Immunoglobulin Class Switching

In Vitro Induction and Analysis
  • Sven Kracker
  • Andreas Radbruch
Part of the Methods in Molecular Biology book series (MIMB, volume 271)

Abstract

During an immune response, B lymphocytes can switch expression of immunoglobulin (Ig) class (isotype) from IgM to IgG, IgE, or IgA. This Ig class switch is based on a deoxyribonucleic acid (DNA) recombination event that results in an exchange of the gene segments coding for the constant region of the Ig heavy chain, although the Ig heavy chain variable region is retained. This process changes the effector functions of the corresponding antibody (Ab). Much of our current understanding of the molecular mechanisms of class switch recombination is based on the analysis of B cells induced to switch class of Ig in vitro. In vitro, murine and human naive B cells can be activated with bacterial lipopolysaccharides, anti-CD40 or CD40L, to undergo class switch recombination. Cytokine signals can direct class switch recombination to distinct classes; for example, interleukin-4 will target murine IgG1 and IgE, and human IgG4 and IgE. Here we describe the technologies for the isolation of B lymphocytes, their activation to class switching, and the analysis of Ig class switching.

Key Words

Lipopolysaccharide LPS CFDA-SE CFSE IgG1 IgG3 IgE interleukin-4 IL-4 proliferation intracellular staining surface staining antibody class switch recombination Ig isotypes B cell B lymphocyte 

References

  1. 1.
    Nossal, G. J. V., Szenberg, A., Ada, G. L., and Austin, C. M. (1964) Single cell studies on 19S antibody production. J. Exp. Med. 119, 485–502.PubMedCrossRefGoogle Scholar
  2. 2.
    Honjo, T. and Kataoka, T. (1978) Organization of immunoglobulin heavy chain genes and allelic deletion model. Proc. Natl. Acad. Sci. USA 75, 2140–2144.PubMedCrossRefGoogle Scholar
  3. 3.
    Kataoka, T., Kawakami, T., Takahashi, N., and Honjo, T. (1980) Rearrangement of immunoglobulin γ1-chain gene and mechanism for heavy-chain class switch. Proc. Natl. Acad. Sci. USA 77, 919–923.PubMedCrossRefGoogle Scholar
  4. 4.
    Rabbitts, T. H., Forster, A., Dunnick, W., and Bentley, D. L. (1980) The role of gene deletion in the immunoglobulin heavy chain switch. Nature 283, 351–356.PubMedCrossRefGoogle Scholar
  5. 5.
    Kataoka, T., Miyata, T., and Honjo, T. (1981) Repetitive sequences in class switch recombination regions of immunoglobulin heavy chain genes. Cell 23, 357–368.PubMedCrossRefGoogle Scholar
  6. 6.
    Radbruch, A., Burger, C., Klein, S., and Müller, W. (1986) Control of immunoglobulin class switch recombination. Immunol. Rev. 89, 69–84.PubMedCrossRefGoogle Scholar
  7. 7.
    Sablitzky, F., Radbruch, A., and Rajewsky, K. (1982) Spontaneous immunoglobulin class switching in myeloma and hybridoma cell lines differs from physiological class switching. Immunol. Rev. 67, 59–72.PubMedCrossRefGoogle Scholar
  8. 8.
    Nakamura, M., Kondo, S., Sugai, M., Nazarea, M., Imamura, S., and Honjo, T. (1996) High frequency class switching of an IgM+ B lymphoma clone CH12F3 to IgA+ cells. Int. Immunol. 8, 193–201.PubMedCrossRefGoogle Scholar
  9. 9.
    Kearney, J. F., Cooper, M. D., and Lawton, A. R. (1976) B cell differentiation induced by lipopolysaccharide IV: development of immunoglobulin class restriction in precursors of IgG synthesizing cells. J. Immunol. 117, 67–72.Google Scholar
  10. 10.
    Anderson, J., Coutinho, A., Lernhardt, W., and Melchers, F. (1977) Clonal growth and maturation to immunoglobulin secretion in vitro of every growth-inducible B lymphocyte. Cell 10, 27–36.CrossRefGoogle Scholar
  11. 11.
    Esser, C. and Radbruch, A. (1990) Immunoglobulin class switching: molecular and cellular analysis. Annu. Rev. Immunol. 8, 717–735.PubMedCrossRefGoogle Scholar
  12. 12.
    Stavnezer, J. and Sirlin, S. (1986) Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. EMBO J. 5, 95–102.Google Scholar
  13. 13.
    Yancopoulos, G. D., DePinho, R. A., Zimmermann, K. A., Lutzker, S. G., Rosenberg, N., and Alt, F. W. (1986) Secondary genomic rearrangement events in pre-B cells: VHDJH replacement by a LINE-1 sequence and directed class switching. EMBO J. 5, 3259–3266.PubMedGoogle Scholar
  14. 14.
    Radbruch, A., Muller, W., and Rajewsky, K. Class switch recombination is IgG1 specific on active and inactive IgH loci of IgG1-secreting B-cell blasts. Proc. Natl. Acad. Sci. USA 83, 3954–3957.Google Scholar
  15. 15.
    Jung, S., Rajewsky, K., and Radbruch, A. (1993) Shutdown of class switch recombination by deletion of a switch region control element. Science 259, 984–987.PubMedCrossRefGoogle Scholar
  16. 16.
    Hein, K., Lorenz, M. G., Siebenkotten, G., Petry, K., Christine, R., and Radbruch, A. (1998) Processing of switch transcripts is required for targeting of antibody class switch recombination. J. Exp. Med. 188, 2369–2374.PubMedCrossRefGoogle Scholar
  17. 17.
    Muramatsu, M., Sankaranand, V. S., Anant, S., et al. (1999) Specific expression of activation-induced cytidine deaminase (AID), a novel member of the RNA-editing deaminase family in germinal center B cells. J. Biol. Chem. 274, 18,470–18,476.PubMedCrossRefGoogle Scholar
  18. 18.
    Muramatsu, M., Kinoshita, K., Fagarasan, S., Yamada, S., Shinkai, Y., and Honjo, T. (2000) Class switch recombination and hypermutation require activation-induced cytidine deaminase (AID), a potential RNA editing enzyme. Cell 102, 553–563.PubMedCrossRefGoogle Scholar
  19. 19.
    Petersen-Mahrt, S. K., Harris, R. S., and Neuberger, M. S. (2002) AID mutates E. coli suggesting a DNA deamination mechanism for antibody diversification. Nature 418, 99–103.PubMedCrossRefGoogle Scholar
  20. 20.
    Petersen, S., Casellas, R., Reina-San-Martin, B., et al. (2001) AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching. Nature 414, 660–665.PubMedCrossRefGoogle Scholar
  21. 21.
    Rolink, A., Melchers, F., and Andersson, J. (1996) The SCID but not the RAG-2 gene product is required for Sµ-S heavy chain class switching. Immunity 5, 319–330.PubMedCrossRefGoogle Scholar
  22. 22.
    Manis, J. P., Dudley, D., Kaylor, L., and Alt, F. W. (2002) IgH class switch recombination to IgG1 in DNA-PKcs-deficient B cells. Immunity 16, 607–617.PubMedCrossRefGoogle Scholar
  23. 23.
    Bosma, G. C., Kim, J., Urich, T., et al. (2002) DNA-dependent protein kinase activity is not required for immunoglobulin class switching. J. Exp. Med. 196, 1483–1495.PubMedCrossRefGoogle Scholar
  24. 24.
    von Schwedler, U., Jack, H. M., and Wabl, M. (1990) Circular DNA is a product of the immunoglobulin class switch rearrangement. Nature 345, 452–456.CrossRefGoogle Scholar
  25. 25.
    Chu, C. C., Max, E. E, and Paul, W. E. (1993) DNA rearrangement can account for in vitro switching to IgG1. J. Exp. Med. 178, 1381–1390.PubMedCrossRefGoogle Scholar
  26. 26.
    Kühn, R., Rajewsky, K., and Müller, W. (1991) Generation and analysis of interleukin-4 deficient mice. Science 254, 707–710.PubMedCrossRefGoogle Scholar
  27. 27.
    Sedgwick, J. D. and Holt, P.G. (1983) A solid-phase immunoenzymatic technique for the enumeration of specific antibody-secreting cells. J. Immunol. Methods 57, 301–309.PubMedCrossRefGoogle Scholar
  28. 28.
    Sedgwick, J. D. and Holt, P. G. (1986) The ELISA-plaque assay for the detection and enumeration of antibody-secreting cells. J. Immunol. Methods 87, 37–44.PubMedCrossRefGoogle Scholar
  29. 29.
    Manz, R., Assenmacher, M., Pfluger, E., Miltenyi, S., and Radbruch, A. (1995) Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix. Proc. Natl. Acad. Sci. USA 92, 1921–1925.PubMedCrossRefGoogle Scholar
  30. 30.
    Manz, R. A., Lohning, M., Cassese, G., Thiel, A., and Radbruch, A. (1998) Survival of long-lived plasma cells is independent of antigen. Int. Immunol. 10, 101,703–101,711.CrossRefGoogle Scholar
  31. 31.
    Hodgkin, P. D., Lee, J. H., and Lyons A. B. (1996) B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med. 184, 277–281.PubMedCrossRefGoogle Scholar
  32. 32.
    Unkeless, J. C. (1979) Characterization of a monoclonal antibody directed against mouse macrophage and lymphocyte Fc receptors. J. Exp. Med. 150, 580–596.PubMedCrossRefGoogle Scholar
  33. 33.
    Lyons, A. B. and Parish, C. R. (1994) Determination of lymphocyte division by flow cytometry. J. Immunol. Methods 171, 131–137.PubMedCrossRefGoogle Scholar
  34. 34.
    Schmitz, J., Assenmacher, M., and Radbruch, A. (1993) Regulation of T helper cell cytokine expression: functional dichotomy of antigen-presenting cells. Eur. J. Immunol. 23, 191–199.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Sven Kracker
    • 1
  • Andreas Radbruch
    • 1
  1. 1.Deutsches Rheuma-Forschungszentrum (DRFZ)BerlinGermany

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