Lipids and cell fusion in vitro: effect of amphotericin B.

Fusion rate of Cl-1D cells mediated by Sendai virus was decreased by preincubating the cells or the virus in the presence of lysolecithin. However, a significant increase occurred when the cells were preincubated in the presence of amphotericin B. This increase was suppressed by exogenous cholesterol.     

Control of human B lymphocyte responsiveness: enhanced suppressor T cell activity after in vitro incubation.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) after a period of preincubation in vitro. When fresh PBM were co-cultured with preincubated PBM their response to PWM was inhibited, indicating that enhanced suppressor activity developed in the aged PBM concomitant with the loss of PWM responsiveness. Suppressor cell activity of aged PBM was present within the T lymphocyte population. The suppressor T cell inhibited PWM responsiveness of autologous and homologous PBM to an equivalent degree. The action of the suppressor cell was abrogated by inhibitors of DNA synthesis or by hydrocortisone. A suppressor T cell population with similar characteristics was found in freshly prepared PBM before in vitro incubation. Expansion of this suppressor T cell population during preincubation required cell division. There was no change in the functional capability of the helper T cell population as a result of similar in vitro culture. These observations indicate that a T cell population capable of suppressing PWM-induced generation of ISC can be selectively expanded by in vitro incubation of normal human PBM without additional mitogenic stimulation. Moreover, these data emphasize that control of B lymphocyte differentiation involves a critical interrelationship between T lymphocyte subpopulations exerting both positive and negative influences.     

An analysis of in vitro B cell immune responsiveness in human lymphatic filariasis

The immunoregulatory mechanisms involved in B cell function in patients with varying clinical manifestations of bancroftian filariasis were examined by studying the ability of peripheral blood mononuclear cells (PBMC) or PBMC subpopulations from patients with elephantiasis, asymptomatic microfilaremia (MF), and acute tropical pulmonary eosinophilia (TPE) to produce polyclonal and parasite-specific antibody in vitro, both spontaneously and in response to a mitogen (PWM) and to parasite antigen. When the spontaneous or mitogen-driven polyclonal responses were examined, all groups produced significant amounts of IgM and IgG; those with TPE produced extremely high levels. However, when in vitro parasite antigen-specific responses were examined, those with MF were unable to produce filaria-specific antibody either spontaneously or in response to PWM or parasite antigen; in contrast, patients with chronic lymphatic obstruction or TPE produced large quantities. Removal of neither adherent cells nor T8+ T cells affected the parasite-specific B cell anergy seen in those with MF. This absent or severely diminished capacity to produce antibody on parasite antigenic stimulation in patients with MF is likely responsible for the low levels of parasite-specific antibody seen in this most common clinical manifestation of bancroftian filariasis. Its inability to be reversed by the removal of "suppressor elements" suggests a state of B cell unresponsiveness to the parasite.     

Heterogeneity of B cell clones: immunoglobulin-secreting subsets

Staphylococcus aureus Cowan I bacteria (SpA CoI) is known to be a polyclonal B-cell activator of human lymphocytes. In this study, we investigated which of the B-cell subsets SpA CoI could stimulate and induce immunoglobulin (Ig) production. B-Cell subsets were separated from peripheral blood and tonsil lymphocytes by rosette formation with E, EA_IgG, EAC, anti-Ig-conjugated ox erythrocytes (OE-anti-Ig), and protein A-conjugated OE (OE-Pro A), or on a bovine serum albumin (BSA) discontinuous density gradient. The cells responding to SpA CoI included E^?, C3 receptor-positive (C3R⁺), Fc receptor-negative (FcR^?), and surface Ig-positive (SIg⁺) B-cell subsets. These B-cell populations responded well to SpA CoI and produced significant amounts of IgG, IgM, and a lesser amount of IgA. Among SIg⁺ B cells, IgG, IgA, and IgM⁺ B-cell subsets responded to SpA CoI and produced large amounts of Ig belonging to each corresponding Ig class. IgD⁺ B cells failed to produce Ig of any class, except for minimal amounts of IgG and IgM. While both the protein A receptor-positive (Pro A · R⁺) and negative (Pro A · R^?) cells responded well to SpA CoI, Pro A · R⁺ B cells produced IgG mainly and Pro A · R^? B cells produced IgM. Fractionation of B cells on a BSA gradient revealed that comparatively small-sized and denser B-cell subsets responded well to SpA CoI and produced every class of Ig.     

Differentiation of B-cell subsets after antigen priming.

Short-term DNP-Ficoll priming results in an increased number of in vitro TNP-LPS sensitive cells. This increase is less pronounced after TNP-LPS priming. Both effects are additive, indicating the existence of separate subpopulations of B cells responding to LPS and Ficoll. A similar phenomenon occurs with TNP-LP priming, being more evident in C3H/Tif mice (which have different subsets of B cells responding to LP and LPS). The evolution in the number of anti-TNP precursors after a similar priming schedule is consistent with the above results. These data are in line with the hypothesis of an antigen-driven B1 → B2 switch which affects the synthesis of carrier receptors.     

Nicotinamide modulation of rat pancreatic islet cell responsiveness in vitro.

The influence of nicotinamide (NA), a highly suitable precursor substrate for NAD synthesis in various tissues, on islet cell responsiveness was determined. After a 30 minute perifusion with this compound, nicotinamide, in a dose-dependent manner, potentiated glucose-induced insulin secretion. Maximal potentiation (approximately 250%) was observed at 20 mM NA and the threshold for potentiation was 3 mM. In the absence of glucose, NA did not affect basal secretion rates. Mannoheptulose blocked the primary stimulant action of glucose and the potentiating effects of NA. NA did not alter the rate of glucose usage by isolated islets. These results further underscore the possible importance of pyridine nucleotides in stimulated secretion.     

T8 cell regulation of human B cell responsiveness: regulatory influences of CD45RA+ and CD45RA- T8 cell subsets

The immunoregulatory functions of human T8 cell subpopulations defined by mAb to the CD45RA molecule (2H4) were examined. Both CD45RA+ and CD45RA- T8 cells that had been treated with mitomycin C provided help for the production of immunoglobulins by B cells in cultures stimulated with immobilized mAb to CD3 (64.1). In contrast, both CD45RA+ and CD45RA- T8 cells that had not been treated with mitomycin C suppressed B cell responses in anti-CD3-stimulated cultures, although CD45RA+ T8 cells were more effective in this regard. Interleukin 2 (IL2) enhanced suppression by anti-CD3-activated CD45RA- T8 cells, whereas suppression by CD45RA+ T8 cells was almost maximal and not as much increased by IL2. The differentiation into suppressor-effector cells in this system appeared to involve the production of IL2, but not the production of interferon (INF)-gamma. Thus, CD45RA+ T8 cells produced higher amounts of IL2 but lower amounts of IFN-gamma than CD45RA- T8 cells in anti-CD3-stimulated cultures. Moreover, addition of mAb to the p55 component of IL2 receptor (anti-Tac) inhibited the generation of suppressor activity from CD45RA+ and CD45RA- T8 cells. The pattern and magnitude of suppression of B cell responses by CD45RA+ and CD45RA- T4 cells were similar to that by CD45RA+ and CD45RA- T8 cells in this system. Finally, preactivated CD45RA+ T8 cells that had lost CD45RA expression suppressed the B cell responses as effectively as fresh CD45RA+ T8 cells. The results indicate that both CD45RA+ and CD45RA- T8 cells can help or suppress B cell responses. More importantly, the data suggest that the suppressor-effector function of human T cells may rather be related with the stages of the post-thymic differentiation as evidenced by the expression of the CD45RA molecule than represent the fully differentiated T cell subsets, such as T4 and T8 cells. In addition, the CD45RA molecule appeared not to be involved in the suppressor-effector function, but to determine the stage of post-thymic differentiation.     

Flow sorting of antigen-binding B cell subsets

Antigen binding was used as a probe in the definition of functional B cell heterogeneity. Unprimed, anti-Thy 1 and complement-treated spleen cells were stained with fluorescent trinitrophenylated bovine serum albumin (FL-TNP-BSA). These cells were sorted, fluorescence negative from fluorescence positive, by using a multiparameter cell sorter and assayed for precursor frequency in antibody responses to TNP by limiting dilution analysis. The cells that bound FL-TNP-BSA were demonstrated to be enriched for antibody-forming precursors to the antigens TNP-lipopolysaccharide (LPS), TNP-sheep red blood cells (SRBC), and TNP- or DNP-Ficoll, whereas the fluorescence negative cells were depleted for these responses. B cells that bound FL-TNP-BSA were then sorted into populations that bound a moderate or high amount of FL-TNP-BSA. The B cells responsive to TNP-LPS and TNP-SRBC were present in both the moderate and high binding populations. In contrast, the B cells responsive to TNP- or DNP-Ficoll were present only in the cells that bound a moderate amount of FL-TNP-BSA. These experiments suggest that there is a population of B cells in adult mouse spleen that binds large amounts of antigen, and that can respond to antigen carried on LPS or SRBC but not carried on Ficoll.     

Differential effects of IL-27 on human B cell subsets

IL-27 is a novel heterodimeric cytokine of the IL-12 family that plays an important role in the regulation of T cell responses. Its role on human B cells has not been previously studied. In this study, we show that both chains of the IL-27 receptor complex, IL-27R and gp130, are constitutively expressed at the surface of naive and memory human tonsillar B cells, and are induced on germinal center B cells following CD40 stimulation. In naive B cells, IL-27 induced strong STAT1 and STAT3 phosphorylation, whereas it induced moderate STAT1 and low STAT3 activation in memory B cells. IL-27 induced T-bet expression in naive and memory B cells stimulated by CD40 or surface Ig engagement, but induced significant IL-12Rbeta2 surface expression in anti-Ig-stimulated naive B cells only. In anti-Ig-stimulated naive or memory B cells, IL-27 also induced CD54, CD86, and CD95 surface expression. In addition, IL-27 increased proliferation of anti-Ig-activated naive B cells and of anti-CD40-activated naive and germinal center B cells, but not of CD40-activated memory B cells. These data indicate that the B cell response to IL-27 is modulated during B cell differentiation and varies depending on the mode of B cell activation.     

Reduced responsiveness of immature B cells to the B cell mitogen, lipoprotein.

When pre-B cells are isolated from bone marrow and incubated in vitro for several days, they spontaneously differentiate into LPS-reactive B cells. These newly produced B cells do not respond to another B cell mitogen, lipoprotein (Lp). The lack of Lp reactivity of newly produced B cells represents a qualitative difference between these cells and B cells in peripheral tissues that respond equally well to both LPS and Lp. Thus, responsiveness to different mitogens provides unique markers to identify B cells at early stages of development.     

Effects of interferon-alpha on human B cell responsiveness: biphasic effects in cultures stimulated with Staphylococcus aureus.

Although interferon-alpha (IFN-alpha) has been found to be involved in the immune regulation in vivo, the effects of IFN-alpha on human B cells have not yet been clarified because of conflicting results in the literature. The present study therefore examined the effects of several subtypes of IFN-alpha (natural, alpha 1, alpha 2a, alpha 2b) on B cell responsiveness in detail by comparing different experimental conditions. Highly purified B cells from normal human individuals were cultured with Staphylococcus aureus (SA) + IL-2 or with immobilized anti-CD3-activated T4 cells in the presence or absence of IFN-alpha. IFN-alpha enhanced the immunoglobulin (Ig) production induced by immobilized anti-CD3-activated T4 cells. By contrast, IFN-alpha (5-50,000 IU/ml) suppressed the Ig production induced by SA + IL-2. The suppression by IFN-alpha was dependent on the concentration of SA. The inhibitory effects of IFN-alpha in SA-stimulated cultures were exerted in the first 72 hr of cultures and required the presence of IL-2, whereas IFN-alpha enhanced the maturation of B cells when it was added after 72 hr of cultures. The suppressive effects of IFN-alpha were overcome by addition of immobilized anti-CD3-preactivated T cells that had been treated with mitomycin C, but not by the addition of fresh T cells or soluble factors produced by activated T cells. Of interest, IFN-alpha did not inhibit the expression of IL-2R, but inhibited that of intercellular adhesion molecule-1 (ICAM-1) on B cells after stimulation with SA + IL-2, suggesting that the suppressive effects of IFN-alpha might be related to the regulation of B cell-B cell contacts through ICAM-1. There was no significant difference in effects on B cells among various subtypes of IFN-alpha. These results suggest that the effects of IFN-alpha on human B cell responsiveness may be different depending on the nature of stimulation. Moreover, the data indicate that IFN-alpha enhances the differentiation of activated B cells irrespective of the activation signals.     

Impact of cigarette smoke on T and B cell responsiveness

Although its direct effects cannot be discounted, tobacco's effects on the immune system have been proposed to play a key role in mediating its deleterious health impact. Studies in rats using high levels of smoke exposure have suggested that tobacco smoke exhausts cellular signal transduction cascades, making lymphocytes unresponsive to stimulation. In the present study, we show that purified B or T cells, and total lymphocytes from the lungs, lymph nodes and spleens of smoke-exposed mice fluxed calcium, proliferated, and secreted immunoglobulin or IFN-gamma similarly to control mice when stimulated with ligands including anti-IgM, and anti-CD3. Importantly, we recapitulated these findings in PBMCs from human smokers; cells from long-term smokers and never-smokers proliferated equivalently when stimulated ex vivo. Previous reports of lymphocyte unresponsiveness in rats are inconsistent with these findings, and may reflect a phenomenon observed only at levels of smoke exposure well above those seen in actual human smokers.     

Inhibitory influence of IL-4 on human B cell responsiveness

The role of IL-4 in human B cell activation, proliferation, and differentiation was examined. rIL-2, but not rIL-4, was able to promote maximum proliferation and generation of Ig-secreting cells in cultures of highly purified B cells stimulated with Cowan I Staphylococcus aureus (SA). Addition of rIL-4 to rIL-2-supported cultures of SA-stimulated peripheral blood, spleen, or lymph node B cells dramatically suppressed both proliferation and differentiation. Results from experiments in which rIL-4 was added to culture at progressively later times indicated a requirement for rIL-4 to be present during the first 2 days of a 5-day incubation to cause inhibition of responsiveness. When a two-stage culture system was utilized, rIL-4 was found to support proliferation or differentiation of B cells initially activated with SA for 2 days only minimally. However, rIL-4 did not inhibit responses of SA preactivated B cells supported by IL-2. The presence of rIL-4 during the initial 48-h activation of B cells with SA and rIL-2 resulted in a profound inhibition of the ability of the activated B cells to respond subsequently to rIL-2 or lymphokine-rich T cell supernatants. A similar 48-h incubation with rIL-4 alone without SA had no effect on subsequent B cell responsiveness. The presence of rIFN-gamma during B cell activation decreased the inhibitory effect of IL-4. Other cytokines including IFN-alpha, IL-1, and commercially available low m.w. B cell growth factor also diminished the inhibitory effect of IL-4. These results indicate that IL-4 inhibits the capacity of human B cells to be activated maximally by SA and rIL-2 and therefore suggest a new immunomodulatory role for this cytokine.     

T cell regulation of polyclonal B cell responsiveness. I. Helper effects of T cells.

Polyclonal activation of murine splenic B lymphocytes to secrete immunoglobulin was shown to be subject to regulation by splenic T cells. By admixture of separated B and T cell populations it was demonstrated that normal fresh splenic T cells were able to augment polyclonal B cell responsiveness to LPS up to several-fold. Optimal collaboration between these two cell types ensued when they were co-cultured in equal numbers. T cell-mediated enhancement of polyclonal B cell responses was dependent upon the ability of T cells to divide and was manifested upon T cell interaction with B cells soon after culture initiation. Originally expounded as a one-signal phenomenon, polyclonal activation of lymphocytes by LPS is, under the circumstances described, attributable instead to two distinct, nonspecific signals acting in concert. The observation that T cells from LPS-nonresponder (C3H/HeJ) mice were deficient in the capacity to enhance polyclonal B cell responsiveness of B cells derived from responder (C3H/HeN) mice implied a direct action of LPS on the involved T cells as well as an active role for the T cell signal in this immunoregulatory event. The novel observation of a functional T cell defect in LPS responsiveness in the C3H/HeJ mouse is discussed in terms of its other cellular defects.     

Difference in B cell mitogen responsiveness between closely related strains of mice.

Spleen cells from C3H/HeJ mice respond very poorly to the mitogenic action of endotoxin (LPS) in vitro whereas cells from a very closely related strain, C3H/HeN respond very well. An analysis of the genetic similarity of these two strains was performed. There is no significant MLR measurable between cells of these strains under a variety of culture conditions. They do not manifest a significant graft-vs-host response nor do they reject reciprocal skin grafts. Because of this genetic similarity, it is possible to reconstitute the LPS response of nonresponder mice by an adoptive transfer of spleen cells from C3H/HeN mice. This strain pair should therefore prove useful in analyzing the genetic and cellular basis of endotoxin reactivity.     

The role of antigen-presenting B cells in T cell priming in vivo. Studies of B cell-deficient mice

Mice rendered B cell deficient by treatment with rabbit anti-mouse IgM (anti-mu) antibodies from birth fail to respond when primed with soluble protein antigens in CFA, as measured by T cell proliferation when challenged with antigen in vitro. The role of B cells in T cell priming in vivo was examined by adoptively transferring hapten-specific B cells into anti-mu mice, followed by immunization with haptenated Ag in CFA. The T cell proliferative response to OVA of anti-mu BALB/c mice was partially restored by the administration of TNP or FITC-specific B cells and immunization with TNP-OVA or FITC-OVA, respectively. This reconstitution was Ag-specific, inasmuch as hapten-binding B cells restored the T cell responses to OVA in mice immunized with the same hapten coupled to OVA. The mechanism of B cell reconstitution of T cell priming in anti-mu mice was addressed using parental to F1 B cell transfers. The Ia restriction pattern of the activated T cells from these mice indicated that both direct presentation of Ag by transferred B cells and antibody-mediated enhancement of Ag presentation by non-B, host Ag-presenting cells occurred. Thus, Ag-specific B lymphocytes play a critical role in priming of T cells in vivo.