Enhancement of the B-cell response to Staphylococcus aureus Cowan strain 1 by natural human gamma interferon.

The requirements for a primary, antigen-specific in vitro immunization of human peripheral lymphocytes using haemocyanin, a T-cell dependent antigen, have been studied. In order to obtain a specific response in vitro the peripheral lymphocytes had to be separated into B, T, accessory (A) and dendritic (D) cells. These cells were activated and reconstituted to give a population with a B:T ratio of 1:2. If the induction was supported by MHC-restricted, radioresistant T cells, this cell population could then be antigen-specifically activated using haemocyanin. The immunization had also to be supported by cytokines, such as B-cell growth and differentiation factors, interleukin-2 and gamma-interferon. A 5-day in vitro immunization using 2 micrograms haemocyanin/ml resulted in 200-300 cells secreting anti-haemocyanin-specific antibodies per 10(6) B cells.     

The induction of human B-cell activation, proliferation and differentiation by anti-CD3-stimulated T cells--a model of T cell/B cell collaboration

It is clear that anti-CD3-activated T cells provide all the signals necessary for polyclonal activation, proliferation and differentiation of human B cells. B-cell activation, proliferation and differentiation are initiated by a complex series of receptor-ligand interactions, of which LFA-1-ICAM-1 ligation plays a central, but not exclusive role. In addition, the action of a number of cytokines, most prominently IL2, is essential for B-cell proliferation and differentiation. It is apparent from the results obtained using this model system that the conjugate formation that occurs as part of antigen presentation and T-cell activation is not required for the subsequent collaboration between activated T cells and B cells necessary to trigger B-cell responses. The implication of these studies is that after initial antigen recognition and T-cell activation, T cell/B cell collaboration leading to antibody production is antigen non-specific and potentially capable of inducing polyclonal B-cell responses. Conjugate formation that develops as part of antigen presentation may tend to focus the response and limit the B cells that can be induced to respond. Alternatively, T-cell recognition of antigen may deliver a signal to the B cell by cross-linking MHC molecules bearing the recognized antigenic fragment that makes it more capable of responding to the T-cell-derived antigen-non-specific signals and cytokines promoting B-cell activation, proliferation and differentiation.     

T cells and follicular dendritic cells in germinal center B-cell formation and selection

Summary: Germinal centers (GCs) are specialized microenvironments formed after infection where activated B cells can mutate their B-cell receptors to undergo affinity maturation. A stringent process of selection allows high affinity, non-self-reactive B cells to become long-lived memory B cells and plasma cells. While the precise mechanism of selection is still poorly understood, the last decade has advanced our understanding of the role of T cells and follicular dendritic cells (FDCs) in GC B-cell formation and selection. T cells and non-T-cell-derived CD40 ligands on FDCs are essential for T-dependent (TD) and T-independent GC formation, respectively. TD-GC formation requires Bcl-6-expressing T cells capable of signaling through SAP, which promotes formation of stable T:B conjugates. By contrast, differentiation of B blasts along the extrafollicular pathway is less dependent on SAP. T-follicular helper (Tfh) cell-derived CD40L, interleukin-21, and interleukin-4 play important roles in GC B-cell proliferation, survival, and affinity maturation. A role for FDC-derived integrin signals has also emerged: GC B cells capable of forming an immune synapse with FDCs have a survival advantage. This emerges as a powerful mechanism to ensure death of B cells that bind self-reactive antigen, which would not normally be presented on FDCs.     

Immunoglobulin and cytokine production by neonatal lymphocytes.

Growth and differentiation of cord blood B cells were studied using T cell-depleted populations. In the absence of in vitro activation, cord blood B cells proliferated in response to cytokines including interleukin-2 (IL-2) and interleukin-4 (IL-4); anti-mu-stimulated cord B cells had a lesser response to IL-2 than adult cells. IgM synthesis by cord blood B cells was enhanced by interleukin-6 (IL-6) and decreased by IL-2. In cultures activated by Staphylococcus aureus Cowan I (SAC), cord blood B cells produced lesser increases in IgM than adult B cells regardless of the cytokine added. Cord blood B cells produced no IgG or IgA with any cytokine preparation with or without SAC activation. Supernatants of cord blood T cells pulse-stimulated with phytohaemagglutinin and phorbol myristate acetate contained less IL-2 and IL-6 and had less growth and differentiation activity than adult T cell supernatants. The results confirm a limited cord blood B cell response and also suggest a limitation in production of B cell stimulatory lymphokines by cord blood T cells.     

Development and characterization of a human monoclonal antibody probably detecting the leukocyte differentiation antigen CD39

A human monoclonal IgM antibody, referred to as TU223, has been produced. The reactivity of TU223 was tested in various cells and cell lines by complement-dependent microcytotoxicity test and fluorescence-activated cell sorter analysis. The antigen defined by TU223 was expressed on Epstein-Barr virus-transformed B-cell lines and on some Burkitt's lymphoma cell lines, but was not expressed on normal T cells, B cells or erythrocytes. In addition, expression of the antigen defined by TU223 was also induced on B cells activated by Epstein-Barr virus or pokeweed mitogen, and on T cells activated by phytohemagglutinin, concanavalin A, pokeweed mitogen or recombinant interleukin-2. However, no expression of the antigen detected by TU223 was induced at all on recombinant interleukin-4-treated B cells or macrophage-like cell line U937. When the ability of TU223 and various mouse monoclonal antibodies to bind to human differentiation antigens was compared, interestingly, the reactivity of TU223 was found to be very similar to that of mouse monoclonal antibody CD23 (H107), which reacts with Fc epsilon receptor II. Two-color analysis revealed that the antigen defined by TU223 is expressed on the cell surface of certain lymphoid cells expressing CD23 antigen. However, it can be concluded that the antigen defined by TU223 is clearly distinct from Fc epsilon receptor II, based on assay of cross-blocking between H107 and TU223. The surface antigen on B85 cells recognized by TU2232 had the molecular size of 80-82 kiloDaltons as determined by immunoblotting analysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct induction of human B-cell differentiation by recombinant interleukin-2.

Recombinant interleukin-2 (rIL-2) induced highly purified human tonsillar B cells to differentiate into immunoglobulin (Ig)-producing cells in vitro. The B-cell response was not due to rIL-2-contaminating substances, but reflected the activity of IL-2 itself, since it was inhibited by addition to the cultures of anti-TAC monoclonal antibody. The rIL-2-induced B-cell response was apparently not mediated by factors released by residual T cells present in B-cell suspensions at undetectable levels, since supernatants (SN) from unstimulated autologous T cells cultured at concentrations even much higher than those possibly contaminating B-cell suspensions did not induce any detectable Ig production. In addition, the Ig production by B cells cultured with SN prepared from high numbers of autologous T cells stimulated with rIL-2, as well as from allo-activated or mitogen-stimulated T cells, was of the same magnitude as the Ig production resulting from direct addition of rIL-2 concentrations comparable with those present in the supernatants. After centrifugation on Percoll density gradients, most of the tonsillar B cells responsive to rIL-2 were recovered in the lower density cell fraction containing a number of larger activated B cells. Moreover, B-cell enriched suspensions from peripheral blood (PB) (which usually contains a lower number of in vivo activated B cells than tonsil) showed poor or no response to rIL-2 alone, but displayed significant Ig production when rIL-2 was added to the cultures in the presence of Staphylococcus aureus Cowan I (SAC) bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

T cells or their products are required for the differentiation of precursor B cells into antibody-secreting cells specific for a supposed T-independent self-antigen.

From our experiments and those of others in which cells were cultured at a density of 100,000 cells per well, it has been suggested that autoantibody production against mouse bromelain-treated erythrocytes (mouse brom-RBC) was independent of T cells, and further, was enhanced by the removal of T cells from responsive cell populations. Here it is shown in limiting dilution cultures that the autoimmune response is highly dependent on T cells or their products. B cells purified from the peritoneal cavities of untreated mice did not differentiate in vitro into autoantibody-secreting cells unless provided with signals from at least one of two types of accessory cells. These were plastic adherent cells and T cells, derived either from the peritoneal cavity or from established cell lines. Here it is shown that peritoneal T cells or T cells from the LBRM-33 cell line stimulated the differentiation of purified B cells in vitro in the absence of added mitogens. The accessory cell effect could be transferred in supernatants derived from T-cell cultures but not filler-cell cultures. Recombinant interleukin-2 (rIL-2) added to culture medium did not stimulate B cells directly, but could increase precursor frequencies when added to unfractionated peritoneal cell cultures, or B-cell cultures to which cells from a T-cell line had been added. From these results, it is concluded that the differentiation of precommitted peritoneal B cells in vitro into autoantibody secretors is at least partially dependent on T cells or lymphokines derived from them. Therefore, any proposed mechanisms for regulation of this autoimmune response should encompass the requirement for T cells or their products in the final differentiation stages to autoantibody secretion.     

Regulation of IgA B-cell development in the mucosal immune system

The factors regulating the differentiation of IgA B cells have been of great interest to mucosal immunologists as well as those generally interested in B-cell differentiation. It is now clear that such differentiation involves two major steps: first, isotype switch differentiation of surface IgM-bearing B cells into surface IgA-bearing B cells and, second, terminal differentiation of IgA B cells into IgA-producing plasma cells. Both of these steps are regulated processes that are under the influence of various cytokines and lymphokines. This paper presents data that define the role of cytokines and lymphokines in the regulation of IgA B-cell differentiation. A model of IgA B-cell differentiation is described in which the first step involves activation of the C alpha gene, while the latter is in germline configuration and thus the induction of surface IgM-bearing B cells partially committed to IgA expression. This occurs in Peyer's patches as a result of as yet incompletely defined signals from patch “switch cells.” The second step consists of conversion of the partially committed B cells to fully IgA-committed B cells and thus the completion of isotype switch differentiation. This step may be under the control of interleukin-4 (IL-4). The last step of the model involves the activation of IgA B cells (by antigen or mitogen) followed by the appearance on the cell surface of receptors which allow the cell to interact with cytokines or lymphokines (particularly IL-5). Such interaction results in cells capable of secreting IgA. Evidence is presented that an important adjuvant for mucosal immune responses, cholera toxin, acts to augment IgA production by promoting IgA B-cell differentiation at the isotype switch step.     

Interleukin-2 effects on human B cells activatedin vivo

While activated B cells, as well as T cells, can express functional interleukin-2 receptors (IL-2R), the physiologic role of IL-2 in B-cell responses is still in doubt. Accordingly, we have examined the role of recombinant IL-2 (rIL-2) in the proliferative response, IL-2R expression, and the terminal differentiation of tonsillar B cells in comparative studies with T cells from the same source. For these analyses of physiologically activated lymphocytes, the B and T cells were first separated, then divided into subpopulations enriched for either resting or preactivated cells on the basis of relative cell density or activation antigen expression. As expected, the relatively low-density fraction of T cells was enriched for IL-2R-positive (Tac⁺) cells and displayed vigorous proliferative responses to IL-2 along with heightened Tac antigen expression. Moreover, limiting dilution analysis revealed that growth of these activated T cells could be perpetuated with the addition of IL-2. By comparison, relatively few tonsillar B cells expressed the Tac antigen. The addition of rIL-2 to cultured B cells of relatively low density resulted in an increase in Tac⁺ cells but only a minimal proliferative response. The subpopulation of tonsillar B cells expressing the Bac-1 activation antigen contained most of the IL-2 inducible Tac⁺ B cells, and rIL-2 induced an efficient but transient proliferative response by these activated B cells. The rIL-2-induced Tac⁺ B cells were noted to be relatively large. A fraction of these Tac⁺ cells, but not the Tac^– cells, produced and secreted immunoglobulins. Incubation with rIL-2 enhanced the Ig secretion, and the anti-Tac antibody blocked this enhancement. Time-course analysis revealed that rIL-2 induced transient Tac expression, whereas mature plasma cells in 6-day cultures no longer expressed detectable Tac antigen. In conclusion, these observations suggest that IL-2 transiently upregulates expression of IL-2R, via which it induces the terminal growth and differentiation of activated B cells into plasma cells.     

MHC-restricted B-cell antigen presentation in memory B-cell maintenance and differentiation

Memory B-cell development, maintenance, and differentiation have been believed to be tightly regulated by T cells through major histocompatibility complex (MHC)-II-restricted cognate interaction. However, recent studies have indicated that memory B cells are a heterogeneous population with various ontogeny and with derivations from T-cell-independent as well as T-cell-dependent response. In addition, several studies highlight T-cell-independent maintenance and differentiation of memory B cells by innate polyclonal stimuli associated with Toll-like-receptors (TLRs). Thus, both T-cell-dependent and T-cell-independent mechanisms with possibly different physiological functions are required for maximal memory B-cell maintenance and differentiation. In this review, we discuss the mechanisms of memory B-cell maintenance and differentiation by re-examining the requirement for MHC-II-restricted B-cell antigen presentation based on our recent study in a mouse model that lacks MHC-II on memory B cells. We propose that MHC-II-restricted memory B-cell maintenance and differentiation are the indispensable mechanisms by which we achieve health and the maximum quality of memory response with specific antibody production, while preventing plasma cell differentiation from self-reactive memory B cells under the control of T-helper cells and possibly regulatory T cells.     

In vitro activation of leukaemic B cells by interleukin-4 and antibodies to CD40.

Chronic lymphocytic leukaemia (CLL) B cells have the phenotype of mature, partially activated B cells, but are relatively resistant to stimulation through cell-surface receptors with agents such as antibodies to immunoglobulin M. In the present study we have shown that culture of CLL cells with interleukin-4 (IL-4) and antibodies to CD40 caused significant DNA synthesis as assessed by incorporation of thymidine. This effect was largely mediated by antibody binding to CD40, a molecule known to induce B-cell activation and to mediate survival signals to germinal centre B cells. Epstein-Barr virus (EBV) infection of CLL cells stimulated with IL-4 and anti-CD40 did not affect the state of differentiation of the cell, augment the proliferative capacity, prolong cell survival or cause virus-driven immortalization.     

Role of interleukin-7 in T-cell development from hematopoietic stem cells

Summary: All lymphocytes are derived from hematopoietic stem cells (HSC). The interleukin-7 receptor (IL-7R) transduces non-redundant signals for both T and B-cell development from HSC. The upregulation of the IL-7R occurs at the stage of the clonogenic common lymphoid progenitor, a recently identified population that can give rise to all lymphoid lineages (T, B and natural killer cells) at a single cell level. The IL-7R plays a critical role in the rearrangement of immunoglobulin heavy chain genes required for B-cell development, IL-7R expression is critically regulated in developing thymocytes; thytnocytes that fail the positive selection process down-regulate the IL-7R, but those undergoing positive selection upregulate or maintain IL-7R expression. Recent data indicate that IL-7 signaling enhances the survival of developing thymocytes and mature T ceils, presumably by its upregulating Bcl-2. Detailed analysis of the signaling cascades activated by the IL-7R may help to reveal the differential roles of IL-7 signaling in T and B-cell development.     

The induction of resting B cell differentiation does not require T cell contact.

A T cell clone as well as immediately ex vivo CD4+ lymph node T cells are shown to support the differentiation of co-cultured resting B cells in the absence of T cell-B cell contact. Antibodies specific for class II products of the major histocompatibility complex inhibit the transactivation of resting but not activated B cells. This differential inhibition pattern indicates that the responses obtained from resting B cell populations are not due to their contamination with B cell blasts. Further, supernatants prepared from an activated T cell clone induce resting B cell differentiation. Two lines of evidence suggest that the activity contained in these supernatants can be attributed to interleukin (IL)-5. Activity is neutralized by monoclonal anti-IL-5; and both recombinant and affinity-purified IL-5 induce the differentiation of immediately ex vivo resting and activated B cells with comparable efficiency. Taken together, these results demonstrate that contact with T cells does not provide prerequisite signals for the induction of resting B cell differentiation.     

Cytoskeleton protein 4.1R regulates B-cell fate by modulating the canonical NF-κB pathway

During the immune response, B cells can enter the memory pathway, which is characterized by class switch recombination (CSR), or they may undergo plasma cell differentiation (PCD) to secrete immunoglobulin. Both of these processes occur in activated B cells, which are reported to relate to membrane-association proteins and adaptors. Protein 4.1R acts as an adaptor, linking membrane proteins to the cytoskeleton, and is involved in many cell events such as cell activation and differentiation, and cytokine secretion. However, the effect of 4.1R on regulating B-cell fate is unclear. Here, we show an important association between B-cell fate and 4.1R. In vitro, primary B cells were stimulated with lipopolysaccharide combined with interleukin-4; results showed that 4.1R-deficient (4.1R^–/–) cells compared with wild-type (4.1R^+/+) B cells augmented expression of activation-induced cytidine deaminase and germline, resulting in increased IgG1⁺ B cells, whereas the secretion of IgG1 and IgM was reduced, and CD138⁺ B cells were also decreased. Throughout the process, 4.1R regulated canonical nuclear factor (NF-κB) rather than non-canonical NF-κB to promote the expression of CSR complex components, leading to up-regulation of B-cell CSR. In contrast, 4.1R-deficient B cells showed reduced expression of Blimp-1, which caused B cells to down-regulate PCD. Furthermore, over-activation of canonical NF-κB may induce apoptosis signaling to cause PCD apoptosis to reduce PCD number. In summary, our results suggest that 4.1R acts as a B-cell fate regulator by inhibiting the canonical NF-κB signaling pathway.     

Modulation of expression of the antigen identified by FMC7 upon human B-lymphocyte activation: evidence for differences between activation in vivo and in vitro.

The antigen detected by the monoclonal antibody (mAb) FMC7 is expressed by a proportion of B lymphocytes, and previous information, based on leukaemias, cell lines and some normal cell studies, suggested that FMC7 reacts with relatively differentiated B cells. In this study, it is shown that cells in the high-density 'resting' fraction of tonsil B lymphocytes have a lower expression of FMC7 than cells in the low-density 'activated' fraction. However, activation of 'resting' B cells with anti-immunoglobulin (anti-IgM) together with IL-4 resulted in a decrease in FMC7 reactivity, whilst recognized markers of activation showed up-regulation, as expected. Activated cells which were cultured for an extra 3 days to allow return to a 'resting' stage showed an increase in FMC7 expression. Culture of activated B cells with low molecular weight B-cell growth factor (LMW-BCGF), tumour necrosis factor (TNF) or interleukin-6 (IL-6), did not lead to further changes in FMC7 reactivity, but stimulation with phorbol ester had an effect similar to that of anti-IgM + IL-4. These results suggest that FMC7 is a marker of differentiation rather than activation, and identifies a relatively mature subfraction of the pool of functionally mature B cells. Consistent with this interpretation, FMC7 and surface IgD tended to be expressed by reciprocal B-cell subpopulations.     

The kinetic study on cell surface antigens of a hybridoma between human B cells and a mouse B-cell line

Human B cells obtained from tonsils were fused with a mutant clone of a murine B-cell line in the presence of polyethylene glycol and dimethyl sulfoxide. THT12.58, a subclone of the human-mouse B-cell hybridoma, was shown to express human B-cell surface antigens on the cell membrane, namely B1, B2, I2, human IgM and IgD derived from human B cells by analysis of flow microfluorometry (FMF), as well as murine B-cell markers; the amount of these human B-cell markers on THT12.58 did not change for more than 1 year after establishment. Interestingly, the expression of the human B-cell antigens significantly decreased after treatment of the cells with B-cell stimulatory factors (BSF) obtained from the supernatant of the culture of PHA-P activated T cells; this was followed by significant enlargement in cell size compared with the control. A marked decrease in the amount of each antigen on the hybrid was observed when treated with Staphylococcus aureus Cowan I strain (SAC) in addition to BSF. In contrast, the expression of these markers on the cells increased after exposure of the cells to recombinant human interferon-gamma (rINF-gamma). Additionally, the effect of BSF on the generation of IgM-secreting cells by human B cells markedly decreased after absorption of BSF with the hybrid cells. These results suggest that THT12.58 may possess a receptor for BSF on the cell surface, and be capable of differentiating into much more mature stage of B-cell lineage after exposure to BSF. Thus, this kind of a human-mouse B-cell hybridoma with human B-cell differentiation antigens can be a good model to investigate the kinetics of cell surface antigens and characterization of a receptor for BSF on human B cells.     

Monoclonal antibody NIM-R3 substitutes for B-cell growth factor.

A rat monoclonal antibody (NIM-R3) was found to be reactive with activated mouse B cells but neither activated T cells nor small resting lymphocytes, T or B. The antibody stains antibody-forming cell precursors within 48 hr of primary or secondary immunization in vivo, but not at longer times (greater than 14 days) immunization. When added to cultures of spleen cells responding in vitro to dinitrophenylated bovine, IgG, the number of antibody-forming cells was increased. NIM-R3 also maintained the proliferation of purified B blasts in the absence of lipopolysaccharide, but did not activate small resting B cells in the presence of anti-Ig. NIM-R3 could replace B-cell growth factor (BCGF II) in cultures of the murine B-cell lymphoma BCL1 inducing proliferation but not differentiation. Finally, competition was demonstrated for binding sites on BCL1 cells between BCGF II and NIM-R3, but not between NIM-R3 and T-cell replacing factor (B15-TRF). We suggest that NIM-R3 may represent a novel specificity and may be directed against the cell surface receptor for BCGF II, and in turn this receptor may be independent of the B15-TRF and BSF1 receptors.