Specific Antibody-producing Cells Research Articles

Generation of monoclonal antibody against 6-Keto PGF1α and development of ELISA for its quantification in culture medium

Prostacyclin or prostaglandin I2 (PGI2), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI2 in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF1α, a stable PGI2 metabolite, and its quantification to determine the role of PGI2 in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF1α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF1α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF1α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI2 regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI2 in maintaining homeostasis and adipocyte differentiation.

Human-type artificial lymphoid tissues (organoids) induce antigen-specific immune responses upon antigen-stimulation

Abstract We had previously reported that the immunologically active artificial lymphoid tissues (aLTs) could be stably generated in mice by implantation of lymphoid stromal cell-embedded biocompatible scaffold (collagen sponge) first in 2004 (Suematsu and Watanabe) as well as later by applying the chemokine/lymphokine-trapped gel-embedded scaffold in 2016 (Kobayashi and Watanabe). The generation of the functional human-type aLTs might be beneficial in future for clinical application as the immunotherapy against cancers as well as the normalization of diseases caused by malfunction of human immune system. It may be applied for the evaluation of vaccines. We first established the human stromal cell lines suitable for the formation of the human aLTs. Then, spheroids composed of the human stromal cells were prepared. The mixtures of stromal cell spheroids and human peripheral blood lymphocytes (PBMCs) were transplanted into renal subcapsular spaces of the immunodeficient NOG-dKO mice. After 2–3 weeks, human type aLTs (hPBMC-aLTs) were stably formed. The human type aLTs are composed of the clusters of human B and T cells including scattered human DC cells. The human type aLTs were immunized with the inactivated varicella zoster virus (VZV) or with NP-conjugated ovalbumin (NP-OVA) in adjuvant. Anti-VZV or anti-NP-hapten specific antibody-producing cells were strongly induced in the human-aLTs. IFNg was produced from human T cells in aLTs upon the immunization. These data indicate that the immunologically functional human aLTs could be successfully generated. We are currently investigating anti-cancer activity of the human aLTs as well as attempting to construct them in vitro system.

Recombinant HIV envelope trimer selects for quaternary-dependent antibodies targeting the trimer apex.

Broadly neutralizing antibodies (bnAbs) targeting the trimer apex of HIV envelope are favored candidates for vaccine design and immunotherapy because of their great neutralization breadth and potency. However, methods of isolating bnAbs against this site have been limited by the quaternary nature of the epitope region. Here we report the use of a recombinant HIV envelope trimer, BG505 SOSIP.664 gp140, as an affinity reagent to isolate quaternary-dependent bnAbs from the peripheral blood mononuclear cells of a chronically infected donor. The newly isolated bnAbs, named "PGDM1400-1412," show a wide range of neutralization breadth and potency. One of these variants, PGDM1400, is exceptionally broad and potent with cross-clade neutralization coverage of 83% at a median IC50 of 0.003 µg/mL. Overall, our results highlight the utility of BG505 SOSIP.664 gp140 as a tool for the isolation of quaternary-dependent antibodies and reveal a mosaic of antibody responses against the trimer apex within a clonal family.

Cytolytic immune lymphocytes in the armamentarium of the human host

The author of the monograph "Cytolytic Immune Lymphocytes..." (published in 2008 by Schenk Buchverlag Campus Dialog, Budapest, Passau, Pécs) proposed several research projects and described certain clinical events that require further elaboration and documentation. In this article the author provides what is required and has since become available. The first subject matter in question concerns the fusogenic viruses. The ancient fusogenic viruses might have created the first eukaryotic cell(s) by uniting archaeabacterial and prokaryotic/protobacterial protospheroplasts. Extant fusogenic viruses either produce tumor cell syncytia and lyse them, thus practicing viral oncolysis. Or, create chimaeric fusion products, the so-called "natural hybridomas", of lymphoma cells exhibiting transmembrane budding of retrovirus particles or envelope proteins, and anti-viral specific antibody-producing plasma cells. The second topic concerns the horizontal-lateral mode of acquisition of those genes, which were "present in the waiting" in the amphioxus, sea urchin, and the agnathans, and met in the primitive gnatostomata sharks to encode in unison the entire adaptive immune system. The consensus of opinion is such that these genes derived from newly acquired transposons/retrotransposons. The author points out that the extant Epstein-Barr virus harbors genes displaying sequence homology with those genes from the sharks up to mammals that regulate the somatic hypermutation of specific antibody production. The author proposes that an ancient herpesvirus might have propagated the V(D)J and RAG genes from sea urchins to sharks. The third area is that of lymphocytes cytotoxic/cytolytic to virally infected or malignantly transformed host cells. This discovery led to the adoptive immune lymphocyte therapy of tumors. Installed in the adaptive immune system are regulatory T cells and myeloid-derived suppressor cells for he protection of "self". Tumor cells masquerading as "self" are protected by these cells from attacks launched by immune T cells. The author supports the replacement of IL-2 by IL-15, inasmuch as IL-2 stimulates not only immune T cells, but also regulatory T cells expressing the CD25 IL-2 receptor. The administration of low dose whole body radiotherapy prior to immune lymphocyte therapy increases the efficacy of immune lymphocyte therapy. The author observed this phenomenon in the mid-1960s. The explanation of this phenomenon revealed itself just recently. In pre-irradiated hosts the intestinal wall becomes permeable to the gut flora; the intestinal bacteria activate the entire innate immune system in the mesenteric lymph nodes and a rapid activation of the adaptive immune faculties follows.

Evaluation of immunomodulatory and anti-inflammatory effects and phytochemical screening of Alternanthera tenella Colla (Amaranthaceae) aqueous extracts

Alternanthera tenella Colla extracts are used in Brazilian traditional folk medicine to treat a variety of infectious diseases as well as inflammation and fever. In this work, the immunomodulatory, anti-inflammatory and potential toxic effects of cold (CAE) and hot (HAE) aqueous extracts of A. tenella were investigated in vivo. In addition, we analyzed the phytochemical properties of both extracts. BALB/c mice were immunized in vivo with sheep red blood cells and concomitantly inoculated intraperitoneally (i.p.) with each extract (50, 100 or 200 mg/kg). Specific antibody-producing cells were enumerated using plaque-forming cell assays (PFC) and anti-SRBC IgG and IgM serum levels were measured via enzyme-linked immunosorbent assay. Body and lymphoid organ weights were determined after treatments in order to evaluate toxic effects. Carrageenan-induced paw edema was employed to investigate anti-inflammatory activity in mice inoculated i.p. with CAE or HAE (200 or 400 mg/kg). Phytochemical screening was performed using spectrometric and chromatographic approaches and revealed that CAE possessed higher tannin and flavonoid levels than HAE. PFC numbers were increased after treatment with CAE (100 mg/kg) four days after immunization, as were the serum antibody titers after four and seven days, suggesting immunostimulatory activity through modulation of B lymphocyte functions. Body and organ weights did not show major changes, suggesting that extracts administered to mice did not induce significant toxicity. Both extracts had significant anti-inflammatory activity in the paw edema assay. These results suggested that aqueous extracts from A. tenella contained several chemical compounds that possess positive and/or negative modulator effects on the immune system, which appeared to correlate with tannin and flavonoid levels in those extracts. In summary, these studies provide important insight into the biological activities of A. tenella.

Immune response and disease resistance in Atlantic salmon (Salmo salar L.) fed three levels of dietary vitamin E and the effect of vaccination on the liver status of antioxidant vitamins

Atlantic salmon were fed 40, 300 or 1100 mgall-rac-α-tocopheryl acetate kg feed−1 for 12 weeks.After 6 weeks half of the fish in each group were marked and vaccinated (i.p.injection) against furunculosis and vibriosis. Liver α-tocopherol levelsreflected the dietary input after 6 and 12 weeks of feeding. Noimmunomodulatoryeffects of dietary vitamin E on baseline levels of the immune parameters beforevaccination were detected in this study, as evaluated by antibody dependent andspontaneous plasma complement activities. In general, vaccination increasedplasma complement activities and the number of antigen specific antibodyproducing cells as compared with unvaccinated control fish, but with nodifferences with respect to the vitamin E regimes. Also the ability ofunvaccinated fish to withstand experimental furunculosis was unaffected bydietary vitamin E. The concentrations of α-tocopherol and ascorbic acidinthe liver were, however, negatively affected 6 weeks post vaccination comparedto respective unvaccinated fish.

Measurement of immunoglobulin synthesis using the ELISPOT assay.

We describe a method for the detection of specific antibody-producing cells from either in vitro or in vivo immunization. These techniques are especially useful for detecting antibodies from developing hybridomas. We have successfully used the system to detect isotype-specific antibodies to a variety of bacterial antigens which were produced by heterohybridomas.

Human immune responses to influenza virus vaccines administered by systemic or mucosal routes

Healthy adult volunteers were immunized by parenteral or oral routes with trivalent inactivated influenza vaccine (A/Chile/1/83 (H1N1), A/Mississippi/1/85 (H3N2), and B/Ann Arbor/1/86), or intranasally with live attenuated, cold-adapted influenza type A/Texas/1/85 (H1N1) reassortant virus. In all volunteers, cells spontaneously secreting IgA, IgG or IgM antibodies specific to influenza virus were detected in peripheral blood on days 6–13 after immunization, and specific IgA, IgG and IgM antibodies to influenza vaccine were measured in sera and external secretions (saliva and nasal lavage). Following systemic immunization, a raise in specific antibodies of all isotypes was observed in sera beginning on day 13. Although small variations in IgA and IgM antibodies in saliva and nasal lavages were detected, antigen-specific IgG significantly increased between days 13 and 27. Intranasal administration of attenuated virus induced IgA and IgG antibodies in serum as well as in secretions. Serum antibodies were not substantially influenced by oral immunization, only a small increase in all isotypes was observed in volunteers' sera 21 days after ingestion of vaccine. However, in secretions, antigen-specific IgA and IgG responses were detected one week after immunization and reached a peak response on day 20. These studies show that different routes of immunization can be effective for the induction of specific antibodies, and support the concept of the common mucosal immune system in humans by demonstrating that the oral or intranasal administration of antigen-induced specific antibodies of IgA isotype in external secretions, preceded by the transient appearance in peripheral blood of specific antibody-producing cells.

Testosterone Alters the Immune Response of Chinook Salmon, Oncorhynchus tshawytscha

We examined the effects of the high concentrations of sex steroid hormones detected in the plasma of sexually maturing chinook salmon (Oncorhynchus tshawytscha) on the functioning of the immune system. Leukocytes from the anterior kidney of juvenile spring chinook salmon were incubated in the presence of steroid, and their ability to form specific antibody-producing cells (plaque-forming cells) was used as a measure of immunocompetence. Testosterone and cortisol, but not 17β-estradiol or aldosterone, were found to significantly reduce the plaque-forming response in vitro. Testosterone and cortisol administered together had a significantly greater effect than did either administered alone. The plaque-forming response was significantly affected by season. The magnitude of the steroid-induced immunosuppression was reduced during winter and increased again in spring.

Fetal intestinal transplant model for mucosal immune responses

A rat fetal intestinal transplant model was developed for long-term study of intestinal immune responses. For the model, fetal small intestine is transplanted into the dorsal subcutaneous tissue of synergic adults and allowed to mature, providing an accessible site, isolated from the intestinal stream. We previously demonstrated normal histologic maturation of the transplant. Specific antibody-producing cells appeared in the lamina propria of both transplant and native in situ intestine following intraluminal immunization of the transplant with cholera toxin, and conversely, in transplants after immunization of in situ intestine. An enterointestinal lymphocyte migratory pathway (originating in intestine and migrating to another region of the intestine) was thus demonstrated unequivocally. We found a bacterial flora in the transplant, and showed normal villus morphology in scanning electron microscopy. Less than 200 pg, i.e., a 10 −7 fraction, of 2 mg macromolecular lipopolysaccharide placed in the transplant lumen was absorbed per plasma lipopolysaccharide half-life. Immunization of the transplant with cholera toxin resulted in specific IgA and IgG antibody in the transplant lumen and in bile, and specific IgG, but not IgA, antibody in serum. A second dose of antigen gave an anamnestic rise in intra-transplant antibody. Intestinal immune tolerance was also demonstrated: sheep red blood cells (SRBC) administered into the transplant for 7 days suppressed splenic IgM plaque forming responses to subsequent intraperitoneal challenge with SRBC. These studies further demonstrate that the fetal intestinal transplant behaves immunologically like native intestine, and therefore provides a useful model for investigation of the intestinal immune system.

Comparison of human interleukin-1β and its 163–171 peptide in bone resorption and the immune response

Human interleukin-1 beta (IL-1 beta) caused a dose- and time-dependent enhancement of the release of 45Ca from prelabeled mouse calvaria in organ culture. In addition, IL-1 beta dose-dependently stimulated the formation of prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha in the calvarial bones. However, IL-1 beta-induced 45Ca release was only partially inhibited by blocking the PGE2 response with indomethacin, suggesting that enhanced PGE2 formation in response to IL-1 beta is not necessary to obtain a bone resorptive effect, but that prostaglandins potentiate the action of IL-1 beta. The synthetic nonapeptide VQGEESNDK, corresponding to the fragment 163-171 of human IL-1 beta, administered simultaneously with antigen (SRBC) to C3H/HeN male mice, induced a dose-dependent enhancement of specific antibody-producing cells in the spleen (PFC). The degree of PFC stimulation was comparable to that caused by native human IL-1 beta. In mouse bone cultures, neither 45Ca release nor prostanoid formation was stimulated by fragment 163-171. These data indicate that (1) IL-1 beta-induced stimulation of bone resorption is dissociable from IL-1 beta-induced increase of prostanoid biosynthesis and (2) the epitope of the IL-1 beta molecule involved in the immunostimulatory effects may be different from that involved in the stimulatory effects on bone resorption.

Antibody-producing cells in peripheral blood and salivary glands after oral cholera vaccination of humans

We examined whether immunization with a newly developed oral cholera vaccine would elicit gut-derived antibody-producing cells in the blood and in distant mucosal tissues, such as the minor salivary glands, in 30 adult Swedish volunteers. The results of this study demonstrated that this vaccine indeed induced production of specific antibody-producing cells against the cholera toxin B subunit in both peripheral blood and salivary glands. The response in blood, which after primary and booster immunizations comprised both immunoglobulin A (IgA) and IgG antibody-forming cells, was highly transient and preceded the response in salivary glands; the latter response was restricted to the IgA isotype. The results provide further evidence of the existence of a common mucosal immune system in humans. Furthermore, these findings support previous observations that in animals, the cholera toxin B subunit may be a useful carrier protein for preparing enteric vaccines against pathogens encountered at intestinal and extraintestinal mucosal sites.

Contents Volume 180 · 1990

By incubating tissue sections with antigen (or hapten)-enzyme conjugates, followed by enzyme cytochemistry1–4, N. van Rooijen and his colleagues have been able to study the development and localization of specific antibody-producing cells in the spleen of animals after a single intravenous antigen injection. Variations in this method5–11 allowed them to compare the localization patterns of cells producing different antibodies and to determine simultaneously their Ig isotype. Studies on primary and secondary immune responses against thymus-dependent and thymus-independent antigens lead them to postulate that there is one ‘differentiation pathway’ for antibody-forming cells in the spleen, independent of the antigen and the type of immune response.

Differential activity of interleukin 1α and interleukin 1β in the stimulation of the immune response in vivo

The biological activities of human recombinant interleukin (IL) 1 alpha and IL 1 beta were compared in different biological systems. The two IL 1 forms were equally active in vitro in inducing proliferation of murine thymocytes and of the murine T helper clone D10.G4.1, and in triggering release of prostaglandin E2 from human skin fibroblasts. In vivo, IL 1 alpha and IL 1 beta were similarly pyrogenic both in rabbits and mice, and could equally increase the circulating levels of the acute phase protein serum amyloid A in mice. However, only IL 1 beta showed immunostimulatory activity in vivo, as it could enhance the number of specific antibody-producing cells in the spleen of mice immunized with either a T-dependent or a T-independent antigen. Although devoid of immunostimulatory activity, IL 1 alpha could efficiently compete immunostimulation induced by IL 1 beta, suggesting an effective interaction with the IL 1 receptor. Thus, IL 1 beta appears to have an important role in the positive regulation of immune responses, while IL 1 alpha may act as down-regulator of the IL 1 beta effect.

Stress alters immune function and disease resistance in chinook salmon (Oncorhynchus tshawytscha).

We examined the effects of acute stress on the immune system and disease resistance of juvenile chinook salmon (Oncorhynchus tshawytscha) in laboratory and clinical trials. Immune function, as measured by the ability of lymphocytes from the anterior kidney to generate specific antibody-producing cells (APC) in vitro, was depressed 4 h after stress, when plasma cortisol levels were highest. At the same time, resistance to the fish pathogen, Vibrio anguillarum, was also depressed. Compared with controls, plasma cortisol and APC of stressed fish were unchanged after 24 h, and disease resistance was enhanced as evidenced by higher survival rate and longer mean time to death of mortalities. After 7 days, even though numbers of APC were depressed, plasma cortisol concentration and disease resistance did not differ from controls. This pattern was generally the same, independent of the type of stress applied: i.e. being held out of water in a dipnet for 30 s, manipulation during hatchery operations for 4 h, or transportation for 9 h. These and earlier findings suggest that similar endocrine-immune interactions operate in the mammalian and salmonid systems during acute stress.

Specific antibody synthesis and biliary secretion by the rat liver after intestinal immunization with cholera toxin

This study was designed to investigate the location, specificity, and significance of antibody-containing cells in the liver. After intestinal administration of cholera toxin, high numbers of specific antibody-containing cells appeared in the liver during the early priming period and after boosting. In contrast, a significant number of specific antibody-containing cells appeared in the lamina propria of the intestine only after boosting. In the liver, the specific antibody-containing cells were predominantly located in the sinusoidal region of zone 1 of the liver lobules. About 80% of the specific antibody-producing cells in the liver synthesized anticholera toxin antibody of the immunoglobulin A class. During the priming period, the concentration of immunoglobulin A anticholera toxin in bile paralleled the increase and decrease in the number of specific antibody-containing cells in the liver. Liver perfusion experiments indicated that during the priming period at least 70% of the biliary immunoglobulin A anticholera toxin antibody was synthesized within the liver, whereas 30% was synthesized in the liver after boosting. Thus, during the early immune response, the liver seems to be the major source for specific biliary antibody to intestinally administered cholera toxin.

In vitro stimulation of presensitized mouse spleen cells with poliovirus type 1, Mahoney, and enhancement of poliovirus-specific hybridomas.

In vivo immunization of BALB/c mice with poliovirus type 1, strain Mahoney, or with its purified polypeptides resulted in 0.2 to 0.5 antigen-specific hybridoma microcultures per 10(6) spleen cells. Stimulation of spleen cells from mice immunized with poliovirus or with its polypeptides in vitro with poliovirus 6 days prior to fusion with the myeloma cells led to a six- to 20-fold increase in the number of positive microcultures, i.e. after stimulation the yield of poliovirus-specific hybridomas was up to 3.8 antigen-specific microcultures per 10(6) spleen cells. The in vitro stimulation of spleen cells primed in vivo was demonstrated by the detection of poliovirus-specific antibody-producing cells 6 days after in vitro cultivation in the presence of poliovirus as antigen. Only spleen cells stimulated under these conditions in vitro gave rise to specific antibody-producing cells and yielded antigen-specific hybridomas after somatic hybridization.

Is there a single differentiation pathway for all antibody-forming cells in the spleen?

By incubating tissue sections with antigen (or hapten)-enzyme conjugates, followed by enzyme cytochemistry 1–4, N. van Rooijen and his colleagues have been able to study the development and localization of specific antibody-producing cells in the spleen of animals after a single intravenous antigen injection. Variations in this method 5–11 allowed them to compare the localization patterns of cells producing different antibodies and to determine simultaneously their Ig isotype. Studies on primary and secondary immune responses against thymus-dependent and thymus-independent antigens lead them to postulate that there is one ‘differentiation pathway’ for antibody-forming cells in the spleen, independent of the antigen and the type of immune response.

Antigen‐specific adherence of antibody‐producing hybridoma cells: Application to neural and nonneural antigens

Antibody-producing hybridoma cells specifically bind to microgram quantities of antigen molecules adsorbed onto the surface of plastic microtiter plates. The binding of hybridoma cells to nonantigen is optimally below 5%, similar binding of non-antibody-producing cells is 4-7%, compared to the binding of the hybridomas to their antigen. There is a difference in the kinetics of binding hybridomas to antigen compared to nonantigen. The number of bound cells depends on the amount, i.e., the surface density, of the antigen molecules and shows typical saturation effects. Preincubation of hybridomas with excess free antigen and saturation of the antibody binding site on the surface with the hybridoma-produced antibody reduces binding of the hybridoma cells to the antigen. Treatment of cells with trypsin reduces binding to antigen-coated plastic surfaces. Drugs such as sodium azide, cytochalasin B, colchicine, vinpocetine, and vincristine sulfate reduce binding to the antigen. Hybridoma cells adhering to the antigen produce more antibody than nonadhering cells. The results reported in this paper show that antigen molecules adsorbed to include a plastic surface and hybridoma cells interact specifically. This system forms a suitable model to study the interaction of antigen with antigen-specific cells and may be useful as a separation method for specific antibody-producing cells.

The influence of the route of antigen administration on the development of specific antibody-producing cells in the follicles of the popliteal lymph nodes of rabbits.

Rabbits received an injection of liposome-associated human serum albumin (HSA) and six weeks later a booster injection of HSA free in solution. Anti-HSA antibody-forming cells were demonstrated in the popliteal lymph nodes by use of a HSA-horseradish peroxidase (HRP) conjugate for their detection. After a single subcutaneous injection into the footpads, specific antibody-forming cells were found, but after a single intravenous injection no anti-HSA-forming cells were observed in the lymph nodes. During the secondary immune response specific anti-HSA antibody-producing cells were found in the lymph nodes of all animals. The localization pattern of anti-HSA antibody-forming cells within the lymph nodes depended on the route of antigen administration used for the primary and booster injection. Anti-HSA-forming cells were mainly found in the cords of the medulla and in the outer cortex. During the secondary immune response, however, they were also detected in the follicles but only when at least one of the two antigen injections was given subcutaneously.