Pneumococcal polysaccharides induce antibody formation by human B lymphocytes in vitro.
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Adult↗
Biomedical subjects
D E Mosier
Publications and source records attributed to D E Mosier.
Negative selection in vivo reveals expression of strong Mls determinants in mice with X-linked immunodeficiency.
Evidence is presented that mice with X-linked immunodeficiency (xid) express strong Mlsa,d determinants, a putative marker of the mature subset of B cells. Although young (3-5 wk) (CBA/N X DBA/2)F1 male (xid+) mice stimulated only very weak mixed lymphocyte reactions (MLR) to Mlsa,d determinants, older mice (greater than 7 wk) regularly elicited conspicuous responses, despite being totally unresponsive to TNP-Ficoll. Expression of Mlsa,d determinants by xid+ mice was also detected by the procedure of negative selection in vivo. Thus BALB/c T cells were totally depleted of Mlsa,d reactivity after blood to lymph recirculation through 10-wk old irradiated xid+ (CBA/N X DBA/2)1 male mice. Significantly, a marked (90%) reduction in the anti-Mlsa,d response also occurred with T cell filtration through 3-wk xid+ mice, i.e., mice that elicit only minimal primary MLR; filtration through 3-wk xid- normal female mice led to near-complete (99%) negative selection. Collectively these data indicate either, (a) that xid+ mice contain appreciable numbers of cells with at least some of the properties of mature B cells, or (b) that the expression of Mlsa,d determinants is not restricted to mature B cells. In either case, B cells from xid mice cannot be viewed as a simple model for immature normal B cells.
Aging↗
Interaction of helper T cells and Lyb5+ B cells responding to phosphorylcholine is MHC-restricted.
The requirements for T cell/B cell interaction for the induction of primary in vitro antibody responses to phosphorylcholine (PC)-keyhole limpet hemocyanin (KLH) were examined. Long-term helper T cell lines derived from KLH-primed (CBA/N X BALB/c) F1 female mice (H-2k/d) were able to support a T15-idiotype dominant, IgM anti-PC response of BALB/c (H-2d) B cells and macrophages, but could not activate PC-specific responses by BALB.B (H-2b) B cells, even in the presence of irradiated H-2k/d antigen-presenting cells. Polyclonal IgM secretion in the same cultures did not appear to depend upon a major histocompatibility complex (MHC)-restricted T-B interaction. Since IgM anti-PC responses seem to be entirely derived from the Lyb5+ B cell subpopulation, we conclude that at least some Lyb5+ B cells can only be activated by MHC-restricted T-B interactions. We also found that xid B cells from (CBA/N X BALB/c) F1 male mice could be polyclonally activated by helper T cell lines by an apparently MHC-unrestricted interaction. Our data thus suggests that residence in the Lyb5- or Lyb5+ B cell subset does not determine the T:B interaction requirements for antibody synthesis.
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Activation of B lymphocytes by monovalent anti-Lyb-2 antibodies.
Although activation of B lymphocytes by antigen or anti-Ig antibody has been shown to require cross-linking of surface Ig molecules, cross-linking is not necessary for B cell activation by anti-Lyb-2 monoclonal antibody (MAb). Monovalent Fab' fragments of anti-Lyb-2 MAb are as effective as the intact antibody in inducing blast cell transformation of small B cells and B cell proliferation in the apparent absence of T cells and adherent cells. In the presence of factors from T cells, B cells activated by Fab' fragments of anti-Lyb-2 MAb were induced to mature into Ig-secreting cells. Since monovalent Fab' fragments probably cannot induce receptor aggregation, it appears that receptor occupancy is sufficient to induce B cell activation with anti-Lyb-2 MAb.
Animals↗
Separation of macrophages on plastic and glass surfaces.
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Animals↗
Helper T lymphocytes from xid and normal mice support anti-phosphocholine antibody responses with equivalent T15, 511, and 603 idiotypic composition.
We have examined the idiotypic composition of secondary adoptive transfer antibody responses to phosphocholine (PC) supported by KLH-primed helper T cells derived from normal mice or xid mice. CBA/N x BALB/c F1 male xid mice have diminished anti-PC responses and virtually undetectable levels of the T15 idiotype; xid mice do express the 511 and 603 idiotypes. Nonetheless, we find helper T cells derived from such mice are indistinguishable from T cells primed in a normal environment in their ability to cooperate with B cells producing anti-PC antibody bearing the T15, 511, or 603 idiotype markers. This result is in contrast to a previously published report from this laboratory. T cells from xid mice did support more IgG PFC than normal T cells, but serum IgG anti-PC antibody levels were similar in both groups. The IgM anti-PC response was predominantly of the T15 idiotype, whereas the 511 idiotype was associated with a minor fraction of IgG1 antibodies. The majority of the secondary IgG "anti-PC" antibody response bore none of the idiotypic markers associated with PC-binding myeloma or hybridoma antibodies, and was directed against phenyl-PC rather than PC. The phenomenon of T15 clonal dominance in the anti-PC response therefore is largely confined to the IgM response. We would conclude that the idiotype levels in the T cell priming environment do not influence the subsequent ability of such primed T cells to support anti-PC antibody responses.
Animals↗
Induction of B lymphocyte proliferation by monoclonal anti-Lyb 2 antibody.
Monoclonal antibody to Lyb 2, a differentiation antigen present on all B cells, has been used to study the role of Lyb 2 molecules in B cell activation. Monoclonal anti-Lyb 2 antibody (m-anti-Lyb 2) transforms resting B cells into blast cells and induces proliferation in these activated B cells. The proliferative response to anti-Lyb 2 is a property of the Lyb 5+ subset of B cells, since the antibody fails to stimulate B cells from mice expressing the CBA/N immune defect. B cells activated by anti-Lyb 2 mature into antibody-secreting cells in the added presence of T cell-replacing factors contained in the supernatants from concanavalin A-activated T cells. Thus, Lyb 2 molecules may participate in the delivery of stimulatory signals during the early phases of B lymphocyte activation.
Animals↗
Lyb-8.2: A new B cell antigen defined and characterized with a monoclonal antibody.
A DBA/1 B10.D2-specific monoclonal antibody (CY34) is described which defines a new murine B lymphocyte differentiation antigen designated Lyb-8.2. The ontogeny, strain distribution, and cell-surface density of the antigen were studied by radioimmunoassay and by fluorescence-activated cell sorter (FACS) analysis. Lyb-8.2 appears to be expressed on pre-B cells and on all mature B lymphocytes. Lyb-8.2 molecules immunoprecipitated from surface labeled B10.D2 spleen cells migrated in polyacrylamide gels with an apparent mol. wt. of 95,000-105,000 daltons and were bound by lentil lectin. The expression of Lyb-8.2 is controlled by a locus on chromosome 7 that is closely linked to Gpi-1 and RP-2. Added Lyb-8.2-specific antibody did not measurably impair B lymphocyte function in several in vitro systems studied.
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Lyb antigens and their role in B lymphocyte activation.
Anti-Lyb 7 antibodies selectively inhibit in vitro immune responses to the TI-2 antigens but not to TI-1 antigens. The Lyb 7 differentiation antigen is distinct from Lyb 5 and is coded by genes linked to IgCH loci. Lyb 8 is a new B cell differentiation antigen defined by a monoclonal antibody. It is a molecule of 95 kd and is specified by genes linked to Gpi on chromosome 7. Since Lyb 8 is found on pre-B cells and since it has no demonstrable function in B cell activation, its current role is as an early marker that signifies commitment of stem cells to the B cell lineage. m-anti-Lyb 2 antibodies inhibit in vitro antibody responses to SRBC and TI-2 antigens but not to TI-1 antigens, m-anti-Lyb 2 induces B cell proliferation only in the Lyb 5+ subset of B cells. Lyb 2 antigens are suggested to play the role of a receptor for a physiological T cell-derived soluble mediator required at one stage of activation of B cells into antibody-forming cells.
Animals↗
Antigen-specific helper T cells required for dominant idiotype expression are not H-2 restricted.
Two synergizing antigen-specific helper T (Th) cell populations are required for an optimal TEPC15 (T15)-dominated antiphosphorylcholine (PC) plaque- forming cell response . In these studies, the two Th cell sets are shown to differ in their requirements for recognition of self-major histocompatibility complex (MHC)-encoded determinants by testing the ability of Th cells from F(1) {arrow} parent bone marrow chimeras to collaborate with PC-specific B cells bearing MHC-encoded determinants of either parental haplotypes. Previous studies have shown that one antigen-specific Th cell population is required for T-dependent anti-PC responses and activates PC-specific B cells only if the hapten, PC, is physically linked to the priming antigen. This Th cell, referred to as ThMHC, induces anti-PC responses that are mainly non-T15 in character, and it appears to be identical to the conventional antigen- specific Th cell. In these experiments, using T cells from (A X B)F(1) {arrow} parent A chimeras, ThMHC cells requiring hapten-carrier association provide help for F(1) and parent A B cells but not for B cells from parent B, thus confirming that the activity of the conventional Th cell is H-2 restricted . The second antigen-specific Th cell population, whose function is measured in the presence of the ThMHC cell set, preferentially activates T15-bearing B cells. This Th cell set (ThId) is missing in mice expressing low levels of T15-bearing antibody and can be restored by the addition of antigen-specific T cells from donors expressing high levels of circulating T15 Id. These studies demonstrate that T cells from F(1) {arrow} parent chimeras that express substantial levels of T15-bearing anti-PC antibody could provide ThId cell activity for the selective activation of T15-bearing B cells of F(1) and both parental H-2 types. These results imply that whereas the activity of conventional, ThMHC, cells is clearly H-2 restricted, ThId cells from the same chimeric donors are not required to recognize antigen in association with self-MHC-encoded determinants for successful T-B collaboration .
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Primary in vitro antibody responses by purified murine B lymphocytes in serum-free defined medium.
A serum-free medium formulation that allows a primary in vitro antibody response to the nonmitogenic thymic independent antigen TNP-AECM-Ficoll is described. Purified murine B lymphocytes and adherent accessory cells are required for optimal specific responses as well as for optimal B cell survival. The medium is based on Iscove's modification of Dulbecco's modified Eagle's medium and Ham's F-12 nutrient mixture. The serum-replacing components are insulin, transferrin, and progesterone. The survival of unstimulated murine T cells in this Iscove/F12 medium is poor, but B cells survive for relatively long periods of time. This medium should prove useful in defining the molecular requirements for B lymphocyte activation by antigen.
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Absence of an antigen-specific helper T cell required for the expression of the T 15 idiotype in mice treated with anti-mu antibody.
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Animals↗
Cell cooperation during in vivo anti-hapten antibody responses. VI. Evidence for an allogeneic effect replacing one of two helper T cells.
T and B lymphocyte activity in adoptive secondary antibody responses was assessed by cell titration in mice of the X-linked immune defective CBA/N strain and mice of their normal partner strain CBA/CaJ. No quantitative differences could be detected in either T or B cell activity in these experiments, in which 2,4-dinitrophenyl was used as the hapten. Both of the helper T cells detected in such assays were present in both strains. During these experiments, it was found that a subline of CBA/N maintained at Yale may have diverged from the CBA/N NIH stock. This was detected by an ability of irradiated mice of the Yale subline to increase the antibody response of B cells from the NIH subline, provided specific helper T cells were also present. This allogeneic effect apparently replaced the activity of one helper T cell, but did not replace the requirement for a carrier-specific helper T cell.
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High anti-TNP plaque-forming cell potential of residual mIg+ cells in a T cell population.
In the course of experiments designed to study the immune response of purified populations of B lymphocytes to thymus-independent (TI) antigens, a variety of cell purification procedures were followed. In using anti-immunoglobulin-coated dishes to separate lymphocytes bearing membrane immunoglobulin (mIg) from mIg- lymphocytes, it was found that the nonadherent fraction, which was predominantly mIg-, complement receptor negative, and nonresponsive to the B cell mitogen lipopolysaccharide, gave very substantial anti-TNP plaque-forming cell responses to 2 TI antigens. These responses could be inhibited by incubation of such cells in the presence of anti-mu and thus appeared to be attributable to mIg+ cells. The evidence suggests the existence of a population of B lymphocytes that constitute a minor component of mIg+ cells having a high potential to make in vitro antibody responses. Users of techniques that utilize anti-Ig as a tool for separating B and T lymphocytes should carefully assess the extent to which residual B lymphocytes in the mIg- population contribute to antibody responses being studied.
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Cellular requirements for antigen presentation in the induction of a thymus-independent antibody response in vitro.
The ability of various cell populations to bind and present the thymus-independent antigen TNP-Ficoll to a responding cell population was assessed. The in vitro antibody response to TNP-Ficoll depends upon the presence of B lymphocytes and plastic-adherent accessory cells, but does not require T lymphocytes. Purified B cells were the most effective population in binding and presenting TNP-Ficoll, and adherent cells did not perform this function. Antigen binding and presentation was antigen specific and could be blocked with anti-mu antibody, but not by antibodies directed against other immunoglobulin classes. Spleen cells from mice genetically unresponsive to TNP-Ficoll (CBA/N X BALB/c F1 males) were equally effective as normal spleen cells in antigen binding and presentation. We conclude that the initial events in the induction of the antibody response involves antigen binding by B cells, and that subsequent activation of the subset of B cells that can respond to TNP-Ficoll proceeds either via B cell-B cell interaction or B cell-dependent transfer of antigen to macrophage-like cells.
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Mice whose B cells cannot produce the T15 idiotype also lack an antigen-specific helper T cell required for T15 expression.
The X-linked CBA/N defect in B cell function precludes an antibody response to phosphorylcholine (PC). Accordingly, (CBA/N X BALB/c)F1 male mice are unresponsive to PC and lack circulating immunoglobulin bearing the T15 idiotype characteristic of BALB/C anti-PC antibody. In contrast, (CBA/N X BALB/c)F1 female mice respond to PC and greater than 80% of the anti-PC antibody is T15+. No T-cell abnormalities are known to be associated with the CBA/N mutation. These experiments compared the ability of helper T cells from either (CBA/N X BALB/c)F1 male (T15-) or F1 female (T15+) mice to help F1 female B cells respond to PC and to influence the level of T15 expression. The results indicate that although F1 male T cells collaborated with F1 female B cells just as efficiently as F1 female T cells for the total anti-PC response, the percentage of T15 expression induced by F1 male T cells fell dramatically. The (CBA/N X BALB/c)F1 male thus appear to lack a helper T-cell subset required for dominant idiotype production. This helper T cell defect could be repaired by adding F1 female T cells primed to a second carrier to F1 male T cells and restimulating the cell mixture with PC coupled to the antigen used to prime the F1 male cells plus free second carrier. This result implies that conventional helper T cells derived from the F1 male donor can collaborate with a distinct helper T-cell subset from the F1 female donor which recognizes both carrier and idiotype to induce an anti-PC antibody response dominated by the T15 clonotype.
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Role of a nonimmunoglobulin cell surface determinant in the activation of B lymphocytes by thymus-independent antigens.
Lyb 5 is a B-cell alloantigen which is expressed on 50-60% of B cells. It was defined originally on the basis of cytotoxicity. We have described a new reactivity within the anti-Lyb 5 serum on the basis of selective inhibition of antibody responses in vitro by this antiserum in the absence of complement. This inhibitory activity of anti-Lyb 5.1 serum appears to be due to recognition of antigenic determinants different from the prototype antigens detected in the cytotoxicity assay. Anti-Lyb 5 serum incorporated into spleen cell cultures selectively inhibits antibody responses to a class of thymus-independent antigens (TI-2) previously characterized by their failure to elicit antibody formation in immature mice or in the defective CBA/N strain. Responses to optimal concentrations of TI-1 antigens, which can induce antibody synthesis in these mice, are unaffected by the addition of anti-Lyb 5.1 serum. The B-cell alloantigen defined by this functional assay is designated tentatively Lyb 7 and it is shown to be distinct from cell surface immunoglobulins. Lyb 7 appears to have a role in the activation of B lymphocytes by the TI-2 class of thymus-independent antigens.
Antibody Formation↗
Ontogeny of susceptibility of mouse splenic B cells to tolerance induction in vitro by TNP-D-GL.
The susceptibility of mouse spleen cells to hapten-specific tolerance induction of a primary in vitro thymus-independent antibody response was examined. Both the induction of tolerance by 2,4,6-trinitrophenyl-D-glutamic acid-D-lysine (TNP96D-GL) and of antibody formation (elicited by TNP-Brucella abortus) in neonatal spleen cell cultures were unaffected by anti-Thy-1.2 plus complement treatment. Spleen cells from neonatal mice were only slightly more sensitive to TNP96D-GL tolerance induction than were cells from adult mice. The difference in susceptibility to tolerance induction was not nearly as great as that predicted by "clonal abortion"-type theories of B cell tolerogenesis.
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