Quantification of T-cell dynamics from telomeres to DNA labelling

1
Quantification of T-cell dynamics from
telomeres to DNA labelling
José Borghans Dpt. Immunology University Medical
Center Utrecht
2
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?

3
What is the difficulty?

• How to follow a lymphocyte from its birth to its
  death?
• Extrapolation from mice to men v.v.

4
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
5
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)
Michie et al. Nature 1992
Caution Cells have DNA damage, and cell numbers
are low
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Static versus dynamic markers of T-lymphocyte
turnover

• Dynamic
• Natural markers
• T-cell telomere lengths
• T-cell receptor excision circles
• Labelling
• (CFSE labelling)
• BrdU labelling
• Stable isotope labelling

• Static
• Ki67-expression
• (protein expressed in G1,S,G2,M phase)
• Annexin V staining
• (stains phosphatidylserine translocation)

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Changes in telomere lengths are no direct measure
of T-cell division
Weng et al. PNAS 1995
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Changes in TRECs do not directly reflect thymus
output
Douek et al. Nature 1998
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Static versus dynamic markers of T-lymphocyte
turnover

• Dynamic
• Natural markers
• T-cell telomere lengths
• T-cell receptor excision circles
• Labelling
• (CFSE labelling)
• BrdU labelling
• Stable isotope labelling

• Static
• Ki67-expression
• (protein expressed in G1,S,G2,M phase)
• Annexin V staining
• (stains phosphatidylserine translocation)

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Dynamic markers (1) BrdU Labelling
BrdU 5-bromo-2-deoxyuridine Nucleoside
analog incorporated instead of thymidine
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Determine percentage BrdU cells by FACS analysis
Kovacs et al. 2001
How to quantify leukocyte turnover?
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Model for BrdU labelling
up-labelling determined by pd
down-labelling determined by p-d
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Expected changes in the percentage of BrdU cells
pd
Kovacs et al. 2001
p-d
See todays exercise
But possible toxicity, almost only done in mice,
only short-term labelling
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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
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Stable isotope labelling
2H2-glucose 2H2O
Intravenously 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
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Enrichment in DNA of cells
Fraction labeled DNA (!) L changes by
Yields two parameters p and d
p from upslope, d from up- and downslope
2H2O labeling does not distinguish between
production in thymus and periphery!
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Paradox d is typically larger than p
Asquith et al. 2002
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Short labelling periods give higher estimate of d
Asquith et al. 2002
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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
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BrdU Potentially toxic Measures labeled cells Up
pd Down p-d
Deuterium labeling Non-toxic (non-radioactive) Mea
sures labeled DNA strands Up p (and d) Down
-d
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NaiveT-cell dynamics of mice and men
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From mice to men...(How) can we extrapolate?

• How to scale?

2 years
mice
men
80 years
2 years
mice
men
80 years

• Effect of thymic involution during aging?

• Young/old mouse young/old human?

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Of mice... Naive T-cell dynamics using 2H2O
H-O-H
Label acquisition - proliferation - thymic
output Loss of label - cell death -
differentiation/migration
H-O-H
4w
2H2O
C57Bl/6
labeled
time
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Short lifespan of naive T cells in mice
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And men... T-cell dynamics using 2H2O
H-O-H
H-O-H
labeled
400 ml D2O on day 0 50 ml D2O daily during 9
weeks
time
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T-cell turnover in healthy individuals
Vrisekoop et al. PNAS 2008
Lifespan 6.0 9.4
0.6 1.0 year
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T-cell turnover in young healthy men
Lifespan 6.0 9.4
0.6 1.0 year
Vrisekoop et al. PNAS 2008
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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
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Scaling from mice to men...
2 years
mice
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
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From mice to men...(How) can we extrapolate?

• How to scale?

2 years
mice
80 years
men

• Effect of thymus involution during aging?

• Young/old mouse young/old human?

31
Thymus involution in mice and men
Mice
Men
Steinman et al. 1985
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2H2O labeling does not distinguish between
production in thymus and periphery
Label acquisition - thymic output -
proliferation Loss of label - cell death -
differentiation/migration
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Distinguishing between T-cell proliferation and
thymic output
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Naive T-cell dynamics after thymectomy
Naive CD4 (spleen)
Naive CD8 (spleen)
Control
Control
Tx
Tx
e 2.8 10-2 rn 3.0 10-3 dn 1.7
10-9
e 2.8 10-2 rn 2.0 10-3 dn 6.8
10-10
0.9 of thymocytes per day
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Contribution of thymic output and T-cell
proliferation in mice
Thymic output
Peripheral proliferation
Total Naive T-cell production Naive CD4 ? p
0.28 106 / day Naive CD8 ? p 0.13 106 /day
Heavy water
Loss
Naive T-cell production in mice almost completely
due to thymic output!
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If everything depends on thymic output, what
happens when thymic output declines?
Young mice12 weeks
Old mice 85 weeks

42 days
43 days
?
91 days
77 days
37
Naive T-cell dynamics in mice hardly change with
age
Young mice12 weeks
Old mice 85 weeks
43 days
42 days
91 days
77 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!
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This is completely different in humans!

• Evidence for naive T-cell proliferation in men

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TREC decline with age originally interpreted to
reflect thymus decline
Child
Adult
Douek et al. Nature 1998
Rodewald Nature 1998
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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
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CD31 proposed as marker for non-divided naive CD4
T cells
Naive
Memory
thymus
CD31
CD31-
CD31 naive T cells have consistently higher TREC
content than CD31- Suggestion CD31 naive T
cells represent (non-divided) thymic emigrants
CD31- naive T cells have divided
Kimmig et al J. Exp Med 2002
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However, even CD31 naive T cells divide in
humans!
TREC content per cell
Age (in years)
See also Kilpatrick et al. 2008
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Fewer thymus emigrants (TE) than CD31 naive T
cells
Naive
Memory
thymus
CD31
CD31-
Naive
Memory
CD31-
thymus
CD31
TE
non-TE
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Which part of T-cell production comes from the
thymus?
T cells TRECs TREC content
Adapted from Hazenberg et al. 2000
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Use TREC content of cord blood or SP thymocytes
to measure c
TREC content per cell
Age (in years)
See also Kilpatrick et al. 2008
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In human adults lt10 of naive T cells is produced
by the thymus
Based on CD31
Fraction naive cells produced in thymus
Based on TREC contents
Age (in years)
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Extrapolating from mice to men
Prediction without proliferation there should
be no TREC dilution in mice
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And indeed... no TREC decline in naive T cells
from mice
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NaiveT-cell dynamics of mice and men
Quantification of T-cell dynamics from
telomeres to DNA labelling
The best-laid schemes o' mice an' men, Gang aft
agley The most carefully prepared plans may go
wrong