The conjunctiva as a site for investigation of human mucosal immunology in situ – elucidating the mechanisms of immune escape in adenovirus-induced

epidemic keratoconjunctivitis (EKC)

Makoto Yawata1,2, Kevin John Selva2, Jay Siak3, Liu Yu Chi3, Louis Tong3,4, Jodbhir S. Mehta3,4, Nobuyo Yawata2,3,4

1Department of Pediatrics, School of Medicine, National University of Singapore 2Singapore Institute for Clinical Sciences, Agency for Science Technology and Research 3Singapore Eye Research Institute 4Duke-NUS Graduate Medical School

Mucosal Immunology and Vaccine development, Melbourne, July 28 2016

Inflammation of the ocular mucosal surface caused by adenovirus infection

– EKC (epidemic keratoconjunctivitis)

Human NK cell populations and their regulation

Profiling NK cells in the conjunctiva mucosa over the course of EKC

Elucidating the mechanisms of immune subversion by adenoviruses

Topics

Group D human adenoviruses (HAdV) cause

epidemic keratoconjunctivitis (EKC)

Human adenovirus types are classified into seven groups

Subepithelial keratitis Pseudomembrane

Group Type Clinical diseases

A 12, 18, 31

B 3, 7, 11, 14… Conjunctivitis

Pharyngitis

Pneumonia

C 1, 2, 5, 6 Pharyngitis

Pneumonia

D 8, 9, 19, 37, 53, 54… EKC

E 4 Conjunctivitis

Pneumonia

F 40, 41 Gastroenteritis

G 52 Gastroenteritis

Severe conjunctivitis

0 7

Conjunctivitis (non EKC)

Conjunctivitis (EKC)

Mechanisms?

Clinical features of epidemic keratoconjunctivitis (EKC)

Severe and prolonged inflammation Group D Human adenoviruses (HAdV) cause EKC

Inflam

mation

Pseudomembrane Severe conjunctivitis

Natural Killer cells provide initial protection

against virus infections and prime adaptive immunity

2 7 10

NK cells

days

Innate immunity

T cells

Cytotoxicity

Cytokine production

IgM

IgG

Adaptive immunity

Cytotoxicity

Cytokine production

Viral load Infection

B cells

Immunoglobulin

Prime Th1 anti-viral response

NK cells are controlled through a balance in signaling from

inhibitory and activating factors

Killer Cell Immunoglobulin-like Receptors

(KIR2DL1/2/3, 3DL1)

NKG2A

LILRB1

2DS1, 3DS1

NKG2C

NKG2D

DNAM-1

NKp30/46

CD16

Cell contact factors

Soluble factors

IL-10

TGF- IL-12, IL-15, IL-18

IFN-

IL-2

HLA-C?

?

HLA-G

HLA-E

MICA&B/ULBP

Fcγ

HLA-C

CD48

BAT3

HA

HA

CD112/155

Activating receptors Receptor Ligand

KIR2DL1 HLA-C2

KIR2DL2/3 HLA-C1

KIR3DL1 HLA-B Bw4

KIR2DL4 HLA-G

KIR2DL5A,5B ?

KIR3DL2 HLA-A3/11

KIR3DL3 ?

NKG2A/CD94 HLA-E

LILRB1 LILRB2 HLA-A,B,C,G

KIR2DS1,2,3,4,5

KIR3DS1

KIR2DL4

NKG2C

NKG2D

CD16

CD160

2B4

NKp30

NKp44

NKp46

DNAM-1

Inhibitory receptors Receptor Ligand

Variegated expression Homogeneous expression

Inhibitory receptors Activating receptors Yawata et al. Blood 2009

Vari

ab

le

Co

nserv

ed

HLA class I-specific inhibitory receptors

create NK cell heterogeneity

‘Missing-self’ response is unique to NK cells

NK

Reduced inhibition of NK cells through

cognate KIR-HLA class I interaction

cytolysis

cytokine production

Abnormal cells virally infected cells cancer cells

NK

Activating ligand

HLA class I /HLA-E

(inhibitory ligand)

iKIR/NKG2A

Normal cells

Activating receptor

Human natural killer cell subsets have distinct function

CD56dim CD56bright

CD56

CD3

Mature Immature

Cytotoxicity +++ +

Cytokine production by monokines + +++

Chemokine receptors CXCR1, CX3CR1 CXCR3, CCR5, CCR7

HLA specific inhibitory receptors KIR, NKG2A NKG2A

ADCC CD16++ CD16-

NK cell populations in human organs

Decidua: CD56bright

Angiogenic cytokines

Lower reproductive tract: CD56dim

Lymph node: CD56dim < CD56bright

PBMC/spleen: CD56dim > CD56bright

IFN-g

Tonsil: CD56dim < CD56bright

IL-22 & IFN-g

Intestine: CD56bright

IL-22

The eye: ?

Lung: CD56dim

Mature CD56dim NK cells are the dominant type in the conjunctiva

Exp

ressio

n f

req

uen

cie

s

on

CD

56

dim

NK

cells (

%)

PBMC

Conjunctiva n=8

SSC-H

SS

C-A

CD45

Dea

d c

ell

CD

3

CD56 FSC-A

SS

C-A

PBMC

Conjunctiva

51.7

0.7

2.4 0.08

CD

16

CD56

lymphocyte

monocyte

NK cells

CD56dimNK cells

CD56brightNK cells T

he

ra

tio

of

CD

56

bri

gh

t /C

D56

dim

NK

ce

lls

Yawata et al., Mucosal Immunology 2015

Fre

qu

en

cie

s in

CD

3- C

D5

6+ c

ell

s(%

)

Ocular surface NK cells produce anti-viral cytokines,

similar to those in peripheral blood

PBMC

Conjunctiva

n=3

PMA(2ng/ml)

Ionomycin (1ug/ml)

5 hrs

Yawata et al., Mucosal Immunology 2015

CD56bright NK cells increase

in acute adenovirus-induced conjunctivitis

18.2

7.3

FSC-A

SS

C-A

CD56

CD

3

Acute phase

Convalescent

phase

Mild conjunctivitis

(HAdV-3)

Acute phase

Convalescent

phase

Severe conjunctivitis

(HAdV-8)

16.3

1.4

0.4

29.6

11.6

0.8

60.2

5.2

PBMC

CD56

CD

16

lymphocyte

monocyte

NK cells CD56dimNK cells

CD56brightNK cells

Yawata et al., Mucosal Immunology 2015

CD56bright NK cells increase

in adenovirus-induced conjunctivitis

n=12

CD

45

+ in

to

tal

ce

lls

(%

)

Fre

qu

en

cie

s in

CD

45

+ c

ell

s (

%)

Th

e r

ati

o o

f

CD

56

bri

gh

t /C

D56

dim

NK

ce

lls

Yawata et al., Mucosal Immunology 2015

Multiplex beads assay

Chemokines attracting CD56bright NK cells (CXCR3, CCR5)

Chemokines attracting CD56dim NK cells (CXCR1, CXCR2, CXCR4)

Acute phase

Convalescent phase

Healthy control

Elevated levels of chemokines that recruit CD56bright NK cells

are identified in the tear fluid in the acute phase of adenovirus-infected conjunctiva

n=13

Yawata et al., Mucosal Immunology 2015

PBMC PBMC

Donor 2

left day2 right day5

Donor 3

left day6 right day8

Iso

typ

e c

on

tro

l

CX

CR

3

CC

R5

C

CR

2

FSC

Donor 1

left day1

Fre

qu

en

cie

s in

NK

ce

lls

(%

)

12.4

4.6

15.4

63.3

64.7

61.2

4.0

54.8

77.8

1.4

21.1

60.5

15.4

39.0

46.4

46.2

34.1

51.4

The CD56bright, immature NK cells express the receptors

CXCR3 and CCR5 in the acute phase of viral inflammation

The conjunctiva NK cells recruited to the conjunctiva in adenovirus infection

(CD56bright NK cells and NKG2A+NK cells) display are functionally suppressed

MF

I o

f H

LA

-DR

HL

A-D

R+ c

ell

s (

%)

MF

I o

f H

LA

-DR

HL

A-D

R+ c

ell

s (

%)

HLA-DR

NK

G2

A

CD

56

Convalescent phase Acute phase

Yawata et al., Mucosal Immunology 2015

Summary from ex vivo study

1. Mature NK cells are the dominant NK cell type in the conjunctiva.

2. Immature NK cells are recruited during adenovirus infection.

3. NKG2A+NK cells increase in severe conjunctival inflammation.

4. Mature NKG2A-NK cells display an activated state.

HAdV

A549

lymphocytes IFN-g Viral protein

CD56dim NK cells produce IFN-g against HAdV-infection

0 102

103

104

105

Intracellular IFN-γ (%)

CD

56

Mock HAdV-3(B)

0 102

103

104

105

0

102

103

104

105

0 102

103

104

105

HAdV-8(D)

15.2% 5.2% 0.7%

Intr

ace

llula

r IF

N-γ

(%

) n=3

0 102

103

104

105

0

102

103

104

105

0 102

103

104

105

0 102

103

104

105

0

102

103

104

105

Viral protein (hexon)

HAdV-3(B) Mock

SS

C

0 50K 100K 150K 200K 250K

0

102

103

104

105

Epithelial cells HAdV-8(D)

PBMC

FSC

63% 45%

Cell contact-dependent NK cell activation in HAdV infection In

tra

ce

llu

lar

IFN

-g (

%)

Yawata et al., Mucosal Immunology 2015

Inhibitory receptor–ligand interactions?

Activating receptor–ligand interactions?

IF

N-γ

+N

K c

ell

s (

%)

Weaker IFN-g production by NKG2A+NK cells

Mock HAdV-3 HAdV-8

NKG2A+ NK cells showed

lower response against HAdV-infected cells

Positive control

Non-licensed NK cells

NKG2A+ NK cells

NKG2A- KIR+ NK cells

IFN-g+ NK cells (%)

Yawata et al., Mucosal Immunology 2015

inhibition

Self specific iKIR

or NKG2A

HLA class I

HLA-E

Normal cells

Activation <

Abnormal cells

Activation

HLA class I HLA-E Ligand for NKG2A Ligand for KIR

Mock

HAdV-3

HAdV-8

Weaker IFN-g production by NKG2A+NK cells

Lower IFN-g response by NK cells against group D HAdVs

IFN

-+N

K c

ell

s (

%)

D B E C

Yawata et al., Mucosal Immunology 2015

DNAM-1 and NKG2D are key for NK cell activation

against HAdV infection

Intr

ac

ell

ula

r IF

N-g

(%

)

n=3

NKG2A-NK cells NKG2A+NK cells

Yawata et al., Mucosal Immunology 2015

Ex

pre

ssio

n f

req

ue

nc

ies

on

CD

56

dim

NK

ce

lls

(%

)

CD155 (Ligand for DNAM-1)

CD112 (Ligand for DNAM-1)

MICA/B (Ligand for NKG2D)

ULBP-1 (Ligand for NKG2D)

ULBP-2 (Ligand for NKG2D)

ULBP-3 (Ligand for NKG2D)

Mock

HAdV-3

HAdV-8

Isotype control

Ligands for DNAM-1 are reduced

in HAdV-8 infection

CD

155 M

FI

Group D HAdVs down-regulate specific activating ligands

on infected epithelia and dampen NK cell responses

D HAdV group B E C

*

D B E C

CD

112 M

FI

Yawata et al., Mucosal Immunology 2015

Infected epithelial cells

Ocular surface mucosa Peripheral blood

CD56dim NKG2A-

NKG2A

CD56dim NKG2A- NK cells are not activated.

HLA-E inhibits NKG2A+NK cells.

HLA-E

KIR

a

CD56dim NKG2A+

a

CD56bright NKG2A+

CD56dim

CD56bright

HLA class I, DNAM1 ligands

a

Immature CD56brightNK cells and NKG2A+mature CD56dimNK cells are inhibited

by up-regulated inhibitory ligand on HAdV-infected epithelium.

Group D HAdV escape from mature NK cell anti-viral response

by down-regulating activating ligands on infected cells.

Summary

Acknowledgements

Singapore Eye Research Institute (SERI)

Woon Kaing

Jessie Lim

Kevin John Selva

Yu-Chi Liu

Jodhbir Singh Mehta

Louis Tong

Wanwen Lan

Singapore National Eye Centre (SNEC)

Jay Jyh Kuen Siak

Anshu Arundhati

Singapore Institute for Clinical Sciences (SICS)

Nobuyo Yawata

National University of Singapore (NUS)

Naoki Yamamoto

Hokkaido University

Koki Aoki

Hidemi Watanabe

Shigeaki Ohno

Fukushima Medical University

Hisatoshi Kaneko

National institute of Infectious diseases

Tsuguto Fujimoto

Agency for Science, Technology and Research (A*STAR) National Medical Research Council (NMRC), Singapore