High resolution of exhausted HBV specific CD8+ T cells in chronic HBV infection

Robert Thimme

Clinic for Gastroenterology, Hepatology, Infectious Diseases and Endocrinology

University of Freiburg

Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic

hepatitis B

Fisicaro et al, Nature Med 2017

Characteristics of exhausted CD8+ T cells

High expression of inhibitory receptors

(e.g. PD1high)

BCL2low

Functionally impaired

(e.g. cytokines, proliferation)

Impaired T-cell homeostasis

(e.g. CD127low)

Eventually depleted

by apoptosis

Eomeshigh

T-betlow

Moskophidis, Nature 1993Galimore, JEM 1998Zajac, JEM 1998Wherry, J Virol 2003Wherry, Immunity 2007Paley, Science 2012Doering, Immunity 2012Buggert, PloS Path 2014Utzschneider, Immunity 2016Im, Nature 2016Fisicaro, Nat. Med. 2017Bengsch, Immunity 2017/2018Wieland, Nat. Communication 2016

Heterogeneous

populations

Altered metabolism

Distinct transcriptional signature

Distinct epigenetic profile

Relevant in defining

therapeutic targets

Subsets of exhausted virus-specific CD8+ T cells

What is meant by different subsets? - Lessons from HCV

CD

127

PD1

TCF1+

CD127+

BCL2high

KLRG1+

PD1+

Eomeslo / T-betlo

TCF1-

CD127-

BCL2+

KLRG1+

PD1high

Eomeshigh / T-betlo

CD39+

TOX

HCV-specific CD8+ T cells Phenotypic/functional analyses

Memory-like

phenotype

severely

exhausted

associated with

expansion capacity

in vitro

Wieland et al., Nat Commun 2017Alfei et al, Nature 2019

1

2

3

CD

127

PD1

Single cell

RNA sequencing

Chronic HCV

infection

Memory-like

CD127hi PD1+

Terminally exhausted

CD127- PD1hi

Intermediate

Diffusion Pseudotime

Heterogeneity of HCV specific CD8+ T cellsby single cell RNA sequencing

C1 → C2 → C3

Bcl-2

CD127

TCF-1

GZMA

IFITIM1

NKG7

HLA-DRA

Differentiation pathway:memory-like→ intermediate → terminally exhausted

DAA-mediated

HCV clearance

Chronic HCV

infection

CD

127

PD1

HCV

CD

127

PD1

HCV

Maintenance of a memory-like

T-cell population

- With remaining chronic signature

- With specific gene regulation

CD127+

PD1+

CD127-

PD1high

Antigen

removal

Fate of HCV specific CD8+ T cells after DAA elimination

Wieland et al., Nat Commun 2017

Memory-like cells before and after DAA therapy are quite similar→maintenance?!

Fate of HCV specific CD8+ T cells after DAA elimination

Il7r – CD127

0

25

50

75

100** **

% C

D1

27

su

bse

ts

Quadratic programmingHCV-specific CD8+ T cells after versus before DAA therapy

(measure of similarity)

before DAA

after DAA

Rehermann/ Thimme, Gastroenterology 2019

Maintenance of a memory-like T-

cell population

•Antigen-independent survival

• Increased functionality

•Recall expansion

Fate of HCV specific CD8+ T cells after DAA elimination

conventional memory

During and afterchronic HCV infection

Cluster 1

Cluster 2

Low input RNAseq - unsupervised clustering of CD127/PD1 subsets

severely exhausted

memory-like

conventional memory

HCV specific CD8+ T cells display chronic scar after DAA elimination

Also different subsets in HBV infection?

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

Memory-like subsets dominate HBV-specific CD8+ T-cell populations withdifferent frequencies depending on the targeted epitope

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+

***

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

Epitope specific differences in HBV

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

Schuch et al., Gut 2019

Gut 2019

How different are core- and pol-specific CD8+ T cells?

core- and pol-specific CD8+ T cells show different transcriptomes

Single cell RNAseq

core18-specific CD8+ T cellspol455-specific CD8+ T cells

Single cell RNAseq – compiled data of 4 patients

Transcriptomic profiles of core- versus pol-specific CD8+ T cells

pol-specific CD8+ T cells

core-specific CD8+ T cells

Transcriptomes of core- and pol-specific CD8+ T cells are clearly distinct

Differentiation status of core- and pol-specific CD8+ T cells

pol-specific CD8+ T cellscore-specific CD8+ T cells

→ terminal CTL differentiation→ precursor CTL differentiation

Nature 2019

Mann, Kaech Nature Immunology 2019

Is there an exhaustive signature in core- and pol-specific CD8+ T cells?

HBVcore18 HBVpol455 cHCV

0

20

40

60

80

100

%TOX

%T

OX

cHBVHBeAg+

cHBVHBeAg-

0

20

40

60

80

100

%TOX

%T

OX

HBVpol455

HBVcore18

→ low TOX expression...

...is associated with clinical state

...is associated with epitope

pol-specific CD8+ T cells

core-specific CD8+ T cells

TOX

Summary

High resolution of exhausted virus-specific CD8+ T cells in chronic HBV and HCV infection reveals:

• Heterogeneity of HCV specific CD8+ T cells linked to antigen recognition

• Differential subset distribution in HBV versus HCV with lack of terminally exhausted CD8+ T cells in HBV

• HBV specific CD8+ T cells differ in transcriptional, phenotypical and functional profile on an antigen-specific level (core versus pol)

• Impact of clinical state (HbeAg, viral load) on TOX expression of HBV specific CD8+ T cells

Complex immunological and virological determinants ofvirus-specific CD8+ T cell exhaustion/ differentiation in chronic HBV

Acknowledgements

Maike HofmannFranziska DaulKatharina DörnbrackKatharina GrützmannKathrin Heim

Nina Hensel

Sebastian Merker

Laura Polcik

Anita Schuch

Özlem Sogukpinar

Catrin Tauber

Dominik Wieland

Elisa Wüstrich

Sebastian Zehe

Christoph Neumann-Haefelin

Janine Kemming

Valerie Oberhardt

Elahe Salimi Alizei

Isabel Schulien

Department of Medicine II - University of Freiburg

All patients

and

volunteers!

Tobias Böttler

Benedikt Csernalabics

Sabine Ehrlich

Rafael Käser

Saskia Killmer

Maike Smits

Katharina Wild

Katharina Zoldan

Bertram Bengsch

Patricia Otto-MoraFrances Winkler

Zheng Zhang

Nico Büttner

Ralf Bartenschlager

Naveed Ishaque / Zuguang Gu

Dominic Grün / Sagar

Christian Konrad / Katharina JechowPeter HasselblattAnna Globig

Thank you for your attention!

Impressions from Freiburg in the black forest!