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Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections.

Fanconi anemia (FA) is characterized by a progressive bone marrow failure and an increased incidence of cancer. FA patients have high susceptibility to immune-related complications such as infection and posttransplant graft-versus-host disease. In this study, we investigated the effect of FA deficiency in B cell function using the Fancc mouse model. Fancc−/− B cells show a specific defect in IgG2a switch and impaired Ab-secreting cell (ASC) differentiation. Global transcriptome analysis of naive B cells by mRNA sequencing demonstrates that FA deficiency deregulates a network of genes involved in immune function. Significantly, many genes implicated in Wnt signaling were aberrantly expressed in Fancc−/− B cells. Consistently, Fancc−/− B cells accumulate high levels of β-catenin under both resting and stimulated conditions, suggesting hyperactive Wnt signaling. Using an in vivo Wnt GFP reporter assay, we verified the upregulation of Wnt signaling as a potential mechanism responsible for the impaired Fancc−/− B cell differentiation. Furthermore, we showed that Wnt signaling inhibits ASC differentiation possibly through repression of Blimp1 and that Fancc−/− B cells are hypersensitive to Wnt activation during ASC differentiation. Our findings identify Wnt signaling as a physiological regulator of ASC differentiation and establish a role for the Wnt pathway in normal B cell function and FA immune deficiency.

Ab-secreting cell (ASC) expansion and survival are important processes in optimizing vaccines and controlling autoimmunity. The microenvironment of the medullary cords is positioned to control these key processes. Previously, we imaged and characterized ASC differentiation and migration by intravital microscopy in the lymph node (LN) by transferring and activating B cells expressing yellow fluorescent protein only in the ASC compartment. In this study, we observed that yellow fluorescent protein+ ASCs in the medullary cords migrated along myelomonocytic cells and arrested in contact with them. Acute ablation of myeloid cells using the human diphtheria receptor system (diphtheria toxin receptor [DTR]) expressed in Lysmd1-cre–positive cells increased ASC and Ab production by 2-fold. Increases in ASC numbers were associated with cell proliferation based on Ki-67 staining, rather than reduced apoptosis, or changes in egress from the LN. Using DTR-mediated ablation targeted to Ccr2-expressing myeloid cells also generated increases in ASCs. In contrast, neither the depletion of Gr-1–positive cells with an Ab nor the ablation of cells using a cd11c-DTR resulted in any change in ASCs. IL-6 cytokine signaling can enhance ASC production and has been implicated in dampening ASCs in lupus mouse models through myeloid cells. Using mixed bone marrow chimeras, we observed that IL-6 enhances ASC production, but IL-6 production was not required by myeloid cells to dampen ASCs in the LN. Inhibition of ASCs by these myeloid cells in the LN provides a new regulatory mechanism with implications for tuning Ab responses.

Although studies investigating the nature of Ab-secreting cells (ASCs) during acute infection with influenza or dengue virus found that the ASC response was dominated by virus-specific IgG secretion, the Ag specificity and phenotype of ASCs during primary acute viral infection were not identified. To this end, we investigated the nature of ASCs in direct ex vivo assays from patients with acute hepatitis A caused by primary infection with hepatitis A virus (HAV). We found that the frequency of CD27highCD38high ASCs was markedly increased in the peripheral blood during the acute phase of HAV infection. Moreover, substantial numbers of ASCs were non-HAV–specific and dominantly secreted IgM. We detected HAV-specific ASCs by staining with fluorochrome-tagged HAV-VP1 protein. As compared with HAV-specific ASCs, non-HAV–specific ASCs were Ki-67lowCD138highCD31highCD38high, demonstrating that non-HAV–specific ASCs had a bone marrow plasma cell–like phenotype whereas HAV-specific ASCs had a phenotype typical of circulating plasmablasts. These data suggest that non-HAV–specific ASCs might be mobilized plasma cells from the bone marrow or the spleen, whereas HAV-specific ASCs were newly generated plasmablasts. In this study, we provide evidence that pre-existing plasma cells are released into the circulation and contribute to Ag-nonspecific secretion of IgM during primary HAV infection.

Nucleic acid (NA)–sensing TLRs (NA-TLRs) promote the induction of anti-nuclear Abs in systemic lupus erythematosus. However, the extent to which other nonnuclear pathogenic autoantibody specificities that occur in lupus and independently in other autoimmune diseases depend on NA-TLRs, and which immune cells require NA-TLRs in systemic autoimmunity, remains to be determined. Using Unc93b13d lupus-prone mice that lack NA-TLR signaling, we found that all pathogenic nonnuclear autoantibody specificities examined, even anti-RBC, required NA-TLRs. Furthermore, we document that NA-TLRs in B cells were required for the development of antichromatin and rheumatoid factor. These findings support a unifying NA-TLR–mediated mechanism of autoantibody production that has both pathophysiological and therapeutic implications for systemic lupus erythematosus and several other humoral-mediated autoimmune diseases. In particular, our findings suggest that targeting of NA-TLR signaling in B cells alone would be sufficient to specifically block production of a broad diversity of autoantibodies.

Ab responses in early life are low and short-lived; therefore, induction of protective immunity requires repeated vaccinations. One of the major limitations in early-life immunity is delayed maturation of follicular dendritic cells (FDCs), which play a central role in mediating the germinal center (GC) reaction leading to production of Ab-secreting cells (AbSCs). We assessed whether a nontoxic mutant of Escherichia coli heat-labile enterotoxin (LT-K63) and CpG1826 as model adjuvants could accelerate FDC maturation and immune response in neonatal mice, using a pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) as a model vaccine. In neonatal NMRI mice, a single dose of Pnc1-TT coadministered with LT-K63 enhanced Pnc1-TT–induced GC reaction. In contrast, CpG1826 had no effect. Accordingly, LT-K63, but not CpG1826, accelerated the maturation of FDC networks, detected by FDC-M2+ staining, characteristic for adult-like FDCs. This coincided with migration of MOMA-1+ macrophages into the GCs that can enhance GC reaction and B cell activation. The FDC-M2+ FDC networks colocalized with enhanced expression of TNF-α, which is critical for the maintenance of mature FDCs and is poorly expressed in neonates. The accelerated maturation of FDC networks correlated with increased frequency and prolonged persistence of polysaccharide- and protein-specific IgG+ AbSCs in spleen and bone marrow. Our data show for the first time, to our knowledge, that an adjuvant (LT-K63) can overcome delayed maturation of FDCs in neonates, enhance the GC reaction, and prolong the persistence of vaccine-specific AbSCs in the BM. These properties are attractive for parenteral vaccination in early life.

Regulatory B cells that are functionally defined by their capacity to express IL-10 (B10 cells) downregulate inflammation and autoimmunity. In studies using well-defined IL-10 reporter mice, this rare B10 cell subset was also found to maintain a capacity for plasma cell differentiation. During a transient period of il10 transcription, the blimp1 and irf4 transcription factors were induced in B10 cells, whereas pax5 and bcl6 were downregulated as a significant fraction of B10 cells completed the genetic and phenotypic program leading to Ab-secreting cell differentiation in vitro and in vivo. B10 cell-derived IgM reacted with both self- and foreign Ags, whereas B10 cells generated Ag-specific IgG in response to immunizations. Moreover, B10 cells represented a significant source of serum IgM and IgG during adoptive-transfer experiments and produced Ag-specific, polyreactive and autoreactive Ab specificities that were consistent with their expression of a diverse AgR repertoire. Thereby, B10 cells limit inflammation and immune responses by the transient production of IL-10, and may facilitate clearance of their eliciting Ags through an inherent capacity to quickly generate polyreactive and/or Ag-specific Abs during humoral immune responses.

Germinal centers (GCs) are specialized microenvironments that generate high-affinity Ab-forming cells (AFCs) and memory B cells. Many B cells undergo apoptosis during B cell clonal selection in GCs. Although the factors that regulate the AFC and GC responses are not precisely understood, it is widely believed that dysregulated AFCs and GCs contribute to autoimmunity. The Mer receptor tyrosine kinase (Mer) facilitates macrophage clearance of apoptotic cells. The Tyro-3, Axl, and Mer receptors, including Mer, suppress TLRs and cytokine-mediated inflammatory responses. We report in this study that tingible body macrophages (TBMϕs) in GCs express Mer. Compared to C57BL/6 (B6) controls, Mer-deficient (Mer−/−) mice had significantly higher AFC, GC, and Th1-skewed IgG2 Ab (especially IgG2c) responses against the T cell-dependent Ag (4-hydroxy-3-nitrophenyl) acetyl-chicken γ globulin. Mer−/− mice had a significantly higher percentage of GC B cells on days 9, 14, and 21 postimmunization compared with B6 controls. Significantly increased numbers of apoptotic cells accumulated in Mer−/− GCs than in B6 GCs, whereas the number of TBMϕs remained similar in both strains. Our data are the first, to our knowledge, to demonstrate a critical role for Mer in GC apoptotic cell clearance by TBMϕs and have interesting implications for Mer in the regulation of B cell tolerance operative in the AFC and GC pathways.

We have recently reported that the sublingual (s.l.) mucosa is an efficient site for inducing systemic and mucosal immune responses. In this study, the potential of s.l. immunization to induce remote Ab responses and CD8+ cytotoxic responses in the female genital tract was examined in mice by using a nonreplicating Ag, OVA, and cholera toxin (CT) as an adjuvant. Sublingual administration of OVA and CT induced Ag-specific IgA and IgG Abs in blood and in cervicovaginal secretions. These responses were associated with large numbers of IgA Ab-secreting cells (ASCs) in the genital mucosa. Genital ASC responses were similar in magnitude and isotype distribution after s.l., intranasal, or vaginal immunization and were superior to those seen after intragastric immunization. Genital, but not blood or spleen, IgA ASC responses were inhibited by treatment with anti-CCL28 Abs, suggesting that the chemokine CCL28 plays a major role in the migration of IgA ASC progenitors to the reproductive tract mucosa. Furthermore, s.l. immunization with OVA induced OVA-specific effector CD8+ cytolytic T cells in the genital mucosa, and these responses required coadministration of the CT adjuvant. Furthermore, s.l. administration of human papillomavirus virus-like particles with or without the CT adjuvant conferred protection against genital challenge with human papillomavirus pseudovirions. Taken together, these findings underscore the potential of s.l. immunization as an efficient vaccination strategy for inducing genital immune responses and should impact on the development of vaccines against sexually transmitted diseases.

Although the mucosal and the systemic immune compartments are structurally and functionally independent, they engage in cross-talk under specific conditions. To investigate this cross-talk, we vaccinated mice with tetanus toxoid together with cholera toxin with s.c. priming followed by intrarectal (IR) boosting. Interestingly, higher numbers of Ag-specific IgA and IgG Ab-secreting cells (ASCs) were detected in the lamina propria of the large intestine of mice vaccinated s.c.-IR. Ag-specific ASCs from the colon migrated to SDF-1α/CXCL12 and mucosae-associated epithelial chemokine/CCL28, suggesting that CXCR4+ and/or CCR10+ IgA ASCs found in the large intestine after s.c.-IR are of systemic origin. In the colonic patches-null mice, IgA ASCs in the large intestine were completely depleted. Furthermore, the accumulation of IgA ASCs in the colonic patches by inhibition of their migration with FTY720 revealed that colonic patches are the IgA class-switching site after s.c.-IR. Most interestingly, s.c.-IR induced numbers of Ag-specific IgA ASCs in the large intestine of TLR2−/−, TLR4−/−, MyD88−/−, and TRIF−/− mice that were comparable with those of wild-type mice. Taken together, our results suggest the possibility that cross-talk could occur between the large intestine and the systemic immune compartments via the colonic patches without the assistance of innate immunity.

We previously demonstrated that B cells expressing a transgenic BCR with “dual reactivity” for the hapten arsonate and nuclear autoantigens efficiently complete development to follicular phenotype and stably reside in follicles in vivo. These B cells express very low levels of surface IgM and IgD, suggesting that they avoid central deletion and peripheral anergy by reducing their avidity for autoantigen via surface BCR (sBCR) down-regulation. Since a variety of states of B cell anergy have been previously described, a thorough examination of the functional capabilities of these B cells was required to test this hypothesis. In this study, we show that surface Ig cross-linking induces amounts of proximal BCR signaling in these B cells commensurate with their reduced sBCR levels. Functionally, however, they are comparable to nonautoreactive B cells in cell cycle progression, up-regulation of activation and costimulatory molecules, and Ab-forming cell differentiation when treated with a variety of stimuli in vitro. In addition, these B cells can efficiently process and present Ag and are capable of undergoing cognate interaction with naive TCR-transgenic T cells, resulting in robust IL-2 production. Together, these data reveal a lack of intrinsic anergy involving any known mechanism, supporting the idea that this type of antinuclear Ag B cell becomes indifferent to cognate autoantigen by down-regulating sBCR.

The development of a safe and effective mucosal adjuvant is a crucial step toward a mucosal HIV/AIDS vaccine. This study seeks to determine the promise of a nontoxic mutant of cholera toxin (mCT; E112K) as a mucosal adjuvant in nonhuman primates. HIV-1 gp120 was nasally administered together with mCT E112K or native CT (nCT) as adjuvant on five to six occasions over a 6- to 8-wk period to groups of four rhesus macaques and alone to two monkeys that acted as controls. Macaques given nasal gp120 with either mCT E112K or nCT showed elevated gp120-specific IgG and IgA Ab responses with virus-neutralizing activity in both their plasma and mucosal external secretions, as well as higher numbers of gp120-specific IgA Ab-forming cells in their mucosal and peripheral lymphoid tissues and of IL-4-producing Th2-type CD4-positive (CD4+) T cells than did controls. Even though significant mucosal adjuvanticity was seen with both mCT E112K and nCT, neuronal damage was observed only in the nCT-treated, but not in the control or mCT E112K-treated groups. These results clearly show that mCT E112K is an effective and safe mucosal adjuvant for the development of a nasal HIV/AIDS vaccine.

Autoreactive B cells are not completely purged from the primary B cell repertoire, and whether they can be prevented from maturation into memory B cells has been uncertain. We show here that a population of B cells that dominates primary immune responses of BALB/c mice to influenza virus A/PR/8/34 hemagglutinin (HA) are negatively selected in transgenic mice expressing PR8 HA as an abundant membrane-bound Ag (HACII mice). However, a separate population of B cells that contains precursors of memory B cells is activated by PR8 virus immunization and is subsequently negatively selected during the formation of the memory response. Negative selection of PR8 HA-specific B cells altered the specificity of the memory B cell response to a mutant virus containing a single amino acid substitution in a B cell epitope. Strikingly, this skewed reactivity resulted from an increase in the formation of memory B cells directed to non-self-epitopes on the mutant virus, which increased 8-fold in HACII mice relative to nontransgenic mice and precisely compensated for the absence of autoreactive PR8 HA-specific memory B cells. Negative selection of PR8 HA-specific B cells was a dominant process, since B cells from HACII mice could induce negative selection of PR8 HA-specific B cells from BALB/c mice. Lastly, HA-specific memory responses were unaffected by self-tolerance in another lineage of HA-transgenic mice (HA104 mice), indicating that the amount and/or cell type in which self-Ags are expressed can determine their ability to prevent autoreactive memory B cell formation.

Lupus nephritis is characterized by immune complex deposition and infiltration of inflammatory cells into the kidney including Ab-producing cells (AbPCs). Although AbPCs play a central role in the pathogenesis of immune complex glomerulonephritis in lupus, the specificity and pathogenic role of AbPCs infiltrating into the kidneys in lupus are poorly understood. To characterize AbPCs present in lupus kidneys, we isolated AbPCs from diseased MRL/MpJ-Faslpr (MRL/lpr) mouse kidneys. ELISPOT assays, using glomerular Ag (GA) extracts as Ag, demonstrated significant enhancement of anti-GA AbPCs in the kidneys as compared in peripheral blood or spleen of the same mouse. We isolated hybridomas with anti-GA specificity from MRL/lpr mouse kidneys. All the anti-GA mAbs had polyreactive binding to ssDNA, dsDNA, and IgG (i.e., rheumatoid factor), but not to histones or Sm. Sequence analysis of anti-GA Abs suggested the occurrence of somatic mutations and amino acid replacement in complementarity-determining regions with a high replacement to silent ratio resulting in charged amino acids. Intravenous administration of the monoclonal anti-GA Abs into BALB/c mice resulted in graded deposition in glomeruli paralleling their ELISA anti-GA reactivity. These results suggest that AbPCs infiltrating the kidneys in MRL/lpr mice accumulate as a result of Ag selection and likely play a pathologic role in lupus nephritis.

Peritoneal cavity B-1 cells are believed to produce IgM natural Abs. We have used α1,3-galactosyltransferase-deficient (GalT−/−) mice, which, like humans, produce IgM natural Abs against the carbohydrate epitope Galα1,3Gal (Gal), to demonstrate that peritoneal cavity B-1b cells with anti-Gal receptors produce anti-Gal IgM Abs only after LPS stimulation. Likewise, peritoneal cavity cells of GalT−/− and wild-type mice do not produce IgM Abs of other specificities without LPS stimulation. Development of Ab-secreting capacity is associated with loss of CD11b/CD18 (Mac-1) expression. In contrast, there are large numbers of cells producing anti-Gal and other IgM Abs in fresh splenocyte preparations from GalT−/− and (for non-Gal specificities) wild-type mice. These cells are Mac-1− but otherwise B-1b-like in their phenotype. We therefore hypothesized a pathway wherein peritoneal cavity B cells migrate into the spleen after activation in vivo and lose Mac-1 expression to become IgM Ab-producing cells. Consistent with this possibility, splenectomy reduced anti-Gal Ab production after immunization of GalT−/− mice with Gal-positive rabbit RBC. Furthermore, splenectomized B6 GalT−/−, Ig μ-chain mutant (μ−/−) (both Gal- and B cell-deficient) mice produced less anti-Gal IgM than nonsplenectomized controls after adoptive transfer of peritoneal cavity cells from B6 GalT−/− mice. When sorted GalT−/− Mac-1+ peritoneal cavity B cells were adoptively transferred to B6 GalT−/−, μ−/− mice, IgM Abs including anti-Gal appeared, and IgM-producing and Mac1− B cells were present in the spleen 5 wk after transfer. These findings demonstrate that peritoneal cavity Mac-1+ B-1 cells are precursors of Mac-1− splenic IgM Ab-secreting cells.

The factors limiting neonatal and infant IgG Ab responses to T-dependent Ags are only partly known. In this study, we assess how these B cell responses are influenced by the postnatal development of the spleen and lymph node microarchitecture. When BALB/c mice were immunized with alum-adsorbed tetanus toxoid at various stages of their immune development, a major functional maturation step for induction of serum IgG, Ab-secreting cells, and germinal center (GC) responses was identified between the second and the third week of life. This correlated with the development of the follicular dendritic cell (FDC) network, as mature FDC clusters only appeared at 2 wk of age. Adoptive transfer of neonatal splenocytes into adult SCID mice rapidly induced B cell follicles and FDC precursor differentiation into mature FDC, indicating effective recruitment and signaling capacity of neonatal B cells. In contrast, adoptive transfer of adult splenocytes into neonatal SCID mice induced primary B cell follicles without any differentiation of mature FDC and failed to correct limitations of tetanus toxoid-induced GC. Thus, unresponsiveness to lymphoid-mediated signals at the level of neonatal FDC precursors delays FDC maturation and GC induction, thus limiting primary Ab-secreting cell responses to T-dependent Ags in early postnatal life.

Secretory IgA (SIgA) is the most characteristic component of the mucosal immune system and has long been considered the major protective factor that prevents pathogens from invading hosts through the mucosae. Recent studies, however, have suggested that complete immunity against a range of mucosal bacterial and viral pathogens can be achieved in the absence of IgA. Therefore, to further dissect the role of SIgA, we generated mice deficient in the polymeric Ig receptor (pIgR−/− mice). As a result of an inability to transport dimeric IgA to the secretions, pIgR−/− mice are deficient in SIgA and accumulate circulating dimeric IgA, with serum levels 100-fold greater than those observed in normal mice. Examination of lamina propria mononuclear cells showed that pIgR−/− mice had ∼3 times as many IgA-secreting cells as C57BL/6 mice. Further analysis showed that these cells displayed the differentiated IgA+ B220− phenotype and accounted for a 2-fold increase in the number of lamina propria blast cells in the pIgR−/− mice. Subsequent experiments showed that OVA-specific CD4+ T cell expansion following OVA feeding was not elevated in pIgR−/− mice. Furthermore, no differences in CD8+ T cell tolerance or induction of influenza virus-specific CD8+ T cells were detected in pIgR−/− mice compared with controls. Therefore, while SIgA is clearly involved in maintaining some parameters of mucosal homeostasis in the intestine, the mechanisms associated with its barrier function and the clinical consequences of its deficiency are yet to be identified.

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4+ T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4+, CD45RB+ T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer’s patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.

In the present work, we have dissected the mechanisms responsible for the impaired humoral responses in aging. We found that there was a substantially higher level of Ab-forming cells in the spleens of aged mice than that of young controls. However, the number of high-affinity, class-switched Ab-forming cells was severely decreased in the spleen of aged mice. The accumulation of low-affinity IgM Ab-forming cells in the spleens of aged animals was not due to a deficiency in isotype switching because the number of total IgG1 splenic plasma cells was not significantly reduced. Remarkably, plasma cells of both low and high affinity were significantly diminished in the bone marrow of aged mice compared with that of young mice. The results from reconstitution experiments showed that aged bone marrow was less supportive for plasma cells derived from young splenic B cells. These findings suggest that humoral immune deficiency in aging results from at least two mechanisms: the inability to generate sufficient numbers of high-affinity Ab-forming cells, which is a result of diminished germinal center reaction, and the defective bone marrow environment that has diminished ability to support the selection and survival of long-term Ab-forming cells.

Previous studies suggest that lymphocyte trafficking to head and neck lymph nodes, also referred to as cranial-, oral-, nasal-associated lymphoid tissue (CONALT), is L-selectin (L-Sel) dependent, despite coexpression of α4β7, resulting in their marked reduction in L-Sel-deficient (L-Sel−/−) mice. Consequently, early phase (16 days) Ab responses to cholera toxin (CT) are diminished. The following studies reveal that lack of mucosal effector responses is not caused by loss of inductive immune responses in the L-Sel−/− CONALT. Indeed, there was an increased accumulation of total IgA, but not Ag-specific IgA Ab-forming cells (AFC) in L-Sel−/− CONALT. This increased accumulation was not evident in L-Sel+/+ CONALT. Identification of lymphocyte-homing receptors on L-Sel−/− and L-Sel+/+ CONALT lymphocytes revealed no significant differences in expression of α4β7, which might contribute to lymphocyte homing in the absence of L-Sel. Studies of CONALT responses during the late phase (6 wk post-intranasal immunization) revealed the number of lymphocytes recovered from L-Sel−/− CONALT was less than L-Sel+/+ CONALT; however, L-Sel−/− CT-specific and total AFC did not vary from 16-day responses, suggesting a defect in CT-specific B cell export. No significant differences in α4β7 expression between L-Sel−/− and L-Sel+/+ CONALT were noted. Yet, these increases in CONALT AFC correlated with restoration of immunity in L-Sel−/− nasal passages and reproductive tracts.

Infection by the neurotropic JHM strain of mouse hepatitis virus produces an acute demyelinating encephalomyelitis. While cellular immunity initially eliminates infectious virus, CNS viral persistence is predominantly controlled by humoral immunity. To better understand the distinct phases of immune control within the CNS, the kinetics of humoral immune responses were determined in infected mice. Early during clearance of the JHM strain of mouse hepatitis virus, only few virus-specific Ab-secreting cells (ASC) were detected in the periphery or CNS, although mature B cells and ASC without viral specificity were recruited into the CNS concomitant with T cells. Serum antiviral Ab and CNS virus-specific ASC became prominent only during final elimination of infectious virus. Virus-specific ASC peaked in lymphoid organs before the CNS, suggesting peripheral B cell priming and maturation. Following elimination of infectious virus, virus-specific ASC continued to increase within the CNS and then remained stable during persistence, in contrast to declining T cell numbers. These data comprise three novel findings. Rapid recruitment of B cells in the absence of specific Ab secretion supports a potential Ab-independent effector function involving lysis of virus-infected cells. Delayed recruitment relative to viral clearance and subsequent maintenance of a stable CNS ASC population demonstrate differential regulation of T and B lymphocytes within the infected CNS. This supports a critical role of humoral immunity in regulating viral CNS persistence. Lastly, altered antiviral ASC specificities following clearance of infectious virus suggest ongoing recruitment of peripheral memory cells and/or local B cell differentiation.

Development of vaccines capable of preventing the transmission or limiting the severity of sexually transmitted viruses, such as HSV and HIV, will likely be dependent on the induction of potent long-lasting mucosal immune responses in the genital tract. Recently, synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs were shown to serve as potent adjuvants for the induction of mucosal immune responses. Here, we show that intranasal immunization with CpG ODN, plus recombinant glycoprotein B (rgB) of HSV-1, results in significantly elevated levels of specific anti-gB IgA Abs in vaginal washes that remained high throughout the estrous cycle. Additionally, dramatically elevated numbers of specific IgA Ab-secreting cells were present and persisted in the genital tract in response to intravaginal (IVAG) HSV-2 challenge. HSV-2-specific CTL were observed at moderate levels in the spleens of CpG or non-CpG ODN-immunized mice. In contrast, strong CTL responses were observed locally in the genital tissues of both groups following IVAG HSV-2 challenge. Interestingly, mice immunized intranasally with rgB plus CpG ODN, but not non-CpG ODN, were significantly protected following IVAG HSV-2 challenge. Measurement of virus in protected CpG-immunized mice revealed a log lower level of replication within the first few days after infection. In conclusion, these results indicate that intranasal immunization with CpG ODN plus protein mediates immunity in the female genital tract capable of protecting against a sexually transmitted pathogen.

RANTES is produced by lymphoid and epithelial cells of the mucosa in response to various external stimuli and is chemotactic for lymphocytes. The role of RANTES in adaptive mucosal immunity has not been studied. To better elucidate the role of this chemokine, we have characterized the effects of RANTES on mucosal and systemic immune responses to nasally coadministered OVA. RANTES enhanced Ag-specific serum Ab responses, inducing predominately anti-OVA IgG2a and IgG3 followed by IgG1 and IgG2b subclass Ab responses. RANTES also increased Ag-specific Ab titers in mucosal secretions and these Ab responses were associated with increased numbers of Ab-forming cells, derived from mucosal and systemic compartments. Splenic and mucosally derived CD4+ T cells of RANTES-treated mice displayed higher Ag-specific proliferative responses and IFN-γ, IL-2, IL-5, and IL-6 production than control groups receiving OVA alone. In vitro, RANTES up-regulated the expression of CD28, CD40 ligand, and IL-12R by Ag-activated primary T cells from DO11.10 (OVA-specific TCR-transgenic) mice and by resting T cells in a dose-dependent fashion. These studies suggest that RANTES can enhance mucosal and systemic humoral Ab responses through help provided by Th1- and select Th2-type cytokines as well as through the induction of costimulatory molecule and cytokine receptor expression on T lymphocytes. These effects could serve as a link between the initial innate signals of the host and the adaptive immune system.

Deficiency in CD21/CD35 by disruption of the Cr2 loci leads to impaired humoral immune responses. In this study, we detail the role of CD21/CD35 on Ab responses to the hapten (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken gamma-globulin. Surprisingly, Cr2−/− mice generate significant Ab responses and germinal center (GC) reactions to low doses of this Ag in alum, although the magnitude of their responses is much reduced in comparison with those of Cr2+/− and C57BL/6 controls. Increasing Ag dose partially corrected this deficit. In situ study of the somatic genetics of GC B cells demonstrated that VDJ hypermutation does not require CD21/CD35, and Cr2−/− mice exhibited enhanced affinity maturation of serum Ab in the post-GC phase of the primary response. On the other hand, Cr2−/− mice displayed accelerated loss of serum Ab and long-lived Ab-forming cells. These observations suggest that B cell activation/survival signals mediated by CD21 and/or the retention of Ag by CD21/CD35 play important roles in the generation, quality, and maintenance of serum Ab.

Signaling through CD40 in B cells leads to B cell proliferation, Ig and IL-6 secretion, isotype switching, and up-regulation of surface molecules. TNF receptor-associated factor (TRAF) proteins associate with the cytoplasmic tail of CD40 and act as adapter molecules. Of the six TRAFs identified to date, TRAFs 2, 3, 5, and 6 are reported to associate directly with the cytoplasmic tail of CD40, but previous studies have principally examined transient overexpression of TRAF6 in cells that do not normally express CD40. Thus, we examined the role of TRAF6 in CD40-mediated B lymphocyte effector functions using two approaches. We produced and stably expressed in mouse B cell lines a human CD40 molecule with two cytoplasmic domain point mutations (hCD40EEAA); this mutant fails to bind TRAF6, while showing normal association with TRAFs 2 and 3. We also inducibly expressed in B cells a transfected “dominant-negative” TRAF6 molecule which contains only the C-terminal TRAF-binding domain of TRAF6. Using both molecules, we found that TRAF6 association with CD40 is important for CD40-induced IL-6 and Ig secretion, and that TRAF6 mediates its effects on CD40-stimulated Ig secretion principally through its effects on IL-6 production by the B cell. TRAF6 association with CD40 was also found to be important for B7-1 up-regulation, but not for up-regulation of other surface molecules. Interestingly, however, although we could show TRAF6-dependent CD40-mediated activation of NF-κB in 293 kidney epithelial cells, no such effect was seen in B cells, suggesting that TRAF6 has cell-type-specific functions.

Whether memory B cells possess altered differentiative potentials and respond in a qualitatively distinct fashion to extrinsic signals as compared with their naive precursors is a current subject of debate. We have investigated this issue by examining the participation of a predominant anti-arsonate clonotype in the primary and secondary responses in the spleens of A/J mice. While this clonotype gives rise to few Ab-forming cells (AFC) in the primary response, shortly after secondary immunization its memory cell progeny produce a massive splenic IgG AFC response, largely in the red pulp. Extensive clonal expansion and migration take place during the secondary AFC response but Ab V region somatic hypermutation is not reinduced. The primary and secondary germinal center (GC) responses of this clonotype are both characterized by ongoing V gene hypermutation and phenotypic selection, little or no inter-GC migration, and derivation of multiple, spatially distinct GCs from a single progenitor. However, the kinetics of these responses differ, with V genes containing a high frequency of total as well as affinity-enhancing mutations appearing rapidly in secondary GCs, suggesting either recruitment of memory cells into this response, or accelerated rates of hypermutation and selection. In contrast, the frequency of mutation observed per V gene does not increase monotonically during the primary GC response of this clonotype, suggesting ongoing emigration of B cells that have sustained affinity- and specificity-enhancing mutations.

Though it has been shown that TGF-beta 1 directs B cells to switch to IgA in vitro, no studies have assessed TGF-beta 1 effects on mucosal vs systemic immunity in vivo. When the B cell functions of TGF-beta 1 gene-disrupted (TGF-beta 1-/-) mice were analyzed, significantly decreased IgA levels and increased IgG and IgM levels in serum and external secretions were observed. Further, analysis of Ab forming cells (AFC) isolated from both mucosal and systemic lymphoid tissue showed elevated IgM, IgG, and IgE, with decreased IgA AFC. A lack of IgA-committed B cells was seen in TGF-beta 1-/- mice, especially in the gastrointestinal (GI) tract. Splenic T cells triggered via the TCR expressed elevated Th2-type cytokines and, consistent with this observation, a 31-fold increase in serum IgE was seen in TGF-beta 1-/- mice. Thus, uncontrolled B cell responses, which include elevated IgE levels, a lack of antiinflammatory IgA, and an excess of complement-binding IgG and IgM Abs, will promote inflammation at mucosal surfaces in TGF-beta 1-/- mice and likely contribute to pulmonary and GI tract lesions, ultimately leading to the early death of these mice.

Along humoral immune responses, different stimuli drive the differentiation of B lymphocytes to Ig-secreting plasma cells in discrete microenvironments. The Blimp-1 transcription factor is up-regulated early during the transition of mature B cells to IgM-secreting plasma cells. In the present study, we have examined the requirement of Blimp-1 in plasma cell formation after both T cell-independent (LPS) and -dependent (CD40 + IL-4, Th cell lines) stimulation of spleen B cells. B lymphocyte-induced maturation protein (Blimp-1) was expressed early after in vitro LPS stimulation, mainly in a population of IgM+Syndecan+CD43+ preplasma cells. In contrast, the BSAP transcription factor expressed in mature B cells was down-regulated during the differentiation to plasma cells. Treatment of these cultures with Blimp-1-specific antisense phosphorothioate oligonucleotides suppressed both Blimp-1 protein levels and the emergence of IgM+Syndecan+ cells and plasma cells. However, T-B cell cocultures of spleen B cells from C3H/HeJ (H-2k) mice and syngeneic autoreactive SR.10 Th2 cells submitted to the anti-Blimp-1 therapy did not show any significant reduction in IgM- and IgG1-secreting plasma cell formation. Spleen B cells treated with anti-CD40 mAb + IL-4 differentiated to IgG1-secreting cells without significant transcription of the Blimp-1 gene; anti-Blimp-1 treatment subsequently did not have any effect in the later cultures. Altogether, these results suggest that Blimp-1 transcription factor specifically promotes T cell-independent B cell differentiation to plasma cells, probably at preplasma cell stages. In contrast, T cell-dependent plasma cell formation likely evolves through Blimp-1-independent pathways.

The inactivated poliovirus vaccine (IPV) is used for protection against poliomyelitis in The Netherlands. It is not clear, however, whether IPV vaccination can lead to priming of the mucosal immune system and the induction of IgA. It has been demonstrated that IPV vaccination is able to induce strong memory IgA responses in the serum of persons who have been naturally exposed to wild-type poliovirus. This has led to the hypothesis that IPV vaccination is able to induce poliovirus-specific IgA at mucosal sites in persons who have been previously primed with live poliovirus at mucosal sites. To test this hypothesis, the kinetics of the IgA response in serum and saliva after IPV vaccination were examined in persons previously vaccinated with oral poliovirus vaccine (OPV) or IPV. ELISA and enzyme-linked immunospot assays were used for the detection of poliovirus-specific IgA responses. In addition, B cell populations were separated on the basis of the expression of mucosal (alpha4beta7 integrin) and peripheral homing receptors (L-selectin). Parenteral IPV vaccination was able to boost systemic and mucosal IgA responses in previously OPV-vaccinated persons only. None of the previously vaccinated IPV recipients responded with the production of IgA in saliva. In agreement with this finding, a large percentage of the poliovirus-specific IgA-producing lymphocytes detected in previous OPV recipients expressed the alpha4beta7 integrin. It is concluded that IPV vaccination alone is insufficient to induce a mucosal IgA response against poliovirus. In mucosally (OPV-) primed individuals, however, booster vaccination with IPV leads to a strong mucosal IgA response.

Female rhesus macaques were nasally immunized with p55gag (p55) of SIV and cholera toxin as a mucosal adjuvant. Nasal immunization induced Ag-specific IgA and IgG Abs in mucosal secretions (e.g., cervicovaginal secretions, rectal washes, and saliva) and serum. Furthermore, high numbers of p55-specific IgA and IgG Ab-forming cells were induced in mucosal effector sites, i.e., uterine cervix, intestinal lamina propria, and nasal passage. p55-specific CD4+ T cells in both systemic and mucosal compartments expressed IFN-gamma and IL-2 (Th1-type)- as well as IL-5, IL-6, and IL-10 (Th2-type)-specific mRNA. Moreover, p55-specific CTL activity was demonstrated in lymphocytes from blood, tonsils, and other lymphoid tissues. These results show that nasal immunization with SIV p55 with cholera toxin elicits both Th1- and selective Th2-type cytokine responses associated with the induction of SIV-specific mucosal and serum Abs, and CTL activity. These results offer a promise for the development of protective mucosal immunity to SIV.

In the first week of the primary immune response to the (4-hydroxy-3-nitrophenyl)acetyl (NP) hapten, plasmacytic foci and germinal centers (GCs) in C57BL/6 mice are comprised of polyclonal populations of B lymphocytes bearing the lambda1 L-chain (lambda1+). The Ig H-chains of these early populations of B cells are encoded by a variety of VH and D exons undiversified by hypermutation while later, oligoclonal populations are dominated by mutated rearrangements of the VH186.2 and DFL16.1 gene segments. To assess directly Ab affinities within these defined splenic microenvironments, representative VDJ rearrangements were recovered from B cells participating in the early immune response to NP, inserted into Ig H-chain expression cassettes, and transfected into J558L (H-; lambda1+) myeloma cells. These transfectoma Abs expressed a remarkably wide range of measured affinities (Ka = 5 x 10(4)-1.3 x 10(6) M(-1)) for NP. VDJs recovered from both foci and early GCs generated comparable affinities, suggesting that initial differentiation into these compartments occurs stochastically. We conclude that Ag normally activates B cells bearing an unexpectedly wide spectrum of Ab affinities and that this initial, promiscuous clonal activation is followed by affinity-driven competition to determine survival and clonal expansion within GCs and entry into the memory and bone marrow plasmacyte compartments.

Staphylococcal superantigens, including staphylococcal enterotoxin B (SEB), promote vigorous T cell-dependent Ig responses at low dose (0.01 ng/ml). In contrast, more mitogenic high dose SEB (100 ng/ml) profoundly inhibits the Ig responses. To assess the contribution of CD8+ T cells to this inhibition, high dose SEB-dependent killing of activated B cells and down-regulation of Ig responses were determined. Rapid killing (4 h) of activated B cells was effected by high dose SEB-activated CD8+ T cells (CD8*), but not by high-dose SEB-activated CD4+ T cells (CD4*), and required the presence of high dose SEB during the cytotoxicity assay. This killing was abrogated by chelation of extracellular calcium or by treatment with concanamycin A but was only modestly affected by treatment with brefeldin A, suggesting a perforin-based pathway of killing. Despite their widely disparate abilities to rapidly kill activated B cells, CD8* and CD4* demonstrated similar quantitative abilities to effect high dose SEB-dependent down-regulation of Ig responses. Antagonist anti-CD95 mAb substantially reversed high dose SEB-dependent downregulation effected by CD8* but had no appreciable effects on high dose SEB-dependent killing of activated B cells. These observations strongly suggest that the small fraction of activated B cells that secrete Ig are selectively sensitive to CD95-based killing but resistant to CD95-independent killing. This finding may help explain why clinical autoimmunity associated with increased titers of autoantibodies is a predominant feature of defects in CD95 or CD95 ligand.

We describe a unique, stable pre-pro-B cell line (YS-PPB) derived from AA4.1+ yolk sac cells from day 10 mouse embryos. This cell line, discovered fortuitously during the course of studies of in vitro B cell differentiation, is independent of IL-7 supplementation for long term expansion in vitro. YS-PPB cells as well as clonal sublines expressed AA4.1, CD43, B220, Sca-1, CD19, heat stable antigen, MHC class I, IL-7R, and FcyR, but did not express cytoplasmic mu-chain, surface IgM (sIgM), or MHC class II molecules. PCR analysis showed that the cells expressed TdT, lambda5, and RAG-1 genes, but that their Ig genes were still in germline configuration. The cell line was dependent on direct contact with S17 stromal cells for growth, but, in contrast to bone marrow stem cells, required no additional growth factors for maintenance and expansion. When stimulated with IL-7 and LPS, YS-PPB cells and cells from all tested clonal sublines differentiated into sIgM+ B cells in vitro. Irradiated mice reconstituted with YS-PPB cells yielded spleens containing 38% sIgM+ donor-derived B cells, demonstrating that YS-PPB cells, although stably arrested in development at the boundary between pre-pro-B and pro-B stages of B cell differentiation, still retain their competence to differentiate into mature, Ig-producing B cells when transferred to a normal host environment. Thus, this new cell line can provide a reproducible source of B cell precursors arrested at that critical time in B cell differentiation when the machinery for Ig gene rearrangement is in place but rearrangement has not yet occurred.

The present study compares the location and phenotype of B lineage lymphocytes in tissues from SCID mice engrafted with PBMC of human, chimpanzee, and pig-tailed macaque origin. In mice repopulated with both human and nonhuman primate lymphocytes, plasma cells were found in the peritoneal cavity in vascularized structures located in the mesentery near the pancreas, intestines, and spleen. The predominant isotype of the plasma cells was IgG; IgM and IgA cells were also present. Kappa and lambda light chains were expressed by 62% and 38% of the Ig-containing cells, respectively. J chain expression occurred in most cells irrespective of the Ig isotype. In the SCID mice engrafted with human lymphocytes, a few IgM-containing cells were found in the spleen; plasma cells were not found in other tissues, including the intestine. The aggregation of plasma cells did not appear to be a result of infection with EBV. T cells were rarely found in the lymphoid aggregates but were recovered from the spleen and peritoneal lavage. Human Ig levels in the serum of engrafted mice reflected the isotype distribution of the cells with IgGIgMor = IgA.

We have previously shown that in vivo nasal challenge with diesel exhaust particles (DEP) induces both quantitative and qualitative changes in local IgE production and stimulates generalized local cytokine production. We have now investigated the combined effects of intranasal challenge with DEP plus ragweed allergen on local humoral immune responses. We collected nasal lavages from ragweed sensitized subjects at different times after nasal challenge. As compared with challenge with ragweed alone, challenge with both DEP and ragweed induced markedly higher ragweed-specific IgE but not total IgE levels or IgE-secreting cell numbers. Total and specific IgG4 levels also were enhanced, while total IgG levels were not. Synergy was also observed between the DEP and ragweed in altering the profile of epsilon mRNAs generated by alternative splicing, mRNAs that code for different expressed IgE proteins. Intranasal challenge with ragweed alone induced inconsistent and low levels of mucosal cytokine mRNAs. In contrast, challenge with both ragweed plus DEP resulted in decreased expression for Th1-type cytokines (IFN-gamma and IL-2) but elevated expression of mRNA for other cytokines (IL-4, -5, IL-6, IL-10, IL-13). This synergy between DEP and natural allergen exposure is suggested as a key feature in increasing allergen-induced respiratory allergic disease.

Specific Ab-secreting cells (ASC) appear in the human blood as a response to oral and parenteral vaccination. The actual contribution of these cells to the defense of the body depends on their final effector site. The homing potentials of mucosally and parenterally induced ASC were compared by examining the homing receptor (HR) expression of circulating specific ASC in the blood of volunteers vaccinated orally or parenterally with the same Ag, Salmonella typhi Ty21a. Circulating lymphocytes were separated into receptor-positive and -negative populations, and the numbers of specific ASC were assayed. The alpha4 beta7 integrin, which acts as a gut HR, was expressed on all (99%) of the mucosally activated ASC, but on only 58% of the parenterally induced ASC or 58% of all Ig-secreting cells of the unvaccinated controls. L-selectin, the peripheral lymph node HR, showed an inverse distribution; it was found on 42% of mucosally activated ASC and on 86% of parenterally induced ASC. These results reveal that all of the circulating ASC after oral vaccination are committed to migrate to the mucosal compartment of the immune system, strongly arguing for a recirculation of activated mucosal cells in humans. By contrast, ASC induced by parenteral vaccination with the same Ag are mostly directed to the systemic compartment, yet a part of them has mucosal homing attitudes as well. These differences indicate that the site of Ag encounter determines the homing potential of the cell.

In this study, we examine the relationship between primary and secondary T cell-dependent immune responses using the response of xid mice to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) as an experimental model. The reduced serologic primary immune response of xid mice was demonstrated to be caused by a substantially decreased Ab-forming cell (AFC) generation. Furthermore, the germinal center reaction in the primary xid immune response was diminished and the frequency of NP-specific memory B cells prior to secondary immunization was reduced 10-fold. Despite the poor primary response of xid mice, secondary exposure to Ag generated a response that was qualitatively and quantitatively equal to that of wt mice. The number of IgG1 AFCs in spleen and bone marrow increased equally in both groups, as did the proportion of AFCs secreting high affinity Ab in both locations. The extent and distribution of somatic mutations in the V(H) genes of xid secondary response B cells was also found to be normal, indicating that the xid gene product is not critical for the processes that result in affinity maturation. Thus, although xid mice generate memory B cells of normal phenotype but at a substantially lower frequency, this does not limit the magnitude of the secondary response. Therefore, our results imply that the reduced memory B cell frequency in xid mice is still above some threshold value necessary for a normal secondary immune response.

A single 250-micrograms dose of the Mel-14 mAb to L-selectin greatly diminished the extent of L-selectin expression on lymphocytes and decreased (60 to 90%) the massive cellular recruitment to the cervical and mediastinal lymph nodes that follows intranasal infection of naive C57BL/6 mice with Sendai virus. The numbers of CD8+ CTL precursors in the mediastinal lymph nodes were considerably reduced on day 7, when compared with virus-infected mice given a control rat IgG2a, but potent CTL effectors were present in the lungs of both groups by day 10 after infection, and the overall magnitude of CTL precursor generation was not obviously compromised. The early dominance of Sendai virus-specific IgM Ab-forming cells was prolonged in the Mel-14-treated mice, whereas plasma cells producing virus-specific IgA were abnormally prominent in the lymph nodes but not in the spleen. The kinetics of virus-specific Ab-forming cells generation and the serum Ab response for the various IgG isotypes were also delayed. Thus, though L-selectin is clearly important for the localization of naive lymphocytes to regional lymph nodes, the Mel-14-treated mouse can still deal effectively with a virus that causes productive infection only in the respiratory tract. The spleen, where L-selectin does not determine lymphocyte trafficking, is a major site for the compensatory T cell and B cell responses.

We have examined the role of passively transferred maternal Abs in the ontogeny of "natural" mucosal IgA responses before weaning of suckling mice by comparing the immune status of gut-associated lymphoid tissue (GALT) (Peyer's patches, mesenteric lymph nodes, and lamina propria) in 7- to 25-day-old F1 severe combined immunodeficient (scid)/+ mice generated through reciprocal crosses of C.B17 scid/scid and normal congenic (+/+) adult mice. We have also examined the ability of prenatal vs postnatal transfer of maternal immunity to forestall the development of natural neonatal mucosal IgA responses by swapping litters of F1 scid/+ pups at birth between +/+ and scid/scid mothers. Our results demonstrate that F1 scid/+ pups born to or nursed by scid/scid mothers undergo an accelerated development of natural IgA responses that include germinal center reactions in both Peyer's patches and mesenteric lymph nodes. These early IgA responses are evident as: 1) increased frequencies of IgA-producing GALT organ cultures; 2) increased mean IgA output by GALT organ cultures; 3) increased frequencies (1 log) of IgA-secreting cells from GALT detected by ELISPOT at 16 days of age; and 4) germinal center development by 17 days of age detected by in vivo bromodeoxyuridine incorporation. Finally, FACS analyses of enteric bacteria isolated from F1 scid/+ pups and stained for the presence of surface-bound mouse IgA demonstrate that the bacterial flora is a major target of both maternal secretory IgA and of the earliest IgA Abs produced in the neonatal GALT of pups deprived of maternal immunity.

This study investigated whether or not the localization of persisting Ag influenced the localization of memory B and/or of Ab-forming cells (AFC). As a model Ag, we used vesicular stomatitis virus, which does not measurably replicate extraneuronally in adult mice after peripheral infection. Our results show that memory B cells and AFC are induced at the site where Ag is present; induced memory B cells then recirculate throughout the lymphoid system independently of Ag localization. In contrast, AFC are induced only at the sites of Ag persistence. These triggered Ab producing cells do not recirculate through the lymphoid tissue but migrate to the bone marrow.

The size of the Ab repertoire has been estimated to comprise theoretically somewhere between10(10) and approximately 10(4) specificities, dependent on the criteria used. In an attempt to estimate the anti-viral protective Ab repertoire of the mouse the B cell and Ab-forming cell (AFC) frequencies and protective neutralizing Ab levels during the course of an infection with vesicular stomatitis virus (VSV) were analyzed. Determination of AFC frequencies, limiting dilution assays, and adoptive transfer experiments to SCID mice revealed that during the acute phase (day 8) of the immune response, more than 50% of all IgG2a-producing AFCs were specific for VSV, most of them recognizing the neutralizing determinant. In a later phase (days 21 or 50), 10 to 20 times fewer VSV-specific AFCs were present, corresponding to a frequency of approximately 1:10(4) spleen cells. Finally, in a protection assay in SCID mice, adoptively transferred protective Ab concentrations were found to be approximately 1 to 10 micrograms Ab/ml mouse serum. Because during the memory phase of the anti-VSV response usually 10(4) AFC/mouse are engaged to maintain a high level of memory IgG against the neutralizing determinant on VSV and if one assumes a total number of about 10(6) AFCs/mouse, these data suggest a rather limited neutralizing anti-viral protective memory-AFC repertoire of 10(2) to 10(4) different specificities.

Two antigenically related porcine coronaviruses, transmissible gastroenteritis virus (TGEV) which infects primarily the intestinal tract and causes severe diarrhea, and porcine respiratory coronavirus (PRCV) which infects the respiratory tract and causes subclinical or mild respiratory infections, presented a unique opportunity to study the interrelationship of gut-(GALT) and bronchus-associated lymphoid tissues (BALT) and their contribution to protective immunity against TGEV infection. Pigs were inoculated oral-nasally with TGEV or with PRCV at eleven days of age and challenged 24 days later with TGEV. All pigs initially given TGEV developed diarrhea and were completely protected against disease upon challenge. In contrast, pigs given PRCV had no clinical disease and shed virus in nasal secretions only; after challenge, 5 of 12 pigs developed diarrhea. Virus-specific IgG and IgA Ab-secreting cells (ASC) were enumerated by ELISPOT in the mesenteric and bronchial lymph nodes, spleens, and gut lamina propria at challenge and various post challenge days. Before challenge, in pigs exposed to TGEV, IgA-ASC in the duodenum and jejunum constituted the major ASC response. Conversely, PRCV-exposed pigs had mainly IgG-ASC in bronchial lymph nodes, with low ASC responses in the gut. After challenge, numbers of IgG-ASC increased rapidly in the gut lamina propria and mesenteric lymph nodes of only PRCV-primed pigs. Our results suggest that virus-specific IgG-ASC precursors derived in BALT of PRCV-primed pigs may migrate to the gut in response to TGEV challenge and contribute to the partial protection observed. The presence of IgA-ASC in the gut lamina propria of TGEV-primed pigs at the time of challenge correlated with complete protection against TGEV challenge. Thus a dichotomy exists in the BALT and GALT ASC responses; immunization via BALT induced a systemic type of response (IgG-ASC) and provided imperfect protection against an enteric pathogen, whereas immunization via GALT induced IgA-ASC and provided complete protection.

Highly conserved enkephalin containing peptides (ECPs) are selectively processed from proenkephalin, which is synthesized in both the neuroendocrine and immune systems. The reported regulatory effects within the central nervous system and the biologic release patterns from both activated lymphocytes and stimulated adrenal chromaffin cells suggest the ECPs may act as regulatory factors of the immune system. We tested the effects of three of the ECPs, Peptides F, E, and B, on the in vitro Ab-forming cell (AFC) response murine splenocytes to antigenic challenge. In contrast to the immunosuppressive effects of the pentapeptide enkephalins, physiologic concentrations of the ECPs significantly enhanced the AFC response to both T cell-dependent and T cell-independent Ags. The effects are not sensitive to competition by the opiate receptor antagonist, naloxone, suggesting cell surface interactions that do not involve classical opiate receptors. These studies provide evidence that the effects are mediated through T cells rather than B cells. Peptide F-treated splenocytes also showed a significant enhancement of the AFC response to suboptimal Ag concentrations, suggesting a mechanism of action in which the ECPs may act to lower the threshold of activation of the effector cell. These results suggest that the ECPs are physiologically important modifiers of the humoral immune response. Given their release patterns and demonstrated action on the in vitro immune response, the proenkephalin-derived ECPs have the potential to be involved in both paracrine and autocrine regulatory networks within the immune system and as a positive immunoregulatory effect from the neuroendocrine system.

Mouse Abs against a bacterial epitope, the phosphorylcholine (PC) hapten are encoded by the T15 genes VH1(S107) and V kappa 22. It has been shown that PC-specific hybridomas from aged animals often express IgV gene families other than T15. To determine the extent of this age-dependent molecular shift in the anti-PC response, we examined antibody-forming cells (AFC) in individual young (2 to 4 month) and aged (20 to 24 month) mice by an in situ RNA hybridization. Mice were immunized either with PC coupled to keyhole limpet hemocyanin or with a Streptococcus pneumoniae strain R36a vaccine. Frozen splenic sections were prepared, and the clusters of PC-specific AFC (i.e., antibody foci) were identified by immunocytochemical staining. The adjacent splenic sections were hybridized with digoxigenin-labeled VH1(S107) and V kappa 22 DNA probes and with a C mu DNA probe as a control. The splenic sections were examined for 1) the number of Ab foci hybridized with the T15 probes, and 2) the estimated proportion of VH1+ and V kappa 22+ AFC within each focus. The results were comparable regardless of the form of PC Ag administered. Virtually all Ab foci (85%) in young mice hybridized with the T15 probes and were occupied by the VH1+/V kappa 22+ AFC. In aged mice, the fraction of PC-binding Ab foci that hybridized with a given T15 probe varied from 35% to85%; T15+ AFC always represented a minor population of the focus (50%), the remaining PC-specific AFC being C mu + but T15-. Also, there appeared to be a greater loss of the V kappa 22 expression relative to the VH1(S107). Thus it appears that the T15+, PC-reactive B cells in aged mice responded to the Ag but that they could not dominate the response. The possibility of an intrinsic molecular change in the aging B cells in discussed.

A cDNA library was prepared from the murine Th cell line Th2 D.10 and used to clone the murine homologue of Ox40 by polymerase chain reaction. Comparison of the mouse sequence with the rat revealed greater than 90% homology between the two sequences at both the DNA and protein level. Northern blot analysis found that, as in the rat, Ox40 expression appears to be restricted to activated T cells. A chimeric receptor globulin was prepared to include the mouse Ox40 extracellular domain coupled to the hinge-CH2-CH3 domains of human IgG1 (Ox40-Ig). This soluble form of the molecular was then used to identify cells bearing a ligand for Ox40. FACS analysis revealed that Ox40-Ig bound to a subset of peritoneal B cells as well as to a fraction of LPS-activated splenic B cells. Immunostaining of spleen sections using an Ag-specific conjugate and Ox40-Ig found a significant proportion of antibody-forming cells co-stained with Ox40-Ig. Immunoprecipitation of cell-surface radiolabeled peritoneal B cells suggests a specific interaction with a protein of 70 kDa.

Antibody-secreting cells (ASC) were enumerated in gut- and bronchus-associated lymphoid tissues of pigs exposed to three antigenically related coronaviruses: virulent transmissible gastroenteritis virus (TGEV), attenuated TGEV, and porcine respiratory coronavirus (PRCV). Exposure of 11-day-old pigs to virulent TGEV resulted in severe gastroenteritis and virus shedding mainly in feces but also to a limited extent in nasal secretions. PRCV and attenuated TGEV exposure produced no clinical signs and only one pig given a high dose of attenuated TGEV shed virus in feces, but virus was shed from the nasal passages. Nasal virus titers were highest after PRCV inoculation of pigs. Mononuclear cells were isolated from spleens, mesenteric, and bronchial lymph nodes of pigs and assayed for virus-specific IgG and IgA antibody secretion by an enzyme-linked immunospot assay. Virus-specific ASC peaked at postinoculation days 12 to 24 and IgG-ASC outnumbered IgA-ASC in all tissues tested. The greatest numbers of ASC were in mesenteric lymph nodes of virulent TGEV-exposed pigs and in BLN of PRCV-exposed pigs. Attenuated TGEV induced intermediate ASC responses in the gut and respiratory tract. Secondary in vitro ASC responses to inactivated TGEV or PRCV paralleled the primary responses except in BLN where the numbers of memory ASC were high for both TGEV- and PRCV-exposed pigs. We conclude that: 1) a single exposure of pigs to PRCV either oral-nasally or by aerosol leads to potent systemic and bronchus-associated, but not gut-associated, ASC responses; 2) a high dose of attenuated TGEV (4 x 10(8) plaque-forming units) is more effective than PRCV (6 x 10(5) or 2 x 10(8) plaque-forming units) or a lower dose of attenuated TGEV (7 x 10(6) plaque-forming units) in eliciting gut-associated ASC; 3) although virulent and a high dose of attenuated TGEV induce high numbers of ASC in the tissues tested, virulent TGEV induces the most ASC in the gut and IgA-ASC in all lymphoid tissues; and 4) virus replication in the gut or respiratory tract is a major factor affecting the magnitude of an ASC response at that site and may be necessary for the recruitment of IgG- and IgA-ASC and memory cells in large numbers from other mucosal inductive sites. This unique model of mucosal immunity using antigenically related viruses with distinct tissue tropisms may help to clarify interactions of the various components of the common mucosal immune system.

Oligoclonal bands in the cerebrospinal fluid indicate intrathecal synthesis of Ig of restricted heterogeneity and are associated with a number of central nervous system inflammatory diseases. To gain better insight into the persistence of oligoclonal bands found in the central nervous system we studied mice infected with Sindbis virus (SV), a RNA virus that causes an acute, nonfatal encephalitis in mice. SV was inoculated intracerebrally into weanling mice and brains and spleens were harvested at various time points long after the acute encephalitis had resolved. A modified enzyme-linked immunoassay was used to study cultured B cells separated from the brain and spleen for their Ig isotype expression and specificity for SV. We used the polymerase chain reaction technique to detect SV RNA in brain. Three mo after inoculation 47% of the B cells found in brain are secreting antibody specific for SV structural proteins. By 1 yr 62% are SV specific. B cells secreting IgG2a predominate. Polymerase chain reaction data indicate that despite complete clearance of infectious virus by 7 days SV RNA is still present in brain at least 6 mo after infection. The data indicate that B cells in brain secrete antibody to SV long after the acute encephalitis has resolved. The persistence of SV RNA suggests that viral protein may continue to be made, providing the impetus for the continued presence of SV-specific B cells in the brain.

The organs in which B cells bearing membrane-bound IgE (sIgE+) and benzylpenicilloyl (BPO)-specific IgE antibody-forming cells (AFC) first appeared were determined in BALB/c mice given BPO-keyhole limpet hemocyanin (10 micrograms) in aluminum hydroxide by various routes (i.p, gavage, s.c., i.v., or i.m.). In mice immunized by the i.p. route, the numbers and location of sIgE+ B cells and asialo GM1 ganglioside (AsGm1+) cells, the location of IgE/CD23 immune complexes, and the numbers of BPO-specific IgE AFC in lymphoid organs were determined. With all routes of immunization, no sIgE+ B cells or BPO-specific IgE AFC were ever detected in any organ before day 8. On day 8, with the s.c., i.v., or i.m. routes, sIgE+ B cells and IgE AFC appeared exclusively in Peyer's patches (PP); with the i.p. or gavage routes, sIgE+ B cells simultaneously appeared in both PP and mesenteric lymph nodes, whereas IgE AFC appeared only in PP. In mice immunized by the i.p. route, IgE/CD23 immune complexes and strikingly increased numbers of AsGm1+ cells transiently appeared only in PP after the appearance and preceding the "disappearance" of the sIgE+ B cells and IgE AFC. The data suggest that specific IgE responses originate in gut-associated lymphoid tissue and appear later in spleen. The data also associate the appearance of IgE/CD23 immune complexes and AsGm1+ cells with the "disappearance" of sIgE+ B cells and IgE AFC from PP.

The development of T cells and the selection of the TCR repertoire in the absence of exogenous antigenic stimulation were investigated. For this purpose germfree BALB/c mice fed an ultrafiltered solution of chemically defined low m.w. nutrients (GF-CD) were used. Previous studies on B cell development and differentiation in GF-CD mice have demonstrated a high reduction in the number of cells secreting Ig of the non-IgM isotypes but an Ig-VH gene usage and a B cell specificity repertoire that is substantially different from that observed in conventional adult mice and more closely resembles that of neonatal conventional mice. In contrast, the present comparison of the various lymphocyte populations in the thymus, lymph nodes, and spleen from GF-CD and conventional mice using flow cytometry analysis revealed no significant differences. Analysis of the TCR-V beta expression on both mature thymocytes and lymph node T cells showed a high degree of similarity between GF-CD and conventional mice. These findings indicate a marked difference in the influence of exogenous antigenic stimulation on the development of B and T cells. Additionally, development in an environment free of exogenous antigenic stimulation allows for full functional maturation of T cells to occur, because MLC showed that GF-CD splenic T cells could mount allogeneic responses in a way similar to T cells generated in a conventional environment. Most importantly, full Th cell function is generated, because activation of GF-CD spleen cells by cross-linking with mAb against CD3 resulted in the induction of cells secreting IFN-gamma and Ig of the non-IgM isotypes, which cannot be detected in GF-CD sera. These findings demonstrate that functional T and B cells develop in mice that have not been exposed to exogenous Ag, and that the TCR repertoire, in contrast to the B cell compartment, is predominantly shaped by endogenously expressed Ag.