Influence of HLA-DR polymorphism and allergic sensitization on humoral immune responses to intact pneumococcus in a transgenic mouse model.

Asthma is independently associated with HLA-DR3 and increased risks of pneumococcal diseases. We aimed to determine whether HLA-DR polymorphism (HLA-DRB1*03), sensitization to house dust mite (HDM), or their interaction affects humoral immune responses to pneumococcal polysaccharide and protein antigens of intact pneumococci. Induction of serum titers of anti-pneumococcal polysaccharide and anti-surface protein IgM and IgG in response to immunization with intact pneumococci (Pn) serotype 14 was determined using humanized HLA-DR3 and DR2 transgenic mice. Transgenic mice were sensitized by injecting HDM and challenged with intranasal HDM. Mice were subsequently immunized with heat-killed Pn14 at day 24. Serum titers of anti-phosphorylcholine (PC) IgM and IgG, anti-pneumococcal polysaccharide, capsular type 14 (PPS14) IgM and IgG, and anti-pneumococcal surface protein A (PspA) IgG were measured. We included a total of 44 mice (22 DR3 and 22 DR2 mice) and half of mice in each group were sensitized with HDM (i.e. 22 HDM-sensitized and 22 control mice). HDM-sensitized mice, irrespective of HLA-DR polymorphism, had significantly lower humoral immune responses. HLA-DR3 mice, irrespective of HDM sensitization, elicited a significantly lower anti-PC IgG response. In contrast, the anti-PspA IgG response was higher in DR3 relative to DR2 mice. The effect of HDM sensitization on lowering humoral immune responses to Pn14 was observed in DR3 mice regardless of the nature of the antigen, whereas such decreases were observed only for the anti-PPS14 IgG and anti-PC IgM responses in DR2 mice. HDM sensitization lowered humoral immune responses to intact pneumococcus and this effect was significantly modified by the HLA-DR polymorphism.

Distinct types of T-cell help for the induction of a humoral immune response to Streptococcus pneumoniae.

Studies have indicated that purified soluble polysaccharide antigens can elicit T cell-independent Ig responses in vivo, although these responses can be modulated by T cells in a noncognate manner. Relatively little is known, however, concerning the parameters that regulate polysaccharide-specific, as well as protein-specific, Ig isotype responses to an intact extracellular bacterium. Using the murine in vivo humoral response to intact Streptococcus pneumoniae as a model it can be shown that CD4+ T-cell receptor alphabeta+ T cells deliver help for both polysaccharide- and protein-specific Ig responses. However, these responses differ fundamentally in their mechanism of action.

B7 requirements for primary and secondary protein- and polysaccharide-specific Ig isotype responses to Streptococcus pneumoniae.

The requirements for B7 costimulation during an in vivo humoral response to an intact extracellular bacteria have not been reported. In this study we immunized mice with Streptococcus pneumoniae (R36A) to determine the B7 requirements for induction of Ig, specific for two determinants on R36A, the phosphorylcholine (PC) determinant of C-polysaccharide and pneumococcal surface protein A (PspA). We show that the primary anti-PspA response, the development of PspA-specific memory, and the induction of the secondary anti-PspA response in primed mice were completely dependent upon B7 costimulation. Of note, costimulation was required only briefly after the secondary immunization compared with after the primary immunization for optimal induction of Ig. Blockade of B7 costimulation at the time of secondary immunization also completely abrogated the established state of memory, but did not induce tolerance. In contrast to the anti-PspA response, the primary anti-PC response involved only a very short period of B7 costimulation. Whereas B7-2 alone was required for induction of the primary anti-PspA and anti-PC responses, a redundant role for B7-1 and B7-2 was noted for the PspA-specific secondary response. CTLA4Ig blocked both the anti-PC and anti-PspA responses equally well over a wide range of bacterial doses. These studies demonstrate a critical, but variable, role for B7-dependent costimulation during an Ig response to an extracellular bacteria.

B-cell activation by T-cell-independent type 2 antigens as an integral part of the humoral immune response to pathogenic microorganisms.

Antigens that are expressed on the surface of pathogens in an organized, highly repetitive form can activate specific B cells by cross-linking of antigen receptors in a multivalent fashion. B cells respond to these multivalent antigens in the absence of MHC class II-restricted T-cell help by a mechanism that depends on the expression of a functional Bruton's tyrosine kinase (Btk). Accordingly, this class of immunogens has been designated T-cell-independent type 2 (TI-2) antigens. The unique properties of the B-cell response to TI-2 antigens are critically dependent on the formation of a small number of antigen receptor clusters, each of which contains approximately 10 to 20 antigen-bound membrane Ig (mIg) molecules. These clusters induce local membrane association of multiple activated Btk molecules, which results in long-term mobilization of intracellular ionized calcium. Such persistent calcium fluxes efficiently recruit transcription factors and thereby induce T-cell-independent B-cell activation and proliferation. While this first signal of multivalent mIg cross-linking can induce B-cell proliferation, we propose that a second signal is required for a TI-2 Ig secretory response. We have found that engagement of members of the Toll-like receptor (TLR) family could provide second signals that selectively induce Ig secretion in B cells that were activated by multivalent, but not by bivalent, antigen receptor engagement. This finding demonstrates a general mechanism by which TLRs recognize molecular motifs on the surface of pathogens and provide the TI-2-activated B cell with a second signal. In addition, TLR-dependent recognition of these non-self motifs by cells of the innate immune system can induce these cells to provide alternative and/or additional second signals in the TI-2 response. The complement system provides another link between the B cell and the innate immune system, and facilitates the mIg signal transduction by recruitment of CD21 in the immune response. Thus, the TI-2 response provides the host with a combination of "the best of both worlds": the recruitment of the fine specificity of the adaptive immune response and the utilization of both the speed of the innate immune system and the wealth of cytokines produced by its member cells upon stimulation by pathogenic organisms or their products. By combining these two pathways, the TI-2 response enables the host to rapidly produce antigen-specific Ig effector molecules that can be secreted at a sufficient rate to keep up with the rapid multiplication of invading infectious microorganisms, and will also prevent the intracellular spreading of a significant part of this population.

T(h)1 versus T(h)2 cytokine profile determines the modulation of in vitro T cell-independent type 2 responses by IL-4.

We have previously demonstrated that stimulation of B cells by multivalent membrane Ig cross-linking, using dextran-conjugated anti-IgD mAb (alpha delta-dex), in the presence of cytokines, is an in vitro model for T cell-independent type 2 (TI-2) Ig secretory responses. Earlier studies have shown that IL-4 enhances IgM secretion upon stimulation with alpha delta-dex plus IL-5 and induces IgG1 isotype-switching, without altering the proliferative response to alpha delta-dex. Here we show that IL-4 can have both stimulatory and inhibitory effects on alpha delta-dex-induced Ig secretion. Both the kinetics and time of exposure to IL-4, and the nature of the cytokine additions, T(h)1 versus T(h)2, determine whether stimulation or inhibition is observed. Preincubation of sort-purified B cells with IL-4 caused a 6- to 8-fold increase in Ig secretory responses to subsequent stimulation with alpha delta-dex plus IL-1, IL-2 or a combination of both. However, the continued presence of IL-4 during B cell stimulation suppressed responses to all cytokine combinations tested, except for those which included IL-5. Of 11 cytokines tested, only IL-4 showed this dual effect of enhancement and suppression. The stimulatory effect of IL-4 required a minimum of 4 h of preincubation and could be inhibited by the addition of IFN-gamma. Thus stimulation of non-MHC class II-dependent T or non-T cells by multivalent antigens to secrete IL-4 may regulate the response to these antigens, such that early and brief exposure of B cells to IL-4 will enhance a subsequent TI-2 response in the presence of T(h)1-dependent cytokines, while continuous exposure will result in inhibition of the response.

NF-kappaB/p50 and NF-kappaB/c-Rel differentially regulate the activity of the 3'alphaE-hsl,2 enhancer in normal murine B cells in an activation-dependent manner.

The enhancer complex located 3' to the C(H)alpha gene in the IgH locus (3alphaE) may regulate B cell function through its ability to act as a locus control region. Multiple, functionally relevant NF-kappaB binding sites are located within the 3'alphaE. NF-kappaB subunits, especially p50 and c-Rel, have also been shown to play critical and differential roles in regulating B cell proliferation, Ig secretion, germline C(H) transcription and Ig class switching. Thus, NF-kappaB could regulate B cell function in part through modulation of 3'alphaE activity. In this study we determined whether p50 and/or c-Rel regulate 3'alphaE activity in normal murine B cells and whether this depends on the nature of the B cell activator. For this purpose, we crossed p50- and c-Rel-deficient mice with mice that are transgenic for a 3'alphaE-hsl,2-human beta-globin reporter gene, and established p50(-/-) or c-Rel(-/-) mice homozygous for the enhancer transgene. We show, using optimal stimulating conditions, that p50 selectively augments 3'alpha E-hsl,2 activity in lipopolysaccharide-activated B cells, whereas c-Rel is required for optimal 3'alphaE-hs1,2 induction in B cells activated through CD40.

Multivalent cross-linking of membrane Ig sensitizes murine B cells to a broader spectrum of CpG-containing oligodeoxynucleotide motifs, including their methylated counterparts, for stimulation of proliferation and Ig secretion.

We have previously reported that B cells that are activated by multivalent but not bivalent membrane Ig cross-linking ligands synergize with various B cell activators culminating in enhanced B cell proliferation. In this study we asked whether B cells that are activated by a multivalent mIg cross-linking agonist could respond to oligodeoxynucleotides (ODN) containing non-stimulatory motifs. Earlier reports have shown that ODN containing a CpG motif in which the cytosine is unmethylated and is flanked by two 5' purines and two 3' pyrimidines induce high levels of B cell activation, while ODN whose CpG are methylated or flanked by sequences other than the optimal two 5' purines and two 3' pyrimidines were non-stimulatory. In this manuscript we show that when B cells are stimulated in vitro with dextran-conjugated anti-IgD antibodies (anti-IgD-dex), as the multivalent mIg ligand, their proliferation is enhanced and they can be induced to secrete Ig in response to ODN containing various non-optimal motifs, both methylated and non-methylated. Furthermore we could induce synergistic levels of proliferation with concentrations of anti-IgD-dex that were in the picomolar concentration range and with concentrations of ODN that were 10- to 100-fold less than previously reported to be necessary for mitogenic activity. These data provided a model to explain how low concentrations of a multi-epitope-expressing microorganism in the context of mammalian (methylated) or microorganism (non-methylated) DNA can lead to dysregulated B cell proliferation and Ig secretion.

In vivo polysaccharide-specific IgG isotype responses to intact Streptococcus pneumoniae are T cell dependent and require CD40- and B7-ligand interactions.

In vivo Ig responses to soluble, haptenated polysaccharide (PS) Ags are T cell independent and do not require CD40 ligand (CD40L). However, little is known regarding the regulation of in vivo PS-specific Ig responses to intact bacteria. We immunized mice with a nonencapsulated, type 2 Streptococcus pneumoniae (R36A) and compared the parameters that regulated in vivo Ig isotype responses to the bacterial cell wall C-PS determinant, phosphorylcholine (PC), relative to Ig responses to the cell wall protein, pneumococcal surface protein A. Consistent with previous reports using soluble PS and protein Ags, the anti-PC and anti-pneumococcal surface protein A responses differed in that the anti-PC response was induced more rapidly, had a distinctive Ig isotype profile, and failed to demonstrate boosting upon secondary challenge with R36A. However, in contrast to previous studies, the IgG anti-PC response was TCR-alphabeta+ T cell dependent, required CD40L, and was blocked by administration of CTLA4 Ig. The nature of the T cell help for the anti-PC response had distinct features in that it was only partially blocked by CTLA4 Ig and was dependent upon both CD4+ and CD8+ T cells. Surprisingly, whereas the IgM anti-PC response was largely T cell independent, a strong requirement for CD40L was still observed, suggesting the possibility of an in vivo T cell-independent source for CD40L-dependent help. These data suggest that the regulatory parameters that govern in vivo Ig responses to purified, soluble PS Ags may not adequately account for PS-specific Ig responses to intact bacteria.

Heterogeneity in the ability of cytotoxic murine NK cell clones to enhance Ig secretion in vitro.

We recently described a panel of cytotoxic murine NK cell clones that also enhanced Ig secretion by B cells activated in an in vitro model of T cell-independent type 2 (TI-2) responses. We employed dextran-conjugated anti-IgD (alphadelta-dex) as a model antigen. Here we study the mechanism of Ig induction by these clones. Addition of the various NK clones to sort-purified B cells stimulated with alphadelta-dex and IL-2 resulted in a markedly heterogeneous increase in Ig secretion, which varied from 3-fold, as mediated by clone PKO 56, to 15-fold, as induced by clone PKO 101. The other NK cells showed intermediate levels of Ig induction. Furthermore, while addition of as few as 0.04% of PKO 101 cells stimulated significant increases and 1% induced near maximum Ig production, a 3% addition of PKO 56 cells was required for significant enhancement of Ig secretion. Supernatant material collected from the NK clones mediated Ig production at levels that mirrored the induction by the corresponding cells. Cytokine analysis showed that while all members of the NK panel produced IFN-gamma only two secreted granulocyte macrophage colony stimulating factor and that the levels of Ig induction mediated by the NK clones correlated only with their levels of IFN-gamma secretion. Culture of B and NK cells in the presence of anti-IFN-gamma demonstrated that IFN-gamma was the critical cytokine in NK-induced Ig production. These findings establish heterogeneity in the ability of NK cells to increase Ig secretion in vitro and show that NK-produced IFN-gamma is an important factor in determining this heterogeneity.

The p65 subunit of NF-kappa B is redundant with p50 during B cell proliferative responses, and is required for germline CH transcription and class switching to IgG3.

B cells lacking individual NF-kappa B/Rel family members exhibit defects in activation programs. We generated small resting B cells lacking p65 or p50 alone, or lacking both p50 and p65, then evaluated the ability of these cells to proliferate, secrete Ig, and undergo Ig class switching. B cells lacking p65 proliferated well in response to all stimuli tested. However, these cells demonstrated an isolated defect in switching to IgG3, which was associated with a decrease in gamma 3 germline CH gene expression. Whereas, previously reported, B cells lacking p50 alone had a severe proliferative defect in response to LPS, a moderate defect in response to CD40 ligand (CD40L), and normal proliferation to Ag receptor cross-linking using dextran-conjugated anti-IgD Abs (alpha delta-dex), B cells lacking both p50 and p65 exhibited severely impaired proliferation in response to LPS, alpha delta-dex, and CD40L. This defect could be overcome by simultaneous administration of alpha delta-dex and CD40L. In response to this latter combination of stimuli, B cells lacking both p50 and p65 secreted Ig and underwent isotype switching to IgG1 as efficiently as B cells lacking p50 alone. These data demonstrate a role for the p65 subunit of NF-kappa B in germline CH gene expression as well as functional redundancy between p50 and p65 during proliferative responses.

Phenotypic and functional characterization of a panel of cytotoxic murine NK cell clones that are heterogeneous in their enhancement of Ig secretion in vitro.

NK cells not only function as cytotoxic effector cells, but also have immunoregulatory roles including the enhancement of Ig secretion. To have a stable and uniform population of NK cells to study their role in Ig secretion, we generated murine NK clones. Thus, culture of splenocytes from mice that were homozygous for a mutation in the p53 tumor suppressor gene (p53-KO) with IL-2 and poly(IC) resulted in a long-term NK line, from which four stable clones were derived. This approach also yielded a long-term NK line from splenocytes of normal C57BL/6 mice. Identification of the clones as members of the NK lineage was based on large granular morphology, expression of NK-TR and absence of TCR gene rearrangement. Flow cytometry revealed that all clones expressed IL-2R alpha and beta, chains and B220, but no CD3, NK1.1, DX5 or Ly-49. RT-PCR analysis showed heterogeneity in NK1.1 gene expression, and demonstrated expression of perforin and several granzymes in all clones. Three out of four clones lysed YAC-1, but not P815 target cells, corresponding to a pattern of NK specificity. All NK clones enhanced Ig secretion in an in vitro model for T cell-independent type 2 antigens, albeit to varying degrees. We found no correlation between the degree of helper activity of the NK clones and the level of their cytotoxic activity on YAC-1 targets. Thus, we established murine NK clones, and show that they mediate both cytotoxicity and enhancement of Ig secretion.

Efficient recombination of a switch substrate retrovector in CD40-activated B lymphocytes: implications for the control of CH gene switch recombination.

Maturing B lymphocytes possess a recombination activity that switches the class of heavy chain Ig. The nature of the recombination activity, its molecular requirements and regulation remain elusive questions about B lymphocyte biology and development. Class switch recombination is controlled by cytokine response elements that are required to differentially activate CH gene transcription before their subsequent recombination. Here, we show that cultures of purified murine and human B cells, stimulated only by CD40 receptor engagement, possess a potent switch recombination activity. CD40 ligand-stimulated murine and human B lymphocytes were infected with recombinant retroviruses containing Smu and S gamma 2b sequences. Chromosomally integrated switch substrate retrovectors (SSRs), harboring constitutively transcribed S sequences, underwent extensive recombinations restricted to their S sequences with structural features akin to endogenous switching. SSR recombination commenced 4 days postinfection (5 days poststimulation) with extensive switch sequence recombination over the next 2 to 3 days. In contrast, endogenous S gamma 2b and S gamma 1 sequences did not undergo appreciable switch recombination upon CD40 signaling alone. As expected, IL-4 induced endogenous Smu to S gamma 1 switching, while endogenous Smu to S gamma 2b fusions remained undetectable. Surprisingly, IL-4 enhanced the onset of SSR recombination in CD40-stimulated murine B cells, with S-S products appearing only 2 days postinfection and reaching a maximum within 2 to 3 days. The efficiency of switch recombination with SSRs resembles that seen for endogenous C(H) class switching.

Multiple hemopoietic defects and lymphoid hyperplasia in mice lacking the transcriptional activation domain of the c-Rel protein.

The c-rel protooncogene encodes a member of the Rel/nuclear factor (NF)-kappaB family of transcriptional factors. To assess the role of the transcriptional activation domain of c-Rel in vivo, we generated mice expressing a truncated c-Rel (Deltac-Rel) that lacks the COOH-terminal region, but retains a functional Rel homology domain. Mice with an homozygous mutation in the c-rel region encoding the COOH terminus of c-Rel (c-relDeltaCT/DeltaCT) display marked defects in proliferative and immune functions. c-relDeltaCT/DeltaCT animals present histopathological alterations of hemopoietic tissues, such as an enlarged spleen due to lymphoid hyperplasia, extramedullary hematopoiesis, and bone marrow hypoplasia. In older c-relDeltaCT/DeltaCT mice, lymphoid hyperplasia was also detected in lymph nodes, liver, lung, and stomach. These animals present a more severe phenotype than mice lacking the entire c-Rel protein. Thus, in c-relDeltaCT/DeltaCT mice, the lack of c-Rel activity is less efficiently compensated by other NF-kappaB proteins.

Enhanced protective antibody responses to PspA after intranasal or subcutaneous injections of PspA genetically fused to granulocyte-macrophage colony-stimulating factor or interleukin-2.

Antibody to pneumococcal surface protein A (PspA) has been shown to be protective for Streptococcus pneumoniae infections in mice. In an attempt to define a model for inducing protective antibody to PspA in the absence of adjuvant, we designed two genetic fusions, PspA-interleukin-2 [IL-2]) and PspA-granulocyte-macrophage colony-stimulating factor (GM-CSF). These constructs maintained high cytokine function in vitro, as tested by their activity on IL-2 or GM-CSF-dependent cell lines. While intranasal immunization with PspA induced no detectable anti-PspA response, both PspA-IL-2 and PspA-GM-CSF stimulated high immunoglobulin G1 (IgG1) antibody responses. Interestingly, only the PspA-IL-2, not the PspA-GM-CSF, construct stimulated IgG2a antibody responses, suggesting that this construct directed the response along a TH1-dependent pathway. Comparable enhancement of the anti-PspA response with similar isotype profiles was observed after subcutaneous immunization as well. The enhancement observed with PspA-IL-2 was dependent on IL-2 activity in that it was not seen in IL-2 receptor knockout mice, while PspA in alum induced high-titer antibody in these mice. The antibody was tested for its protective activity in a mouse lethality model using S. pneumoniae WU-R2. Passive transfer of 1:90 dilutions of sera from mice immunized with PspA-IL-2 and PspA-GM-CSF elicited protection of CBA/N mice against intravenous challenge with over 170 50% lethal doses of capsular type 3 strain WU2. Only 0.17 microg or less of IgG antibody to PspA was able to provide passive protection against otherwise fatal challenge with S. pneumoniae. The data demonstrate that designing protein-cytokine fusions may be a useful approach for mucosal immunization and can induce high-titer systemic protective antibody responses.

Nuclear factor (NF)-kappa B2 (p100/p52) is required for normal splenic microarchitecture and B cell-mediated immune responses.

The nfkb2 gene is a member of the Rel/NF-kappa B family of transcription factors. COOH-terminal deletions and rearrangements of this gene have been associated with the development of human cutaneous T cell lymphomas, chronic lymphocytic leukemias, and multiple myelomas. To further investigate the function of NF-kappa B2, we have generated mutant mice carrying a germline mutation of the nfkb2 gene by homologous recombination. NF-kappa B2-deficient mice showed a marked reduction in the B cell compartment in spleen, bone marrow, and lymph nodes. Moreover, spleen and lymph nodes of mutant mice presented an altered architecture, characterized by diffuse, irregular B cell areas and the absence of discrete perifollicular marginal and mantle zones; the formation of secondary germinal centers in spleen was also impaired. Proliferation of NF-kappa B2-deficient B cells was moderately reduced in response to lipopolysaccharide, anti-IgD-dextran, and CD40, but maturation and immunoglobulin switching were normal. However, nfkb2 (-/-) animals presented a deficient immunological response to T cell-dependent and -independent antigens. These findings indicate an important role of NF-kappa B2 in the maintenance of the peripheral B cell population, humoral responses, and normal spleen architecture.

B cells genetically deficient in the c-Rel transactivation domain have selective defects in germline CH transcription and Ig class switching.

The Ig heavy chain locus contains a number of binding sites for the transcriptional activator, c-Rel. In this study, we evaluated the capacity of B cells from mice made genetically deficient in the C-terminal, transactivation domain of the c-Rel protein (delta c-Rel) to undergo Ig class switching. Flow-cytometric and digestion circularization PCR analyses revealed that delta c-Rel B cells failed to switch to IgG3 in response to LPS alone, or to IgG1 or IgE in response to LPS + IL-4. This failure to switch to IgG3 or IgG1 was associated with a corresponding loss of germline CH gamma 3 or CH gamma 1 RNA. However, the defective switching to IgE in delta c-Rel B cells was associated with normal levels of germline CH epsilon RNA relative to control B cells. The ability of delta c-Rel B cells to switch to IgG1, in response to LPS + IL-4, could be restored through the action(s) of additional stimuli, and this was associated with induction of normal levels of germline CH gamma 1 RNA relative to controls. In contrast, LPS-activated B cells from delta c-Rel mice underwent normal switching to IgA in the presence of TGF-beta, relative to control B cells. This was associated with equivalent steady state levels of germline CH alpha RNA between the two B cell populations. These data are the first to demonstrate a key and selective role for c-Rel in the regulation of Ig class switching. Furthermore, distinct differences are revealed in the Ig isotype induction profiles of B cells lacking c-Rel activity vs those deficient in p50/nuclear factor-kappa B.

Regulation of Ku expression in normal murine B cells by stimuli that promote switch recombination.

DNA-dependent protein kinase, the catalytic subunit associated with the Ku heterodimer (Ku70/Ku86), has been implicated in switch recombination. Therefore, we tested whether certain stimuli known to promote switch recombination may act in part by inducing Ku expression. We find that resting B cells contain relatively low levels of nuclear Ku, but that Ku expression can be up-regulated by culturing the cells with two switch stimuli. First, IL-4 and CD40 engagement in combination, but neither of these stimuli acting alone, strongly induce Ku expression; Ku levels rise within 24 h, about 2 days before switch recombination is detected. Second, dextran-conjugated anti-IgD Abs strongly induce Ku expression, which is variably enhanced by IL-5, but not by IL-4. Our data suggest that switch recombination may be regulated, at least in part, through changes in the nuclear expression of Ku.

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.

Restoration of T cell-independent type 2 induction of Ig secretion by neonatal B cells in vitro.

The humoral immune response of neonates to T cell-independent type 2 (TI-2) Ags is markedly defective. We previously demonstrated that multivalent membrane Ig cross-linking, using dextran-conjugated anti-Ig Abs (anti-Ig-dextran), is an in vitro model for membrane Ig-dependent TI-2 induction of Ig secretion. In this work, we demonstrate that highly purified neonatal B cells are intrinsically defective in IgM secretion in response to anti-Ig-dextran and cytokines in vitro, as well as other modes of B cell activation, relative to adult B cells. However, costimulation of anti-Ig-dextran-activated neonatal B cells with either CD40-ligand, a recombinant bacterial lipoprotein, or LPS restores the IgM secretory response of neonatal B cells to adult levels. Analysis of Ig isotype secretion indicates that neonatal B cells have an enhanced capacity to secrete IgE and IgA relative to other Ig isotypes. These data suggest that neonatal B cells are competent to secrete Ig in response to TI-2 Ags if adequate costimuli are provided, and thus may have particular relevance for the design of vaccine strategies in the immunodeficient host. The data also suggest that neonatal B cells are programmed to secrete relatively enhanced amounts of IgE and IgA, which may be relevant for antimicrobial resistance at mucosal surfaces.