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Chronic inflammation characterized by T cell and macrophage infiltration of visceral adipose tissue (VAT) is a hallmark of obesity-associated insulin resistance and glucose intolerance. Here we show a fundamental pathogenic role for B cells in the development of these metabolic abnormalities. B cells accumulate in VAT in diet-induced obese (DIO) mice, and DIO mice lacking B cells are protected from disease despite weight gain. B cell effects on glucose metabolism are mechanistically linked to the activation of proinflammatory macrophages and T cells and to the production of pathogenic IgG antibodies. Treatment with a B cell-depleting CD20 antibody attenuates disease, whereas transfer of IgG from DIO mice rapidly induces insulin resistance and glucose intolerance. Moreover, insulin resistance in obese humans is associated with a unique profile of IgG autoantibodies. These results establish the importance of B cells and adaptive immunity in insulin resistance and suggest new diagnostic and therapeutic modalities for managing the disease.

EBV is a human tumor virus that infects and establishes latency in the majority of humans worldwide. In vitro, EBV growth transforms primary B lymphocytes into lymphoblastoid cell lines with high efficiency. We have used cDNA subtraction cloning to identify cellular target genes required for growth transformation and identified a new C(2)H(2) (Krüppel-type) zinc finger gene, ZNF(EB), that is trans-activated early following EBV infection. In this study, we characterize ZNF(EB), including its intronless locus, and human and mouse protein variants. The gene is transiently expressed during normal lymphocyte activation, and its expression is sustained in EBV-positive but not EBV-negative B cell lines. There is limited expression in nonhemopoietic tissues. Its critical role in the growth transformation of B lineage cells is indicated by the abrogation of transformation with antisense strategies. ZNF(EB) maps to chromosome 18q12, a region with mutations in numerous, predominantly hemopoietic malignancies.

We have used an efficient cDNA subtraction library procedure to identify newly induced genes in human B lymphocytes infected for 6 h with Epstein-Barr virus (EBV). Among the genes identified by automated sequencing of a random subset of clones from this library, one coded the EBV BCRF1 open reading frame, which specifies the viral interleukin 10 gene (vIL-10). This molecule is highly homologous to human (h)IL-10 and was previously thought to represent a "late" viral gene expressed only during the lytic phase of virus replication. Using gene amplification by reverse transcriptase polymerase chain reaction of B cell RNA obtained at varying times after infection, we detected vIL-10 expression within a few hours of EBV infection, followed, 20-30 h later by expression of hIL-10. Expression of both genes continued beyond the initial transformation phase (5-10 d) and was present in all transformed cell lines tested. When added at the time of viral infection, antisense (but not sense) oligonucleotides for vIL-10 mRNA (cytosolic half-life, approximately 6 h) prevented subsequent B cell transformation. The antisense effect was highly specific, leaving the expression levels of other transformation-related genes intact. Addition of exogenous (h)IL-10 rescued the transformation process in antisense-treated cells. Our observations establish vIL-10 as a new latency gene with a directly transformation-prerequisite function.

Following Epstein-Barr virus (EBV) binding to its CD21 cell surface receptor, virus internalization, nuclear translocation, and circularization of the viral episome were found to occur within 30 min, immediately preceding the expression of EB nuclear antigen (EBNA)-1 and -2 and latent membrane protein (LMP)-1 and -2 genes. Early viral gene expression was unaffected by blockade of the virus induced, transformation-prerequisite cellular activation pathway (Ca2+ currents, tyrosine phosphorylation, induction of p56lck, hsp70, and hsp90). Despite life times of only 3 h, antisense (but not sense) oligonucleotides for the above latency genes prevented subsequent transformation. Any one antisense oligonucleotide dramatically depleted transcripts not only of the target gene, but of all other latency genes. The blocking effect of antisense oligonucleotides allowed us to identify a new transformation-prerequisite latency gene near the fused termini. The concerted regulation of EBV gene expression is highly unusual and unexplained but our results imply critical, perhaps regulatory roles for initial latency gene transcripts themselves.

Epstein-Barr virus (EBV) is a latent human herpes virus associated with a range of malignant and non-malignant disorders. EBV binds to CD21 virus receptors on B lymphocytes and growth transforms these cells; in susceptible (e.g., immunodeficient) hosts such cells rapidly expand into fatal lymphomas. Virus binding and infection trigger a cascade of cellular events which are transformation prerequisite and analogous to non-oncogenic cell activation events but which differ in several quantitative or qualitative respects. Unique trans-membrane Ca2+ currents, Na+/H+ exchange, as well as tyrosine phosphorylation and p56lck-gene induction suggest that even early on the transformation process has oncogenic specificity. In this report we describe that two additional cellular gene families, the stress proteins hsp70 and hsp90, are coordinately induced at mRNA and protein levels and, quite different from hsp induction by thermal stress, this induction is dependent on EBV-induced trans-membrane Ca2+ currents. Blockade of hsp induction prevents transformation. The kinetics and induction prerequisites set this response well apart from reported responses to thermal or viral stress protein induction. Like p56lck-, hsp induction is purely a post-receptor binding event and not dependent on expression of any viral gene. The induction kinetics, with a peak at approximately 12-16 hr and subsequent decline to control levels, considerably extend the chronological map of elements in the CD21-dependent branch of the transformation pathway and suggest a specific role of induced hsp different from the cell cycle-related functions observed in other cell systems.

Analysis of VH gene segments deleted in the process of immunoglobulin heavy chain (IGH) variable region assembly in three series of monoclonal B cell lines has been used to determine the human VH region organization. A deletion map of the relative positions of 21 different VH gene segments has been determined. The characterization of B cell lines from three unrelated adults of two racial groups yielded the same relative VH gene segment order, suggesting that the overall order of VH genes in the normal population is constant. This VH gene segment order was consistent with what we had previously generated from physical mapping techniques. DH segments from the second DH cluster, distinct from the major DH locus 3' of the VH region, were not observed to be used in 32 different rearrangements. Approximately 77% of the VH-(D)JH rearrangements involved VH gene segments within 500 kb of the JH region, indicating that human B cell lines preferentially rearrange JH-proximal VH gene segments. The switch, observed in mice, from the fetal use of JH-proximal VH gene segments to an adult VH use dependent upon VH family size may therefore not occur in humans. This detailed map of the VH gene segments is a necessary prerequisite for understanding VH usage in development and disease.

Epstein-Barr virus (EBV) exposure of human B lymphocytes induces rapid, Ca(2+)-dependent tyrosine phosphorylation of two cytosolic proteins, one likely the CD21 EBV receptor and another unknown species of 55-60 kDa. We now identify the latter protein as the tyrosine kinase lck (p56lck). In T cells many activation events reduce the high constitutive p56lck expression levels typical for that lineage, and they induce the appearance of a 60-kDa lck species. We now demonstrate that in B cells exposed to EBV the at best low constitutive p56lck expression levels are rapidly and transiently up-regulated without generation of 60-kDa lck. lck-specific antisense oligonucleotides block p56lck induction and prevent subsequent B cell activation and immortalization whereas B cell activation by nononcogenic agents was unaffected. We propose that p56lck superinduction is a transformation prerequisite which signals entry into the oncogenic growth transformation process.

During development, B lymphocytes have the ability to switch from synthesis of IgM to immunoglobulins of another isotype such as IgG, IgA, or IgE. This class switching mechanism has been shown to involve DNA rearrangement and concomitant deletion of intervening CH genes. In our report, an EBV-transformed B lymphoblastoid cloned cell line is described that simultaneously expressed and secreted both IgM and IgE. DNA analysis showed the (nonproductive) rearrangement of one allele to gamma and (productive) rearrangement of the other allele to mu. Germ-line arrangement of the C epsilon gene was preserved on both alleles.

EBV growth-transforms primate B lymphocytes and directly causes mono/multiclonal B cell lymphomas in vulnerable hosts. In this report we demonstrate that the degree of B cell transformability is not quantitatively determined at the level of either the saturable, transformation-prerequisite virus receptors or of the actual viral cell entry process. Instead, post-receptor binding events [Na+/H+ exchange, Ca2+ flux, tyrosine phosphorylation of two proteins (55-60/130-140 kd)] were identified as critical determinants of transformability. The presence of competent virus in transformable cells was per se insufficient for transformation: blockade of Ca2+ fluxes (or the antiport) generates virus-loaded cells that express viral genes but remain untransformed. Delayed induction by ionomycin of appropriately sized Ca2+ fluxes ([Ca2+]i greater than 180 less than 400 nM) re-starts transformation processes in EGTA-blocked, virus-loaded cells, perhaps providing a model for the study of virus re-activation. Overall, EBV induces unique cellular activation events different from non-oncogenic lymphocyte mitogens/activators, and, given the oncogenic potential of transformed cells in susceptible hosts, we hypothesize that these events describe a novel oncogenic transformation pathway.

Fresh and transformed human B lineage cells were found to be sensitive to the cytotoxic action of Shiga-like toxin (SLT), a bacterial cytotoxin. The toxin was specifically bound by the glycolipids globotriosylceramide and galabiosylceramide expressed on the surface of sensitive cells. Mutant Daudi cells selected for resistance to SLT cytotoxicity (SLTR20) were deficient in SLT-binding glycolipids and failed to bind SLT to their surface, suggesting a role for these glycolipids in the mediation of SLT cytotoxicity. Of a number of normal and transformed lymphoid and myeloid cells screened for SLT sensitivity, only B lymphoid cells were susceptible to SLT action. Moreover, B lymphoid cells were the only cells expressing the SLT binding glycolipids. In vitro B cell activation studies with Epstein-Barr virus and pokeweed mitogen both indicated that the vast majority of SLT-sensitive B cells belong to the IgG and IgA committed subset, whereas most IgM and IgM/D producing cells were resistant to SLT toxicity. The selective elimination of IgG and IgA committed cells may explain the production of only IgM class anti-SLT antibodies in Shigella-infected humans leading to the failure of long-term immunity to dysenteric disease.

The capacity of the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), and Staphylococcus protein A (SpA) to induce B cell proliferation and differentiation was compared with the B cell mitogen, formalinized Staphylococcus aureus (STA). Lymphocyte subpopulations from normal donors and patients with various immunodeficiency diseases were studied. In the presence of the T cell mitogens, irradiated T cells were capable of providing a helper cell activity that enabled co-cultured B lymphocytes to proliferate in response to these mitogens and to differentiate into IgM-secreting (direct) hemolytic plaque-forming cells (PFC). In the PFC response, radioresistant T-helper and radiosensitive T-suppressor cell activities could be demonstrated. T-suppressor cell activity outweighed helper activity only in nonirradiated co-cultures stimulated with Con A. Patients with severe combined immunodeficiency lacked mitogen-induced helper T cells, whereas patients with various forms of humoral immune deficiency were normal in this respect. These findings and the tissue distribution of the helper activity is aquired early in post-thymic T cell differentiation. The data suggest that experiments with cell lineage-specific lymphocyte mitogens should be considered in the context of more complex cell-cell interactions.

Possible explanations for the immune deficiency usually associated with graft-versus-host disease include defective lymphocyte differentiation and active suppressor cell mechanisms. The present observations in a patient with chronic graft-versus-host disease delineate yet another possible mechanism: a failure of successful interaction between competent patient lymphocytes. Thus, helper and suppressor T cells as well as precursor B cells for the generation of specific in vitro plaque-forming cell (PFC) responses were present among patient lymphocytes and could interact normally with cells from four unrelated normal donors and produce PFC responses. In contrast, patient cells failed to interact successfully with each other and generate PFCs. This suggests a highly (self-) specific block in cell interaction that could explain the inability of the patient to mount a PFC response in vitro as well as a number of antibody responses in vivo during this phase of his disease.

Short-term treatment of normal human B lymphocytes with low concentrations of papain resulted in selective and reversible removal of sIgD determinants, whereas HLA and Ia-like antigens, sIgM as well as receptors for E, C3, and FcIgG were unaffected. When studied for their capacity to generate antigen-specific direct PFC, papain-treated (delta-) B cells were highly sensitive to inactivation by even low concentrations of antigen. In addition, these cells were impaired in their ability to cooperate normally with T-helper cells or their humoral product(s).

The 8-substituted guanosine derivatives (8-sGs), mercaptoguanosine and bromoguanosine, are shown to induce immunoglobulin production and proliferation in a subset of normal human B lymphocytes. Limiting dilution analysis indicated that the 8-sG-responsive B cell pool is approximately 10 times larger than that activated by pokeweed mitogen (PWM), 10 times smaller than that activated by Epstein-Barr virus (EBV), and contains approximately equal proportions of cells committed to the expression of IgG, IgA or IgM isotypes. Although some B cells seem able to respond to 8-sGs in the absence of T helper cells, maximal immunoglobulin production is only observed in the presence of T cells. The 8-sG response pattern of human B lymphocytes appeared similar but not identical to that reported for rodent cells. The mitogenic 8-sGs are unique activators as they bypass surface membrane interactions obligatory for other agents including anti-IgM, Staphylococcus aureus particles, PWM, and EBV.

In response to hypotonic stress, human T and B cells vary in the ability to adjust their volume. Whereas T cells exhibit a regulatory volume decrease (RVD) under these conditions, B cells do not. We studied the ability of peripheral blood-derived natural killer (NK) cells to regulate their volume in this way. Percoll density gradient purified NK subpopulation showed variable RVD which suggested the presence of mixtures of regulatory and non-regulatory cells within these subgroups. Further purification by flow cytometry into Leu 7+ and Leu 11+ cells showed that these NK cells displayed RVD similar to thymocytes but distinct from B cells and more mature T cells. These data support the hypothesis that NK cells may be derived from T cell precursors which, upon differentiation to NK cells, retain the RVD characteristic of pre-T cells. This finding may also be useful in further characterizing neoplastic clones which display NK-like activity but phenotypic heterogeneity.

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.

A series of 75 EBV-transformed pre-B and B-cell lines from fetal bone marrow at 14 to 18 wk of gestation was cloned for phenotypic, functional and molecular genetic studies. B-cell type volume regulation in response to hypotonic stress, low level CD9 (BA2), and, perhaps biased by our use of EBV, functional EBV receptors with expression of CD21 (B2) determinants characterized the most immature cells detected. These "B-progenitors" contained EBV genomes, maintained Ig H and L chain (as well as TCR) constant region genes in germ-line configuration and did not express other B, T, or myeloid lineage-associated surface markers including CD20 and MHC class II determinants. Such cells may represent B progenitor cells preceding classical pre-B-lymphocytes in pathways of B cell differentiation. Reminiscent of Abelson virus-induced transformation of immature murine B cells, EBV transformability together with the above properties may be the earliest markers of B lineage commitment in man. The expression of MHC class II Ag, CD20, C mu-H and then L chain rearrangements and expression followed in less immature pre-B lymphocytes and permitted a classification of lines into discrete subgroups of increasing maturity. The physical organization of the H chain locus in a given line was a stable characteristic. However, fetal pre-B cell lines showed considerable intraclonal heterogeneity with respect to H chain gene expression and that of differentiation markers such as CD20. Subcloning experiments indicated that this heterogeneity reflected clonally stable expression patterns distributed among subclones in an all-or-none fashion. The induction, by IL-6, of L chain expression in some but not all of these clones was linked to the presence of C mu transcription products, consistent with a possible regulatory role of mu protein in L chain rearrangement and expression. Although pre-B cell differentiation likely follows inherent programming, external signals seem able to hasten development along prescribed, hierarchical differentiation pathways.

T cells from highly atopic individuals spontaneously secrete in vitro a factor that specifically induces IgE synthesis from normal human B cells. We investigated the effects of such T cell supernatants derived from atopic individuals (TCSN-A) on functionally distinct B cell subsets to determine at what developmental stage B cells become responsive to this IgE-enhancing activity. B cells from normal and allergic donors were separated into subsets of small resting and large activated cells by density centrifugation or unit gravity sedimentation. When stimulated by TCSN-A, large activated B cells made more IgE than small resting B cells. The difference was as much as 3300% in comparing these subsets from allergic donors. Similarly, resting B cells stimulated by Staphylococcus aureus Cowan I (SAC) made 52 to 125% more IgE in response to TCSN-A than unstimulated small resting B cells. However, IgE production from large B cells, already activated in vivo, was not enhanced by the addition of SAC. Notably, the IgE level synthesized by in vivo large activated B cells from allergic persons was markedly greater than that seen with similar cells from normal donors, whereas resting B cells purified from allergic and normal donors produced comparable levels of IgE in response to TCSN-A. These results suggest that this enhancing activity functions as an IgE differentiation factor for activated B cells. This was further confirmed by the effects of TCSN-A on the IgM- and IgE-secreting EBV-transformed human B cell line K1D5. TCSN-A specifically enhanced IgE synthesis from these cells; TCSN from normal donors, IL 2, IFN-gamma, and BCGF did not. These results confirm that this activity functions as an IgE-specific differentiation factor, directly influencing activated B cells to synthesize IgE.

The staphylococcal cell wall component protein A (SpA) and formalinized, Cowan I strain Staphylococcal organisms (STA) were compared with the lectins phytohemagglutinin, concanavalin A, and pokeweed mitogen for their ability to trigger proliferation of normal human lymphocytes, lymphocyte subpopulations, and cells from patients with primary immune deficiency diseases. SpA was found to be a potent T cell mitogen, very similar to the other lectins tested. It failed to stimulate purified non-T cells and peripheral blood lymphocytes from patients with different forms of severe combined immunodeficiency disease (SCID). STA, treated to prevent the leakage of soluble SpA during culture, exclusively stimulated non-T cells: the responding cell population was characterized to be E-rosette negative but positive for C3 receptors, surface Ia, a receptor for STA itself, and likely carried surface immunoglobulin. Normal responses to STA were found in patients with the adenosine deaminase-positive form of SCID. In 18 patients with humoral immune deficiency syndromes, the presence of STA responses was correlated with the presence of circulating, surface immunoglobulin-bearing cells. A commercial STA preparation was rendered B cell specific after reformalinization, a procedure that eliminated the shedding of soluble SpA under culture conditions.

Limiting dilution analysis, hemolytic plaque assay, and ELISA procedures were used to study the recruitment, clonal expansion, and antibody secretion in human TNP-specific B cells activated in the presence of TNP-ovalbumin (TNP-OA), pokeweed mitogen (PWM), or regulatory T cells. TNP-OA-responsive, hapten-specific PFC precursor cells occupy approximately 0.5% of all sIgM+/sIgD+ B cells in cord blood, bone marrow, peripheral blood, and tonsil. The PWM-responsive, hapten-specific PFC precursor pool is 70 to 90% smaller and does not express sIgD. Antigen-reactive B cells go through a minimum of three divisions in culture (six to nine PFC per clone), and antibody secretory rates of about 10(4) molecules IgM/cell/hr are achieved. In contrast, PWM-induced clone sizes were at least 60 PFC per clone, with antibody secretory rates of approximately 6 to 7 X 10(4) molecules IgM/cell/hr. Addition of high-dose carrier-primed suppressor T cells to limit dilution cultures reduced PFC precursor cell recruitment by up to 99%. However, in the few clones escaping from suppression, both clonal expansion and antibody secretory rates were much higher than in suppressor cell-free cultures, generating 30 to 60% of the antibody secreted in controls but with consequently much more restricted clonal diversity. When limiting dilution cultures were compared with standard microcultures of 2 X 10(5) cells, both clonal expansion and antibody secretory rates were much lower than expected, with a culture efficiency calculated to be 10 to 20% of that in low-density cultures. Our data suggest that the B cell subsets activated by antigen and by mitogen differ in their abilities for clonal expansion and antibody secretion. The hapten-specific and -responsive B cell family is expressed early in ontogeny, and in adults it is distributed evenly throughout the body. These limiting dilution experiments revealed that the primary effect of regulatory T cells is a drastic reduction in clonal diversity, and much less a mere reduction in overall response magnitude.

Immunological and biochemical studies were carried out in two patients with the Lesch-Nyhan syndrome. No abnormalities of T- or B-lymphocyte function could be demonstrated in the presence of hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency.

Cultures of peripheral blood lymphocytes (PBL) in which specific hemolytic plaque-forming cells (HcPFC) had been induced were labeled with 14C-amino acids. Antigen-specific products in the culture supernatants were characterized by using indirect immune precipitation in conjunction with specific immunoabsorbents and/or gel filtration followed by SDS-polyacrylamide gel electrophoresis. After 5 days of culture with antigen (sheep red blood cells or ovalbumin) newly synthesized IgM and specific IgM antibody were demonstrated in culture supernatants from normal donors and from four out of five patients with congenital agammaglobulinemia (cAgamma). Secreted products bound specifically to antigen and pretreatment of labeled supernatants with anti-mu and anti-L chain antisera, but not with anti-gamma antiserum, prevented binding. Typical mu- and L chains constituted only a proportion of the anigen-binding peptides recognized by the anti-mu reagents. Induction of IgM antibody synthesis was dependent on the presence of antigen and was correlated with the generation of HcPFC. No major differences between the antigen-induced products of cAgamma and normal PBL were observed. These findings suggest that in the absence of terminal B cell differentiation in vivo, certain patients with cAgamma possess precursor cells that can respond to antigen in vitro with the synthesis of specific humoral products, including IgM antibody.

Despite the absence of B lymphocytes, peripheral blood lymphocytes (PBL) from four of five patients with congenital agammaglobulinemia (cAgamma) generated a specific hemolytic plaque-forming cell (HcPFC) response in vitro to sheep red blood cells and ovalbumin. The kinetics, antigenic, and cellular requirements were similar to normals, but significantly less HcPFC were found in patient cultures. Normal but not patient HcPFC-precursor cells were inactivated by treatment with anti-mu antisera whereas generated HcPFC in both controls and patients were sensitive to treatment with anti-mu. Pokeweed mitogen (PWM) and dextran sulfate (DXS) enhanced the HcPFC-response of normal PBL; cAgamma-cells were unresponsive to DxS and, in the presence of PWM, the development of HcPFC was inhibited. These findings indicate the presence of B lymphocyte precursors in the majority of patients with cAgamma investigated.

Mononuclear cells (MNC) obtained from adult rib specimen were investigated for their capacity to produce immunoglobulin (Ig) in vitro. Using limiting dilution analysis 3 populations of B lineage cells could be distinguished. The first produces Ig in culture without any intentional activation. These cells are strictly nonproliferating and sustain extraordinary secretory rates of 5 X 10(7)-10 X 10(7) molecules IgG, IgA or IgM/cell/h for at least 2 weeks. They occur at frequencies of 1 X 10(-4)-10 X 10(-4) (marrow MNC) or represent 1-5% of marrow B cells. Following exposure to infectious Epstein-Barr virus (EBV), these cells do not (a) proliferate, (b) express EBV-determined nuclear antigens (EBNA), (c) enhance their secretory rates or (d) show secretory activity for prolonged time. These cells are therefore EBV resistant. If these cells produced their antibody at similar rates in vivo, then their frequencies would suggest that they could provide up to 2/3 of daily synthetic rates for IgG and IgA and 20-30% of the daily IgM production. The second population of marrow B cells is exclusively committed to IgM production. Following exposure to EBV these cells proliferate, express EBNA and begin to secrete IgM. The third population represents 90% of marrow B cells. In our hands, these cells are unable to produce Ig in vitro.

The addition of fresh peripheral blood mononuclear cells (PBL) from four of seven patients with agammaglobulinemia to generated hemolytic plaque-forming cells (PFC) resulted in a dose-dependent suppression of PFC. This spontaneous suppressor cell activity (SSA) was restricted to the four patients who could generate a PFC response in vitro. SSA was mediated by a small subset of E-rosetting T lymphocytes characterized by theophylline-sensitive E-receptors and surface receptors for Fc-IgG. The effects of SSA were temperature dependent and reversible, and pokeweed mitogen could prevent the rapid decline of SSA observed during culture. Augmentation of SSA was achieved by agents known to increase intracellular levels of cyclic AMP, whereas lithium chloride abrogated SSA, including the drug-induced effects. Cells mediating SSA may play a role in preventing the normal transition of pre-B cells to B cells in patients with agammaglobulinemia without B lymphocytes.

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)

We have followed the pathway of the IgE-committed B lymphocyte from fresh, unstimulated peripheral blood, through EBV activation, transformation, and eventual cloning. Using cell sorting in conjunction with limiting dilution culture systems, we found that: (a) cells that are selected in the cell sorter and secrete IgE in culture are sIgM+/sIgD+. They secrete all three isotypes after EBV activation and continue to do so stably in culture; (b) individual IgE+ cells in culture coproduce IgM, IgD, and IgE and cytoplasmic Ig of each isotype can be detected in single cells; (c) no rearrangement was observed of VDJ to epsilon in any of six lines tested. DNA between the rearranged VDJ-mu and -epsilon appears to be overall intact, including a region 10.5 kb upstream and 18 kb downstream of the 2-kb epsilon coding region and; (d) mRNA of mu and epsilon species is of normal and comparable size. In contrast to IgG- and IgA-producing clones, multiple isotype expression appears to be both frequent and stable in cells committed to IgE production. We propose that IgE-committed cells represent a unique B cell sublineage whose differentiation pathway may be more strictly regulated than that of other isotypes with regard to the signals required for classical, deletional switch recombination that has been observed in rare IgE-producing myeloma cell lines.

Extracellular nucleotide degradation was studied in intact human B and T lymphocyte subpopulations and in lymphoblastoid cell lines. Cells of B lymphocyte lineage showed high nucleotide degrading activity, whereas T lymphocytes were unable to degrade extracellular nucleotides. The external surface of B cells contained active sites of ecto-triphosphonucleotidase (ecto-ATPase), ecto-diphosphonucleotidase (ecto-ADPase), and ecto-monophosphonucleotidase (ecto-AMPase). The expression of all three ectoenzyme activities seemed closely associated with B cell development. ATPase and ADPase activities increase continuously during B cell maturation, ecto-AMPase activity, on the other hand, reaches maximal activity in late pre-B cells. These results combined with our previous studies of intracellular ATP catabolism (Barankiewicz, J., and Cohen, A. (1984) J. Biol. Chem. 259, 15178-15181) provide evidence that extracellular ATP catabolism may represent exclusive source for adenosine in lymphocytes. It is suggested that adenosine may serve as a means of communication between B and T cells in lymphoid organs, B lymphocytes being the sole producers of adenosine and T lymphocytes being the recipients of this signal.

The induction of in vitro IgE production in human B cells from normal, nonatopic donors has been difficult and somewhat controversial. We report that IgE production is consistently observed in limiting dilution cultures of in vitro Epstein-Barr virus (EBV)-infected normal human B lymphocytes. The frequency of IgE-committed, EBV-responsive cells ranged from 1/810 to 1/10 000 B lymphocytes, and it was similar in peripheral (blood, tonsil) and central (bone marrow) tissue sites. Poisson distribution analysis of these limiting dilution cultures suggested that IgE-committed B cells comprise 0.1-1% of all EBV-responsive B lymphocytes.

A system for the induction of specific, hemolytic plaque-forming cells from normal human lymphocytes in vitro (HcPFC) has been established and cells from various normal lymphoid tissues have been investigated. Normal values for anti-SRBC HcPFC responses in cultures of 107 Ficoll-Hypaque separated lymphocytes range from 2000 (bone marrow) to 7000 (spleen) and 15,000 (tonsillar and peripheral blood lymphocytes). HcPFC responses to ovalbumin were lower by factor of 2 to 4. Anti-SRBC as well as anti-ovalbumin responses required the cooperation of T lymphocytes and IgM-bearing B lymphocytes and the magnitude of the response was antigen dose dependent. Addition of adherent cells as well as of 2-mercaptoethanol enhanced the response. On the basis of the data obtained in experiments examining the role of B and T lymphocytes, a tentative model of cellular interaction has been postulated, suggesting a major role for antigen concentration in the modulation of the response via reactive T lymphocytes.

The activation of human IgE-secreting B lymphocytes was difficult until the use of low cell density limiting dilution cultures, which are more efficient and eliminate regulatory growth restraints, became possible. The transformation of IgE-producing cells by Epstein-Barr virus unexpectedly required the presence of small supplements of mature T lymphocytes early in culture. Interleukin 2 (IL2, 10 U/ml) was able to completely replace T-cell supplements when added during a brief (1-2 cell cycles) but critical period, 5-8 days after infection with the virus. The observation that IgE-committed B cells are principally able to express receptors for and respond to IL2 is of experimental utility and implies the possibility that this T-cell product could be part of the complex T-cell controls which govern IgE-committed B cells.