1. Symposium: 70th Birthday of Max Cooper B Lymphopoiesis II Birmingham, Alabama 2003 25 Minutes

2. Division of Molecular Immunology University of Erlangen-Nürnberg Friedrich-Alexander-University Erlangen-Nürnberg Selection of Functional B-Lymphocytes Symposium: 70th Birthday of Max Cooper B Lymphopoiesis II Birmingham, Alabama 2003

3. Selection of Functional B Lymphocytes - 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

4. Discovery of the Pre-B Cell

6. 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 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)

7. ? Ig Receptors μHC λ5λ5 Igαβ VpreB Igαβ IgL μHC

8. Late Pro-B Late Pre-B Immature B cell Early Pre-B Stem cell Early Pro-B  fct ? L ? Mature B cell Pairing of Ig chains 1 2 Autoreactivity 3 Quality Control of B-Lymphoid Precursors Quality Control of B-Lymphoid Precursors At Developmental Checkpoints D  JHD  JH V H  DJ H V L  J L

9. λ5 +/+ λ5 -/- μHC B220 Spleen SLC-Independent Generation of B Cells

10. SL-chain independent signals mediated by μH chains? Question

11. The μHC-inducible Mouse Pro-B - Tet + Tet µ Rag -/- tTA + tetop-µH Double Transgenic Mouse (dTg) = Recombination activating gene Tet-op  H-Gen tTA Tet tTA + Tet Tet-off System of Gossen und Bujard + - Rag2 -/-

12. dTg Rag2 - λ5 + dTg Rag2 - λ5 - Fluorescence intensities (FI) No. λ5λ5 VpreB 50% 48% µHC dTg Rag2 - λ5 + FI (B220) No. FI (cyto μHC) FI (CD25) 195.7 205.1 C57Bl/6 Control of µH-Chain Expression by Tetracycline μ/ no λ5 μ/λ5μ/λ5

13. In Vivo Signaling of a µH-chain in the absence of λ 5 μ / λ5 dTg/Rag2 - /λ 5 + μ / no λ5 dTg/Rag2 - /λ5 - no μ tTA/Rag2 - /λ5 - c-kit CD25 CD19 29.0 16.4 4.6 40.4 11.9 10.0 9.9 0.8 7.6 9.1 1.3

14. Signaling of a µH-chain in the absence of λ 5 Rag2 -/- / dTg / λ 5 +/+ Rag2 -/- / dTg / λ 5 -/- Rag2 -/- ‚Inducible μ mouse‘ (dTg) µ - + 1234 5 20 10 15 25 012345 + IL7 (10ng/ml) Culture time [days] + Tet Cell count x10 4 + tet - tet λ5 +/+ λ5 -/- +μ+μ 012345 0.1 1.0 - IL7 Pro-B +/- IL7 +μ+μ -μ-μ -μ-μ -μ-μ +μ+μ +μ+μ

15. Analysis of µH-Chain Surface Expression Indirect immunostaining of surface μHC with monoclonal rat anti-mouse µH antibodies (mAb b7.6) membrane cytoplasmic 6,2 22,5 6,1 12,6 Grey: + Tet Black: - Tet μ/λ5 dTg/Rag2 - /λ 5 + μ / no λ5 dTg/Rag2 - /λ5 -

16. TK - TK.μ F (81X) Cyto Surface CD43 0.3 15.8 10.7 0.3 17.3 18.1 B220 10 0 10 2 10 3 10 4 10 1 μHC Surface Expression is Required for B Cell Development J H +/+ J H -/- 81XF J H -/- Bone Marrow Pre-B Cell Lines μHC Events

17. μH-Chain Signaling in the Absence of λ 5 A μH-Chain in the absence of λ5 ocan be expressed on the surface of in- vitro cultivated pre-B cells omust be transported to the cell surface to aquire signal competency oinduces signals in-vivo and in-vitro These findings could explain owhy B cell maturation is leaky in a λ5 knock-out mouse

18. Pre-BCR Signal Initiation ?

19. Models: Initiation of Pre-BCR Signals ER Constitutive from ER ER cytoplasm ? Constitutive from cell surface Ligand cytoplasm ER Ligand induced cytoplasm ? ? ?

20. 0123456 10 6 10 5 Pro-B cells (dTg/Rag2 -/- / 5 -/- ) 10 5 10 4 123 - tet + tet No. days - tet + tet Pro-B cells (dTg/Rag2 -/- ) +/- stroma cells (SC) Signaling of a µH-chain in the absence of λ 5 0 1 23 10 5 10 4 Pro-B cells (dTg/Rag2 -/- ) +SC ( λ5 + ) +SC (λ5 - ) -SC (λ5 + ) Rag2 -/- / dTg / λ 5 +/+ Rag2 -/- / dTg / λ 5 -/- Rag2 -/- ‚Inducible μHC mouse‘ (dTg) + Tet Pro-B µ

21. Ligand-Induced Pre-BCR Signaling ? Flow cytometry soluble pre-BCR FITC ligand ST2 + Heparin + Trypsin + Heparitinase Events Control: (Fab-like BCR) Fluorescence Intensity (pre-BCR)

22. Schematic Aligments of Murine VpreB and λ 5 with a Conventional λ Chain V J C  chain VpreB 5 non-Ig like tail NH3 + COO - NH3 + COO - - - + + + +

23. VpreB plus 5(  8) CDR1 CDR2 V L domain - + Computer Modelling of a SL Chain In cooperation with Dr. Harald Lanig, Chemie Computer Center, FAU Erlangen-Nürnberg + CDR1 CDR2 CDR3

24. λ5 Tail is Critical for Pre-BCR/Stroma Cell Interaction Events F (pre-BCR) VpreB/ 5 anti-VpreB VpreB/ 5 VpreB  T/ 5 VpreB/ 5  T VpreBTU/ 5  T anti- 5  IP-Ab 14 - 21- 30-  Blot-Ab  VpreB  T  VpreB A VpreB  T/ 5  T VpreB/ 5  T VpreB  T/ 5

25. GST GST- λ5Tail GST- VpreBTail + Heparin Binding of GST-Tail-Fusionproteins to ST2 cells

26. The interaction of a pre-BCR with stroma cells is mediated by the tail of λ 5 and a stroma cell-associated heparan sulfateConclusion

27. Model for Selection of Early Pre-B Cells Pre-BCR levels below signaling threshold Pre-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

28. Thanks – Jäck Lab Anja Haude Wolfgang Schuh Christian Vettermann Harald Bradl Silke Meister Edith Roth

29. 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) Reagents Collaborators

30. Happy Birthday Max

31. dTg Rag2 - 5 + dTg Rag2 - 5 - 21 15 34% 58% - + - µHC - 30% 64% - + µHC FI (cyto µHC) FI (membrane µHC) FSC No. + Tet- Tet 134 96

32. 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-BCR