee A Gilhar et al

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

MCs in healthy perifollicular

human scalp skinMCs in lesional AA skin

Perifollicular MCs switch from an immuno-inhibitory

to a pro-inflammatory phenotype in lesional AA skin

MHCI

Heparin-Histamine

c-Kit

PD-L1

OX40L

TGFβ-1

Tryptase

IL-10

CD30L

Mast Cell

4-1BBL

ICAM-1

c-Kit

MHCI

Heparin-Histamine

4-1BBL

OX40L

PD-L1

IL-10

TGFβ-1

Tryptase

CD30L

Mast Cell

AgICAM-1

Bertolini et al PLoS ONE 2014

Does this MCs switch promote amp maintain IP collapse

Do these MCs regulate (auto-)antigen specific CD8+ T cells in AA

Mice possible in principle StelekatiPausBulfone-Paus et al Immunity 2009

IP guardians up

IP guardians down

Profinflammatory signals up

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Frontiers in HF immune privilege

No MHC I

No szlig2mg TAP

bull No autoantigen

presentation

TGFszlig12 aMSH IGF-1

SST cortisol MIF

CGRP PD-L1 VIP

bull bdquoIP guardiansldquo

create immuno-

inhibitory milieu

No MHC class II+ LCs

antigen-presentation

to CD4+ TCs impaired

Low level of NKG2D ligands

(MICA ULBP3)Insufficient stimulation of

NKG2D+ cells

(NKs NKTs CD8 gdTCs) Y Uchida M Bertolini

immunoinhibitory function

perifollicular mast cellsPeripheral tolerance

to anagen HF-associated

(auto-)antigens

Role of Tregs

M Rosenblum

Oelert et al Exp Dermatol 2016

AIRE gene

Kumar et al AJP 2011 HF co-local isation with K17

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells

switch toProinflammatory

phenotype

Promotion of

autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

Mast cells switch toProinflammatory phenotype

Promotion of autoantigen-specific

CD8+ T cells responses

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

IFNg-inducible chemokines

eg CXCL10 CXCR3

Ito T et al JDS 2013 Dai et al JI 2016

Xing et al Nat Med 2014

Perceived stress amp

neurogenic inflammation

SP mast cells

Peters et al AJP 2007 (human)Siebenhaar et al JID 2007 (mice)

MHC II Secondary autoimmune responses

epitope spreading

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Multiple factors promote IP collapse in AA

MHC I szlig2mg TAP

(Auto-)antigen

presentation

TGFszlig12 aMSH IGF-1

Cortisol VIP-R MIF

Autoimmmunity-

promoting milieu

MHC II Secondary autoimmune

responses epitope spreading

bdquostressedldquo HFs

NKG2D ligandsMICA (ULBP3) LiSinclair et al JID 2016

Activation of

NKG2D+ cells

NKs CD8+ TCs gdTCs

Mast cells switch to

proinflammatory phenotype

Key role of excessive

IFNg signaling via JAK

Loss of peripheral tolerance

to HF (auto-)antigens

AA

IFNg-inducible chemokines

eg CXCL10 CXCR3

AIRE SNPs WengrafTazi-Ahnini 2008

Treg defect

Perceived stress amp

neurogenic inflammation

SP mast cells

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

gd T-cells and HF biology

bull gd T-cell number fluctuates significantly during murine hair cyclePaus et al BJD 1994 + JID 1998

bull Only extremely few gd T-cells inaround human HFs Christoph et al BJD 2000

bull gd TC KO mice show catagen retardation followed by accelerationof HF cycling Kloumlpper et al JID 2013

bull FGF9-secreting gd T-cells key role in wounding-induced HF neogenesisis GayCotsarelis et al 2013

bull gd T-cells are involved in stress surveilance in murine skinHayday 2009

HYPOTHESIS

Can excessive stress immunosurveillance activities of

gd T-cells induce HF IP collapse and

thus contribute to AA pathogenesis

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

N=15-21 HFsgroup from 6-7 AA patients or healthy donors

Vd1+T-cells infiltrate in around AA hair bulbs

A

HS = healthy skinNL = non-lesional AA skinAA = lesional AA skin

hellip also seen in experimentally induced

AA in human skinxenotransplants in vivo

(Gilharlsquos humanizedAA mouse model)

Youhei Uchida et alunpublished

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Vd1+T-cells show increased NKG2D

amp IFNg expression within AA lesions

E

Do Vd1+T cells operate as

stress sentinels around human HFs

NKG2D IFNg

HS = healthy skin NL = non-lesional AA skinAA = lesional AA skin

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Ki67TUNEL

staining

bull Reduced hair matrix

amp outer root sheath

KC proliferation

bull Massive outer root

sheath KC apoptosis

bull Signs of direct HF

cytotoxicity

incl

enhanced LDH release

loss of intercellular adhesion

pigmentary abnormalities

(eg melanin clumping)

Co-culture of ldquostressedrdquo scalp HFs with autologous

dermal Vd1+T-cells induces HF dystrophy ex vivo

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Autologous dermal Vd1+T-cells induce HF IP collapse

and premature catagen in ldquostressedrdquo HFs

bull uarr MHC class I uarr MICA

bull darr IP guardians αMSH and TGFszlig2 = AA phenocopy

bull premature catagen HF dystrophy

MHC class IαMSH

TGFβ2

Hair cycle

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Autologous dermal Vd1+T-cells become activated and

up-regulate IFNg and NKG2D when co-cultured with

ldquostressedrdquo human scalp HFs

Isotype control non-co-cultured Vd1 T cellsVd1 T cells co-cultured with ldquostressedrdquo healthy (non-AA) scalp HFs

n= 3 experiments from 3 donors

None of the above phenomena seen with

bdquonon-stressedldquo human scalp HFs

Abrogated by anti- IFNg CD1d or MICA antibodies

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Conclusions 1

bull New physiological role of human gd T cells (Vd1)

in human skin biology

scouting for

distress signals

from stressed HFs

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Conclusions 2

bull These physiological stress sentinel functions of dermal Vd1 T cells

can become pathological by inducing IP collapse amp promoting AA

AA

response

pattern

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

Youhei Uchida

Marta BertoliniJennifer GherardiniJeacutereacutemy Cheacuteretet al

Special thanks toAmos Gilhar HaifaAlfredo Rossi Rome

Acknowledgements

gd TC study

Kagoshima Univ

Japan

Jeremy Jen

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

bull Therapeutically protecting amp restoring HF IP is crucial

amp has the widest success chance among AA therapies

works in all AA pathobiology scenarios but is not curative

bull Antagonizing IFNgJAK signalling makes perfect sense but isnot the only sensible strategy amp is also not curative

bull Identifying the elusive CD8+MHC class I-presented (auto-) antigens in AA and restoring peripheral toleranceremains critical for curative AA therapy

bull Yet this strategy will counteract only one of several IP collapsepathways leading to the AA response pattern

understand roles of Tregs NKTregs chemokines AIRE in human AA target also mast cells NK cells excessive gdTCs activities restore IP guardians (aMSH VIP CGRP antagonize SP)

Multimodal AA therapy needed

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA

T cell induced-self MHC class Ipeptide complexes may enable de novo tolerance induction to neo-antigens outside of the thymusOelert T Gilhar A Paus R Exp Dermatol 2016 Oct [Epub ahead of print]

HF epithelium can present self-antigens to cognate CD8+ T cells incl hair cycle-dependent immunogenic proteins including HF-specific neo-antigens

HYPOTHESIS

IFNg derived from antigen-specific T cells spotting self-peptides may induce amp alter self-antigen presentation (induced-self) Initially this silences autoreactive T cells including neo-epitope-specific T cells Since the thymus cannot recapitulate neo-epitopes evolving in the periphery HF-specific induction of MHC molecules that present these neo-epitopes to self-peptide-reactive CD8+ T cells is a key element of self-tolerance Subsequently however the local perpetuation and modification of this crosstalk induces HF IP collapse and AA

complements thymus-based regulation models of selfnon-self-discrimination induced-self in HF maintains peripheral self-tolerance in the case of danger Can backfire leading to AA