Immunofluorescent studies of the development of pre‐B cells, B lymphocytes and immunoglobulin isotype diversity in humans

Immunofluorescent techniques were used to examine several aspects of B cell ontogeny in humans. Large lymphoid cells containing intracytoplasmic IgM (pre‐B cells) were present in fetal liver as early as 7 weeks of gestation, approximately 2 weeks prior to the appearance of surface IgM positive (sIgM+) B lymphocytes. Pre‐B cells outnumbered sIgM+ B lymphocytes in fetal liver up until the 13th week of gestation. In fetuses older than 13 weeks, pre‐B cells and sIgM+ B lymphocytes were present in approximately equal proportions in liver and bone marrow. Pre‐B cells in fetal liver, and fetal and adult marrow, were large and small (indicating a heterogeneous population of cytoplasmic IgM+. SIg‐ cells in these sites), while only the small pre‐B cells were present in fetal spleen, blood and lymph node. Lymphocytes bearing sIgG were detected earlier than those bearing sIgD or sIgA, which were present by the 12th gestational week. Using double‐staining techniques, we determined that during fetal life, (a) the proportion of B lymphocytes bearing only sIgM, as opposed to those bearing both sIgM and sIgD, was much higher in liver and bone marrow than in spleen, blood and lymph node, and (b) sIgG, sIgA and sIgD appear independently on lymphocytes bearing sIgM. Studies of the frequency of double‐stained cells for each combination of the four sIg isotypes indicated that B lymphocytes from neonatal humans may simultaneously bear three or more sIg isotypes, whereas sIgG+ and sIgA+ B lymphocytes in adult blood usually express only the single isotype. Lower concentrations of anti‐y antibodies were required for modulation of sIgM from B lymphocytes of fetal liver and adult bone marrow than for equivalent removal of sIgM from circulating B cells of mature individuals. In conjunction with data obtained in mice, our observations indicate that (a) the presence of large and small pre‐B cells, (b) a high ratio of sIgM single to sIgM.sIgD double B lymphocytes, and (c) increased sensitivity to modulation of B cell sIgM by divalent anti‐μ antibodies define the fetal liver and adult bone marrow as bursa‐equivalent sites in humans. Our results are consistent with a model of isotype diversification in which immature sIgM+ cells give rise to B cell sublines devoted to synthesis of each of the Ig classes, and sIgD is secondarily expressed on unstimulated B cells of all of these sublines.