- Memory T cell compartments are maintained through a balance of input from precursors and turnover of existing memory cells. - Experiments tracking donor cells in host mice found the rate of input into memory compartments differs between environments. - A "two-compartment" model of fast- and slow-replenishing memory cells best explains the data from both clean and dirty environments. - The environment influences the establishment of memory during development but has less impact on steady-state maintenance once compartments are established.

1. Sources and mixtures - recipes for
CD4 T cell memory
Benedict Seddon
Thea Hogan
Melissa Verheijen
Andrew Yates
Sanket Rane
Maria Nowicka
Data driven systems medicine
11-12 June 2019
Cardiff University Brain Research Imaging Centre

2. T cells are essential for normal immunity
First line defence Specific response

3. T cells are essential for normal immunity
First line defence Specific response

4. Memory is a feature of Adaptive immunity
Innate
Adaptive
Pathogen Pathogen re-encounter

5. Stability of T cell compartments over the life course
0 100 200 300 400
104
105
106
107
108
mouse age (days)
cellnumber(spleen+LNs)
CD4 Naive
0 100 200 300 400
104
105
106
107
108
CD4 Central Memory
0 100 200 300 400
104
105
106
107
108
CD4 Effector Memory
0 10
3
10
4
10
5
<BV786-A>: CD44
0
10
3
10
4
105
<BUV737-A>:L-sel
14wk old C57BL6
TCRb+CD4+CD25-NK1.1-
Central
Memory
Effector
Memory
Naive
CD62L
CD44

6. Naive Tcm Tem Naive Tcm Tem
0
20
40
60
%Ki67+ve
CD8 CD4
Memory pools highly dynamic
12.1
34.3
2.69
273.17
SSc
Ki67
Naive MemoryCD4 T cells:
dynamism vs stability
n=14
0 100 200 300 400
104
105
106
107
108
mouse age (days)
cellnumber(spleen+LNs)
CD4 Naive
0 100 200 300 400
104
105
106
107
108
CD4 Central Memory
0 100 200 300 400
104
105
106
107
108
CD4 Effector Memory

7. What are the rules for maintenance of T cell memory compartments ?
Naive
Central Memory
CD62Lhi CD44hi
Apoptosis
Effector Memory
CD62Llo CD44hi
Is replacement
- random?
- conveyor belt-like (first in, first out)?
- age before beauty (first in, last out)?
- some/all of the above?
- Role of replenishment ?
?

8. Temporal fate mapping - a clear window upon tonic replenishment
Hogan, T., Gossel, G., Yates, A. J. & Seddon, B. Temporal fate mapping reveals age-linked heterogeneity in naive T lymphocytes in
mice. Proceedings of the National Academy of Sciences 112, E6917–E6926 (2015).
RADIATION
BUSULFAN
DONOR
bone
marrow
Haematopoetic
system
HOST
Alkylating agent that causes DNA damage
to dividing cells - thought to be more toxic
to cells in G1 phase
Used in the clinic as a chemotherapeutic for
CML, and as a conditioning agent prior to
bone marrow transplant
Eliminates HSC in bone marrow
Also eliminates peripheral
lymphocytes
BMT gives stable, high levels of
chimerism
Eliminates HSC in bone marrow
Minimal/no effect on peripheral
lymphocytes
BMT gives stable but partial chimerism
(+ immunosuppresion)

9. Switch from host to donor development
week 2 week 4 week 6
0 102
103
104
105
<Pacific Orange-A>: CD8
0
10
3
104
105
<PerCP-Cy5-5-A>:CD4
DN
2.12
DP
86.6
SP4
6.8
SP8
1.85
CD4 thymocytes :
Ly5.1
host
Ly5.2
donor
CD4
CD8
BMT
0 10 20 30 40 50
0.0
0.2
0.4
0.6
0.8
1.0
SP thymocytes Naive Memory
wks
fractionreplaced

10. Kinetic heterogeneity: a two-compartment model of “fast” and “slow” memory
input
CD4 memory
loss
“fast”
renewal
loss
“slow”
renewal
(6-10%
per week)
Gossel, G., Hogan, T., Cownden, D., Seddon, B. & Yates, A. J. eLife Sciences 6, 596 (2017).
“How” but not “what”

11. Q. What are the immune stimuli for generation of memory compartments ?
Age
Memorycellnumber
Establishment of the memory
compartment
Maintenance of the memory
compartment
1. input
3. turnover
2. input
0 100 200 300 400
104
105
106
107
108
CD4 Central Memory

12. A natural environmental experiment - moving mouse house
Institute of Immunity and Transplantation
Royal Free Hospital
** accommodation
Open cage
Tap drinking water
**** accommodation
IVC cages
Irradiated food
Autoclaved water
NIMR
UCL

13. NIMR vs UCL thymic reconstitution
Characterising thymocyte dynamics in UCL/NIMR chimeras
• We performed linear regression on log counts of DP1, SP4 and SP8. Numbers in NIMR mice fall more rapidly with age than at UC
(p-value 0.008, 0.003 and 0.006, respectively) which suggests faster thymic involution in NIMR mice.
• NIMR mice show higher numbers of DP1, SP4/8 cells than UCL mice at 7 weeks (p-value 0.00 for all).
DP thymocytes SP4 thymocytes

14. How does environment influence replenishment of memory ?
Age
Memorycellnumber
Establishment of the memory
compartment
Maintenance of the memory
compartment
1. input
3. turnover
2. input
0 100 200 300 400
104
105
106
107
108
CD4 Central Memory

15. Distinct memory compartment infusion by donor cells in different environments
0 10 20 30 40 50 600 10 20 30 40 50 600 10 20 30 40 50 60
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
Naive donor fraction, normalised CM donor fraction, normalised EM donor fraction, normalised
Age (weeks)
Weeks post BMTWeeks post BMTWeeks post BMT
Age (weeks)Age (weeks)
D
10 20 30 40 50 60 70010 20 30 40 50 60 700 10 20 30 40 50 60 700
103
10
103
10
103
10
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
1.00
0.75
0.50
0.25
0.00
+ +
+
Donor infiltration
UCL
NIMR
UCL
NIMR
UCL
NIMR

16. Distinct memory compartment infusion by donor cells in different environments
Donor naive CD4+
T cells Donor central memory CD4+
T cells Donor effector memory CD4+
T cells
Total naive CD4+
T cells Total central memory CD4+
T cells Total effector memory CD4+
T cells
Age (weeks)Age (weeks)Age (weeks)
CD45.2 donor
bone marrow
Busulfan
2 x 10 mg/kg
CD45.1
host
Time
HSC
THYMUS
PERIPHERY
A
B
C
108
107
106
109
106
105
104
107
106
105
107
108
10 20 30 40 50 60 70010 20 30 40 50 60 700 10 20 30 40 50 60 700
Clean
Clean
Dirty
Dirty
Clean
Dirty
0 10 20 30 40 50 600 10 20 30 40 50 600 10 20 30 40 50 60
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
Naive donor fraction, normalised CM donor fraction, normalised EM donor fraction, normalised
Age (weeks)
Weeks post BMTWeeks post BMTWeeks post BMT
Age (weeks)Age (weeks)
D
10 20 30 40 50 60 70010 20 30 40 50 60 700 10 20 30 40 50 60 700
103
10
103
10
103
10
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
1.00
0.75
0.50
0.25
0.00
+ +
+
Donor infiltration
Compartment sizes
UCL
NIMR
UCL
NIMR
UCL
NIMR
UCL
NIMR
UCL
NIMR
UCL
NIMR

17. small memory at UCL = clean ?
Large memory compartment at NIMR = dirty ?

18. Models of CD4 memory infusion
λ λ
γ
Mfast(t)
Mslo w(t)
λfast
λslo w
S(t)
Precursor
population
(source)
S(t)
Precursor
population
(source)
Force of
recruitment
Net
loss rate
Net
loss rates
ϕ
Transition rate
Force of
recruitment
ϕ
M(t)
λ
CD4 T cell
memory
subset
A B
S(t) M(t) ϕS(t)
ϕ
λ 1/λ
ϕ
M (t) λ
M (t) γ
Single homogenous
compartment
Two compartments

19. Two compartment models best explain both UCL and NIMR reconstitution data
108
10 20 30 40 50 60 70
10 20 30 40 50 60 70 10 20 30 40 50 60 70
10 20 30 40 50 60 70 10 20 30 40 50 60 70
10 20 30 40 50 60 70
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
107
106
105
104
108
107
106
105
104
Clean
Total cell numbers
Central memory CD4+
T cells Effector memory CD4+
T cells
A
B Fraction donor cells in clean mice,
normalised to thymus
Fraction donor cells in dirty mice,
normalised to thymus
Fraction donor cells in dirty mice,
normalised to thymus
Fraction donor cells in clean mice,
normalised to thymus
Age (weeks)
Age (weeks)
Age (weeks)Age (weeks)
Age (weeks)
Age (weeks)
Total cell numbers
Clean
Dirty
Dirty
< 10
> 16
10-12
13-16
Age @ BMT (wk)
< 10
> 16
10-12
13-16
Age @ BMT (wk)
Age @ BMT = 8 wk Age @ BMT = 8 wk
108
10 20 30 40 50 60 70
10 20 30 40 50 60 70 10 20 30 40 50 60 70
10 20 30 40 50 60 70 10 20 30 40 50 60 70
10 20 30 40 50 60 70
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
1.00
0.75
0.50
0.25
0.00
107
106
105
104
108
107
106
105
104
Total cell numbers
Central memory CD4+
T cells Effector memory CD4+
T cells
A
B Fraction donor cells in clean mice,
normalised to thymus
Fraction donor cells in dirty mice,
normalised to thymus
Fraction donor cells in dirty mice,
normalised to thymus
Fraction donor cells in clean mice,
normalised to thymus
Age (weeks)
Age (weeks)
Age (weeks)Age (weeks)
Age (weeks)
Age (weeks)
Total cell numbers
< 10
> 16
10-12
13-16
Age @ BMT (wk)
< 10
> 16
10-12
13-16
Age @ BMT (wk)
Age @ BMT = 8 wk Age @ BMT = 8 wk
Clean
Clean
Dirty
Dirty
One compartment Two compartments
UCL
NIMR
TOTAL
Nos
ΔAIC (low best) 0 0107 42

20. Constant rates of constitutive memory cell generation irrespective of environment
1.00 1.00 1.00
Naive donor fraction, normalised
Donor naive CD4+
T cells Donor central memory CD4+
T cells Donor effector memory CD4+
T cells
Total naive CD4+
T cells Total central memory CD4+
T cells Total effector memory CD4+
T cells
CM donor fraction, normalised EM donor fraction, normalised
Age (weeks)Age (weeks)Age (weeks)
Age (weeks)Age (weeks)Age (weeks)
bone marrowhost
Time
HSC
THYMUS
PERIPHERY
B
C
D
108
107
106
109
106
105
104
107
106
105
107
108
10 20 30 40 50 60 70010 20 30 40 50 60 700 10 20 30 40 50 60 700
10 20 30 40 50 60 70010 20 30 40 50 60 700 10 20 30 40 50 60 700
105
103
10
107
105
103
10
107
105
103
10
107
Clean
Clean
Dirty
Dirty
Clean
Dirty
Clean
Dirty
Clean
Dirty
Clean
Clean
Dirty
1.00
λ λ
γ
Mfast(t)
Mslo w(t)
λfast
λslo w
S(t)
Precursor
population
(source)
Net
loss rate
Net
loss rates
Transition rate
Force of
recruitment
ϕλ
B
S(t) M(t) ϕS(t)
λ 1/λ
ϕ
λ
M (t) γ
λ < λ
Force of recruitment into memory
per source cell per day,
Net los
with
Central
memory CD4
Centr
memory
Effector
memory CD4
0.010
0.1
0.01
1
0.005
0.005
0
A B
0.001
0.0001
Clean Dirty
_
ϕ
−1

21. Input at steady state is insensitive to environment
Age
Memorycellnumber
Establishment of the memory
compartment
Maintenance of the memory
compartment
1. input
3. turnover
2. input

22. What about establishing memory ?
Q. How does memory generation during ontogeny compare with steady state ?
Age
Memorycellnumber
Establishment of the memory
compartment
Maintenance of the memory
compartment
1. input
3. turnover
2. input

23. Q. Can rates of memory generation in adult predict ontogeny of memory ?
0 10
Age (weeks)
20
0 10
Age (weeks)
20
0 10
Age (weeks)
20
0 10
Age (weeks)
20 0 10
Age (weeks)
20
Central memory CD4+
T cells in clean mice Effector memory CD4+
T cells in clean mice
Central memory CD4+
T cells in dirty mice Effector memory CD4+
T cells in dirty mice
B
C
103
107
106
105
104
103
107
106
105
104
103
107
106
105
104
103
107
106
105
104
106
105
Chimeras
Predicted
trajectory
WT
Chimeras
Predicted
trajectory
WT
WT
Chimeras Chimeras
Predicted
trajectory
Predicted
trajectory
S(t)
0 10
Age (weeks)
20
0 10
Age (weeks)
20
0 10
Age (weeks)
20
Naive CD4+
T cells in clean WT mice
Central memory CD4+
T cells in clean mice Effector memory CD4+
T cells in clean mice
Central memory CD4+
T cells in dirty mice Effector memory CD4+
T cells in dirty mice
A
B
103
107
106
105
104
103
107
106
105
104
108
107
106
105
Chimeras
Predicted
trajectory
WT
Chimeras
Predicted
trajectory
WT
WT
input :
𝜑 clean
𝜑 dirty
Larger memory populations in dirty mice derive from early antigen exposure

24. How does environment influence memory ontogeny ?
Age
Memorycellnumber
Establishment of the memory
compartment
Maintenance of the memory
compartment
1. input
3. turnover
2. input
GF vs SPF vs UCL vs NIMR

25. A role for micro-biome in establishment of memory ?
… but also a role for spMHC signals to establish memory ?
Counts (Spleen + lymph nodes)
% Ki67 expression % Ki67 expression
Counts (Spleen + lymph nodes)
Chim
eraNIM
RW
T
NIM
R
Chim
eraUCL
SPFOxfordGFOxford
W
T
UCL
Chim
eraNIM
RW
T
NIM
R
Chim
eraUCL
SPFOxfordGFOxford
W
T
UCL
1 x 10 6
1.5 x 10 6
Dirty
Dirty
Clean Clean
5 x 10 5
0
6 x 10 6
8 x 10 6
2 x 10 6
0
4 x 10 6
Dirty
Clean
ND
Clean
40
60
40
60
Central memory CD4+
T cells Effector memory CD4+
T cells
A
B

26. Does chronic microbiome exposure drive turnover ?
Age
Memorycellnumber
Establishment of the memory
compartment
Maintenance of the memory
compartment
1. input
3. turnover
2. input
Measure Ki67 as indicator of cell division

27. Turnover of memory cells insensitive to environment
0 100 200 300 400
0
20
40
60
80
100
mouse age (days)
%Ki67+ve
NIMR
UCL
Germ Free

28. Intrinsic and extrinsic antigens shape memory compartments
Age
Memorycellnumber
Establishment of the memory
compartment
Maintenance of the memory
compartment
input
turnover
input
spMHC
microbiome

29. Acknowledgements
Benedict Seddon
Thea Hogan
Melissa Verheijen
Fiona Powrie
Claire Pearson
Andrew Yates
Sanket Rane
Maria Nowicka
Graeme Gossel
Daniel Cownden
Edward Lee