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Expression of the interleukin (IL)-10/BCRF1 gene was studied by in situ hybridization in tissue samples from acquired immunodeficiency syndrome (AIDS) lymphomas using a BCRF1 probe which also recognizes the human IL-10 sequence. Hybridization was detected in 8 out of 15 lymphomas. In contrast, the IL-10/BCRF1 gene expression was detected in only 1 out of 11 lymphomas from human immunodeficiency virus (HIV)-seronegative patients (p = 0.05). In AIDS lymphomas, the number of cells labeled with a BCRF1-specific probe was dramatically lower than that of cells labeled with the IL-10/BCRF1 probe. Thus, the IL-10 rather than the BCRF1 gene was expressed. Production of IL-10 was associated with that of IL-10 mRNA, as shown by immunodetection of the protein in numerous cells. In contrast, BCRF1-producing cells were rarely detected. Both in situ hybridization and immunochemical experiments indicated that malignant cells were involved in this IL-10 synthesis. IL-10 production in AIDS lymphomas was associated with the presence of Epstein-Barr virus (EBV) in lymphomatous cells (p = 0.02). As IL-10 is a potent growth factor for human B lymphocytes, these results suggest that IL-10 may stimulate the proliferation of malignant cells in an autocrine pathway in a number of AIDS lymphomas, and that EBV and HIV may synergistically trigger its production.

We analyzed the effects of interleukin 2 (IL 2) and IL 4 isolated and in association on the specific response of human B cells triggered by trinitrophenylated polyacrylamide beads (TNP-PAA). IL 2 induced an increase (more than 10 times) in the number of hapten-binding cells [detected by a rosette-forming cell (RFC) assay] as well as the generation of antibody-producing cells [detected by a plaque-forming cell (PFC) assay]. IL 4 induced an isolated RFC response without PFC response. We verified that the IL 4 (as well as 12)-induced RFC were hapten specific and mediated through membrane IgM. Density fractionation experiments showed that IL 4-induced RFC were equally distributed between high-density and intermediate-density B cells. IL 2 appeared to drive more B cells into the intermediate density fraction. IL 2-induced PFC belonged to the RFC population and were intermediate-size B cells. IL 2 drove more RFC into an activated stage and it induced the differentiation of a number of them into antibody-producing cells. The evaluation of the proportion of RFC able to incorporate thymidine showed that both IL induced a substantial proliferation of antigen-activated B cells. However, IL 4 inhibited the IL 2-dependent PFC without affecting the number of RFC nor the proportion of proliferating RFC induced by this IL. These results directly demonstrate that human IL 4 triggers the expansion of antigen-activated B cells and selectively inhibits the IL 2-induced differentiation.

The C3d receptor is a specific marker of B lymphocytes. Recently we have shown that C3d receptor activity is carried by a gp 140 membrane antigen. A polyclonal antibody has been prepared by immunizing a rabbit with highly purified gp 140 molecule isolated from membranes of the human B lymphoblastoid cell line Raji and its high specificity was previously demonstrated. We tested the effect of this antibody to the C3d receptor on the B cell proliferative response. Purified B cells from human blood were induced to proliferate by a B cell growth factor (BCGF)-containing partially purified supernatant from activated T cells. The anti-C3d receptor F(ab')2 enhanced the BCGF-dependent B cell proliferation. This effect was dose dependent, was observed in the presence of different concentrations of BCGF and did not correspond to a change in the time course of the response. The anti-C3d receptor F(ab')2 had no mitogenic effect in the absence of T cell supernatant. In contrast the undigested anti-C3d receptor IgG suppressed the BCGF-dependent B cell proliferation. These results emphasize the potentialities of anti-gp 140 F(ab')2 to explore the involvement of the C3d receptor in the regulation of B cell response to T cell products.

Nonspecific T cell factors produced by lectin-activated human peripheral blood lymphocytes (PBL) were used to restore the T-dependent B cell response to trinitrophenyl-polyacrylamide (TNP-PAA). Preincubation experiments with the particulate antigen TNP-PAA and/or a soluble TNP-protein conjugate show that a first specific signal provided by the antigen and nonspecific lymphokines sequentially acts on B cells. By gel filtration the T cell-replacing factor (TRF) activity is present in the 30-15-kDa fraction of T cell supernatants and is associated to interleukin 2 (IL2). However, absorption of IL2 does not abolish the TRF activity. Moreover, chromatofocusing of this 30-15-kDa material allows the obtaining of an IL2-free fraction containing a differentiation factor (with an isoelectric point of 5.7 +/- 0.2). The ability of this fraction to restore the anti-TNP response is manifest in the presence of a 50-kDa B cell growth factor. This latter, prepared by a combination of absorption on concanavalin A-Sepharose and gel filtration, was IL2 free and unable to support the anti-TNP response. We thus directly demonstrate that in the absence of IL2 three separate signals (the antigen, T cell-derived growth and differentiation factors) are involved in human-specific B cell response.

Upon in vitro activation by Staphylococcus aureus Cowan I strain (SAC) human peripheral blood B cells produce only marginal amounts of Ig when cultured in the presence of interleukin 2 (IL2; 10 U/ml). This response is only moderately increased by the addition of monocytes or of IL1. In the presence of dexamethasone (DM; 10(-7)-10(-8) M) microgram amounts of both IgM and IgG are produced in co-cultures of B cells and monocytes. This response is not modified by inhibitors of cyclooxygenase and is specifically inhibited by a monoclonal antibody interfering with the binding of IL2 to its receptor. This enhancing effect of DM is not observed in the absence of monocytes even if IL1 is added to the cultures. Moreover, monocytes pretreated with DM stimulate the response of B cells cultures in the absence of DM. Enhancement of Ig production by DM and monocytes could be demonstrated with B cells obtained from a patient suffering from a hyperlymphocytic form of B cell type chronic lymphocytic leukemia, and in this case only IgM was produced. Importantly, DM fully inhibited the IL2-dependent proliferation of these monoclonal B cells. Thus, physiological concentrations of DM can modulate monocytic function to enhance the differentiative effect of IL2.

We tested the effect of interleukin (IL) 4 on the specific IgM antibody response induced by trinitrophenylated-polyacrylamide beads (TNP-PAA) in cultures of human B cells. T cell help was provided by exogeneous IL2. IL4 profoundly suppressed the response to optimal concentrations (50 U/ml) of IL2, with a 50% inhibitory concentration of 6 U/ml. This was due neither to a shift in the kinetics nor to a switch to an IgG response. The production of anti-TNP antibody (as measured by an enzyme-linked immunosorbent assay in the culture supernatant) was inhibited to the same extent as the generation of plaque-forming cells. The effect of IL4 was completely abolished by a neutralizing antibody toward IL4. Kinetic studies showed that IL4 had to be present during the first 48 h of culture to fully inhibit the response. The sequential stimulation of B cells by antigen and by IL2 showed that IL4 does not negatively interfere with signaling through membrane Ig but counteracts the effect of IL2 on antigen-activated B cells.

In order to analyze the effects of interleukin (IL) 6 on human in vitro Ig production B lymphocytes were activated by Staphylococcus aureus Cowan strain I (SAC) in the presence of low concentrations of IL2 (1 U/ml) and dexamethasone (10(-7) M). Previously we showed that this model of B cell response is completely monocyte dependent. We here demonstrate that, under these experimental conditions, IL6 is able to replace monocytes and stimulate Ig production provided IL1 is also present. Dose-effect curves show that these two monokines act synergistically. This synergy is demonstrable only in the presence of dexamethasone, when B lymphocytes are activated (by SAC) and when T cell help (provided by IL2) is present. It results in the production of both IgM and IgG. Both IL1 and IL6 have to be present during the first 48 h of culture to exert an optimal effect. These results show that IL6 may act on early (as well as on late) stages of normal B lymphocyte differentiation. Moreover, glucocorticosteroids potentiate the synergistic effect of IL1 and IL6 on their B lymphocyte target, an effect comparable to that exerted on hepatocytes.