Microenvironmental studies of pre-B and B cell development in human and mouse fetuses

Immunofluorescence techniques were used to trace the development of cells expressing mu heavy chains in humans and mice. IgM B cells were distinguished from pre-B cells by their additional expression of kappa or lambda light chains. Generation of pre-B and progeny B cells was evident in hemopoietic fetal liver and bone marrow, but not in thymus, heart, lung, spleen, kidney, and placental tissues. Pre-B and B cells, in a ratio of 2 to 1, were abundant in sections of hemopoietic liver and in bone marrow from 12- to 15-wk-old human fetuses, whereas these cells were rare in nonhemopoietic liver samples obtained beyond the 34th week. In mouse fetal liver mu+ cells appeared first around the 12th day of gestation and increased in frequency throughout the third trimester. On day 17 of gestation, kappa light chain expression by 1% of mu+ cells was noted, and the percentage of kappa+/mu+ cells increased progressively to more than 80% by 5 days after birth. Pre-B and B cells were interspersed among myeloid and more abundant erythropoietic cellular elements in the extrasinusoidal areas adjacent to hepatic cords. A loose clustering or "starburst" distribution pattern of pre-B cells became evident around day 17. These observations suggest a model for in situ generation of pre-B and progeny B cells in the hemopoietic fetal liver. In the midst of more numerous erythropoietic elements, immunoglobulin-negative precursors divide to generate a loose colony of mu+ pre-B cells that divide again before giving rise to a wave of IgM B cells.     

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.     

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.     

Ontogeny of terminal deoxynucleotidyl transferase-positive cells in lymphohemopoietic tissues of rat and mouse.

The ontogeny of hemopoietic cells which contain the enzyme terminal deoxynucleotidyl transferase (TdT) was studied in rats and mice. During fetal life, TdT-positive cells were first detected in the thymus, where they appeared on or about day 17 of gestation. TdT-positive cells were not found in fetal liver, spleen, or bone marrow, but appeared in bone marrow and spleen on the day after birth. In the rat, peak levels of TdT-positive cells were attained at 3 to 4 weeks of age in thymus, bone marrow, and spleen, accounting for 67, 3.9, and 2.3% of nucleated cells, respectively. The percentages of TdT-positive cells in thymus and bone marrow decreased gradually thereafter, whereas, TdT-positive cells in spleen were no longer detectable by 7 weeks of age. Normal percentages of TdT-positive cells were found in bone marrow and spleen from neonatally thymectomized rats and congenitally athymic (nu/nu) mice. Dexamethasone treatment resulted in a marked decrease in TdT-positive cells. The results are discussed with respect to the putative role of TdT-positive hemopoietic cells as thymocyte progenitors.     

Ontogeny of IgE-bearing lymphocytes in the rat

IgE-bearing lymphocytes were detected by immunofluorescence in the spleen of neonatal Hooded Lister strain rats within 24 hr after birth. The same cells were detected in the bone marrow as early as the 4th day after birth. Both fetal spleen and liver obtained 1 day before birth contained IgM-bearing cells but no detectable IgE-bearing cells. The proportion of IgE-bearing cells in the spleen and bone marrow increased during the neonatal period and reached an adult level within 3 to 4 weeks after birth. In adult Hooded Lister rats, IgE-bearing cells were 3 to 6% of total spleen cells and 1.5 to 2.2% of bone marrow cells. Most of the IgE-bearing cells from bone marrow cells. Most of the IgE-bearing cells from both newborn and adult animals carried IgM determinants on their surface. Capping experiments showed that epsilon chain determinants and mu chain determinants belonged to separate molecules. IgG2a-bearing lymphocytes were detected in the neonatal spleen as early as the 4th day after birth, but a significant number of these cells was not detected in the bone marrow until the 4th week. In newborn spleen the percentage of IgE-IgM double bearing cells was higher than that of IgG2a-bearing cells.     

Ontogeny of Nk-1+ natural killer cells. I. Promotion of Nk-1+ cells in fetal, baby, and old mice

Using anti-Nk-1.1 serum, the alloantiserum specific for murine natural killer (NK) cells, we followed the ontogenetic development of Nk-1+ cells in fetal thymus, liver, and spleen. A transient population of Nk-1+ cells in fetal thymus was observed on day 14 but not on day 16 of gestation. On day 16 of gestation, Nk-1+ cells were detected only in liver and spleen. The proportion of Nk-1+ cells in spleen remained high (20 to 30%) at birth and persisted until 2 to 3 wk old. The Nk-1+ cells in "baby" (1 to 2 wk old) spleen bound to YAC cells but failed to lyse them in 51Cr-release assay. Upon induction with interferon (IF), the proportion of Nk-1+ cells increased, but the lytic activity remained low, suggesting that the "baby" NK-1+ cells are immature in lytic function. In old mice (12 to 14 mo), Nk-1+ cells were also detectable, even though NK activities were lower compared with those of the young adult (6 to 8 wk old) mice. The Nk-1+ cells of old mice were readily induced by IF to exhibit activities, and the induced NK cells were Nk-1+. We have thus established Nk-1.1 antigen as an early hemopoietic differentiation antigen. Splenic Nk-1- cells could be induce by IF to become NK-1+ cells, which could be inactive or active in NK assays, dependent on the age of the mice.     

A granulocytosis-inducing tumor inhibits the production of B lymphocytes in murine bone marrow

Mice bearing a transplantable CE mammary carcinoma have been shown to have greatly augmented rates of neutrophil production coupled with a marked diminution of bone marrow lymphocytes. The objective of the present study was to test whether the loss of lymphocytes, and especially of B cells, from the bone marrow and spleen of tumor-bearing animals was due to a reduced rate of cell production and if so, at what level this response was regulated. A modified 3H-TdR pulse and chase analysis was used to assess the rates of production of small lymphocytes and B cells (stained for c mu and s mu) at weekly intervals after CE tumor transplantation. 3H-TdR was infused continuously for 24 hr, and radioautographs were prepared of bone marrow and spleen cells 0, 24, and 48 hr after termination of the infusion. Pre-B cells (c mu+s mu-) essentially disappeared from the femoral bone marrow by the end of 1 wk of tumor growth, followed by a great reduction in the number of c mu+s mu+ cells in the marrow and s mu + cells in the spleen. Although pre-B cells appeared in the peripheral marrow (caudal vertebrae, metatarsal bones) and spleen of tumor-bearing mice, these cells could not compensate for the continued decrease in the numbers of more mature B cells. In normal mice, during the 48-hr chase period, newly formed, 3H-TdR-labeled, small lymphocytes and s mu+ cells continued to emerge from the prelabeled precursor compartment at a steady rate, but after 1 wk of tumor growth, the number of small lymphocytes and s mu+ cells emerging from the precursor compartment fell steadily during the 48-hr chase period. During the second and third weeks of tumor growth, a steady state appears to have been reached in B cell production, which was at a level approximately 10 times below that of normal. Because pre-B cells are normally maintained by a less mature precursor population (2), the initial disappearance of c mu+s mu- cells suggests that the CE mammary carcinoma exerts its modulatory influence on primary B cell production by inhibiting or eliminating the cells that eventually feed into the pre-B compartment. The nature of the regulatory factors apparently secreted by the tumor and the more precise identity of the target cells are under investigation.     

Pre-B cell generation potentiated by soluble factors from a bone marrow stromal cell line

Two bone marrow stromal cell lines isolated from the adherent layer of a Dexter-type long term bone marrow culture differ markedly in their hemopoietic support capacity. S17 supports myelopoiesis and the differentiation of early B cell precursors into B lymphocytes while S10 supports myeloid cell differentiation and not B lymphopoiesis. The identification of a stromal cell line with B cell support capacity prompted an investigation of whether the effects of S17 were mediated via soluble factors. Results presented herein indicate that medium conditioned by S17 but not S10 contains an activity that can induce the expression of the 220,000 m.w. 14.8 antigen and cytoplasmic mu H chain of Ig in B lymphocyte progenitors that have not yet expressed these markers. Bone marrow cells were depleted of 14.8+, cytoplasmic mu+ pre-B cells on antibody-coated petri dishes. After 24-h liquid culture newly generated pre-B cells were enumerated as cells that expressed cytoplasmic mu H chain of Ig but not Ig L chains by immunofluorescence. Expression of Ly5(220) was monitored by 14.8 antibody binding. This pre-B cell differentiation activity was abrogated by digestion with pronase, aminopeptidase, or carboxypeptidase. Isoelectric focusing data revealed the activity to have isoelectric point of 5.9 to 6.2. S17-conditioned medium was fractionated using HPLC and each fraction tested for pre-B cell-generating activity. Fractions collected from a Superose 12 gel filtration column were found to have two peaks of activity associated with molecules of apparent m.w. of approximately 60,000 and 10,000. Virtually identical peaks of activity were observed when medium conditioned by heterogeneous stromal cell cultures was fractionated. Separation of S10-conditioned medium revealed no cryptic activity. S17-conditioned medium was further characterized by anion exchange chromatography and the majority of the pre-B cell generating activity shown to be associated with the void volume that eluted from a MonoQ column. These fractions were rechromatographed on Superose and the activity again found to be associated with two fractions corresponding to apparent m.w. of 60,000 and 10,000. The S17 pre-B cell differentiation activity appears to result from the presence of a novel molecule because other well characterized mediators had no activity in this short-term liquid culture system. No pre-B cell-generating activity was observed when IL-1 or conditioned medium containing IL-2, IL-3, or IL-4 (B cell stimulatory factor 1) were added to cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

Proliferation activity of stromal stem cells (CFU-f) from hemopoietic organs of pre- and postnatal mice

Stromal stem cells (CFU-f assay) from hemopoietic organs of fetuses, in contrast to adult animals, exhibit a high proliferation activity. This implies that these CFU-f are radiosensitive and potential target cells after radioactive contamination of fetuses. Furthermore, the percentage of CFU-f in DNA synthesis is correlated with the hemopoietic activity in liver, spleen, and bone marrow. As hemopoiesis starts, high numbers of CFU-f are in S phase. In fetal liver, spleen, and bone marrow, values of 70, 43, and 58%, respectively, are reached. As hemopoietic activity decreases in liver and stabilizes in spleen and bone marrow, mitotic activity of these stromal stem cells becomes undetectable.     

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.     

Mechanisms of lymphocytic choriomeningitis virus-induced hemopoietic dysfunction.

Results of this study showed that lymphocytic choriomeningitis virus infection causes a marked activation of natural killer (NK) cells not only in the spleen but also in the bone marrow. This activity reached its peak at about day 3 of infection and declined after days 6 to 7. Enhanced NK cell activity was found to correlate with decreased receptivity for syngeneic stem cells in bone marrow and spleen, with the notable exception that decreased receptivity persisted longer in bone marrow. Treatment of infected recipients with anti-asialo GM1 (ganglio-N-tetraosylceramide) significantly increased the receptivity for syngeneic hemopoietic cells. These findings are consistent with the hypothesis that NK cell activation causes rejection of syngeneic stem cells, thus resulting in hemopoietic depression. To understand the mechanisms behind the prolonged decrease in bone marrow receptivity (and bone marrow function in the intact mouse) mentioned above, we followed the changes in the number of pluripotential stem cells (CFU-S) circulating in the peripheral blood and in endogenous spleen colonies in irradiated mice, the limbs of which were partially shielded. It was found that following a marked early decline, both parameters increased to normal or supranormal levels at about day 9 after infection. Because the bone marrow pool of CFU-S is only about 20% of normal at this time after infection, a marked tendency for CFU-S at this stage in the infection to migrate from the bone marrow to the spleen is suggested. It seems, therefore, that as NK cell activity declines, the spleen regains the ability to support growth of hemopoietic cells and the bone marrow resumes an elevated export of stem cells to the spleen. This diversion of hemopoiesis could explain both the long-standing deficiencies of the bone marrow compartment and the prolonged decrease in the receptivity of this organ.     

Migration and differentiation of bone marrow lymphocytes: development of surface immunoglobulin, Fc receptor, complement receptor, and functional responsiveness as studied with anti-allotype serum

Immunofluorescent studies using fluorescein isothiocyanate-conjugated mouse anti-allotype antibody were carried out to study the migration pattern and the development of surface Ig (SIg), Fc receptor for IgG (FcR gamma), and complement receptor (CR) or mouse bone marrow lymphocytes following intravenous injection into congenic mice. After transfer of bone marrow cells from CSW mice into untreated congenic CWB mice, the absolute number of donor-type SIg-bearing (SIg+) cells and the proportion of either FcR gamma- or CR-bearing (FcR gamma+ or CR+) cells in donor-type SIg+ cells were evaluated in the recipient spleen and the results were compared with those obtained after the transfer of CSW spleen cells. After injection of donor bone marrow cells, detectable donor-type SIg+ cells, although few initially, increased from day 1 to Day 2 and reached a plateau thereafter. The proportion of FcR gamma+ cells in donor-type SIg+ cells, although very low in the donor marrow inoculum, increased progressively after 1 day to reach a maximum at Day 5 (90%). On the other hand, following the transfer of spleen cells, the proportion of FcR gamma+ cells remained at high levels (90%) for 5 days after transfer. Likewise, the proportion of CR+ cells in donor-type SIg+ cells was very low (less than 1%) in the original donor bone marrow cells but high (60%) in the donor spleen cells. However, in transferring bone marrow cells this proportion also increased in the recipient spleen to reach a maximum (49%) at Day 5 although it was lower compared to the percentage of FcR gamma+ cells in donor SIg+ cells. Furthermore, the ability of functional responsiveness to antigen was also examined in the same system by detecting plaque-forming cells (PFC) from donor origin. In transferring donor bone marrow cells into recipient, the participation of donor cells in the PFC response was very low when the recipients were primed with sheep red blood cells at Day 3 after transfer. However, when the recipients were primed at Days 7 to 21 after transfer, increasing numbers of the donor marrow-derived cells were involved in the PFC response. Thus, the present study demonstrates that the bone marrow-derived lymphocytes, albeit lacking both distinctive surface receptors (IgM, FcR gamma, CR) and the functional responsiveness to antigen, continue their development along the B-cell lineage after migrating into the spleen, as evidenced by the surface receptor expression and participation in the antibody response.     

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.     

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.     

Age-dependent changes in B lymphocyte lineage cell populations of autoimmune-prone BXSB mice

BXSB mice, a recently developed autoimmune strain, develop a human lupus-like disease with B cell hyperplasia in peripheral lymphoid organs. Unlike other experimental models of autoimmunity and human lupus, BXSB male mice manifest accelerated autoimmune phenomena through the influence of a Y chromosome-linked enhancing factor. The present studies were performed to investigate the features of B lymphopoiesis in BXSB mice and to determine whether differences exist between BXSB males and females in this respect. B lineage cell populations in the marrow of BXSB mice were identified phenotypically by studying the cytoplasmic mu-heavy chains of IgM (c mu), and functionally by their ability to acquire clonability and sIg in short-term liquid cultures. Male BXSB mice became deficient in both the precursors of functional B cells and c mu + pre-B cells by the age of 8 to 12 wk. This followed a transient increase in this population, which peaked when the mice were 2 to 4 wk old. In females, substantial numbers of functional B cell precursors and c mu + cells were maintained until more than 4 mo of age. Cells lacking Ig but bearing a B lineage cell antigen (14.8) were elevated in numbers in both BXSB males and females until 16 wk of age when compared to normal strains of mice. At the time pre-B cells and functional B precursors were elevated in numbers, some sIg- cells were shown to form colonies in mitogen-stimulated semisolid agar cultures without a period of preculture. Most of these sIg- cells seemed to bear the B lineage cell antigen (14.8). They were independent of both G-10 adherent regulatory cells and Thy-1+ cells for their colony formation. These results indicate that B lymphocyte formation may be maintained in a hyperactive state in BXSB females, whereas males become deficient in B cell precursors very early in life. This early decline might be related to the accelerated development of autoimmune disease in BXSB mice. Bone marrow transplantation studies showed that these unusual characteristics of B lymphopoiesis were reciprocally transferable with unseparated bone marrow cells between BXSB males and females. This finding indicates that sex hormones are not a critical variable in abnormal B lymphocyte formation in this strain, and that the premature deficiency of immediate B precursors in males may be regulated by a genetic factor(s) located on the Y chromosome.     

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.     

Phenotypic and functional characterization of murine B lymphocyte precursors isolated from fetal and adult tissues

Murine fetal liver and adult bone marrow cells identified by monoclonal 14.8 antibody were enriched on antibody-coated polystyrene petri dishes. Cell surface immunoglobulin (sig)-bearing cells were depleted before this enrichment procedure, and the resulting preparations of 14.8+, slg- cells were characterized as to morphology, immunoglobulin gene expression, and functional potential in vivo and in vitro. All cells with detectable mu chains of IgM in the cytoplasm (cmu) were found to be included in the 14.8+ population. The enriched cells did not contain significant numbers of committed granulocyte-macrophage progenitor cells or putative hemopoietic stem cells. Selected cells from 16-day fetal liver were large, a majority of the cells had a lobulated rather than a spherical nuclear outline, and less than 1% had detectable cmu. Enriched cells from 19-day fetal liver were on the average smaller than those from 16-day-gestation liver and had a more typical lymphoid morphology; 30% were cmu+. Adult bone marrow 14.8+, slg- cells were similar to 19-day fetal liver cells in morphology, and approximately half were cmu+. These selected precursor cells retained the capacity to mature in vivo and in vitro. Fetal and adult 14.8+, slg- cells were efficient in generating newly formed B cells in vivo, and this maturation step appeared to be dependent on the presence of microenvironmental accessory cells. However, the ability of positively selected cells to mature in vitro was markedly decreased, and this potential was not rescued by providing known sources of accessory cells. Possible reasons for this difference are considered. This technique for positively selecting cells has allowed us to directly compare for the first time B cell precursors from fetal and adult tissues and will be invaluable for resolution of the cell compartments in the differentiation of B lymphocyte precursors, in the study of accessory cells known to facilitate this process, in the definition of humoral factors which may act on pre-B cells, in the study of immunoglobulin gene rearrangements which take place during normal differentiation, and for further comparative studies of fetal and adult lymphopoiesis.     

A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure

The derivation of a new human pre-B cell line from the leukocytes of a girl in the leukemic phase of lymphoblastic lymphoma is reported. Cells of this line, termed SMS-SB, synthesized mu but not light chains. These mu-chains were found only in the cytoplasm and were not secreted. SMS-SB cells bore HLA-DR determinants. They did not express the common ALL antigen or terminal deoxynucleotidyl transferase and, therefore, differed from previously described human pre-B leukemia cell lines. SMS-SB cells resembled certain Abelson leukemia virus-transformed mouse bone marrow cells. Except for the lack of formed mouse bone marrow cells. Except for the lack of mu secretion, the phenotype of SMS-SB cells was the same as the major subpopulation of marrow pre-B cells.