Isolation and characteristics of tonsil centroblasts with reference to Ig class switching

Most tonsil B cells have high levels of surface CD44 but this molecule is either expressed at low levels or is absent from germinal centre B cells (GCB). On average 62% of isolated GCB were found to be CD44- and the remainder CD44low. Most CD44- GCB were in cell cycle, indicating that they were centroblasts, while centrocytes, non-dividing GCB, were mainly CD44low. Immunohistological analysis confirms that centrocytes, which are located in the light zone of germinal centres, express low levels of CD44, while centroblasts, cells of the dark zone, are CD44-. While most CD77high GCB are centroblasts and CD77low GCB centrocytes, many centroblasts and centrocytes express intermediate levels of CD77, making this less reliable than CD44 for discriminating between these cells. Most CD44low and CD44- GCB were shown to have undergone Ig switch recombination in vivo. This indicates that switch recombination is independent of the maturation of centroblasts to centrocytes and precedes the signals that induce GCB to differentiate to plasma cells or memory B cells. The average rate of entry of the CD44- GCB fraction to apoptosis on culture at 37 degrees C was faster than that of the total GCB preparation. It is suggested that this may reflect strict stromal-dependence of centroblasts while centrocytes have to survive for long enough to have the chance of receiving antigen-specific selection signals. Inhibition of apoptosis by CD40 mAb with IL-4 or phorbol myristate acetate with ionomycin was similar in the CD44- and CD44low preparations.