Polyclonal activation of the murine immune system by an antibody to IgD. II. Generation of polyclonal antibody production and cells with surface IgG.
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Animals↗
Biomedical subjects
I Scher
Publications and source records attributed to I Scher.
B lymphocyte subpopulation defined by a rat monoclonal antibody, 14G8.
14G8 is monoclonal rat antibody that recognizes an antigen found on 30 to 40% of B lymphocytes from normal mice and on approximately 65% of B lymphocytes from mice with the xid-determined immune defect. 14G8+ B cells from normal mice resemble B cells from mice with the xid-determined defect in that the median amount of membrane IgM expressed per cell is much larger than that of 14G8- B cells. The frequency of 14G8+ cells is highest in neonatal mice (approximately 55% of all spleen cells) and falls with age to approximately 25% of all spleen cells in adult mice. Relatively few lymph node or bone marrow B cells express the antigen recognized by 14G8. 14G8 also reacts with 50% of resident peritoneal cells and with red blood cells. 14G8+ and 14G8- B cell preparations were obtained by fluorescence-activated cell sorting and by adherence to 14G8 coated dishes. 14G8+ cells responded with in vitro proliferation to both anti-mu and to LPS. Cell cycle analysis indicated that approximately 33% of these cells entered S phase in response to LPS and 38% in response to anti-mu. In contrast, 14G8- cells responded poorly to LPS (7% of cells entered S phase) although they showed good responses to anti-mu (40% of cells entered S phase). Thus, 14G8+ B cells, despite their similarity to B cells from mice with the xid defect, can proliferate to anti-mu, which B cells from defective mice fail to do. 14G8 provides a monoclonal antibody valuable in the description of functional B cell subpopulations.
Aging↗
Antibodies from the Lyb-5- B cell subset predominate in the secondary IgG response to phosphocholine.
The X-linked CBA/N immune defect was used to investigate the role of the Lyb-5- B cell subset in phosphocholine- (PC) specific memory responses. Immune-defective mice, which express only the Lyb-5- B cell subset, are unable to mount a primary or secondary T15+, IgM response to PC but can produce a substantial secondary IgG response. The majority of these IgG anti-PC antibodies are T15- and can be inhibited by phenylphosphocholine, but not PC. Normal mice, which possess Lyb-5+ and Lyb-5- B cells, produce both IgM and IgG anti-PC antibodies; however, there is a striking difference in the idiotype and fine specificity of antibodies expressed by these two classes. The IgM anti-PC antibodies are T15+ and PC-inhibitable, whereas the IgG antibodies are identical to those observed in the immune-defective mice, i.e., T15- and PC-noninhibitable. This unexpected difference in both idiotype and fine specificity between IgM and IgG anti-PC antibodies results from activation of different B cell subsets. Lyb-5+ B cells produce T15+, PC-inhibitable IgM antibodies, whereas T15-, PC-noninhibitable IgG antibodies are produced by Lyb-5- B cells. These data indicate that a majority of the thymus-dependent, anti-PC IgG memory responses arises from Lyb-5- B cells.
Animals↗
Neonatal suppression with anti-Ia antibody. I. Suppression of murine B lymphocyte development.
To investigate the role of sIa in B lymphocyte development, mice were injected from birth with hybridoma anti-Ia antibody. There was on the average a 95% reduction in the number of sIgD+ cells, and 85% diminution in the number of sIgM+ cells, a small but significant increase in the number of sIgM+ sIgD- sla- B cells, as well as a dramatic increase in the number of sIgM- cells bearing a B lineage antigen. Exposure of cells to high doses of anti-Ia antibody in vitro or in vivo for 24 hr did not affect the number of sIg+ lymphocytes. Thus, it appears that chronic anti-Ia administration critically alters normal B lymphocyte development.
Animals↗
Regulation of T15 idiotype dominance. I. Mice expressing the xid immune defect provide normal help to T15+ B cell precursors.
The immune response to phosphocholine (PC) in many strains of mice is dominated by the T15 idiotype family of anti-PC antibodies. By introducing the CBA/N X-linked immune defect (xid gene) into these mice, one profoundly alters their ability to make a T15-predominant, IgM anti-PC response. This loss of T15 dominance in mice expressing the xid gene is not due to the presence of suppressor T cells or the lack of T15 idiotype-specific helper cells in these mice. Thus, one can reconstitute a T15 idiotype-dominant response in immune defective mice with B cells from normal mice, and in adoptive transfer assays the primed T helper cells from immune-defective mice provide qualitatively the same help to normal B cells as the T helper cells from normal mice. T15 idiotype dominance appears to be controlled by the expression and activation of Lyb-5+ PC-specific B cells. Thus, the majority of T15+ B cell precursors are restricted to this B cell subset, whereas the Lyb-5- B cell subset contains predominantly T15-, anti-PC B cell precursors, which produce mainly IgG antibodies after activation by PC-containing antigens.
Animals↗
Immunoglobulin determinants on the surface of mouse splenic lymphocytes from Trypanosoma musculi-infected mice.
The splenic B-lymphocyte population of C57BL/6 mice was analyzed by flow microfluorometry to determine the relative density of surface immunoglobulin (sIg) during the course of infection with Trypanosoma musculi and after the injection of T. musculi derivatives. Cells were stained with fluorescent-conjugated antisera directed against 7S mouse immunoglobulins or the major sIg components, IgM and IgD. Evaluation of relative density of sIg was made through observation of histograms plotted by the Fluorescence Activated Cell Sorter (FACS). The FACS also provided the percentage of fluorescence-positive cells detected with each stain. The relative density of sIg was altered in all experimental groups as evidenced by abnormal histograms. These alterations in relative density of sIg persisted only on B cells of the trypanosome-infected and not the T. musculi-derivative-treated animals. The changes in sIg presumably resulted from sIgD modulation, because the histograms resulting from cells stained with anti-IgM remained unchanged. In the trypanosome-infected animals, there also was a reduction in the percentage of sIgD-positive cells near the end of the parasitemia. Observations of spleen sizes showed the same pattern of change with splenomegaly as with relative density of sIg. These data may be the result of the persistence of the parasite in host tissues after clearance of the blood infection.
Animals↗
Antibodies against liposomes in normal and immune-defective mice.
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Animals↗
Role of the major histocompatibility complex in T cell activation of B cell subpopulations Lyb-5+ and Lyb-5- B cell subpopulations differ in their requirement for major histocompatibility complex-restricted T cell recognition.
This report has examined the requirements for T helper (T(H)) cell recognition of major histocompatibility complex (MHC) determinants expressed by B cells for the activation of unprimed Lyb-5(+) and Lyb-5(-) B cell subpopulations . The generation of primary T(H) cell-dependent plaque-forming cell responses in vitro microculture required the presence of Lyb-5(+) B cells because B cell populations that were deprived, either genetically or serologically, of the Lyb-5(+) subpopulation were not activated in these responses. Cell-mixing experiments in which A X B {arrow} A chimeric T(H) cells were mixed with purified populations of parental accessory cells and parental B cells demonstrated that the in vitro activation of Lyb-5(+) B cells did not require T(H) cell recognition of B cell MHC determinants, although it did require T(H) cell recognition of accessory cell MHC determinants . In contrast to the failure of Lyb-5(-) B cells to be activated in primary T(H) cell-dependent responses in vitro microculture, isolated populations of Lyb-5(-) B cells were triggered by T(H) cells in vivo in short-term adoptive transfer experiments . By the use of A X B {arrow} A chimeric T(H) cells and parental strain B adoptive hosts, it was possible in vivo to distinguish genetically restricted T(H) cell recognition of B cells from genetically restricted T(H) cell recognition of accessory cells. Similar to the results obtained in vitro, the activation in vivo of unfractionated (Lyb-5(+) plus Lyb-5(-)) B cell populations did not require T(H) cell recognition of B cell MHC determinants . In contrast, in the same in vivo responses activation of isolated populations of Lyb-5(-) B cells did require T(H) cell recognition of B cell MHC determinants. The most straightforward interpretation of these experiments is that T(H) cell recognition of B cell MHC determinants is required for the activation of Lyb-5(-) B cells but is not required for the activation of Lyb-5(+) B cells . To better understand why T(H) cell activation of one B cell subpopulation is genetically restricted, whereas activation of another subpopulation is not, the response of Lyb-5(+) and Lyb-5(-) B cells to the soluble activating factors present in concanavalin A-induced spleen cell supernates (Con A SN) was examined. It was observed that Lyb-5(-) B cells, as opposed to Lyb-5(+) B cells, were unable to respond in microculture to the nonspecific T(H) cell- activating factors present in Con A SN, even though they were able to nonspecifically respond under the same conditions to trinitrophenyllipopolysaccharide. It was observed that the ability of B cell subpopulations to respond to nonspecific soluble T cell factors paralleled their ability to be activated by T(H) cells in a genetically unrestricted manner. Thus, the present experiments demonstrate that activation by T(H) cells of Lyb-5(-) B cells is MHC restricted, whereas activation of Lyb-5(+) B cells is not. These experiments suggest that one possible explanation for such differences is that activation of Lyb-5(+) B cells does not require direct interaction with T(H) cells because they can be activated by soluble activation signals that T(H) cells secrete.
Antibody-Producing Cells↗
Autologous rosette-forming T cells regulate responses of T cells. Phenotypic and functional analysis of suppressor cells generated from autologous rosette-forming T cells after autologous mixed lymphocyte reactions.
An average of 5--9% of human peripheral blood of T lymphocytes from rosettes with autologous erythrocytes (ARFT). This population responded only slightly against autologous and allogeneic non-T cells. In contrast, T cells that did not form rosettes with autologous erythrocytes (NRFT) proliferated to a greater degree in auto- and allogeneic mixed lymphocyte reactions (MLR) and also in reactions to trinitrophenyl (TNP) modified autologous non-T cells (TNP-auto-MLR) as compared with ARFT or unfractionated T cells. The ARFT populations could suppress the increased allogeneic (allo)MLR and TNP-auto-MLR of NRFT when the ARFT were added to the NRFT at the beginning of the cultures. Fluorescence-activated cell-sorter (FACS) analysis of these freshly obtained T cell fractions using monoclonal antibodies to subpopulations of T cells did not demonstrate any selective gain or less of T cell subsets in the ARFT and NRFT as compared with unfractionated T cells. But when each T cell fraction was cultured separately for a week in the presence of autologous non-T cells (auto-MLR) and the cells were again analyzed by fluorescence-activated cell sorter, there was an increase in OKT8-positive cells (suppressor/cytotoxic subset) only in the ARFT fraction. The above findings strongly suggest that suppressor T cells are generated from the ARFT fraction during an auto-MLR, these may then regulate the responses on NRFT.
Antigens, Surface↗
Role of accessory cells in B cell activation. IV. Ia+ accessory cells are required for the in vitro generation of thymic independent type 2 antibody responses to polysaccharide antigens.
It has been previously shown that the in vitro antibody response to TNP-Ficoll requires the presence of adherent accessory cells. In order to determine if this characteristic was unique to TNP-Ficoll or a general feature of the TI-2 antibody responses, responses to the polysaccharide antigens TNP-Levan and TNP-Dextran were studied. Also, it was determined if the functionally relevant accessory cell expresses Ia determinants. Passage of spleen cells over Sephadex G-10 abrogated the response to TNP-Levan and TNP-Dextran as well as to TNP-Ficoll. Addition of adherent accessory cells to the G-10 passed spleen cells reconstituted the response to all 3 antigens. Pretreatment of the adherent accessory cells with a specific anti-Ia serum plus complement abrogated the ability of these cells to provide accessory cell function in the responses to all 3 antigens. Thus, an Ia-positive adherent accessory cell is required for the generation of TI-2 antibody responses to these polysaccharide antigens. This raises the possibility that genetic restrictions may exist between the Ia-positive accessory cell and the lymphocytes involved in the responses to TNP-Ficoll, TNP-Dextran, and TNP-Levan.
Animals↗
Surface membrane kappa and lambda light chain expression on spleen cells of neonatal and maturing normal and immune-defective CBA/NB mice: the kappa:lambda ratio is constant.
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Aging↗
Activation of mouse lymphocytes by anti-immunoglobulin. III. Analysis of responding B lymphocytes by flow cytometry and cell sorting.
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Animals↗
Genetically conferred defect in anti-Salmonella antibody formation renders CBA/N mice innately susceptible to Salmonella typhimurium infection.
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Animals↗
Altered idiotype response to phosphocholine in mice bearing an x-linked immune defect.
The x-linked CBA/N defect results in an altered idiotype expression among the anti-phosphocholine (PC) antibodies produced after antigenic challenge with the thymic dependent antigen PC-KLH but does not preclude the response to this hapten as previously suggested. The majority of immune-defective F1 male mice can be divided into 2 groups based on their T15 idiotype profile. Group 1 mice fail to produce anti-PC antibodies bearing the T15 idiotype in either a primary or secondary response, whereas group 2 mice produce low levels of T15 idiotype; however, this idiotype often appears only after secondary immunization. These responses are distinct from the anti-PC response of normal F1 females, which is predominantly of the T15 idiotype. In addition to the altered idiotype expression, F1 male mice exhibited a greatly reduced primary anti-PC response compared to normal mice, and secondary responses were approximately one-third that of normal mice. The delayed expression of anti-PC antibodies in immune defective mice appears to be due to their inability to produce IgM anti-PC antibodies in either a primary or secondary response to PC-KLH.
Animals↗
Role of accessory cells in B cell activation. III. Cellular analysis of primary immune response deficits in CBA/N mice: presence of an accessory cell-B cell interaction defect.
The effect of the X-linked CBA/N genetic defect on the ability of mice to generate primary responses to thymic-dependent and thymic-independent antigens was assessed by comparing the ability of abnormal (CBA/N x DBA/2)F1 male mice and normal (DBA/2 x CBA/N)F1 male mice to generate 2,4,6-trinitrophenyl (TNP)-specific plaque-forming cell responses to TNP-keyhole limpet hemocyanin (KLH), TNP-conjugated Ficoll (TNP-Ficoll), TNP-Brucella abortus (BA), and TNP-lipopolysaccharide (LPS). The reciprocal F1 combinations used in this study differ genetically only in the origin of their X chromosome, but differ immunologically in that (CBA/N x DBA/2)F1 male mice express all the CBA/N immune abnormalities, whereas (DBA/2 x CBA/N)F1 male mice are immunologically normal. Analysis of thymic-dependent responses to TNP-KLH revealed that abnormal F1 mice were capable of generating primary responses in vivo to high doses of TNP-KLH, but failed to generate responses to suboptimal doses of TNP-KLH that were still immunogenic for normal F1 mice. Furthermore, under limiting in vitro micro-culture conditions, the abnormal F1 mice failed to generate primary thymic-dependent responses to any dose of TNP-KLH, even though under the identical conditions normal F1 mice consistently responded to a wide antigen dose range. The cellular basis of the failure of abnormal F1 mice to respond in vitro to TNP-KLH was investigated by assaying the ability of purified populations of accessory cells, T cells, and B cells from these mice to function in responses to TNP-KLH. The results of these experiments demonstrated that helper T cells and antigen-presenting accessory cells from abnormal F1 mice were competent and functioned as well as the equivalent cell populations from normal F1 mice. Instead, the failure of CBA/N mice to generate primary in vitro responses to TNP-KLH was solely the result of a defect in their B cell population such that B cells from these mice failed to be triggered by competent helper T cells and/or competent accessory cells. Similarly, the failure of abnormal F1 mice to respond either in vivo or in vitro to TNP-Ficoll was not the result of defective accessory cell presentation of TNP-Ficoll, but was the result of the failure of B cells from these mice to be activated by competent TNP-Ficoll-presenting accessory cells. In contrast to the failure of B cells from abnormal F1 mice to be activated in vitro in response to either TNP-KLH or TNP-Ficoll, B cells from abnormal F1 mice were triggered to respond to TNP-BA and TNP-LPS, antigens that did not require accessory cell presentation. The specific failure of B cells fron abnormal F1 mice to be activated in responses that required antigen-presentation by accessory cells suggested the possibility that the X-linked CBA/N genetic defect resulted in B cell populations that might be deficient in their ability to interact with antigen-presenting accessory cells...
Animals↗
Augmentation of in vitro humoral immune responses in the mouse by an antibody to IgD.
Heterologous anti-delta-chain antibodies have an adjuvant effect on specific in vivo humoral immune responses to simultaneously, or subsequently, injected antigens in the rat and rhesus monkey. We have used a hybridoma-secreted antibody that binds murine delta-chain of the allotype (4.22aM delta a) to study this phenomenon in the mouse and to investigate the mechanism of this effect. Injection of 4.22aM delta a into BALB/c mice removes almost all surface IgD (sIgD) from splenic B lymphocites. sIgD does not reappear until the serum level of 4.22aM delta a decreased 5-7 d after injection. 4.22aM delta a fails to induce detectable proliferation or to raise total serum Ig levels substantially above control values. However, 4.22aM dalta a injected 24 h before antigen elicits an approximately twofold enhancement of serum IgM and a 3- to 10-fold enhancement of serum IgG anti-trintriphenyl (TNP) antibodies in response to immunization with optimal doses of TNP-Ficoll or TNP-sheep red blood cells (TNP-SRBC). 4.22aM delta a injected 1 wk before or 3 d after TNP-SRBC, however, has no effect on IgG anti-TNP levels. The adjuvant effect of anti-delta-chain antibody was markedly decreased when suboptimal antigen doses were used. Furthermore, even in the case of TNP-Ficoll, a relatively T-independent antigen, the ability of 4.22aM dalta a to enhance the anti-TNP antibody response was T cell dependent. Our data suggest that the binding of anti-delta-chain antibody to cell sIgD may partially activate B lymphocytes and make them more capable of differentiating into antibody-secreting cells when stimulated by antigen-specific T cell help.
Animals↗
Susceptibility to in vitro tolerance induction of adult B cells from mice with an X-linked B-cell defect.
Previous studies from this laboratory have indicated that the susceptibility to in vitro tolerance induction is restricted to B cells early in their development (12,14). In this study, a modification of the in vitro splenic focus technique was used to determine whether 2,4-dinitrophenyl (DNP)-specific splenic B cells from adult (CBA/N X DBA/2)F1 males are susceptible to in vitro tolerance induction. The results demonstrate that greater than 50% of the DNP-specific B cells in the adult F1 male are tolerizable and therefore immature by this criterion. Moreover, the findings define at least two subpopulation in adult CBA/N mice, one of which is tolerizable. These findings are consistent with the hypothesis that the lymphoid population in the adult CBA/N mouse is characteristic of a neonatal B-cell population.
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