Sequential expression of immunoglobulin on developing mouse B lymphocytes: a systematic survey that suggests a model for the generation of immunoglobulin isotype diversity.

Paired immunofluorescent staining with antibodies specific for the major isotypes of mouse immunoglobulin was used to study the ontogenetic expression of diversity of cell surface immunoglobulin. The first B lymphocytes to emerge, derived from cytoplasmic IgM+ precursors, express sIgM exclusively. Between birth and 3 days of age separate populations of sIgM+ B lymphocyte acquire a second isotype: sIgD, one of the subclasses of sIgG, or sIgA. At 3 days, all splenic B lymphocytes that bear sIg or sIgA also express sIgM, but virtually none stain for sIgD. By 7 days, a substantial porportion of sIgG+ or sIgA+ lymphocytes in spleen and most of those in lymph node express both sIgM ans sIgD. Anti-mu antibody treatment from birth prevented development of B lymphocytes expressing any isotype. These observations suggest that the immature sIgM+ B lymphocyte is the pivotal cell in the generation of the different sublines of B cells and that sIgD ig or IgA. The frequency of lymphocytes bearing only sIgG or sIgA is higher in old than in young mice, suggesting that sIgD and sIgM may be lost after stimulation by antigens. The occurrence of a nearly identical distribution of sIg isotypes on B lymphocytes from athymic, pathogen-free mice suggests that primary expression of isotype diversity does not require T cells.     

Induction of isotype switching and Ig production by CD5+ and CD10+ human fetal B cells.

In the present study the capacity of early fetal B cells to produce Ig was investigated. It is shown that B cells from fetal liver, spleen, and bone marrow (BM) can be induced to produce IgM, IgG, IgG4, and IgE, but not IgA, in response to IL-4 in the presence of anti-CD40 mAb or cloned CD4+ T cells. Even splenic B cells from a human fetus of only 12 wk of gestation produced these Ig isotypes. IFN-alpha, IFN-gamma, and transforming growth factor-beta inhibited IL-4-induced IgE production in fetal B cells, as described for mature B cells. The majority of B cells in fetal spleen expressed CD5 and CD10 and greater than 99% of B cells in fetal BM were CD10+. Highly purified CD10+, CD19+ immature B cells and CD5+, CD19+ B cells could be induced to produce Ig, including IgG4 and IgE, in similar amounts as unseparated CD19+ B cells. Virtually all CD19+ cells still expressed CD10 after 12 days of culture. However, the IgE-producing cells at the end of the culture period were found in the CD19-,CD10- cell population, suggesting differentiation of CD19+,CD10+ B cells into CD19-,CD10- plasma cells. Pre-B cells are characterized by their lack of expression of surface IgM (sIgM). Only 30 to 40% of BM B cells expressed sIgM. However, in contrast to sIgM+,CD10+,CD19+ immature B cells, sorted sIgM-,CD10+,CD19+ pre-B cells failed to differentiate into Ig-secreting cells under the present culture conditions. Addition of IL-6 to these cultures was ineffective. Taken together, these results indicate that fetal CD5+ and CD10+ B cells are mature in their capacity to be induced to Ig isotype switching in vitro as soon as they express sIgM.     

sIgD-negative B cells from neonatal mice do not respond to the thymus-independent antigen TNP-BA in limiting dilution cultures

The majority of B lymphocytes in adult mice express both IgM and IgD on their surface (sIgM and sIgD). A small percentage of sIgM+ splenic B cells lack (or express very low levels of) sIgD. These cells have been termed "mu-predominant" (mu p) B cells. In neonatal mice (5 to 12 days of age), mu p B cells account for more than 50% of the sIg+ cells. There is conflicting evidence concerning the immunocompetency of mup cells in vitro. To study this question further, splenocytes from neonatal BALB/c mice were depleted of sIgD+ B cells by a panning procedure. A portion of the nonadherent (mu p) cell population was analyzed for residual sIgD+ cells by using indirect immunofluorescence in conjunction with the fluorescence-activated cell sorter (FACS). Such cells were then tested for their responsiveness to the thymus-independent (TI) antigen, trinitrophenyl Brucella abortus (TNP-BA), by using a limiting dilution culture system. Results indicate that the depletion of sIgD+ B cells and the decrease in the precursor frequency of splenocytes responding to TNP-BA are very similar, suggesting that virtually all of the responding B cells bear sIgD.     

Effects of neonatal anti-delta antibody treatment on the murine immune system. I. Suppression of development of surface IgD+ B cells and expansion of a surface IgM+ IgD- B lymphocyte population

Lymphocytes that bear surface (s) IgD make up the majority of B cells in mature mice and are the precursors of most antibody secreting cells in primary immune responses made by these mice. In order to study the functional capabilities of the minority sIgD- B lymphocyte population and to gain insight into the possible roles of sIgD, we attempted to abort the development of sigD+ B cells and to expand the sigM+IgD- B cell population by treating mice from birth with affinity-purified rabbit antibodies specific for mouse IgD (RaM delta). RaM delta-suppressed mice had no detectable sIgD+ spleen, lymph node, or bone marrow cells and, on average, only 20% as many sIgM+Ia+ splenic B cells as control mice but had normal numbers of splenic T cells. Lymph nodes from anti-delta suppressed mice were even more depleted of B cells than were spleens from these mice, whereas the percentage of bone marrow B cells in these mice was relatively normal. Germinal centers of anti-delta suppressed mice were fairly normal in appearance, whereas follicular mantle layers, the locus of most sIgD+ B cells in normal mice, were greatly depleted. In addition to their lack of sIgD, splenic B cells of anti-delta suppressed mice differed from those found in control mice in that they bore, on average, twice as much sIgM as control cells, and in that they included an increased percentage of large, DNA synthesizing cells as compared with spleen cells from control mice. However, most sIgM+IgD- splenic B cells from anti-delta suppressed mice were small, nonproliferating cells. B cells from anti-delta suppressed mice insert little or no sIgD into their cell membranes since they continued to bear no detectable sIgD 2 days after in vivo neutralization of RaM delta and since, unlike B cells from control mice, they failed to be activated by a single in vitro injection of a goat anti-mouse delta antibody. Despite their lack of sIgD+ B cells, anti-delta suppressed mice had relatively normal levels of serum IgG as well as normal to increased levels of serum IgM. Thus, sIgM+IgD- B cells appear to have the potential of differentiating into Ig secreting cells in vivo without acquiring sIgD.     

Deletion of Cmu genes in mouse B lymphocytes upon stimulation with LPS.

Mouse B lymphocytes can be activated polyclonally by bacterial lipopolysaccharide (LPS) to differentiate into plasmablasts. Within several days many cells perform immunoglobulin (Ig) class switching in vitro. We have purified LPS blasts expressing IgM or only IgG3 on the cell surface and analysed the DNA of these cells by Southern hybridisation blotting to detect rearrangement or deletion of CH genes. Quantitative evaluation of the Southern blots suggests that populations of surface IgG3+ (sIgG3+) cells from 6-day and sIgM+ cells from 8-day-old cultures contain only about half as many Cmu genes as spleen cells. Cmu deletion is nearly complete in populations of sIgG3+ cells from 9-day-old cultures. Therefore, upon stimulation with LPS, within a few days Cmu is deleted in most sIgG3+ cells from both chromosomes.     

Heterogeneity of EBV-transformable human B lymphocyte populations

Although most human B cells express receptors for Epstein Barr virus (EBV), few (usually less than 1%) are readily transformed into B lymphoblastoid cell lines after exposure to EBV. Transformable cells previously have been found to be mostly resting B lymphocytes. We recently developed a limiting dilution culture system which permits the growth of EBV-transformed B lymphocytes with high efficiency. Because in this system up to over 30% of peripheral blood- or tonsil-derived B cells respond to EBV, we re-examined the properties of EBV-transformable cells. Frequencies of transformable lymphocytes were determined by Poisson analysis. EBV-susceptible B cells committed to IgM, IgG, or IgA secretion were found to occur in the range of 3 to 27, 0.1 to 6, and 0.1 to 5 per 100 B cells, respectively. Under our culture conditions, a major proportion of the IgM-committed cells derived from large lymphocytes which appeared to have entered the cell cycle. This population contains most of the EBV-responsive cells detected and, therefore, most of the additional cells responding in our culture system. In contrast, precursors of IgG- or IgA-producing lymphoblast lines were small cells with DNA contents typical for the G0/G1 phases of the cell cycle. Anti-immunoglobulin antibodies were used in panning experiments to separate B cell subpopulations which expressed different immunoglobulin isotypes on their surface. In limiting dilution cultures of these purified B lymphocytes subsets, it was found that virtually all precursors of IgM-producing cell lines expressed surface IgM (sIgM) before their infection and transformation by EBV. The "cloning efficiency" of positively selected, large sIgM+ cells approached 100%. In contrast, sIgG or sIgA were found only on cells committed to the production of IgG or IgA, respectively. The expression of sIgD was examined by using sequential panning procedures. Virtually all IgM-committed lymphocytes and a subset of cells committed to IgA secretion were found among the sIgD+ cells. The majority of cells committed to IgA production and all IgG-committed cells were found in the sIgD- B cell population. Our findings indicate that the EBV-susceptible B cell subset of normal lymphocytes is heterogeneous with respect to cell size, cell cycle, sIg determinants, and efficiency of transformation. On the basis of our findings and previous reports, we propose a model in which transformability is a B cell-inherent property. Factors unrelated to the virus but present in our culture system appear responsible for the enhanced vulnerability to viral transformation in some cells which entered into the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Preparation and analysis of antigen-specific memory B cells

A procedure has been developed for the enrichment of TNP-binding memory B cells (TNP-MABC) from spleens of immunized mice. More than 75% of the cells expressed surface IgM (sIgM) and IgD (sIgD) and about 9% expressed surface IgG (sIgG). The TNP-MABC consisted of small resting lymphocytes with high affinity antigen-binding receptors. These cells expressed increased densities of Ia antigens and decreased densities of sIgD. Adoptive transfer of the cells into irradiated, carrier-primed syngeneic recipients resulted in their differentiation into IgG anti-TNP antibody-secreting cells. TNP-MABC secreted high affinity IgG anti-TNP antibodies when cultured in vitro with carrier-primed T cells and antigen. Limiting dilution analysis revealed that TNP-MABC contained a relatively low frequency of precursors for IgG-secreting cells that had an exceptionally large clone size. These results show that a highly enriched population of antigen-specific memory B cells can now be prepared and used to analyze their activation requirements.     

Quantitative analysis of Fc gamma receptors on murine spleen cell populations by using dual parameter flow cytometry

The expression of Fc gamma R on subsets of mouse spleen cells was examined by dual parameter flow microfluorometry. B cells were detected by labeling them with antibodies against sIgM, sIgD, sIgG, or I-A; essentially all B cells expressed Fc gamma R. The number of Fc gamma R per cell on the sIgD+, sIgM+, and I-A+ cells averaged 2 X 10(4) receptors, and no correlation between the levels of expression of Fc gamma R and the B cell markers was evident. The sIgG+ B cells, however, expressed more Fc gamma R (8 X 10(4) receptors/cell) than sIgM+ and sIgD+ B cells. Fc gamma R on splenic macrophages were examined by double labeling spleen cells for Fc gamma R and Mac-1. The Mac-1+ cells (2 to 16% of the spleen cells) were 100% Fc gamma R+ and expressed threefold to fivefold higher numbers of Fc gamma R per cell than the sIgM+ or sIgD+ B cells. The Fc gamma R on T cells were studied on cells double labeled for Fc gamma R and Thy-1, Lyt-1, or Lyt-2. An average of 20% of the T cells expressed Fc gamma R and at least two subsets of Fc gamma R+ T cells were evident: Lyt-2- cells, most of which expressed intermediate (2 X 10(4) Fc gamma R/cell) levels of Fc gamma R, and Lyt-2+ cells, which expressed mainly high (8 X 10(4) Fc gamma R/cell) amounts of Fc gamma R. The levels of expression of Fc gamma R and sIgM increased dramatically in response to infection and were elevated in mice with genetic defects. We conclude that the level of Fc gamma R expression is a characteristic property of subsets of spleen cells from normal and infected mice.     

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.     

The use of haptenated-immunoglobulin molecules to induce tolerance in B cells from neonatal mice

The role of the Fc region of trinitrophenylated (TNP)-immunoglobulins (Ig) in their ability to induce tolerance in immature B cells was examined. With the use of B cells from neonatal mice, tolerogens that could or could not bind to Fc receptors were assessed for their ability to induce tolerance. This was accomplished by tolerizing spleen cells in bulk culture and assessing the degree of tolerance by challenging the cells with the thymus-independent antigen TNP-Brucella abortus (TNP-BA) in limiting dilution cultures. It was found that by using tolerogens containing 10 to 11 haptens per Ig molecule, immature B cells were very susceptible to tolerance induction. Mature B cells were not as susceptible. This increased susceptibility was independent of the Fc portion of the tolerogen, because TNP11-HGG and a TNP10-F(ab')2 induced equivalent degrees of unresponsiveness. When the TNP density was lowered to approximately five haptens per Ig molecule, those Ig molecules that contained Fc portions were superior tolerogens with the use of B cells from 6-day-old mice. Thus, a TNP4-HGG, TNP7-mouse IgG1, and TNP6-mouse IgG2a were more effective tolerogens than either TNP5-F(ab')2 or TNP6-mouse IgG3. These results confirm previous findings that immature B cells are inherently more susceptible to tolerance induction than mature B cells. They also suggest that very lightly haptenated Ig molecules may depend on Fc receptor-binding for effective tolerance induction. Finally, by means of a cytofluorograph, the surface IgD (sIgD) and sIgM phenotypes of splenic B cells from neonates of increasing age were determined. When comparing the phenotype of maturing cells with their tolerance susceptibilities, a correlation between the appearance of sIgD and the acquisition of resistance to tolerance was observed.     

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.     

Pre-B cells: bone marrow persistence in anti-mu-suppressed mice, conversion to B lymphocytes, and recovery after destruction by cyclophosphamide.

Chronic treatment of mice from birth with anti-mu antibodies aborts development of B lymphocytes and plasma cells. In these studies we show that bone marrow from anti-mu-treated mice contains a population of cells with cytoplasmic IgM, but which lack detectable cell-surface IgM. These cells are analogous to pre-B cells, defined in ontogenetic studies as the immediate precursors of B lymphocytes. Pre-B cells from bone marrow of anti-mu treated mice retain their functional integrity, as evidenced by their ability to give rise to sIgM+, LPS-responsive lymphocytes in culture. We also show that cyclophosphamide treatment destroys pre-B cells and that recovery of pre-B cells in bone marrow precedes the regeneration of sIgM+ B lymphocytes. Generation of B lymphocytes in adult mice apparently occurs exclusively in the bone marrow because induction of extramedullary hemopoiesis in spleen was not accompanied by the appearance of pre-B cells in that organ.     

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.     

Presence of cells in B-cell lineage in mixed (GEMM) colonies from murine marrow cells

Recent progress in clonal cell culture techniques makes it possible to detect pluripotent hemopoietic precursors from murine marrow cells. The precursors can proliferate, differentiate and form mixed colonies containing erythroblasts, granulocytes, macrophages and often megakaryocytes in viscid culture medium. In the present investigation, the presence of cells of B-cell lineage in mixed colonies was investigated. Experiments on colonies containing cIgM, cIgG, sIgM and sIgG bearing cells using goat IgG fluorescein-conjugated anti-mouse IgM, goat F(ab')2 fraction fluorescein-conjugated anti-mouse IgG and immunobeads revealed the presence of cytoplasmic IgM bearing cells in 47% of the colonies and surface IgM bearing cells in 74-84% of the colonies. Mixed colonies, however, did not contain either cIgG bearing cells or sIgG bearing cells. The results may indicate that some CFU-MIX proliferate and differentiate along B-cell lineage to sIgM or cIgM bearing cells in vitro.     

In vitro activation of murine antigen-specific memory B cells by a T-dependent antigen

A procedure that generates an enriched population (60 to 85%) of memory B cells specific for TNP (TNP-MABC) was employed. The activation requirements of TNP-MABC for the T-dependent antigen TNP-KLH and carrier-primed helper T (Th) cells were compared to those for TNP-binding cells from nonimmune mice (TNP-ABC). Proliferation and differentiation of TNP-MABC in response to cognate recognition of antigen requires less antigen and fewer carrier-primed Th cells than the activation of TNP-ABC. Furthermore, responses of the TNP-MABC were of a greater magnitude. Non-cognate activation induces a low level of proliferation of both TNP-ABC and TNP-MABC, but induces differentiation of TNP-MABC only. Percoll density fractionation of spleen cells prior to enrichment for TNP-MABC suggests that the small, dense cell population responds to cognate, but not to non-cognate activation. FACS separation of TNP-MABC by surface Ig isotype reveals that approximately 80% of the secondary IgG response is derived from cells expressing sIgG. Such cells constitute less than 10% of the total number of TNP-MABC. Limiting dilution studies with sorted TNP-MABC indicate that sIgG+ TNP-MABC are enriched for precursors that give rise to a large clone size. The in vitro results indicate the existence of three putative pathways for antigen-specific memory B cell activation by a T-dependent antigen: 1) sIgG+ cells differentiating into IgG-secreting cells; 2) sIgM+ cells differentiating into IgG-secreting cells; and 3) sIgM+ cells differentiating into IgM-secreting cells.     

Changes in B lineage cell population in liver and spleen of normal neonatal mice

The frequency and characteristics of B lymphocyte lineage cells in neonatal murine liver and spleen were studied during the first 10 days after birth. These were distinguished as B cells with surface IgM (slgM), immediate precursors of B cells (pre-B cells) lacking slgM but containing micron-heavy chains of IgM, and earlier precursors that did not synthesize immunoglobulin but could be detected with monoclonal 14.8 antibody. Experiments were also done to relate these to cells capable of clonal proliferation in mitogen-containing semisolid agar cultures and cells that acquire this function only after preculture in liquid medium. Newborn liver contained large numbers of early precursors as well as pre-B cells, and culture studies revealed that a majority of the colony-forming B cells present at that time were slg-. Adherent accessory cells in newborn liver suspensions facilitated the maturation of these into functional B cells in vitro. At most ages, however, numbers of slg+ B cells detected in that tissue were surprisingly low. Possible explanations for this include a rapid exit of newly formed B cells and their immediate precursors from liver and/or a high rate of abortive lg gene rearrangements during the neonatal period. In contrast, whereas the spleen contained early precursors and pre-B cells at birth, these cells steadily declined in number with age as the numbers of slgM+ B cells increased. Adherent cells in liver but not spleen of immunodeficient CBA/N mice suppressed B lymphocyte formation in semisolid or liquid cultures. These observations document population dynamics in B lineage cells during a critical period of development.     

The signal for tolerance in B cells is not transmitted through antigen-specific immunoglobulin receptors

Studies of B cell tolerance at the single-cell level require a ready source of antigen-specific B cells that are uncontaminated by T cells or accessory cells. We have isolated normal dinitrophenyl (DNP)-specific B cells from spleens of unprimed mice and propagated these cells in vitro. These B cells are uncontaminated by T cells or macrophages. Long-term cultures of these cell lines contain pre-B cells that are surface (s) IgM-, B cells with sIgM alone, and more mature B cells with sIgM, sIgD, and Ia antigens. Using the cell line lymphocytes we have shown that the early binding of the tolerogenic form of hapten to B cell receptor on mature B cells induces the same activation signal as antigen, and the negative signal induced by tolerogen occurs after B cell activation. Exposure of maturing B cells to DNP bound to murine IgG2a (MGG) for 30 days does not inhibit growth or receptor expression, but does induce tolerance that is reversible when DNP-MGG is removed. A 45-day exposure to DNP-MGG also induces a reversible tolerance.     

Phenotype, frequency, and EBV responsiveness of human marrow B and pre-B cells

We have purified subpopulations of B lineage cells from human adult (rib) bone marrow by cell sorting and panning. Limiting dilution analysis was then used for a clonal analysis of cells able to secrete IgG, IgA, or IgM spontaneously or after infection with EBV. Nonproliferating, high rate IgG or IgA producers occurred at frequencies of about one per 1000 marrow mononuclear cells. Their frequency and Ig production was unaffected by EBV, and they appeared not to express EBNA after exposure to EBV. These cells were Ia+, B1+, and over 85% expressed sIg of the IgM/D (up to 75%) and/or IgG/A isotypes (40 to 60%). B cells committed to the secretion of IgM represent 2 to 10% of marrow B lymphocytes. They were found to be Ia+/B1+/B2+/CALLA- and C3b receptor (CR3)-cells, and most (greater than 90%) required infection with EBV and proliferation to develop into IgM-producing lymphocytes. Thirty to 40% of these cells did not express Ig (H or L chain) on their surface, and therefore resembled pre-B cells at the beginning of the 4- to 5-wk culture period. Proliferating pre-B cells from adult human marrow have been described, but their conversion into IgM-producing cells has not been formally demonstrated. Although EBV induces IgM production, the expression of EBNA, and several rounds of cell division in these cells, the induction of stable (greater than 5 wk) growth transformation represents a rare event in these pre-B cells: in several thousand limiting dilution wells, not a single culture of sIg-cells showed stable growth transformation. The dichotomy between EBV-induced high-rate IgM responses and absent growth transformation discriminates activation and transformation as distinct aspects of EBV-induced B cell "responses", and suggests that cellular properties play critical roles for viral transformation. We propose a model in which cellular target genes for transforming sequences in the EBV genome are transiently expressed during B cell differentiation.     

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.     

Changes in the expression of B cell surface markers on complement receptor-positive and complement receptor-negative B cells induced by phorbol myristate acetate

The ability of phorbol myristate acetate (PMA) to induce changes in the expression of B cell surface markers on CR- and CR+ B cells from normal mice in an in vitro culture system was examined. The markers studied were CR, sIgM, sIgD, and sIa. CR- B cells acquired the CR after overnight incubation with PMA. A twofold increase in sIa expression on CR- and CR+ B cells was also noted, whereas the staining intensity of sIgM and sIgD decreased on both B cell populations. These changes in the expression of surface markers took place without detectable increases in cell proliferation, cell size, or RNA content. Furthermore, the same effects were observed when CR- and CR+ B cells were prepared from a small B cell population purified by elutriation. It therefore appears that PMA can exert its effect directly on small, resting B cells.