Quantification of T-cell dynamics
from telomeres to DNA labelling
José Borghans
Dpt. Immunology
University Medical Center Utrecht
How long does a T cell live?
• Estimates vary widely
• How is T-cell turnover disturbed
in HIV infection, leukemia,
or after stem cell transplantation?
What is the difficulty?
How to follow a lymphocyte from its
birth to its death?
Extrapolation from mice to men v.v.
Experiments of nature (1):
T-cell reconstitution after chemotherapy
3 years of age
23 years of age
Underestimate?: Cells may also die during reconstitution
Overestimate?: Cells undergo little competition
Mackall et al. 1995
Experiments of nature (2):
Loss of T cells with chromosome damage
Immunological memory resides in a population with rapid turnover:
Memory T cells have a shorter lifespan (~250 d) than naive T cells (~1000 d)
CD45RA (“naive”)
CD45RO (“memory”)
Michie et al.
Nature 1992
Caution: Cells have DNA damage, and cell numbers are low
Static versus dynamic markers
of T-lymphocyte turnover
Static
• Ki67-expression
(protein expressed in
G1,S,G2,M phase)
• Annexin V staining
(stains
phosphatidylserine
translocation)
Dynamic
Natural markers:
• T-cell telomere lengths
• T-cell receptor excision
circles
Labelling:
• (CFSE labelling)
• BrdU labelling
• Stable isotope labelling
Changes in telomere lengths are no
direct measure of T-cell division
Weng et al. PNAS 1995
Changes in TRECs do not directly reflect thymus output
Douek et al. Nature 1998
Static versus dynamic markers
of T-lymphocyte turnover
Static
• Ki67-expression
(protein expressed in
G1,S,G2,M phase)
• Annexin V staining
(stains
phosphatidylserine
translocation)
Dynamic
Natural markers:
• T-cell telomere lengths
• T-cell receptor excision
circles
Labelling:
• (CFSE labelling)
• BrdU labelling
• Stable isotope labelling
Dynamic markers (1)
BrdU Labelling
BrdU = 5-bromo-2’-deoxyuridine
Nucleoside analog
incorporated instead of thymidine
Determine percentage BrdU+ cells
by FACS analysis
Kovacs et al. 2001
How to quantify leukocyte turnover?
Model for BrdU labelling
up-labelling:
determined by p+d
down-labelling
determined by p-d
Expected changes in the percentage
of BrdU+ cells
p+d
Kovacs et al. 2001
p-d
See today’s exercise
But… possible toxicity, almost only done in mice,
only short-term labelling
Dynamic markers (2)
Stable isotope labelling
Deuterium (2H) replaces hydrogen in DNA of proliferating cells
Measure deuterium-enrichment using GC/MS
Main advantage: no interference with cell dynamics
Stable isotope labelling
2H
2-glucose
2H
2O
Intravenously
Long-term administration
Hellerstein et al. 1999, 2003
McCune et al. 2000
Mohri et al. 2001
Ribeiro et al. 2002
Macallan et al. 2003, 2004
Wallace et al. 2004
Hellerstein et al. 2003
Vrisekoop et al. 2008
Enrichment in DNA of cells
Fraction labeled DNA (!) L changes by:
U(t)
Label
p - dL
- dL
Yields two parameters: p and d
p from upslope, d from up- and downslope
2H O
2
labeling does not distinguish between production in thymus and periphery!
Paradox: d is typically larger than p
d
p
Typically: p < d
Asquith et al. 2002
Conclusion: d is no good measure of average turnover rate
It represents the turnover of labelled cells
Short labelling periods give higher
estimate of d
Asquith et al. 2002
Average turnover (p) needs data during
up-labeling period
D-glucose
D2O
Advantages of heavy water: long-term labeling possible
many data points during labeling
BrdU
Potentially toxic
Measures labeled cells
Up: p+d
Down: p-d
Deuterium labeling
Non-toxic (non-radioactive)
Measures labeled DNA strands
Up: p (and d)
Down: -d
NaiveT-cell dynamics
of mice and men
From mice to men...
(How) can we extrapolate?
• How to scale?
mice
2 years
men
mice
men
• Effect of thymic involution during aging?
• Young/old mouse = young/old human?
80 years
2 years
80 years
Of mice...
Naive T-cell dynamics using 2H2O
H-O-H
Label acquisition:
H-O-H
- proliferation
- thymic output
Loss of label:
4w 2H2O
C57Bl/6
% labeled
- cell death
- differentiation/migration
time
Short lifespan of naive T cells in mice
Naive CD4
60
Expected lifespan:
p = 0.023
d* = 0.022
40
Naive CD4: 6 weeks
Percentage labeled
20
0
Naive CD8
60
p = 0.013
d* = 0.011
40
Naive CD8: 11 weeks
20
0
0

50
100
150
Time (days)
Young mice (12 week old)
200
And men...
T-cell dynamics using 2H2O
H-O-H
% labeled
H-O-H
~ 400 ml D2O on day 0
~ 50 ml D2O daily during 9 weeks
time
T-cell turnover in healthy individuals
Vrisekoop et al.
PNAS 2008
Lifespan
6.0
9.4
0.6
1.0 year
T-cell turnover in young healthy men
Naive CD4
60
40
Percentage
labeled
20
0
Naive CD8
60
40
20
0
0
50
100
Time (days)
150
200
Young adult mice
(12 week old)
Lifespan
6.0
9.4
0.6
1.0 year
Vrisekoop et al. PNAS 2008
Labeled naive T cells present
3 years after stop of label
For all fits of naive T cells: p = d
All naive T-cells are very long-lived in humans
Scaling from mice to men...
mice
2 years
80 years
men
Mice live 80 weeks
naive CD4 T cells 6 weeks, CD8 11 weeks
Humans live 80 years
naive CD4 T cells 6 years, CD8 10 years
From mice to men...
(How) can we extrapolate?
• How to scale?
mice
men
• Effect of thymus involution during aging?
• Young/old mouse = young/old human?
2 years
80 years
Thymus involution in mice and men
Mice
Men
Steinman et al. 1985
2H O
2
labeling does not distinguish between
production in thymus and periphery
Proliferation
Differentiation
Thymic output
Naïve
Label acquisition:
- thymic output
- proliferation
Loss of label:
- cell death
- differentiation/migration
Fraction labeled DNA
Death
Time in days
Distinguishing between T-cell
proliferation and thymic output
C57BL/6
ATx or ShamTx at
7 weeks of age
Isolation of spleen, PLNs and thymus
FACS analysis
Mathematical modelling
Naive T-cell dynamics after thymectomy
N’ = ε f(t) + rnN – dnN2
Naive T cells:
Naive CD4
(spleen)
Naive CD8
(spleen)
Cells (x106)
Control
Control
Tx
Tx
Mouse age (weeks)
ε = 2.8 10-2
rn = – 3.0 10-3
dn = 1.7 10-9
0.9% of thymocytes per day
ε = 2.8 10-2
rn = – 2.0 10-3
dn = 6.8 10-10
Contribution of thymic output and T-cell
proliferation in mice
Thymic
output
Total Naive T-cell production
Naive CD4  p = 0.28 106 / day
Naive CD8  p = 0.13 106 /day
Peripheral
proliferation
Heavy
water
Thymectomy
Loss
Thymic
output
Loss
Thymic output:
CD4 thymic emigrants  0.35 106 / day
CD8 thymic emigrants  0.12 106 / day
Peripheral
proliferation
Naive T-cell production in mice almost completely
due to thymic output!
If everything depends on thymic output, what
happens when thymic output declines?
Young mice:12 weeks
p = 0.023
Old mice: 85 weeks
p = 0.024
42 days
43 days
?
p = 0.013
p = 0.011
77 days
Time (days)
91 days
Naive T-cell dynamics in mice hardly
change with age
Young mice:12 weeks
p = 0.023
Old mice: 85 weeks
p = 0.024
43 days
42 days
p = 0.013
p = 0.011
77 days
91 days
Time (days)
The naive pool of a mouse is a big homogeneous pool of thymus emigrants
Naive T-cell proliferation hardly occurs at any age in mice!
This is completely different in
humans!
Evidence for naive T-cell proliferation in men…
TREC decline with age originally interpreted to
reflect thymus decline
Child
Adult
Douek et al. Nature 1998
Rodewald Nature 1998
If naive T-cells proliferate homeostatically,
TREC contents do decline
Naive TREC decline in humans suggests that naive T-cell
proliferation contributes to the naive T-cell pool in humans
Hazenberg et al. 2000, Dutilh et al. 2003
CD31 proposed as marker for nondivided naive CD4 T cells
Naive
Memory
thymus
CD31+
CD31-
CD31+ naive T cells have consistently higher TREC content than CD31Suggestion: CD31+ naive T cells represent (non-divided) thymic emigrants
CD31- naive T cells have divided
Kimmig et al J. Exp Med 2002
However, even CD31+ naive T
cells divide in humans!
CD4 SP
cell
percell
TRECSjcontent
TREC content/
1
CD31+
0.1
0.01
CD31-
0.001
0.0001
0
10
20
30
40
50
Age (years)
Age (in years)
See also Kilpatrick et al. 2008
60
70
80
Fewer thymus emigrants (TE) than
CD31+ naive T cells
Naive
Memory
thymus
CD31+
CD31-
Naive
thymus
CD31+
TE
non-TE
Memory
CD31-
Which part of T-cell production comes
from the thymus?
T cells:
TRECs:
TREC content:
s(t)
A/c =
pN + s(t)
Adapted from Hazenberg et al. 2000
Use TREC content of cord blood
or SP thymocytes to measure c
CD4 SP
cell
percell
TRECSjcontent
TREC content/
1
CD31+
0.1
0.01
CD31-
0.001
0.0001
0
10
20
30
40
50
60
70
80
Age (years)
Age (in years)
s(t)
A/c =
pN + s(t)
See also Kilpatrick et al. 2008
In human adults <10% of naive T cells
is produced by the thymus
Fraction naive cells produced
RTE
thymus
inFraction
1.1
1.0
0.9
Based on CD31
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Based on TREC contents
0.0
0
10
20
30
40
50
60
Age (years)
Age (in
years)
70
80
90
100
Extrapolating from mice to men
mice
2 years
80 years
men
Proliferation
RTE
Men
Proliferation
RTE
Prediction:
without proliferation there should be no TREC dilution in mice…
And indeed... no TREC decline in naive
T cells from mice
1
CD4N
CD4E/M
CD8N
CD8E/M
0.01
0.001
Age [weeks]
15
0
12
5
10
0
75
50
25
0.0001
0
TRECs/cell
0.1
Quantification of T-cell dynamics
from telomeres to DNA labelling
NaiveT-cell dynamics
of mice and men
“The best-laid schemes o' mice an' men,
Gang aft agley”
The most carefully prepared plans
may go wrong