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