The human lymph node germinal center cell: Characterization and isolation by using two-color flow cytometry

The Journal of Immunology (Impact Factor: 4.92). 10/1986; 137(5):1486-94.
Source: PubMed


The germinal center of lymphoid tissues is a critical microenvironmental site of B cell activation and differentiation in response to antigenic stimuli. However, characterization of germinal center cells (GCC) in tissue sections has proved technically difficult. Therefore, we have employed two-color flow cytometric analysis of suspended human tonsillar lymphocytes in order to define more precisely the immunologic features of GCC. These cells were identified in suspension by virtue of their specific surface binding of the lectin peanut agglutinin (PNA), confirmed by tissue immunoperoxidase studies. Phycoerythrin-labeled lectin was used in combination with a variety of fluorescein-labeled antibodies in order to identify subpopulations of tonsillar lymphocytes. The majority of PNA+ cells were B cells, and both PNA+ and PNA- B cells stained for surface immunoglobulin light chains. PNA+ cells lacked surface IgD, but included cells with surface IgG and IgM. Both PNA+ and PNA- cells stained for B1, B2, BA-1, Leu-12, Leu-14, CR-I, and HLA-DR antigens, whereas CALLA was present only on PNA+ cells. There were differences between PNA+ and PNA- cells in the relative expression of B1 and B2 antigens, possibly reflecting differences in B cell activation or maturation. A small proportion of T cells were PNA+, including both helper/inducer and suppressor/cytotoxic phenotypes. PNA+ cells included both small and large lymphoid cells, and almost all DNA synthetic activity was associated with the large PNA+ cells. PNA+ B cells isolated by cell sorting had morphologic features characteristic of GCC. Therefore, PNA+ cells in suspension appeared to represent GCC, and features of these cells that cannot be convincingly shown in tissue section studies were demonstrated by flow cytometry.

8 Reads
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Systemischer Lupus Erythematodes (SLE) und verschiedene andere Kollagenosen gehen mit Autoantikörperproduktion gegen Kernbestandteile einher, und es mehren sich die Hinweise, dass B-Lymphozyten in der Pathogenese des SLE eine entscheidende Rolle spielen. Deshalb wurde in vorliegender Studie der periphere B-Lymphozytenpool von 18 SLE-Patienten, 17 gesunden Kontrollpersonen und 13 Personen mit anderen Autoimmunerkrankungen als Krankheitskontrollgruppe durchflusszytometrisch untersucht. Dabei wurden folgene B-Zellsubpopulationen unterschieden und charakterisiert: Naive B-Zellen (CD19+, CD21+, CD27-) unterschieden sich bei Patienten mit Autoimmunkrankheiten weder in der Häufigkeit noch im Phänotyp von gesunden Kontrollpersonen. Bei SLE-Patienten waren die IgM-Gedächtnis-B-Zellen (CD19+, CD21+, CD27+) prozentual vermindert, bei der Krankheitskontrollgruppe sowohl IgM- als auch klassengewechselte Gedächtniszellen. Phänotypisch zeigten die Gedächtniszellen eine erhöhte Expression von CD27. Des weiteren fand sich sowohl bei SLE-Patienten als auch bei der Krankheitskontrollgruppe ein positiv mit der Krankheitsaktivität korrelierender Anteil an Plasmablasten (CD19low, CD21low, CD27+). Daneben wurde bei SLE-Patienten eine statistisch hochsignifikant expandierte B-Lymphozytenpopulation beschrieben, die möglichen Keimzentrumsgründer- oder Transitional-B-Zellen (CD19+, IgMhigh, IgD+, CD24high, CD38high, CD10+)entsprach. Interessanterweise konnte überdies bei Patienten mit SLE eine statistisch hochsignifikant und bei der Krankheitskontrollgruppe signifikant erhöhte B-Zellsubpopulation mit aktiviertem Phänotyp (CD19high, CD21-, CD86+) und möglichem extralymphatischen Homingpotential (CXCR6+) charakterisiert werden, deren prozentualer Anteil an den Gesamt-B-Lymphozyten mit der Krankheitsdauer korrelierte. Zusammenfassend versucht die vorliegende Untersuchung den peripheren B-Lymphozytenpool in Autoimmunität und Gesundheit möglichst umfassend zu beschreiben und zu vergleichen.
  • [Show abstract] [Hide abstract]
    ABSTRACT: New methods for double and triple colour labelling using monoclonal antibodies to the proliferation-associated markers 5-bromo-2′-deoxyuridine (BrdU) and Ki67 are described. In order to make incorporated BrdU accessible to most anti-BrdU antibodies, mild denaturation of the DNA is needed, and this is usually obtained by exposing the cells to acid or base. This procedure destroys most cellular antigens, including nuclear TdT and Ki67. In this study we show that fixation in cold methanol, instead of 70% ethanol, for 30 min followed by immersion in 7 × 10−3 N NaOH for 10–15 s allows BrdU staining with the simultaneous detection of nuclear, cytoplasmic and membrane antigens as well as preservation of morphological detail. This method is optimal for detection of nuclear Ki67 and TdT. These reagents together with antibodies to membrane antigens can be included in triple colour labelling using second layers conjugated to FITC, TRITC and colloidal gold. With these methods it is now possible to characterize the phenotype of dividing cell populations such as precursors in central lymphoid tissues and germinal centre blasts in peripheral lymphoid organs.
    Journal of Immunological Methods 02/1988; 107(1-107):79-88. DOI:10.1016/0022-1759(88)90012-9 · 1.82 Impact Factor

  • Clinica Chimica Acta 03/1988; 171(2-3):125-73. DOI:10.1016/0009-8981(88)90142-8 · 2.82 Impact Factor
Show more

Similar Publications