Search

Antibodies are an essential component of our immune system, underpinning the effectiveness of both the primary immune response to microbial pathogens and the protective and long-lived immunity against re-challenge. All antibodies are produced by relatively rare populations of plasmablasts and plasma cells, collectively termed antibody-secreting cells (ASCs). It is now apparent that ASCs are unique in the body in terms of their gene expression program and metabolic pathways that enable these cells to have an extraordinary rate of immunoglobulin gene transcription, translation, assembly and secretion. In this review we will discuss the cellular, metabolic and molecular specialization that allows ASCs to maintain such high rates of antibody production, in some cases for the life of the individual. Throughout the review we will link these exquisite cellular and molecular adaptations to the major regulators of ASC gene expression, in an attempt to define how the ASC phenotype and function is genetically programmed.

Being the sole source of antibody, plasmablasts and plasma cells are essential for protective immunity. Due to their relative rarity, heterogeneity and the loss of many canonical B-cell markers, antibody-secreting cells (ASCs) have often been problematic to identify and further characterize. In the mouse, the combination of the expression of CD138 and BLIMP-1, has led to many insights into ASC biology, although this approach requires the use of a GFP reporter strain. In the current issue of the European Journal of Immunology, two independent studies by Wilmore et al. and Pracht et al. provide alternative approaches to identify all murine ASCs using antibodies against the cell surface proteins, Sca-1 and TACI, respectively. Here we will discuss the advantages of these new approaches to identify ASCs in the context of our emerging knowledge of the cell surface phenotype and gene expression program of various ASC subsets in the murine and human systems.

Hypergammaglobulinemia and production of autoantibodies occur during many viral infections, and studies have suggested that viral antigen-presenting B cells may become polyclonally activated by CD4 T cells in vivo in the absence of viral engagement of the BCR. However, we have reported that CD4 cells in lymphocytic choriomengitis virus (LCMV)-infected mice kill adoptively transferred B cells coated with LCMV class II peptides. We report here that most of the surviving naïve B cells presenting class II MHC peptides undergo an extensive differentiation process involving both proliferation and secretion of antibodies. Both events require CD4 cells and CD40/CD40L interactions but not MyD88-dependent signaling within the B cells. B cells taken from immunologically tolerant donor LCMV-carrier mice with high LCMV antigen load became activated following adoptive transfer into LCMV-infected hosts, suggesting that B cells present sufficient antigen for this process during a viral infection. No division or activation of B cells was detected at all in virus-infected hosts in the absence of cognate CD4 T cells and class II antigen. This approach, therefore, formally demonstrates and quantifies a virus-induced polyclonal proliferation and differentiation of B cells, which, due to their high proportion, would mostly have BCR not specific for the virus.

Immunoglobulin E (IgE) is the key effector element in allergic diseases ranging from innocuous hay fever to life-threatening anaphylactic shock. Compared to other Ig classes, IgE serum levels are very low. In its membrane-bound form (mIgE), IgE behaves as a classical antigen receptor on B lymphocytes. Expression of mIgE is essential for subsequent recruitment of IgE-secreting cells. We show that in activated, mIgE-bearing B cells, mRNA for the membrane forms of both murine and human epsilon (epsilon) heavy chains (HC) are poorly expressed compared to mRNA for the secreted forms. In contrast, in mIgG-bearing B cells, mRNA for the membrane forms of murine gamma-1 (gamma1) and the corresponding human gamma4 HC are expressed at a much higher level than mRNA for the respective secreted forms. We show that these findings correlate with the presence of deviant polyadenylation signal hexamers in the 3' untranslated region (UTR) of both murine and human epsilon genes, causing inefficient processing of primary transcripts and thus poor expression of the proteins and poor recruitment of IgE-producing cells in the immune response. Thus, we have identified a genetic steering mechanism in the regulation of IgE synthesis that represents a further means to restrain potentially dangerous, high serum IgE levels.

In contrast to T cells, information on skin-homing B cells expressing the cutaneous lymphocyte antigen (CLA) is sparse. CLA expression on human B cells was investigated among circulating immunoglobulin-secreting cells (ISC) and among antigen-specific antibody-secreting cells (ASC) elicited by parenteral, oral or rectal primary immunization, or by parenteral or oral secondary immunization with Salmonella typhi Ty21a. CLA expression was examined by combining cell sorting with an enzyme-linked immunospot assay. Among all ISC, the proportion of CLA(+) cells was 13-21%. Parenteral immunization induced antigen-specific ASC of which 13% were CLA(+), while oral and rectal immunizations were followed by only 1% of CLA(+) ASC (p

Polyreactive antibodies are naturally occurring antibodies, primarily of the IgM isotype, that are capable of reacting with a wide variety of different self and non-self antigens. Previously, we reported that a B cell capable of making polyreactive antibody has Ig receptors on its surface that can bind different antigens. The present investigation was initiated to characterize these polyreactive antigen-binding B cells further. A panel of fluorescein isothiocyanate-labeled antigens (insulin, IgG Fc fragment or beta-galactosidase) served as probes to select polyreactive antigen-binding B cells by cell sorting. Our experiment revealed that these polyreactive antigen-binding B cells were mainly of the IgD isotype. They expressed high levels of CD40 and major histocompatibility complex class II molecules, but little or no B7-1, B7-2, or Fas. In contrast to the binding of antigens to monoreactive receptors (usually high affinity), the binding of antigens to polyreactive receptors (usually moderate or low affinity) did not up-regulate the expression of B7-1 or B7-2. Antigens that bound to polyreactive receptors, however, were internalized and degraded, although not as efficiently as antigens that bound to monoreactive receptors. Despite the ability of these B7- cells to process antigens, they were not able to activate T cells in a mixed leukocyte reaction. It is concluded that polyreactive antigen-binding B cells have properties that are consistent with the ability to induce immunological tolerance.

The first product of the humoral response to antigen is low-affinity antibody, produced by extrafollicular foci of antibody-forming cells (AFC) in organs such as spleen and lymph node. These cells proliferate rapidly but then undergo an equally rapid decline, so that they are present in only small numbers 14 days after immunization. We have used 6-parameter flow cytometry to isolate and examine the characteristics of (4-hydroxy-5-nitrophenyl)acetyl-specific AFC, looking in particular for those markers that might differentiate them from cells of the intrafollicular (germinal center) arm of the T-dependent immune response. At day 7 of the primary response, most AFC were found to express surprisingly low levels of B220, high levels of syndecan, and retain significant levels of surface IgG1. We then used enzyme-linked immunospot assays to demonstrate that the rapid decline of these cells was not likely to be due to migration to organs such as the bone marrow. Their decline could, however, be explained by apoptosis in situ, which was demonstrated immunohistologically by nick-end labeling.

The present experiments were initiated to see if cells capable of binding antigens could make polyreactive antibodies. Fluorescein isothiocyanate-labeled self and non-self antigens were incubated with B cells from normal individuals. Antigen-binding cells were separated from non-antigen-binding cells by flow cytometry, immortalized with Epstein-Barr virus and analyzed at the clonal level for their capacity to make polyreactive antibodies. Four to six times more cells making polyreactive antibodies were found in the B cell subset that bound antigens than in the B cell subset that did not bind antigens. The majority of the polyreactive antibodies were of the immunoglobulin (Ig)M isotype. Immunoflow cytometry revealed that cell lines making polyreactive antibodies bound a variety of antigens (e.g., insulin, IgGFc and beta-galactosidase), whereas cell lines making monoreactive antibodies bound only a single antigen. The binding of antigens to B cell lines that made polyreactive antibodies could be inhibited (range, 28%-57%) by both homogeneous and heterogeneous antigens. Both CD5+ and CD5- antigen-binding B cells made polyreactive antibodies, but the frequency was slightly higher in the CD5+ antigen-binding (85%) as compared to the CD5- antigen-binding (50%) population. Comparison of CD5+ B cells that bound antigens with CD5+ B cells that did not bind antigens showed that approximately 86% of the former, but only 15% of the latter, made polyreactive antibodies. It is concluded that cells capable of binding a variety of different antigens can make polyreactive antibodies and that antigen binding is a good marker for identifying polyreactive antibody-producing cells.

The disposition of teleost memory and plasma cells (PCs) has essentially been un-explored. As the organization of the teleost immune system differs significantly from that of mammals (i.e. no bone marrow or lymph nodes, hematopoietic anterior kidney), this disposition could be essential in understanding how comparable functions are achieved. To address this question, the primary and secondary antibody-secreting cell, B memory cell, and antibody responses to T-independent and T-dependent antigens were analyzed in trout. Although the TI and TD antibody responses did not differ substantively from one another, the secondary responses to both were significantly prolonged compared with the primary responses. Logarithmic increases in titer and affinity were achieved for both antigens during the primary, with only modest increases during the secondary response. Antibody-secreting cells, both PCs and plasmablasts, quantitatively paralleled antibody production, with antibody-secreting cells skewing to the hematopoietic anterior kidney for both antigens. However, the enhanced antigen-inducible response of immune fish (indicative of the memory pool) skewed to the peripheral blood and spleen. This pattern of memory versus PC disposition parallels that observed in mammals even though the organization of the respective immune systems differs considerably.

Interleukin-5 (IL-5) is a cytokine that participates in the regulation of antibody secretion, in particular promoting the secretion of IgA at mucosal sites. In this report, recombinant vaccinia viruses expressing IL-5 have been inoculated into mice and the appearance of antibody-secreting cells in the spleen and lungs investigated. Although vaccinia virus-expressed IL-5 did not increase the level of IgA in serum, antibody-secreting cells, measured in an enzyme-linked immunosorbent spot assay, appeared earlier in lungs when the immunizing virus expressed IL-5. These early B cells secreted either IgM or IgG1.

Plasma cells can be classified as long- or short-lived. The lifespan of a plasma cell largely depends on whether it arises from a germinal center or an extrafollicular locus and most importantly whether it can find a survival niche in the spleen or BM. In systemic lupus erythematosus (SLE) patients, long-lived plasma cells are believed to be responsible for the production of anti-RNA and anti-cardiolipin antibodies, whereas short-lived plasma cells, which are more susceptible to anti-proliferation therapies, are the main producers of anti-DNA antibodies. A previous study showed that transient overexpression of interferon-α (IFN-α), a cytokine that plays a pathogenic role in SLE, accelerates disease onset in lupus-prone NZB/W mice. In this issue of the European Journal of Immunology, the same group report that IFN-α induces large numbers of short-lived plasma cells, accompanied by high titers of anti-dsDNA antibodies in NZB/W, but not BALB/c, mice. Our commentary discusses this interesting observation in the context of the previous data regarding plasma cell differentiation and conveys our view about the clinical implications with respect to therapies that target plasma cells in SLE patients.

The immune response to cholera toxin B subunit given orally was studied in 13 human volunteers. A serum IgG and IgA antitoxin response was observed, which was boosted by a second immunization. Using an immunospot assay, cells spontaneously secreting anti-toxin IgG and IgA, but not IgM appeared transiently in the blood after immunization. There were 105 IgG- and 87 IgA-secreting cells per 2 x 10(6) mononuclear cells 7 days after the first immunization, and 282 IgG- and 413 IgA-secreting cells 5 days after the second immunization. A polyclonal increase in total IgM-secreting cells was observed. Few anti-toxin-secreting cells were observed in the bone marrow at the peak of the circulating cell response, which could be accounted for by contamination of the sample with peripheral blood, suggesting that the bone marrow is not a significant site of anti-toxin-secreting cells after oral immunization.

The pathogenesis of multiple sclerosis (MS) could involve an autoimmune response to proteolipid protein (PLP). Immunization of experimental animals with this major myelin protein can lead to experimental allergic encephalomyelitis. To identify a possible role of PLP as target antigen in MS, we evaluated T cell immunity to PLP in blood and cerebrospinal fluid (CSF) from patients with MS and controls by counting cells which in response to PLP in short-term cultures secreted interferon-gamma. The PLP-specific B cell response was analyzed by counting cells secreting anti-PLP antibodies. PLP-reactive T cells were detected in blood of most MS patients (mean value 1 per 20,408 mononuclear cells), and at 41-fold higher numbers in CSF (mean 1 per 500 CSF cells). Anti-PLP IgG antibody-secreting cells were detected in blood from most MS patients (mean 1 per 30,303 cells), but such cells were 49-fold more frequent in CSF (mean 1 per 625 cells). PLP-reactive T and B cells were also detected in blood and CSF from control patients, but at much lower numbers. A strong and persistent autoimmune response to PLP as well as to other myelin proteins, enriched in CSF, is proposed to be pathogenetically important in MS.

We analyzed the effects of interleukin 2 (IL 2) and IL 4 isolated and in association on the specific response of human B cells triggered by trinitrophenylated polyacrylamide beads (TNP-PAA). IL 2 induced an increase (more than 10 times) in the number of hapten-binding cells [detected by a rosette-forming cell (RFC) assay] as well as the generation of antibody-producing cells [detected by a plaque-forming cell (PFC) assay]. IL 4 induced an isolated RFC response without PFC response. We verified that the IL 4 (as well as 12)-induced RFC were hapten specific and mediated through membrane IgM. Density fractionation experiments showed that IL 4-induced RFC were equally distributed between high-density and intermediate-density B cells. IL 2 appeared to drive more B cells into the intermediate density fraction. IL 2-induced PFC belonged to the RFC population and were intermediate-size B cells. IL 2 drove more RFC into an activated stage and it induced the differentiation of a number of them into antibody-producing cells. The evaluation of the proportion of RFC able to incorporate thymidine showed that both IL induced a substantial proliferation of antigen-activated B cells. However, IL 4 inhibited the IL 2-dependent PFC without affecting the number of RFC nor the proportion of proliferating RFC induced by this IL. These results directly demonstrate that human IL 4 triggers the expansion of antigen-activated B cells and selectively inhibits the IL 2-induced differentiation.

Neurotropic coronavirus infection of mice results in acute encephalomyelitis followed by viral persistence. Whereas cellular immunity controls acute infection, humoral immunity regulates central nervous system (CNS) persistence. Maintenance of serum Ab was correlated with tissue distribution of virus‐specific Ab‐secreting cells (ASC). Although virus‐specific ASC declined in cervical lymph node and spleen after infectious virus clearance, virus‐specific serum Ab was sustained at steady levels, with a delay in neutralizing Ab. Virus‐specific ASC within the CNS peaked rapidly 1 wk after control of infectious virus and were retained throughout chronic infection, consistent with intrathecal Ab synthesis. Surprisingly, frequencies of ASC in the BM remained low and only increased gradually. Nevertheless, virus‐specific ASC induced by peripheral infection localized to both spleen and BM. The data suggest that CNS infection provides strong stimuli to recruit ASC into the inflamed tissue through sustained up‐regulation of the CXCR3 ligands CXCL9 and CXCL10. Irrespective of Ag deprivation, CNS retention of ASC coincided with elevated BAFF expression and ongoing differentiation of class II+ to class II–CD138+CD19+ plasmablasts. These results confirm the CNS as a major ASC‐supporting environment, even after resolution of viral infection and in the absence of chronic ongoing inflammation.

The biology of antibody-secreting cells is still a matter of controversial discussion. One of the key questions is how the migration and lifespan of antibody-secreting cells is linked to their generation. A paper in this issue of the European Journal of Immunology provides further information on the generation of long-term mucosal immune responses; however, the study leaves many questions unanswered as discussed in this brief Commentary.

It is generally accepted that memory B cells can be defined by their ability to produce, upon antigenic challenge, somatically mutated antibody molecules characterized by an increased affinity and by the expression of a downstream heavy chain isotype. However, the inability to isolate this particular B cell compartment has precluded the study of memory B lymphocyte physiology in man. We previously reported on the identification of an IgD- B cell subset in human tonsils that we defined as CD38- B cells, whose phenotype is highly reminiscent of that of memory B lymphocytes from the splenic marginal zone of rodents. In the present study, we developed a model of the measles virus (MV)-specific secondary antibody response in vitro to assess the presence of memory B lymphocytes in different B cell subsets isolated from human tonsils and explore the activation requirements of human memory B cells. Our findings show that the memory B cell pool resides in the CD38- B cell subpopulation and that the differentiation of MV-activated memory B cells into antibody-secreting cells can be achieved upon co-stimulation with interleukin (IL)-2 and IL-10, but does not require engagement of CD40. Interestingly, the CD40-mediated signal was found to synergize with Ig-cross-linking agents for the proliferation of memory B cells, but strongly suppressed their capacity to differentiate along the plasmacytoid pathway. Collectively, our results suggest that the CD40 signaling pathway is instrumental for the clonal expansion of the memory B cell pool, but does not operate in the later phase of the response, which allows their maturation into antibody-secreting cells.

CD45 exon 6-deficient mice show a profound block of thymocyte development at a transitional differentiation step from immature CD4+ CD8+ to mature single-positive cells. Only a few T lymphocytes are observed in the periphery of such animals, but B cell development is not affected. We investigated whether mature B lymphocytes in CD45 exon 6-deficient mice have normal functions in the absence of CD45 expression and show here that CD45-defective B lymphocytes from CD45 exon 6-deficient mice have intact B lymphocyte functions, including T-dependent and T-independent antigen-specific antibody production and class switching in vivo if normal CD4+ T lymphocytes were adoptively transferred into the mutant mice. From these results, we conclude that CD45 expression on B lymphocytes is not essential for B cell responses following antigen stimulation in vivo. However, CD4+ T helper function was severely diminished in CD45 exon 6-deficient mice, suggesting that the requirements of CD45 molecules in antigen-specific response and development are different between T and B lymphocytes.

Circulating spontaneous antibody-secreting cells (ASC) induced by mucosal and systemic immunizations in human volunteers have been characterized with respect to differentiation stage and homing commitments. Irrespective of the immunization route, the large majority of ASC co-expressed CD19 and HLA-DR, which are normally lost during the transition of plasmablasts to plasmocytes, as well as CD38, a marker of activated B cell blasts, expressed also by plasmocytes. However, these cells expressed neither CD28, a molecule acquired by plasmocytes, nor CD22 and CD37, which are lost during the transition of plasmablasts to plasmocytes. Therefore, the large majority of ASC found in peripheral blood after oral and parenteral immunizations are terminally differentiated B cells, but not fully differentiated plasmocytes. As a whole, the mucosally derived ASC population seemed to be more homogenously differentiated. CD25 was detected on few ASC, whereas ASC expressing CD71 were more numerous, especially among systemically derived ASC. Almost all ASC expressed the adhesion molecules CD44 and alpha 4-integrins, irrespective of immunization route. However, virtually all systemically derived ASC expressed L-selectin, recognizing the peripheral lymph node addressin, whereas only a minority of mucosally induced blood ASC expressed L-selectin. These studies are the first to demonstrate in humans that circulating precursors of mucosal B cell immunoblasts utilize organ-specific recognition mechanisms distinct from those of corresponding systemic B cells and appear to be more advanced in the B lineage maturation pathway. Specialization of receptor expression could explain both the unification of immune responses in diverse mucosal sites and the physiologic segregation of mucosal from non-mucosal immune mechanisms in humans.

Human B cells capable of spontaneous IgG secretion are commonly found in circulation and in lymphoid tissues such as tonsil and bone marrow (BM). The present study compares the mechanisms that regulate tonsil, blood and BM B cells capable of spontaneous IgG secretion. The BM cell subset produced IgG during a markedly longer period of time (14 days) than did tonsil and blood cell subsets (2-3 days). Blood and BM, but not tonsil, B cell IgG secretion depended on the presence of adherent cells, as demonstrated by adherent cell depletion and re-addition experiments. Stromal BM cells supported linear IgG secretion by non-adherent BM cells for 2 weeks, but were unable to prolong the short-term IgG secretion by tonsil and blood cells. Different factors induced IgG secretion in each of the three B cell populations as optimal IgG secretion by tonsil, blood or BM cell subsets required either tumor necrosis factor-alpha, interleukin-6 or fibronectin + interleukin-6, respectively. Finally, these populations also showed differences in the expression of adhesion molecules; the tonsilar cell subset was PNA+/- CD44+ CD49d+ CD49e- Leu-8+/-, the blood cell subset was PNA- CD44+/- CD49d+ CD49e- Leu-8+ and the BM cell subset was PNA- CD44+/- CD49d+ CD49e- Leu-8-. These results suggest that the mechanisms controlling the final differentiation and the expression of adhesion molecules in these B lymphocytes exhibit territorial specificity.

Single antibody-forming cells (AFC) specific for alpha(1-3) dextran (Dex) from i.p.-immunized BALB/c mice were enumerated in soft agar cultures by blotting on antigen-precoated membranes and subsequent staining via enzyme-coupled anti-IgM antibodies. Short cultures (2 h) revealed AFC as harvested ex vivo, while in long-term cultures (4 days), in the presence of lipopolysaccharide (LPS) as B cell mitogen, cells or colonies developed by differentiation in vitro. Whereas the spleen contained most AFC ex vivo in a sharp-peak response at 4 and 5 days after i.p. injection of Dex in aqueous solution, peritoneal exudate cells (PEC) contained only very few AFC. However, the same PEC population developed Dex-specific cells or colonies after 4 days of culture. The isotype of antibodies was IgM. The frequency of these Dex-specific LPS-inducible precursor cells rose exponentially in the course of the immune response to a broad plateau and was still, 11 weeks after Dex injection, approximately 40-fold higher than in non-immunized mice. Since these cells increased in frequency after antigen injection, and since they could not be detected as AFC during 2 h ex vivo, they were regarded as memory cells. They seemed to be arrested in vivo, but could be induced to differentiation and/or proliferation in vitro. Although these cells had the functional characteristics of memory cells as defined above, they produced anti-Dex antibodies of IgM isotype. Their population might be critical for the protection of the peritoneal cavity against microbial invasion from the intestines, and it may be significant in this context that we could evoke a peritoneal memory cell response only when antigen was injected intraperitoneally, but not intravenously. In athymic BALB/c-nu/nu mice only few of these Dex-specific memory cells were found. It is possible that T cells exert a regulatory influence on this pathway of differentiation.

We have studied a secondary IgG anti-dinitrophenyl response in dissociated mouse spleen cell cultures which is comparable in magnitude to the responses detected after adoptive transfer. The data indicate that the responsive precursor cell is not a recirculating memory cell but is an intermediate B cell type which appears in vivo after antigen challenge of primed mice. Both antigen and T helper cells are required for in vitro stimulation of the intermediate cell type into the division and maturation steps leading to IgG antibody secretion. The buoyant density of precursor cells has been analyzed by bovine serum albumin density gradient centrifugation, and is consistent with their recent origin by division from memory cells.

A single injection of mice with a low dose of sheep red blood cells results in a cyclic appearance of antibody-forming cells of the IgG classes. The peaks of the responses occur at the same time in the spleen and the thoracic lymph nodes. We have investigated the phenomenon with respect to IgG2a response by using the isoelectric focusing overlay assay and have assumed that in the great majority of cases, after immunization with limiting amounts of antigen, an individual spectrotype represents a “clonal” product. Enumeration of spectrotypes which arise only in the first peak and those which arise only in the second, suggests that the second peak of plaque-forming cells can be accounted for on the basis of the stimulation of a second population of precursor cells. Several possible mechanisms whereby new antigen-sensitive precursor cells can be turned on late in a primary response are discussed.

A rat anti-mouse monoclonal antibody, DNL 1.9, has been produced and shown to bind to all cells of the B lineage in mice. By quantitative immunofluorescence, DNL 1.9 binds to approximately 50% of spleen cells, 20-30% of lymph node cells, but no to thymocytes. In bone marrow and neonatal liver, 70-80% of the small cells and 10-20% of the large cells were identified by this antibody. Two-color immunofluorescence studies showed that all immunoglobulin-positive cells were also DNL 1.9-positive, while Thy-1.2-positive cells did not bind the monoclonal antibody. All pre-B cells in bone marrow , as identified by cytoplasmic IgM staining, were labeled by the DNL 1.9 antibody. A significant proportion of lymphocytes in the bone marrow which did not express cell surface immunoglobulin did bind DNL 1.9. Both direct and indirect plaque-forming cells were also shown to bind DNL 1.9 antibody. The antigen recognized by this monoclonal antibody was not immunoglobulin nor was the antigen present in normal mouse serum. All seven mouse strains tested showed similar patterns of antigen expression. These results show that all of the cells indentifiable as being of the B lineage were identified by this monoclonal antibody. It is possible that the cells which bind DNL 1.9 but do not express cell surface immunoglobulin may be very early precursors of B lymphocytes.

We have previously established human T cell hybridomas which produce IgE-binding factors. Incubation of one of the T cell hybridomas, 166A2, with human IgE dimer in the presence of 1 microgram/ml bradykinin resulted in the formation of IgE-binding factors having affinity for lentil lectin. The factors selectively enhanced both IgE-forming cell responses of rat mesenteric lymph node (MLN) cells and spontaneous IgE synthesis by human peripheral blood B cells of atopic patients, without affecting the IgG response. The same factors that enhanced IgE synthesis of B cells from atopic patients also enhanced IgE synthesis induced under bystander conditions by activated alloreactive T cells. Fractionation of the affinity-purified IgE-binding factors by gel filtration revealed three molecular mass species, i.e., 60 kDa, 30 kDa and 15 kDa. The 60-kDa and 15-kDa IgE-binding factors selectively enhanced both the spontaneous IgE synthesis by B cells of atopic patients and IgE response of rat MLN cells. In contrast, the 30-kDa IgE-binding factors had only marginal enhancing effects on the IgE synthesis by both human B cells and rat MLN cells. When the 166A2 hybridoma cells were incubated with IgE dimer in the presence of glycosylation-inhibiting factor (GIF), essentially all IgE-binding factors formed by the cells had affinity for peanut agglutinin (PNA) but for neither lentil lectin nor concanavalin A. All of the 60-kDa, 30-kDa and 15-kDa species, having affinity for PNA, selectively suppressed the potentiating factor-enhanced IgE response of rat MLN cells. The factors also suppressed the IgE synthesis of human B cells from atopic patients when the synthesis was enhanced by IgE-potentiating factor. The results indicate that human IgE-binding factors regulate IgE synthesis by both human and rat lymphocytes.

We identified the presence in the Aleutian skate, Bathyraja aleutica, of two classes of immunoglobulins (Ig), a high molecular weight Ig analogous to mammalian IgM and a low molecular weight Ig, similarly to the spiny rasp skate, Raja kenojei, (Kobayashi, K. et al., Mol. Immunol. 1984. 21: 397), using an immunological cross-reaction with the specific antisera to the spiny rasp skate Ig components. The antigenic similarity of the heavy chains of the Ig of the Aleutian skate and those of the spiny rasp skate was less than that between their light chains. Two types of Ig-producing cells, one producing the high molecular weight and the other forming the low molecular weight Ig, were present in the spleen of embryos and adults in the Aleutian skate at a ratio of 5-6:4-5. The number of these Ig-producing cells increased with advancing development of the embryos but was 1/20 to 1/50 of those of adults. Cells, each of which were capable of forming both classes of Ig, were found in the spleen of embryos but not in that of adults. These results suggested that the spleen of the Aleutian skate is the primary lymphoid organ for B lymphocyte differentiation and proliferation, possibly equivalent to the bursa of birds.

Mononuclear cells (MNC) obtained from adult rib specimen were investigated for their capacity to produce immunoglobulin (Ig) in vitro. Using limiting dilution analysis 3 populations of B lineage cells could be distinguished. The first produces Ig in culture without any intentional activation. These cells are strictly nonproliferating and sustain extraordinary secretory rates of 5 X 10(7)-10 X 10(7) molecules IgG, IgA or IgM/cell/h for at least 2 weeks. They occur at frequencies of 1 X 10(-4)-10 X 10(-4) (marrow MNC) or represent 1-5% of marrow B cells. Following exposure to infectious Epstein-Barr virus (EBV), these cells do not (a) proliferate, (b) express EBV-determined nuclear antigens (EBNA), (c) enhance their secretory rates or (d) show secretory activity for prolonged time. These cells are therefore EBV resistant. If these cells produced their antibody at similar rates in vivo, then their frequencies would suggest that they could provide up to 2/3 of daily synthetic rates for IgG and IgA and 20-30% of the daily IgM production. The second population of marrow B cells is exclusively committed to IgM production. Following exposure to EBV these cells proliferate, express EBNA and begin to secrete IgM. The third population represents 90% of marrow B cells. In our hands, these cells are unable to produce Ig in vitro.

The object of the present study was to compare the fine specificity of helper T cell receptors to that of humoral antibodies, the combining sites of which are presumed to represent replicas of antibody-forming cell precursor (AFCP) receptor combining sites. The main elements of our experimental system were a series of cross-reacting serum albumins coupled to the 4-hydroxy-5-iodo-3-nitrophenacetyl (NIP) hapten and a cell transfer system, in which albumin-specific helper cells mediate the secondary anti-hapten response of hapten-primed AFCP to hapten-albumin conjugate. Dose response analyses showed that the response in the transfer system was limited by two components only. Two experimental approaches, independent of each other, are described in this paper. The first consisted of establishing serological cross-reaction patterns in the albumin system by an extensive analysis in radioactive binding assays and comparing this pattern to the cross-stimulation pattern, which was obtained by determining helper activity of albumin-primed helper cells towards hapten conjugates of the various cross-reacting albumins in the cell transfer system. Since native serum albumins are particularly stable proteins, which possess similar physicochemical properties and can be expected to behave similarly in the body, the cross-stimulation patterns reflect, in all likelihood, cross-reactions at the level of the helper cell receptors. A possible interference of circulating anti-albumin antibody with the specificity of cross-stimulation was excluded in experiments in which column-purified helper T cells were used. This was corroborated by the paralysis experiments summarized below. In a second approach aimed at comparing the heterogeneity in helper and AFCP receptors, anti-albumin antibodies and helper cells were classified into subpopulations of defined specificities. Immunosorbent techniques were used for this purpose at the level of the antibodies, and paralysis experiments for the classification of helper cells. In this system, in contrast to the first approach, we assess the specificity distribution in unprimed T helper cells. A consistent picture emerged from the two experimental approaches. The discriminating power of the receptor system in primed and unprimed T helper cells was similar and of a high order, and a striking similarity to antibody specificity, in particular to that of highly avid antibody, was observed. Two cases of asymmetrical serological cross-reactions repeated at the level of the helper cells. The data strongly support the concept that AFCP and T helper cells express the same system of antigen-binding receptors. In addition, the experiments provide clear examples of how the distribution of receptor specificities can be selectively modulated in the helper cell population during ontogeny. Whereas in a normal mouse, bovine serum albumin (BSA) and sheep serum albumin cross-react strongly at the levels of both T helper and B cell receptors, in an animal low zone tolerant to BSA, this cross-reaction disappears selectively in the T cell compartment. This result shows that when differences in the specificity of T and B cell recognition are encountered, this does not necessarily imply receptors of a different nature on the two cell types. It also excludes the possibility that helper cell receptors are passively adsorbed, conventional humoral antibody.

The study was undertaken to elucidate some functional characteristics of T cell subsets in human cord blood. A comparison of the cellular interactions involved in the in vitro regulation of pokeweed (PWM) vs. Epstein-Barr virus (EBV)-driven B cell differentiation was done in vitro in short-term cultures of lymphocytes from newborns or adults. T cell subsets were isolated using the monoclonal antibodies OKT4+ and OKT8+. OKT4+ but not OKT8+ T lymphocytes from adults as well as neonates suppressed EBV-induced immunoglobulin (Ig) secretion of B lymphocytes from adults. This inhibition was mediated through gamma-type interferon (IFN-γ). B cells from newborns were not inhibitable by OKT4+ lymphocytes as a result of their insensitivity to IFN-γ. Helper activity for PWM-induced Ig secretion was exclusively contained within the OKT4+ population from adult T cell donors. This function was normally not detectable in any of the neonatal T cell subsets. OKT8+ cells from both adults and neonates suppressed PWM-induced Ig secretion, but required the collaboration with cells within the OKT4+ population. The suppressor activity in the PWM system was not IFN-γ-mediated. Thus, suppressor functions for EBV-vs. PWM-induced Ig synthesis were mediated through different pathways. There was no evidence of a unique suppressor system in the newborn. In the neonate, suppressor T cell activities are developed before T helper functions, a circumstance for which there is good evolutionary reason.

The role of antibody-forming cell (AFC) blockade in the genesis of hapten-specific tolerance induced by hapten (2,4-dinitrophenyl)-coupled pneumococcal polysaccharide (DNP-lys-S3) was investigated. Tolerizing doses of DNP-lys-S3 given to mice making antibodies to DNP-hemocyanin a day before assay markedly reduced numbers of detectable anti-DNP AFC. Furthermore, anti-DNP AFC were specifically inhibited by brief in vitro incubation with DNP-lys-S3, followed by extensive washing. This inhibition requires multivalent binding of tolerogen to immunoglobulin receptors on AFC, and appears to result from a decrease in the rate of antibody secretion per cell. A variety of data indicate that IgM AFC are more susceptible to blockade than IgG AFC, and the susceptibility of both cell types decreases with time after immunization. This probably reflects differences in the density and in the rate of loss of surface immunoglobulin receptors on IgM and IgG AFC and their immediate precursors. However, we conclude that under “conventional” tolerizing conditions, i.e. when tolerogen is given prior to immunogen, DNP-lys-S3 suppresses antibody formation primarily by inactivating DNP-specific precursor cells. AFC blockade is therefore probably only relevant in the suppression of (primary?) IgM responses by large doses of tolerogen. The relative importance of precursor inactivation and AFC blockade in tolerogenesis of IgM responses to T cell-independent antigens (such as 53) remains to be elucidated.

F1 male offspring of CBA/N female mice carry an X-linked immune defect, one manifestation of which is their inability to mount plaque-forming cell (PFC) responses to certain thymus-dependent (TD) and thymus-independent (T1) antigens. Reconstitution of PFC responses in (CBA/N × BALB/c)F1 (NBF1) male mice with immature, immunocompetent neonatal liver cells requires preparative host irradiation. The need for irradiation is not absolute since the radiosensitive “barrier” can be overridden if a sufficiently large number of donor cells are transferred, or if one waits a sufficiently long period of time between transfer and challenge. An additional characteristic is that this resistance distinguishes between subsets of trinitrophenyl (TNP)-specific and phosphorylcholine (PC)-specific B cell progenitors. Comparisons of lethally irradiated NBF1 females with males indicate that the two are qualitatively similar as hosts: responses to TD PC antigens appear before those to TI PC antigens, and quantal responses can be elicited under the appropriate conditions. In sublethally irradiated male recipients, however, the NBF1 male environment seems to operationally slow down the development of antigen-reactive B cells; this is reflected as the transplantation barrier, which discriminates between compartments of B cell progenitors. It is precisely this differential delay of maturation which has enabled us to observe that the emergence of antigen-specific responses proceeds in cycles of quantal, all-or-none, events. We discuss how the resistance of the immunodefective host environment to transplanted immature B cells may influence their differentiation.

Suppression of the antibody response by supraoptimal numbers of T helper cells was studied in vitro and found to have both a specific and a nonspecific component. Suppression did not depend on direct cell contract of T and B cells, as supernatants of activated T cells were just as inhibitory. Suppression by excess T cells (or T cell supernatant) was abrogated by the addition of macrophages. By using a tolerance induction protocol, it was shown that T cell supernatants induce partial tolerance in both T and B cells. This tolerance occurred in three different experimental settings – if adherent cells were physically removed by surface adherence, in the presence of anti-macrophage serum, or in the presence of very high concentrations of T cell supernatant. All three conditions stress the importance of the interaction between T cell supernatants and macrophages. The nonspecific form of suppression was not analyzed in detail here, but was also shown to be partly abrogated by macrophages, suggesting that it may be analogous to antigenic competition, one model of which has recently been shown to be abolished by addition of macrophages. The results suggest the presence of homeostatic feedback loops, due to excess T cell function – T cell products suppress the function of T cells, preventing the induction of further helper cells, and also suppress B cells directly. The balance between T cell suppression and cooperation is affected by macrophage function.

Removal of just the three N-terminal residues Lys-Val-Phe (TIP) on hen egg white lysozyme (HEL), by aminopeptidase cleavage, eliminates an antigenic determinant which is a recurrent and dominant focus of primary but not secondary antibody responses to HEL in a variety of mouse strains. We have generated an anti-idiotypic rabbit antiserum against such a TIP-dependent monoclonal antibody (mAb). This antiserum reacts with many different primary anti-HEL mAb, but fails to react with all of a variety of secondary anti-HEL mAb. The idiotype defined by this antiserum, termed IdXE, is a common feature of early anti-HEL antibody responses but does not appear in secondary responses. Although the presence of IdXE and TIP dependence is correlated in primary responses, studies of idiotype expression on mAb and on plaque-forming cells (PFC) using mixed erythrocyte monolayers clearly show that at the single-cell level the properties are separable, i.e., not all TIP-recognizing PFC display IdXE and a sizable proportion of cells producing non-TIP-dependent antibodies are IdXE+. The restricted idiotypy and specificity of early antibody responses to HEL occur in each of eight diverse mouse strains tested: it is not associated with a particular MHC haplotype, heavy chain allotype or light chain allotype. The finding of such strain-independent restriction in the early response pattern to a typical protein antigen is novel and suggests the involvement of highly conserved, potent regulatory mechanisms which are manifested as a limitation in the initial expression of the available repertoire.

After suicide of the anti-dinitrophenyl (DNP) clone E21 in a previous experiment (Eur. J. Immunol. 1975. 5: 58) several new clones (S clones) appeared promptly in the recipients. There was compelling evidence that they were then resistant to suicide, relative to E21. We now report further studies on these S clones and their antibodies. The antibodies of half of the S clones had affinities ten to one hundred times higher than E21 (3 × 10−7 M); none was lower. The four tested were also able to bind DNP on the particular conjugate used for the suicide procedure. Three of the S clones were serially propagated; they showed a very great capacity for proliferation, transferring into 510, 85 and 110 recipients each. When their cells were tested at subsequent passages, they recovered early after partial inhibition by radioactive antigen. Two S clones may still have been somewhat refractory to suicide but, probably in all three, rapid proliferation was largely responsible for the recovery of the few cells escaping suicide, and helped them to appear suicide resistant.

We studied the pattern of responsiveness of normal spleen cells to three different B cell mitogens: dextran sulfate (DS), lipopolysaccharide (LPS) and purified protein derivative of tuberculin (PPD). Activation by DS results in extensive proliferation of cells, most of which are not high-rate antibody-secreting cells and can be reactivated. PPD activates high-rate antibody synthesis in cells which are more restricted in mitotic capacity. LPS seems to activate both of these subsets of B cells. LPS exhibits strong mitogenicity in vivo and a large fraction of LPS-activated cells are high-rate antibody-secreting end cells. It is suggested that these distinct subsets of B cells include cells at different stages of differentiation.

Human B cells were cultured without added lymphokines, with interleukin 2 (IL2) or interferon-gamma (IFN-gamma) alone, or with a combination of IL2 and IFN-gamma. Treatment with IL2 alone induced differentiation of the B cells, as shown by an increase in the number of immunoglobulin (Ig)-containing cells and plaque-forming cells, and by a larger amount of Ig secreted into the medium. In most of the induced Ig-containing cells, heavy chains but not light chains were detectable by cytoplasmic staining. Treatment with IFN-gamma alone did not stimulate B cell differentiation. However, a combination of IFN-gamma and IL2 was more effective than IL2 alone in inducing B cell differentiation, as measured by further increases in the number of plaque-forming cells and in total Ig secretion. Furthermore, after treatment with both IL2 and IFN-gamma, most cells that contained cytoplasmic heavy chains also contained cytoplasmic light chains. We conclude that IFN-gamma acts in synergy with IL2 in B cell differentiation by enhancing light chain synthesis, leading to secretion of Ig molecules.

Rabbits were completely suppressed for kappa chain allotype b4.2, and autoantibodies against b4.1 or b4.2 could be raised in 2 out of 3 animals. The animal immunized with b4.1 produced anti-b4 and anti-Ms3, two activities which have never as yet been found together in one antiserum. Both autoantisera lacked the capacity to bind b4.2-IgM, whereas they precipitated b4.2-IgG very well. In one animal, anti-b4 IgM activity appeared after the sixth immunization, at the age of 14 months. Allotype suppression was maintained in both rabbits till the end of their lives whereas all control animals recovered within 6 months. The autoantisera which were specific for b4 IgG could not induce suppression in vivo. However, specific inhibition of b4 IgG secretion was observed in vitro.

The production of anti-self red cell antibodies has been studied in mice bearing the CBA/N X-linked B lymphocyte defect and their phenotypically normal littermates. Defective mice produced anti-self red blood cell (RBC) antibodies at markedly reduced levels after multiple injections of rat RBC. They also failed to show significant numbers of plaque-forming cells against enzyme-treated isologous RBC. These findings are discussed in terms of the nature of the CBA/N defect and the acquisition of a complete specificity repertoire.

Mice were i.v. immunized with various 2,4,6-trinitrophenyl (TNP)-carrier conjugates, and the development and localization of specific (i.e. anti-TNP) antibody-forming cells (AFC) in the spleen was studied. The nature of the carrier used, thymus dependent (TD) or thymus independent type 1 or type 2 (TI-1, TI-2), had marked effects on the number and time of appearance of AFC. However, no influence of the different carriers on the localization of specific AFC in the spleen was observed. By using a double immunocytochemical technique we could simultaneously determine specificity (anti-TNP) and isotype (class and subclass) of the AFC. The TNP-carrier conjugates evoked (intracellular) immunoglobulins with carrier-characteristic isotype distribution. No carrier-dependent localization pattern, in the lymphoid compartments of the spleen, of these AFC was found. These results show for the first time the actual in situ effects of various TI and TD carriers on the development of specific AFC. Based on the present findings, no direct evidence for a special role of the marginal zone in the immune response against TI-2 antigens could be demonstrated. We suggest that this new method for in vivo investigation, in combination with existing standard enzyme- and immunohistochemical techniques, can provide more insight into the precise role of lymphoid and nonlymphoid cells in TD and TI immune responses.

It has been demonstrated in the preceding report (Bach, M. A., Beckmann, E. and Levitt, D., Eur. J. Immunol. 1984. 14: 589) that phosphorylcholine (PC) on the bacterium Streptococcus pneumoniae R36a stimulated polyclonal as well as anti-PC plaque-forming cells (PFC) in mouse spleen in vivo. In this study, red blood cells from BALB/c mice (MRBC) were either conjugated with PC, 2,4,6-trinitrophenyl (TNP) or treated with phospholipase A2 (PLA2) to expose PC on the cell membrane (determined by hemagglutination with the anti-PC myeloma HOPC8). When BALB/c mice were immunized i.v. with the conjugated or enzyme-treated MRBC, a significant polyclonal antibody response occurred (p less than 0.05) using PC-MRBC or PLA2-treated MRBC, but not with TNP-MRBC or sham-treated MRBC. No anti-PC or anti-MRBC immunoglobulin-secreting cells developed after immunization. Repeated immunization with PC-MRBC resulted in similar levels of protein A PFC after each immunization but no anti-PC, anti-MRBC or anti-PC-MRBC PFC. Thus, PC on R36a or isologous RBC stimulated increased numbers of splenic plaque-forming cells. In the case of R36a, 10-25% of these PFC produced antibodies directed towards PC. In contrast, PC-MRBC or PLA2-treated MRBC, failed to evoke any anti-PC antibody responses.

The antibody response to a defined protein antigen, hen egg white lysozyme (HEL) has been investigated using an aminopeptidase-treated HEL molecule, des-1,2,3-HEL (AP-HEL). Surprisingly, removal of these three N-terminal residues eliminates an epitope which is a dominant B cell determinant recognized in the primary antibody response to HEL. Thus, the initial antibody response focuses on a very small region of the molecule. Even more striking is the observation that removal of this epitope markedly reduces the immunogenicity of HEL. Therefore, the epitope is not only the focus of the primary antibody response, but is essential for the initiation of the response. This report demonstrates that a selective mechanism must be activated during the response to this protein antigen. Of the multitude of B cell determinants present on HEL, only a limited number are focused upon by the immune system.

This paper investigates the validity of plaque morphology as a simple marker for distinguishing single cells producing antibody with different specificities. Plaques on an indicator mixture of related erythrocytes may be clear, with both red cell types lysed, or partial with only one lysed, or “sombreros” where both target erythrocytes are lysed but to different extents. The technique can be adapted for use with defined antigens by testing cells against indicator erythrocytes half of which are coupled at high density with the antigen and half at low density: again the population is split into clear and partial plaques. Various arguments and control experiments have been put forward to support the claim that plaque morphology depends on antibody specificity, and hence V region structure, and not on amount of antibody or C region changes: morphology remains the same at different temperatures of incubation, and as a plaque grows at 37 °C, and is not affected by different concentrations of complement in the medium.

Mice immunized with an optimal (10 μg), but not suboptimal, dose of levan (LE) became specifically unresponsive to a second dose given 2–8 weeks later. No evidence which could implicate an active suppression mechanism was found. In particular (a) spleen cells from unresponsive donors did not inhibit normal cells in a transfer system; (b) equilibration of serum antibody following 2 days celomic parabiosis between LE-treated and normal mice did not impair responsiveness of the latter. It was concluded that B cells were exhausted in the absence of memory cell accumulation. LE can therefore induce specific B cell deletion, not only directly with 1 mg, but also following immunization with 10 μg. The exhaustive capacity of another polysaccharide, SIII, was found to be much weaker. Cyclophosphamide (CP) (150 mg/kg) completely suppressed the responses to LE and SIII when injected with them. Recovery following CP alone was nearly complete 2 weeks later, but simultaneous injection of an immunizing amount of LE was succeeded by dose-related specific tolerance. Total suppression followed only 100 ng LE + CP, a 10 000-fold reduction in the “high zone” tolerizing dose. The corresponding reduction in SIII experiments was only 5-fold. The times for recovery following transfer of spleen cells from donors tolerant of LE in the exhaustion and CP models were 2 and 4 weeks, respectively; periods commensurate with B cell renewal. The basis for these further differences in the tolerogenic properties of LE and SIII are discussed.

To investigate the histocompatibility requirements for the macrophage-T cell interaction in the secondary antibody response, splenic T cells from antigen (carrier)-primed F1 hybrid mice were restimulated in vitro with carrier-pulsed F1, parental or allogeneic macrophages. Surviving T cells were cocultured with hapten-primed F1 or parental “B cells” and restimulated with the appropriate hapten-carrier conjugate. The IgG antibody-forming cell response was then measured using a plaque assay. Mapping of the genetic restriction was performed by use of different strain combinations. T helper cells could be restimulated in the presence of macrophages only provided they shared the I-A subregion of the major histocompatibility complex with the F1 T cells. T cells from F1 hybrids restimulated with parental or I-A-identical macrophages were shown to only cooperate with parental B cells of the same I-A haplotype as the macrophages used for restimulation. The defect was at the level of the macrophage, as addition of macrophages of the I-A haplotype used for the restimulation culture reconstituted the ability of F1 helper cells to cooperate with the I-A-nonidentical B cells.

The activity of rabbit antisera against nu/nu BALB/c lymphocytes was estimated in vivo and in vitro. It was found that anti-lymphocyte serum (ALS) against nu/nu lymph node cells suppressed the alloantigen reaction and the spontaneous rosette-forming cell (sRFC) or plaque-forming cell (PFC) formation for T-dependent (sheep red blood cells) and T-independent (lipopolysaccharide) antigens. ALS against nu/nu spleen cells affected only the sRFC and PFC for T-independent antigen. The former serum exhibited a high cytotoxicity for the suspensions enriched or depleted in B cells, while the latter was more cytotoxic for the suspension enriched in B cells. This may indicate that ALS anti-nu/nu spleen cells is specific for B lymphocytes, and ALS anti nu/nu lymph node cells is directed not only to B cells but also to a subpopulation of T lymphocytes. It may suggest the existence of a subpopulation of T lymphocytes in nu/nu lymph node cells.

Human peripheral blood mononuclear cells were depleted of surface IgM+ or IgD+ cells and assayed for mitogen-induced differentiation to immunoglobulin-secreting cells (ISC) of IgM, IgG and IgA classes. Stimulatory agents included T cell-dependent pokeweed mitogen, B cell mitogen Staphylococcus aureus bacteria strain Cowan I, and a combination of the two which gives uniform, high levels of ISC from all normal donors. Depletion of either IgM- or IgD-bearing B lymphocytes resulted in loss of cells bearing the other Ig class and blocked most of the mitogenic reactivity to anti-IgM and anti-IgD. Proliferative responses to Cowan I in these depleted populations were about 20% that of unfractionated mononuclear cells. Depletion of T cells increased the mitogenic response to Cowan I and to the two antibody preparations, showing that they are T-independent mitogens. Depletion of IgD+ cells caused partial loss of mitogen-induced IgM ISC (22%-60% of unseparated controls) but no loss of IgG or IgA ISC. Depletion of IgM-bearing cells caused complete loss of IgM ISC, but no loss of IgG or IgA ISC. We previously demonstrated that anti-IgM ISC, but no loss of IgG or IgA ISC. We previously demonstrated that anti-IgM antibody blocked mitogen induction of Ig secretion of these three classes in spleen cells, but only IgM secretion in blood mononuclear cells. Together, the results suggest that the majority of cells in normal blood responding to mitogens to mature to IgG or IgA production belong to IgM-, IgD- B cell subjects, in contrast to precursors of secreting cells for these isotypes in the spleen. Thus, these blood precursors appear to be more mature than the corresponding spleen cells.

The role of IgD (delta) in the induction of tolerance in murine B lymphocytes was explored in the in vivo adoptive antibody response to bovine serum albumin (BSA). Delta+ or delta- B cells purified on the fluorescence-activated cell sorter were injected, with or without purified T cells, into lethally irradiated intermediate hosts, which were rendered tolerant by an injection of deaggregated BSA one day after the cell transfer. Spleen cells from the intermediate hosts were treated with anti-Thy-1.2 antiserum and complement mixed with normal T cells and were transferred to final hosts. The ability of the B cells of the final hosts to respond to the tolerogen BSA and to dinitrophenylated human gamma globulin antigen was examined. This approach enabled examination of the function of the B cell subpopulations in an environment free of tolerogen and of induced T suppressor cells. The results revealed that in the presence of T cells the delta+ subpopulation was highly resistant to tolerance induction, whereas the delta- subpopulation was highly susceptible to tolerance induction. However, there was no difference in susceptibility to tolerance induction between the two subpopulations when tolerance was induced in the absence of T cells.

The immune response of mice to the synthetic polypeptides (T,G)-A–L and (H,G)-A–L is genetically controlled by the Ir-1 locus. In an attempt to characterize the cell type(s) involved in this control, DNP-conjugates of (T,G)-A–L and (H,G)-A–L were used to study the carrier function of these polypeptides. In adoptive cell transfer experiments carried out in C57BL and C3H mice it was found that in contrast to the respective high responder strain, low responder mice appear to be incapable of recognizing these polypeptides as carriers. This is compatible with the hypothesis that low responders are defective with respect to the specific stimulation of thymus-dependent lymphocytes by (T,G)-A–L or (H,G)-A–L, or to the cooperation of these cells with the precursors of antibody-producing cells.

The increase in affinity of anti-2, 4-dinitrophenyl (DNP) antibodies of IgM class in tadpoles was followed as a function of time after administration of antigen by the inhibition of inactivation of DNP-bacteriophage T4. A 10-fold increase in binding affinity was observed over a period of 3–4 weeks after injection.

A reverse hemolytic plaque assay utilizing protein A-coated sheep red cells and a specific rabbit anti-rat IgE preparation has been adapted for the enumeration of rat IgE-secreting cells derived from the IR-162 rat plasmacytoma and from rats infected with Nippostrongylus brasiliensis. Under optimal conditions, approximately 10-15% of the viable plasmacytoma cells were scored as plaque-forming cells. In rats infected with 5000 Nippostrongylus brasiliensis larvae, a maximum of 2 x 10(6) IgE-secreting cells were found in the mesenteric lymph nodes, and no IgE plaque-forming cells in their spleens. The kinetics of the mesenteric lymph node plaque-forming cell responses closely coincided with total serum IgE levels, with maximum responses occurring 15-16 days after infection. There was a high degree of correlation between the mesenteric lymph node IgE plaque-forming cell responses and total serum IgE levels of individuals rats. It was concluded that the IgE-secreting cells in the mesenteric lymph nodes contributed, in a large way, to the elevated levels of IgE found in the circulation of these rats.