Polyclonal activation of the murine immune system by a goat antibody to mouse IgD. IX. Induction of a polyclonal IgE response

The possibility that injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) that stimulates polyclonal IgG1 secretion might also stimulate differentiation of B cells into IgE-secreting cells was suggested by the observation that such treatment induces T cells from those mice to secrete a lymphokine, B cell stimulatory factor 1 (BSF-1), that can stimulate both IgG1 and IgE secretion in vitro. Studies described in this paper show that injection of BALB/c mice with 200 to 3200 micrograms of GaM delta greatly increased the quantity of splenic epsilon chain-encoding mRNA, the number of spleen cells with cytoplasmic IgE, and the concentration of serum IgE 7 days after injection. Serum IgE levels obtained in these mice were approximately 100 times baseline levels and were comparable with those found in mice infected with the nematode parasite Nippostrongylus brasiliensis, but were approximately 2000-fold less than the peak serum IgG1 levels induced by GaM delta injection. Both IgE and IgG1 secretion in GaM delta injected mice were T dependent (blocked by anti-L3T4 antibody). These observations are consistent with the hypothesis that BSF-1 may play a role in the in vivo stimulation of IgE secretion and provide an easy to apply model for the investigation of in vivo regulation of IgE responses.     

Polyclonal activation of the murine immune system by an antibody to IgD. VIII. Stimulation of IgD secretion

The low levels of serum IgD found in mice and the lack of a typical DNA switch sequence between C delta and C mu raise the possibility that the generation of murine IgD-secreting cells results from a chance "mistake" rather than a controlled process. The recent observation that injection of mice with purified IgD upregulates IgD receptor expression on helper T cells and enhances the ability of these T cells to induce B cells to differentiate into antibody secreting cells led us to look for evidence of controlled differentiation of B cells into IgD-secreting cells. To do this, we injected mice with a goat antibody to IgD (GaM delta), because this antibody stimulates large increases in IgM, IgG1, IgG2a, and IgE secretion. Mice injected with GaM delta demonstrated a large increase in splenic content of mRNA specific for the secreted form of delta-chain, as well as a greater than 100-fold increase in the percentage of splenic IgD-containing plasmablasts. The secretory IgD response was totally T-dependent. Production of the secretory form of IgD was not seen until 7 days after GaM delta injection, and peaked sharply on day 8, whereas by day 6 IgM secretion had already peaked and IgG1 and IgG2 secretion had attained substantial levels. This observation suggests that: 1) either cells that synthesize large quantities of the secretory form of delta-chain, unlike cells that synthesize large quantities of the secretory forms of gamma-, epsilon-, or alpha-chains, do this without deleting C mu, or, despite the absence of a typical DNA switch sequence between C mu and C delta, controls must exist to effect the C mu deletion and VDJ-C delta joining; and 2) if secreted IgD has a role in the regulation of a humoral immune response it most likely is involved in later processes, such as memory cell generation or response termination, rather than in relatively early processes, such as helper T cell activation.     

Production of BSF-1 during an in vivo, T-dependent immune response

BSF-1, a cytokine produced by some T lymphocyte tumors, has been shown to act with anti-Ig antibodies to stimulate B lymphocyte proliferation, to independently induce resting B lymphocytes to increase their expression of surface Ia antigen, and to induce some activated B lymphocytes to differentiate into IgG1- or IgE-secreting cells. To determine whether BSF-1 might be secreted by normal lymphoid cells in the course of a physiologic immune response, BALB/c mice were injected with an affinity-purified goat antibody to mouse IgD (GaM delta), which induces the generation of a large, polyclonal T-dependent IgG1 response; 4-hr culture supernatants of spleen cells from these mice were prepared, and these supernatants were assayed for BSF-1 activity by analyzing their ability to induce BALB/c nu/nu spleen cells to increase their expression of cell surface Ia in vitro. Culture supernatants of unfractionated spleen cells removed from mice 4 to 8 days after GaM delta antibody injection induced substantial increases in B lymphocyte surface Ia expression; these increases were blocked by a monoclonal anti-BSF-1 antibody. Culture supernatants of spleen cells from untreated BALB/c mice or from untreated or GaM delta antibody-treated BALB/c nu/nu mice induced small to moderate increases in B cell surface Ia expression, and GaM delta antibody itself induced large increases in B cell surface Ia expression; however, these increases were not significantly blocked by a monoclonal anti-BSF-1 antibody. A culture supernatant of T cell-enriched spleen cells from untreated mice induced small increases in B cell surface Ia expression that were inhibited by anti-BSF-1 antibody, as was the larger increase in B cell Ia expression induced by a culture supernatant of T cell-enriched spleen cells from mice sacrificed 3 days after GaM delta injection. On the other hand, T cell-depleted spleen cells from BALB/c mice injected with GaM delta antibody 7 days before sacrifice failed to generate culture supernatants with BSF-1 activity. Supernatants prepared from spleen cells taken from untreated mice or mice treated with GaM delta antibody 1 to 3 days before sacrifice did not block the ability of purified BSF-1 to induce an increase in B cell surface Ia expression, and thus did not contain inhibitors of BSF-1 activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rapid stimulation of large specific antibody responses with conjugates of antigen and anti-IgD antibody

Injection of mice with goat anti-mouse IgD antibody stimulates a large IgG1 anti-goat IgG antibody response, as well as polyclonal IgG1 production. To determine if this phenomenon could be used to induce large antibody responses to other Ag, covalent conjugates were produced between BSA or other Ag and H delta a/1, a mAb specific for IgD of the a allotype, and between BSA and AF3.33, a mAb specific for IgD of the b allotype. Injection of H delta a/1-BSA into BALB/c mice, which express Ig of the a allotype, or into (BALB/c x CB20)F1 mice (a x b allotype heterozygotes) induced IgG1 anti-BSA antibody responses that peaked 8 to 9 days after injection, and were more than 1000 times larger than those induced by injection of BSA alone, and 100 times larger than those induced by injecting unconjugated BSA plus H delta a/1. H delta a/1-BSA was no more immunogenic than unconjugated BSA when injected into CB20 mice, which express Ig of the b allotype, while AF3.33-BSA greatly enhanced anti-BSA antibody production in CB20, but not in BALB/c mice. Mice serially immunized with three different Ag conjugated to H delta a/1 made large antibody responses to all three Ag, provided that the mouse strain used did not recognize allotypic determinants on H delta a/1 as foreign and produce a neutralizing antibody response. Intravenous and s.c. routes of inoculation produced responses of similar magnitude and relatively low variability; responses to footpad or intramuscular inoculation were more variable, and i.p. inoculation induced smaller responses. Injection of BALB/c mice i.v. with 100 micrograms of H delta a/1-BSA induced an IgG1 anti-BSA response of 5.6 mg/ml, which was approximately 70% of the total IgG1 response. Anti-BSA responses to 30 micrograms of conjugate or less were much smaller, but could be considerably enhanced by adding unconjugated H delta a/1 to the inoculum. This system will be useful for the rapid stimulation of large antibody responses to biologically important Ag, and for investigating mechanisms of Ag processing and B and T cell activation.     

Polyclonal activation of the murine immune system by an antibody to IgD. VII. Demonstration of the role of nonantigen-specific T help in in vivo B cell activation

Previous studies from this laboratory have shown that the injection of mice with an affinity-purified goat antibody to mouse IgD (GaM delta) induces T-independent polyclonal increases in: 1) B cell DNA synthesis, and 2) expression of surface Ia antigen and receptors for T helper factors, 1 to 2 days after injection. In addition, T-independent polyclonal increases in B cell number and IgG1 secretion are observed 6 to 7 days after injection. The administration of normal goat IgG (GIgG) along with GaM delta has been shown to augment GaM delta-induced polyclonal IgG1 secretion. To obtain information about the characteristics of the T help that is required for polyclonal antibody production in this model system, we investigated: 1) the length of the period during which GaM delta must be present to induce day 7 polyclonal antibody production, and 2) the kinetics of the induction of splenic T cell DNA synthesis. We found that GaM delta can be neutralized 3 days after injection by the administration of IgD without decreasing day 7 polyclonal IgG1 secretion, as long as mice are given GIgG at the time that GaM delta is neutralized. In contrast, polyclonal IgG1 secretion is greatly inhibited if GaM delta is neutralized 1 to 2 days after injection or if GaM delta is neutralized 3 days after injection, but GIgG is not administered at this time. Because GIgG can stimulate activated GIgG-specific T cells to secrete helper factors, but, unlike GaM delta cannot focus GIgG-specific T help polyclonally onto B cells, these findings suggest that nonspecific T help, rather than antigen-specific T help, is required in this system after day 3 for the induction of polyclonal IgG1 secretion. Determination of the kinetics of the induction of T cell DNA synthesis in this system by in vivo [3H] thymidine incorporation studies, as well as dual laser fluorescence-activated cell sorter analysis of T and B cell DNA content, indicate that T cells are induced to synthesize DNA 2 days after GaM delta injection and reach plateau rates of DNA synthesis 3 days after injection. Taken together, the GaM delta neutralization experiments and DNA synthesis studies suggest that one reason that GaM delta is required for 3 days in this system is to allow maximal activation of GIgG-specific T cells, which when stimulated later by GIgG secrete nonantigen-specific helper factors that induce GaM delta-activated B cells to secrete IgG1.     

Unidirectional IgG allotype- and isotype-specific suppressor cells in congeneic mice

By the use of allotypic markers on immunoglobulin molecules of isotypes IgG1 and IgG2a, in transfers of spleen cells between Igh haplotype congeneic partner strains BALB/c (Igha) and CB20 (=BALB/c-Ighb), the expression of donor and recipient lymphocytes could be followed differentially. BALB/c donor's allotype a was produced in nonirradiated CB20 recipients for months. By contrast, CB20 donor's allotype b disappeared in nonirradiated BALB/c recipients shortly after transfer. These BALB/c recipients of CB20 spleen cells ("CB20-primed") developed lymphocytes which were able to suppress the autochthoneous allotype b production of CB20 irradiated or CB20 nu/nu or neonatal F1 (BALB/c female X CB20 male) recipients immediately after transfer. Titers decreased with a half life of about 4 days, resembling that of immunoglobulin molecules. The suppression was restricted to the IgG2a isotype of allotype b. Neither the other isotype IgG1 of allotype b, nor, in the reciprocal transfer experiment, IgG1 or IgG2a of allotype a was affected. Analogous transfers between Igh congeneic partners on a C57B1/6 genomic background revealed the same susceptibility of allotype b-producing cells from C57B1/6 donors toward suppression by C57B1/6-Igha mice as recipients. Allotype suppression, induced by cell transfer, is thus unidirectional in that Igha haplotype mice react against allotype b but not vice versa, and it is isotype-specific, only directed against IgG2a, and not IgG1.     

Physiology of IgD. V. Enhancement of antibody responses in vivo by allo anti-IgD is due primarily to an indirect effect on B cells

Although responses of BALB/c mice to TNP-Ficoll or TNP-Brucella abortus are usually decreased by injection of allo anti-IgD (anti-Igh-5a) given 1 day before antigen, increased responses are obtained if a lymphokine mixture (SN) containing IL 2 is also injected. Simultaneous injection of anti-IgD and SN 4 days after priming with TNP-KLH induces an increase in antibody production similar to that induced by a second antigen injection. Injected together with a second injection of TNP-KLH at that time, anti-IgD and SN cause a synergistic enhancement of the secondary response. In allotype heterozygous (BALB/c X SJL)F1 mice injected with anti-IgD directed against one allotype, this enhancement of the secondary response is seen predominantly in the alternate allotype, because the IgG response of linked allotype specificity is slightly suppressed by the anti-IgD alone and is less enhanced than the alternate allotype by anti-IgD plus SN. Cells from unprimed heterozygous mice, incubated with anti-Igh-5a in vitro and transferred, together with antigen, to TNP-KLH-primed recipients, cause a much greater enhancement of the IgG responses of the Igb than of the Iga allotype in recipients. If, however, SN is also injected into the recipients, the anti-TNP response of both IgG allotypes is greatly enhanced.     

Increased expression of the B lymphocyte receptor for transferrin is stimulated by in vivo crosslinking of cell surface IgD

Expression of a receptor for the serum protein transferrin has been shown to be a characteristic of several cell lineages and increased transferrin receptor (TFR) expression to reflect cellular activation. In vitro studies of human B lymphocytes stimulated with antibodies to IgM have demonstrated that these cells have the ability to express TFR and that increased B-cell TFR expression is seen first sometime after these cells enter the G1 phase of the cell cycle. It also has been shown that TFR expression is necessary for proliferation to occur and may be regulated by a factor produced by mitogen-activated T lymphocytes. To examine expression of TFR by activated B lymphocytes in vivo, and to determine the kinetics of induction of TFR expression, we have studied the effects of injecting mice with an affinity-purified goat antibody to mouse IgD (GaM delta) on TFR expression. This antibody previously has been shown to activate polyclonally mouse splenic B cells in vivo in a T-independent fashion. Results show that there is a small but definite quantity of TFR on resting splenocytes, at 24 hr after injection nearly all B cells but not T cells express increased amounts of TFR, TFR is increased to nearly the same extent in congenitally athymic BALB/c nu/nu mice as in their normal nu/+ littermates and therefore GaM delta-induced increased B lymphocyte TFR expression is relatively T independent, TFR expression increases as early as 3 hr after injection of 800 micrograms of GaM delta and increases steadily until it peaks 24-48 hr later, and TFR expression in GaM delta-injected mice increases concomitantly with surface Ia antigen and cell size.     

Autoimmunity after induction of neonatal tolerance to alloantigens: role of B cell chimerism and F1 donor B cell activation

BALB/c mice rendered tolerant by the neonatal injection of semiallogeneic (C57BL/6 X BALB/c)F1 spleen cells develop features of autoimmune disease. The possible mechanisms involved in autoantibody production, particularly anti-DNA antibodies, were investigated. In the first 5 wk, there was polyclonal B cell activation, as indicated by marked hypergammaglobulinemia, with a predominance of IgG1 and an increased production of antihapten antibodies. IgG1 anti-SSDNA and anti-DSDNA antibodies were detected with similar kinetics, but at higher titers than the anti-hapten antibodies. Also, there was a correlation between the effective induction of tolerance, as evaluated by the measurement of alloantigen-specific cytolytic T lymphocyte precursors, the persistence of B cell chimerism, and the production of anti-DNA antibodies. Anti-DNA antibodies were observed only in mice exhibiting a persistence of immunoglobulins bearing the donor's allotype. To determine the origin of anti-DNA antibodies, experiments were conducted whereby newborn BALB/c (Igh-1a) mice were injected with F1 cells from mice resulting from a crossing between Igh congenic BALB/c mice bearing the IgCHb allotype and conventional C57BL/6 mice (Igh-1b). All anti-DNA and anti-hapten antibodies exhibited the Igb allotype and thus were produced by the F1 donor B cells. The initial phase of tolerance induction was apparently associated with an allogeneic helper effect, because DNP-KLH-primed F1 donor cells transferred to newborn BALB/c could be stimulated after challenge with DNP-BGG. The triggering of persisting auto-reactive F1 donor B cells may reflect an activation by "incompletely" tolerant semiallogeneic T cells.     

Polyclonal activation of the murine immune system by an antibody to IgD. XI. Contribution of membrane IgD cross-linking to the generation of an in vivo polyclonal antibody response

The injection of mice with a foreign, polyclonal antibody to IgD sequentially induces: 1) activation of B cells by cross-linking of their cell membrane (m) IgD; 2) B cell processing and presentation of the bound anti-IgD antibody to T cells; 3) activation of these T cells; and 4) T-dependent stimulation of B cell differentiation into IgG1 secreting cells. To determine whether the cross-linking of B cell membrane IgD and/or the resulting B cell activation that follows contribute to the generation of the polyclonal IgG1 response, we examined the abilities of three sets of anti-delta mAb or mAb fragments to stimulate polyclonal IgG1 production. Within each set mAb were matched for species and Ig isotypic determinants, but differed in avidity for IgD or in ability to cross-link IgD. In addition, experiments were performed to determine whether the anti-delta mAb had to be foreign to the immunized mouse to stimulate an IgG1 response. Results of these experiments indicate that: 1) recognition of the injected anti-delta antibody as foreign is required for the induction of a polyclonal IgG1 response; 2) the cross-linking of B cell membrane Ig, which directly activates B cells, can contribute considerably to the generation of in vivo IgG1 production; and 3) that even relatively weak cross-linking of membrane Ig by ligands that bind it with low avidity can make this contribution.     

Characterization of a B cell defect in the NZB mouse manifested by an increased ratio of surface IgM to IgD.

In an effort to define the cellular basis of abnormalities in polyclonal B cell activation previously noted in NZB mice, the surface immunoglobulin (sIg) isotypes of spleen cells from NZB mice were examined. After lactoperoxidase-catalyzed radioiodination, the cell surface immunoglobulins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Spleen cells from 8- to 10-week-old NZB mice were found to have an increased ratio of cell surface IgM/IgD compared to cells from 11 control strains. The altered ratio of sIg isotypes was not a consequence of increased proteolytic activity present in NZB cell suspensions or of the presence of cytophilic antibody or autoantibody. Ontogenetic studies of the sIgM/sIgD (mu/delta) ration on splenocytes from NZB and BALB/c mice revealed that the former cells had higher mu/delta ratios as early as 2 weeks after birth. By 4 weeks of age the mu/delta ratios were equivalent. Between 4 weeks and 1 year of age, the mu/delta ratios on NZB splenocytes remained constant whereas those on BALB/c splenocytes decreased and reached adult levels at 6 weeks.     

Role of antigen-specific T cell help in the generation of in vivo antibody responses. I. Antigen-specific T cell help is required to generate a polyclonal IgG1 response in anti-IgD antibody-injected mice.

A system in which injection of mice with an antibody to mouse IgD that they recognize as foreign stimulates a large, T cell-dependent IgG response was used to study whether Ag-specific T cell help is required to stimulate polyclonal (non-Ag-specific) IgG production in vivo. Igha x Ighb allotype heterozygous mice were injected with a conjugate of a foreign Ag coupled to a mAb specific for one of the two IgD allotypes expressed in these mice. This conjugate cross-links mIgD on B cells that express the recognized allotype. These cells process the conjugate and present the foreign Ag to Ag-specific T lymphocytes, which become activated. Thus, B cells of the recognized allotype can be stimulated by cross-linking of their mIgD, Ag-specific T cell help, non-Ag-specific cytokines, and non-Ag-specific contact with activated T cells. In contrast, B cells that express the Igh allotype not recognized by the Ag-anti-IgD antibody conjugate (bystander B cells) can be stimulated in this system only by non-Ag-specific cytokines and non-Ag-specific contact with activated T cells. Although both recognized and bystander B cells in conjugate-injected mice demonstrated substantial increases in size and Ia expression, only the recognized B cells were induced to synthesize DNA and to make a substantial polyclonal Ig response. Bystander B cells still failed to secrete IgG when mice were injected with an anti-IgD-Ag conjugate specific for the other Igh allotype as well as a mAb that cross-linked IgD of the bystander B cell allotype. These observations demonstrate that although non-Ag-specific cytokine and contact-mediated T cell help are sufficient to induce B cells to increase in size and Ia expression in anti-IgD antibody-injected mice, Ag-specific T cell help is required to stimulate the generation of an IgG response in these mice.     

Murine B cells expressing Thy-1 after in vivo immunization selectively secrete IgE

IL-4 induces Thy-1 expression and IgE secretion by LPS--and T cell-stimulated murine B cells in vitro. IFN-gamma inhibits both of these IL-4-mediated effects. IL-4 and IFN-gamma are often exclusively produced by different CD4+ T cell subsets. Injection of mice with a polyclonal goat anti-mouse IgD antibody (GaM delta) stimulates large increases in the serum concentration of IgE through the production of IL-4. Neutralization of endogenous IFN-gamma production in GaM delta-injected mice leads to further increases in serum IgE levels. We show that IL-4 and IFN-gamma, produced after GaM delta injection, respectively, stimulate and inhibit the number of splenic B cells expressing Thy-1 in vivo. Increased numbers of Thy-1-expressing B cells are observed concomitantly with the onset of enhanced IgE secretion. These Thy-1-expressing B cells are highly and selectively enriched for IgE-secreting cells.     

Allotype-specific immunoregulation of autoantibody production by host B cells in chronic graft-versus host disease

A chronic graft-vs-host (GVH) reaction induced in nonautoimmune mice by the transfer of Ia-incompatible spleen cells results in a syndrome that closely resembles SLE in the spectrum of autoantibodies and immunopathology. We have utilized Ia- and Igh-congenic strains to study the immunoregulation of autoantibody-producing B cells in this model. We have found that the autoantibodies are produced almost entirely by the host B cells. The transferred donor B cells contributed neither to the autoimmune response nor to the total serum Ig, with rare exceptions. The donor cell population did, however, exert an Igh allotype-specific immunoregulatory effect on the host B cells. For example, in allotype-heterozygous recipients, the autoantibodies were preferentially made by those host cells that expressed the donor allotype, whereas those host B cells that expressed nondonor allotype were relatively suppressed. In allotype-homozygous recipients, the donor cells frequently suppressed the host IgG2a allotype completely. This suppression sometimes prevented the IgG antichromatin response, although in other cases the response occurred with the use of a different isotype. In a final set of experiments, a chronic GVH reaction was induced in which both the donors and the recipients were Igh allotype heterozygous and yet differed at Ia. In this case, no donor influence on allotype should be expected; yet the IgG2a autoantibodies were clearly skewed toward the b allotype. These results show that host B cells play a unique role in the GVH autoimmune syndrome. In addition, they are immunoregulated in allotype-specific manners, some of which presumably involve interaction with donor T cells.     

Effects of neonatal anti-delta antibody treatment on the murine immune system. I. Suppression of development of surface IgD+ B cells and expansion of a surface IgM+ IgD- B lymphocyte population

Lymphocytes that bear surface (s) IgD make up the majority of B cells in mature mice and are the precursors of most antibody secreting cells in primary immune responses made by these mice. In order to study the functional capabilities of the minority sIgD- B lymphocyte population and to gain insight into the possible roles of sIgD, we attempted to abort the development of sigD+ B cells and to expand the sigM+IgD- B cell population by treating mice from birth with affinity-purified rabbit antibodies specific for mouse IgD (RaM delta). RaM delta-suppressed mice had no detectable sIgD+ spleen, lymph node, or bone marrow cells and, on average, only 20% as many sIgM+Ia+ splenic B cells as control mice but had normal numbers of splenic T cells. Lymph nodes from anti-delta suppressed mice were even more depleted of B cells than were spleens from these mice, whereas the percentage of bone marrow B cells in these mice was relatively normal. Germinal centers of anti-delta suppressed mice were fairly normal in appearance, whereas follicular mantle layers, the locus of most sIgD+ B cells in normal mice, were greatly depleted. In addition to their lack of sIgD, splenic B cells of anti-delta suppressed mice differed from those found in control mice in that they bore, on average, twice as much sIgM as control cells, and in that they included an increased percentage of large, DNA synthesizing cells as compared with spleen cells from control mice. However, most sIgM+IgD- splenic B cells from anti-delta suppressed mice were small, nonproliferating cells. B cells from anti-delta suppressed mice insert little or no sIgD into their cell membranes since they continued to bear no detectable sIgD 2 days after in vivo neutralization of RaM delta and since, unlike B cells from control mice, they failed to be activated by a single in vitro injection of a goat anti-mouse delta antibody. Despite their lack of sIgD+ B cells, anti-delta suppressed mice had relatively normal levels of serum IgG as well as normal to increased levels of serum IgM. Thus, sIgM+IgD- B cells appear to have the potential of differentiating into Ig secreting cells in vivo without acquiring sIgD.     

Facilitated antigen presentation by B cells expressing IgD when responding T cells express IgD-receptors

In vitro studies have confirmed that cognate interactions between T and B cells are required to demonstrate enhanced helper activity using T cells with upregulated IgD-receptors (IgD-Rs). We studied the mechanism by which IgD-R+ T cells facilitate antibody responses by examining whether T cells also benefit from their expression of IgD-R. Experiments were designed to determine whether upregulation of IgD-R on T cells facilitates antigen presentation by IgD+ B cells. Goat Ig-primed splenic T cells from BALB/c mice were tested for their ability to respond to antigen-presenting B cells treated with goat anti-mouse (GAM) IgM or GAM IgD. T cell responses to GAM IgM and GAM IgD presented by B cells were significantly higher when goat Ig-primed cells were induced to express IgD-R by exposure to oligomeric IgD compared with goat Ig-primed control T cells. This effect was inhibited when monomeric IgD was added to the cultures. No differences in T and IgD-R+ T cell responses were seen using adherent cells as APCs. B cells from IgD-/- mice were also tested. Such B cells present antigen to IgD-R+ T cells without promoting enhanced responses compared with B cells from heterozygous IgD+/- mice. These studies suggest that IgD may play a costimulatory role during antigen presentation. We conclude that when T cells are induced to express IgD-R, these lectin-like receptors can ligate B cell membrane IgD during antigen presentation to facilitate responses of each of the cells engaged in cognate interaction, yielding enhanced antigen-specific T cell and B cell responses.     

IgD allotypic determinants. I. Determinants expressed on murine IgD of the a allotype

Allotypes of membrane IgD of 27 strains of mice were determined with a series of anti-delta reagents, each capable of binding to the IgD of BALB/c spleen cells. One of these reagents is a newly defined allospecific rabbit anti-mouse-delta a. Typing with these reagents reveals the strain distribution of the previously described IgD.36 (Ig5.1) and IgD.43 (Ig5.4) specificities and demonstrates the existence of a new specificity, IgD.45 (Ig5.5). The results confirm the previous subdivision of the Igh-Ce haplotype into Igh-Ce, to which A mice are assigned, and Igh-Cn, to which NZB and NZW mice are assigned. In addition, it is shown that the Igh-Cd haplotype must also be subdivided. AKR mice, which express the a allele at the Igh-5(delta) locus, are designated as possessing the Igh-Cd haplotype while AL/N and C.AL20 mice, which have the e allele at the Igh-5(delta) locus, are assigned a new Igh-C haplotype designation, o.     

Regulation of the anti-alpha(1----3) dextran IgG antibody response of BALB/c mice by idiotype-specific T suppressor lymphocytes.

The immune response of BALB/c mice against the so-called thymus-independent bacterial Ag alpha(1----3) dextran (Dex) is restricted to the expression of few major idiotypes (Id). It is furthermore under the control of T lymphocytes which regulate the isotype expression in such a way that they prevent anti-Dex IgG antibody production upon immunization. At the same time these T cells are part of a regulatory system for Dex-specific B cell memory formation. The underlying Ts cell activity has previously been analyzed by using euthymic and athymic congenic animals. Now we have isolated CD4-positive Id-specific T cell lines and clones which by several criteria are representatives of the above Ts cells. They inhibit in vitro proliferation and antibody secretion of Dex-specific hybridoma B cells. They prevent Id-restricted in vivo IgG anti-Dex antibody formation in T cell-reconstituted BALB/c nu/nu mice. At the same time they enforce, again Id-specific, accumulation of Dex-specific B memory cells. As has been shown previously under the influence of splenic Ts cells, these B memory cells are arrested in the original host but can be expanded and activated for anti-Dex IgG antibody formation upon adoptive transfer into X-irradiated allotype congenic nonresponder BALB.Ighb mice. The data show that the regulatory influence of T cells on the anti-Dex response is Id specific. It can now be studied by means of cloned Ts cells.     

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.     

In vivo and in vitro expression of an interleukin 2 receptor by murine B and T lymphocytes

Interleukin 2 (IL 2), which is well established to be a T cell growth factor, has more recently been shown to stimulate B lymphocyte growth and differentiation in vitro. Responsiveness of B and T cells to IL 2 has been associated with expression of a cell membrane IL 2 receptor (IL 2R). To investigate the role of IL 2 in B cell growth and differentiation in vivo, a system was used in which the injection of mice with a goat antibody to mouse IgD (GaM delta) induces polyclonal T-independent B cell proliferation first, and later induces polyclonal T-dependent B cell proliferation and IgG secretion. IL 2R expression by splenic B and T lymphocytes from GaM delta injected mice was studied by a dual label immunofluorescence technique. Although GaM delta was found to be a strong inducer of B cell IL 2R expression in vitro, even in serum-free medium, and stimulated up to 50% of splenic T cells to express considerable quantities of IL 2R in vivo, it failed to induce more than minimal B cell IL 2R expression in vivo. Concanavalin A and bacterial lipid A also induced B cells to express IL 2R to a much greater extent in vitro than in vivo. Although these agents and GaM delta acted synergistically to stimulate B cell IL 2R expression both in vitro and in vivo, a single agent induced B cell IL 2R expression to a considerably greater extent in vitro than did all three agents acting together in vivo. In vitro GaM delta-induced B cell IL 2R expression was not suppressed by inclusion of IL 2 in the culture medium but was suppressed by the presence of 10% normal mouse serum or plasma. These observations suggest that polyclonal T-dependent B cell proliferation and antibody secretion may not require an interaction between B cells and IL 2; the in vivo environment may downregulate IL 2R expression by B cells: and in vivo B cell IL 2R expression and consequently, induction of B cell responsiveness to IL 2, may require stimuli beyond those sufficient to induce B cell IL 2R expression and IL 2 responsiveness in vitro.