Beverley L. Pike

Spontaneous reverse haemolytic plaque formation. I. Technical aspects of the protein A assay

Abstract The technical factors affecting the detection and measurement of spontaneous immunoglobulin secretion by circulating normal human lymphocytes with a protein A plaque assay have been investigated. Each of the parameters influencing the numbers of plaques obtained, protein A coupling to indicator red cells, antibody and complement concentrations and mixing conditions are considered and a recommended procedure for optimum plaque formation is suggested.

Cell Fractionation Methods and the Target Cells for Clonal Abortion of B Lymphocytes

Since we last reviewed the work from our laboratory on B lymphocyte differentiation and the tolerance problem (Nossal et al. 1977), further confirmation of the exquisite sensitivity of immature, differentiating B cells to negative signals has come from a number of laboratories (Venkataraman &. Scott 1977, Szewczuk & Siskind 1977, Elson 1977, Kincade et al. 1978). As is to be expected in a field with many complexities, some differences in results and interpretation have emerged. The purpose of the present paper is to present new results in the tolerance field which we have obtained in the last year, and to discuss these in the light of any controversies that have arisen. We have previously proposed that the physiologically most important mechanism of tolerance in the B cell population is the existence of a differentiation stage where the preferred reaction with antigen is the receipt of an irreversible negative signal. Under such a mechanism, potentially self-reactive clones would be aborted by contact with self antigens. They would thus never reach the stage of being triggerable and a danger to the body. We obtained evidence in favor of such a clonal abortion mechanism in the mouse with B cell populations from newborn spleen (Stocker 1977, Nossal et al. 1977); adult bone marrow {Nossal & Pike 1975a) and the spleens of lethally irradiated adult mice were repopulated with fetal liver stem cells {Nossal & Pike 1975b). The biggest single problem in coming to grips with the cellular and molecular mechanisms underlying clonal abortion arises from the fact that all these tolerizable maturing B cell populations represent heterogeneous cell mixtures. They are heterogeneous in terms of celt maturity, each containing B cells, pre-B cells with intracytoplasmic Ig but no surface Ig receptors (Owen et al. 1977) and less differentiated cells in-

An ELISA assay efficiently detects clonal antibody formation by single, hapten-specific B lymphocytes

Spleen cells from non-immunized adult mice were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B cells. These B cells were cultured either singly or in very small numbers in 10 microliter microcultures with 0.1 microgram/ml of the T cell-independent (TI) antigen FLU-E. coli lipopolysaccharide (FLU-LPS). Cultures were either filler cell-free, or supported by the addition of 10(5) CBA/N thymus cells per well. At 4-6 days, culture supernatants were assayed for the presence of anti-FLU antibody either by an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). With the filler cell-free cultures, B cell proliferation was scored microscopically before removal of culture supernatant. The cultured cells from each well were assayed for their capacity to form directly hemolytic FLU-specific plaques. In the filler cell-free system, the ELISA was much more sensitive than the plaque assay in identifying antibody-forming cell (AFC) clones, with over 10% of fractionated B cells yielding clones secreting detectable antibody, though with a low mean optical density (OD). This value represented over 80% of the proliferating clones. In the more efficient, filler cell-supported system, the difference between the 2 read-out methods was smaller. Here, one half of the hapten-specific B cells formed AFC clones, the highest cloning efficiency yet reported for an antigen-driven system. Comparative studies showed the RIA to be only marginally more sensitive than the ELISA, and not nearly as convenient for routine use.

Current problem areas in the study of B lymphocyte differentiation.

If the understandable excitement about the presence and functional significance of IgD receptors on the surface of a large percentage of B lymphocytes is to be placed in suitable historic perspective, it can only be in the context of the wider field of B lymphocyte differentiation. Knowledge about the many life stages, both preand post-antigenie, during which B cell development can be observed has accumulated rapidly, chiefly because of improvements in a variety of tissue culture and cloning assays and the increasing availability of cell surface markers and biophysical separation techniques to help in the identification of various B cell subsets. As is common in an emerging field, overall understanding is hampered by competing nomenclatures, and by the fact that most of the leading laboratories use differing operational criteria to describe, enumerate and assess the function of the various differentiation stages. The object of this paper is to present an overview of B cell differentiation from the viewpoint of a group of linked laboratories that has been investigating various facets of the problem for the past two decades. The analysis will reveal that the number of identifiably different cell sets which need to be understood is surprisingly large. Special emphasis will be placed on a phase of non-specific B cell activation that has not yet been the object of extensive study outside the Hall Institute., and on mechanisms of tolerance induction. We hope to show that despite the undoubted complexity of the problem, a definitive framework for classification and further research is emerging. The chief objects of the review are to suggest a provisional nomenclature and to highlight problems that are amenable to experimental approach in the near to medium term.

Differentiation of B lymphocytes from stem cell precursors.

The ready capacity of mouse B lymphocytes to bind 125I-labeled anti-globulin has been used as a marker for a lymphocyte’s B cell status. Fetal liver stem cells lack this marker, and have been used to reconstitute lethally irradiated animals. When 2 x 106 fetal liver cells are injected, and the spleens of the host mice are monitored for appearance of B cells, it is seen that no reconstitution occurs before 8 days. This is surprising, as erythropoiesis and myelopoiesis are by then proceeding very rapidly. After eight days, repopulation occurs exponentially, with a doubling of the B cell content each 1. 5 days. Reconstitution is complete only three to four weeks after irradiation. Functional reconstitution of the immune response to DNP-flagella follows a basically similar but slightly slower pattern.

A microculture method for the generation of primary immune responses in vitro

A microculture method for the generation and study of the primary immune response of murine spleen cells to defined antigens in vitro is described. Many of the variable parameters which occur in culture systems have been studied in an attempt to define the optimal culture conditions for this system. Cultures of 10(6) CBA spleen cells consistently produced an immune response of 300-600 hapten-specific plaque-forming cells after 3 days of incubation with the T cell-independent antigens DNP-POL and NIP-POL. Cultures were set up in Microtest II tissue culture plates in a volume of 0.2 ml of medium containing 10(-4) M 2-mercaptoethanol. The system described has the advantages of being highly efficient and reproducible and utilises small amounts of cells, medium and antigen. It provides a simple, economic and reliable approach for the systematic study of the immune response in vitro.

The response of human B cells to interleukin 4 is determined by their stage of activation and differentiation

The effect of purified‐ recombinant human interleukin 4 (IL‐4) on proliferation and IgM secretion of normal and malignant human B cells was studied. IL‐4 was found to co‐stimulate the proliferation of splenic B cells in the presence of anti‐Ig coupled to polyacrylamide beads (anti‐Ig beads) for a period of 4 days In contrast. IL‐4 had little co‐stimulatory effect on the proliferative response of splenic B cells to the more potent mitogen Staphylococcus aureus Cowan strain I (SAC) Moreover, IL‐4 inhibited interleukin: 2(IL‐2) induced proliferation of cells co‐stimulated with SAC Mitogen‐induced pre‐activation of B cells in the presence of IL‐4 resulted in a reduction in subsequent IL‐2‐induced IgM secretion without significantly affecting proliferation Human B‐cell tumours were also cultured over a 2‐3 day period in the presence of anti‐Ig beads plus IL‐2. or IL‐4orboth IL‐2 and IL‐4. IL‐4 inhibited IL‐2‐induced proliferation in all cases of B‐cell chronic lymphocytic leukaemia (B‐CLL)and the majority of cases of low‐grade lymphoma (LGL)and hairy cell leukaemia (HCL). These findings suggest that IL‐4 has stimulatory actions on resting B cells, most evident in the presence of submaximal co‐mitogenic signals, and inhibitory actions on actuated B cells, especially antagonism of the effects of IL‐2.

A simple semi-automated plaque method for the detection of antibody-forming cell clones in microcultures.

A simple semi-automated method for the assay of large numbers of replicate microcultures for the presence of antibody-forming cell clones is described. The supernatant medium is removed from microcultures by a single sharp flick on inverting the tray. The cultured cells are mixed with 0.05 ml of a plaque-revealing mix containing indicator erythrocytes and complement and then transferred to new flat-bottomed 96-well microculture trays, using a multichannel pipette or 96-channel replicator. The tray is centrifuged, the indicator erythrocytes and cultured cells forming an even monolayer on the bottom surface of the well. Trays are held at 37 degrees C for 1-1 1/2 h to allow plaque development. Using a dissecting microscope, the number of plaques in each well is counted, or in the case of limiting dilution analysis, each well is simply scored as positive or negative. This assay procedure provides a simple, rapid and inexpensive means of assaying large numbers of microculture trays for the detection and enumeration of antibody-forming cell clones. There is no loss in sensitivity compared with the standard hemolytic plaque assay methods. The method is particularly useful for limiting dilution analysis which necessitates the assay of large numbers of replicate cultures for either the presence of absence of a clone of antibody-forming cells.

Surface immunoglobulin isotype, activation, and tolerance susceptibility of B lymphocytes of New Zealand black mice

Abstract Fluorescence-activated cell sorter (FACS) analysis of B-lymphocyte surface isotype expression, and limiting dilution B-lymphocyte cloning techniques, have been used to establish characteristics of B lymphocytes from New Zealand Black (NZB) mice which might contribute to the predisposition of this strain to autoimmune disease. The NZB mice and two other strains (BALB/c and CBA) used were either specific pathogen free (SPF) or germ free (GF). The NZB B lymphocytes differed from the normal strains in the following respects: they showed considerably higher spontaneous conversion into antibody-forming cell clones in the absence of antigen or mitogen; substantially higher cloning efficiency at optimal antigen or mitogen concentration; a slightly lower antigen concentration optimum; and an abnormally high proportion of large cells among the s-IgM + , s-IgD − lymphocyte subset. All these differences argue for a heightened excitability of the NZB B cell to triggering stimuli. Although there was a small-to-moderate increase in the proportion of s-IgM + , s-IgD − b cells, the differences in μ:δ ratio were less than those previously reported. Finally, only a minor and barely significant resistance to tolerance induction in vitro was observed. The results suggest that the heightened B-cell excitability is only one factor in the etiology of autoimmune disease.

INTERFERON-GAMMA DOWNREGULATES THE PROLIFERATIVE RESPONSE OF HAPTEN-SPECIFIC B CELLS STIMULATED BY ANTIGEN AND CYTOKINES

IFN‐γ plays a role in many aspects of cellular interactions, both positive and negative. Among its functions during the immune response, the antagonistic effects of IFN‐γ and IL‐4 are well documented. Observations in our laboratory suggested that IFN‐γ could also interfere with the activation of single, antigen‐specific B cells by antigen and other cytokines. Closer examination revealed that IFN‐γ reduced the number of proliferating cell clones in response to antigen and a variety of cytokines, alone or in combination. Cell viability remained at the initial level and the cells were still able to produce Ig, albeit to a lesser extent than in the absence of IFN‐γ. On the other hand, the frequency of IgM secreting clones was not affected, whereas the total amount of secreted IgM was lower in the presence of IFN‐γ, probably due to the reduced cell number and a decrease in Ig production. In addition, proliferation was prevented when B cells were pre‐incubated with IFN‐γ and then stimulated by other cytokines. Kinetic studies revealed that INF‐γ had to be present from the onset of culture because delayed addition did not inhibit the proliferation of the B cells. After its initial action, IFN‐γ could be removed without abolishing the negative signal for proliferation. From these results it can be concluded that IFN‐γ transmits a signal that causes B cells to stop proliferating and prevents them from forming large clones.