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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
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Targeting mitochondrial dysfunction can restore antiviral activity of exhausted HBV-specific CD8 T cells in chronic
hepatitis B
Fisicaro et al, Nature Med 2017
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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
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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
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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
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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
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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
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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
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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?
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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
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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
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Gut 2019
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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
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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
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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
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Nature 2019
Mann, Kaech Nature Immunology 2019
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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
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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
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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
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Thank you for your attention!
Impressions from Freiburg in the black forest!