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In this study, we investigated the expression of CD5 molecules on the surface of mature human peripheral B lymphocytes. Human CD5+ B cells were isolated from tonsils and peripheral blood. Their terminal differentiation into plasma cells was induced with IL-2. In the presence of this lymphokine, a subset of CD5+ B cells ceased to express CD5 proteins on their surface. When CD5+ B cells and non-B cells were removed from the suspension. CD5 antigens were spontaneously reexpressed on the surface of CD5- B cells. This suggests that the CD5- phenotype of a subset of B cells in these suspensions resulted from pressure(s) exerted on them by the other cellular components of the suspensions. B cells which have reexpressed membrane CD5 in the absence of CD5+ B cells and non-B cells retained their ability to reprogress to CD5- phenotype and to undergo terminal differentiation into plasma cells when stimulated with IL-2. A short exposure (4 hr) of CD5- B cells to 5 nM IL-2 resulted in the loss of their capability to spontaneously reexpress surface CD5 in the absence of other lymphoid cells. Taken together, these data suggest that the expression of CD5 antigens on B cell membrane is governed by a network-type balance between all cellular compartments within the suspension. Fluctuations of this equilibrium results in the shuttling of B cells between CD5+ and CD5+ phenotypes until they become irreversibly engaged in the terminal differentiation pathway. The interconversion CD5+<==>CD5- on B cell membrane does not argue for CD5 molecule as the marker of a distinct B cell lineage.

We have reported that one of the currently known receptors for interleukin-2 (IL-2), the p70 protein, is constitutively expressed on resting T lymphocyte membrane. We demonstrated that exposure of these cells to high concentrations of IL-2 resulted in the transcription of genes whose expression occurred early during cell activation such as cmyc, cmyb protooncogenes, and the Tac gene itself. IL-2 is thought to exert its biological effects by binding to its high-affinity receptors on cell membrane. Recent studies using B cell lines emphasized that within the high-affinity receptor complexes, p55 serves to allow high-affinity binding, and p70, to transduce signals. In this study, we prepared human unstimulated B cells devoid of detectable Tac antigen, and consequently, of the high-affinity receptor complexes. We designed experiments such that no in vitro de novo expressed receptors, if any, could interact with IL-2, so that any biologic events triggered by IL-2 must have been mediated in the absence of the high-affinity receptors. Under these conditions, we demonstrated that a short and unique exposure (1 hr) of these cells to high concentrations (5 nM) of IL-2 allowed its binding to cell membrane and resulted in a competent signal leading to the generation of a majority of 25S Tac mRNA, and a progression signal allowing B cell terminal differentiation into plasma cells.

We have developed a system to study transcriptional regulation of the lambda immunoglobulin gene in a natural setting -- lambda light chain producing lymphoid cells. This assay system has allowed the detection of an enhancer element located 3' of the lambda gene coding sequence. The enhancer can stimulate transcription from the lambda promoter as well as from other immunoglobulin and unrelated promoters. Like all enhancers, the lambda enhancer can function in either orientation with respect to a promoter, but it is significantly more active in one orientation than in the other. The lambda enhancer is unusual in spanning at least 4000 bp of DNA sequence and containing several distinct subelements that have independent enhancer activity. The enhancer is also remarkable because it functions in lambda light chain producing cells but not in kappa chain producing cells. This fact can be interpreted to support a model of immunoglobulin gene rearrangement in which rearrangement follows and depends on transcriptional activation.

The aim of this paper was to review the action of the receptors for the Fc portion of Ig on the polyclonal activation of human B lymphocytes. The IgG Fc fragments were shown to be digested by monocytes into peptides which triggered B-cell differentiation and the release of a T-cell replacing factor termed (Fc)TRF. In the presence of PWM and monocytes, unbound aggregated IgG suppressed B-cell differentiation into Ig-synthesizing cells. This suppression was not isotype-specific. After having bound aggregated IgG, T cells were able to display an isotype-restricted suppression of B-cell differentiation induced by PWM. Finally, soluble Fc gamma R released from T or B lymphocytes or neutrophils were found to inhibit both the synthesis of IgG and the maturation of B cells into IgM or Ig A-secreting cells.

Receptors for the Fc part of IgG were isolated by affinity chromatography from supernatants of unstimulated human lymphocytes or polymorphonuclear neutrophils. When added to cultures stimulated by polyclonal activators, these soluble receptors selectively depressed the maturation of B lymphocytes into IgG-producing cells, whereas the number of IgM-producing cells was either increased or unchanged. The number of IgA-containing cells was not changed. The data support the concept of class-specific control of Ig synthesis by Fc receptors.

During short incubation in serum-free medium, polymorphonuclear neutrophils (PMN) release soluble material that can be characterized as receptors for Fc IgG (Fc gamma R) on the following evidence: it agglutinates erythrocyte-IgG antibody (EAG) complexes, it prevents the binding of EAG to EAG-rosette-forming cells, and it binds to EAG-rosette-forming cells after modulation of their Fc gamma R, allowing the formation of 'passive' rosettes. These Fc gamma R were isolated by affinity chromatography on sepharose 4B-IgG. This material was shown to interfere with the differentiation of peripheral blood B cells into Ig-secreting cells in cultures stimulated by pokeweed (PWM) or Nocardia opaca (NOC) extracts. The number of Ig-secreting cells determined by a reverse hemolytic plaque assay using protein A-coated sheep erythrocytes was decreased by addition of Fc gamma R over a wide range of dilutions. The number of Ig-containing cells was diminished in PWM-stimulated cultures, but not in cultures stimulated with NOC. Fc gamma R did not affect cell viability, nor did they interfere with plaque-forming cell assay. Fc gamma R was not suppressive when added before the 3rd day or after the 6th day of culture. The suppressor factor was shown to remain associated with Fc gamma R after elution at acidic pH; it was removed by absorption on Sepharose 4B-IgG, but not on pepsin-digested F(ab')2 fragments. The suppressive factor as well as the capacity to restore EAG rosette formation by modulated lymphocytes were destroyed by heating (56 degrees C, 30 min) or by freezing and thawing. Properties of Fc gamma R released from PMN in this system are similar to those of Fc gamma R released from unstimulated human peripheral blood lymphocytes (PBL) and to those of mouse Ig-binding factor produced by alloactivated T cells or T cell lines.

Enhancers are DNA sequences that stimulate transcription from eukaryotic promoters. This stimulatory effect can be exerted over large distances and from a position either 5' or 3' of a promoter. Enhancers have been found in the genomes of many viruses, and in some cellular genes such as those encoding immunoglobulin heavy chain and kappa light chain. An important feature of both viral and cellular enhancers is the ability of each enhancer to stimulate transcription from many promoters other than the one with which it is found associated. However, the question of whether cellular enhancers stimulate their 'own' promoter more efficiently than other promoters has apparently not been investigated. We show here that the kappa light-chain enhancer stimulates a kappa promoter about 20-fold more than it stimulates either the simian virus 40 (SV40) early promoter or a metallothionein (MT) promoter, two promoters that are very sensitive to other enhancers. Similarly, the heavy-chain enhancer stimulates a heavy-chain promoter much more than it stimulates the SV40 and MT promoters. This synergism between immunoglobulin enhancers and promoters might be due to the action of a protein that binds specifically to each of the regulatory elements.

Human IgG binding factors (IgG BF) were prepared by immunopurification on IgG immunosorbents from cell-free supernatants of unstimulated peripheral blood mononuclear cells (PB MNC). The suppressive effects of IgG BF was studied using PB MNC stimulated by pokeweed mitogen or by nocardia delipidated cell mitogen. At the end of the culture three parameters of B cell activation were measured: (1) the numbers of IgM-, IgG-, or IgA-containing cells (CC) using direct immunofluorescence, (2) the numbers of IgM, IgG, or IgA plaque-forming cells (PFC) using a Protein A hemolytic plaque assay, and (3) the concentrations of IgM, IgG, or IgA in culture supernatants using an enzyme-linked immunosorbent assay. Addition of IgG BF at the third day of culture resulted in a selective decrease of IgG CC, while IgM CC and IgA CC were increased or unchanged. Conversely, IgG BF induced a nonselective diminution of the number of PFC and of the amount of secreted Ig of the three major Ig classes. Therefore the results demonstrate two distinct effects of IgG BF: (1) an isotype-specific suppression of cells producing IgG, demonstrated by the parallel decrease of IgG CC and IgG PFC, and (2) a blocking of the late stages of B cell maturation evidenced by the discrepancy between normal or elevated Ig CC and decreased Ig PFC of the IgM and IgA classes.

The relative proportions of cells synthesizing the three major Ig classes or one of the four IgG subclasses in cultures stimulated with pokeweed mitogen (PWM) or Nocardia-delipidated cell mitogen (NDCM) were investigated. In cultures of human peripheral blood mononuclear cells (PB MNC) stimulated with PWM, the number of IgG-containing cells (CC) was higher than the number of IgM-CC, and a substantial number of IgA-CC was found. Conversely, in NDCM-stimulated PB MNC cultures IgM-CC outnumbered IgG-CC and only few IgA-CC were detected. In those cultures, the removal of T cells resulted in an increase in the number of IgM-CC concomitant with a decrease in the number of IgG-CC. A substantial number of cells containing simultaneously IgG or IgA in addition to IgM could be found in PWM-stimulated cultures. These cells were virtually absent in NDCM-stimulated cultures. The relative proportions of IgG subclass-CC were IgG1-CC greater than IgG2-CC greater than IgG3-CC greater than or equal to IgG4-CC in PWM-stimulated and IgG2-CC greater than IgG1-CC greater than IgG3-CC greater than or equal to IgG4-CC in NDCM-stimulated cultures. The removal of T cells from NDCM-stimulated cultures did not result in major alteration of this distribution. The role of T cells and of the genomic order of the Igh-C genes in their phenotypic expression triggered in vitro by PBA is discussed.

In vitro human plasma cell generation induced by both T-dependent (PWM) and T-independent (NWSM) mitogens was found to be suppressed by peripheral blood lymphocytes preincubated with human aggregated IgG. T cells, but not B lymphocytes, were able to mediate the suppressive activity; since aggregated (Fab)'2 fragments were found unable to generate suppressor cells, it was concluded that the suppressor cell was a T lymphocyte bearing Fcgamma receptors. These cells appeared to be largely radiosensitive. In most cases the proliferative responses remained unchanged. Since NWSM-induced activation is not dependent on the presence of T cells, these results show that, at least in this case, T cells exert their suppressor function directly on B lymphocytes. Whether PWM-induced B cell differentiation is suppressed by the same mechanism or/and by inactivation of T helper lymphocytes remains under investigation.

Human peripheral blood mononuclear cells as well as T-cell-enriched or T-cell-depleted populations were found to proliferate in response to recombinant interleukin-2 (IL-2) in vitro in the absence of a lectin preactivation signal. This proliferative response was detected at Day 2, peaked at Day 5, and was dependent on the concentration of IL-2 used. At the initiation of culture, these cells did not appear to be activated as determined by the expression of Tac antigens. In cultures of unfractionated T-cell-enriched suspensions, high concentrations of IL-2 resulted in preferential expansion of the OKT8+ population, although both OKT4+ and OKT8+ cells proliferated in response to IL-2 when cultured alone. These studies demonstrate that human lymphocytes obtained by standard fractionation procedures from peripheral blood are capable of proliferation in response to IL-2 without in vitro preactivation signals given by the addition of mitogens or antigens to cultures. These findings suggest that in vivo IL-2, in the absence of other exogenous stimuli, may directly influence immune responses and thus may have a potential role as a clinical immunopharmacologic agent.

The regulation of IgG subclass production by polyclonally activated human B cells was investigated by using two systems previously shown to selectively suppress the generation of IgG-containing cells (CC) but not that of IgMCC or IgACC. The first one involved a brief exposure of peripheral blood mononuclear cells (PBMNC) to heat-aggregated human IgG (Agg-IgG) followed by repeated washings and culture with untreated autologous PBMNC. The second one was achieved by addition of human IgG-binding factor(s) (IgGBF) prepared by affinity chromatography from supernatants of unstimulated PBMNC. Pokeweed mitogen (PWM) and Nocardia opaca delipidated cell mitogen (NDCM) were used as polyclonal B cell activators. The latter can induce the terminal differentiation of peripheral B lymphocytes into plasma cells in the absence of helper T cells. After 6 days of culture, the number of cells containing IgM, IgG, or IgA was determined by direct immunofluorescence, and that of cells containing IgG1, IgG2, IgG3, or IgG4 was determined by indirect immunofluorescence with the use of subclass-specific monoclonal antibodies. After stimulation with PWM, exposure of PBMNC to Agg-IgG resulted in a selective diminution of the number of IgG4CC. With NDCM-stimulated cultures the same procedure induced a selective suppression of the generation of IgG2CC and IgG4CC. Conversely, the addition of IgGBF at the third day of culture was found to induce a 30 to 40% decrease in the number of cells containing each of the four IgG subclasses. Because of their differential pattern of IgG subclass suppression, Agg-IgG and IgGBF are likely to trigger distinct regulatory pathways.

High concentrations of interleukin 2 (IL 2) were shown to produce a delayed but pronounced proliferation of purified resting T cells in the apparent absence of other activation signals. Because these stimulatory effects of IL 2 occurred in the absence of detectable Tac+ cells, the possibility that IL 2 might be initially interacting with an IL 2 binding protein distinct from the Tac protein was studied. Chemical cross-linking studies with 125I-IL 2 revealed the presence of an IL 2 binding protein distinct from the Tac protein on the surface of these unstimulated T cells. This second IL 2 receptor has an estimated molecular size of 70,000 daltons, lacks reactivity with the anti-Tac antibody, and appears to be identical to the p70 protein recently proposed as a component of the high affinity IL 2 receptor. Scatchard analysis of IL 2 binding assays performed with the unactivated T cells revealed approximately 600 to 700 p70 sites per cell and an apparent Kd of 340 pM. These data indicate that the p70 protein present on resting T cells binds IL 2 with an intermediate affinity compared with the previously recognized high and low affinity forms of the receptor and may account for the high concentration of IL 2 needed to induce resting T cell proliferation. To investigate the early biologic consequences of IL 2 binding to the p70 protein, potential changes in the expression of genes involved in T cell activation were examined. Northern blotting revealed the rapid induction of c-myc, c-myb, and Tac mRNA after stimulation of resting T cells with a high concentration of IL 2. The anti-Tac antibody did not inhibit IL 2 induced expression of these genes, suggesting that the p70 protein rather than the Tac antigen or the high affinity IL 2 receptor complex mediated this signal. However, in contrast to these early activation events, the anti-Tac antibody significantly inhibited IL 2 induced T cell proliferation. This finding implicates the high affinity form of the IL 2 receptor in the proliferative response of the IL 2 activated T cells. Thus these data support a two step model for the induction of resting T cell proliferation by high doses of IL 2 involving the initial generation of an activation or "competence" signal through the p70 protein and a subsequent proliferation or "progression" signal through the high affinity form of the receptor.

We show in this report that the transcription induced by interleukin-2 or pokeweed mitogens of the kappa MOPC 41 immunoglobulin light-chain gene transfected into primary human or murine B lymphocytes initiates from a previously unobserved start site about 26 base pairs upstream of the start site used in myeloma cell lines.