804 W.E. Gathings, A.R. Lawton and M.D. Cooper zyxwvuts Eur. J. Immunol. 1977.7: 804-810 z W.E. Gathings, AR. Lawton’ and M.D. Cooper Immunofluorescent studies of the development of pre-B cells, B lymphocytes and immunoglobulin isotype diversity in humans* Departments of Pediatrics and Microbiology and the Comprehensive Cancer Center University of Alabama in Birmingham, Birmingham 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 de- tected 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 inde- pendently 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+ 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-p 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. 1. Introduction Mammalain B cell development has been best studied in mice. In this species, small lymphocytes bearing surface IgM (sIgM+) are not detectable by immunofluorescence in fetal liver until 16- 17 days of gestation zyxwvutsrqp [ 1-41, but large sIgM-negative cells containing cytoplasmic IgM (cIgM+.sIgM-) are found 4-5 days earlier [ 51. Large IgM-synthesizing cells have also been demonstrated in 12 to 16-day fetal liver by other techniques. [I zyxwvutsr 18351 * This work was supported in part by a grant from the National Founda- tion (1-354) and by grants CA 16673 and A1 11502 from the National Institutes of Health. zyxwvutsrq O Recipient of a Research Career Development Award, A1 70780 from the National Institutes of Health. Correspondence: William E. Gathings, University of Alabama in Birmingham, University Station, 224 Tumor Institute, Birmingham, Alabama 35294, USA Abbreviations: slg: Surface Ig cIg: Cytoplasmic Ig HBSS: Hanks’ balanced salt solution PBS: Phosphate-buffered saline FCS Fetal calf serum FITC Fluorescein isothiocyanate RITC Tetramethyl- rhodamine isothiocyanate F/P: Fluorescein to protein molar ratio P/k Protein to rhodamine ratio S.E.: Standard error Melchers et al. observed biosynthesis of 8 S IgM by a rapidly sedimenting population of cells. These cells had sIgM which was shed and resynthesized at a rate considerably greater than that characteristic for mature B lymphocytes [6]. More recent- ly, Rosenberg and Parish have defined a population of large lymphoid cells in 12-day liver that adhere to carbonyl iron and bear small amounts of sIg, detectable by formation of small rosettes with anti-Ig-coated erythrocytes [7]. These clearly differ from the small, nonadherent, sIgM+ B lymphocytes which appear at day 16. It seems that all three techniques detect the same population of precursors of B lymphocytes (pre-B cells). From a functional viewpoint, pre-B cells clearly lack stable sIgM receptors, since their development cannot be inhibited by exposure to high concentrations of bivalent anti-p anti- bodies either zyxwv in vitro [5] or in vivo [8, zyx 91. On the contrary, immature B lymphocytes in fetal liver and in bone marrow are much more easily suppressed by anti-p antibodies (poly- clonal inhibition) or with multivalent antigen (“clonal abor- tion’’) than are mature B lymphocytes from peripheral tis- sues [ 10-141. This increasing resistance of sIgM+ cells to in- hibition may be related to the acquisition of sIgD, I region- determined antigens (Ia) or other maturational changes [15, 161.