Antibody-forming Cell Precursors Research Articles

B cell modulation in transplantation.

B cells are cells of the lymphoid lineage characterized by the surface expression of antibody/immunoglobulin (Ig) as the B cell receptor (BCR). B cells are the precursors of antibody-forming plasma cells. It has recently been appreciated that subpopulations of B cells may also act as immunoregulatory cells. These ‘regulatory’ B cells were first described in the mouse and subsequently in humans 1,2, and mediate immunoregulation via the production of interleukin (IL)-10 and IL-35 3. B cells bind antigen via their BCR, and the outcome of this interaction depends upon the affinity of the BCR for the antigen and the environmental context. For example, B cell activation can be facilitated by the presence of complement components (C3b and C3d binding to CD21/CR2). B cells may also receive proactivation and survival signals via cytokines, including BAFF (B cell-activating factor belonging to the tumour necrosis factor family, also known as BLyS). Over-expression of BAFF is sufficient to drive abnormal B cell survival, hypergammaglobulinaemia and a lupus-like autoimmune disease in mice independent of T cell help 4. If the BCR aggregation is sufficient to induce signalling, the B cell internalizes bound antigen, processes it and presents in the context of major histocompatibility complex (MHC) class II. At the border between the B cell follicle and T cell zone of secondary lymphoid organs, the B cell MHC–peptide complex may be recognized by a cognate T cell. Following this T–B interaction, B cells return to the follicle to form the germinal centre, where they undergo somatic hypermutation and class-switch recombination. Here, B cells with a higher affinity for antigen are positively selected and differentiate into either memory B cells or plasma cells. A subset of CD4 T cells located within B cell follicles and characterized by expression of the transcription repressor Bcl-6 [known as T follicular helper (Tfh) cells] are essential for the development of germinal centre B cells 5. A small proportion of plasma cells arising from the germinal centre become established as long-lived plasma cells in the bone marrow. Given the potent and widespread effects of B cell activation, it is critical that there are also stringent control measures to prevent inappropriate B cell responses. To this end, the B cell expresses a number of inhibitory receptors, for example FcγRIIB, CD22, CD72 and PIR-B 6. In recent years, there has been increasing interest in how B cells, plasma cells and their associated antibody respond to allografts 7. Sensitized patients with preformed human leucocyte antigen (HLA) antibodies have an increased risk of acute and chronic antibody-mediated rejection (AMR), which significantly impacts allograft longevity 8. Furthermore, it is now well established that the appearance of de-novo donor-specific antibodies (DSAs) is associated with AMR and chronic allograft attrition 9. Outside their remit of antibody production, there is an appreciation that B cells may play a role in acute cellular rejection, also known as T cell-mediated rejection (TCMR). In contrast to these negative effects of B cells and antibody on the allograft, there is a growing body of evidence that B cells may be beneficial for long-term graft survival; a number of studies have shown a B cell transcriptomic signature in tolerant transplant recipients 10,11 and an up-regulation of B cell biomarkers in rejection-free transplant recipients 12 that may be due to the effects of ‘regulatory’ B cells. Possible strategies to target B cells in transplantation include: (i) B cell depletion; (ii) modulation of B cell activation; (iii) increase B cell inhibition; and (iv) enhancing the generation of regulatory B cells.

Antigen‐specific clone selection in vivo

THYMUS NEGATIVE SELECTION In 1959–1961, I was far from Burnet’s influence studying the pathogenesis of virus-induced mouse leukaemia in London, UK. As a result of this work, I discovered the immunological function of the thymus.1 How that came about has been recounted elsewhere.2 Grafting thymus tissue reversed the immune defects of neonatally thymectomized mice but when thymus came from a strain of mice allogeneic to the thymectomized recipients, these were able to reject skin grafts from the allogeneic strains tested but not from the thymus donor strain (Figure 1). This led me to suggest, ‘when one is inducing a state of immunological tolerance in a newly born animal, one is in effect performing a thymectomy, not a complete thymectomy but a partial, selective or immunological thymectomy. In accordance with selective theories of antibody formation, injected antigenic material might make contact with certain cell types differentiating in the thymus and in some way prevent these cells from maturing to a stage when they would be capable of reacting immunologically.3 In other words, the thymus could negatively select antigenspecific clones in vivo and might be the seat where self-tolerance was induced. It was, therefore, a great surprise to me when Burnet asked me to see him during his visit in London in early 1962 and I was immensely pleased to show him the thymus graft results that were in press in the Proceedings of the Royal Society.3 He was, at that time, one of the few immunologists convinced of the immune function of the thymus, as was evident in the lecture he later gave in London.4 The finding that thymus-deficient mice were much more prone than normal mice to develop tumours after treatment with certain carcinogenic chemical agents and oncogenic viruses5,6 provided some experimental support for Burnet’s concept of immunological surveillance.7 Although T cells can, in some experimental situations, act as killer cells against tumour cells, I believe that if tumour antigens do cross-react with self-antigens that are ectopically expressed in thymus medullary epithelial cells, one will not be able to prime the immune system to these antigens.2 POSITIVE SELECTION OF ANTIGEN-SPECIFIC T-CELL CLONES In 1966, I headed the Experimental Pathology Unit of the Walter and Eliza Hall Institute of Medical Research following the invitation of the then director, Gustav Nossal. With my first PhD student, Graham Mitchell, we gathered more evidence for in vivo selection of antigen-specific clones. As admirably recounted by Graham Mitchell in this symposium,8 we used genetically marked cells and adoptive transfer to established unequivocally and for the first time: (a) that thymusderived cells could be activated specifically by antigen9 (b) that they were not the precursors of antibody-forming cells,10,11 (c) that they were essential to help, via some type of collaboration, other lymphocytes derived from bone marrow to respond to antigen by producing antibody.11 These results were initially greeted with surprise and scepticism (for example, ref12), a common criticism being that two rare, clonally individuated

A form of immunologic tolerance through impairment of germinal center development.

Primary immunization with the T-cell-dependent antigen (4-hydroxy-3-nitrophenyl)acetyl (NP) coupled to human serum albumin results in the development of two pathways of B-cell development, the extrafollicular pathway and the germinal center pathway. Soluble, deaggregated NP-human serum albumin injected before immunization results in a marked diminution of clonable higher-affinity antibody-forming cell precursors--i.e., a form of immunologic tolerance within the secondary B-cell repertoire. We describe here the cellular changes in the spleen that underlie this tolerance. Using multiparameter flow cytometry, we show that tolerant mice develop far fewer NP-binding, peanut agglutinin-positive, or germinal center cells than the control immunized mice; 14 days after challenge control spleens have approximately 2 x 10(5) such cell per spleen, whereas the tolerant mice have approximately 1 x 10(4) cells. Furthermore, we demonstrate by immunohistology a reduction in the number of germinal centers containing lambda-bearing cells, characteristic of the response of C57BL/6 mice to NP. Taken together, these data suggest an impairment of germinal center development in the tolerant mice.

Soluble antigen profoundly reduces memory B-cell numbers even when given after challenge immunization.

The splenic B-cell repertoire of unimmunized C57BL/6 mice can be examined for anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) B cells of relatively high affinity by using a dual strategy. First, limiting numbers of splenocytes are polyclonally activated by Escherichia coli lipopolysaccharide and a mixture of interleukins 2, 4, and 5 in the presence of 3T3 filler cells, thus ensuring that many B-cell clones switch to IgG1 antibody production. Second, an enzyme-linked immunosorbent assay is geared to register only higher-affinity antibody by (i) detecting only bivalent IgG1 antibody and ignoring IgM and (ii) using a lowly substituted NP-conjugated protein as the capture layer. Naive spleens contain very few higher-affinity anti-NP B cells thus defined, but thymus (T)-dependent immunization causes the appearance of approximately 10(5) per spleen within 2 weeks. The development of these clonable anti-NP antibody-forming cell precursors can be virtually eliminated by a single injection of 1 mg of soluble, freshly deaggregated NP2-human serum albumin (HSA). This toleragen works not only if injected prior to challenge immunization, but even if given up to 6 days later. Soluble HSA works partially but not nearly as well as NP2-HSA, suggesting the possibility that the toleragen must act on T and B cells. NP conjugated to irrelevant carriers achieved partial tolerance in only one of four experiments. The studies demonstrate the need for continuing T-cell help throughout the process of memory B-cell generation. They also show that those recently activated T cells involved in this process can be silenced in vivo by soluble toleragen.

Changes in follicular dendritic cell and CD8+ cell function in macaque lymph nodes following infection with SIV251.

By virtue of their extensive meshlike network of dendritic processes expressing C3b, C3d and Fc receptors, follicular dendritic cells (FDC) found in lymphoid follicles are able to trap and retain unprocessed antigen in the form of Ag/Ab complexes (1-4). As such these cells provide the microenvironment for germinal centre reactions in which antigen specific B cells proliferate, differentiate into memory cells and antibody forming cell precursors and undergo class switching and affinity maturation. To date FDCs appear to serve two major functions. Firstly to present immunogen in the form of iccosomes to B cells and secondly as a source of of a costimulatory signal(s) required for B cell proliferation (Tew et. al. and Burton et.al., this volume). Although T cells are also required for germinal centre formation in vivo and for cluster formation and proliferation in vitro, little is known about FDC/T cells interactions. An understanding of these roles is achieved by dissecting the cellular and molecular interactions leading to the spontaneous cluster formation and proliferation observed in in vitro cultures of FDC-enriched lymphoid cells 5,6).KeywordsSimian Immunodeficiency VirusFollicular Dendritic CellCostimulatory SignalSimian Immunodeficiency Virus InfectionGerminal Centre Formation

Memory cell generation ablated by soluble protein antigen by means of effects on T- and B-lymphocyte compartments.

Adult C57BL/6 mice were injected with 100 micrograms of soluble, freshly deaggregated human serum albumin (HSA) to produce partial immunologic tolerance. Uninjected normal control (N) mice contain only approximately 100 B cells in their spleens with the capacity to (i) be activated in vitro into clonal proliferation by Escherichia coli lipopolysaccharide plus interleukins 2, 4, and 5, (ii) form IgG1 as well as IgM antibody, and (iii) display specificity for HSA when only IgG1 is allowed to score in an enzyme-linked immunosorbent assay (ELISA). Such N mice generate approximately 50,000 clonable anti-HSA IgG1 antibody-forming cell precursors in their spleens after T-dependent immunization with HSA absorbed onto alum and given with Bordetella pertussis adjuvant. Mice preinjected with soluble HSA (TOL) generate far fewer anti-HSA IgG1 antibody-forming cell precursors, termed anti-HSA memory cells. Splenocytes were transferred from N or TOL mice into lethally irradiated syngeneic recipients together with syngeneic bone marrow. Whereas N splenocytes generated plentiful memory cells within 2 weeks in antigenically challenged recipients, TOL splenocytes did not. Work with Ly-5 congenic mice ruled out memory cell generation from either the host or the bone marrow inoculum within this limited time. N T cells plus TOL B cells showed consistently lowered memory cell generation. TOL T cells plus N B cells showed an even greater lowering of adoptive memory cell generation. Thus the lowered response capacity of TOL mice resided in the T- and B-cell compartments. Attempts to show a suppressor component within the T-cell population were inconclusive, but a profound defect in capacity to respond to HSA in vitro was exhibited by the CD4+ T cells of TOL mice. B lymphocytes were harvested from T-dependently immunized mice 5 days after challenge, incubated with soluble HSA for 18 hr, and then adoptively transferred together with N T cells. The recently activated B cells were not rendered tolerant by this manipulation. The results argue for a major T-cell component in the process whereby soluble protein antigens ablate affinity maturation and memory cell generation.

Increase of ovalbumin (OVA)-specific B cells in the peripheral blood of egg-allergic patients

Allergen-specific B cells were detected by antigen-coated magnetic beads. The frequency of ovalbumin (OVA)-specific B cells was significantly higher in patients with egg white-allergy than in age-matched nonallergic individuals. These B cells, isolated by the immunomagnetic-beads method, produced anti-OVA antibodies of mostly IgM class when they were transformed by Epstein-Barr virus and cultured for about 2 weeks. Based on a chronologic analysis, the increase of OVA-specific B cells was found to precede the increase of IgG and IgE anti-OVA antibodies in the serum. These observations indicated that OVA-binding B cells in the peripheral blood are already committed to producing IgM antibody and probably are the precursors of antibody-forming cells of the IgG or IgE class.

Quantitative clonal analysis of the B cell repertoire in human lupus

To gain further insight into the origin of autoantibody hyperproduction in human lupus, we quantitated the B cell repertoire toward exogenous and self-antigens. Using the Spot-ELISA method and two panels of nine exogenous and 10 self-antigens, we found that the normal human immune repertoire comprises a high frequency of B cell precursors secreting IgM antibodies to self- and exogenous determinants. This repertoire was markedly deficient in precursors producing IgG able to bind self-antigens. In lupus patients, the absolute numbers of clone precursors of the immune repertoire expressing IgM receptors whose paratopes impart affinity to self- and exogenous determinants were higher than in control individuals. Additionally, IgG antibody-forming cell precursors with binding specificity for lupus-associated antigens were detectable in the repertoire of these patients. Based on these results, we propose that hyperproduction of human lupus-associated autoantibodies arises in a two-stage mechanism whereby a general activation of the multireactive immune B cell repertoire precedes an oligospecific expansion of selected B cell clonotypes.

Clonal repertoire analysis of murine B cells specific for repeat sequence antigens of Plasmodium falciparum

Clonal analysis of the murine B-cell repertoire has been used to investigate the possible role of tandem repeat sequence epitopes of Plasmodium falciparum in immune evasion. A limiting dilution culture system was used whereby murine spleen cells were stimulated with the B-cell mitogen lipopolysaccharide (LPS) in the presence of 3T3 fibroblast filler cells. One in three B cells were shown to produce clones secreting immunoglobulin measurable by an ELISA. The frequency of antibody forming cell precursors (AFCp) specific for the 3' repeat epitopes of the ring injected erythrocyte surface antigen (RESA) was estimated in non-primed mice and found to be low. However, an accurate frequency determination was not possible using this method since the detection of the few positive cultures was found to depend on the presence of more than one AFCp or its products. Limiting dilution analysis was used to assess the frequency and repertoire of splenic AFCp at various times after immunization with a synthetic peptide of the RESA 3' repeat epitope (8 x 4-mer), presented in various ways. There was no marked increase in LPS-responsive AFCp specific for this antigen at the level of either IgM or IgG secretion. This was in marked contrast to the antibody response in vivo, where moderate IgG antibody titres, normally indicative of a secondary response, were seen in the serum of the same mice used for AFCp assay. This discrepancy between serum titre and AFCp frequency following immunization was not apparent with a non-malarial antigen, keyhole limpet haemocyanin (KLH). It was concluded that the LPS-stimulated limiting dilution culture system was not registering RESA-specific memory AFCp. These results raise the possibility that the malarial antigens are deficient in memory B-cell generation, or that secondary responses to these determinants may arise from a distinct B-cell progenitor which is non-responsive to LPS in vitro.

Clonal analysis of autoantibody-producing cell precursors in the preimmune B cell repertoire.

Evidence for anti-self-reactivity in the preimmune B cell repertoire has been well documented. The present study aimed to determine the frequency of antibody-forming cell precursors in this repertoire whose Ig V regions impart reactivity to "self" or autologous Ag. Clones were activated in vitro with LPS and their secreted IgM antibody was assayed for reactivity by direct binding to cell surface or intracellular Ag. An IL-4-containing lymphokine mixture was added to the clonal cultures to induce the secretion of IgG1. The reactivity of secreted IgG1 with Ag would more closely resemble the binding required to activate B cells through their monomeric surface IgM and/or IgD. The results indicate a high frequency of precursors secreting IgM with reactivity to intracellular Ag, namely 1 in 37 +/- 6 B cells, with a marked paucity of response to cell surface molecules. The repertoire was markedly deficient in precursors secreting IgG1 able to bind to intracellular Ag, with only one clone detected by the screening of 3.0 x 10(6) spleen cells. No positives were detected for cell surface Ag. This suggested that the frequency of clones in the preimmune repertoire that express IgR with sufficient affinity to bind "self" molecules must be very low.

Analysis of the B lymphocyte repertoire by polyclonal activation Hindrance by clones yielding antibodies which bind promiscuously to plastic

We here describe a form of ‘noise’ in the ELISA as commonly performed on antigen-coated microtiter trays that represents a major hindrance to the accurate enumeration of infrequent antibody-forming cell (AFC) precursors (AFCp) specific for epitopes on monomeric proteins. Supernatants from cultures of lipopolysaccharide-stimulated murine splenocytes, when split into aliquots and separately assayed, scored as positive in parallel on ELISA trays coated with unrelated proteins and on uncoated trays. Some properties of such coincident false positives (CFP) noted were: (1) optical density (OD) ranges for CFP and non-CFP overlapped; (2) different members of CFP triplets on differently coated assay trays usually had similar OD values; (3) CFP-generating culture supernatants did not contain unusually high immunoglobulin concentrations; and (4) numbers of CFP-forming supernatants increased with increasing input cells/culture consistent with causation by single AFCp present at an approximate mean frequency of 1 in 6600 CBA splenocytes. It is proposed that CFP are due to AFC clones that secrete antibody reactive with some epitope(s) present in the assay tray itself. Repertoire elements with such ‘anti-plastic’ characteristics are rarer than anti-keyhole limpet hemocyanin (KLH) AFCp, but at least as frequent as anti-bovine serum albumin (BSA) or anti-transferrin (TFN) AFCp.

Antigen-specific drug-targeting used to manipulate an immune response in vivo.

The administration of dextran-conjugated cytosine arabinonucleoside (araC) to BALB/c mice at various times prior to but not subsequent to immunization with native dextran renders mice unresponsive to this thymic-independent antigen. These results demonstrate that the primary immune response to an antigen can be selectively and efficiently suppressed or eliminated in vivo by the delivery of a single dose of an appropriate antigen-cytotoxic drug conjugate. Evidence presented here indicates that the dextran-araC conjugate (toxogen) acts directly and selectively upon unprimed dextran-specific antibody-forming cell precursors, presumably by binding to their receptors and subsequent internalization of the resultant receptor-toxogen complexes. The resistance of antigen-primed mice to the cytotoxic effect of the toxogen could result from the failure of dextran-primed cells to reexpress antigen-specific receptors, from an alternative processing of the toxogen, or from the inability of the antigen-primed cells to internalize a second round of receptor-ligand complexes. We also determined that B cells responding to thymic-dependent antigens were not affected by the prior exposure to a toxogen. The inability to eliminate or suppress the primary response to a thymic-dependent antigen via the administration of a cytotoxic drug-antigen conjugate distinguishes the thymic-independent set of B cells from the thymic-dependent B-cell repertoire. The difference between these two B-cell compartments could be due either to differences in the amount of ligand bound to receptors or to differences in the trafficking patterns of receptor-ligand complexes within each cell type.

Growth of antibody-producing cell clones in microcultures

Publisher Summary This chapter describes growth of antibody-producing cell clones in microcultures. The objective of the microculture technique is to analyze the frequency of rare cells involved in the immune response. The precursors of antibody-forming cells (B cells), helper T cells, and suppressor T cells are considered rare cells. The microculture assay allows the assessment of the average clone size of the progeny of precursors, the class, the allotype, and other characteristics of the products of B cells. Many small aliquots of lymphoid cells are cultured in the presence of antigen. The number of cells in each aliquot is chosen so that a considerable fraction of cultures may not contain any precursor cell. From the fraction of non responding cultures, using the Poisson formula, it is possible to calculate the frequency of precursor cells.

Effect of antigen-induced suppressor B cells on development of memory B cells, carrier-specific helper T cells, and antibody-forming cells

This paper studies the presence of an Fc receptor on the surface of antigen-induced suppressor B cells (AISB), their sensitivity to antiproliferative agents, their interaction with other immunoregulator cells and with the precursors of antibody-forming cells (AFC). Mice were used in the experiments and were irradiated in a dose of 8.5 Gy (/sup 137/Cs). The effect of AISB on memory B cells is shown as is the effect of AISB on development of carrier-specific helper T cells. Also presented is the effect of AISB on development of AFC.

Role of antigen-binding cells in the formation of producers of antibodies and antigen-dependent nonspecific immunoglobulins

The role of antigen-binding cells in the formation of antigen-dependent nonspecific immunoglobulin producers in the Mishell-Dutton system was investigated. It was shown that the main part of antigen-dependent nonspecific immunoglobulin producers arise from the cells bearing antigen-specific receptors (as well as antibody producers). The nature of antigen-specific receptors of the precursors of antibody-forming and antigen-dependent nonspecific immunoglobulin-forming cells is discussed.

Effect of cyclosporin A on the induction and activation of B memory cells by thymus-independent antigens in mice.

The effect of cyclosporin A on the induction and activation of B memory cells by thymus-independent (TI) antigens was investigated. Studies were carried out in C57BL/6 mice, a strain in which TI.1 trinitrophenyl-lipopolysaccharide (TNP-LPS) and TI.2 dinitrophenyl-(DNP)-Ficoll antigens can elicit a secondary response. Evidence is presented that cyclosporin A does not adversely affect the primary or secondary response to TNP-LPS. In contrast, this fungal metabolite prevents the triggering of virgin B lymphocytes and TNP-LPS-induced memory cells by DNP-Ficoll. Cyclosporin A does not interfere with the generation of hapten-specific B memory cells by TNP-LPS or DNP-Ficoll. These findings are discussed in terms of B cell lineages leading to antibody-forming cell precursors and memory cells.

Idiotype-anti-idiotype network. II. Activation of silent clones by treatment at birth with idiotypes is associated with the expansion of idiotype-specific helper T cells.

BALB/c mice immunized with bacterial levan (BL) produce a vigorous antibody response that fails to include antibodies expressing the idiotype of the beta 2 leads to 6 fructosan-binding myeloma protein ABPC48 (A48). Treatment of newborn BALB/c mice at 1 d of age with 0.1-10 microgram of either the A48 myeloma protein or monoclonal proteins that share idiotopes with the A48 family, followed by immunization with BL 2-4 wk later, produces an anti-BL response that is dominated by the A48Id. Various degrees of activation of the A48Id BL response were observed by injecting mice with A48 monoclonal protein only up until 3 wk of age. Activation of the A48Id clones by treating with A48 monoclonal protein was ineffective in mice who were older than 4 wk. Elicitation of an A48Id BL response required specific antigenic stimulation with either beta 2 leads to 6 or beta 2 leads to 1 fructosan epitopes, because it does not occur after injection with TNP-Ficoll in spite of the A48 treatment. The expansion of A48Id clones in mice treated at birth with A48 monoclonal protein is associated with an increase in A48Id-specific helper T cells. The binding specificity of these cells was demonstrated by infusing them into nu/nu BALB/c mice and observing that they rendered help that enalbed the animal to mount an anti-TNP response after immunization only with A48-TNP, but not with MOPC384-TNP conjugates. The helper activity of these cells is sensitive to the effects of treatment with anti-Lyt-1.2 antibodies plus complement. A predominantly A48Id BL-specific response can be transferred into lethally irradiated mice by infusing them with purified T and B cells from A48-treated mice. The transfer of this response can be ablated by treating the T cells with anti-Lyt-1.2 antibodies plus complement. These results indicate that A48Id-specific helper cells possess the ability to select the A48Id-bearing B cell precursors for expression, thus exerting a fine-tuning effect on the idiotypic expression of the anti-BL repertoire. We propose that this idiotype-induced idiotype response, which can be, in principal, induced by idiotypes provided by the mother, plays an important role in the expansion of precursors of antibody-forming cells during embryonic as well as postnatal life.

Plasma Cells and their Precursors: II. Kinetics of B-Memory Cell Production in Rabbits

Rabbits were irradiated with 4.5 Gy in order to eliminate completely preexisting antibody-forming cell precursors. Sheep red blood cells were administered 24 h or 8 days after irradiation in order to induce the production of IgG B-memory AFCP. Resulting B-memory cells were triggered into antibody synthesis by a second dose of SRBC given 8 days after the challenge; the resulting IgG antibody clones were analyzed by isoelectric focusing. Memory IgG antibody clones were detectable from the third day after secondary immunization onward. It is concluded that antigen administered as early as 24 h after the irradiation induces B-memory cell production equally well as primary immunization 8 days after the irradiation. This B-memory cell production proceeds in the absence of detectable primary IgG antibody formation. Irradiated non-immunized rabbits showed spontaneous reappearance of IgG-AFCP with specificities to SRBC. In sharp contrast to the specifically induced production of B-memory IgG-AFCP mentioned above, this process took more than two months to reach potentialities comparable to those of «preexistent» AFCP present in normal, control rabbits.

The effect of x-rays on the precursors of antibody forming cells (B cells) as measured with the in vitro limiting dilution assay.

The effect of X-irradiation upon murine antibody-forming cell precursors (B cells) was established in cultures of spleen cells stimulated with the B cell mitogen lipopolysaccharide (LPS). At day 5 and 7 the numbers of IgM- and IgG2-secreting cells were determined in cultures of irradiated and nonirradiated spleen cells. From these numbers a Do of 0.6-1.2 Gy for the IgM, and of 0.9-2.1 Gy for the IgG2 response was calculated. Similar Do values were obtained under limiting dilution culture conditions. In the limiting dilution assay the effect of irradiation upon the size of the IgM-producing clones could also be determined. It was found that irradiation reduced the number of LPS-reactive B cells without affecting the size of the clones produced by the surviving cells.

Antibody-forming foci in soft-agar cultures of human peripheral blood cells

Human peripheral blood lymphocytes (PBL) from healthy blood donors were grown in soft-agar gel with sheep red blood cells (SRBC) and autologous plasma as a source of complement. After 4–6 days incubation, foci of proliferating hemolysin-forming cells, surrounded by a lytic area of 0.2–0.5 mm, were detected on the surface of the plates. The response was antigen specific, since new hemolytic areas were observed on pouring a fresh agar-SRBC mixture over the surface of primary cultures, but not on pouring a mixture containing rat or rabbit erythrocytes. The antibody response was significantly increased by addition to the cultures of polyethylene glycol 6000 (PEG), 8% final concentration. The mean number of foci was 8.4±2.2 in cultures without PEG and 36.2±2.3 in PEG+ cultures, both containing 9 × 10 6 lymphocytes. This finding is in agreement with observations on the frequency of precursors of antibody-forming cells among lymphoid populations. The explanation of the mechanism by which PEG 6000 modified the immune reactivity of PBL is not clear. However, we think that this technique provides a reliable methodology for PBL antigenic stimulation in vitro.