Synergy between anti-CD40 MAb and Epstein-Barr virus in activation and transformation of human B lymphocytes.

For human B lymphocytes, Epstein-Barr virus (EBV) is a polyclonal activator, inducing both proliferation and Ig secretion. It is also a transforming virus capable of generating immortalized B cell lines. These early and late functions of EBV are not apparently connected. The receptor for EBV, CD21, also serves as a receptor for some complement components and is called CR2. This molecule associates with CD19 and TAPA-1 on the surface of B cells. This complex is involved in signaling B cells and participates in many responses. We have observed that simultaneous ligation of CD40 and the CD21 complex, by exposure to anti-CD40 MAbs and EBV, enhances both the short-term proliferation as well as the long-term transformation rate of human B lymphocytes. B cell proliferation shows synergy between anti-CD40 MAb and EBV. CD19 also appears to be involved in the synergistic activation of B cells through CD40 and CD21, since ligation of CD19 with anti-CD19 MAbs, either prior to or concomitant with exposure to anti-CD40 and EBV, markedly inhibits both proliferation and subsequent B cell transformation. These observations do not elucidate the mechanisms of B cell transformation employed by EBV but the do suggest a relationship between early proliferation and later transformation induced by the virus. Anti-CD40 enhances both these effects and anti-CD19 is capable of inhibiting both.

Comparison of anti-TNF alpha autoantibodies in plasma and from EBV transformed lymphocytes of autoimmune and normal individuals.

To examine the ability of normal and autoimmune individuals to produce circulating anti-TNF alpha antibodies, plasma samples from 10 RA patients, 10 SLE patients and 5 normal subjects were assessed for anti-TNF alpha antibody. While every individual tested demonstrated circulating IgM anti-TNF alpha antibody, IgG anti-TNF alpha autoantibody was seen predominantly in autoimmune patients. Only 1 of 5 normal individuals, but 15 of 20 autoimmune individuals had plasma IgG anti-TNF alpha antibodies. To examine the ability of normal and autoimmune individuals to produce anti-TNF alpha autoantibody from their circulating lymphocytes, EBV transformation was performed. Oligoclonal immortal cell lines were successfully established from 13 patients and each one secreted detectable IgM anti-TNF alpha autoantibody. Transformed cells from only 1 of 5 normal individuals secreted IgM anti-TNF alpha autoantibody. These results indicate a higher prevalence of anti-TNF alpha autoantibody production among autoimmune individuals although normal individuals are also capable of producing these autoantibodies.

A novel monoclonal human IgM autoantibody which binds recombinant human and mouse tumor necrosis factor-alpha.

Several monoclonal human IgM antibodies to recombinant human tumor necrosis factor-alpha (rhTNF alpha) have been generated and partially characterized. The F78-1A10-B5 monoclonal antibody (mAb) (B5) binds to rhTNF alpha with a titer comparable to three high-affinity neutralizing mouse mAbs, when tested by ELISA. However, the B5 mAb binds relatively weakly to soluble rhTNF alpha. It appears to bind to epitopes on rhTNF alpha distinct from those bound by the mouse mAbs for three reasons. First, preincubation of plate-bound rhTNF alpha with mouse mAbs does not decrease or compete subsequent B5 mAb binding. Second, rhTNF alpha complexed to the mouse mAbs can still be bound by B5 mAb. Third, the mouse mAbs neutralize TNF alpha cytotoxicity whereas the B5 mAb does not. Binding analyses indicate that this human IgM autoantibody binds to both human and mouse recombinant TNF alpha, but not to other antigens commonly recognized by polyreactive natural IgM autoantibodies. The high level of amino acid identity between the human and mouse TNF alpha molecules suggest that the B5 mAb is monospecific for a given epitope shared by these two forms of TNF alpha. This spectrum of characteristics makes B5 a novel mAb.

The B5 monoclonal human autoantibody binds to cell surface TNF alpha on human lymphoid cells and cell lines and appears to recognize a novel epitope.

A human IgM monoclonal antibody (B5) recognizing human TNF alpha was established from peripheral blood lymphocytes by transformation with Epstein-Barr virus and subsequent cell fusion. The B5 monoclonal antibody (mAb) binds to cell surface TNF alpha (csTNF alpha) on human T cells, B cells, and monocytes. In addition, this autoantibody binds to csTNF alpha on a variety of lymphoid and monocyte lineage cell lines of human origin, as well as astrocytomas, a breast carcinoma, and a melanoma. Interestingly, the B5 mAb also binds to chimpanzee lymphocytes and to mouse T lymphoma cell line csTNF alpha. Many neutralizing mouse anti-TNF alpha mAbs do not exhibit comparable binding to csTNF alpha. This is consistent with the previous demonstration that B5 recognizes an epitope on TNF alpha distinct from those recognized by three neutralizing mouse anti-TNF alpha mAbs. B5 binding to csTNF alpha is specific since it can be inhibited by TNF alpha. No inhibition of B5 binding was seen by a neutralizing mouse anti-TNF alpha mAb. The B5 autoantibody appears to recognize the transmembrane form of TNF alpha and most likely also recognizes TNF alpha associated with its receptor. The unique specificity of this B5 autoantibody provides some additional insight into the complex physiology of cell surface-associated TNF alpha.

Induction of interleukin-5 responsiveness in resting B cells by engagement of the antigen receptor and perception of a second polyclonal activation signal.

Experiments were performed to examine the nature of agents which could induce IL-5 responsiveness in small, resting splenic B lymphocytes. First, IL-5 increased plaque forming cell responses to the TI-1 antigen TNP-LPS. A second set of experiments using anti-IgM + LPS which allowed limiting dilution analysis showed induction of IL-5 responsiveness in about 20% of the resting B cell population. In the same system, IL-4 increased the percentage of proliferating cells by about 40%. A third system using the TI-2 analog conjugate anti-IgD-dextran (anti-delta-dextran) also rendered small, resting B cells responsive to IL-5. An additional system employing anti-IgM plus dextran sulfate, which also allowed limiting dilution analysis, induced IL-5 responsiveness in at least 10% of resting B cells. The features common to all four systems inducing B cell IL-5 responsiveness are at least twofold. Each system directly accesses the B cell antigen receptor and causes crosslinking. Second, each system also provides an additional polyclonal activating moiety, some of which may be similar to those in thymus independent antigens. These results suggest that some resting B cells may become IL-5 receptive after perception of at least two kinds of signals one of which perturbs sIg and the second being nonspecific and polyclonally activating.

Interleukin 5 (IL-5) can act in G0/G1 to induce S phase entry in B lymphocytes from normal and autoimmune strain mice and in transformed leukemic B cells.

In addition to its ability to enhance antibody secretion, Interleukin 5 (IL-5) enhances murine B lymphocyte proliferation. This so-called growth factor activity has been amply demonstrated by many laboratories assessing thymidine incorporation or cell recovery. Attempts to actually quantitate the fraction of fresh splenic B cells responding to IL-5, by limiting dilution analysis or other means, with few exceptions have yielded disappointingly small numbers--generally between 1 and 5%, or perhaps less. We have recently identified the peritoneal cavity as a reservoir rich in IL-5-responsive B cells. In this report, we provide independent corroboration of this high IL-5 reactivity by means of cell cycle analysis. Low-density peritoneal B cells, more than 90% of which are in G0 and G1 phases, were stimulated with polyclonal activators in the presence of mitotic inhibitors. Frequencies of IL-5-responsive B cells were measured by observing the differences in the proportions of cultured cells entering S and later phases in the presence, compared to the absence, of IL-5. Some 10 to 20% more of the low-density peritoneal B cells from normal mice entered S phase when IL-5 was present with LPS + DXS. A similar IL-5-mediated elevation in the frequency of S phase entry was seen with peritoneal B cells from the autoimmune mouse strain NZB. Furthermore, a measurable fraction of peritoneal B cells from these mice were even capable of responding to IL-5 alone. These IL-5-induced increases could be blocked by anti-IL-5 mAb. About 30% of the BCL1 leukemic B cell line initiated DNA replication when stimulated with IL-5 alone. Hence, IL-5-responsive B cell fractions have been measured for some normal, autoimmune strain and transformed leukemic B cell phenotypes. In addition to quantitating the proportion of IL-5-responsive B cells, these experiments formally demonstrate that IL-5 can act in the G1 phase to increase S phase entry.

Interleukin 5 regulation of peritoneal B-cell proliferation and antibody secretion.

The influence of recombinant interleukin 5 (rIL-5) on murine peritoneal B-cell proliferation and antibody secretion was examined. Larger, low buoyant density peritoneal B cells proliferated better with rIL-5 than the smaller resting B cells. this was also true for splenic B cells; however, comparison of the respective populations showed the large peritoneal B-cell responses to be superior. Limiting dilution analyses showed that from 25% to about 40% of large peritoneal B cells proliferated in response to rIL-5 when lipopolysaccharide (LPS) was present. No detectable difference in the fraction of proliferating splenic B cells was seen in the presence of rIL-5. These results are consistent with expression of IL-5 receptors on about 70% of low-density peritoneal B cells as determined by fluorescent staining with anti-Il-5 receptor monoclonal antibody (MoAb). IL-5 also enhanced spontaneous and mitogen-driven IgM secretion by both peritoneal and splenic B lymphocytes; the increases exhibited by peritoneal B cells, however, were at least twice those exhibited by splenic B cells. Spontaneous and mitogen-driven secretion of auto-antibodies to bromelain-treated mouse erythrocytes (BrMRBC) by peritoneal B cells were also increased by this interleukin. Furthermore, rIL-5 enhanced peritoneal B-cell plaque-forming cell (PFC) responses to TNP-LPS but not to TNP-Ficoll. Both an anti-IL-5R MoAb and an anti-IL-5 MoAb blocked the rIL-5-induced enhancement of proliferation and auto-antibody PFC responses. Hence, IL-5 appears to be important for the regulation of proliferation and antibody secretion by many murine peritoneal B cells.

Interleukin 5 regulation of peritoneal Ly-1 B lymphocyte proliferation, differentiation and autoantibody secretion.

The in vitro effects of recombinant interleukin (IL) 5 on proliferation and maturation of mouse Ly-1 B cells were studied. Most freshly isolated peritoneal Ly-1 B cells expressed high levels of IL5 receptor (R). Limiting dilution analyses showed that mitogens could reveal IL5 responsiveness in more than half of low density peritoneal Ly-1 B cells. IL 5 was able not only to increase the proportion of these Ly-1 B cells induced to proliferate, but it also shifted the clone size distribution of proliferating cells towards larger clone sizes. Splenic Ly-1 B cells also proliferated in response to mitogens plus IL5. Spontaneous and polyclonal activator-induced plaque-forming cell responses of Ly-1 B cells were increased by IL5. Furthermore, IL5 increased the frequency of peritoneal Ly-1 B cells induced to secrete certain autoantibodies. IL5 was certainly the agent responsible since its effects on both proliferation and differentiation were inhibited by either anti-IL5R monoclonal antibodies or by anti-IL5 monoclonal antibodies. Hence, Ly-1 B cells, IL5 and the IL5R appear to constitute a system of cellular proliferation, differentiation and some autoantibody production. Strategies specifically targeting the interleukin and receptor elements of this system might afford external control of these cellular responses.

The immunomodulatory effects of interferon-gamma on mature B-lymphocyte responses.

Interferon-gamma (IFN-gamma) exerts a broad spectrum of activities which affect the responses of mature B-cells. It strongly inhibits B-cell activation, acts as a B-cell growth factor (BCGF), and also induces final differentiation to immunoglobulin (Ig) production. IFN-gamma is deeply involved in the differential control of isotype expression, as it enhances IgG2a production and suppresses both IgG1 and IgE production. Although it is now possible to draw a general scheme of the effects of IFN-gamma on B-cells, a number of paradoxical results still exist in the field. In this manuscript, different experimental systems are analyzed in an attempt to explain these apparent paradoxes.

Partial purification and characterization of a factor from a cloned thymic epithelium cell line.

The culture supernatant from a cloned line of thymic epithelium (TEPI) is shown to enhance the response of thymocytes to alloantigen as measured by cell-mediated lympholysis. The supernatant has no effect on the spleen cell response to alloantigen as measured by cell-mediated lysis and does not contain interleukin 1, interleukin 2, interleukin 3, or interferon-gamma activity. The activity is shown to have an apparent m.w. of 160,000 by Sephacryl S-200 gel permeation chromatography, to have an isoelectric point of 6.5, and to elute from DEAE-Sepharose at 0.07 M NaCl.

Evidence for two distinct activation states available to B lymphocytes.

The sites and modes of action of several B cell mitogens and interleukins were examined. Cell cycle analyses of B cell responses to several polyclonal activators including LPS, DXS, LPS plus DXS, and anti-immunoglobulin were performed. Two different states of B cell activation distinguished by RNA content, DNA content, and cell size were observed. LPS promoted transitions throughout the cell cycle, whereas DXS primarily caused exit from G0. Synergy between LPS and DXS was observed in elicitation of exit from G0. Activation by anti-immunoglobulin was found to be influenced by the antibody dose and the cell density of culture. Interleukins could influence anti-IgM-induced responses by increasing G0 exit, and by increasing commitment to DNA synthesis. A model of B cell activation in which cells are stimulated to a stage with intermediate RNA levels (G1A) by polyclonal activators is suggested. Some interleukins appear to be involved in this process. At G1A, cells are receptive to signals delivered by interleukins or some polyclonal activators that drive them to late G1 and DNA synthesis. After cell division, cells re-enter G1A in which interleukins or mitogens are necessary for a continued response.

Partial purification and characterization of a BCGFII from EL4 culture supernatants.

Two B cell growth factor activities have been previously described. One activity, present in the culture supernatants of PMA-induced EL4 is active in a co-stimulator assay with normal B cells and anti-immunoglobulin. The other activity is present in the culture supernatants of the alloreactive T cell line C.C3.11.75 and can be assayed in a co-stimulator assay with normal B cells and dextran sulfate or with BCL1 in vivo line B cell tumor. We have termed the first activity BCGFI and the second BCGFII. We have now shown that a very similar BCGFII activity can be obtained from EL4 culture supernatants induced by PMA. This (EL4)BCGFII has an apparent m.w. of 55,000, is eluted from DEAE Sephacel at 0.05 M NaCl, and has a pI of 5.5, which is clearly distinct from the properties of (EL4)BCGFI activity. (EL4)BCGFII activity is similar to but not identical to (DL)BCGFII. It differs from (DL)BCGFII in chromatographic behavior and in the kinetics of the response of BCL1 to the two factors. (EL4)BCGFII activity can be detected in 18 to 24 hr by virtue of its ability to cause in vitro proliferation of in vivo BCL1 tumor B cells.

Induction of T-cell maturation by a cloned line of thymic epithelium (TEPI).

A cloned cell line of thymic origin has been characterized as epithelial in nature. A description of the procedures for derivation and cloning of the cell line includes use of epidermal growth factor. The thymic epithelial (TEPI) cell line is Ia antigen positive, forms desmosomes, and produces an extracellular fibronectin matrix. The supernatant from confluent monolayers of TEPI was tested for its ability to promote thymocyte functional activity. TEPI supernatant (TEPI SN) was demonstrated to greatly enhance the response of peanut agglutinin-positive thymocytes to alloantigen, as measured by cell-mediated lympholysis. Furthermore, preincubation of peanut agglutinin-positive thymocytes with TEPI SN prior to allostimulation resulted in marked enhancement, thus distinguishing it from interleukin 2. Finally, TEPI SN was demonstrated to induce interleukin 2 production by peanut agglutinin-positive thymocytes in the presence of concanavalin A. This activity was demonstrated not to be due to interleukin 1, which is absent in TEPI SN. Preliminary biochemical analysis indicates that the biological activity is associated with a Mr 50,000 entity. The data suggest that TEPI produces a soluble factor capable of inducing function of an immature thymocyte subpopulation into an IL 2 producer.

Evidence for two distinct classes of murine B cell growth factors with activities in different functional assays.

Several previously described B cell growth factor (BCGF) activities from a number of mouse monoclonal T cell sources were compared in different functional assays. The results indicate that there are two distinct classes of BCGF defined by functional activity and source. BCGF I, whose prototype is (EL4)BCGF, synergized with anti-Ig in the proliferation of normal splenic B cells but had no activity when dextran sulfate (DXS), rather than anti-Ig, was used to costimulate the same source of B cells. BCGF I also failed to directly stimulate BCL1 tumor B cells. In contrast, BCGF II, whose prototype is (DL)BCGF, showed a reciprocal pattern of activity. BCGF II failed to synergize with anti-Ig-costimulated normal B cells to give good proliferative responses. Sources of BCGF II also directly stimulated (no anti-Ig or DXS added) B cells of the BCL1 tumor-carrying mice. These results suggest that the two BCGF may have activity on two subsets of B cells that respond differentially to induction with the two polyclonal B cell activators, anti-Ig and DXS. The possibilities that these different patterns of response occur in separate lineages of B cells and/or in B cells in different states of differentiation is discussed.

Growth of single B cell clones is enhanced by products of a T cell line.

In this paper we show the presence of a B cell growth-promoting activity in T cell replacing factor (TRF) supernatants from a monoclonal T cell line and polyclonally activated splenic T cells. The target cell of this activity is indisputably shown to be the B cell, which indicates that T cell-derived factors can act directly on B cells. The effect of monoclonal TRF-containing supernatant from the C.C3.11.75 Dennert cell line, (DL)TRF, which demonstrates B cell growth-promoting activity, is to increase the frequency of B cell clones stimulated by mitogens as opposed to increasing B cell clone sizes. (DL)TRF B cell growth enhancement is observed when B cells are activated by fetal calf serum mitogens, lipopolysaccharide (LPS), dextran sulfate (DXS), or LPS + DXS. The growth-promoting activity of (DL)TRF appears to be that of a costimulator rather than a classical growth factor because (DL)TRF alone is not sufficient to maintain clonal growth of activated B lymphoblasts.

A monoclonal T cell-replacing activity can act directly on B cells to enhance clonal expansion.

We have used a B cell cloning system in which the response of a single isolated B cell to lipopolysaccharide and dextran sulfide can be followed. We have shown that culture supernatants from the Dennert long-term alloreactive T cell line C.C3.11.75 increase the frequency of B cells stimulated to clonal expansion by mitogens. These culture supernatants are devoid of interleukin 1 and 2 but contain the T cell-replacing factor activity (DL)TRF. These experiments provide unequivocal proof that a T cell-derived factor or factors can act directly on a B lymphocyte in the absence of any other cell.