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Symposium: 70th Birthday of Max Cooper B Lymphopoiesis IIBirmingham, Alabama 2003
25 Minutes
Division of Molecular ImmunologyUniversity of Erlangen-Nürnberg
Friedrich-Alexander-UniversityErlangen-Nürnberg
Selection of Functional B-Lymphocytes
Symposium: 70th Birthday of Max Cooper B Lymphopoiesis IIBirmingham, Alabama 2003
Selection of Functional B LymphocytesSelection of Functional B Lymphocytes- Function of the Pre-B Cell Receptor -- Function of the Pre-B Cell Receptor -
Pro-B Pre-B B
Discovery of pre-B cells and pre-BCR Function of the surrogate light chain Ligand-induced pre-BCR signaling
Discovery of the Pre-B CellDiscovery of the Pre-B Cell
Discovery of the Pre-B CellDiscovery of the Pre-B Cell
Lambda 5, a new light-chain-related locus selectively expressed in pre-B lymphocytes.Sakaguchi N. and Melchers F.
Nature 324:579-82 (1986).
Discovery of the Surrogate Light Chain GenesDiscovery of the Surrogate Light Chain Genes
A second gene, VpreB in the lambda 5 locus of the mouse, which appears to be selectively expressed in pre-B lymphocytes.
Kudo A and Melchers F.Basel Institute for Immunology, Switzerland.
EMBO J. 6(8):2267-72 (1987)
?
Ig ReceptorsIg Receptors
μHC
λ5
Igαβ
VpreB
Igαβ
IgL
μHC
LatePro-B
LatePre-B
ImmatureB cell
Early Pre-B
Stemcell
EarlyPro-B
fct?
L ?
MatureB cell
Pairing of Ig chains
1 2Autoreactivity
3
Quality Control of B-Lymphoid PrecursorsQuality Control of B-Lymphoid PrecursorsAt Developmental CheckpointsAt Developmental Checkpoints
D JH VH DJH VL JL
λ5+/+
λ5-/-
μHC
B2
20
Spleen
SLC-Independent Generation of B CellsSLC-Independent Generation of B Cells
SL-chain independent signals mediated by μH chains?
QuestionQuestion
The The μμHC-inducible MouseHC-inducible Mouse
Pro-B - Tet
+ Tet
µ
Rag-/-tTA+
tetop-µH
DoubleTransgenic
Mouse (dTg)
= Recombination activating gene
Tet-op H-Gen
tTATet
tTA
+ Tet
Tet-off System of Gossen und Bujard
+ -
Rag2-/-
410
310
210
110
010
48
36
24
12
04
103
102
101
100
10
48
36
24
12
0
dTg Rag2- λ5+
dTgRag2- λ5-
Fluorescence intensities (FI)
No.
λ5 VpreB
410
310
210
110
010
48
36
24
12
04
103
102
101
100
10
48
36
24
12
0
410
310
210
110
010
48
36
24
12
0
50%
48%
µHC
dTg Rag2- λ5+
FI (B220)
No.
FI (cyto μHC)
FI (
CD
25)
195.7
205.1
C57Bl/6
Control of µH-Chain Expression by TetracyclineControl of µH-Chain Expression by Tetracycline
μ/ no λ5
μ/λ5
In Vivo Signaling of a µH-chain in the absence of In Vivo Signaling of a µH-chain in the absence of λλ55
μ / λ5 dTg/Rag2-/λ 5+
μ / no λ5 dTg/Rag2- /λ5-
no μtTA/Rag2- /λ5-
c-ki
tC
D25
CD19
29.0
16.4
4.6
40.4
11.9
10.0
9.9
9.9
0.8
7.6
9.1
1.3
Signaling of a µH-chain in the absence of Signaling of a µH-chain in the absence of λλ55
Rag2-/- / dTg / λ5+/+ Rag2-/- / dTg / λ5 -/-
Rag2-/-‚Inducible μ
mouse‘(dTg)
µ
-
+
1 2 3 4
5
20
10
15
25
0 1 2 3 4 5
+ IL7 (10ng/ml)
Culture time [days]
+ Tet
Cel
l cou
nt x
104
+ tet
- tet
λ5+/+
λ5-/-
+μ
0 1 2 3 4 5
0.1
1.0
- IL7
Pro-B
+/- IL7
+μ
-μ -μ-μ+μ
+μ
Analysis of µH-Chain Surface ExpressionAnalysis of µH-Chain Surface Expression
Indirect immunostaining of surface μHC with monoclonal rat anti-mouse µH antibodies (mAb b7.6)
membranecytoplasmic
410
310
210
110
010
48
36
24
12
04
103
102
101
100
10
48
36
24
12
0
410
310
210
110
010
48
36
24
12
04
103
102
101
100
10
48
36
24
12
0
6,2
22,5
6,1
12,6
Grey: + Tet
Black: - Tet
μ/λ5 dTg/Rag2-/λ 5+
μ / no λ5 dTg/Rag2- /λ5-
TK-
TK.μ
F (81X)
Cyto Surface
CD
43
0.3
15.8
10.7
0.3
17.3
18.1
B220
100
10
21
031
04
101
μμHC Surface Expression is Required HC Surface Expression is Required for B Cell Developmentfor B Cell Development
JH+/+
JH-/-
81XF
JH-/-
Bone MarrowPre-B Cell Lines
μHC
Eve
nts
μμH-Chain Signaling in the Absence of H-Chain Signaling in the Absence of λλ55
A μH-Chain in the absence of λ5o can be expressed on the surface of in-
vitro cultivated pre-B cells
o must be transported to the cell surface to aquire signal competency
o induces signals in-vivo and in-vitro
These findings could explain o why B cell maturation is leaky in a
λ5 knock-out mouse
Pre-BCR Signal Initiation ?
Models: Initiation of Pre-BCR SignalsModels: Initiation of Pre-BCR Signals
ER
Constitutive from ER
ER
cytoplasm ?
Constitutivefrom cell surface
Ligand
cytoplasm
ER
Ligand induced
cytoplasm
?
?
?
0 1 2 3 4 5 6
106
105
Pro-B cells(dTg/Rag2-/-/5-/-)105
104
1 2 3
- tet
+ tet
No
.
days
- tet
+ tet
Pro-B cells(dTg/Rag2-/-)
+/- stroma cells (SC)
Signaling of a µH-chain in the absence of Signaling of a µH-chain in the absence of λλ55
0 1 2 3
105
104
Pro-B cells(dTg/Rag2-/-)
+SC (λ5+) +SC (λ5-)-SC (λ5+)
Rag2-/- / dTg / λ5+/+ Rag2-/- / dTg / λ5 -/-
Rag2-/-‚Inducible μHC
mouse‘(dTg)
+ Tet
Pro-B µ
Ligand-Induced Pre-BCR Signaling ?Ligand-Induced Pre-BCR Signaling ?
Flow cytometry
soluble pre-BCR
FIT
C
ligand
ST2
+ Heparin
+ Trypsin
+ Heparitinase
Eve
nts
Control: (Fab-like
BCR)
Fluorescence Intensity (pre-BCR)
Schematic Aligments of Murine VpreB and Schematic Aligments of Murine VpreB and λλ5 5 with a Conventional with a Conventional λλ ChainChain
V J Cchain
VpreB
5
non-Iglike tail
NH3+ COO-
NH3+
NH3+
COO-
COO-
--
++
+
+
VpreB plus 5(8)
CDR1
CDR2
VL domain
- +
Computer Modelling of a SL ChainComputer Modelling of a SL Chain
In cooperation with Dr. Harald Lanig, Chemie Computer Center, FAU Erlangen-Nürnberg
+
CDR1
CDR2
CDR3
λλ5 Tail is Critical for Pre-BCR/Stroma Cell Interaction5 Tail is Critical for Pre-BCR/Stroma Cell Interaction
Eve
nts
F (pre-BCR)
VpreB/5
anti-VpreB
Vpr
eB/
5
Vpr
eBT
/5
Vpr
eB/
5T
Vpr
eBT
U/
5T
anti-5 IP-Ab
14 -
21-
30-
Blot-Ab
VpreBT VpreB
A
VpreBT/5T
VpreB/5T
VpreBT/5
GST GST- λ5Tail GST- VpreBTail
+ Heparin + Heparin + Heparin
Binding of GST-Tail-Fusionproteins to ST2 cellsBinding of GST-Tail-Fusionproteins to ST2 cells
The interaction of a pre-BCR with stroma cells is mediated by the tail of λ5 and a stroma cell-associated heparan sulfate
ConclusionConclusion
Model for Selection of Early Pre-B Cells Model for Selection of Early Pre-B Cells
Pre-BCR levels below signaling thresholdPre-BCR levels above threshold
μH levels above signaling threshold
IL7
Pre
Survival ?
Expansion
Pre
BM stroma cell
protein backbone
Heparan sulfate(HS) chain
Expansion
IL7
Pre
IL7
HS chain
Thanks – Jäck LabThanks – Jäck Lab
Anja Haude
WolfgangSchuh
ChristianVettermann
HaraldBradl
SilkeMeister
EdithRoth
Thanks - Collaborators Thanks - Collaborators
• Harald Lanig (Erlangen)• N. Klinman (Scripps)• Gill Wu (Toronto) • C. Raman (UAB)• Michael Karras (Frankfurt)• Matthias Wabl (San Francisco)
• J. Jongstra (Toronto)• F. Melchers (Basel)• M. Cooper (UAB)• J. Kearney (UAB)
ReagentsCollaborators
Happy Birthday
Max
dTgRag2- 5+
dTgRag2-
5-
21
15
34% 58%
- +-
µHC-
30% 64%
- +µHC
FI (cyto µHC) FI (membrane µHC)
FS
C
No.
+ Tet - Tet - Tet
134
96
33 29 22 19 15
14-
21-
45-
31-
66-
anti-galectin-1
HeLa clones
anti-actin
gal-1
No.
F (SLC)
kDa
clone 15
clone 19
clone 33
HeLa
No.
F (pre-BCR)
+ maltose
+ lactose
A B
c
Galectin does not interact with murine pre-BCRGalectin does not interact with murine pre-BCR