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Journal ArticleDOI

Selection of a Single Antibody-Forming Cell Clone and Its Propagation in Syngeneic Mice

TL;DR: This report describes the history of one clone of cells (E9) producing IgG antibody to dinitrophenyl, which is long-lived and has been maintained for five transplant generations (over 6 months) by serial transfer of spleen cells into irradiated syngeneic mice.
Abstract: A single antibody-forming cell clone has been selected from primed mice by sequential transfer of limited numbers of spleen cells into irradiated syngeneic mice. The original spleen cell donors had been immunized with dinitrophenylated bovine gamma globulin. Specific antibody molecules in sera of recipient mice were separated by isoelectric focusing on polyacrylamide gels and visualized by 131I-hapten binding and autoradiography. This method provided a marker for antibody-forming cells derived from a single cell clone. This report describes the history of one clone of cells (E9) producing IgG antibody to dinitrophenyl. Clone E9 is long-lived and has been maintained for five transplant generations (over 6 months) by serial transfer of spleen cells into irradiated syngeneic mice. Clone E9 has the following properties: (1) Antibody production strictly depends on antigen, presented either in vivo or in vitro; (2) Induction of E9 anti-dinitrophenyl shows specificity for the carrier protein; (3) Antibody is produced in amounts (2-3 mg/ml serum) comparable with myeloma-protein production by murine plasmacytomas; (4) In the absence of antigen, memory cells have a lifetime exceeding 28 days.
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Journal ArticleDOI
20 Nov 1987-Science
TL;DR: The repertoire of antibody variable (V) regions has been subject to evolutionary selection, affecting both the diversity of V region genes in the germline and their expression in the B lymphocyte population and its subsets.
Abstract: The repertoire of antibody variable (V) regions has been subject to evolutionary selection, affecting both the diversity of V region genes in the germline and their expression in the B lymphocyte population and its subsets. In ontogeny, contact with an antigen leads to the expansion of B cells expressing antibodies complementary to it. In a defined phase of B cell differentiation, new sets of V regions are generated from the existing repertoire through somatic hypermutation. Cells carrying advantageous antibody mutants are selected into the memory compartment and produce a stable secondary response upon reexposure to the antigen.

397 citations

Journal ArticleDOI
TL;DR: It is concluded that hapten‐binding cells found in the marginal zones are memory B cells i.e. they have been derived from B cells which have undergone antigen‐driven proliferation, they are no longer in cell cycle but can be induced to re‐enter cell cycle by subsequent exposure to antigen.
Abstract: Specific hapten-binding B cells were identified in the splenic marginal zones following immunization with hapten-protein conjugates. Hapten binding by marginal zone B cells does not appear to be due to passive absorption of anti-hapten antibody. For double immunization with two haptens, 2,4-dinitrophenyl (DNP) and 2-phenyloxazalone (Ox) each conjugated to hemocyanin, resulting in the appearance of discrete DNP-binding cells and Ox-binding cells in the marginal zone. Very few cells were identified which bound both haptens. The hapten-binding cells in the marginal zones have a phenotype characteristic of other marginal zone B cells. They express surface IgM but not IgD. Occasional cells also have surface IgG2c. All hapten-binding cells possessed the antigen recognized by the monoclonal antibody HIS 14 but lacked those identified by HIS24 and HIS22. Hapten-binding B cells were shown to have been in cell cycle shortly before entering the marginal zone but were no longer in cell cycle after arriving at that site. Once in the marginal zone hapten-binding cells were shown to remain in that site for upwards of 2 weeks. Following reimmunization with DNP-hemocyanin, DNP-binding but not Ox-binding cells were lost from the marginal zone. At the same time DNP-binding cells arrived in the periarteriolar lymphocytic sheath and to a lesser extent the follicles. These cells were in active cycle and appeared to give rise both to plasma cells and marginal zone hapten-binding cells. It is concluded that hapten-binding cells found in the marginal zones are memory B cells i.e. they have been derived from B cells which have undergone antigen-driven proliferation, they are no longer in cell cycle but can be induced to re-enter cell cycle by subsequent exposure to antigen. Good antibody responses were obtained following immunization with hapten-polysaccharides; however, no hapten-binding cells appeared in the marginal zones in response to these T cell-independent type 2 antigens.

269 citations

Journal ArticleDOI
TL;DR: Analysis of in vitro stimulation of clonal precursors of anti-2,4-dinitrophenyl (DNP) antibody-producing cells derived from both immune and nonimmune mice indicates that carrier-specific enhancement is obligatory for stimulation of primary precursor cells and increases both the size and number of detectable foci derived from secondary precursor.
Abstract: Cell transfers to carrier-immunized irradiated mice have permitted an analysis of the in vitro stimulation of clonal precursors of anti-2,4-dinitrophenyl (DNP) antibody-producing cells derived from both immune and nonimmune mice. The results indicate that: (a) carrier-specific enhancement is obligatory for stimulation of primary precursor cells and increases both the size and number of detectable foci derived from secondary precursors. (b) This carrier-specific enhancement is most apparent in the stimulation of precursors of high-affinity antibody producer cells. (c) The antibody produced by primary foci, like that of secondary foci, appears homogeneous. (d) The frequency of clonal precursors in normal spleens is 38% that in spleens from mice 4-8 months after immunization, and the number of such precursors in normal spleens can be reduced fivefold by specific suppression of donor mice with soluble antigen. (e) The average of association constants of primary monofocal antibodies, like that of primary serum antibody produced in carrier-primed mice, is less than 10-fold lower than that of secondary clonal or serum antibody. (f) The affinity of primary monofocal antibodies shows a slight dependence on stimulating antigen concentration; however, a minimum threshold affinity consonant with stimulation is apparent. (g) Free hapten inhibits antigenic stimulation of primary precursor cells at a much lower concentration than is required for the inhibition of secondary precursors. These results are interpreted as indicating that (a) primary stimulation, like secondary stimulation, results from the selective stimulation by antigen of a population of cells differing from one another in their potential antibody product but each having only a single such product; (b) the antigen receptors of primary cells interact with antigen as if they are monovalent while receptors of secondary cells evidence multivalence; (c) antigenic stimulation appears to require both a relatively high affinity of receptors for bound antigen and an interlinking of receptors through such antigen; stimulation is thus seen as resulting from a stabilization of receptors within antigen-receptor aggregates to the cell surface; (d) T-cells appear to serve both in cross-linking antigens and in amplifying the size of stimulated clones.

257 citations


Cites background from "Selection of a Single Antibody-Form..."

  • ...--The notion that single cells and the clonal progeny of a single precursor cell produce a single immunoglobulin product has been reinforced by both studies on the immunoglobulin product of single cells (24) and the analysis of the antibody product of transferred cells where, statistically, a single clonal precursor was stimulated (1-6)....

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  • ...Recent evidence suggests that the heterogeneous antibody population produced in response to stimulation by an antigenic determinant represents the antibody product of numerous clones of cells, each cell and cell clone producing homogeneous antibody (1-6)....

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Journal ArticleDOI
TL;DR: The current understanding of the generation and maintenance of B cell and T cell memory is reviewed.

252 citations