Assays for B Cell and Germinal Center Development

Jack J.H. Bleesing1

1 Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 7.35
DOI:  10.1002/0471142735.im0735s63
Online Posting Date:  November, 2004
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Abstract

The study of B cell development is highly relevant for a better understanding of human disorders in which B cells are involved. B cell development in humans appears to proceed largely along a linear pathway and occurs in three compartments: bone marrow, peripheral blood, and lymphoid tissue. The focus of this unit is on the detailed immunophenotypic evaluation of cell suspensions obtained from these compartments. These protocols are based on routine methodology, commonly used by clinical flow laboratories. Special emphasis will be placed on the approach to the study of B cell development, including current knowledge regarding immunophenotypic identification of B cell subsets. Multiparameter flow cytometry provides powerful analytical tools, as long as attention is paid to careful design and proper execution of flow cytometric acquisition and analysis. The goal of this unit is to provide a guide to the flow cytometric study of B cell development in humans.

Keywords: B cells; B cell development; bone marrow; peripheral blood; lymphoid tissue; germinal center; flow cytometry; immunohistochemistry

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of Cell Suspensions from Bone Marrow Aspirates
  • Basic Protocol 2: Isolation of Cell Suspensions from Peripheral Blood
  • Basic Protocol 3: Isolation of Cell Suspensions from Tonsillar Tissue
  • Basic Protocol 4: Immunofluorescence Staining and Analysis of B Cell Subsets by Flow Cytometry
  • Basic Protocol 5: Identification of B Cell Subsets Representing Different Phases of B Cell Development
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of Cell Suspensions from Bone Marrow Aspirates

  Materials
  • Bone marrow (BM) sample
  • Heparin anticoagulant (liquid form)
  • PBS ( appendix 2A), pH 7.4
  • Disposable thermometer, optional
  • Additional reagents and equipment for BM aspiration, trypan blue viability stain ( appendix 3B), and cell counting ( appendix 3A)

Basic Protocol 2: Isolation of Cell Suspensions from Peripheral Blood

  Materials
  • Peripheral blood (PB) sample
  • Heparin or EDTA anticoagulant
  • PBS ( appendix 2A), pH 7.4
  • Pipets, sterile
  • Disposable thermometer, optional
  • 15‐ or 50‐ml conical tubes (e.g., Falcon)
  • Additional reagents and equipment for blood drawing ( appendix 3F), trypan blue viability stain ( appendix 3B), and cell counting ( appendix 3A)

Basic Protocol 3: Isolation of Cell Suspensions from Tonsillar Tissue

  Materials
  • Fresh tonsillar tissue
  • Isotonic nutrient medium: HBSS or PBS ( appendix 2A), calcium and magnesium free, pH 7.2
  • RPMI‐1640 with antibiotics ( appendix 2A)
  • FBS (heat inactivated), optional
  • Enzymatic digestion solution: 1 mg/ml type 1 or 3 collagenase and 1 mg/ml type 1 deoxyribonuclease (Worthington), or equivalent, freshly made in sterile isotonic nutrient medium (optional)
  • Sterile container containing saline‐moistened gauze
  • 2 × 2–in. gauze
  • 60 × 15–mm or 150 × 25–mm petri dishes
  • Straight scissors
  • 250‐ml polycarbonate Erlenmeyer flask with cap, optional
  • Plastic syringes and 25‐G needles
  • 50‐ml conical tubes (e.g., Falcon)
  • 50‐µm or greater nylon mesh
  • Centrifuge (Beckman J6‐M centrifuge with JS‐4.2 rotor or equivalent)
  • Additional reagents and equipment for trypan blue viability stain ( appendix 3B) and cell counting ( appendix 3A)

Basic Protocol 4: Immunofluorescence Staining and Analysis of B Cell Subsets by Flow Cytometry

  Materials
  • Cell suspension prepared from bone marrow (BM), peripheral blood (PB), or lymphoid tissue (LT; see Basic Protocols protocol 11 to protocol 33)
  • 2% and 5% (v/v) FBS (heat inactivated) in PBS ( appendix 2A), pH 7.4
  • Unconjugated or directly conjugated monoclonal antibodies (MAbs) and polyclonal antibodies (e.g., goat F(ab′) 2 anti‐human IgM and IgD; appendix 4A4)
  • PBS, pH 7.4
  • Conjugated secondary antibodies (for unconjugated primary antibodies only)
  • Plasmacytoma‐derived murine immunoglobulin of appropriate subclass, for sequential use of unconjugated and directly conjugated antibodies only
  • Lysing solution (e.g., unit 4.8 or BD FACS lysing solution; Becton Dickinson Biosciences), for samples contaminated with red blood cells
  • Flow cytometry buffer: 1% to 2% (v/v) heat‐inactivated FBS and 0.1% (w/v) sodium azide in PBS, pH 7.4
  • 1% paraformaldehyde, pH 7.4
  • IntraPrep permeabilization solution (Beckman Coulter) or equivalent
  • Centrifuge (Beckman J6‐M centrifuge with JS‐4.2 rotor or equivalent)
  • 12 × 75–mm round‐bottom polystyrene centrifuge tubes (e.g., Falcon)
  • Flow cytometer, equipped for multicolor flow cytometry
  • Additional materials for immunofluorescence staining of cells (unit 5.3), flow cytometry (units 5.1& 5.3), and analysis of data (unit 5.2)
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Figures

Videos

Literature Cited

   Bleesing, J.J.H. and Fleisher, T.A. 2003. Human B cells express a CD45 isoform that is similar to murine B220 and is down‐regulated upon acquisition of the memory B cell marker CD27. Clin. Cytometry 51B:1‐8.
   Caldwell, C.W., Poje, E., and Helikson, M.A. 1991. B cell precursors in normal pediatric bone marrow. Am. J. Clin. Pathol. 95:816‐823.
   Campana, D., Janossy, G., Bofill, M., Trejdosiewicz, L.K., Ma, D., Hoffbrand, A.V., Mason, D.Y., Lebacq, A.M., and Forster, H.K. 1985. Human B cell development. I. Phenotypic differences in the bone marrow and peripheral lymphoid tissue. J. Immunol. 135:1524‐1530.
   de Vries, E., de Bruin‐Versteeg, S., Comans‐Bitter, W.M., de Groot, R., Boerma, G.J., Lotgerink, F.K., and van Dongen, J.J. 1999. Correction of erythroid cell contamination in microassay for immunophenotyping of neonatal lymphocytes. Arch. Dis. Child. Fetal Neonatal Ed. 80:F226‐F229.
   Dono, M., Zupo, S., Colombo, M., Massara, R., Gaidano, G., Taborelli, G., Ceppa, P., Burgio, V.L., Chiorazzi, N., and Ferrarini, M. 2003. The human marginal zone B cell. Ann. N.Y. Acad. Sci. 987:117‐124.
   Dworzak, M.N., Fritsch, G., Fleisher, C., Printz, D., Froschl, G., Buchinger, P., Mann, G., and Gadner, H. 1997. Multiparameter phenotype mapping of normal and post‐chemotherapy B lymphopoiesis in pediatric bone marrow. Leukemia 11:1266‐1273.
   Fleisher, T.A., Bleesing, J.J.H., and Marti, G. 2001. Flow Cytometry. In Clinical Immunology: Principles and Practice, Vol. 2 (R.R. Rich, T.A. Fleisher, W.T. Shearer, B.L. Kotzin, and H.W. Schroeder, eds.) pp. 121.1‐121.12. Mosby, New York.
   Ghia, P., ten Boekel, E., Sanz, E., de la Hera, A., Rolink, A., and Melchers, F. 1996. Ordering of human bone marrow B lymphocyte precursors by single‐cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci. J. Exp. Med. 184:2217‐2229.
   Ghia, P., ten Boekel, E., Rolink, A., and Melchers, F. 1998. B cell development: A comparison between mouse and man. Immunol. Today 19:480‐485.
   Groeneveld, K., te Marvelde, J.G., van den Beemd, M.W.M., Hooijkaas, H., and van Dongen, J.J.M. 1996. Flow cytometric detection of intracellular antigens for immunophenotyping of normal and malignant leukocytes. Leukemia 10:1383‐1389.
   Guzman‐Rojas, L., Sims‐Mourtada, J.C., Rangel, R., and Martinez‐Valdez, H. 2002. Life and death within germinal centres: A double‐edged sword. Immunology 107:167‐175.
   Kincade, P.W., Medina, K.L., Payne, K.J., Rossi, M.I.D., Tudor, K.‐S., Yamashita, Y., and Kouro, T. 2000. Early B lymphocyte precursors and their regulation by sex steroids. Immunol. Rev. 175:128‐137.
   King, L.B. and Monroe, J.G. 2000. Immunobiology of the immature B cell: Plasticity in the B cell antigen receptor‐induced response fine tunes negative selection. Immunol. Rev. 176:86‐104.
   Klein, U., Rajewsky, K., and Kuppers, R. 1998. Human immunoglobulin (Ig)M+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188:1679‐1689.
   Kuppers, R., Zhao, M., Hansmann, M.‐L., and Rajewsky, K. 1993. Tracing B cell development in human germinal centers by molecular analysis of single cell picked from histological sections. EMBO J. 12:4955‐4965.
   LeBien, T.W. and Villablanca, J.G. 1990. Ontogeny of normal human B cell and T cell precursors and its relation to leukemogenesis. Hematol. Oncol. Clin. North Am. 4:835‐847.
   LeBien, T.W., Wormann, B., Villablanca, J.G., Law, C.‐L., Steinberg, L.M., Shah, V.O., and Loken, M.R. 1990. Multiparameter flow cytometric analysis of human fetal bone marrow B cells. Leukemia. 4:354‐358.
   Liu, Y.‐J. and Arpin, C. 1997. Germinal center development. Immunol. Rev. 156:111‐126.
   Liu, Y.J., de Bouteiller, O. Arpin, C., Briere, F., Galibert, L., Ho, S., Martinez‐Valdez, H., Banchereau, J., and Lebecque, S. 1996. Normal human IgD+IgM‐ germinal center B cells can express up to 80 mutations in the variable region of their IgD transcripts. Immunity 4:603‐613.
   Loken, M.R., Shah, V.O., Dattilio, K.L., and Civin, C.I. 1987. Flow cytometric analysis of human bone marrow. II. Normal B lymphocyte development. Blood 70:1316‐1324.
   Lucio, P., Parreira, A., van den Beemd, M.W.M., van Lochem, E.G., van Wering, E.R., Baars, E., Porwit‐MacDonald, A., Bjorklund, E., Gaipa, G., Biondi, A., Janossy, G., van Dongen, J.J.M., and San Miguel, J.F. 1999. Flow cytometric analysis of normal B cell differentiation: A frame of reference for the detection of minimal residual disease in precursor‐B ALL. Leukemia 13:419‐427.
   Noordzij, J.G., de Bruin‐Versteeg, S., Comans‐Bitter, W.M., Hartwig, N.G., Hendriks, R.W., de Groot, R., and van Dongen, J.J.M. 2002. Composition of precursor B cell compartment in bone marrow from patients with X‐linked agammaglobulinemia compared with healthy children. Pediatr. Res. 51:159‐168.
   Pascual, V., Liu, Y.‐L., Magalski, A., de Bouteiller, O., Banchereau, J., and Capra, J.D. 1994. Analysis of somatic mutation in five B cells subsets of human tonsil. J. Exp. Med. 180:329‐339.
   Robinson, J.P., Darzynkiewicz, Z., Dean, P.N., Hibbs, A.R., Orfao, A., Rabinovitch, P.S., and Wheeless, L.L. (eds.) 2004. Current Protocols in Cytometry. John Wiley & Sons, New York.
   Rolink, A., Andersson, J., Ghia, P., Grawunder, U., Kalberer, C., Karasuyama, H., Oka, Y., ten Boekel, E., Winkler, T.H., and Melchers, F. 1999. Molecular mechanisms guiding B cell development. In Primary Immunodeficiency Diseases. (H.D. Ochs, C.I.E. Smith, and J.M. Puck, eds.) pp. 55‐65. Oxford University Press, New York.
   Simone, N.L., Bonner, R.F., Emmert‐Buck, M.R., and Liotta, L.A. 1998. Laser‐capture microdissection: Opening the microscopic frontier to molecular analysis. Trends Genet. 14:272‐276.
Key References
   National Committee for Clinical Laboratory Standards (NCCLS). 1992. Clinical Applications of Flow Cytometry: Quality Assurance and Immunophenotyping of Peripheral Blood Lymphocytes; Tentative Guideline. NCCLS Document H42‐T. Wayne, Penn.
  Describe detailed guidelines for immunophenotyping of blood, bone marrow, and tonsillar tissue.
   National Committee for Clinical Laboratory Standards (NCCLS). 1998. Clinical Applications of Flow Cytometry: Immunophenotyping of Leukemic Cells; Approved Guideline. NCCLS Document H43‐A. Wayne, Penn.
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