A stathmokinetic study of B lymphocytopoiesis in rat bone marrow: proliferation of cells containing cytoplasmic mu-chains, terminal deoxynucleotidyl transferase and carrying HIS24 antigen

In rat bone marrow (BM), the B lineage surface antigen HIS24 is expressed by all surface mu chain-bearing (s mu+) B cells, by cytoplasmic mu chain-containing (c mu+s mu-) pre-B cells and TdT+ cells, and by lymphoid cells lacking both mu and TdT. Because TdT+ and HIS24+TdT-mu- cells may represent stages in B lymphocytopoiesis before mu chain expression, we investigated their kinetics. The metaphase arrest method was combined with immunofluorescence staining to detect proliferation and to quantitate cell production in the BM pre-B, TdT+, and HIS24+TdT-mu- compartments. Their apparent cell cycle times (tC(a)) were 38, 36, and 19 hr, and the number of cells produced per hour per femur were 58, 9, and 41 X 10(4), respectively. The HIS24+ compartments showed further phenotypic heterogeneity. Six percent of TdT+ cells expressed mu chains and were therefore pre-B cells. Twenty percent of HIS24+TdT-mu- cells expressed Ig other than mu chains, with size distribution and kinetics similar to HIS24+TdT-Ig- cells. Thus, the rate of production in the truly Ig-HIS24+ compartment was about 40 X 10(4)/hr/femur (8.5 by TdT+mu- and 33 by TdT-Ig-). In each phenotypic compartment, mitoses were confined to subsets of large (greater than 11 to 12 micron) cells with tC(a) of 13 to 15 hr. Surface mu+ B cells were essentially non-cycling. To quantitate whole body BM cell production, the recovery of marrow from bone and the distribution of BM were measured in 59Fe distribution experiments. The number of cells produced by whole body BM was estimated as follows: for pre-B cells, 4.5 X 10(8)/day; for TdT+mu-, 0.7 X 10(8)/day; and for HIS24+TdT-Ig- 2.6 X 10(8)/day. From the derived cell flux in these compartments we suggest that 1) many more pre-B cells are produced than needed by the peripheral B cell pool; 2) if TdT is an obligatory stage in B cell genesis, there must be at least two cell cycles in the pre-B cell compartment; 3) if it is not, the TdT+ stage may be bypassed, with HIS24+TdT-Ig- cells perhaps feeding directly into the pre-B cell compartment.     

Phenotypic features and proliferative activity of B cell progenitors in X-linked agammaglobulinemia

In this study, we applied mAb and heterologous antisera in double marker combinations to investigate the phenotype and the proliferative activity of immature B lineage cells in XLA. Bone marrow (BM) samples from eight male adult patients with no circulating B lymphocytes were studied. The proportions and the phenotype of the earliest identifiable B cell progenitors, expressing nuclear terminal deoxynucleotidyl transferase (TdT), cytoplasmic CD22, and membrane CD19 and CD10 were identical to those observed in normal BM. In XLA these cells represented 1.2% to 22% of BM mononuclear cells; 5% to 42% and 1% to 45% of such cells weakly expressed CD20 and CD37, respectively, and invariably lacked CD13 and CD33. Cytoplasmic mu+ sIg- pre-B cells were seen in low numbers (0.1% to 0.3%) in four samples and were undetectable in the remaining four. Consequently, the ratio TdT+/c mu+ was greater than 100 in five out of eight samples studied in contrast to the less than 10 values seen in normal individuals. The proliferative activity of B lineage progenitor cells was studied by using Ki67 and anti-bromodeoxyuridine mAb. Although the proliferation of TdT+ cells in XLA was comparable with that seen in normal BM samples (24% to 59% of TdT+ were Ki67+ and 11% to 27% incorporated bromodeoxyuridine), this was dramatically reduced in the c mu+ cells (no c mu+, Ki67+ seen in three samples where pre-B cells were observed). Thus, the abnormalities of B cell differentiation in XLA are first seen at the c mu+ pre-B stage and suggest a maturation block in the transition between TdT+, c mu- pre-pre-B cells and c mu+ pre-B cells. The severity of this block may be variable, allowing the generation of a near normal number of pre-B cells in some patients, which nevertheless have a defective proliferative activity. Finally, our study further supports the concept that the effects of the "XLA gene" are confined within the B lineage by demonstrating that the proportions of T cells bearing TCR-alpha beta and TCR-gamma delta in XLA are similar to those seen in normal individuals.     

An in situ study of B-lymphocytopoiesis in rat bone marrow. Topographical arrangement of terminal deoxynucleotidyl transferase-positive cells and pre-B cells

To understand bone marrow (BM) as a site of B-lymphocytopoiesis, insight into the topographical arrangement of developing B cells and their relationships to the microenvironment in vivo is required. To study the spatial distribution of B lymphocyte progenitors defined by intracellular markers (cytoplasmic mu H chain and nuclear terminal deoxynucleotidyl transferase (TdT], we developed a technique to cut frozen femurs of rat, yielding cross-sections with intact subendosteal and central marrow. By using (double) immunofluorescence staining techniques we located pre-B and TdT+ cells, and IgM+ B cells in those sections. Of the B cells present in BM, one-third was accumulated in the lumen of blood sinuses. The rest were in the BM parenchyma, as were virtually all pre-B and TdT+ cells. The subendosteal area was twice as rich in pre-B and TdT+ cells as the central area, and within the subendosteal area a profound positive gradient toward the bone was evident. B cells showed an equal distribution over the center and the periphery of the BM. The distribution patterns of B lineage cells in the BM parenchyma were analyzed and shown in part to deviate from random distribution. Additional study of clonal development and microenvironmental factors in hematopoiesis will have to clarify the underlying mechanisms for the observed distribution patterns of B cell precursors in BM.     

The CD24 antigen discriminates between pre-B and B cells in human bone marrow.

When bone marrow (BM) lymphoid cells from 12 adult healthy donors were labeled by CD24 antibodies and analyzed by flow cytometry, two positive populations of cells were demonstrated in each sample (by a separated bimodal specific immunofluorescence). One population had intermediate CD24-Ag density (termed CD24+ cells) whereas the other had high CD24-Ag density (termed CD24(2+) cells). CD24+ cells represented 5.8 +/- 2.7% of the total lymphoid BM cells and CD24(2+) cells 5.6 +/- 2.5%. Using dual fluorescence analysis on eight samples, all CD24+ cells expressed the CD21 and CD37 mature B cell Ag and also surface IgM (sIgM), but this population lacked CD10 Ag. These cells also expressed CD19 Ag, and at a higher density than CD24(2+) cells. They were also positive for HLA-DR Ag. Conversely, CD24(2+) cells were shown to be early cells of the B cell lineage. While all the CD24(2+) cells were HLA-DR+ and CD19+, 64 +/- 16% of them expressed CD20 Ag (at a lower density than CD24+ cells), 65 +/- 21% CD10 Ag, and 22 +/- 8% were positive for cytoplasmic mu-chains (c mu). None of these cells expressed the CD21 and CD37 mature B cell Ag or sIgM. Additional experiments on four different healthy donors demonstrated that 30 +/- 9% of the CD24(2+) cells expressed the CD34 Ag and that the CD24+ cells did not express it. Thus, the CD24 Ag permits discrimination between two populations of the B cell lineage present in adult BM: 1) A CD24(2+) cell population including "pre" pre-B cells (HLA-DR+, CD19+, CD10+/-, CD20-, CD21-, CD34+, CD37-, c mu-), "intermediate" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu-), and "true" pre-B cells (HLA-DR+, CD19+, CD10+, CD20+, CD21-, CD34-, CD37-, c mu+). 2) A CD24+ cell population including B cells of the standard phenotype (HLA-DR+, CD19+, CD10-, CD20+, CD21+, CD34-, CD37+, c mu-, sIgM+).     

Terminal transferase-positive human bone marrow cells exhibit the antigenic phenotype of common acute lymphoblastic leukemia.

Combined immunologic assays for TdT enzyme and membrane markers show that TdT+ cells in nonleukemic human bone marrow carry ALL-associated and Ia-like antigens but no thymocyte markers or surface Ig. These cells could be precursors involved in acute lymphoblastic leukemia of the "common" or non-T, non-B type and in lymphoid blast crisis of Ph' positive chronic myeloid leukemia. A few TdT+, Ia+ cells express cytoplasmic IgM, indicating that some pre-B cells may be TdT positive.     

Regulation of early precursor B cell proliferation in mouse bone marrow: stimulation by exogenous agents mediated by macrophages in the spleen.

An increase in pre-B cell proliferation and B lymphocyte production in mouse bone marrow has previously been shown to follow the administration of various foreign agents in vivo. The responses of early precursor B cells before the expression of mu chains (pro-B cells) have now been examined, using double immunofluorescence labeling for terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein as detected by monoclonal antibody 14.8. A single injection of sheep red blood cells (SRBC) was followed by an increase in the number of cells in three defined populations of early precursor B cells lacking mu chains (TdT+ 14.8- cells, TdT+ 14.8+ cells, and 14.8+ mu- cells) as well as cytoplasmic mu-bearing pre-B cells and surface mu-bearing B lymphocytes. An accompanying increase in proliferative activity was indicated by the numbers of 14.8+ mu- cells and pre-B cells which accumulated in metaphase after inducing mitotic arrest with vincristine. These effects were all abrogated either by treating mice with silica to depress macrophage function or by splenectomy. In mice given multiple injections of SRBC for 4 weeks the elevated levels of early precursor B cell production and B cell genesis were sustained. The work demonstrates that the in vivo production of early precursor B cells, putatively including those at the stage of Ig heavy chain gene rearrangement, can be stimulated by exposure to external agents acting indirectly by a silica-sensitive, spleen-dependent mechanism. The findings suggest that the level of pro-B cell proliferation and primary B cell genesis normally taking place in mouse bone marrow may reflect the level of exposure to potential stimulants in the external environment mediated by activation of splenic macrophages. The possibility that abnormally high levels of macrophage activation could predispose to dysregulations of the B cell lineage is raised.     

Heterogeneity of bone marrow lymphocytes: radioautographic detection of pre-B cells bearing cytoplasmic mu chains, and of B and T lymphocytes, and characterization of null lymphoid cells

A radioautographic immunolabeling technique has been developed to detect pre-B cells bearing cytoplasmic mu chains among populations of bone marrow lymphoid cells identified by conventional hematologic stains. 125I-Anti-mu antibody was applied either to fixed marrow smears, labeling total mu chains both in the cytoplasm (c mu) and at the cell surface (s mu), or to cell suspensions, labeling s mu alone. In stained radioautographs the incidence of c mu+ s mu- pre-B cells was derived both indirectly by subtracting values for s mu+ cells from those for total mu+ cells of various sizes in normal mice and directly by the total mu chain labeling in mice depleted of s mu+ cells by anti-IgM treatment in vivo. Binding specificity was demonstrated by the displacement of labeling by nonradioactive anti-mu antibody. The c mu+ s mu- cells showed a bimodal size distribution. They accounted for 40% of the large lymphoid cells and 30% of the small lymphocytes in the marrow. A further 50% of the small lymphocytes were B lymphocytes (s mu+) and 8% were T lymphocytes (Thy 1.2+). Thus, the technique demonstrates the presence of c mu+ s mu- pre-B cells among both proliferating large lymphoid cells and nondividing small lymphocytes, as classically defined in marrow smears. In addition, the results reveal a broad size distribution of mu- lymphoid cells, including a subset of small lymphocytes which lack c mu, s mu, and Thy 1.2 and thus cannot be assigned to either B or T lineage by these criteria. The findings suggest that in addition to B cells the marrow may produce other types of lymphoid cells, yet to be defined.     

Association of Ig L chain-like protein lambda 5 with a 16-kilodalton protein in mouse pre-B cell lines is not dependent on the presence of Ig H chain protein

Using a polyclonal rabbit antiserum against recombinant mouse lambda 5 protein, we determined that the pre-B cell specific mouse lambda 5 gene encodes a 22-kDa protein. The lambda 5 protein, which is related to conventional Ig lambda L chain proteins forms a complex with Ig mu H chain protein and an as yet unidentified 16-kDa protein (p16) in mu+ pre-B cell lines carrying a functionally rearranged VH-DH-JH allele. In pre-B cell lines which carry DH-JH rearrangements and do not express mu H chain protein, lambda 5 protein is associated with p16. Thus the expression of lambda 5 protein precedes the expression of intact mu H chain protein. This suggests the existence of developmentally regulated protein complexes involving the Ig L chain-like protein lambda 5 and p16 in mu- pre-B cells; lambda 5, p16, and Ig H chain protein in mu+ pre-B cells and Ig H chain and conventional Ig L chain proteins in B cells and plasma cells.     

Mouse hepatitis virus 3 pathogenicity expressed by a lytic viral infection in bone marrow 14.8+ mu+ B lymphocyte subpopulations

Mouse hepatitis virus type 3 (MHV3) provides an excellent model for studying viral-B lymphocyte interaction in the immune system, which plays an important role in the outcome of an acute disease. Bone marrow B lymphocyte subpopulations, at various times postinfection, were studied in genetically C57BL/6 and resistant A/J mice, infected with pathogenic L2-MHV3 and its nonpathogenic variant, YAC-MHV3. B lineage cell subpopulations were identified by double immunofluorescence assays using mAb of terminal deoxynucleotidyl transferase, 14.8 and cytoplasmic (cu) or surface (su) Ig mu-chains. Results revealed diminished percentage and absolute number in the bone marrow 14.8+ mu+ B lymphocyte subpopulations, including pre-B (cu+ su-) and B (cu+ su+) cells of L2-MHV3-infected susceptible C57BL/6 mice; whereas, slight or no increase was evident in the cell subpopulations of L2-MHV3 infected resistant A/J mice or in YAC-MHV3 infected in both strains of mice. Abnormal large-sized forms of the 14.8+ mu+ cells occurred, at 48-h postinfection, in L2-MHV3-infected susceptible C57BL/6 mice only. In contrast, no change in the percentage and absolute number of precursor cells (terminal deoxynucleotidyl transferase positive) and pre pre-B cells (14.8+ mu-) were detected in all infected mice. In vitro L2-MHV3 infection of C57BL/6 bone marrow purified B lineage cell subpopulations showed that pre-B (cu+ su-) and B (cu+ su+) cells became abnormally large in size and depleted in number as a result of a productive and lytic viral replication. Low L2-MHV3 viral replication occurred in these cell subpopulations of A/J mice but no YAC-MHV3 virus was produced in the cells of both strains of mice. Pre pre-B (14.8+ mu-) cells in both strains were not permissive to L2-MHV3 or YAC-MHV3 viral replication. These results are discussed with regard to the role of humoral immunodeficiency in the pathogenic process.     

Cell surface antigens expressed by subsets of pre-B cells and B cells

A large number of monoclonal antibodies, produced by immunizing rats with mouse pre-B cell lines, have been analyzed for their ability to define cell surface antigens expressed by B cells at early stages of differentiation. Whereas many antibodies recognized antigens on pre-B cell lines, only two clones detected cell surface antigens that were distinguished by their restricted distribution among a panel of continuous cell lines and cells from various tissues. Monoclonal antibody clone AA4.1 recognized a cell surface antigen found on all pre-B lymphomas and on one of three B lymphomas tested. This antigen was found on cells at highest frequency in the bone marrow. Adult spleen and fetal liver also have detectable numbers of AA4.1+ cells. Cells that did not express this antigen include plasmacytomas, two of three B lymphomas, T lymphomas, a stem cell line, adult liver, brain, thymus, and lymph node cells. Clone GF1.2 detected an antigen on some pre-B cell lines, one of three B lymphomas tested, and a small fraction of cells from adult bone marrow, spleen, lymph node, and fetal liver. Plasmacytomas, some pre-B lymphomas, two B lymphomas, T lymphomas, adult liver, brain, and thymus cells were negative. In adult bone marrow, AA4.1 bound to all cytoplasmic IgM+ pre-B cells, whereas GF1.2 detected one-half of these cells. Both antibodies recognized approximately 50% of surface IgM+ (sIgM+) bone marrow cells. A small population of bone marrow cells lacking any detectable Ig (surface or cytoplasmic) also reacted with these antibodies. Depletion of AA4.1 or GF1.2 antigen-bearing cells from bone marrow reduced the ability of bone marrow B cells to respond to LPS by 50 to 65%. Experiments with a cloned pre-B lymphoma demonstrate that AA4.1+ pre-B cells become sIgM+ GF1.2+ B cells after activation with LPS. These antibodies recognize cell surface determinants with restricted distribution among the B lymphocyte lineage because they detect antigens displayed by normal and transformed immature B lymphocytes.     

Development and distribution of B lineage cells in the domestic cat: analysis with monoclonal antibodies to cat mu-, gamma-, kappa-, and lambda-chains and heterologous anti-alpha antibodies

To trace the development and distribution of B lineage cells in the domestic cat (Felis catus), we have produced monoclonal antibodies against mu-, gamma-, kappa-, and lambda-chains of feline immunoglobulins (Ig). Goat antibodies against human mu-, alpha-, and lambda-chains, which are reactive with shared determinants on their feline counterparts, were used in conjunction with the panel of mouse monoclonal antibodies. Cytoplasmic mu+ pre-B cells and surface IgM+ B lymphocytes were observed in 42 day fetal liver in which pre-B cells were more abundant than IgM+ B cells. Pre-B cells also were found in bone marrow in young cats, and continued to be generated in the marrow throughout life. In the spleen, adult levels of B cells were attained by 12 wk of age, at which time the frequencies of surface IgM+, IgG+, and lambda+ cells were 49, 3, and 40%, respectively. The distributions of Ig isotypes also were determined among plasma cells as a function of age and tissue localization. IgM plasma cells were predominant in the bone marrow of 1-wk-old cats, whereas IgG plasma cells were the prevalent isotype in adult bone marrow. In the mesenteric lymph nodes of adult animals, the frequency distributions of IgM, IgG, and IgA plasma cells were similar to the frequency distributions of IgM, IgG, and IgA isotypes among bone marrow plasma cells. IgA+ plasma cells predominated in the intestinal lamina propria, in which IgG+ and IgM+ plasma cells were relatively infrequent. In the tissues of both young and adult animals, the ratio of lambda:kappa expression was approximately 3:1. We conclude that the pattern of B cell development in the cat resembles that found in other mammals, except that the kappa to lambda ratio is reversed.     

Mouse pre-B cells synthesize and secrete mu heavy chains but not light chains

The immunoglobulins produced by the earliest recognizable B cell precursors (pre-B cells) were characterized in the mouse and human. Immunofluorescent analysis revealed no evidence of surface IgM components, and only mu heavy chains could be detected intracytoplasmically in pre-B cells. Surface IgM components could not be isolated from intact fetal liver cells that lacked sIgM+ B lymphocytes but possessed pre-B cells. Pre-B cells were shown to synthesize and secrete mu heavy chains but not light chains by immunochemical analysis. These mu chains constituted less than 0.01% of TCA precipitable protein synthesized and secreted by fetal liver cells during an 8 hr labelling period. Migration of both intracellular and secreted mu chains on SDS-PAGE suggested that they were smaller than mu chains secreted by mouse and human plasmacytomas. These data indicate that mu chain synthesis precedes light chain expression during B cell ontogeny and suggest a new role for pre-B cells in the generation and expression of a diverse immunoglobulin repertoire.     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.     

Pre-B cells in mouse bone marrow: in vitro maturation of peanut agglutinin binding B lymphocyte precursors separated from bone marrow by fluorescence-activated cell sorting

Peanut agglutinin (PNA) binding by mouse bone marrow cells and fractionation by the fluorescence-activated cell sorter have previously been shown to separate high concentrations of pre-B cells, as identified by cytoplasmic mu-chains (c mu). PNA+ and PNA- marrow cell fractions have now been assayed for the presence of functional pre-B cells able to generate mature B cells in culture, as defined by three criteria, the appearance of cell surface mu-chains (s mu), immunoglobulin secretion in response to bacterial lipopolysaccharides, and B cell colony formation. Small PNA+ cell fractions contained pre-B cells that developed into mature B lymphocytes in 1/2 to 1 day but did not sustain B cell production. Large PNA+ cells included pre-B cells that gave rise to mature B lymphocytes after an interval of 1 1/2 to 3 days and were able to sustain B cell genesis in vitro for at least 3 to 5 days thereafter. PNA- cell fractions contained mature B cells but lacked pre-B cell activity. The results demonstrate that PNA binding allows the separation of functional subsets of pre-B cells from bone marrow and that the three in vitro assays used in this study are closely comparable with one another as functional pre-B cell criteria. The findings suggest correlations between functional assays, c mu expression, PNA receptors, and cell size in characterizing stages of pre-B cell development.     

Characterization of rat bone marrow cells. II. Analysis of surface antigens in small lymphocytes with particular reference to thymus antigen-carrying cells.

Rat bone marrow (BM) small cells were enriched by velocity sedimentation, further separated by means of free-flow electrophoresis, and characterized using T- and B-cell-specific surface markers. More than 80% of these cells were small lymphocytes by morphological criteria and reacted with lymphocyte-specific antisera. A minority of cells had high electrophoretic mobility (EPM), carried surface antigens characteristic of mature T cells, and lacked B-cell markers. These cells may represent recirculating T cells. A small number of cells were found with rat B lymphocyte-specific antigen (RBLA) and surface immunoglobulin (sIg) and had medium EPM. These cell fractions also contained “null” cells which were devoid of T- and B-cell-specific antigens. More than 80% of the BM small cells had low EPM and carried the three subspecificities of the Thy-1 antigen complex and the Thy-A antigens. These antigens were found at several-fold higher concentration on the surface of all thymocytes, but are lacking in most other lymphocytes. The thymus antigen-carrying BM cells of low EPM do not carry other T- and B-cell-specific markers found in thymocytes and peripheral T and B lymphocytes. These markers comprise the T-cell antigens RTLA (rat T-lymphocyte-specific antigen) and RHLA (antigens specific for rat T cells of high EPM) and the B-cell markers RBLA and sIg. Thus the majority of rat BM lymphocytes differ from all other lymphocytes of the T- and B-cell series which makes any classification on this basis purely speculative.     

Functional maturation of murine B lymphocyte precursors. I. Selection of adherent cell-dependent precursors from bone marrow and fetal liver

A cell culture assay is described which is suitable to explore interactions between cells of the bone marrow (BM) microenvironment on one side and B lymphocyte progenitors on the other. First, a heterogeneous adherent BM (aBM) cell population was established on Cytodex 1 microcarriers. Then, adherent cell and surface IgM+(sIgM+) cell-depleted BM precursors or adherent cell-depleted day 12 fetal liver cells were added. The generation of B cells in these cultures was monitored by staining with fluorochrome-labeled anti-mu-chain antibody and by lipopolysaccharide (LPS) induction of protein A plaque-forming cells at limiting dilution. In the absence of aBM cells, some B cells arose after 24 hr from BM precursors but not from day 12 fetal liver cells. With aBM cells, BM precursors gave rise to a distinct second wave of B cells starting after 5 days of culture. When fetal liver cells were cultured on aBM cells, B cells appeared after a delay of 4 to 5 days. By using Ig allotype-congenic mouse strains (C.AL 20, BALB/c) and an allotype-specific plaque assay, we established that mature B cells originate from the putative progenitors and not from the aBM cell population. In an attempt to eliminate the aBM cell-independent progenitor subset, mice were pretreated with 5-fluorouracil 5 days before BM cells were collected. The remaining cells still contained B cells, but the frequency of c mu+ sIgM- pre-B cells was less than 10(-5). Remaining B cells were removed by anti-mu panning. In cultures of this precursor cell population, LPS-responsive B cells appeared after a delay of about 1 wk, and their generation was totally aBM cell-dependent and was maintained for more than 2 wk.     

IL-4-dependent IgE switch in membrane IgA-positive human B cells

IgE responses by human B cells, separated according to membrane Ig classes, were analyzed in a clonal assay using EL-4 thymoma cells as helper cells, T cell supernatant, and rIL-4. In cultures seeded by means of the autoclone apparatus of the FACS, IgE responses were generated frequently by either IgM (mu+/gamma-alpha-) or IgA (alpha +/mu-)-positive B cells (16 and 14% of the Ig producing wells, respectively), but rarely by IgG (gamma +/mu-)-positive B cells (1.3% of Ig producing wells). The total amounts of Ig secreted by IgM-, IgG-, or IgA-positive cells and the total proportions of responding autoclone wells (23-27%) were comparable. All IgE secretion was IL-4 dependent. When the Ig secretion patterns from alpha +/mu- vs alpha +/mu-epsilon- B cells were compared, most autoclone wells from both types of cells produced IgA only, and similar proportions of IgA producing wells (6.2 and 6.0%) also secreted IgE. In addition, IgE restricted responses occurred 6 times more frequently with alpha +/mu- than with alpha +/mu-epsilon- cells, which suggests that membrane IgA+E double-positive, IgE committed B cells occur in vivo. The isotype pattern generated by alpha +/mu-epsilon- B cells cannot be explained by a chance assortment of separate IgA and IgE precursors or by cytophilic antibody. Thus, IL-4 dependent switch to IgE occurred frequently in IgM- or IgA-positive, but rarely among total IgG-positive, B cells. This could be relevant to IgE production in mucosal tissues rich in IgA expressing B cells.     

Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains.

During B cell development V kappa gene rearrangement seems to occur only in mu-positive pre-B cells. To study the role of the mu chain in the activation of the Ig kappa locus, we introduced expression vectors carrying different forms of the mu gene into null pre-B cells. The activation of the Ig kappa locus followed the expression of the membrane form (micron) of the mu chain. The expression of the secreted form (microS) did not result in the activation of the Ig kappa locus. We further show that both forms of the mu chain differ in their intracellular transport in pre-B cells.     

Expression, distribution and specificity of Fc receptors for IgM on murine B cells

Subpopulations of normal adult murine splenic B cells and a panel of murine B cell tumors were examined for their ability to bind murine IgM specifically. By using two-color flow cytometric analyses, we have demonstrated that 90 to 95% of surface (s)IgD+ B cells express surface membrane receptors for IgM (Fc mu R). The binding of pentameric murine IgM to splenocyte Fc mu R was IgM-specific since it was totally inhibited by other polymeric IgM proteins, but not by Ig of other H chain classes or by mAb specific for the murine IgG or IgE FcR. Binding of IgM to splenic cells was saturable. Fc mu R were co-expressed with the Fc gamma R as well as the Fc epsilon R on the majority of splenic B cells. Minor populations of splenic mononuclear cells expressed only an Fc mu R, Fc gamma R or Fc epsilon R. In a survey of B tumor cell lines representing different stages of B cell development, we observed that the Fc mu R was expressed on pre-B cell lines and that Fc mu R detection was maximal on immature B cell lines that expressed sIgM and low amounts of sIgD and Ia. Fc mu R were not detected on cell lines that had switched from sIgM to the expression of another sIg, or on plasmacytomas and hybridomas. The studies with normal splenocytes establish that the majority of sIgD+ B lymphocytes in adult BALB/c mice express surface membrane receptors that specifically bind IgM. The studies with B lineage tumor cells suggest that the expression of Fc mu R on B cells is developmentally regulated and that the pattern of expression exhibited by Fc mu R during B cell ontogeny differs from the patterns that have been previously found for IgG and IgE FcR. These observations raise the possibility that Fc mu R might have a functional significance in some aspect of B cell maturation and activation. By using a family of IgM H chain constant region domain deletional mutants, we have further demonstrated that, like the T cell Fc mu R, the B cell Fc mu R also requires a C mu 3 domain for binding to occur, raising the possibility that the T and B cell Fc mu R in mice may be structurally related molecules.     

Human bone marrow cells positive for terminal deoxynucleotidyl transferase (TdT), HLA-DR, and a T cell marker may represent prothymocytes

Recent evidence suggests that prothymocytes, which occur in a low frequency in murine bone marrow (BM), are already committed to thymocyte differentiation and discrete from precursor B cells as well as pluripotent hematopoietic stem cells. Furthermore, it was suggested that, in rodents, prothymocytes are positive for the nuclear enzyme terminal deoxynucleotidyl transferase (TdT) and a T cell surface antigen. The human prothymocyte has not been identified as yet. We analyzed human BM cells by double immunofluorescence staining for TdT and the T cell surface markers Tp41 (recognized by the monoclonal antibodies WT1 and 3A1), T11, T1, and T6. In the BM samples tested, neither T1+/TdT+ nor T6+/TdT+ cells were detected, but Tp41+/TdT+ and T11+/TdT+ cells were present in low frequencies. In childhood BM, the frequency was about two to five in 10,000, whereas in adult BM and regenerating BM, these cells were not always detectable, but if detected, their frequency was five- to 10-fold lower. In a triple staining, using fluorescein, rhodamine, and colloidal gold particles as labels, it appeared that all Tp41+/TdT+ cells were also positive for HLA-DR. These Tp41+/HLA-DR+/TdT+ cells were also detectable in low frequencies in the thymus, and occasionally Tp41+/TdT+ and T11+/TdT+ cells were detected in the peripheral blood (PB), suggesting a migration from the BM to the thymus via the PB. The malignant counterpart of the Tp41+/HLA-DR+/TdT+ cell was detected in a patient with acute lymphoblastic leukemia with the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- phenotype and germ-line immunoglobulin heavy chain genes. We postulate that the Tp41+/T11+/HLA-DR+/TdT+/T1-/T6- cell represents a human prothymocyte.