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02.10.09(d): B-Cell Development


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School's M1 Immunology sequence

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  • 1. Attribution: University of Michigan Medical School, Department of Microbiologyand ImmunologyLicense: Unless otherwise noted, this material is made available under the termsof the Creative Commons Attribution–Noncommercial–Share Alike 3.0License: We have reviewed this material in accordance with U.S. Copyright Law and have tried to maximize your ability to use, share, and adapt it. The citation key on the following slide provides information about how you may share and adapt this material. Copyright holders of content included in this material should contact with any questions, corrections, or clarification regarding the use of content. For more information about how to cite these materials visit Any medical information in this material is intended to inform and educate and is not a tool for self-diagnosis or a replacement for medical evaluation, advice, diagnosis or treatment by a healthcare professional. Please speak to your physician if you have questions about your medical condition. Viewer discretion is advised: Some medical content is graphic and may not be suitable for all viewers.
  • 2. Citation Key for more information see: + Share + Adapt { Content the copyright holder, author, or law permits you to use, share and adapt. } Public Domain – Government: Works that are produced by the U.S. Government. (USC 17 § 105) Public Domain – Expired: Works that are no longer protected due to an expired copyright term. Public Domain – Self Dedicated: Works that a copyright holder has dedicated to the public domain. Creative Commons – Zero Waiver Creative Commons – Attribution License Creative Commons – Attribution Share Alike License Creative Commons – Attribution Noncommercial License Creative Commons – Attribution Noncommercial Share Alike License GNU – Free Documentation LicenseMake Your Own Assessment { Content Open.Michigan believes can be used, shared, and adapted because it is ineligible for copyright. } Public Domain – Ineligible: Works that are ineligible for copyright protection in the U.S. (USC 17 § 102(b)) *laws in your jurisdiction may differ { Content Open.Michigan has used under a Fair Use determination. } Fair Use: Use of works that is determined to be Fair consistent with the U.S. Copyright Act. (USC 17 § 107) *laws in your jurisdiction may differ Our determination DOES NOT mean that all uses of this 3rd-party content are Fair Uses and we DO NOT guarantee that your use of the content is Fair. To use this content you should do your own independent analysis to determine whether or not your use will be Fair.
  • 3. B Cell Development M1 – Immunology SequenceWinter 2009
  • 4. 1.  Receptors and cells in innate immunity.2.  The two fundamental stages of B cell differentiation.3.  The steps in antigen-independent B cell differentiation.4.  How is possible for a human to express more than ten million antibodies?5.  How are the genes for antibody variable regions organized?
  • 5. Innate immunity is that protection againstpathogens which is rapid and does notrequire specific recognition of the pathogen.Toll-like receptors (TLRs) are used torecognize bacteria and viruses in innateimmunity. They are named after similarreceptors in Drosophilia. One function ofthese receptors in Drosophilia is to induceimmunity against fungi.
  • 6. Receptor Ligand(s)TLR1 triacyl lipoproteinTLR2 lipoproteins, peptidoglycan, lipoteichoic acidsTLR3 double-stranded RNATLR4 lipopolysaccharideTLR5 flagellinTLR6 diacyl lipoproteinsTLR7, TLR8 single-stranded RNATLR9 unmethylated CpG DNATLR 11 profilinTLR12, TLR13 unknown
  • 7. TLR recognition displays only limitedspecificity. TLRs bind to pattern moleculesthat are not expressed by humans, but areshared by groups of pathogens.TLRs are expressed by many cells, includingleukocytes.
  • 8. Binding to pattern molecules results in signaltransduction from the TLR to the nucleus.Consequences of TLR signaling:• Production of cytokines and chemokinesand subsequent inflammation.• Production of Type I interferons (α and β)• Killing or inhibition of viruses andintracellular bacteria.• Upregulation of co-stimulatory moleculesthat help to activate T and B lymphocytes.
  • 9. NOD receptors are another set of receptorsassociated with innate immunity. They areexpressed in the cytoplasm.NOD: containing a nucleotide-binding/ oligomerization domain
  • 10. Several cells are associated with innate immunity:• Macrophages and neutrophils•  B1 B cells—make most of the antibody in serum.These antibodies tend to bind common epitopes onpathogens.• Dendritic cells, γδ T cells, NK T cells (Dr. Chang)The innate B and T cells tend to have a set ofantigen receptors with a limited diversity.
  • 11. NK cells are lymphocytes that may be a little largerand more granular than T and B lymphocytes. Theykill cells that do not express MHC class I molecules(for example, those infected with viruses or tumorcells). Their killing is regulated by a complexinteraction among several inhibitory (for example,binding to some class I MHC inhibits killing) andactivating receptors.Some NK cells have an Fc receptor that allows themto bind antibody that is bound to a cell, and kill thatcell by a process called antibody-dependent, cell-mediated cytotoxicity (ADCC).
  • 12. Immature MatureStem cell Pro B cell Pre B cell B cell B cell Y Y Y µ IgD and IgM µ chain in IgM on on cellAbsent Absent cytoplasm cell surface surface University of Michigan Department of Microbiology and Immunology
  • 13. B cell differentiation occurs in the bone marrowfrom pluripotent stem cells.Pro B cells can be distinguished from stem cellsby the expression of several CD antigens: CD19,CD20, etc.B cell development depends on the adherent cellpart of bone marrow (stromal cells) andcytokines (IL-7).The final product is a mature B cell, which hasnever been exposed to antigen (it is naïve).
  • 14. CD molecules (cluster of differentiation).These molecules are found on the surface of agroup of cells that are at the same state ofdifferentiation. The group of cells can be large(all lymphocytes) or small (CD8+ T cells).Thus, the expression of a CD molecule can beused to define the state of development of acell. They are detected by monoclonalantibodies.
  • 15. Flow Cytometry (FACS).This instrument is used to detect the cellsurface expression of CD molecules.Bind an antibody that is tagged with afluorescent molecule (fluorescein,phycoerythrin, or rhodamine, for example) to acell surface molecule.Analyze amount of antibody bound, andtherefore expression of the cell surfacemolecule, in the flow cytometer.
  • 16. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  • 17. If one uses two different fluorescent tags on twodifferent antibodies, excites them with twodifferent lasers, and detects them at twodifferent emission wavelengths, then one canmonitor two cell surface molecules at once. (Oreven more.)Data presentation: Each cell is represented asone dot. For single colors, data are oftenpresented as the number of cells versusrelative fluorescence.
  • 18. Normal Immunodeficient Immunodeficient Minegishi et al., J. Clin Invest. 104: 1115-21 (1999)Pro and Pre B cellsB cells Stem cells
  • 19. Antigen-independent B cell differentiationfrom a pre B cell to an immature B celldepends on positive signaling through areceptor with the mu heavy chain only(the pre B cell receptor).
  • 20. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  • 21. Bruton s agammaglobulinemia• Boys with repeated infections by encapsulated bacteria or sometimes viruses• X-linked• Failure to produce antibodies• Almost no production of immature B cells in thebone marrowMutation in Bruton s tyrosine kinase (btk) that isactivated upon engagement of the pre B cellreceptor. A signal from mu on the surface of preB cells in these boys does not reach the nucleus—positive signaling fails.
  • 22. Antigen-independent B cell differentiationin the bone marrow results in ten milliondifferent clones of B cells, each with anantibody on their surface that binds adifferent epitope. (The sequences ofheavy chain variable region and the lightchain variable region are different forevery B cell clone.) Thus, the receptorrepertoire is ten million.(A human has more than 1012 B cells).
  • 23. Each antibody is made by one clone of Bcells. Hence, antibodies are clonallydistributed . Regents of the University of Michigan
  • 24. Immature B cells with animmunoglobulin on their surfacethat binds to a self antigen sends anegative signal, resulting in deletion ofthe immature B cells in the bonemarrow. Clonal deletion This is part of self-tolerance for B cells
  • 25. Regents of the University of Michigan
  • 26. Allelic exclusion: In a B cell clone, only one of the twoantibody loci (one of the two homologouschromosomes) is expressed as antibody protein.Therefore, even though a B cell could express two muheavy chains, it only expresses one. Even though a Bcell could express four light chains, it expresses onlyone.
  • 27. B cell diversity of more than ten million clones isgenerated during antigen-independent B celldevelopment. How is this diversity generated?A hint comes from the fact that antibodies are two-partproteins.Constant region--one gene. If there were two genes,single amino acid changes would occur and therewould soon be two isotypes: λI, λII, λIII, etc.Variable region--many genes for the three kinds ofvariable regions: Vκ, Vλ, and VH.
  • 28. Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Vκ encodes amino acids 1-95Jκ encodes amino acids 96-107Cκ encodes amino acids 108-214
  • 29. Human Vκ1500 kb of DNA. V genes are 5 to 20 kbapart. Most 3 Vκ is 23 kb 5 of Cκ.Five Jκ regions, encoding amino acids 96to 107, lie 1.2 kb 5 of Cκ.
  • 30. Of the 79 Vκ genes, almost one-half arepseudogenes--genes that cannot beexpressed as a light chain, because theyhave an in frame stop codon, lack aninvariant amino acid, or are truncated atthe 5 end.
  • 31. V codon 95 CACAGTG--spacer--GGTTTTTGTACAAAAACC--spacer--CACTGTG J codon 96Each V is followed by CACAGTG--spacer--GGTTTTTGT (recombination signal sequence),or a slight variant of it, and each J segment is preceded by ACAAAAACC--spacer--CACTGTG, or a slight variant of it.This is true for the variable regions and J segments associated with heavy chain, kappa, and lambda light chain genes.
  • 32. orange arrow: CACAGTG blue arrow: GGTTTTTGTJaneway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997
  • 33. V(D)J recombination is mediated by thelymphoid specific recombination activating genesRAG1 and RAG2.Mutations in the recombination activating geneslead to severe combined immunodeficiency (no Bor T cells) or to Omenn s syndrome, a milderimmunodeficiency.
  • 34. Summary1.  Innate immunity involves recognition by TLRs and NODs--receptors for pattern molecules.2.  NK cells kill cells with low expression of MHC class I molecules.3.  Antigen-independent B cell differentiation occurs in the bone marrow. It includes several stages that involve changes in immunoglobulin heavy and light chain expression, as well as other CD antigens. Antigen-independent B cell differentiation results in a repertoire of at least ten million clones of B cells.
  • 35. 4.  Anti-self, immature B cells are deleted.5.  Variable and constant region genes for immunoglobulins are separated in DNA. 6.  Variable regions are encoded by many genes —Germline diversity.7.  V(D)J joining uses specific sequences (CACAGTG--spacer--GGTTTTTGT) and is mediated by the recombination activating gene products.
  • 36. Additional Source Information for more information see: 12: University of Michigan Department of Microbiology and ImmunologySlide 17: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 18: Minegishi et al., J. Clin Invest. 104: 1115-21 (1999)Slide 20: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 23: Regents of the University of MichiganSlide 25: Regents of the University of MichiganSlide 28: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997Slide 32: Janeway. Immunobiology: The Immune System in Health and Disease. Current Biology Ltd./Garland Publishing, Inc. 1997