Antigen-specific Antibody-secreting Cells Research Articles

Delayed and deficient establishment of the long-term bone marrow plasma cell pool during early life.

Early life antibody responses are characterized by a rapid decline, such that antigen-specific IgG antibodies decline to baseline levels within months following infant immunization. This generic observation remains unexplained. Here, we have analyzed the induction and organ-localization of antigen-specific IgG antibody-secreting cells (ASC) following immunization of 1-week-old or adult BALB/c mice with tetanus toxoid (TT), a T-dependent antigen. Early life priming induced only slightly lower numbers of TT-specific IgG ASC in the spleen, and these reached adult levels following repeat immunization. In contrast, early life immunization generated much fewer bone marrow plasma cells than in adults, even after boosting. A similar limitation of the natural development of the bone marrow pool of ASC was observed. Transfer experiments with adult or early life spleen ASC indicated limited homing of TT-specific adult ASC to the bone marrow of 4-week-old mice as compared to adult recipients, whereas homing patterns were similar when early life or adult ASC were transferred into adult recipients. These observations suggest that a limited bone marrow B cell homing capacity and, as a result, relatively deficient bone marrow ASC responses, are critical factors which may explain the limited persistence of IgG antibodies to T-dependent antigens in early life.

Enhanced induction of the IgA response in pigs by calcitriol after intramuscular immunization

In this study, the immunomodulating effect of two steroid hormones namely 1,25-dihydroxyvitamin D 3 [1,25(OH) 2D 3] and dehydroepiandrosterone (DHEA) was examined on the antigen-specific antibody responses by intramuscular immunization of pigs with human serum albumin alone (HSA) or supplemented with 2 μg of 1,25(OH) 2D 3, 40 μg of DHEA or the combination of both steroids. 1,25(OH) 2D 3 significantly enhanced the antigen-specific IgA and IgM serum response. Higher HSA-specific IgA titers were also found in the mucosal secretions (saliva, feces and nasal) of the steroid treated animals, especially in the 1,25(OH) 2D 3 group. Furthermore, 1,25(OH) 2D 3 and DHEA increased the number of antigen-specific IgA and IgG antibody-secreting cells in the local draining lymph nodes, but only low numbers were detected in lymph nodes draining the mucosa. DHEA decreased the IgM serum response and had a tendency to enhance the IgG2 and IgG serum responses. Strong and comparable IgG, IgG1 and IgG2 serum responses were seen in all groups. Combining both steroids did not result in a higher IgA serum response. On the contrary DHEA seems to neutralize the effect of 1,25(OH) 2D 3 on the IgA response. In conclusion, 1,25(OH) 2D 3 significantly enhanced the antigen-specific IgA and IgM response in serum and the number of antigen-specific IgA and IgG ASC in the local draining lymph nodes following intramuscular immunization.

Persistence of plasma cells in the kidneys of autoimmune NZB/W mice

NZB/W mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hyper-gammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice but the role of this organ for the production of antibodies is not clear. Here, we compare the role of bone marrow, spleen and inflamed kidneys of NZB/W mice for the activation of B cells and for the persistence of antibody secreting cells (ASC). ASC are present in the kidneys of mice with full blown disease, as many as in the spleen and bone marrow, and 50 times more than in the kidneys of normal mice. In the kidneys, ASC are located mainly in the outer medulla, close to B- and T cell infiltrates. The specificity of the ASC in the inflamed kidneys is not restricted to self-antigens. After immunization of NZB/W mice with Ovalbumin (OVA), the antigen-specific ASC are found initially exclusively in the spleen. Weeks later, during a period of at least 3 months, OVA-specific ASC are found in stable and high numbers within the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220+/PNA+) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB/W mice. By histology, germinal centers could not be detected in the kidneys, but in the spleen. The lack of B cell activation and the kinetics of the appearance of OVA-specific ASC suggest that in autoimmune NZB/W mice kidneys, plasma cells generated during an immune reaction in secondary lymphoid organs, later accumulate and persist, like in bone marrow. These experiments identify the inflamed kidneys of NZB/W mice as site of prime relevance for the homeostasis of plasma cells, irrespective of their specificity, suggesting that chronically inflamed tissue attracts plasma cells as such and extends the overall capacity of the body for plasma cells, allowing autoreactive plasma cells to survive for long times within the inflamed tissue and to provide exorbitant titers of antibodies locally.

Engineering the microflora to vaccinate the mucosa: serum immunoglobulin G responses and activated draining cervical lymph nodes following mucosal application of tetanus toxin fragment C-expressing lactobacilli.

The delivery of antigens to mucosal-associated lymphoid tissues in paediatric and immunocompromised populations by safe, non-invasive vectors, such as commensal lactobacilli, represents a crucial improvement to prevailing vaccination options. In this report, we describe the oral and nasal immunization of mice with vaccines constructed through an original system for heterologous gene expression in Lactobacillus in which the 50 000-molecular weight (MW) fragment C of tetanus toxin (TTFC) is expressed either as an intracellular or a surface-exposed protein. Our data indicate that L. plantarum is more effective in this respect than L. casei and that, under the experimental conditions investigated, delivery of TTFC expressed as an intracellular antigen is more effective than cell-surface expression. Immunization of mice with live recombinant lactobacilli induced significant levels of circulating TTFC-specific immunoglobulin G (IgG) following nasal or oral delivery of vaccine strains. In addition, following nasal delivery, secretory immunoglobulin A (sIgA) was induced in bronchoalveolar lavage fluids, as were antigen-specific antibody-secreting cells and antigen-specific T-cell activation in draining lymph nodes, substantiating their potential for safe mucosal delivery of paediatric vaccines.

Induction of immune responses in pigs following oral administration of purified F4 fimbriae

An effective way of stimulating the mucosal immune system was examined in piglets, using F4 fimbriae of enterotoxigenic Escherichia coli (ETEC). It was demonstrated that purified F4 fimbriae, as opposed to ovalbumin (OVA), are powerful oral immunogens. Indeed, oral administration of purified F4 induced antigen-specific antibody-secreting cells (ASC) in the Peyer's patches, mesenteric lymph nodes (LN), blood and lamina propria 4, 7, 9 and 11 days postimmunization, respectively, indicating a stimulation of the mucosal immune system, whereas upon oral administration of OVA, no immune response was observed. Moreover, the induced F4-specific IgA and IgG antibody responses were comparable with those obtained upon oral infection with viable E. coli and intramuscular (IM) F4 injection, respectively. Furthermore, a priming of the mucosal immune system is better obtained by oral infection (ASC localized in mesenteric LN) than by IM F4 injection (ASC localized in spleen and retropharyngeal LN) since an oral boost with purified F4 induced a secondary response in the orally infected animal (mainly IgA and IgG ASC, rapid increase of IgA antibodies) while in the IM primed animal a secondary (more circulating antigen-specific ASC than in the unprimed animal) as well as a primary IgM and IgA response (mainly IgM ASC, slow increase of IgA antibodies), suggesting a primary mucosal response, were seen. An oral challenge of the naive control displayed a primary response (mainly IgM ASC, slow increase of IgA and IgG antibodies). The capacity of purified F4 to activate the mucosal immune system on oral administration, is of importance for the development of oral vaccines against ETEC infections.

Induction of Antigen-specific Antibody-secreting Cells in the Nasal Mucosa by Intranasal Immunization of BALB/c Mice

Recent studies have demonstrated that antigen-specific IgA immune responses in nasal cavity are induced by intranasal immunization with several antigens. However, the precise mechanisms regulating such immune responses are unclear. In the present study, mice were immunized intranasally with horseradish peroxidase (HRP) plus cholera toxin into the right nostril, and HRP-specific IgA immune responses were investigated. After the immunization, an enzyme-linked im munospot assay showed an increased number of anti-HRP IgA cells in the nasal mucosa. Histochemical staining demonstrated the presence of anti-HRP antibody-secreting cells, mainly in the nasal subepithelial layer. Interestingly, these cells were induced predominantly on the right nostril, where intranasal immunization was performed. These findings suggest that antigen-specific IgA antibodies in the nasal secretions were produced locally from antibody-secreting cells in the nasal mucosa and that IgA immune responses in the nose were compartmentalized. Furthermore, lectin histochemistry showed an accumulation of peanut agglutinin-positive cells in the nasal-associated lymphoreticular tissue, suggesting the development of germinal centers in the immunized animals.

Immunoreactivity of Roach,Rutilus rutilus,Following Laboratory Exposure to Bleached Pulp and Paper Mill Effluents

In order to study immunomodulation, controlled laboratory experiments were carried out with roach (Rutilus rutilus) exposed to bleached kraft mill effluent (BKME) or spent bleach liquor (SBL) from two pulp and paper mills using elementary chlorine and chlorine dioxide for bleaching. The total number of immunoglobulin-secreting cells (ISC) and the number of specific antibody-secreting cells (ASC) were determined by ELISPOT assay. Exposure to BKME resulted in decreased numbers of ISC in the spleen. To study the capability of response against foreign antigens the fish were immunized with bovine γ-globulin. Exposure to BKME or SBL reduced antigen-specific ASC response in fish. Moreover, the suppression of ASC response did not require exposure prior to immunization. Depuration of fish in clean water reveals that the immunosuppression caused by BKME is reversible. The ASC response in the BKME-pretreated fish returned to the same or even a higher level compared to untreated immunized fish and, in addition, the number of ISC increased greatly. The results of this study verify an earlier finding on poor antibody response due to exposure in a lake contaminated with BKME. Exposure to SBL alone suppresses antibody-mediated immunity, suggesting that compounds formed in bleaching are at least partly responsible for immunotoxic effects.

Nasal lymphoid tissue (NALT) as a mucosal immune inductive site.

Intranasal (i.n.) immunization is a very effective route for inducing mucosal immunity, but the cellular mechanism responsible for regulating and disseminating these responses is not fully understood. The authors studied the role of nasal lymphoid tissue (NALT) as a mucosal inductive site by using highly purified NALT cells obtained by a new method of mechanical isolation. The NALT cells, like Peyer's patch (PP) cells, were smaller in size and less granular than lymphocytes from salivary glands (SG) and small intestinal lamina propria (LP). The NALT cells isolated from i.n. immunized mice contained antigen-specific antibody-secreting cells (ASC) predominantly of immunoglobulin (Ig)A isotype, similar to those also recovered from salivary glands in a time course study. However, the higher proportion of smaller sized sports formed by NALT cells in ELISPOT assays suggested that these cells were less differentiated precursors of those found in salivary glands. This was supported by the fact that after i.n. immunization, IgA ASC appeared in NALT, as well as in mucosal effector sites SG and LP, but none or very few in another mucosal inductive site, PP. In contrast, after intragastric (i.g.) immunization, IgA ASC were detected in PP, along with the SG and LP, but none or very few in NALT. Furthermore, after i.n. immunization, lymphocytes from NALT but not PP proliferated in response to the specific antigen in culture. These findings imply that NALT served as an inductive site for IgA antibody responses at mucosal effector sites.

Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection.

To facilitate the evaluation of vaccines against bovine herpesvirus type 1 (BHV-1), a cotton rat model of intranasal (i.n.) BHV-1 infection was established. Cotton rat lung cells were similar to bovine cells in their ability to support BHV-1 replication in vitro. Furthermore, i.n. inoculation of cotton rats with BHV-1 resulted in pulmonary lesions comparable to BHV-1 infection in cattle. Using this model, the potential of i.n. and gastrointestinal (g.i.) immunization was examined with recombinant human adenoviruses expressing glycoprotein D (gD) of BHV-1 to induce protective immunity against BHV-1. The replication-competent virus (gD-dE3) was more efficient than the replication-defective virus (gD-dE1E3) in inducing gD-specific antibody in the serum and in the respiratory tract. Furthermore, i.n. immunization with gD-dE3 stimulated antigen-specific antibody-secreting cells in the lung 12 weeks following immunization. Protection against BHV-1 challenge correlated with gD-specific antibody levels such that i.n. immunization with gD-dE3 conferred complete protection, while g.i. immunization conferred only partial protection of the lungs of most animals against BHV-1 challenge. In comparison, immunization with gD-dE1E3 by either route resulted in only a partial reduction of BHV-1 titre in the respiratory tract. The results obtained demonstrate that mucosal immunization with replication-competent recombinant adenovirus expressing gD of BHV-1 can induce immunity and protection against BHV-1 challenge.

Standardization of measurement of immunoglobulin-secreting cells in human peripheral circulation.

A sensitive, and at times the most sensitive, measurement of human vaccine immunogenicity is enumeration of antibody-secreting cells (ASC) in peripheral blood. However, this assay, which is inherently capable of measurement of the absolute number of antigen-specific ASC, is not standardized. Thus, quantitative comparison of results between laboratories is not currently possible. To address this issue, isotype-specific ASC were enumerated from paired fresh and cryopreserved mononuclear cell (MNC) preparations from healthy adult volunteers resident in either the United States (US group) or Egypt (EG group). Analysis of fresh cells from US volunteers revealed mean numbers of ASC per 10(6) MNC of 617, 7,738, and 868 for immunoglobulin M (IgM), IgG, and IgA, respectively, whereas EG volunteers had 2,086, 7,580, and 1,677 ASC/10(6) MNC for the respective isotypes. Cryopreservation resulted in a slight reduction in group mean IgM, IgG, and IgA ASC (maximum reduction in group mean, 14%), but in no instance were results obtained with cryopreserved cells significantly lower than those obtained with fresh cells. To determine if cryopreservation affected the number of bacterial antigen-specific ASC detected, cells from a group of US adult volunteers who received a single oral dose of a mutated Escherichia coli heat-labile enterotoxin (LT(R192G)) were tested. There was no significant difference (P > 0.05) in the number of antigen-specific IgA or IgG ASC detected between fresh and cryopreserved MNC. The results support the views that ASC assays can be standardized to yield quantitative results and that the methodology can be changed to make the test more practical.

Activation patterns of murine B cells after oral administration of an encapsulated soluble antigen

Oral administration of soluble protein antigen, ovalbumin (OVA), encapsulated by an acid-resistant acrylic polymer, induced a strong immune response in BDF 1 mice. Mice fed the encapsulated OVA showed activation of antigen-specific IgA, IgG and IgG1 isotype antibody-secreting cells which migrated to various lymphoid tissues via the common mucosal immune system traffic pathway. The highest number of antigen-specific antibody secreting cells was found in the Peyer's patches isolated from the upper segment of the small intestine, with their numbers declining along the length of the intestinal tract. Oral administration of this product suggests activation of lymphocytes exhibiting Th2 phenotype as shown by OVA-specific IgA and IgG1 isotype antibody responses. In conclusion, we demonstrated that oral administration of encapsulated soluble protein, in the absence of adjuvant, can activate the murine common mucosal immune system.

Systemic and pulmonary immune response to intrabronchial administration of ovalbumin in calves

Local immunization of the respiratory tract may be the best way to achieve protection against respiratory pathogens. In order to do so successfully, it is important to fully understand how the immune response to antigen administered via the respiratory route develops. We studied the respiratory and systemic immune response after subcutaneous (SC) and intrabronchial (IB) inoculation of calves with ovalbumin (OVA). Eight calves received two SC inoculations of OVA and eight other calves received two SC and three additional IB inoculations of OVA. The occurrence of OVA-specific antibodies and antibody-secreting cells (ASC) was measured over time using isotype-specific enzyme linked immunosorbent assay (ELISA) and ELISPOT. SC immunization of calves did not result in OVA-specific IgA in bronchoalveolar lavage (BAL) fluid. Subcutaneous priming followed by intrabronchial challenge caused an initial IgG1 response in the bronchoalveolar lavage fluid, followed by a large IgA response. The presence of IgG1-ASCs indicated that the IgG1 was at least partially locally produced. Most of the OVA-specific IgA in the BAL fluid was secreted by pulmonary ASCs as indicated by the large number of IgA-ASCs in BAL samples and the low serum level of OVA-specific IgA. Antigen-specific IgG1 ASCs were detectable among peripheral blood mononuclear cells after culture with OVA.

Activation patterns of murine B cells after oral administration of an encapsulated soluble antigen

Oral administration of soluble protein antigen, ovalbumin (OVA), encapsulated by an acid-resistant acrylic polymer, induced a strong immune response in BDF 1 mice. Mice fed the encapsulate OVA showed activation of antigen-specific IgA, IgG and IgG1 isotype antibody-secreting cells which migrated to various lymphoid tissues via the common mucosal immune system traffic pathway. The highest number of antigenspecific antibody secreting cells was found in the Peyer's pathches isolated from the upper segment of the small intestine, with their numbers declining along the length of the intestinal tract. Oral administration of this product suggests activation of lymphocytes exhibiting Th2 phenotype as shown by OVA-specific IgA and IgG1 isotype antibody responses. In conclusion, we demonstrated that oral administration of encapsulated soluble protein, in the absence of adjuvant, can activate the murine common mucosal immune system.

Generation of specific antibody-secreting cells in salivary glands of BALB/c mice following parenteral or oral immunization with Porphyromonas gingivalis fimbriae

The humoral immune response and induction of antigen-specific antibody-secreting cells in mucosal lymphoid tissues were examined in mice immunized subcutaneously or orally with fimbrial protein purified from Porphyromonas gingivalis strain 381. A group of BALB/c mice was immunized subcutaneously with P. gingivalis fimbriae and semisynthetic adjuvant GM-53 in Freund's incomplete adjuvant on days 0 and 28. Another group of mice was immunized perorally with liposome containing fimbriae and GM-53 on days 0, 1, 27 and 28. In the mice immunized subcutaneously, salivary anti-fimbrial IgM antibodies were detected transiently on day 5, followed by the appearance of specific IgG and IgA antibodies on day 14. Higher concentrations of salivary IgG and IgA anti-fimbrial antibodies were found after the second immunization. Fimbria-specific IgM and IgG spot-forming cells (SFC) were detected in cervical lymph nodes of the immunized mice by the ELISPOT method. Fimbria-specific IgA SFC but not IgM and IgG appeared in the parotid and submandibular glands of subcutaneously immunized mice. On the other hand, mice immunized by gastric intubation generated almost exclusively salivary anti-fimbrial IgA antibodies. In agreement with this finding, increased numbers of antigen-specific IgA but not IgM and IgG SFC were seen in parotid and submandibular glands, but not in cervical lymph nodes of orally immunized mice. It can be concluded that systemic or oral immunization with fimbrial antigen induces distinct immune responses in specific lymphoid tissues or in the salivary glands in respect of their temporal sequences and the numbers of plasma cells secreting antigen-specific and non-specific immunoglobulins.

Immune responses to Shigella dysenteriae 1 and Shigella flexneri lipopolysaccharide and polysaccharide antigens in Bangladeshi patients with shigellosis

The immune responses of adult Bangladeshi patients to diarrhoea caused by Shigella dysenteriae type 1 ( n = 20) and Shigella flexneri ( m = 12) were analysed and compared with that of 20 healthy individuals. Antigen-specific antibody secreting cells (ASC) were estimated by ELISPOT, the peripheral mononuclear cell response was determined by a lymphocyte proliferation assay, and serum and mucosal antibody responses were measured by ELISA. Purified lipopolysaccharides (LPS) and O-polysaccharides (PS) from S. dysenteriae type 1 or S. flexneri Y strains, respectively, were used as antigens. A significant increase ( P < 0.001) in the number of LPS-specific IgA-ASC was observed with a peak 6–8 days after the onset of disease followed by a rapid decline within 16 days. The mucosal IgA responses were similar. Serum IgA titres were highest 9–11 days after the onset of diarrhoea and significantly higher ( P < 0.001) than in the control group. The serum IgG levels in S. dysenteriae 1 group were almost twice ( P = 0.008) the level measured in the control group. The mean IgG titres in the S. flexneri group, however, were only slightly higher ( P > 0.05) than that seen in the control group during the acute phase (3–5 days after onset). A strong specific cellular immune response ( P < 0.001) to the homologous S. dysenteriae 1 and S. flexneri O PS antigens was observed in both groups 6–8 days after the onset of symptoms. The immune responses observed suggest that the patients had previously been exposed to shigella. The study demonstrates a good correlation between humoral and cellular immune responses which may play concomitant protective roles in the host defence against shigellosis. Informed consent was obtained from study patients and controls in accordance with the guidelines of the ethical review committee of International Centre for Diarrhoeal Diseases Research, Bangladesh.

Amplified ELISPOT assay for the detection of HIV-specific antibody-secreting cells in subhuman primates

A novel immunoenzyme amplification technique has been evaluated in an ELISPOT assay for the detection of antigen-specific antibody-secreting cells (ASC) in monkeys. In this assay, mononuclear cells containing putative ASC are incubated for a few hours in antigen-coated wells. Following removal of the cells, zone of solid phase bound antibodies secreted by individual ASC are6visualized in four consecutive steps. First, a primary biotinylated anti-immunoglobulin (Ig) reagent is a added followed by enzyme-labelled avidin. The amplification procedure comprises the addition of biotinylated anti-enzyme antibodies in the third stage, followed by enzyme-conjugated avidin and substrate. When evaluated in a modified ELISPOT assay for the detection of simian B cells secreting antibodies to the envelope gylcoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1), this amplification procedure proved to be suitable even when using anti-human Ig antisera as primary antibody reagents. This development should be useful for other ELISPOT assays where species specific anti-Ig reagents are not always available and, most importantly, for enumerating cells producing immunoreactive substances in such minute amounts that they may escape detection by conventional ELISPOT assays. Furthermore, a functional simian HIV-specific ELISPOT assay could prove valuable for assessing the humoral immunogenicity of future candidate vaccines against the acquired immunodeficiency syndrome (AIDS).

Occurrence of antigen-specific B cells following oral or parenteral immunization with Porphyromonas gingivalis fimbriae.

The induction and distribution of antigen-specific antibody-secreting cells in various tissues were assessed in BALB/c mice immunized with the purified fimbrial protein of the Porphyromonas gingivalis strain 381. Groups of mice were immunized by gastric intubation of liposomes containing fimbriae and GM-53 on days 0, 1, 27, and 28. Additional groups of mice were immunized with P. gingivalis fimbriae and adjuvant GM-53 in Freund's incomplete adjuvant by subcutaneous injection on days 0 and 28. In the latter group of mice, levels of serum IgM anti-fimbria antibodies were first detected on day 7, while high levels of serum IgG anti-fimbria antibodies were seen after secondary immunization. Fimbria-specific spot-forming cells (SFC) were detected in the spleen, circulating blood mononuclear cells (CBMC), and brachial lymph nodes of immunized mice by ELISPOT. Fimbria-specific IgM SFC appeared by day 5 and antigen-specific IgG SFC were seen later in subcutaneously immunized mice. Mice immunized orally exhibited serum anti-fimbria IgG and IgA antibodies after boosting. Although numerical analysis revealed that the numbers of fimbria-specific SFC were generally lower than in subcutaneously immunized mice, significant numbers of antigen-specific IgA SFC were seen in lamina propria and mesenteric lymph nodes of orally immunized mice. In contrast, antigen-specific IgM and IgG SFC were observed mainly in CBMC. The route of immunization with fimbriae and GM-53 also influenced the total numbers of immunoglobulin-secreting cells. Thus, subcutaneous immunization enhanced the total number of IgM and IgG SFC, including fimbria-specific antibody-secreting cells in CBMC and the spleen. (ABSTRACT TRUNCATED AT 250 WORDS)

Plasmodium yoelii: Antibody response in resistant and susceptible mouse strains

The numbers of antigen-reactive antibody-secreting cells, levels of parasite antigen-specific serum antibodies and numbers of red blood cells staining positive for surface immunoglobulin were determined for susceptible and resistant mouse strains following infection with Plasmodium yoelii 17×. As a control, these parameters also were measured using antigen prepared from normal red blood cells. The relatively susceptible C57BL/6 mice produced more antigenspecific antibody-secreting cells and had higher levels of immunoglobulin positive red blood cells than did DBA/2 mice, but the DBA/2 mice had more antigen-specific IgG in their sera. Both mouse strains possessed cells secreting antibody reactive with soluble normal red blood cell antigen; however, C57BL/6 mice had more IgG positive unparasitized RBC than did DBA/2 mice. Despite possessing fewer antibody positive normal RBC, DBA/2 mice had significantly higher levels of serum antibodies that reacted with soluble red blood cell antigen. These data indicate that levels of serum antibody may not reflect the amounts of antibody produced and that use of any single assay to assess the magnitude of the antibody response may give rise to misleading results.

Recombinant human interleukin 6 (rhIL-6) promotes the terminal differentiation of in vivo-activated human B cells into antibody-secreting cells

Antigen-activated peripheral blood B cells were induced by parenteral immunization of healthy individuals with a polyvalent pneumococcal vaccine, or diphtheria toxoid. Seven to nine days after immunization, high frequencies of antigen-specific antibody-secreting cells, representing in vivo activated lymphoblastoid B cells, were detectable in peripheral blood or spleen. The B cellenriched fractions were stimulated for 7 days with different concentrations of rhIL-6. Both the frequency of antibody-secreting cells and the secreted amount of antibody to the immunizing antigen were increased by rhIL-6 in a dose-dependent fashion. Stimulation with rhIL-6 did not alter the isotype distribution of antibody-secreting cells. A polyclonal anti-IL-6 serum completely abrogated the stimulatory effect of rhIL-6 on the in vitro antibody secretion. Fluorescence-activated cell sorter analysis revealed that 25–29% of cells in the large B cell fraction which presumably contained the in vivo activated cells bore the IL-6 receptor. Thus, rhIL-6 enhances the terminal differentiation of in vivo activated B cells into antibody-secreting plasma cells.

Antibody-secreting peripheral blood lymphocytes induced by immunization with a conjugate consisting of Streptococcus pneumoniae type 12F polysaccharide and diphtheria toxoid

Healthy adult volunteers were injected either with one of two conjugates composed of Streptococcus pneumoniae type 12F polysaccharide (Pn12F) covalently coupled to diphtheria toxoid or with Pn12F alone (as a component of Pnu-Imune, a 23-valent pneumococcus vaccine). The conjugates induced Pn12F-specific antibody-secreting cells in peripheral blood with numbers and isotype distribution similar to those induced by Pnu-Imune, with immunoglobulin A (IgA) as the predominant isotype. The conjugates also elicited high numbers of diphtheria toxoid-specific antibody-secreting cells of the IgG class. There was no distinct booster effect, since a second dose of the conjugates induced antibody-secreting cells at significantly lower numbers than after the first dose. In contrast to the cell numbers, the conjugate vaccines induced higher increases of IgA1 Pn12F antibodies in serum than did Pnu-Imune. However, neither the conjugates nor Pnu-Imune induced a secretory antibody response. Antibody levels in serum and saliva correlated poorly with the frequency of antigen-specific antibody-secreting cells. Circulating antibody-secreting cells present 7 days postimmunization were probably not responsible for the high increase of antibodies in serum but rather represented a population of in vivo-activated B cells with the ability to disseminate the humoral response from the antigen recognition site to distant locations of antibody production.