Autoreactive T-cell response to resting or activated B cells.

Cloned autoreactive T-cell lines and hybridomas have been selected in many laboratories. A number of observations have suggested that activation of Ia-positive stimulators may be required for optimal induction of an autoreactive response. We have examined the ability of small resting B cells fractionated by centrifugal elutriation to stimulate the proliferative response of seven cloned autoreactive T-cell lines. Of these, six were efficiently stimulated by resting B cells. One I-Ed-specific Th2-type T-cell clone failed to be stimulated by resting B cells. This clone did, however, respond to this same cell fraction following lipopolysaccharide (LPS) activation. An independent I-E-specific Th2 clone was stimulated by resting B cells. It appears, therefore, that a requirement for activated stimulators is not a general property of either autoreactive T cells or the Th2 helper T-cell subset.     

Cross-linking of surface IgM,but not surface IgD receptors,by soluble monoclonal antibodies primes murine B cells to secrete immunoglobulin in response to lymphokines

We have previously reported the development of a two-step culture system in which soluble anti-μ monoclonal antibodies prime small resting murine B cells to secrete immunoglobulin (Ig) in response to restimulation with a mixture of interleukin-4 (IL-4) and IL-5. Here we have extended these studies to investigate the effects of engaging surface IgD (sIgD).We find that, unlike anti-μ, three different anti-5 monoclonal antibodies did not prime B cells to secrete Ig. In addition, these anti-δ antibodies inhibited anti-μ-stimulated priming for Ig secretion, while enhancing DNA synthesis in response to anti-μ,. Furthermore, anti-δ antibodies still inhibited anti-μ-induced priming when added 24-48 h after anti-μ. These results therefore suggest that triggering of sIgD on B cells induces a dominant inhibitory signal which is not necessarily dependent upon co-ligation of sIgM and sIgD receptors. In addition, these findings raise the possibility that ligating sIgM or sIgD receptors on mature B cells in the absence of Tcell help, may produce different downstream effects.     

Inhibition of lipopolysaccharide-induced activation of immature B cells by anti-mu and anti-delta antibodies and its modulation by interleukin-4.

Soluble anti-Ig or anti-mu antibodies completely abrogate the lipopolysaccharide (LPS)-stimulated proliferation of purified B cells obtained from spleens of 5-7 day old mice. This provides further evidence for the powerful nature of the negative signals transduced by sIgM receptors on immature B cells. In addition, ligation of sIgD receptors (expressed by approximately 30% of these cells) by two out of three monoclonal anti-delta antibodies inhibits the response to LPS by some 50%. Ligation of sIgD on purified sIgD+ B cells (greater than 98% sIgD+) completely inhibited the LPS response of these cells. The inclusion of IL-4 in these cultures partially (with anti-mu), or completely (with anti-delta), restored the proliferative response. Immobilized anti-mu or anti-delta caused comparable levels of inhibition as the soluble antibodies: IL-4 again rescued the inhibition caused by immobilized anti-delta, but not that induced by anti-mu. These results therefore indicate that engaging either sIgM or sIgD receptors on developing B cells delivers negative (tolerogenic) signals. They also implicate IL-4 as a major (although not the only) T cell-derived influence which can modulate these signals. Finally, the data suggest that extensive cross-linking of sIgM (by immobilized anti-mu) causes more profound unresponsiveness in immature B cells, which cannot be rescued by IL-4 alone.     

Induction of resting B cells to DNA synthesis by soluble monoclonal anti-immunoglobulin

A novel monoclonal anti-immunoglobulin is described which in soluble form is comparable to lipopolysaccharide in its ability to induce DNA synthesis in populations of resting B cells. It inhibits lipopolysaccharide-induced B cell maturation to immunoglobulin secretion while not suppressing mitogen-induced proliferative responses. B cells from C3H/HeJ mice are stimulated as well as those from C3H/Tif mice demonstrating that the induction capacity of this monoclonal antibody does not involve functional lipopolysaccharide receptor molecules.     

A noncognate interaction with anti-receptor antibody-activated helper T cells induces small resting murine B cells to proliferate and to secrete antibody

Culture of small resting allogeneic B cells (of an irrelevant haplotype) with two clones of T helper (Th) cells that were activated by the F23.1 anti-T cell receptor antibody led to the activation of B cells to proliferate and to secrete antibody. Th cell supernatants by themselves had no effect on resting B cells (even in the presence of intact F23.1 antibody), but could induce antibody secretion by anti-Ig-preactivated B cells. Both F23.1+ clones (E9.D4 and 4.35F2) and one F23.1- clone (D2.2) could synergize with supernatants from activated E9.D4 T cells to induce B cell activation. F(ab')2 fragments of F23.1 induced E9.D4 to activate B cells as efficiently as intact F23.1 and B cell populations that had been incubated with F23.1 were not activated when cultured with E9.D4, although T cells recognized cell-presented F23.1 and were weakly activated. Reduction of the density of F23.1 adsorbed to plastic resulted in weak T cell activation, and these T cells did not induce B cell responses. Haptenated B cell populations, although recognized by E9.D4, were not activated. Separation of T and B cells by a 0.4-micron membrane prevented T-dependent B cell activation, although Th cell-derived B cell-activating lymphokines would be assayed across these membranes. These results suggest a polyclonal noncognate B cell activation that depends on physical contact between B cells and activated T cells. The requirement for a cognate interaction of Th with B cells for the production and delivery of B help can therefore be overcome by activating Th cells with high densities of T cell receptor ligands.     

Triggering of monoclonal human lymphoma B cells with antibodies to IgM heavy chains: differences of response obtained with monoclonal as compared to polyclonal antibodies

A comparative study of human B lymphoma cells activation by monoclonal (murine hybridoma) antibodies to mu heavy chains (Ma-mu) as compared to polyclonal (rabbit) antibodies to mu heavy chains (Ra-mu) has been carried out. Early events related to calmodulin activation such as 86Rb influx and changes in cell volume at 4 h could be induced by Ma-mu. One antibody (AF6) approached Ra-mu with regard to the strength of response obtained. However, Ma-mus including AF6 were deficient in inducing DNA synthesis under conditions where this was achieved with Ra-mu. Studies in one lymphoma, where stimulation of re-expressed surface IgM could be studied, revealed that Ma-mu was deficient in stimulating re-expressed sIgM. These findings raise questions with regard to polyclonal antibody to surface Ig as a model for B cell triggering by antigen and suggest that antigen-induced B cell triggering may be more complex than indicated by previous studies with polyclonal antibody.     

Characterization of two distinct antigens expressed on either resting or activated human B cells as defined by monoclonal antibodies.

Two antigen systems (L29 & L30) expressed on two distinct human B cell subpopulations were identified by using BL1-4D6 and TB3-7D5 monoclonal antibodies, respectively. L29 was expressed on approximately one-third of B cells in human lymphoid tissues. These B cells associated with L29 were large activated B cells located in the germinal centres of lymphoid follicles. L30, on the other hand, existed on approximately two-thirds of B cells mainly located in the mantle zone of lymphoid follicles, most of which also expressed IgM and IgD on their cell membrane. In addition, L30 was shared on mature granulocytes. With the use of polyclonal activators such as pokeweek mitogen (PWM) and protein A-bearing staphylococci (SAC), L29 antigen was inducible on PWM- or SAC-stimulated B cells in correspondence with the emergence of Tac and T10 antigens of these B cells. In contrast, L30 antigen on the B cells stimulated by the polyclonal activators was decreased in its expression and was finally lost from these B cells. Although none of L29 and L30 was expressed on normal, non-activated human thymus and peripheral T cells, L29 but not L30 was expressed on concanavalin A-activated T cells. Immunochemical studies showed that L30 consist of a single polypeptide with mol. wt of 40,000. L29 antigen is presently under study.     

B lymphocyte activation by insoluble anti-mu antibodies in patients with systemic lupus erythematosus.

In order to study the capacity of anti-immunoglobulin (Ig) antibodies to induce proliferative responses in peripheral blood mononuclear cells (PBC) from patients with systemic lupus erythematosus (SLE), anti-mu coupled to Sepharose beads (anti-mu) was used as a polyclonal activator. In 18 patients, a strong proliferative response was associated with inactive disease, and the response was lower in clinical active disease (P less than 0.02). An inverse correlation could also be observed in six patients who were studied longitudinally (P less than 0.01). These results indicate that anti-mu responsiveness is closely related to disease activity in SLE. In addition, sequential data obtained from two patients during an early stage of clinical deterioration suggest that a low anti-mu response might be an early indicator of a clinical relapse. In the patients investigated, the anti-mu response was not correlated with the response to pokeweed mitogen (PWM), or with the quantity of B cells. When T cell depleted cell fractions were studied, marked increases in the proliferative responses to anti-mu were observed in some patients. These studies suggest that the response to anti-mu might be modified by T cells to a variable extent in patients with SLE.     

The effects of a monoclonal anti-mu chain antiserum on the in vitro growth of normal B cells

Membrane immunoglobulin (Ig) on B cells plays a role in specifically focusing antigen onto the cell surface. An unresolved question is whether the Ig receptor itself has the capacity to deliver a signal to the B cell, and if so what is the mechanism of that signal. To explore these questions we have developed a monoclonal rat anti-mouse mu chain antibody (E4). In this study it is shown that protein A-Sepharose-purified monoclonal anti-mu chain antibody inhibits proliferation of normal B cells in vitro even at the single cell level. This effect was independent of Fc receptor interactions. Furthermore, E4 was shown to inhibit lipopolysaccharide-induced mitogenesis. These results provide evidence that interaction of antibody with only a single determinant on the Ig receptor can deliver a negative signal to the B cell.     

Differential effect of anti-HLA class I monoclonal antibodies on resting and in vivo-induced B lymphocytes

The role of HLA class I antigens in B cell triggering was investigated by analyzing the effect of monoclonal antibodies (MAbs) directed to such molecules on the in vitro function of resting and in vivo-induced lymphoblastoid (LB) B cells. Staphylococcus aureus Cowan I (SAC)-induced proliferation of high-density B lymphocytes was markedly inhibited by W6/32 MAb (reactive with a monomorphic determinant contributed by an alpha-chain and beta 2-microglobulin) but not by FG2/2 MAb (reactive with beta 2-microglobulin). The inhibition was not due to either a toxic effect or a change in the response kinetics. In contrast, LB B cells' spontaneous DNA synthesis and IgG production was not altered by such MAb, although these cells possessed surface HLA class I antigens. These findings suggest that HLA class I molecules are involved in the activation process of resting but not mature B lymphocytes.     

Chlamydia trachomatis stimulates human peripheral blood B lymphocytes to proliferate and secrete polyclonal immunoglobulins in vitro

Infectious Chlamydia trachomatis (LGV strain), obligate intracellular bacteria, stimulated human peripheral blood lymphocytes to proliferate and secrete immunoglobulins in vitro. In contrast, mock-infected preparations were unable to induce similar responses in peripheral blood lymphocytes. Although levels of immunoglobulin secreted into the media of LGV-stimulated cultures were greater than 10 micrograms/ml, we estimated that less than 1% of these molecules were directed against the bacteria itself, suggesting polyclonal antibody production. Since stimulation with Formalin-killed bacteria resulted in comparable numbers of plaque-forming cells (PFC) as infectious particles, we concluded that the polyclonal immunoglobulin response was not dependent on the in vitro chlamydial infectious process. The polyclonal PFC response induced by LGV was highly sensitive to monocyte inhibition. Although LGV induced proliferation of predominantly B cells, the numbers of generated PFC was increased by the addition of autologous T cells. Neither lymphocyte proliferation nor PFC responses of normal human volunteers correlated significantly with the presence or titer of antichlamydial antibodies in their sera.     

Immunogenic and antigenic epitopes of immunoglobulins. XIV. Antigenic variants of IgG4 proteins revealed with monoclonal antibodies

Two IgG4 paraproteins having heavy chains of normal molecular weight are shown to be antigenically distinct in their reactivity profiles with 18 monoclonal antibodies having specificity for the IgG2 or IgG4 subclasses. One protein expresses IgG2 and IgG4 epitopes within the C gamma 2 domain. A second protein is deficient in the expression of IgG4 Fc-specific epitopes. These proteins will be of value in defining the structural basis of Fc effector functions and individual epitope expression.     

Epithelial cells secrete the chemokine interleukin-8 in response to bacterial entry.

Bacterial invasion of mucosal surfaces results in a rapid influx of polymorphonuclear leukocytes. The chemotactic stimulus responsible for this response is not known. Since epithelial cells are among the first cells entered by many enteric pathogens, we investigated the ability of epithelial cells to provide an early signal for the mucosal inflammatory response through the release of chemotactic cytokines. As shown herein, the chemokine interleukin-8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by intestinal and cervical epithelial cells in response to bacterial entry. Moreover, a variety of different bacteria, including those that remain inside phagosomal vacuoles, e.g., Salmonella spp., and those that enter the cytoplasm, e.g., Listeria monocytogenes, stimulated this response. Increased IL-8 mRNA levels could be detected within 90 min after infection. Neither bacterial lipopolysaccharide nor noninvasive bacteria, including Escherichia coli and Enterococcus faecium, induced an IL-8 response. Moreover, tumor necrosis factor alpha, which is known to be expressed by some epithelial cells, was not detected in the culture supernatants after bacterial entry, and addition of anti-tumor necrosis factor alpha antibodies had no effect on the IL-8 response following bacterial entry. These data suggest the novel concept that epithelial cells serve as an early signaling system to host immune and inflammatory cells in the underlying mucosa following bacterial entry.     

Isolation of porcine colostral immunoglobulins and preparation of monospecific anti-gamma, anti-alpha, and anti-mu chain antibodies using agarose-linked immunosorbents.

A simple method for the simultaneous isolation of IgG2, IgG1, IgA and IgM from porcine colostral whey is described. It makes use of DEAE-cellulose chromatography followed by repeated Sepharose 6B exclusion chromatography after the removal of fat, casein and lipoproteins, Fused rocket immunoelectrophoresis is used to show the elution patterns of each immunoglobulin class. In addition, the prepation of monospecific anti-gamma, anti-alpha and anti-mu sera by means of agarose-linked immunoadsorbents is also described.     

Activation of macaque T cells and B cells with agonistic monoclonal antibodies

A series of mouse monoclonal antibodies (mAb) to human differentiation antigens known to have agonistic activity for human T or B cells was found to bind specifically to macaque T or B cell subsets. Most of these mAb also stimulated macaque lymphocyte proliferation, implying that they recognize functional homologues in monkeys. Anti-CD3, anti-CD28 (9.3), and anti-Lp220 (CD45R) mAb stimulated proliferation of both human and macaque T cells; similarly, anti-IgM and anti-CDw40 mAb stimulated both human and macaque B cells. In contrast, anti-CD20 and anti-CD39 mAb, which are known to stimulate human B cells, did not stimulate macaque B cells. A human low-molecular weight B cell growth factor (BCGF) and anti-IgM were co-stimulatory for macaque splenic B cells but not for blood B cells, suggesting that B cell subpopulations may differ in their responsiveness to BCGF. The results show that functional epitopes on some lymphocyte surface molecules such as CD28 or CDw40 are conserved in primate evolution. Functional epitopes on other cell surface molecules such as CD3 and CD20 may have more complex evolutionary constraints.     

CD5 monoclonal antibodies react with human peripheral blood dendritic cells.

CD5 monoclonal antibodies (MAbs) define a 67,000 kd monomeric glycoprotein expressed predominantly by thymocytes, mature T cells and a subpopulation of B cells. CD5 is believed to be an alternative signaling molecules capable of increasing the supply of second messengers and thereby altering the cellular response threshold to other activation stimuli. Human peripheral blood dendritic cells (PBDC) are a circulating component of the immune dendritic cell family, which also includes Langerhans' cells in epithelia and interdigitating cells in the T-cell domains of lymphoid tissues. PBDC comprise less than 1% of the peripheral blood mononuclear cell fraction. They are morphologically, immunophenotypically, and functionally distinct from monocytes. In this study, we report that at least a subpopulation of PBDC react with the anti-CD5 MAbs Leu-1 and UCHT2, which define the two major non-crossblocking CD5 epitopes. In contrast, Langerhans' cells, interdigitating cells, monocytes, and macrophages were uniformly CD5-. These findings suggest that PBDC can express the CD5 molecule. Furthermore, they define an additional feature of many enriched PBDC that distinguishes them from monocytes and certain other mononuclear leukocytes, and may provide insights into their activation pathways.     

B7-1 and B7-2 monoclonal antibodies modulate the severity of murine Lyme arthritis.

We assessed the role of B7-1 and B7-2 costimulatory molecules on the course of murine Lyme borreliosis because experimental Lyme arthritis is dependent, at least partially, upon the development of the host immune response and these costimulatory molecules have been implicated in CD4+ T-cell differentiation. We demonstrated that Borrelia burgdorferi infection upregulated the surface expression of B7-1 and B7-2 in macrophages and B7-2 expression in B cells. Anti-B7-2 monoclonal antibody (MAb) or both anti-B7-2 and anti-B7-1 MAbs produced a dose-dependent increase in the severity of Lyme arthritis in C3H/HeN mice. In contrast, the administration of an anti-B7-1 MAb reduced the degree of arthritis. These effects occurred independently of significant alteration in B. burgdorferi-specific immune responses, including splenocyte proliferative responses to B. burgdorferi, B. burgdorferi antibody levels and specificity, and mRNA levels of gamma interferon, interleukin-4 (IL-4), IL-10, and IL-12 in the spleen. These results demonstrate that signaling delivered by B7-1 and B7-2 plays a role in determining the severity of acute murine Lyme arthritis.