high resolution of exhausted hbv specific cd8+ t cells in...
TRANSCRIPT
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!