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Prevention of T cell ageing will
rejuvenate anti-cancer efficacy
Graham Pawelec
University of Tübingen Medical School
Tübingen
Germany
IABG 10, Cambridge, 20th September 2003
Cancer is a genetic disease:
Cancer cells are altered self by virtue
of aberrant gene expression
As a result of this:
Cancer cells contain proteins not found in normal cells,
or not expressed at high levels in normal cells,
or only expressed in normal cells at certain stages of development
Types of tumour antigens
MHC class II restricted
 Products of translocations, eg. bcr/abl (Pawelec et al 1996)
abl/bcr (Wagner et al 2002)
 Differentiation antigens, eg. gp100 (Halder et al 1997)
 Transcription factors, eg. WT-1 (Knights et al 2002)
 Cancer/testis antigens, eg. HAGE (Knights et al 2003)
Tumour cells are visible to the immune system
The immune system exists to recognise and respond to non-self
Many studies prove beyond doubt:
CANCER IS IMMUNOGENIC
.........and tumour-specific T cells can destroy tumours and
cure mice
Hypothesis
The innate immune
system first recognises
tumour cells and
produces IFN-
Inflammatory cascade
causes limited
tumour cell death and
dendritic cells then
transport tumour
products to the
draining lymph node
(Dunn et al., Nature
Immunol Nov. 2002)
The natural immune system
controls tumour while
specific T cells develop
in the lymph nodes
Specific T cells (mostly CD8)
infiltrate the tumour and
destroy cells expressing
appropriate tumour antigens
So if cancer is immunogenic and
immunosurveillance exists
Why does cancer occur?
Why is the immune response not successful?
In fact,
The immune response is mostly successful
but
Tumours escape from the immune response
Tumour escape is the main hurdle
for immunotherapy
Tumours predominantly employ two escape strategies:
 Antigen loss
 Immunosuppression
Antigen loss
(Khong & Restifo, Nature Immunol Nov 2002)
Immunosuppression
The tumour environment is immunosuppressive
 Production of soluble factors (IL 10, TGF-ß)
 Destruction of tumour-infiltrating cells (fas; free radicals)
 Defusing tumour-infiltrating cells (anergy induction)
 Induction of suppressor cells (Treg)
 Clonal exhaustion (proliferative senescence)
Requirements for T cell-mediated
immune responses
T cell activation
clonal expansion
CD28
Costimulation
IL2 gene
TCR
Ag/MHC
complex
eg. virus
IL2 receptor gene
IL2-receptor
....and
exhaustion
IL2-secretion
autocrine proliferation
We can model this
process in vitro
Longevity of human CD4+ T cell clones
Origin
%CE
Clones/
Expts
Percentage of clones reaching: Max.
20 PD 30 PD 40 PD
longevity
CD3 (young)
CD3 (old)
CD3 (CML)
47
52
49
1355/15
116/2
35/1
47
55
60
24
22
35
15
16
14
170
72
51
CD34 (periph)
CD34 (cord)
55
43
533/6
94/2
31
29
17
15
6
5
60
57
Decreased density of expression of CD28
clone 402-16
100
100
CD3
MFI
75
50
CD95
CD134
CD154
25
75
50
25
CD28
0
40
50
60
70
0
80
PD
Pawelec 2002
Decreased production of TNF- correlates with
recovery of CD28 expression
clone 401-2
100
1500
CD3
75
TNF-
MFI
50
CD95
CD28
TNF-
1000
500
25
0
20
CD134
CD154
30
40
50
60
0
70
PD
Pawelec 2002
Effect of anti-TNF-
402-16 + anti-TNF
402-16
401-2 + anti-TNF
401-2
CPD
50
40
30
50
75
100
125
150
175
Days
(401-2 loses TNF- production capacity at ca. 40 PD; 402-16 retains TNF- production)
Telomerase not induced in old
CD4+ T cell clones
Telomerase activity
3
Young clone, - IL 2
Young clone, + IL 2
Old clone, + IL 2
Old clone, - IL 2
2
1
0
-24
0
24
48
Kinetic, hours
72
96
Engel 2001
Is limited longevity of CD4+ TCC
to do with telomere length?
Decreasing TL in cultured TCC
12.5
399-35
399-37
400-23
400-60
397-19
TL
10.0
7.5
r
P
0.12
0.74
0.95
0.95
0.99
0.65
0.14
0.005
0.028
0.002
5.0
2.5
25
35
45
55
65
75
(MESF x 0.495 = TL kb)
CPD
clones 399 are from a >85 yr old SENIEUR donor
clones 400 are from a 26 yr old healthy donor
clones 397 are from CD34+ bone marrow cells
Brummendorf 2002
Is limited longevity to do with telomere length?
 telomerase induction in all clones seems to decrease with age
 telomere length in most clones decreases with age
 already short telomeres in SENIEUR-derived clones remain stable
 SENIEUR-derived clones are no more long-lived than the others
 but, hTERT transfection can increase CD4 and CD8 TCC longevity
 is the mechanism for this something other than TL maintenance?
 is it to do with DNA repair?
Microsatellite instability
MS status was tested at 6 loci by PCR and sequencing,
comparing CD4+ TCC at 3 different ages
3 SENIEUR-derived TCC acquired MSI with increasing PD
in 5/15 cases (33%)
4 non SENIEUR TCC acquired MSI in 13/22 cases (59%)
Acquisition of MSI reflecting poorer MMR may therefore be
less common in SENIEUR-derived TCC as they age in culture
(Ben Yehuda et al 2003)
DNA damage by Comet assay
Standard oxygen tension
Reduced oxygen tension
(a)
C
l
o
n
e
P
D
C
l
o
n
e
P
D
5
0
3
9
9
3
7
4
0
3
1
2
8
3
9
9
3
7
5
2
4
3
2
9
5
3
3
9
9
3
5
4
0
2
7
4
4
3
9
9
3
5
3
6
2
9
Invitro Age(PD)ofClnes
4
3
4
0
0
2
3
5
0
3
9
2
9
Invitro Age(PD)ofClnes
4
3
4
0
0
2
3
3
6
2
7
6
5
3
8
5
7
5
4
3
3
3
8
5
7
3
0
2
7
0
6
5
5
9
3
8
5
2
4
6
2
8
4
4
3
8
5
2
3
9
3
1
1
0
2
0
3
0
4
0
5
0
0
6
0
(b)
C
l
o
n
e
P
D
4
3
4
0
0
2
3
3
6
2
7
Pyrimidines
Invitro Age(PD)ofClnes
4
4
3
9
9
3
5
3
6
2
9
Invitro Age(PD)ofClnes
5
3
3
9
9
3
5
4
0
2
7
4
0
5
0
6
0
6
5
3
8
5
7
5
4
3
3
3
8
5
7
3
0
2
7
6
5
5
9
3
8
5
2
4
6
2
8
4
4
3
8
5
2
3
9
3
1
1
0
2
0
3
0
4
0
5
0
0
6
0
1
0
2
0
3
0
4
0
5
0
6
0
%
D
N
A
i
n
C
o
m
e
t
T
a
i
l
%
D
N
A
i
n
C
o
m
e
t
T
a
i
l
(c)
C
l
o
n
e
P
D
5
0
4
0
0
2
3
4
3
3
6
2
7
C
l
o
n
e
P
D
Purines
4
3
4
0
0
2
3
5
0
3
9
2
9
3
9
9
3
7
5
2
4
3
2
9
5
3
3
9
9
3
5
4
0
2
7
4
4
3
9
9
3
5
3
6
2
9
Invitro Age(PD)ofClnes
3
9
9
3
7
4
0
3
1
2
8
Invitro Age(PD)ofClnes
3
0
C
l
o
n
e
P
D
3
9
9
3
7
5
2
4
3
2
9
6
5
3
8
5
7
5
4
3
3
3
8
5
7
3
0
2
7
6
5
5
9
3
8
5
2
4
6
2
8
4
4
3
8
5
2
3
9
3
1
0
2
0
4
3
4
0
0
2
3
5
0
3
9
2
9
3
9
9
3
7
4
0
3
1
2
8
0
1
0
%
D
N
A
i
n
C
o
m
e
t
T
a
i
l
%
D
N
A
i
n
C
o
m
e
t
T
a
i
l
1
0
2
0
3
0
4
0
5
0
%
D
N
A
i
n
C
o
m
e
t
T
a
i
l
Duggan et al 2003
6
0
0
1
0
2
0
3
0
4
0
5
0
%
D
N
A
i
n
C
o
m
e
t
T
a
i
l
6
0
Total population doublings achieved
Total Population Doublings Achieved
Clone
Standard Oxygen Tension
Reduced Oxygen Tension
385-2
69.9
61.6
385-7
73.5
71.5
399-35
72.1
45.0
399-37
78.1
51.9
400-23
80.7
51.7
Mean lifespan (PD)
74.9*
56.3*
* Significantly lower, p<0.05
Duggan et al 2003
DNA damage and repair
 DNA repair is better, and better maintained, in SENIEUR-derived TCC
 Accordingly, MSI develops less frequently as SENIEUR TCC age
 Nonetheless, there is the same level of oxidative DNA damage
 Culture in reduced oxygen results in less oxidative damage
 Culture in reduced oxygen nonetheless decreases longevity
 However, culture with PBN does increase longevity
 Therefore a critical amount of free radical production (TCR
signalling!) is required, but you can have too much of a good thing
How can we improve T cell longevity in vitro
(and in vivo?)?
 hTERT transfection
 proteasome reconstitution?
 heat shock protein expression?
 improved DNA repair (mitochondrial)?
 maintain CD28 expression, neutralise inhibitory cytokines,
provide more appropriate growth cocktails
(eg. mix IL 2, IL 7, IL 15)?
All of the above
SENIEUR-derived versus non-SENIEURderived TCC
DNA damage
MSI
DNA repair
Telomere length
Telomerase induction
CD28 level
IL 2 production
Reduced O2 culture
Reduced O2 culture
Culture with PBN
SENIEUR
non-SENIEUR
 with age
 with age
maintained
short, maintained
 with age
maintained
maintained
 DNA damage
 longevity
 longevity (10.6%)
 with age
  with age
 with age
long,  with age
 with age
usually  with age
usually  with age
 DNA damage
 longevity
 longevity (11.2%)
Dysfunctional T cells
Many of the characteristics of the dysfunctional T cells found
associated with tumours are shared with those found in ageing.








shortened telomeres
increased levels of oxidative DNA damage
decreased DNA repair
decreased expression of positive costimulatory receptors
increased expression of negative costimulatory receptors
curtailed proliferative capacity
altered cytokine secretion patterns
changes in apoptosis induction
Chronic antigenic stress
It is suggested that many of these changes are caused by
chronic antigenic stress and oxidative stress
 stimulation by tumour antigens in cancer patients
 stimulation by persistent viruses in the elderly.
 The CD8 cells are characterised by increased resistance to
apoptosis and the CD4 cells by increased susceptibility
 Hence dysfunctional CD8 cells accumulate and specific CD4
cells are clonally deleted; the CD4:8 ratio can become inverted
CMV-specific CD8+ T cells from the elderly
cannot make IFN- as well as the young
but make equivalent amounts of IL-10
Young
Old
IFN-
CD8
IFN-
IL-10
HLA-A2/CMV pp65 tetramer
IL-10
HLA-A2/CMV pp65 tetramer
(Ouyang 2003)
% positive cells
CMV-specific T cells from the old are CD28- …
100
Old
Young
75
50
25
0
CD28
CD45RO
CD45RA
and express the KLRG1 receptor
Old N50
Young 1
Young 7
A2/CMVpp65
Old N45
KLRG1
(Ouyang 2003)
Interferon- production in young and old
measured by cytoplasmic staining
(Ouyang 2002)
Keratin 18-specific CD8+ T cells are expanded
in the blood of renal cell carcinoma patients
HD2
RCC48
0.2%
A2/Keratin 18
HD1
6.9%
CD8
(Gouttefangeas 2003)
Keratin 18-specific CD8+ T cells express markers of effector
cells but do not produce IFN-
RCC48: Gated on Ker18-specific CD8+
CD28
CD57
relative copy numbers
300
230,72
200
100
0
1,00
HIV
HIV
0,84
posmix
posmix
Ker18
Ker18
0,90
1,22
KIA
KIA
Met
Met
real-time RT-PCR mRNA IFN-/CD8
10 0
10 1
10 2
10 3
10 4
(Gouttefangeas 2003)
Can we identify any better biomarkers of
ageing?
For example, using new proteomics techniques?
(Ciphergen SELDI „proteomics-on-a-chip“ in this example)
(Surface-Enhanced Laser Desorption/Ionization)
15
10
400-23.2 y pH 9
5
0
20
8000
15
10000
12000
14000
10
16000
400-23.2 o PH 9
5
0
8000
10000
12000
14000
16000
(Tolson 2002)
Artificial neural networks programs analyse
proteomics data to identify the most important
ions to distinguish aged T cells
Model II Clusters,Top 50 and Top 20 tested on 7
Unseen Clones
100
90
70
60
50
40
30
lu
st
er
1+
2+
3
C
lu
st
er
2+
3
C
lu
st
er
1+
3
C
lu
st
er
1+
2
Model
C
C
lu
st
er
3
lu
st
er
2
C
lu
st
er
1
C
20
To
p
50
20
To
p
% Correct
80
(Tolson 2003)
Acknowledgements
Center for Medical Research
University of Tübingen
Ludmila Müller
Qin Ouyang
Wolfgang Wagner
Ashley Knights
Angeliki Zaniou
Jon Tolson
Arnika Rehbein
Karin Hähnel
Lilly Wedel
2nd Dept Internal Medicine
Tim Brummendorf
Dept of Immunology
University of Tübingen
Cécile Gouttefangeas
Stefan Stevanovic
Steffen Walter
Hans-Georg Rammensee
Royal Free Hospital
Paul Travers
Tony Dodi
University of Ulster
Yonne Barnett
Paul Hyland
Karolinska Institute
Rolf Kiessling
Kalle Malmberg
Hadassah Hospital
Arie Ben Yehuda
University of Bologna
Unilever PLC
Erminia Mariani
Claudio Franceschi
Roz Forsey
Acknowledgements
We are supported by the DFG, Mildred-Scheel
Foundation, VERUM Foundation and the
Dieter Schlag Foundation, as well as the
University of Tübingen Medical School
fortune Program.
....and by
The European Commission
through projects
T-CIA
T Cells in Ageing
QLK6-CT1999-02031
QLK6-CT2002-02283
QLRI-CT2001-01325
http://www.medizin.uni-tuebingen.de
/imagine/
/t-cia/
/estdab/