We have developed a simple method for the fractionation of T and B lymphocytes. Plastic dishes coated with antibodies specific for mouse Ig selectively bind splenic B lymphocytes. The adherent cells are easily removed by gentle pipetting; both adherent and nonadherent populations retain immunologic function. In a typical experiment, when 3 X 107 splenic Iymphocytes were added to a 100 X 15 mm plastic dish coated with microgram quantities of anti-Ig, 98% of the nonadherent cells were Ig negative and 97% of the adherent cells were Ig positive. The method is sufficiently sensitive to allow detection and separation of cell types comprising as little as 2% of the total population and can be modified to allow the selection of cells by a double-antibody procedure. We believe that the plastic dish method will be generally useful for fractionating cells on the basis of their cell surface antigens. The ability to fractionate lymphocytes on the basis of surface phenotype has been a major technical advance in the study of the functional diversity of these cells. Many of the methods currently used exploit antibody specificity to separate cells of one type from a mixed population. Cell lysis with antibody and complement is useful only for cell elimination. Separation by affinity column chromatography (1) or fluorescence-activated cell sorting (2) offers the advantage of positive selection by allowing the recovery of both enriched and depleted cell populations.We report a simple and inexpensive method for the separation of B T cell specificity of the serum was determined by complement-dependent lysis of thymocytes and specific immunoprecipitation of T25 (Thy 1 antigen) from thymocyte membranes (5). The cytotoxic titer of this serum was very low (95% of thymocytes were lysed at an antibody dilution of 1:5). Rabbit anti-mouse Ig was raised by repeated subcutaneous injection of DEAE-cellulose-purified mouse Ig in complete Freund's adjuvant. The Ig fraction was purified by ammonium sulfate precipitation (4) and affinity chromatography on Sepharose 4B conjugated with purified mouse Ig (6).Goat anti-rabbit IgG was purchased as serum from SeraSource, Inc., and purified by ammonium sulfate precipitation and affinity chromatography on Sepharose 4B conjugated with DEAE-cellulose-purified rabbit IgG.Normal rabbit IgG was prepared by ion exchange chromatography on Whatman DE-52 ion exchange resin (7).F(ab')2 fragments were prepared by pepsin digestion and gel filtration through Sephadex G-150 (4).Antibodies conjugated with rhodamine were prepared by the method of Cebra and Goldstein (7). Purity of all antibody reagents was determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (8) under reducing conditions.All immunoglobulin preparations were centrifuged at 80,000 X g for 1 hr to remove aggregates, sterilized by Millipore filtration, and stored at 4°.Fetal Calf Serum. Fetal calf serum, purchased from Grand Island Biological Co., was heat inactivated at 560 for 2 hr and tested for mitogenic activity on splenic l...

The contribution of anergy to silencing of autoreactive B cells in physiologic settings is unknown. By comparing anergic and nonanergic immunoglobulin-transgenic mouse strains, we defined a set of surface markers that were used for presumptive identification of an anergic B cell cohort within a normal repertoire. Like anergic transgenic B cells, these physiologic anergic cells exhibited high basal intracellular free calcium and did not mobilize calcium, initiate tyrosine phosphorylation, proliferate, upregulate activation markers, or mount an immune response upon antigen-receptor stimulation. Autoreactive B cells were overrepresented in this cohort. On the basis of the frequency and lifespan of these cells, it appears that as many as 50% of newly produced B cells are destined to become anergic. In conclusion, our findings indicate that anergy is probably the primary mechanism by which autoreactive B cells are silenced. Thus maintenance of the unresponsiveness of anergic cells is critical for prevention of autoimmunity.

Available evidence indicates that B cell tolerance is attained by receptor editing, anergy, or clonal deletion. Here, we describe a p-azophenylarsonate (Ars)-specific immunoglobulin transgenic mouse in which B cells become anergic as a consequence of cross-reaction with autoantigen in the bone marrow. Developing bone marrow B cells show no evidence of receptor editing but transiently upregulate activation markers and appear to undergo accelerated development. Mature B cells are present in normal numbers but are refractory to BCR-mediated induction of calcium mobilization, tyrosine phosphorylation, and antibody responses. Activation marker expression and acquisition of the anergic phenotype is prevented in bone marrow cultures by monovalent hapten. In this model, it appears that induction of anergy in B cells can be prevented by monovalent hapten competing with autoantigen for the binding site.

Immunization of strain A mice with p- Similarly, the L chain V domain is fashioned by the apposition of VL and JL gene segments. Additional diversity is generated at the junctions between V gene segments during assembly (2-4) and by an unknown mechanism resulting in nucleotide replacements (termed "somatic mutation") (5-8).The latter mechanism amplifies the potential V region diversity to an almost limitless extent. A single B cell synthesizes antibodies with homogeneous V domains and expresses some of these as cell-surface receptors. Binding of antigen to these receptor antibodies constitutes part of a signal that induces proliferation, resulting in a population that synthesizes antibodies encoded by a uniform combination of V gene segments (9-12). While a general scheme of somatically generated antibody diversity has been elucidated, we lack an understanding of how this diversity is utilized during the initial process of clonal selection and the subsequent acquisition of functional immunity. Initial attempts to identify clonally related V regions during an immune response were dependent exclusively on serological methods (idiotype analysis).The idiotype investigated here is reproducibly elicited in strain A mice by immunization with proteins conjugated with the hapten, p-azophenylarsonate (Ars). We sampled B cells participating in the anti-Ars immune response by generating hybridomas during primary and secondary immune responses and characterized the V region structures of the antibodies they secrete with respect to the V gene segment combinations encoding them, somatic mutation, and affinity for Ars. Our results demonstrate a strong correlation between clonal "success"-i.e., a B-cell clone whose Ig product constitutes a significant fraction of serum antibodies-and affinity for Ars. Clonal success appears to be determined by both information encoded in the germ line and information stochastically generated by somatic mutation. MATERIALS AND METHODSAntigens. Keyhole limpet hemocyanin (KLH) was purchased from Calbiochem, and a peptide fragment containing the amino-terminal 102 amino acids of the phage X repressor protein was provided by Robert T. Sauer. Both of these were conjugated with the diazonium salt of Ars as reported (16).Fusions. All fusions were performed between splenic cells and the Sp2/0-Ag-14 cell line (17) as reported (18)