Rationale for Modulating Adaptive Immunity:

Therapeutic Vaccination, Checkpoint Inhibitor

and others

Robert Thimme

Clinic for Gastroenterology, Hepatology, Infectious Diseases and Endocrinology

University of Freiburg

Fibrosis / cirrhosisHCC

Acute

hepatitis

20-30% in 20-30 years 1-5% per year

Viral elimination

Rationale for modulating T cell responses

in viral hepatitis

HBV: >95%

HBV: <5%

Chron. hepatitis

Virus-specific

CD8+ T cellCD8+

weeks after infection

HBV DNA

T-ce

ll re

spo

nse

0 2 4 6 8 10 12 14 16 18 20 22

Maini et al., 2001

Boettler et al., 2006

weeks after infection

HBV DNA

T-ce

ll re

spo

nse

0 2 4 6 8 10 12 14 16 18 20 22

Temporal association of CD8+

T-cell response and viral load

Depletion of CD8+ T-cells

prolongs viremia

Thimme et al., 2003

CD8+ T-cells are the main effector cells against HBV

► Role of HBV-specific CD8+ T cells in strategies aiming at virological cure

of chronic HBV infection!

Key open question II

• Are HBV specific CD8+ T cells present during chronic

HBV infection?

• What are their phenotypical and functional

characteristics?

• Best approach for T cell restoration?

Questions

Methods

Patient cohort 1

• Chronic HBV infection, genotype D (n=70)

- mainly low viral loads

- mainly HBeAg negative

• Resolved HBV infection (n=11)

• Acute HBV infection (n=2)

Rehermann/ Thimme, Gastroenterology 2018, in press

Stimulation of PBMCs with 223 overlapping peptides (18-mers, genotype D)

• 20% of PBMC loaded with peptide x 1 hour, washed

• 80% of PBMC added

• Culture for 10 days

Screen for responses with the overlapping peptides using Elispot

Confirmation of positive responses by intracellular IFN-γ staining using single peptides

HLA-restriction using EBV-transformed B-cells (B-LCL)

Epitope fine-mapping

Methods Patient cohort 1

• Chronic HBV infection, genotype D (n=70)

- mainly low viral loads

- mainly HBeAg negative

• Resolved HBV infection (n=11)

• Acute HBV infection (n=2)

Experimental approach 1

70 patients with chronic HBV infection

(genotype D): 59 CD8+ responses

No detectable

CD8+

response

Detectable

CD8+

response

Functional HBV-specific CD8+ T-cell responses are

present in ~50% of chronically infected patients

CD8

IFN

-γ

Without

peptide

OLP175

LIISCSCPTVQASKLCLG

3.9%

Without

peptide

OLP51

IPRTPARVTGGVFLVDKN

1.4%

Patient 7

Lack of HBsAg-specific responses in chronic infection

Breadth of CD8+ T-cell responses in chronic

infectionn

um

ber

of

IFN

-y+C

D8

+ r

esp

on

ses

20 40 60 80 100 120 140 160 180 200 2200

1

2

3

4

5

6

polymerase1-119

surface120-173

core174-203

X204-225

Chronic HBV infection (n=70)

nu

mb

er

of

IFN

-y+C

D8

+ r

esp

on

ses

20 40 60 80 100 120 140 160 180 200 2200

1

2

3

4

5

6

polymerase1-119

surface120-173

core174-203

X204-225

► Differential targeting compared to acute-resolving infection?

Chronic HBV infection (n=70)

Resolved HBV infection (n=11)

nu

mb

er

of

IFN

-y+C

D8

+ r

esp

on

ses

20 40 60 80 100 120 140 160 180 200 2200

1

2

3

4

5

6

polymerase1-119

surface120-173

core174-203

X204-225

Breadth of CD8+ T-cell responses in

resolved versus chronic infectionn

um

ber

of

IFN

-y+C

D8

+ r

esp

on

ses

20 40 60 80 100 120 140 160 180 200 2200

1

2

3

4

5

6

polymerase1-119

surface120-173

core174-203

X204-225

Chronic HBV infection (n=70)

Resolved HBV infection (n=11)

Breadth of CD8+ T-cell responses in

acute / resolved versus chronic infection

Acute HBV infection (n=2)

nu

mb

er

of

IFN

-y+C

D8

+ r

esp

on

ses

20 40 60 80 100 120 140 160 180 200 2200

1

2

3

4

5

6

polymerase1-119

surface120-173

core174-203

X204-225

Lack of HBsAg-specific responses is unique to persistent

infection!

nu

mb

er

of

IFN

-y+C

D8

+ r

esp

on

ses

20 40 60 80 100 120 140 160 180 200 2200

1

2

3

4

5

6

polymerase1-119

surface120-173

core174-203

X204-225

Are HBsAg specific CD8+ T cells really absent or

undetectable by functional assays?

detectable virus-specific

CD8+ T-cell response

no detectable virus-specific

CD8+ T-cell response

core18

87.0%

n=23

env183

n=32

25.0%

pol455

n=33

66.7%

Core18- and pol455- but not env183-specific CD8+ T cells are detectable in the

majority of analyzed patients

0.35%

CD8

tetr

am

er

before

enrichment

after

enrichment

7.92% 0.0%

before

enrichment

1.22%

after

enrichment

pol455core18

before

enrichment

env183

0.0%

after

enrichment

0.13%

• HBV specific CD8+ T cell present at low frequencies in

most patients

• Specific lack of HBsAg specific CD8+ T cells in chronic

infection

Summary I

Key open question II

• Are HBV specific CD8+ T cells present during chronic

HBV infection?

• What are their phenotypical and functional

characteristics?

• Best approach for T cell restoration?

Questions

Exhausted CD8+ T cells

Expression of exhaustion markers

(e.g. PD1high, CD39+)

BCL2low

Eomeshigh

Functionally impaired

(e.g. cytokines, proliferation)

Impaired T-cell homeostasis

(e.g. CD127low)

Eventually depleted

by apoptosis

T-betlow

Moskophidis, Zinkenagel Nature 1993

Gallimore, Zinkernagel, JEM 1998

Zajac, Ahmed JEM 1998

Wherry, Ahmed J Virol 2003

Paley, Wherry Science 2012

Doering, Wherry, Immunity 2013

Gupta, Hainning PloS Path 2015

Utzschneider, Zehn Immunity 2016

Im, Ahmed Nature 2016

Bengsch, Wherry, Immunity 2018

Heterogeneous

populations

Are exhausted HBV-specific CD8+ T cells comprised

of different subsets?What is meant by different subsets?

CD

127

PD1

TCF1+

CD127+

BCL2high

PD1+

Eomeslo / T-betlo

TCF1-

CD127-

BCL2+

PD1high

Eomeshigh / T-betlo

CD39+

HCV-specific CD8+ T cells Phenotypic/functional analyses

Memory-like

phenotype

severely

exhausted

associated with

expansion capacity

in vitro

Wieland et al., Nat Commun 2017

DAA-mediated

HCV clearance

Chronic HCV

infection

CD

127

PD1

HCV

CD

127

PD1

HCV

Maintenance of a memory-like

T-cell population

•Antigen-independent survival

• Increased functionality

CD127+

PD1+

CD127-

PD1high

Antigen

removal

Wieland et al., Nat Commun 2017

Loss of terminally exhausted HCV-specific CD8+ T

cells after antigen elimination

Rehermann/ Thimme, Gastroenterology 2018, in press

Loss of terminally exhausted HCV-specific CD8+ T

cells after antigen elimination

Are HBV-specific CD8+ T cells comprised of

different subsets?

PD1

CD

12

7

core18 pol455

78.9%

21.1%

89.6%

8.83%

CD8+ CD8+

HBV

memory-like

phenotype

severely

exhausted

CD127+

PD1+

TCF1++

CD127-

PD1+

Eomeshi / T-betlo

HCV specific CD8+ T cells are more exhausted compared to HBV

Cor and polymerase show phenotypical differences

memory-like

phenotype

c o r e p o l

0

2 0

4 0

6 0

8 0

1 0 0

%C

D1

27

+P

D1

+ core18

core141

pol455

pol173

**

PD1

CD

12

7

HCV

41.1%

58.6%

CD8+

Central role for Tox in T cell exhaustion

Tox expression is high in cells with

functional exhaustion (FES)

Bengsch/Wherry Immunity 2018

High Tox

Expression in

Exhausted T

cell subsets

Central role for Tox in virus-specific T cells

in chronic vs acute infection

LCMV HIV/NSCLC

Doering/Wherry Immunity 2013

TOX

pM

HC

I-Te

t HCV HBV

Different Tox expression in HBV versus HCV

TOX expression also reveals less severe exhaustion of HBV-specific CD8+ T cells

HC

V

HC

V D

AA

HB

Vco

re18

HB

Vp

ol4

55

0

2 0

4 0

6 0

8 0

1 0 0

% T O X +

HC

V

HC

V D

AA

HB

Vco

re18

HB

Vp

ol4

55

0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

5 0 0 0

M F I T O X

Differences between cor and pol-specific CD8+ T

cell responses

core

0 2 0 4 0 6 0 8 0 1 0 0

0

1

2

3

4

5

% C D 1 27 +P D 1+

ex

pa

ns

io

n fa

cto

r

LO

G (

EI+

1)

core18

core141

R2=0.2493

p=0.0295

pol

0 2 0 4 0 6 0 8 0 1 0 0

0

1

2

3

% C D 1 27 +P D 1+

ex

pa

ns

io

n fa

cto

r

LO

G (

EI+

1)

pol455

pol173

R2=0.03281

p=0.5183

Expansion of core18/141- but not of pol455/173-specific CD8+ T-cell populations

is linked to the memory-like subset

c o r e p o l

0

2 0

4 0

6 0

8 0

1 0 0

%C

D1

27

+P

D1

+ core18

core141

pol455

pol173

memory-like***

c o r e 18 p o l455

0

1

2

3

4

5

ex

pa

ns

ion

fa

cto

r

LO

G (

EI+

1)

expansion**

What are the differences of memory-like

core18/141- vs. pol455/173-specific CD8+ T cells?

c o r e p o l

0

2 0

4 0

6 0

8 0

1 0 0

%T

CF

1+

of

CD

12

7+

PD

1+

memory-like

TCF1

(proliferative capacity)

TCF1

core18

pol455

CD8+

93.4%

91.6%

48.9%

c o r e p o l

0

2 0

4 0

6 0

8 0

1 0 0

%T

CF

1+

core18

core141

pol455

pol173

Similar TCF1 expression in memory-like core18/141- and pol455/173-specific CD8+ T cells

What are the differences of memory-like

core18/141- vs. pol455/173-specific CD8+ T cells?

c o r e p o l

0

2 0

4 0

6 0

8 0

1 0 0

%B

CL

2h

igh

***

c o r e p o l

0

2 0

4 0

6 0

8 0

1 0 0

%B

CL

2h

igh

of

CD

12

7+

PD

1+

*

memory-like

BCL2

(survival capacity)

BCL2

core18

pol455

CD8+

57.7%

40.3%

53.9%

core18

core141

pol455

pol173

0 2 0 4 0 6 0 8 0 1 0 0

0

1

2

3

4

5

% B C L 2h igh

ex

pa

ns

ion

fa

cto

r

LO

G (

EI+

1)

R2=0.3391

p=0.0071 core18

core141

pol455

pol173

Decreased BCL2 expression of memory-like pol455/173-specific CD8+ T cells...

...correlates with the decreased expansion capacity.

HBV-specific CD8+ T cells are heterogeneous

CD

127

PD1

Memory-like

phenotype

severely

exhausted

• consisting of distinct subsets:

• differing with respect to their targeted epitopes:

Core18/141 Pol355/173

expansion

severly exhausted

memory-like TCF1BCL2 TCF1BCL2

Relevance? – immunotherapies targeting T cells

Summary II

Schuch et al., GUT 2018, in press

Bertoletti, Gut Liver 2017

Rationale for Modulating Adaptive Immunity:

Therapeutic Vaccination, Checkpoint Inhibitor

and others

Bertoletti/ Ferrari, Gut 2012

Rationale for Modulating Adaptive Immunity:

Therapeutic Vaccination, Checkpoint Inhibitor

and others

Env183Core18 Pol355

Absence Mild Exhaustion Sev. Exhaustion

vaccination Antigen reduction PD1 blockade Cytokines for survival?

CD8+ T cell epitope

Mechanims of T cell

failure

Eomeshigh

T-betlow

Epitope specific CD8+ T cell failure may lead to specific therapeutic strategy

Hofmann/ Thimme

Annegrit Decker

Anna Globig

Kathrin Heim

Nina Hensel

Catharina Kramer

Sebastian Merker

Charlotte Rennert

Josefine Schardey

Anita Schuch

Özlem Sogukpinar

Catrin Tauber

Dominik Wieland

Elisa Wüstrich

Britta Zecher

Sebastian Zehe

Xin Zhou

Christoph Neumann-Haefelin

Janine Kemming

Michael Kiraithe

Elahe Salimi

Department of Medicine II - University of Freiburg

AcknowledgementsAll patients and volunteers!

Tobias Böttler

Kristi Basho

Maike Smits

Katharina Wild

Katharina Zoldan

Marissa Russ

FreiburgBasel

Strasbourg

Hepato-Regio-NetTRR 179

Bertram Bengsch

Anna Katharina Braun

Patricia Otto-Mora

Thank you for your attention!

Impressions from Freiburg in the black forest!