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.

3 years of age 23 years of age

Mackall et al. 1995

Experiments of nature (1): T-cell reconstitution after chemotherapy

Underestimate?: Cells may also die during reconstitution

Overestimate?: Cells undergo little competition

Michie et al. Nature 1992

CD45RA (“naive”)

CD45RO (“memory”)

Immunological memory resides in a population with rapid turnover:

Memory T cells have a shorter lifespan (~250 d) than naive T cells (~1000 d)

Experiments of nature (2): Loss of T cells with chromosome damage

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

Weng et al. PNAS 1995

Changes in telomere lengths are no direct measure of T-cell division

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

p+d

See today’s exercise

But… possible toxicity, almost only done in mice, only short-term labelling

Kovacs et al. 2001

Deuterium (2H) replaces hydrogen in DNA of proliferating cells

Dynamic markers (2) Stable isotope labelling

Measure deuterium-enrichment using GC/MS

Main advantage: no interference with cell dynamics

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

Long-term administration

Hellerstein et al. 2003

Vrisekoop et al. 2008

Stable isotope labelling

Enrichment in DNA of cells

Fraction labeled DNA (!) L changes by:

Label

U(t)

p - L - dL

2H2O labeling does not distinguish between production in thymus and periphery!

Yields two parameters: p and d

p from upslope, d from up- and downslope

Asquith et al. 2002

dp

Typically: p < d

Paradox: d is typically larger than p

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?

2 years

80 years

mice

men

2 years

80 years

mice

men

• How to scale?

• Young/old mouse = young/old human?

• Effect of thymic involution during aging?

Of mice... Naive T-cell dynamics using 2H2O

H-O-H

H-O-HLabel acquisition:

- proliferation- thymic output

Loss of label:- cell death- differentiation/migration

C57Bl/62H2O4w

% l

ab

ele

d

time

Short lifespan of naive T cells in mice

Expected lifespan:

Naive CD4: 6 weeks

Naive CD8: 11 weeks

Per

cent

age

labe

led

20

40

60

0

50 100 150 200

Time (days)

20

40

60

00

Naive CD4p = 0.023

d* = 0.022

Naive CD8p = 0.013

d* = 0.011

Young mice (12 week old)

And men... T-cell dynamics using 2H2O

H-O-H

H-O-H

% l

ab

ele

d

time~ 400 ml D2O on day 0

~ 50 ml D2O daily during 9 weeks

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

Vrisekoop et al. PNAS 2008

200

Per

cent

age

labe

led

20

40

60

0

50 100 150Time (days)

20

40

60

00

Naive CD4

Naive CD8

Young adult mice (12 week old)

Lifespan 6.0 9.4 0.6 1.0 year

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...

2 years80 years

mice

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?

2 years

80 years

mice

men

• How to scale?

• Young/old mouse = young/old human?

• Effect of thymus involution during aging?

Thymus involution in mice and men

Mice Men

Steinman et al. 1985

2H2O labeling does not distinguish between production in thymus and periphery

Proliferation

Thymic outputDifferentiation

Naïve

Death

Label acquisition:- thymic output- proliferation

Loss of label:- cell death- differentiation/migration

Fra

ctio

n la

be

led

DN

A

Time in days

Distinguishing between T-cell proliferation and thymic output

ATx or ShamTx at 7 weeks of age

Isolation of spleen, PLNs and thymus

FACS analysis

Mathematical modelling

C57BL/6

Naive T-cell dynamics after thymectomy

N’ = ε f(t) + rnN – dnN2Naive T cells:

Naive CD4 (spleen)

Naive CD8 (spleen)

Tx

Control

Tx

Control

ε = 2.8 10-2

rn = – 3.0 10-3

dn = 1.7 10-9

Cel

ls (

x10

6)

Mouse age (weeks)

ε = 2.8 10-2

rn = – 2.0 10-3

dn = 6.8 10-10

0.9% of thymocytes per day

Naive T-cell production in mice almost completely due to thymic output!

Loss

Thymic output

Heavy water

Peripheral proliferation

Total Naive T-cell production

Naive CD4 p = 0.28 106 / day

Naive CD8 p = 0.13 106 /day

Thymectomy Thymic output

Loss

Peripheral proliferation

Thymic output:

CD4 thymic emigrants 0.35 106 / day

CD8 thymic emigrants 0.12 106 / day

Contribution of thymic output and T-cell proliferation in mice

If everything depends on thymic output, what happens when thymic output declines?

Young mice:12 weeks

43 days

77 days

42 days

91 days

Old mice: 85 weeks

p = 0.023 p = 0.024

p = 0.013 p = 0.011

Time (days)

?

Naive T-cell dynamics in mice hardly change with age

The naive pool of a mouse is a big homogeneous pool of thymus emigrants

Young mice:12 weeks

43 days

77 days

42 days

91 days

Old mice: 85 weeks

p = 0.023 p = 0.024

p = 0.013 p = 0.011

Time (days)

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 non-divided naive CD4 T cells

Naive Memory

CD31+ CD31-

thymus

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

However, even CD31+ naive T cells divide in humans!

See also Kilpatrick et al. 2008

Age (years)

0 10 20 30 40 50 60 70 80

Sj T

RE

C c

on

ten

t/ c

ell

0.0001

0.001

0.01

0.1

1

CD31+

CD31-

CD4 SP

Age (in years)

TR

EC

con

tent

per

cel

l

Fewer thymus emigrants (TE) than CD31+ naive T cells

Naive Memory

CD31+ CD31-

thymus

Naive Memory

CD31-thymus CD31+

TE non-TE

T cells:

TRECs:

TREC content:

Which part of T-cell production comes from the thymus?

Adapted from Hazenberg et al. 2000

tpN + (t)

A/c =

Use TREC content of cord blood or SP thymocytes to measure c

See also Kilpatrick et al. 2008

Age (years)

0 10 20 30 40 50 60 70 80

Sj T

RE

C c

on

ten

t/ c

ell

0.0001

0.001

0.01

0.1

1

CD31+

CD31-

CD4 SP

Age (in years)

TR

EC

con

tent

per

cel

l

tpN + (t)

A/c =

In human adults <10% of naive T cells is produced by the thymus

Age (years)

0 10 20 30 40 50 60 70 80 90 100

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

Fra

ctio

n R

TE

Fra

ctio

n na

ive

cells

pro

duce

d in

thy

mus

Age (in years)

Based on CD31

Based on TREC contents

Extrapolating from mice to men

RTERTE

Proliferation

RTERTE

ProliferationMen

2 years80 years

mice

men

Prediction:

without proliferation there should be no TREC dilution in mice…

0 25 50 75 100

125

150

0.0001

0.001

0.01

0.1

1 CD4NCD4E/MCD8NCD8E/M

Age [weeks]

TR

EC

s/ce

ll

And indeed... no TREC decline in naive T cells from mice

NaiveT-cell dynamics of mice and men

“The best-laid schemes o' mice an' men, Gang aft agley”

The most carefully prepared plansmay go wrong

Quantification of T-cell dynamics

from telomeres to DNA labelling