Download Rhomeo I

Biological background for Project Rhomeo
Benedict Seddon
MRC National Institute for Medical Research
London, U.K.
Outline
 T cells
 Homeostasis
 Experimental philosophy
 Experimental design and preliminary data
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
T lymphocytes in blood . . . .
T cells
(“thymus”)
B cells
(“bone marrow”)
1. T cells
2. Homeostasis
3. Experimental philosophy
. . . and in ‘Lymphoid’ organs
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
TCR recognition of antigen
Antigen presenting cell
MHC
Peptide
CD4/CD8
Coreceptor
TCR
Cognate :
Foreign
Autoantigen
Self in thymus:
Restriction
Positive selection
Self in periphery:
Survival
Lymphopenia
Induced proliferation
T cell
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
TCR signalling
APC
MHC
MHC
Peptide
TCR
CD4/CD8
Coreceptor
LAT
+p
p56lck
+p
Ras
+p
p59fyn
MAPKs
Ca++ flux
Slp76
+p
+p
T-cell
ζζ
ZAP-70
DAG
Cytoskeletal Reorganisation
Activation
Survival
Proliferation
1. T cells
2. Homeostasis
3. Experimental philosophy
Cell division in the Immune System
•
For making new immune cells
– Haematopoetic stem cell divide 12-16
times
•
Immune response
“Clonal expansion”
– T cell divide ~ 10-12 times
•
Fine adjusting immune cell numbers
– Few divisions
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
T cell homeostasis : keeping the immune system the right size
Activated/Effector
T cells
Naïve T cell pool
Thymus
Ag
INPUT
Resting
memory
Resource
competition
Death
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
The homeostatic T cell response to lymphopenia
Activated/Effector
T cells
Naïve T cell pool
Thymus
Ag
INPUT
Memory
Lymphopenia
• Infection, stress, HIV
• Thymectomy/old age
• Clinical intervention (chemo and radio therapy)
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Different signals for different subsets
Naïve T cells
Cytokines + TCR
Memory T cells
Cytokines
CD4
(s)pMHC
spMHC
IL-7
dc
dc
Stromal cell
CD8
IL-7
IL-15
IL-7 - survival
IL-15- proliferation
More division but more death also
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Model systems in mice and men
Man
Mice
T cell donor
Analyse donor T cells
Purify T cells
Host
What can we measure ?
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Cellular phenotype
•
T cell lineages
– αβ vs γδ
– CD4 vs CD8
•
Cell cycle
– DNA content - cells in S phase
•
Cell division history
– Cell dyes
•
T cell subsets
– CD44
• Low on Naïve
• High on activated or
memory
– CD25 (IL2 Receptor)
• Regulatory T cells
– Many others . . .
• CD122 (IL15 R)
•
Cytokine responses
– IL-2
– IFN-γ
•
Cell death
– Very important
– But how too ??
– Dead cells cleared v. quickly
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
CD4
Subsets
CD8
9.9
TCR
CD25
Treg
82.5
7.41
CD44
Naive
Naive
Memory
CD44
Memory
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Tracking cell division in vivo - cell dyes
d1
d2
d3
100%
dye
50%
dye
25%
dye
12.5%
dye
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Counting fluorescent cells
100 T cells
676 T cells
200
200
184
144
7days
100
100
140
150
Cell number
Cell number
150
96
100
64
50
38
50
10
0
1
2
4
8
16
32
Fluorescence
(arbitary units)
64
128
256
0
1
2
4
8
16
32
Fluorescence
(arbitrary units)
Cell division results in expansion
64
128
256
1. T cells
2. Homeostasis
3. Experimental philosophy
Real data
7days
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
WT mouse
‘full’
CD8
Different host environment
Rag1 KO
mouse
No antigen receptor gene rearrangements
No T cells or B cells
Normal lymphoid structure
CD8
CD4
CD4
1. T cells
2. Homeostasis
3. Experimental philosophy
Measuring homeostatic T cell responses in vivo
Lymphopenia Induced Proliferation (LIP)
Dye label T cells
Rag1-/14 d
CFSE
1. Proliferation (CFSE)
2. Survival (cell recovery)
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Heterogeneous LIP response of polyclonal CD8 T cells
Day 13
Day 6
Memory/effector
41%
91%
Naive
CD44
59%
CFSE
9%
Reductionist approach - limit T cell repertoire to 1 clonotype
TCR transgenic mice :
1. Clone αβ TCR from T cell clone
2. Generate transgenic mice expressing TCR in T lineage
3. Prevent endogenous genes rearranging (Rag1-/-)
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Normal development
Thymus
DN
CD4+CD8+ DP
β-selection
+ve selection
Periphery
SP
Naïve Pool
Survival
Homeostatic
proliferation
Lineage
commitment
Memory
TCR
IL-7
Activation and
proliferation
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
TCR transgenic mouse
Thymus
DN
β-selection
CD4+CD8+ DP
+ve selection
Periphery
CD8 SP
Naïve Pool
Survival
No effector or
memory
Express
TCR transgene
Death
1. T cells
2. Homeostasis
3. Experimental philosophy
TCR transgenic mice
CD4
Thymus
F5
CD4
WT
CD8
CD4
Lymph node
CD4
CD8
CD8
CD8
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Proliferative responses by different clones are heterogenous
F5 - low avidity
10
5
10
5
10
4
10
4
3
10
3
10
0
0
0
OT1- high avidity
10
10
3
10
4
10
5
5
10
4
10
3
10
5
10
4
10
3
0
10
3
10
4
10
5
0
10
3
10
4
10
5
0
0
CD44
CD122
0
CFSE
10
3
10
4
10
5
CFSE
Q. What is the relationship between stimulation, division
and function ?
Cell division dependent ?
Signal strength dependent ?
Rate of division ?
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Effector function in response to different agonist ligands
Autopilot Proliferation +
effector function
Strong
Partial
4h
>18h stimulation
IFNγ
IL-2
Upregulation activation
markers
Effector function
Division independent
Good
Poor
Division independent
Division dependent (> 2)
1. T cells
2. Homeostasis
3. Experimental philosophy
Autopilot divisions model
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
Stochastic divisions
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
4. Experimental design
Experimental design
Lymphopenia Induced Proliferation (LIP)
Dye label T cells
Hosts with no T cells
CFSE
Donors
F5 (low avidity)
OT-I (high self avidity)
Cell dose : hi vs lo
Hosts
Rag1-/Cytokine deficient
(IL7, IL15)
Recovery
Time points
Surface phenotype
Cell division
Effector function
(cyokine, killing)
1. T cells
2. Homeostasis
3. Experimental philosophy
Example - d10 OT-1 transfer
CD44
CD44
5 x 106
CD122
IL15R
CFSE
CD44
5 x 105
CD122
CFSE
CFSE
CFSE
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
Proliferation - hi vs lo
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
Timecourse data
Cell no.
Division
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
Phenotype with division and time
4. Experimental design
1. T cells
2. Homeostasis
3. Experimental philosophy
Future
•
Understand LIP in OT1
– Time course
• Division
• Phenotype
• Function
– Control
• Cell density dependent ?
• Do rules change with phenotype ?
• Clonal competition - I.e. with F5
– TCR
– Cytokine
•
Polyclonal responses
– Clonal diversity ?
– Responsiveness ?
– Primary response from memory pool ?
4. Experimental design