Morphological and immunocytochemical examinations on development of B-cells in human embryonic and fetal liver

Morphological characteristics of B cell in human embryonic and fetal livers during the first trimester of gestation were examined. Light microscopically, CD9+, CD10+, CD19+, and CD20+ cells of B cell lineage became detectable as small lymphoid cells from 8 weeks gestation. Tdt+ cells first appeared also as small lymphoid cells on the 43th day of gestation. Ia+ or CD34+ cells in embryonic livers between the 33th and 43th day of gestation were large blastic cells resembling myeloblasts while some of Ia+ or CD34+ cells after the 43th day of gestation as well as Tdt+ cells were similar to lymphocytes. Electron-microscopically, all Ia+, CD10+, and CD19+ cells existed solitarily in intercellular spaces of hepatocytes, but not in intravascular spaces. Ultrastructural aspects of these cells were distinguishable each other. These findings indicate that 1) B cells developed and differentiated in the fetal liver, but the fetal liver during the first trimester was not a lymphoid organ, 2) lymphohemopoietic progenitor cells were derived from Tdt+ cells in the livers between 43th and 56th day of gestation. Ia+ cells detected as a small lymphoid cell in the liver at the 50th day were considered to be progenitor cells of lymphocytic lineage.     

Differential expression of leukocyte common antigen in human fetal lymphoid organs.

To investigate the differential expression of various types of leukocyte common antigen (LCA) isoforms during development, we analyzed human fetal lymphoid organs, including the thymus, liver, spleen, and bone marrow from 14 weeks to 29 weeks of gestational age by immunohistochemical and flow cytometric methods. In fetal thymus, over 90% of thymocytes throughout the entire fetal life expressed CD45RO and CD45RB, while CD45RA was expressed only in less than 5% of thymocytes. This expression pattern of LCA isoforms was established by a gestational age of 14 weeks or earlier, and persisted throughout the fetal period. The tissue distribution was different from each isoform; CD45RO-positive thymocytes were found in both the cortex and medulla at the 14th week with low intensity, but was localized in the cortex with increasing fetal age. CD45RB-positive thymocytes distributed mainly in the medulla from early gestational age. Among extrathymic lymphoid organs, a small portion of lymphoid cells expressing leukocyte common antigens appeared first in the liver at 10-12 weeks of gestational age and was followed by a small number in the spleen and bone marrow by 13-15 weeks. All lymphoid cells in these extrathymic lymphoid organs at this stage were CD19+ B cells. The number of these CD19+ cells increased abruptly during the early period of mid-gestational age. The pattern of tissue distribution of each LCA isoform in the fetal liver and spleen correlated well with the patterns of quantitative analysis by flow cytometry. In summary we found that different LCA isoforms expressed in cell-type-specific pattern and showed different tissue distribution during the period of fetal development, and that LCA was the earliest antigen expressed by lymphocytes in the thymus and extrathymic lymphoid organs in our series.     

Ontogeny of hepatic fatty acid-binding protein immunoreactivity in human liver and intestinal tract

Liver and intestinal tissues of human fetuses at gestational ages between 6 and 30 weeks were immunostained with rabbit antibody against fatty acid-binding protein (FABP) isolated from rat liver, since the antibody crossreacted with human FABP of the hepatic type. FABP-immunoreactive hepatocytes were found in the liver as early as the 7th week of gestation, but not at the 6th week. The frequency of immunoreactive cells was about 80% throughout the gestational period examined. No immunoreactive cells other than hepatocytes were found in liver tissue. In the intestinal tract, ileal, colonic and vermiform appendicular FABP immunoreactivity was demonstrated at the 23rd week of gestation, and duodenal and jejunal immunoreactivity at the 26th week. Positive cells in the jejunum were very few at this stage, but numerous at the 30th week of gestation. The immunoreactive cells were primitive absorptive cells in intestinal villi, and no cryptic epithelial cells were positively immunostained. Thus, FABP immunoreactivity was considered to be a marker for hepatocytes at the early to late fetal stage, and for intestinal absorptive cells at mid- to late fetal stage.     

Human fetal liver as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation

The CFU-GM and T cell contents of human fetal livers were studied at various times between 6-14 weeks of gestation. The number of CFU-GM increased parallel to gestational age, especially after week 10. Cells bearing mature T cell markers, however, were found only in one case out of 35 fetal liver samples. Cryopreservation of fetal liver cells hardly affected the viability and proliferative capacity of CFU-GM in the sample. According to these findings fetal liver is, at least up to the 14th gestational week, practically free of mature T cells but it does contain a considerable amount of CFU-GM (an accepted indicator of pluripotent stem cell content), consequently fetal liver can be considered as a valuable source of haemopoietic stem cells for allogeneic bone marrow transplantation for children.     

ONTOGENY OF HEPATIC FATTY ACID-BINDING PROTEIN IMMUNOREACTIVITY IN HUMAN LIVER and INTESTINAL TRACT

Liver and intestinal tissues of human fetuses at gestational ages between 6 and 30 weeks were immunostained with rabbit antibody against fatty acid-binding protein (FABP) isolated from rat liver, since the antibody crossreacted with human FABP of the hepatic type. FABP-immunoreactive hepatocytes were found in the liver as early as the 7th week of gestation, but not at the 6th week. The frequency of immunoreactive cells was about 80% throughout the gestational period examined. No immunoreactive cells other than hepatocytes were found in liver tissue. In the intestinal tract, ileal, colonic and vermiform appendicular FABP immunoreactivity was demonstrated at the 23rd week of gestation, and duodenal and jejunal immunoreactivity at the 26th week. Positive cells in the jejunum were very few at this stage, but numerous at the 30th week of gestation. The immunoreactive cells were primitive absorptive cells in intestinal villi, and no cryptic epithelial cells were positively immunostained. Thus, FABP immunoreactivity was considered to be a marker for hepatocytes at the early to late fetal stage, and for intestinal absorptive cells at mid- to late fetal stage. ACTA PATHOL JPN 38: 979∼987, 1988.     

Characterization of human lymphocytes separated from fetal liver and spleen at different stages of ontogeny

Membrane markers on human lymphocytes separated from fetal liver and spleen were studied. Depending on the period of intrauterine development, a growing percentage of T- and B-lymphocytes (up to 16% and 45%, respectively) among spleen cells was seen, but in liver the number was low independent of the gestational age (T cells less than 10% and B cells less than 15%). The majority of early CD3+ spleen cells (21st-28th week) expressed TCR alpha beta but not TCR gamma delta, although a significant proportion of these cells was still lacking CD4, CD8, and CD5 differentiation antigens, suggesting their immaturity. Later spleen T cells (29th-36th week) expressed the phenotype as mature adult-type T cells (CD3+TCR alpha beta +CD4/8+CD5+). During ontogeny in fetal spleen, a growing number of B cells could be estimated without any changes in the proportion of subsets, expressing the different light and heavy chains. However, the proportion of CD5+ B cells decreased with gestational age. The results suggest that the functional immaturity of antenatal splenocytes could not be caused by dramatic phenotypical differences in comparison with adult-type splenic lymphocytes.     

口服伤寒杆菌后小鼠肠道黏膜上皮内淋巴细胞的变化

目的研究经口服伤寒杆菌后,小鼠肠道黏膜上皮内淋巴细胞中T细胞亚群的变化,探讨肠道黏膜免疫应答机制。方法将8～10周龄的BALB/C小鼠100只随机分为对照组和实验组。实验组灌胃伤寒杆菌悬液分两次进行免疫,在第二次灌胃后第3、5、7、9、11d分别处死小鼠。通过免疫组化染色对小鼠肠道上皮内淋巴细胞进行表型分析,并按表型进行计数。对照组给予同体积PBS。结果实验组小鼠上皮内淋巴细胞中CD4+、CD8+、TCRαβ+、TCRγδ+亚群在3、5、7、9、11天呈增高趋势,其中从第5天开始CD8+、TCRγδ+T细胞亚群数量(个/100肠上皮细胞)与对照组相比,差异有统计学意义(P<0.01),CD4+、TCRαβ+T细胞数量与对照组相比。差异无统计学意义(P>0.05)。结论肠道黏膜上皮内淋巴细胞中CD8+、TCRγδ+T细胞增殖明显,提示其在抗感染免疫应答中起主导作用。     

Presence of murine fetal liver cells capable of being induced to differentiate in vitro into T cell receptor-positive cells.

Mouse fetal liver cells were analyzed for the surface expression of T cell markers. Fetal liver cells prepared from mouse embryos at 14.5 days of gestation contained a small number of CD4+ cells (1.4%), but virtually no cells positive for any other T cell markers such as CD8, CD3 and T cell receptor (TcR). When a fetal liver cell suspension prepared from BALB/c(male) x AKR(female) F1 embryos at 14.5 days of gestation was cultured in medium supplemented with culture supernatants of both WEHI-3 and concanavalin A-stimulated rat spleen cells, TcR alpha beta+ and CD4+ cells were generated, whereas CD8+ and TcR gamma delta+ cells were hardly detectable. Most of TcR alpha beta+ and CD4+ cells were H-2d+, thus clearly showing their fetal origin. Treatment with anti-CD4, anti-CD3 or anti-TcR alpha beta antibodies plus complement or electronic sorting to remove cells expressing these markers failed to inhibit the generation of T cell marker-positive cells following culture in vitro. On the other hand, depletion of Thy-1.2+ cells reduced their generation. These findings indicate the presence of some progenitor T cells in fetal liver with the Thy-1+, CD3-, CD4-, CD8-, TcR- phenotype, which can be induced to differentiate into TcR alpha beta+ cells in the presence of specific humoral supplements without the influence of the thymus.     

Ontogeny of T lymphocyte differentiation in the human fetus: acquisition of phenotype and functions

Phenotype and functions of cells of the T lymphocyte lineage from fetal liver, thymus, spleen and bone marrow were investigated at various ages. T lymphocyte differentiation was shown to be initiated in the thymus after the 7th week of gestation. In this organ, a large number of cells with a phenotype comparable to that of children thymocytes and with a high proliferative response to phytomitogens was observed at the 14th week. The fetal liver and bone marrow never contained many T-cells and the liver was shown to be virtually devoid of any of these cells before the 13th week. Fetal spleen contained appreciable amounts of T-cells after the 13th week. Helper and suppressor activities of fetal thymocytes and splenocytes were acquired between the 12th and the 16th week, but they were never as complete nor as potent as those of adult lymphocytes. HLA antigens were detected in very low amount in lymphocytes from the various organs at the beginning of the second trimester and their expression was significantly enhanced by in vitro incubation with alpha-interferon (alpha-IFN), a procedure that permits easier HLA typing of fetal cells.     

Contribution of NK, NK T, gamma delta T, and alpha beta T cells to the gamma interferon response required for liver protection against Trypanosoma cruzi

In the present work, we show that intracellular Trypanosoma cruzi is rarely found in the livers of acutely infected mice, but inflammation is commonly observed. The presence of numerous intrahepatic amastigotes in infected gamma interferon (IFN-gamma)-deficient mice corroborates the notion that the liver is protected by an efficient local immunity. The contribution of different cell populations was suggested by data showing that CD4- and CD8-deficient mice were able to restrain liver parasite growth. Therefore, we have characterized the liver-infiltrating lymphocytes and determined the sources of IFN-gamma during acute T. cruzi infection. We observed that natural killer (NK) cells increased by day 7, while T and B cells increased by day 14. Among CD3+ cells, CD4+, CD8+, and CD4- CD8- cell populations were greatly expanded. A large fraction of CD3+ cells were positive for PanNK, a beta1 integrin expressed by NK and NK T cells. However, these lymphocytes were not classic NK T cells because they did not express NK1.1 and showed no preferential usage of Vbeta8. Otherwise, liver NK T (CD3+ NK1.1+) cells were not increased in acutely infected mice. The majority of PanNK+ CD4+ and PanNK+ CD8+ cells expressed T-cell receptor alphabeta (TCRalphabeta), whereas PanNK+ CD4- CD8- cells were positive for TCRgammadelta. In fact, gammadelta T cells showed the most remarkable increase (40- to 100-fold) among liver lymphocytes. Most importantly, intracellular analysis revealed high levels of IFN-gamma production at day 7 by NK cells and at day 14 by CD4+, CD8+, and CD4- CD8- TCRgammadelta+ cells. We concluded that NK cells are a precocious source of IFN-gamma in the livers of acutely infected mice, and, as the disease progresses, conventional CD4+ and CD8+ T cells and gammadelta T cells, but not classic NK-T cells, may provide the IFN-gamma required for liver protection against T. cruzi.     

Expression of the CD2 molecule on human B lymphoid progenitors.

CD2 expression on human B lymphoid progenitor cells was examined. By immunofluorescence analysis, a small fraction of bone marrow B cells was found to express CD2 on their surface. CD2 expression was not demonstrated on peripheral B cells. Epstein-Barr virus-transformed B cell lines derived from fetal liver at 8 weeks of gestation were analyzed to delineate the expression and function of CD2 at the early stage of human B cell development. Characterization of surface and genomic phenotypes of cell lines revealed that the established cell lines represent at least three different phenotypic characteristics of early B lineage cell: B progenitor, pre B, or early B cell. None of the 18 cell lines and 13 subclones with the phenotype of the early B lymphoid cells initially expressed CD2 antigen. However, CD2 expression was induced by the successive cultivation of some cloned B progenitor cell lines. In spite of the expression of CD2, these clones cell lines were unable to form rosettes with sheep red blood cells. By immunoprecipitation analysis, an identical 50 kDa protein was precipitated with anti-CD2 antibody from the lysates of the radioiodinated CD2+B progenitor cell line and peripheral blood T cells. Anti-CD2 antibody induced significant enhancement of proliferation of the CD2+B progenitor subline. These data indicate that human CD2 is expressed on a fraction of B lineage cells at a very early differentiation stage and may play a role in B lymphopoiesis.     

Human thymic epithelium and T cell development: current issues and future directions

The human thymus develops early in fetal gestation with morphologic maturity reached by the beginning of the second trimester. TE3+ cortical thymic epithelium is most likely derived from endodermal third pharyngeal pouch, while A2B5/TE4+ medullary and subcapsular cortical thymic epithelium is likely derived from third pharyngeal cleft ectoderm. Fetal liver and yolk sac CD7+, CD4-, CD8-, surface(s) CD3- T cell precursors begin to colonize the thymus between 7 and 8 weeks of fetal gestation, followed by rapid expression of other T lineage surface molecules on developing thymocytes. CD7+, CD4-, CD8-, sCD3- thymocytes give rise to T cells of both the TCR alpha beta and TCR gamma delta lineages. Human thymic epithelial cells produce numerous cytokines including IL1, IL6, TGF alpha, leukemia inhibitory factor (LIF), M-CSF, G-CSF and GM-CSF- molecules that likely play important roles in multiple stages of thymocyte selection, activation and differentiation. Important areas for future research on human thymic epithelium include study of lymphoid and non-lineage differentiation potentials of CD7+, CD4-, CD8-, sCD3- T cell precursors in response to TE-cell produced cytokines, study of the triggering signals of cytokine release within the thymic microenvironment, and study of TCR-MHC mediated TE-thymocyte interactions.     

Expression of CXCR4, the receptor for stromal cell-derived factor-1 on fetal and adult human lympho-hematopoietic progenitors.

Stromal cell-derived factor-1 (SDF-1) is a CXC chemokine produced by stromal cells that acts as a chemoattractant for human CD34+ progenitor cells. We investigated the expression of CXCR4, the receptor for SDF-1, on CD34+ cells from different hematopoietic sites and developmental stages. CXCR4 was detected by flow cytometry on 37 % of fetal bone marrow (BM) [gestation weeks (gw) 14-23] and 40% of adult BM CD34+ cells. Interestingly, in fetal liver CD34+ cells, CXCR4 was expressed at lower levels at later stages (9%, gw 20-23) compared to early stages of development (39%, gw 7.5-18), suggesting a development-related change in the migratory capacity of progenitors. CXCR4 was detected at similar levels on both phenotypically primitive and committed progenitors from fetal and adult sites. However, B cell lineage progenitor and precursor cells expressed CXCR4 at the highest density (80% of BM CD34+/CD10+ pro-B cells are CXCR4+). CXCR4 was also expressed in the fetal thymus in early T cell precursors and found to be down-regulated during T cell maturation. Finally, we found that stem cell factor, alone or in combination with other cytokines, can up-modulate CXCR4 expression on CD34+ cells by three- to fourfold. In conclusion, our results suggest that CXCR4 may play an important role in the local and systemic trafficking of human CD34+ cells as well as in human B lymphopoiesis and that its expression can be modulated by cytokines.     

T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver

The developmental potential of individual cells in the Lin-c-kit+CD45+IL-7R+ (IL-7R+) population from murine fetal liver was investigated using a clonal assay capable of determining the potential of a progenitor to give rise to myeloid, T, and B cells. Unipotent progenitors generating T cells (p-T) or B cells (p-B) but not other types of progenitors were found in the IL-7R+ population. A large proportion of progenitors at day 12 of gestation are p-T, whereas the frequency of p-T dramatically decreases with gestational age. In marked contrast, p-B are very rare by day 12, but they rapidly increase thereafter. These findings strongly suggest that the commitment of multipotent progenitors to T and B cell lineages occurs independently.     

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.     

Characterization of early lymphoid precursor cells in the human fetus using monoclonal antibodies and anti-terminal deoxynucleotidyl transferase.

Monoclonal antibodies (MoAbs) directed primarily against immature lymphoid cells (VIL-A1, BA-2, OKT10) or recognizing antigens associated with the B cell lineage (VIB-C5, OKI1) were used for the identification of lymphoid cells in liver, bone marrow, spleen and thymus of human fetuses between 8 and 20 weeks of gestational age. Many lymphocytes in liver, bone marrow and spleen reacted with the MoAbs used. In the fetal thymus, however, cells did not bind to the VIL-A1 and VIB-C5 MoAbs and only a few cells were BA-2+ or OKI1+. In the liver and bone marrow the VIL-A1, VIB-C5 and BA-2 MoAbs reacted almost exclusively with terminal deoxynucleotidyl transferase (TdT) containing cells, pre-B and B cells. TdT+ cells were present in liver, bone marrow and thymus, but not in the spleen. In liver and bone marrow the relative numbers of TdT+ cells decreased during gestation, in the thymus they increased. The antigenic make-up of the TdT+ cells in liver and bone marrow was comparable to that of pre-B and B cells in these organs: most of them reacted with VIL-A1, VIB-C5 and OKT10 MoAbs and many were BA-2+ and OKI1+. TdT+ cells in liver and bone marrow did not bind to T-cell-markers, i.e. OKT6 and WT-1. A few lymphoid cells in these organs contained TdT and mu heavy chains. TdT+ cells in the thymus had a completely different phenotype: most of them were OKT6+ and they did not react with the VIL-A1 and VIB-C5 MoAbs. These findings suggest that TdT+ cells in fetal liver and bone marrow are precursors of the B cell lineage, whereas those in the thymus probably belong to the T cell lineage. In the fetal spleen almost all B cells displayed the VIB-C5 and OKI1 antigens. At 12 weeks of gestation greater than 80% of splenic B cells were also VIL-A1+ and BA-2+; with ongoing gestation far less B cells in spleen expressed these antigens, however, indicating that these B cells are more mature than those in fetal liver and bone marrow, but still less mature than the B cells in postnatal blood and bone marrow, which do not display the VIL-A1 and BA-2 markers. These findings suggest that some further maturation of B cell stages takes place in the spleen during human fetal life.     

Development of rat CD45+ 13-day-old fetal liver cells in SCID mouse fetal thymic organ cultures.

A phenotypic analysis of the lympho-hemopoietic cells which occur in the liver of 13-day-old fetal rats was achieved by flow cytometry in an attempt to further characterize the rat lymphoid progenitor cells. A small fraction of rat 13-day-old fetal liver (r13FL) cells, which weakly expressed the leukocyte common antigen CD45, constituted a homogeneous Thy-1(hi), CD71(-), CD44(+), MHC class I+, CD43(+) cell subpopulation negative for CD45RC, CD3, TCRalphabeta, TCRgammadelta, CD2, CD5, CD4, CD8, CD25, CD28, NKR-P1a and sIg. On the contrary, the CD45(-) cells were a heterogeneous cell subset which expressed Thy-1, CD71 and CD44 at distinct levels. After MACS separation, the CD45(+) r13FL cells, but not the CD45(-) cell subset, in vitro repopulated 14-day-old SCID mouse fetal thymic lobes providing rat T cells, both TCRalphabeta and TCRgammadelta, NK cells, and thymic dendritic cells but not B lymphocytes. Interestingly, NKR-P1a(lo) TCRalphabeta+ or TCRgammadelta+ cells developed in the xenogeneic cultures, and a rare CD4(+)CD8(+) double-positive subpopulation among the TCRgammadelta-expressing cells accumulated in the oldest cultures. These results are discussed from the double perspective of the nature of the precursor cells which colonize the fetal thymus and the relevance of the xenogeneic SCID mouse fetal thymic microenvironment for supporting rat lymphopoiesis.     

In Situ Study of Haemopoiesis in Human Fetal Liver

The anatomy of haemopoietic cells in human fetal liver was examined using immunohistological techniques on frozen sections of 31 fetuses (10-28 weeks gestational age). The immunohistological findings were consistent with reported cell suspension data. With regard to the location of haemopoietic activity no particular relationship existed between the various haemopoietic cell lineages. A large number of proliferating cells was present; only a few of these were reactive with haemopoietic progenitor cell monoclonal antibodies (MoAb) CD34. A population of haemopoietic cells expressed CD43 antigen (MoAb MT1) alone or together with anti-vimentin MoAb reactivity; this population needs further delineation. Erythropoiesis and myelopoiesis occurred in clusters around sinusoids and portal triad vessels respectively. Lack of MoAb reacting exclusively with early developmental stages of erythropoiesis and myelopoiesis precluded dissection of these lineages. Lymphopoiesis occurred in a loosely scattered pattern without any sign of focal development. Pre-B and B-cell numbers increased with gestational age. Cells expressing markers of more mature B cells (surface IgD, CD35, and CD21) were rare. Also, few cells reacted with mature T-cell markers, but CD7+ cells were obviously present. This expression of CD7 on haemopoietic fetal liver cells suggests that T-cell precursors develop in fetal liver as well as B cells.     

A human autoantibody to peroxisomes.

Fetal bone marrow liver and spleen of gestational age 15-20 weeks contain CALLA+, HLA-DR+ lymphoid cells. We show that a proportion of them expresses surface membrane immunoglobulins (SIg) as well as B cell differentiation antigens. A multiple phenotypic analysis reveals that CALLA+ fetal B cells are: HLA-DR+, SIg+, FMC8+, BA1+, Y29.55+ or Y29.55-, B2+, TdT-. Tissue specific phenotypic differences concern the expression of B7 and HLA-DC on spleen but not on bone marrow B cells. This study indicates that the distribution of the CALL antigen across the B cell committed lineage is much wider in fetal than neonatal life since CALLA+B cells have not yet been detected in bone marrow and peripheral blood of normal infants and adults. In addition, the interpretation of our phenotypic data suggest that fetal bone marrow B cells are more immature than those present in the spleen, thus, further supporting the evidence that the bone marrow is the organ of B cell lymphopoiesis.