Phenotypic Analysis of B-Cells and Plasma Cells

  • Henrik E. Mei
  • Taketoshi Yoshida
  • Gwendolin Muehlinghaus
  • Falk Hiepe
  • Thomas Dörner
  • Andreas Radbruch
  • Bimba F. Hoyer
Part of the Methods in Molecular Medicine book series (MIMM, volume 136)


B-cells and antibody-secreting plasma cells are key players in protective immunity, but also in autoimmune disease. To understand their various functions in the initiation and maintenance of autoimmune pathology, a detailed dissection of their functional diversity is mandatory. This requires a detailed phenotypic classification of the diversity of B-cells. Here, technologies of immunocytometry and ELISpot are described in detail, and their value for phenotypic characterization of cells of the B lineage, as well as for preparative cell sorting, to further characterize them functionally and on the molecular level are described.

Key Words

B-cells plasma cells flow cytometry intracellular Ig surface Ig secreted Ig ELISpot antigen specific memory B-cells naïve B-cells FACS autoimmunity cytometric secretion assay 


  1. 1.
    Behring, E. and Kitasato, S. (1980) Über das Zustandekommen der Diphterie-Immunität und der Tetanus-Immunität bei Tieren. D. Medizinische Wochenschrift 49, 41–52.Google Scholar
  2. 2.
    Fagraeus, A. (1948) The plasma cellular reaction and its relation to the formation of antibodies in vitro. J. Immunol. 58, 1–3.PubMedGoogle Scholar
  3. 3.
    Nossal, G.J. (2002) One cell, one antibody: prelude and aftermath. Immunol. Rev. 185, 15–23.PubMedCrossRefGoogle Scholar
  4. 4.
    Glick, B., Chang, T.S., Japp, R.G. (1956) The bursa of Fabricius and antibody production. Poultry Sc 35, 224.Google Scholar
  5. 5.
    Cooper, M.D., Raymond, D.A., Peterson, R.D., South, M.A., and Good, R.A. (1966) The functions of the thymus system and the bursa system in the chicken. J. Exp. Med. 123, 75–102.PubMedCrossRefGoogle Scholar
  6. 6.
    Bernard, A.R., Boumsell, L., Dausset, J., Schlossman, S.F. (1984) Leucocyte Typing I., Berlin: Springer-Verlag.Google Scholar
  7. 7.
    Radbruch, A. (1999) Flow Cytometry and Cell Sorting. Berlin: Springer.Google Scholar
  8. 8.
    Herzenberg, L.A. and S.C. De Rosa (2000) Monoclonal antibodies and the FACS: complementary tools for immunobiology and medicine. Immunol. Today 21, 383–90.PubMedCrossRefGoogle Scholar
  9. 9.
    Miltenyi, S. et al (1990) High gradient magnetic cell separation with MACS. Cytometry, 11, 231–238.PubMedCrossRefGoogle Scholar
  10. 10.
    Leyendeckers, H., Odendahl, M., Lohndorf, A., et al. (1999) Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG. Eur. J. Immunol. 29, 1406–1417.PubMedCrossRefGoogle Scholar
  11. 11.
    Janeway, C.A.T., Walport, M., Shlomchik, M. (2001), Immunobiology. 5th ed. New York and London: Garland Publishing.Google Scholar
  12. 12.
    Rolink, A.G., Nutt, S.L., Melchers, F., and Busslinger, M. (1999) Long-term in vivo reconstitution of T-cell development by Pax5-deficient B-cell progenitors. Nature 401, 603–606.PubMedCrossRefGoogle Scholar
  13. 13.
    Nutt, S.L., Heavey, B., Rolin, A.G., and Busslinger, M. (1999) Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562.PubMedCrossRefGoogle Scholar
  14. 14.
    Carsetti, R. (2004) Characterization of B-cell maturation in the peripheral immune system. Methods Mol. Biol. 271, 25–35.PubMedGoogle Scholar
  15. 15.
    Carsetti, R., Rosado, M.M. and Wardmann, H. (2004) Peripheral development of B cells in mouse and man. Immunol. Rev. 197, 179–191.PubMedCrossRefGoogle Scholar
  16. 16.
    Hardy, R.R. and K. Hayakawa (2001) B cell development pathways. Annu. Rev. Immunol. 19, 595–621.PubMedCrossRefGoogle Scholar
  17. 17.
    Iwakoshi, N.N., A.H. Lee, and L.H. Glimcher (2003) The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response. Immunol. Rev. 194, 29–38.PubMedCrossRefGoogle Scholar
  18. 18.
    Johnson, K., Shapiro-Shelef, M., Tunyaplin, C., and Calame, K. (2005) Regulatory events in early and late B-cell differentiation. Mol. Immunol. 42, 749–761.PubMedCrossRefGoogle Scholar
  19. 19.
    Manz, R.A., Hauser, A.E., Hiepe, F., and Radbruch, A. (2005) Maintenance of serum antibody levels. Annu. Rev. Immunol. 23, 367–386.PubMedCrossRefGoogle Scholar
  20. 20.
    Klein, U., Tu, Y., Stolovitzky, G.A., et al. (2003) Transcriptional analysis of the B cell germinal center reaction. Proc. Natl. Acad. Sci. USA 100, 2639–2644.PubMedCrossRefGoogle Scholar
  21. 21.
    Scheffold, A., Miltenyi, S. and Radbruch, A. (1995) Magnetofluorescent liposomes for increased sensitivity of immunofluorescence. Immunotechnology 1, 127–137.PubMedCrossRefGoogle Scholar
  22. 22.
    Wang, Y.H., Stephan, R.P., Scheffold, A., et al. (2002) Differential surrogate light chain expression governs B-cell differentiation. Blood 99, 2459–2467.PubMedCrossRefGoogle Scholar
  23. 23.
    Jerne, N.K. and Nordin, A.A. (1963) Plaque formation in agar by single antibodyproducing cells. Science 140, 405.CrossRefGoogle Scholar
  24. 24.
    Czerkinsky, C.C., Nilsson, L.A., Nygren, H., Ouchterlony, O., and Tarkowski, A. (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J. Immunol. Methods 65,109–121.PubMedCrossRefGoogle Scholar
  25. 25.
    Blink, E.J., Light, A., Kallies, A., Nutt, S.L., Hodgkin, P.D., and Tarlinton, D.M. (2005) Early appearance of germinal center-derived memory B cells and plasma cells in blood after primary immunization. J. Exp. Med. 201, 545–554.PubMedCrossRefGoogle Scholar
  26. 26.
    Jacobi, A.M., Odendahl, M., Reiter, K., et al. (2003) Correlation between circulating CD27high plasma cells and disease activity in patients with systemic lupus erythematosus. Arthritis Rheum. 48, 1332–1342.PubMedCrossRefGoogle Scholar
  27. 27.
    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
  28. 28.
    Boyum, A. (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand. J. Clin. Lab. Invest. Suppl 97, 77–89.Google Scholar
  29. 29.
    Arce, S., Luger, E., Muehlinghaus, G., et al. (2004) CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations. J. Leukoc. Biol.l 75, 1022–1028.CrossRefGoogle Scholar
  30. 30.
    Assenmacher, M., Lohning, M., Scheffold, A., Manz, R.A., Schmitz, J., and Radbruch, A. (1998) Sequential production of IL-2, IFN-gamma and IL-10 by individual staphylococcal enterotoxin B-activated T helper lymphocytes. Eur. J. Immunol. 28,1534–1543.PubMedCrossRefGoogle Scholar
  31. 31.
    Odendahl, M., Mei, H., Hoyer, B.F., et al. (2005) Generation of migratory antigen-specific plasma blasts and mobilization of resident plasma cells in a secondary immune response. Blood 105, 1614–1621.PubMedCrossRefGoogle Scholar
  32. 32.
    Czerkinsky, C., Moldoveanu, Z., Mestechky, J., Nilsson, L.A., and Ouchterlony, O. (1988) A novel two colour ELISPOT assay. I. Simultaneous detection of distinct types of antibody-secreting cells. J. Immunol. Methods 115, 31–37.PubMedCrossRefGoogle Scholar
  33. 33.
    Tangye, S.G., Avery, D.T., Deenick, E.K., and Hodgkin, P.D. (2003) Intrinsic differences in the proliferation of Naive and Memory Human B cells as a Mechanism for Enhanced Secondary Immune Response. J. Immunol. 170, 686–694.PubMedGoogle Scholar
  34. 34.
    Chung, J.B., Silverman, M., and Monroe, J.G. (2003) Transitional B cells: step by step towards immune competence. Trends Immunol. 24, 343–349.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Henrik E. Mei
    • 1
  • Taketoshi Yoshida
    • 1
  • Gwendolin Muehlinghaus
    • 1
  • Falk Hiepe
    • 2
  • Thomas Dörner
    • 3
  • Andreas Radbruch
    • 1
  • Bimba F. Hoyer
    • 2
  1. 1.German Rheumatism Research CentreBerlinGermany
  2. 2.Rheumatology and Clinical Immunology, CharitéClinic for Internal MedicineBerlinGermany
  3. 3.Institute of Transfusion Medicine, CharitéBerlinGermany

Personalised recommendations