Evidence that intestinal IgA plasma cells in {micro},{varkappa} transgenic mice are derived from B-1 (Ly-1 B) cells

B6-Sp6 transgenic mice carry fully rearranged (BALB/c-derived,Igh-C^a allotype) µ. heavy chain and light chain transgenes,specific for trinitrophenyl, on a C57BL background (Igh-C^b allotype).FACS analyses show that the majority of B cells in peripherallymphoid organs and bone marrow(BM) express transgenic IgM exclusively.A small proportion of the B cells, however, express endogenousIgM, usually concomitant with transgenic IgM. Three criteriaestablish that the endogenous IgM expressing B cells belongto the B-1 cell lineage. (I) Endogenous IgM expressing B cellsin B6-Sp6 mice have the same localization pattern as B-1 cellsfrom normal animals: they are enriched in the peritoneal cavity.(II) The endogenous IgM⁺ B cells have the phenotype of B-1 cells:the endogenous IgM⁺ peritoneal B cells express Mac-1 (CD11b)and low levels of IgD, and most also express CD5 (L-1). (III)B6-Sp6 BM poorly reconstitutes endogenous IgM⁺ B cells, justas adult BM from normal mice poorly reconstitutes B-1 cells.In contrast, B cells which only express the transgene are readilyreconstituted by B6-Sp6 BM. The few endogenous IgM⁺ cells inthe B6-Sp6 BM recipients are located in the peritoneal cavityand have the phenotype of B-1b cells (previously the Ly-1 Bsister population), which are known to be reconstituted by adultBM.Two-color immunofluorescence staining of tissue sectionsfrom the gut and from isolated gut lamina propria cells showsthe presence of many IgA containing cells, about one-third ofwhich simultaneously express cytoplasmic (transgenic) IgM. TheC-region of this IgA is produced by endogenous C a genes, becausethe transgene encodes only for C_µ. Furthermore, the majorityof gut IgA containing cells do not express the Idiotype of thetransgene, indicating that most of the gut IgA cells are encodedby endogenous V_H genes and thus the result of an isotype switchfrom endogenous IgM expressing B cells. Since the endogenousIgM⁺ cells are B-1 cells (both B-1a and B-1b), the data stronglyindicate that the intestinal IgA plasma cells also belong tothe B-1 cell lineage.     

The B cell receptor, but not the pre-B cell receptor, mediates arrest of B cell differentiation

B cell development in organ cultures of fetal liver from mice at day 14 of gestation resembles in kinetics and cell numbers generated the one observed in vivo. This development in vitro can be blocked by an IL-7 receptor-specific monoclonal antibody. Monoclonal antibodies specific for the pre-B cell receptor, i. e. for VpreB, lambda5, or muH chains, do not perturb B cell development in these organ cultures up to and including the CD25+ small pre-BII cell stage. However, muH chain-specific antibodies inhibit the appearance of the subsequent surface IgM+ immature B cells. In organ cultures of muH chain allotype heterozygous (muHa x muHb)F1 fetal livers a dose-dependent inhibition by allotype-specific monoclonal antibodies of sIgM+ immature B cells expressing the corresponding, but not the other, allotype was observed. By combining cell sorting with limiting dilution analysis of lipopolysaccharide-reactive cells, the probable target cell of this muH chain-specific inhibition was identified as an IgM+, CD23-immature B cell. Hence, engagement of the pre-B cell receptor by specific antibodies does not influence B cell development, while engagement of the B cell receptor on immature B cells does.     

Non-tolerant B cells cause autoimmunity in anti-CD8 IgG2a-transgenic mice

Using a pair of γ2a/x immunoglobulin genes, transgenic mice were generated to study tolerance induction in B cells that express IgG2a autoantibodies. The transgenic IgG2a specifically binds CD8 α chains of the CD8.2 allotype expressed on the surface of CD8⁺ T cells, but not CD8 molecules expressed by the CD8.1 allele. Thus, IgG2a transgenic mice expressing the CD8.1 allele were used as controls to monitor B cell development and mice expressing CD8.2 were used to study B cell tolerance. Both types of mice showed transgenic γ2a expression on the surface of B cells. Expression of endogenous heavy chain alleles was strongly inhibited in immature B cell subsets, whereas mature B cells co-expressed transgenic γ2a and endogenous IgM/D. The transgenic x chain expression leads only to partial allelic exclusion of endogenous light chains. B cells that express high levels of transgenic CD8.2-specific IgG2a were identified using soluble CD8-Ig. In CD8.1⁺ and in CD8.2⁺ mice, we found no differences in expression and maturation of transgenic anti-CD8.2 IgG2a⁺ B cells. High levels of serum anti-CD8.2 IgG2a antibodies led to the elimination of CD8⁺ T cells, causing a severe defect in cytotoxic immune responses. These results show that tolerance induction is incomplete in the CD8.2⁺ mice, either because IgG2a⁺ B cells are resistant to censoring mechanisms or because the secreted CD8-specific IgG2a antibodies render the CD8 autoantigen inaccessible to the B cells. This contrasts strongly with the efficient induction of B cell tolerance in mice expressing anti-CD8.2 IgM autoantibodies.     

Effects of IgM Allotype Suppression on Serum IgM Levels,B-1 and B-2 Cells,and Antibody Responses ir Allotype Heterozygous F1 Mice

IgM allotype heterozygous F1 mice were independently suppressed for Igh6a or Igh6b to evaluate the contribution of B-1 and B-2 cells to natural serum IgM levels and Ab responses. B-2 B cells expressing IgM of the suppressed allotype were evident in the spleens of suppressed mice 4 to 6 weeks after cessation of the suppression regimen, whereas B-1 B cells of the suppressed allotype were undetectable for up to 9 months. Although serum IgM of the suppressed allotype was initially depleted in mice suppressed for either allotype, by 7 months of age, there were detectable levels of IgM of the suppressed allotype in the serum; however, the levels were significantly below that found in nonsuppressed mice. When mice were immunized with either the T-independent or T-dependent form of phosphorylcholine, those suppressed for either allotype, and consequently depleted of B-1 B cells of that allotype, did not respond with phosphorylcholine-specific IgM of the suppressed allotype. In contrast, when mice were immunized with α1-3 dextran, the Igh6a allotype-suppressed mice were able to produce dextran-specific IgM of that allotype. These results show that allotype-bearing B-1 cells of both allotypes can be effectively suppressed by this suppression protocol and this produces long-lasting effects on B-1 cell levels and serum IgM of the suppressed allotype. These observations reflect the derivation of the majority of B-1 cells from fetal-neonatal precursors, which cannot be replaced by newly emerging B-2 cells of adult origin. Their ablation by antibody treatment results in permanent alterations to the adult B-cell repertoire.     

In vivo generation and function of B cells in the presence of a monoclonal anti-IgM antibody: implications for B cell tolerance

C57BL/6 mice were chronically treated with milligram doses of the noncytotoxic monoclonal anti-mu b antibody MB86 (IgG1, kappa) from birth or from fetal life. The spleens of the manipulated animals contained large numbers (25% as compared to control mice) of B lineage cells which expressed IgMb on the surface after overnight incubation in vitro. The spleens also contained B cells whose surface IgM was unreactive with antibody MB86. A few such cells were immortalized by cell fusion. They included cells secreting mu together with lambda 2 chains which apparently prevent recognition by antibody MB86, and a point mutant in the first constant domain of the mu chain, changing the b to the a allotype. Cells expressing MB86- surface IgM did not selectively expand under MB86 treatment over the first few months of life. Serum Ig levels in the manipulated mice were normal except for IgM which was undetectable in most instances. In some animals low levels of MB86- IgM molecules were produced. At 7 weeks of age, mice treated with MB86 from birth produced normal-size IgG anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) responses with the usual predominance of lambda 1 chain-bearing IgG1 antibodies. At the age of 5-6 months, and also in young mice treated with MB86 from fetal life, the responses were variable and presumably oligoclonal, with a tendency towards the production of antibodies with gamma 3 heavy and lambda 2 or lambda 3 light chains. We interpret these results to mean that B cells hit by antibody MB86 from the time of their generation become unresponsive to T cell-dependent stimulation, but are still able to expand. Occasionally, they escape functional suppression through class switching (to IgG3) upon mitogenic stimulation. At birth, C57BL/6 mice contain a mature B cell population which mediates normal immune responses under MB86 treatment and eventually dies out. Taken as a model of tolerance induction in B cells, the data provide evidence for "tolerant" cells and support the concept of an early phase of sensitivity to tolerance induction in B cell differentiation. The anti-NP response under MB86 treatment differed profoundly from control responses in idiotypic terms, but became normal as the animals recovered from suppression. This may reflect blockade by MB86 of idiotypic selection within the B cell population.     

Endogenous VH gene family expression in immunoglobulin-transgenic mice: evidence for selection of antibody repertoires

VH gene family expression in single cells of the emergent, available and actual B cell repertoires of C57BL/6 mice was compared to that of two immunoglobulin (Ig)-transgenic B6 lines (B6-Sp6 and M54). We found that less than 5% of bone marrow cells of transgenic mice express endogenous VH genes and that the vast majority (95%) of the peripheral, mature B cell repertoire in these animals is composed of cells expressing the VHJ558 transgenic family. Unimmunized transgenic mice, however, diversify VH gene family usage by 'background' Ig-secreting cells in the spleen, greater than 50% of which express endogenous VH genes. The pattern of endogenous VH gene family expression in the actual repertoire of B6-Sp6 mice is indistinguishable from that of normal B6 mice. In contrast, actual repertoires of M54 mice differ by a 4- to 5-fold higher representation of the VHQ52 family. These results demonstrate a powerful positive selection of B cells into the secretory compartments of unimmunized animals, show that actual and available repertoires differ very markedly, and suggest that V region interactions participate in the selection of 'natural antibody' repertoires.     

Inheritance of antibody specificity. The IgM anti-lipopolysaccharide response in mice

Mice of different genotypes were immunized with Salmonella anatum. The cross-reactivity patterns of their IgM anti-S. anatum lipopolysaccharide (LPS_AN) antibodies were characterized by their relative avidity toward heterologous LPS. When the LPS from S. cholera suis (LPS_CHS) was used as the heterologous LPS, clear differences between mouse strains were found. DBA/2 and DBA/1 showed cross-reacting IgM, whereas C57BL/10, C57BL/6, BALB/c-Ig^b and B10.D2 had mainly noncross-reacting IgM. In C3H and C57BL/6-Ig^a, individual mice express either the cross-reacting or the noncross-reacting antibodies. The IgM antibodies from individual mice were further characterized for their cross-reactivity toward the LPS from S. strasbourg (LPS_STR) and S. illinois (LPS_ILL). Only individual patterns with no correlation to the cross-reactivity pattern with LPS_CHS were found. This shows that more than one antibody type is characterized by cross-reactivity. (B 10.D2 × DBA/1)F₁ mice showed a biphasic distribution of cross-reactivity. Of the F₁ x DBA backcross mice 21 % had IgM antibodies which showed no cross-reaction with LPS_CHS. This still is in agreement with one locus controlling this phenotype. This locus segregates independently from Ig allotype since no correlation was found between allotype and cross-reactivity pattern in F₁ x DBA backcross mice.     

Regulation of allotype expression in heterozygous rabbits. II. Concomitant suppression of b4 and b6 allotypes in the same cell.

Cells from heterozygous b4b6 rabbits were treated at 4 degrees with anti-b4 or anti-b6 antibodies and then warmed at 37 degrees. A disappearance of both b4 and b6 allotypes (concomitant modulation) ensued. When cells which had undergone extensive comodulation were cultured overnight we noted that those cells were unable to re-express either allotype at pre-modulation levels. This suppression was likely linked to the initial events which culminated in comodulation. Those cells were not further suppressible when suppressive antibodies were added to the cultures whereas cell cultures which had undergone little or no previous modulation or comodulation were readily suppressed for both allotypes after anti-allotype antibodies had been added to the cultures overnight (concomitant suppression). This indicated that in vitro suppression of allotype may depend on cell surface allotype being present at a sufficiently high density. We present data which show that events at the cell surface may play a role in the regulation of cell surface allotype expression and propose that concomitant suppression may have bearing on cellular mechanisms which control allotype expression and also allotype suppression.     

Evidence for selection of a population of multi-reactive B cells into the splenic marginal zone

Antibody reactivity to self-antigens is a normal component of the immune system. To study the mechanism by which self-reactive B cells are generated and maintained, we analyzed B cell development in transgenic mice that express a rearranged VH81X heavy chain from the pre-immune repertoire. In these mice, > 95% of B cells express the transgene in association with a variety of kappa light chains but V kappa 1 C being the dominant light chain. These transgenic B cells with identical V kappa 1C-J kappa 5 joins do not normally secrete IgM in vivo, but antibodies derived from these B cells, through LPS activation in vitro or after hybridoma immortalization, are self-reactive and recognize an ubiquitous epitope(s) on intracytoplasmic proteins from different tissues. They have the phenotype and localization pattern of long-lived marginal zone B cells and their development in vivo is blocked by injection of soluble VH81X-V kappa 1CJ kappa 5 IgM antibody. The observations in this transgenic mouse provide evidence for positive selection of a population of self-reactive B cells. These B cells enter the peripheral pool of B cells where they localize in the marginal zone of the spleen and, in contrast to other transgene-expressing B cells, do not secrete IgM antibody.      

Effect of Anti-δ Antibodies on Clonal Expansion and Maturation of B Lymphocytes

The effect of a monoclonal anti-δ antibody on the in vitro B cell response to the B cell mitogen lipopolysaccharide (LPS) has been studied.Titration of anti-δ antibody in mass cultures stimulated by LPS resulted in a dose-dependent reduction of thymidine incorporation. IgM and IgG plaque forming cell (PFC) responses were poorly affected at high concentration, and slightly increased at low concentrations of anti-δ antibody.By limiting dilution analysis it was shown that anti-δ antibody inhibit 30-50 % of LPSreactive B cells to grow as IgM secreting clones, while increasing the average size of clones that grew in the presence of anti-δ as compared to control cultures. Anti-δ also results in increased frequencies of IgG secreting clones.By immunofluorescence it was possible to show the presence of a higher relative number of cells containing immunoglobulin as an effect of anti-δ antibodies. Observations made at early times of culture indicate that the cells that do not proliferate in the presence of anti-δ undergo an early maturation to secretion.Experiments performed on LPS blasts suggest that the effects of anti-δ on cell proliferation require the presence of antibodies at early times in the response, while the effects on maturation can be manifested during clonal development.The relevance of membrane IgD and of the IgM-to-IgD ratio in the maintenance versus exhaustion of the clone is discussed.     

Functional maturation of immature B cells accumulated in the periphery by an intraperitoneal administration of a traditional Chinese medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to)

We found that an intraperitoneal (ip) injection of a traditional Chinese herbal medicine, xiao-chai-hu-tang (Japanese name: shosaiko-to), induced accumulation of B lymphocytes (sIgM+) in the peritoneal cavity and spleen. 1) Cell surface marker analysis by a fluorescence-activated cell sorter (FACS) demonstrated that the accumulated B cells on day 4 or 7 after shosaiko-to administration (early phase) were composed mainly of sIgM+IgD- cells and suggested that these B cells maturated into sIgM+IgD+ cells on days 10 or 14 (late phase). Relative decrease of IgM+IgD+ cells at early phase was more profound in peritoneal cells (PC) than in spleen cells. 2) With respect to spleen lymphocytes, antibody responses to a thymus-independent (TI) antigen of type 2 (trinitrophenylated Ficoll) and a thymus-dependent (TD) antigen (sheep erythrocyte) were enhanced at late phase but not at early phase. In contrast, responses to trinitrophenylated lipopolysaccharide (TNP-LPS) as a TI-1 antigen and LPS as a B cell mitogen or a polyclonal B cell activator were enhanced markedly at early phase but declined at late phase. 3) With respect to peritoneal lymphocytes, responses to LPS were suppressed at early phase but recovered at late phase. Enhanced responses to TI and TD antigen at late phase in spleen lymphocytes and suppressed response to LPS at early phase in peritoneal lymphocytes may be explained by increases of IgM+IgD+ mature B cells and IgM+IgD- immature B cells, respectively, at those times. Enhanced responses to TI-1 or LPS in spleen lymphocytes at early phase may be explained by elevated sensitivity of IgM+IgD+ cells which reside in the spleen before shosaiko-to administration and receive the direct stimulation by shosaiko-to, or by acquired responsiveness of IgM+IgD- cells which migrate after stimulation with shosaiko-to.     

Priming of helper T cell-dependent antibody responses by hemagglutinin-transgenic B cells

Mice expressing the hemagglutinin (HA) gene of influenza virus PR8 (H1 subtype) under the control of x light chain promoter and enhancer have been generated. They express HA in and on B cells, and are tolerant to HA. In vitro, only lipopolysaccharide (LPS) blasts but not resting B cells of transgenic mice can stimulate HA-specific helper T cells of HA-specific α/β T cell receptor (TCR)-transgenic mice. Transfer of HA-transgenic LPS blasts into syngeneic, non-transgenic recipients primes HA-specific antibody responses. Resting, small HA-transgenic B cells, which were purified by fluorescence-activated cell sorting, prime lower antibody responses. Host B cells produce the HA-specific antibody response. The donor HA-transgenic B cells need to express major histocompatibility complex (MHC) class II molecules and need to be alive to induce the antibody response in the host. Most notably, the host antibody response never produces detectable levels of IgM, but only of switched IgG isotypes. Neither resting nor activated HA-transgenic B cells induce tolerance in antibody responses. These results suggest that HA-transgenic B cells, presenting both the intact antigen on the cell surface and peptides of the antigen on MHC class II, are effective inducers of helper T cell responses, and as judged by the Ig-isotype response pattern, which is mainly IgG1, of Th2 type.     

Feedback regulation of murine Ly-1 B cell development

Studies presented here, conducted with allotype homozygotes, demonstrate the existence of a feedback mechanism that regulates development of Ly-1 B cells from immature progenitors. In the preceding study (P. A. Lalor et al., Eur. J. Immunol. 1989. 19:501), conducted with allotype heterozygotes, we showed that treating neonates with monoclonal antibody to the paternal allotype IgM depletes roughly half of the neonatal B cell population (i.e. those expressing the paternal IgM allotype) and that paternal allotype Ly-1 B cells specificically remain depleted for the life of the animal. Here we show that treating allotype homozygotes with the same antibody depletes all (rather than half) of the B cells and that, under these conditions, relatively normal numbers of Ly-1 B cells reappear shortly after the treatment antibody disappears. The recovery, we also show, is prevented by restoring allotype-congenic Ly-1 B cells to the treated homozygotes, i.e. by reconstituting treated neonates with allotype-congenic peritoneal cells, sorted Ly-1 B cells or a monoclonal population of Ly-1 B "tumor" cells. These findings in essence reveal a feedback mechanism through which mature Ly-1 B cells prevent further Ly-1 B cell development from Ig- precursors. This feedback regulation is independent of Ig secretion by the mature Ly-1 B cells, since the monoclonal Ly-1 B "tumor" population that prevents endogenous Ly-1 B development does not secrete Ig. Furthermore, it appears to be independent of Ly-1 B surface Ig specificity, since a monoclonal population is sufficient to block all Ly-1 B cell development. This mechanism appears to operate normally to fix the composition of the Ly-1 B population, which survives through self-replenishment in adults, in accord with conditions that influence Ly-1 B development during neonatal life.     

A role for membrane IgD in the tolerance of pathological human rheumatoid factor B cells

Under non-autoimmune conditions, rheumatoid factor (RF) B cells coexist peacefully with their antigen (IgG), or can be transiently activated during secondary immune responses because they can present xenoantigens to specific T cells captured in immune complex form. Such a situation should lead to affinity maturation of RF B cells and potentially dangerous production of high-affinity RF. We used two lines of transgenic mice expressing a somatically mutated pathological human RF in presence (IgM and IgD) or in absence (IgM only) of surface IgD, and confirm that RF B cell tolerance can result from an antigen-induced specific, but incomplete, deletion of naive RF B cells after antigen encounter. This deletion mainly concerns immature, transitional B cells. On the contrary, mature, IgM- and IgD-expressing RF B cells are resistant to such a deletion. These IgM and IgD RF B cells are functional and activable through both B cell receptor dependent (anti-IgM) and independent (LPS) pathways, but they are not fully responsive to human IgG either in vivo or in vitro. Taken together, these results suggest that another mechanism could be involved in the silencing of mature naive IgM and IgD RF B cells.     

c-fos overexpression in splenic B cells augments development of marginal zone B cells

Marginal zone (MZ)-B cells participate in very early immune responses and play a pivotal role in the first-line of defense against blood-borne Ags including bacterial LPS. Since splenic B cells from c-fos transgenic (H2-c-fos) mice are hyper-sensitive to LPS stimulation, we examined LPS-sensitivity of MZ-B cells in the spleen of H2-c-fos mice. Here, we show that proliferation of MZ-B cells from H2-c-fos mice stimulated with LPS was larger than that from control mice. Proliferation and IgM production of the H2-c-fos MZ-B cells were also larger than those of splenic follicular (FO)-B cells from the H2-c-fos mice, suggesting that c-fos overexpression augments LPS-sensitivity of MZ-B cells more than that of FO-B cells. Furthermore, the number of MZ-B cells but not that of FO-B cells in the spleen of H2-c-fos mice was two- to three-fold larger than that in control littermates. The number of transitional type II (T2)-B cells in H2-c-fos mice was also larger than that of control littermates. However, the number of transitional type I (T1)-B cells in the spleen of H2-c-fos mice was not larger than that of control mice. Moreover, this c-fos effect on differentiation of MZ-B cells was intrinsic in B cells by the competitive repopulation assay with hematopoietic stem cells of H2-c-fos and control mice. These results suggest that c-fos overexpression in B cells augments differentiation and accumulation of MZ-B cells.     

Ly6C expression differentiates plasma cells from other B cell subsets in mice.

Plasma cell differentiation is induced in vitro by lipopolysaccharide (LPS) stimulation but can be blocked by including anti-CD40 antibodies. Using subtractive cDNA hybridization we have identified the cell surface protein Ly6C as differentially expressed on B cells stimulated with LPS only. Ly6C has been shown to be expressed on certain T cell subsets and on subsets of macrophages and NK cells, but not on resting B cells. We show that Ly6C is up-regulated upon LPS stimulation of B cells in vitro and that this up-regulation is blocked by anti-CD40 or anti-Ig antibodies. Furthermore, ELISPOT analysis of cells sorted by magnetic-activated cell sorting show that Ly6C is expressed on ex vivo plasma cells from the spleen and bone marrow. Flow cytometric analysis showed that Ly6C is expressed on splenic plasma cells as well as on lamina propria plasma cells. Finally, Ly6C cross-linking positively up-regulated the amount of immunoglobulin produced by LPS-stimulated splenic B cells in vitro.     

Differential sensitivity of B lymphocyte populations to IgM receptor ligation is determined by local factors

Ligation of surface IgM on B cells responding to lipopolysaccharide (LPS) suppresses terminal differentiation and high-rate Ig secretion with no effect on proliferation. As shown here, different B cell populations show characteristic mean values of ligand concentration required for 50% inhibition, with Gaussian distributions of sensitivity to IgM receptor ligation that reflect cellular heterogeneity of 'al-or- none' inhibitions in single cells. Differential sensitivity of B cell populations to IgM ligation seems to be locally determined by the cellular environment and unrelated to the 'maturity' of the responding cells. Thus, while long-lived peritoneal B cells are 3- to 5-fold more resistant than splenic B cells, there is no difference in sensitivity between short- and long-lived B cells in the spleen. Furthermore, while B cells in bone marrow and spleen differ in sensitivity by two orders of magnitude, B cells differentiated in vitro from bone marrow pre-B cells are as resistant as splenic B cells. Moreover, bone marrow cell culture supernatants restore a high level of sensitivity in such cell populations. Differential sensitivity to IgM receptor ligation is reproduced by multivalent nominal antigen, in cell populations that show identical dose-response inhibition curves to direct activation of protein kinase C by phorbol esters. We conclude that the level of sensitivity to IgM ligation is independent of the life span or maturity of the B cell, but differentially regulated in vivo by putative tissue factors.      

Activation of BCL1 Cells by Polyclonal Activators and Inhibition of Growth and IgM Secretion by Anti-IgM

Bacterial lipoprotein (LP) and lipopolysaccharide (LPS) both activated an in vitro line of the B-cell tumour BCL1 to IgM secretion, as determined by the protein A plaque assay. LPS but not LP activation was inhibited by polymyxin B. Activation with both LPS and LP resulted in a less than additive response. Several clones of BCL1 were tested, and all responded to both LPS and LP. Both LPS and LP induced broad dose-response curves in normal lymphocytes, recently cloned BCL1 cells, and cloned and synchronized (G1 phase) BCL1 cells. This suggests that the dose-response curve cannot be due to accumulation of responding cells with different threshold sensitivities for activation. We cannot exclude the possibility that the broad dose-response curve is due to a heterogeneity of the LPS or LP preparation. The results indicate that LPS and LP induce similar signals in BCL1 cells. Furthermore, binding to the cell membrane and activation of BCL1 cells by LPS or LP seem to be separate events. An anti-IgM antiserum inhibited spontaneous DNA synthesis and spontaneous and LP-induced IgM secretion of BCL1 cells. Equal inhibition was observed with F(ab')2 fragments but not with Fab fragments of the antiserum, suggesting that cross-linking of IgM bound to the cell surface membrane-induced inhibition. Supernatants from concanavalin A (Con A)-activated spleen cells induced BCL1 cells to secrete IgM. Fab anti-IgM added alone to BCL1 cells did not induce IgM secretion. Furthermore, Fab anti-IgM plus Con A supernatant did not induce a higher response than the supernatant alone. This suggests that inductive signals via the IgM receptor do not occur in BCL1 cells.     

Neisserial porins may provide critical second signals to polysaccharide-activated murine B cells for induction of immunoglobulin secretion.

Resting B cells stimulated with dextran-conjugated anti-immunoglobulin D (anti-IgD) antibodies (anti-Ig-dex), a model for B-cell activation in response to polysaccharide antigens, proliferate but secrete little if any Ig, unless additional stimuli are present. In order to elucidate the parameters which costimulate T-cell-independent antipolysaccharide antibody responses during bacterial infections, we tested the capacities of highly purified porin proteins from Neisseria meningitidis and Neisseria gonorrhoeae to augment in vitro proliferation and induce Ig secretion by anti-Ig-dex-activated B cells. Resting B cells, from lipopolysaccharide (LPS)-nonresponsive C3H/HeJ mice, proliferated and secreted IgM in response to each of three distinct porins acting alone. Further, porins, even at concentrations that were minimally inductive when acting alone, were strongly synergistic with anti-Ig-dex for proliferation and Ig secretion. Similar synergistic effects of porins with CD40-ligand were also observed. These effects of porins were shown to occur directly at the level of the B cell. The predominant Ig isotype elicited in response to porins plus anti-Ig-dex or CD40-ligand was IgM (>97%), with the remainder comprising IgG. Surprisingly, picogram-per-milliliter amounts of neisserial LPS were also found to be highly synergistic with anti-Ig-dex for induction of IgM secretion by LPS-responsive C3H/HeN, but not C3H/HeJ, B cells. Thus, these data suggest that porins, as well as LPS, may provide critical second signals for T-cell-independent induction of polysaccharide-specific Ig in response to neisserial and other gram-negative porin-expressing bacterial pathogens, without a requirement for the participation of non-B cell types. These data may also help to explain the potent immunopotentiating effects of porins for polysaccharide-specific, as well as protein-specific, humoral responses in vivo.     

Long-lasting thymus-independent immune responses to anti-idiotype lipopolysaccharide conjugates require continuous B cell renewal

In the present investigation, we have studied the antibody response of both C57BL/6 lipopolysaccharide-responder mice (B6 LPS-R) and C57BL/10ScCr LPS-nonresponder mice (B10.Cr LPS-NR) upon immunization with copolymers of LPS and either Ac38 or Ac46 anti-idiotypes. As both strains of mice studied are of the Ighb allotype the antibody response obtained was quantified by estimating the serum levels of the complementary idiotype, B1-8, in immunized mice. The results show that while the injection of the LPS-R strain B6 with copolymers of LPS anti-idiotype resulted in a long-lasting idiotype response the immunization of the LPS-NR strain, B10.Cr, resulted in virtually no response. This finding allowed us to perform cell transfer experiments to study the response of isolated cells from B6 mice transferred into LPS-NR hosts. Using such a cell transfer model, the results obtained suggest that long-lasting idiotype responses observed in the LPS-R strain require continuous B cell production in bone marrow. The relevance of this finding to the understanding of the long-lasting (fluctuating) antibody responses to nondegradable thymus-independent polysaccharides is discussed.