Interactions between B lymphocyte subpopulations. Augmentation of the responses of resting B lymphocytes by activated B lymphocytes

Mouse B lymphocytes were fractionated from normal T lymphocyte-depleted spleen cell populations using discontinuous percoll gradients and were stimulated with rabbit F(ab')2 anti-mouse mu-specific antibodies (anti-mu) plus the supernatant of Con A-stimulated rat spleen cells (SN) as a source of lymphokines. The responses of small (mean volume 120 mu 3), dense (greater than 1.087 specific gravity), resting (least spontaneous thymidine incorporation) B lymphocytes were augmented by irradiated (4000 rad), larger (mean volume greater than 170 mu 3), less dense (less than 1.081 specific gravity), activated (greater spontaneous thymidine incorporation) B lymphocytes. Proliferation was augmented 2- to 4-fold and polyclonal antibody-forming cell responses three- to sixfold. Maximal augmentation of the responses of 5 X 10(4) resting B cells was obtained with 10(4) activated B cells. Augmenting activity was specific for activated B lymphocytes in that responses were not augmented by irradiated thymocytes, T lymphoblasts, macrophages, or additional supernatant. B lymphocytes activated in vitro by LPS or anti-mu also had augmenting activity. Augmentation of responses was maximal only when activated B lymphocytes were added simultaneously with anti-mu. The interaction between activated and resting B lymphocytes did not appear to be genetically restricted. Interestingly, the augmenting activity of activated B cells could be reconstituted by a combination of supernatant and cell membranes from these cells but not by either alone, suggesting that two components are required, one soluble and the other membrane-bound. Thus, a functional interaction has been demonstrated between B lymphocyte subpopulations which differ in their state of activation, and this interaction appears to involve a novel mechanism of action.     

Internalization and acidification of insulin by activated human lymphocytes

The binding and internalization of fluorescein isothiocyanate-conjugated insulin by nonactivated and phytohemagglutinin-activated circulating human lymphocytes was measured by flow cytometry. In confirmation of previous results, negligible binding or internalization was observed for unstimulated cells, while activated lymphocytes showed significant insulin binding. The majority of this insulin was demonstrated to be internalized via receptor-mediated endocytosis and acidified within 60 min after addition of insulin. Dual-fluorescence flow cytometry, using antibodies specific for human T cell subsets, was used to show that the expression of insulin binding sites occurs for at least some cells from both the helper/inducer and cytotoxic/suppressor T cell subsets. Insulin internalization is not an artifact of in vitro stimulation, since more than 90% of the unstimulated lymphocytes from a patient with a helper T cell leukemia are positive for insulin internalization. The usefulness of flow cytometric analysis for measuring lymphocyte activation in unstimulated populations and the therapeutic potential of the reported findings for control of lymphocyte proliferation are discussed.     

A novel polypeptide secreted by activated human T lymphocytes

We have identified two cDNA clones, I-309 and G-26, which define genes expressed abundantly in activated human PBMC, but at low or undetectable levels in resting PBMC. Based upon nucleotide sequence analysis, both clones are predicted to encode small, structurally related polypeptides, each containing a hydrophobic leader sequence characteristic of secreted proteins and a motif of four conserved cysteine residues. Further, I-309 and G-26 are structurally related to a growing family of genes that apparently encode small polypeptides whose secretion is induced upon cell activation. I-309 represents a previously undescribed human gene. We have generated an anti-peptide antiserum to the I-309 gene product which recognizes proteins in culture supernatants of an activated T cell clone and of COS cells transfected with the I-309 cDNA, supporting the idea that I-309 encodes a secreted protein. Because I-309 encodes a small protein secreted by activated T cells that displays structural features similar to other cytokines, we believe that it defines a novel cytokine with as yet unknown function.     

Bacteria induce lymphokine synthesis polyclonally in human B lymphocytes.

We have studied the ability of various bacteria to stimulate human lymphocytes to produce leukocyte migration inhibitory factor (LIF). Mononuclear cells from adult and cord blood as well as purified T and B lymphocytes were stimulated with killed bacteria. The culture supernatants were tested for the presence of LIF by the agarose migration method. All nine bacterial strains tested activated unseparated mononuclear cells and B lymphocytes but not T cells to produce LIF. LIF was also present in cord blood cell cultures suggesting that the stimulation of lymphocytes was polyclonal rather than antigenic. Therefore, we propose that one of the physiologic functions of B lymphocyte lymphokines might be to form part of the nonspecific defense mechanisms against microbial invasion.     

Neutrophil-assisted DNA synthesis by human lymphocytes in response to mevalonic acid; enhancement by cytochalasin B

Mevalonic acid is capable of initiating DNA synthesis, morphologic transformation, and cell division in cultures of human peripheral blood lymphocytes. Pure populations of lymphocytes respond poorly to mevalonic acid, but their response can be enhanced by peripheral blood neutrophils. Addition of cytochalasin B (0.5-1.0 micrograms/ml), but not cytochalasin A, to mixed neutrophil-lymphocyte cultures enhances the response of lymphocytes to both Con A and mevalonate, but the increment in mevalonate-induced DNA synthesis (+343%) far exceeds the enhancement which cytochalasin B produces in the Con A response (+24%). As a consequence, the DNA synthetic response in mevalonate (10(-2) M) containing cultures averages 39% of the response to an optimal dose of Con A. The cytochalasin B-enhanced response of mixed neutrophil-lymphocyte cultures to mevalonate is abolished by prior X irradiation of the lymphocytes, or their pretreatment with mitomycin C, as well as by the addition of hydroxyurea to the cultures but is not altered by prior X irradiation or mitomycin C pretreatment of the neutrophil helper population. These experiments suggest that the enhancing effect of cytochalasin B in the response of mixed neutrophil-lymphocyte cultures to mevalonic acid results from its ability to potentiate a time-dependent conditioning effect on lymphocytes which neutrophils exert. The conditioning effect of neutrophils cannot be achieved by cell-free neutrophil lysosomal enzymes released by exocytosis, and reactive oxygen species potentially generated by neutrophils are not involved. Attempts to demonstrate the production by neutrophils of a soluble mediator induced by mevalonate in the presence of cytochalasin B were without success. In the presence of cytochalasin B, only the metabolically active R(-) enantiomeric form of mevalonate is capable of initiating DNA synthesis in mixed neutrophil-lymphocyte cultures. The cytochalasin B-potentiated response of mixed neutrophil-lymphocyte cultures to mevalonic acid is preferentially displayed in cultures containing E-rosette-negative, as opposed to E-rosette-positive, lymphocytes. These observations add to the growing knowledge about the relationship between mevalonate metabolism, DNA synthesis, and cell replication.     

Phospholipid transmethylation in the membrane of human neutrophils and lymphocytes

Phospholipid transmethylation in the microsomal fraction of stimulated and unstimulated human leukocytes was measured in a recently developed assay system. Microsomal fraction was prepared from neutrophils, unseparated lymphocytes, T lymphocytes, and non-T lymphocytes by sonication and subsequent ultracentrifugation. Two hundred micrograms of microsomal protein was reacted with S-adenosyl-L-[methyl-3H]methionine. In unstimulated cells, incorporation of methyl-3H into phospholipid was 0.60 +/- 0.06 pmol min-1 mg protein in neutrophil membrane, 0.84 +/- 0.075 in unseparated lymphocytes, 1.23 +/- 0.17 in T lymphocytes, and 0.71 +/- 0.085 in non-T lymphocytes (mean +/- SE). Stimulation of neutrophils with opsonized zymosan or concanavalin A (Con A), and of lymphocytes with Con A, phytohemagglutinin, or pokeweed mitogen increased 15 to 30%. The resulting methylated phospholipids were identified and quantitated by two-dimensional thin-layer chromatography. The inhibitor 5'-S-isobutyl-5'-deoxyadenosine (SIBA) inhibited transmethylation 47-55%. This assay system appears to measure specifically the activity of methyltransferases which mediate the transmethylation of membrane phospholipid; the assay should find important applications in the study of membrane lipid metabolism in human health and disease.     

Unscheduled synthesis induced by thiophosphamide in human lymphocytes

An unscheduled DNA synthesis in human nonreplicative lymphocytes is shown to follow cell exposure to thiophosphamide at a dose level of 1-10 microgram/ml. Incorporation of 3H-thymidine in the absence of the mutagen is probably due to spontaneous reparative processes. Significant variations are found of spontaneous and thio-phosphamide-induced levels of a reparative DNA synthesis in normal individuals.     

Interferon acts directly on human B lymphocytes to modulate immunoglobulin synthesis

At different times of exposure, interferon (IFN) enhanced and suppressed pokeweed mitogen- (PWM) induced IgG synthesis by human peripheral blood lymphocytes (PBL). Pretreatment of PBL and IFN frequently increased antibody production by more than 100% when compared with that by untreated PBL. Results of experiments in which PBL were separated into T and B subpopulations indicated that IFN preparations acted directly on B cells. Thus, mixtures of IFN-treated B cells and untreated T cells from 5 of 7 persons tested produced 81% to 500% more IgG than untreated, matched control cells. However, IFN-treated monocytes mixed with untreated B and T cells or IFN-treated T cells mixed with untreated B cells failed to enhance IgG production significantly in similar assays. In contrast to the pretreatment protocol, when IFN was present in the incubation mixture throughout the PWM assay, IgG production decreased. Sephadex chromatography of the IFN and tests of the resulting fractions indicated that the IgG production-enhancing activity was located in the fraction carrying the antiviral activity.     

Induction of glycoprotein biosynthesis in activated B lymphocytes

Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells.     

Bispecific-monoclonal-antibody-directed lysis of ovarian carcinoma cells by activated human T lymphocytes

Summary Two different bispecific hybrid antibodies were established by fusing a hybridoma producing monoclonal antibody (mAb) against the pancarcinoma antigen KS1/4 with either of the two hybridomas OKT3 and 9.3, secreting antibodies reactive with the T cell determinants CD3 and CD28, respectively. The KS1/4 antibody reacts with a 40-kDa cell-surface glycoprotein antigen that is expressed on the surface of a variety of adenocarcinoma cells, including ovarian carcinoma. The ability of the bispecific antibodies 9.3KS1/4 and OKT3KS1/4 to direct peripheral blood mononuclear cells (PBMC) specifically against OVCAR-3 ovarian carcinoma target cells was measured in a 4-h⁵¹Cr-release assay. The bispecific antibodies were four to six times more potent in killing the OVCAR-3 target cells when compared to their parental antibodies either alone or in combination. A dose-dependent response was observed in the 10–10000 ng/ml range. The specificity of the targeting was demonstrated by the complete inhibition of cytotoxic activity following pre-incubation of tumor target cells with the parental mAb and by the lack of killing of KS1/4-negative target cell lines. An evaluation of the efficacy of PBMC from ovarian cancer patients as effector cells revealed that their specific cytotoxicity against OVCAR-3 cells was enhanced severalfold by bispecific antibodies as compared to parental antibodies. Furthermore, stimulation of PBMC with immobilized CD3 and interleukin-2 for 4 days resulted in an enhanced directed killing of human ovarian carcinoma cells by human T effector cells and the bispecific antibodies.     

Activation of human B lymphocytes. IX. Modulation of antibody production by products of activated macrophages.

Human monocytes, after in vitro activation by mixed lymphocyte culture (MLC) supernatants produce a monokine (MK) that enhances the plaque-forming cell (PFC) response of pokeweed mitogen (PWM)-stimulated human B lymphocytes. Technical conditions and kinetics of MK production were established. Irradiation of monocytes (5000 rads) does not abolish MK production but heat-killed cells are unable to release the factor. Highly T cell-depleted monocyte populations still produced the PFC-enhancing factor. The same MK has an inconsistent enhancing effect on the PFC responses of lipopolysaccharide (LPS) and nocardia water-soluble mitogen (NWSM)-stimulated B cells. Other macrophage activators such as LPS, phytohemagglutinin (PHA), and latex particles failed to induce consistently the liberation of the PFC-enhancing MK. The target cell for the MK activity on PWM-stimulated B cells appears to be the B lymphocyte itself. These studies demonstrate that soluble monocyte products can have substantial modulatory effects on human B cell function.     

Alpha1-antitrypsin synthesis by human lymphocytes

$\text{De}$$\text{novo}$ synthesis of alpha₁-antitrypsin (α₁AT) by human peripheral lymphocytes has been demonstrated in the present study. Treatment of the mononuclear cells with concanavalin A(Con A) resulted in a triple increase in the amount of α₁AT synthesized by the untreated cells. A small amount of α₁AT, equivalent to that synthesized by the unstimulated mononuclear cells, was observed in cultures of monocyte-depleted lymphocytes, with or without Con A stimulation. Monocytes treated with or without Con A scarcely synthesized α₁AT. Conditioned media derived from monocyte enriched mononuclear cells treated with Con A enhanced about threefold α₁AT synthesis by the Con A-stimulated lymphocytes. α₁AT is suggested to be synthesized by lymphocytes assisted by monocytes.     

Fatty acid requirement of B lymphocytes activated in vitro

The lipid and fatty acid requirement of B lymphocytes activated in vitro was examined by replacing soybean lipids with various combinations of lecithins, fatty acids and cholesterol. It is reported here that linoleic acid is the sole fatty acid required to support the proliferation of B lymphocytes and maturation to immunoglobulin-secreting cells.     

Interleukin 2 produced by activated B lymphocytes acts as an autocrine proliferation-inducing lymphokine

Normal peripheral blood B cells produce a soluble factor after activation that is functionally indistinguishable from interleukin 2 (IL 2) and can support B cell proliferation in vitro. Purified rabbit peripheral blood B cells, when stimulated with a combination of ionomycin (0.5 microgram/mL) and phorbol myristate acetate (PMA) (1 ng/mL), secreted a soluble factor in the culture medium that supported the IL 2-dependent cell line CTLL-2. The ability of these supernatants to support CTLL-2 growth was almost completely blocked by rabbit antibodies against human recombinant IL 2 and by the anti-IL 2 receptor monoclonal antibody 7D4. These data strongly suggest that the growth factor secreted by rabbit B cells is IL 2. To examine the possibility that the IL 2 activity detected in the B-cell cultures may be derived from residual T cells, B cells were further purified by successive panning with a pan-T-cell monoclonal antibody, L11-135, and goat anti-rabbit IgG. These highly purified B cells produced levels of IL 2 activity comparable to those produced by the initial B cell populations. Comparison of IL 2 production by decreasing numbers of purified T cells and purified B cells also indicated that the B cells were the source of IL 2 activity. Supernatants of activated B cells could support proliferation of B-cell blasts, and this activity could be completely absorbed by CTLL-2 cells, indicating that IL 2 is a major growth factor for B cells.(ABSTRACT TRUNCATED AT 250 WORDS)