B CELL DEVELOPMENT IN THE BONE MARROW

1. B CELL DEVELOPMENT IN THE BONE MARROW

2. a a ORDERED B-CELL DEVELOPMENT immature B cell pre B cell ANTIGEN RECOGNIZING RECEPTOR H2L2 pro B cell H-chain + surrogate L-chain SIGNALING RECEPTOR NO ANTIGEN RECOGNIZING RECEPTOR

3. Pre-B cell receptor expressed Stages of differentiation in the bone marrow are defined by Ig gene rearrangement B CELL STAGE IgH GENE CONFIGURATION Stem cell Early pro-B Late pro-B Large pre-B Germline DH to JH VH to DHJH VHDHJH Ig Light chain gene has not yet rearranged

4. 1. Specific cell-cell contacts between stromal cells and developing B cells 2. Secretion of cytokines by stromal cells Secreted Factors - CYTOKINES B Cell-cell contact Stromal cell Bone marrow stromal cells nurture developing B cells Types of cytokines and cell-cell contacts needed at each stage of differentiation are different

5. Maturing B cells Bone marrow stromal cell

6. B B Stromal cell

7. c-Kit Receptor Tyrosine kinase Stem VLA-4 (Integrin) Stem cell factor Cell-bound growth factor VCAM-1 (Ig superfamily) Cell adhesion molecules Stromal cell Cytokines and cell-cell contacts at each stage of differentiation are different Early pro-B

8. Interleukin-7 receptor Interleukin-7 Growth factor Early pro-B Stromal cell VLA-4 (Integrin) VCAM-1 (Ig superfamily) Cytokines and cell-cell contacts at each stage of differentiation are different Late pro-B Pre-B

9. Heavy chain VHDHJH V-preB l5 CHm Iga & Igb signal transduction molecules Pre- B cell receptor Transiently expressed when VHDHJH CHm is productively rearranged VpreB/l5 - the surrogate light chain, is required for surface expression Ligand for the pre-B cell receptor is unknown

10. Large Pre-B Unknown ligand of pre-B cell receptor Stromal cell Ligation of the pre-B cell receptor 1.Suppresses further H chain rearrangement 2. Triggers entry into cell cycle 1. Ensures only one specificty of Ab expressed per cell 2. Expands only the pre-B cells with in frame VHDHJH joins ALLELIC EXCLUSION

11. Development continues Pre-B cell receptor can ligate to stromal cell Translation in frame 1 MKXLWFFLLLVAAPRWVLSQVHLQESGPGLGKPPELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCXXCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDXXVQFKWYVDGVEVHNAKTKLREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRYTQKSLSLSPGK* Large pre-B Development arrests Development arrests Large pre-B cells need in frame VHDHJH joins to mature Human IgG3 Heavy Chain nucleotide sequence ATGAAACANCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCCAGGTGCACCTGCAGGAGTCGGGCCCAGGACTGGGGAAGCCTCCAGAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCNNNGTGCCCAGCACCTGAACTCTTGGGAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGATACCCTTATGATTTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACCCNNNNGTCCAGTTCAAGTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCTGCGGGAGGAGCAGTACAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGACAGCCCGAGGAGATGACCAAGAACCAAGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAACACCACGCCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACATCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCGCTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Translation in frame 2 (no protein) * Translation in frame 3 ETXVVLPSPGGSSQMGPVPGAPAGVGPRTGEASRAQNPTW*

12. Proliferation Large pre-B Y Immature B cell IgM Small pre-B Large pre-B Large Pre-B Large Pre-B Proliferation stops Pre-receptor not displayed Light chain expressed IgM displayed on surface Large Pre-B Large Pre-B Intracellular VDJCH chain VL-JL rearranges Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Large Pre-B Ligation of the pre-B cell receptor triggers entry into the cell cycle Many large pre-B cells with identical pre-B receptors

13. Heavy chain VHDHJH Light chain VLJLCL CHm Iga & Igb signal transduction molecules B cell receptor L chain is rearranged

14. Stem Cell Early pro-B cell Late pro-B cell Nagy pre-B sejt Large pre-B cell Pre-receptor Peripheral Small pre-B cell Immature B cell Mature B cell Stages of B cell development Y Receptor H+L Each stage of development is defined by IgH and IgL chain genes, expression of adhesion molecules and cytokine receptors

15. ORDER OF REARRANGEMENTS OF IMMUNOGLOBULIN GENE SEGMENTS Surrogate light chain D – J recombination V – DJ recombination V – J recombination VDJ –δ transcription δ translation VJ –  (or VJ - ) transcription  or  translation B-sejt Secreted IgM mIgD mIgM

16. DEVELOPMENT OF B-LYMPHOCYTES IN THE BONE MARROW Limphoid precursor B B B B c-kit/CD44 L rearrangement Surrogate L RAG-1/RAG-2 H rearrrangement  Selection clonal deletion • B cells recognizing self structures • Cell surface molecules • MHC proteins • Common molecules of haemopoetic cells • apoptosis, clonal deletion • Soluble molecules • House keeping genes • Metabolites • functional unresponsiveness • anergia H átrendeződés Other specificites PERIPHERAL LYMPHOID TISSUES

17. RECEPTOR EXPRESSION DURING B-CELL DEVELOPMENT

18. Y B YES YES YES YES IgMk NO NO NO YES YES NO NO YES B Y IgMl NO YES NO B NO B cells have several chances to successfully rearrange Ig genes Early Pro B Late Pro B Pre B Immature B k on first chromosome VH-DJH On first chromosome DH-JH On first chromosome k on second chromosome VH-DJH On second chromosome DH-JH On second chromosome l on first chromosome l on second chromosome

19. Self recognition Clonal deletion Self structure Negative selection of immature B-cells in the bone marrow Potential B-cell repertoire BONE MARROW RNA editing PERIPHERAL LYMPHOID ORGANS Available B-cell repertoire Foreign antigen independent About 30 billion mature naive B cells leave the bone marrow per day to circulate in blood

20. SYNTHESIS OF IMMUNOGLOBULINS Secreted Ig Membrane Ig Golgi ER H and L chains are synthesized on separated ribosomes CHAPERONES Leader sequence Ribosome mRNA

21. RESULT OF SOMATIC GENE REARRANGEMENT AND ALLELIC EXCLUSION • Somatic rearrangement of Ig gene segments in a highly controlled manner • Single B-cells become committed to the synthesis of one unique H-chain and one unique L-chain variable domain, which determine their specificities • In one individual a huge B-cell repertoire is generated consisting of B-cell clones with different H- and L-chain variable domains • This potential B-cell repertoire is able to recognize a wide array of various antigens • Immature B-cells express IgM and IgD surface Ig with the same variable domains

22. MEMBRANE BOUND AND SECRETED IMMUNOGLOBULIN How can immature B-cells express surface IgM and IgD

23. Co-Expression of cell surface IgM and IgG On Mature B-cells is controlled by alternative RNA processing

24. V V V V V V D D D D D D J J J J J J Cm Cd Cg3 Cg1 Cm1 Cd1 Cd2 Cm2 Cm3 Cm4 Cd3 DNA pA2 pA1 RNA cleaved and polyadenylated at pA1 AAA Cm1 Cd1 Cd2 Cm2 Cm3 Cm4 Cd3 Cm IgM mRNA RNA cleaved and polyadenylated at pA2 AAA Cm1 Cd1 Cd2 Cm2 Cm3 Cm4 Cd3 pA1 Cd IgD mRNA Splicing of IgM and IgD RNA Two types of mRNA can be made simultaneously in the cell by differential usage of alternative polyadenylation sites and splicing of the RNA

25. SYNTHESIS OF IMMUNOGLOBULINS Secreted Ig Membrane Ig Golgi ER H and L chains are synthesized on separated ribosomes CHAPERONES Leader sequence Ribosome mRNA