anti-TT Antibody Research Articles

Pertussis toxin enhanced IgG1 and IgE responses to primary tetanus immunization are mediated by interleukin-4 and persist during secondary responses to tetanus alone

Pertussis toxin (Ptx), the major toxin product of Bordetella pertussis, has potent immunologic effects including adjuvant effects on antibody responses and sensitization for anaphylaxis. In order to further define the effect of Ptx on the class and subclass of murine antibody response, we measured total and antigen specific IgG subclasses and IgE in Balb c mice after primary and secondary immunization with tetanus toxoid (TT). Low doses of Ptx (100 ng) given intravenously at the time of primary immunization increased primary IgG1 and IgE anti-TT antibodies as well as total IgG1 and IgE concentrations compared to controls. The increase in IgG1 subclass and IgE response when Ptx was present during primary immunization was even more pronounced after secondary immunization with TT alone 3 weeks or 3 months later. Similar effects were noted after diphtheria toxoid immunization in the presence of Ptx. Administration of the anti IL-4 monoclonal antibody (11B11) suppressed the enhanced total and TT-specific IgE responses but not the enhanced IgG1 responses. The presence of low concentrations of Ptx during primary immunization primes for induction of IL-4 producing T-cell help which enhances IgG1 and IgE responses to the primary exposure as well as to subsequent exposures of the antigen in the absence of Ptx. This phenomenon may have significance for the adjuvant activity of vaccines containing Ptx as well as for the immune response to natural pertussis.

Adjuvant properties of non-phospholipid liposomes (Novasomes ®) in experimental animals for human vaccine antigens

Non-phospholipid liposomes composed of dioxyethylene cetyl ether, cholesterol and oleic acid were evaluated as adjuvants with human vaccine antigens, tetanus toxoid (TT) and diphtheria toxoid (DT), in mice and rabbits. Antigens encapsulated in or mixed with liposomes elicited antitoxin levels similar to those elicited by antigens given with Freund's adjuvant or adsorbed onto aluminum phosphate. All liposomal antigen preparations, antigen given with Freund's adjuvant or adsorbed onto aluminum phosphate, elicited significantly higher IgG antibodies and antitoxin levels than soluble antigens in mice after a single injection and in rabbits after each of three injections. TT encapsulated in liposomes elicited sustained anti-TT IgG antibody levels in mice after a single injection as compared to TT mixed with liposomes. TT mixed with or encapsulated within liposomes containing monophosphoryl lipid Alsqualene or squalene alone, as well as aluminum phosphate adsorbed TT elicited greater primary responses in mice than TT mixed with or encapsulated within plain liposomes. Liposomal TT preparations produced a slightly higher anamnestic response in mice than aluminum phosphate adsorbed TT. Subclass analysis of anti-TT antibodies showed that the majority of the antibodies belong to IgG1 subclass. Liposomal TT preparations, particularly those with encapsulated monophosphoryl lipid Alsqualene or squalene alone, consistently elicited higher levels of anti-TT IgG2a and IgG2b than aluminum phosphate adsorbed or soluble TT. None of the preparations elicited IgG3 or IgM antibodies. It appears that non-phospholipid liposomes are as potent adjuvants as the currently employed adjuvant for human vaccines (aluminum phosphate) or a benchmark adjuvant for experimental immunology (Freund's adjuvant), and may be able to modulate the immune response towards the Th1 type.

Immunologic Response of Extremely Premature Infants to Tetanus, Haemophilus influenzae, and Polio Immunizations

To determine whether extremely premature infants have immunologic responses to tetanus toxoid, Haemophilus influenzae type b polysaccharide and polio vaccines similar to those of full-term infants. Sixteen extremely premature (< 29 weeks, < 1000 g at birth) infants received separate diphtheria-tetanus-pertussis and H influenzae type b oligosaccharide-CRM197-conjugated (HbOC) vaccines at 2, 4 and 6 months of chronologic age, enhanced potency inactivated polio vaccine at 2 months, and oral polio vaccine at 4 months. Serum was obtained for anti-tetanus toxoid (TT), anti-Haemophilus b polysaccharide (HbPs) and polio neutralizing antibody assays before the 2-month vaccination and 4 to 6 weeks after the 6-month vaccination. Comparison sera were obtained from full-term infants immunized with the same lots of diphtheria-tetanus-pertussis (n = 46) and HbOC (n = 66) vaccines or the same sequence of polio vaccines (n = 10). Preterm and full-term infants had similar geometric mean titers of anti-TT antibodies, anti-HbPs antibodies, and neutralizing antibodies to polio serotypes 1, 2, and 3 after the completion of the primary series of vaccines. After vaccination, similar proportions of preterm and full-term infants had protective levels of antibody to TT (preterm 100% vs full-term 100% with levels > 0.01 IU/mL), HbPS (82% vs 87%, > 1.0 microgram/mL), and polio serotypes 1 (85% vs 80%, > or = 1:8) and 2 (100% vs 100%, > or = 1:8). Preterm infants were less likely than full-term infants to have protective levels of neutralizing antibody to polio serotype 3 (31% vs 90%, > or = 1:8). Extremely premature infants have adequate antibody responses to tetanus and HbOC antigens but may have diminished responsiveness to serotype 3 polio vaccine.

Comparison of Adjuvant Activities of Aluminium Phosphate, Calcium Phosphate and Stearyl Tyrosine for Tetanus Toxoid

Abstract. The adjuvant activity of three adjuvants, aluminium phosphate (AIPO 4), calcium phosphate (CaHPO 4) and stearyl tyrosine for tetanus toxoid (TT) were compared to soluble TT in mice at a dose of 0·5 Lf (1/10th of the single human dose) and in guinea-pigs at a dose of 7·5 Lf (1·5 times the single human dose). Three TT preparations varying in purity were used: (1) ammonium sulphate precipitated formalin detoxified tetanus toxin (AS-TT); (2) AS-TT ultrafiltered to remove low molecular weight peptides (UF-TT); and (3) chromatographically purified tetanus toxin subsequently detoxified with formalin (CP-TT). After primary immunization of mice, AIPO 4 adsorbed TTs induced higher toxin-neutralizing and IgG (by ELISA) antibodies than CaHPO 4, stearyl tyrosine adsorbed or soluble TT preparations, but this difference was no longer present after secondary immunization. TT preparations of varying purities showed similar antibody responses after primary and secondary immunizations when adsorbed on each adjuvant. CP-TT preparation showed the highest neutralizing antibody level amongst soluble preparations after the first dose. All the preparations induced mainly IgG1 antibodies. However, stearyl tyrosine adsorbed TT induced relatively higher IgG2a and IgG2b responses than AIPO 4, CaHPO 4 adsorbed or soluble TTs particularly after booster injection. No preparation induced detectable IgG3 or IgM antibodies. AIPO 4 adsorbed preparations induced higher IgE antibodies than CaHPO 4 and stearyl tyrosine adsorbed vaccines. Among the soluble preparations, CP-TT induced lower anti-TT IgE antibodies than standard AS-TT. All these preparations were also tested in the US potency test for adsorbed TT in guinea-pigs. While all the preparations passed this test, AIPO 4 adsorbed TT preparations induced higher neutralizing and IgG antibodies than CaHPO 4 and stearyl tyrosine adsorbed or soluble TT preparations. In these animal models, purified TT was a strong immunogen and traditional AIPO 4 adjuvant gave the highest antibody responses.

Anti-tetanus toxoid antibody production after mismatched T cell-depleted bone marrow transplantation.

We explored B-cell function after tetanus toxoid (TT) immunization in 12 children with severe combined immunodeficiency disease or leukemia who were long-term survivors of an HLA-matched sibling or haplocompatible T cell-depleted parental bone marrow transplant (BMT), 10 of their healthy donors, and 13 normal controls. Specific in vivo and in vitro anti-TT antibody (Ab) production were measured by ELISA. We studied donors' and recipients' peripheral blood mononuclear cells (PBMC) and mixed E- (non-T cells) and E+ cells (T cells) spontaneously and after stimulation by TT in the absence or presence of interleukin-2 (IL-2), IL-4, and IL-6. Five of the 12 patients and all donors and controls responded with in vivo anti-TT Ab. In vitro anti-TT Ab production correlated with the in vivo response. All seven of the nonresponders were either fully engrafted or mixed chimeras (donor T cells but autologous B cells and monocytes). We could not identify a T-cell defect in four of the five nonresponders who were tested. In contrast, E- cells from three of three responders cooperated with fresh donor E+ cells even when they shared only one HLA haplotype. In three of seven nonresponders, in vitro anti-TT Ab production was restored after the addition of IL-4 or IL-6 but not IL-2. Our results suggest that the humoral immunodeficiency that exists post mismatched T cell-depleted BMT is either a B-cell, a monocyte, or a B-cell/T-cell cooperation defect which, in some patients, may be correctible with the addition of a cytokine. Also, it is not necessary to engraft donor B cells to achieve normal antibody responses and the ability to respond does not appear to correlate with pretransplant chemotherapy.

Differential effects of superantigen-induced "anergy" on priming and effector stages of a T cell-dependent antibody response.

The in vitro T cell nonresponsiveness or anergy to restimulation with staphylococcal enterotoxin B (SEB) following the in vivo injection of the superantigen is well characterized. Here we use mice transgenic for a V beta 8.2+ T cell receptor (TcR) (reactive with SEB) to establish a large population of anergic T cells in vivo. As expected, peripheral T cells from the SEB injected transgenic mice failed to proliferate or produce interleukin (IL)-2 following restimulation with the superantigen in vitro. However, in this system superantigen reactivity could be restored by either addition of exogenous IL-2, or stimulation with immobilized anti-TcR antibody. To evaluate the effects of superantigen-induced anergy in vivo, SEB-injected or noninjected control transgenic mice were immunized and boosted with the T cell-dependent antigen tetanus toxin (TT). SEB injection of the V beta 8.2+ transgenic mice 5 days prior to the TT immunization inhibited the anti-TT antibody response as measured over a 100-day period, whereas injection of a superantigen which does not interact with the V beta 8.2% TcR (such as SEA) did not. Furthermore, SEB injection of control nontransgenic mice did not interfere with the induction of a high titer anti-TT antibody response. In contrast to the inhibition seen when SEB was given prior to TT immunization, injection of transgenics with SEB either after the priming TT immunization or after the recall booster injection did not significantly influence the titers of anti-TT antibodies produced. These results demonstrate that the establishment of peripheral T cell anergy to superantigens inhibits the specific antigenic priming of helper T cells in vivo, but does not prevent primed T cells from helping B cells to mount an effective antibody response.

Quantitative and qualitative analysis of anti-tetanus toxoid antibody response in the elderly. Humoral immune response enhancement by thymostimulin

In order to explore the humoral primary and secondary response to tetanus toxoid (TT), and to define the possible immunopotentiating effect of the thymic hormone thymostimulin, we studied 13 elderly people, selected according to the Senieur Eurage protocol, vaccinated against TT, an antigen never encountered before. Six of them were treated with thymostimulin before and during the immunization protocol. Specific anti-TT antibody level measurement and spectrotypic analysis were performed on the sera collected from the subjects at different times over the immunization protocol. In addition, spontaneous in vitro production of anti-TT antibodies as well as cutaneous delayed hypersensitivity reactions were also studied. Only one patient showed a detectable humoral immune response after the first immunization. After the booster, four of six thymostimulin-treated individuals, compared with only two of seven controls, showed in vivo anti-TT humoral response; at the same time, spontaneous anti-TT production was detected in peripheral blood mononuclear cells from five of six thymostimulin-treated individuals but only three of seven untreated controls. These differences were highly significant (p < 0.0001). In addition, only in thymostimulin-treated subjects were the levels of serum anti-TT antibodies 14, 21 and 28 days after the booster significantly (p < 0.05) higher than the baseline values. The spectrotypic analysis of anti-TT antibodies performed by isoelectric focusing and reverse blotting showed total agreement with the results from enzyme-linked immunosorbent assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Optimizing oral vaccines: induction of systemic and mucosal B-cell and antibody responses to tetanus toxoid by use of cholera toxin as an adjuvant

Cholera toxin (CT) is an effective mucosal antigen and acts as an adjuvant when given orally with various antigens; however, few studies have compared the levels of antibody responses to CT and coadministered protein in systemic and mucosal tissues. In this study, we used tetanus toxoid (TT) for assessment of immune responses. Time course and dose-response studies established that 250 micrograms of TT given orally with 10 micrograms of CT three times at weekly intervals induced high serum and gastrointestinal tract anti-TT and anti-CT antibody responses. Oral immunization with TT alone induced no detectable mucosal immunoglobulin A (IgA) antibodies in fecal extracts and only weak serum IgG anti-TT responses. The coadministration of CT and TT induced peak serum IgG anti-TT responses following two oral doses that remained constant after the third oral immunization, while optimal mucosal IgA responses were seen after the third oral immunization. The serum anti-TT response obtained with CT and TT proved protective against TT challenge (100 minimum lethal doses), whereas mice orally given CT or TT alone died. Antigen-specific B-cell responses were assessed with an isotype-specific Elispot assay of isolated lymphoid cells from the spleen, Peyer's patches, and the small intestinal lamina propria. Interestingly, approximately fourfold-higher numbers of IgA anti-CT than of anti-TT antibody-producing (spot-forming) cells occurred in lymphocytes from the lamina propria of mice orally immunized with both TT and CT. The adjuvant CT did not induce polyclonal B-cell responses in mice given CT by the oral route, since no significant differences in total numbers of B cells producing IgA, IgG, or IgM were found compared with the numbers in mice given TT alone. The results clearly indicate that serum and mucosal antibody responses develop with different kinetics and that protective TT-specific antibody responses are generated in the systemic compartment when TT is administered with CT via the oral route.

Immunotherapy with human monoclonal antibodies. Fragment A specificity of polyclonal and monoclonal antibodies is crucial for full protection against tetanus toxin.

To investigate the feasibility of substituting human mAb (HmAb) for human polyclonal preparations in the treatment of infections, we employed anti-tetanus toxin (TT) as a model system. We established a large panel of hybridomas secreting anti-TT HmAb and compared their fine specificities and protectivity with those exhibited by tetanus immune globulin (TIG). Analysis of three different commercial TIG preparations indicated that the majority of anti-TT antibodies is directed against epitopes expressed by the A fragment, the L chain of TT. Absorption of TIG with purified A fragment completely abolished its protective capacity in mice. Absorption with C fragment, the carboxy-terminal portion of the H chain of TT, had no discernible effect, illustrating the crucial importance of anti-A fragment antibody. The vast majority of more than 100 generated TT-specific HmAb showed specificity for the A fragment. Six HmAb with significant neutralizing activity were identified and further characterized. Five of them recognized the A fragment, whereas one, ST12, bound to both the A fragment and the C fragment with equal affinity. ST12 by itself conferred long lasting protection against TT intoxication when singly administered, and the remainder mediated only a delayed death. ST12 conferred a protection of 13.2 IU/100 micrograms IgG. However, when individual HmAb were combined, synergistic effects were observed. Optimal potency (43 IU/100 micrograms IgG) was obtained with a combination of two HmAb. To obtain a 250-IU dose, only 0.7 mg of this mixture was required in contrast to 100 to 170 mg IgG for TIG.

Complement activation by polyclonal immunoglobulin G1 and G2 antibodies against Staphylococcus aureus, Haemophilus influenzae type b, and tetanus toxoid.

To obtain information on effector functions of human immunoglobulin G2 (IgG2), we have measured the complement-activating properties of polyclonal IgG subclass antibodies against bacterial antigens. IgG1 and IgG2 were purified from serum samples from five healthy individuals, and complement activation was measured with different bacterial antigens. We used Staphylococcus aureus Wood 46 (STAW), which is a common antigen, Haemophilus influenzae type b (Hib), which is a common pathogenic microorganism in children, and formaldehyde-inactivated tetanus toxin (TT). Bacteria were incubated with antibodies and then incubated with sera from agammaglobulinemic patients as a complement source, and C3c deposition was measured by enzyme-linked immunosorbent assay. We found that anti-STAW IgG2 activated complement to a level similar to that of anti-STAW IgG1. Anti-Hib IgG1 complement activation was as much as seven times higher than that of anti-Hib IgG2 in four individuals. In one individual, anti-Hib IgG2 was more effective in complement activation than anti-Hib IgG1. Anti-TT antibodies showed patterns similar to those of anti-Hib. Our results indicate that IgG2 antibodies may contribute significantly to antibacterial defense. Also, individual differences in antibody effector functions should be taken into account when evaluating the immune status of patients and during early phase 1 studies of new vaccines.

Immunomodulatory effects of neem ( Azadirachta indica) oil

Immunomodulatory effects of neem oil were studied in mice. The animals were treated intraperitoneally (i.p.) with neem oil; control animals received the emulsifying agent with or without peanut oil. Peritoneal lavage, collected on subsequent days, showed a maximum number of leukocytic cells on day 3 following treatment with neem oil; peritoneal macrophages exhibited enhanced phagocytic activity and expression of MHC class-II antigens. Neem oil treatment also induced the production of gamma interferon. Spleen cells of neem oil-treated animals showed a significantly higher lymphocyte proliferative response to in vitro challenge with Con A or tetanus toxoid (TT) than that of the controls. Pre-treatment with neem oil, however, did not augment the anti-TT antibody response. The results of this study indicate that neem oil acts as a non-specific immunostimulant and that it selectively activates the cell-mediated immune (CMI) mechanisms to elicit an enhanced response to subsequent mitogenic or antigenic challenge.

A single human monoclonal antibody that confers total protection from tetanus.

Protective human monoclonal antibodies (HuMAbs) are superior to hyperimmune sera and murine monoclonal antibodies as far as human immunotherapy is concerned. In this report, we describe the successful generation of triomas secreting HuMAbs to tetanus toxin (tt). Lymphoblastoid cell lines secreting anti-tt antibodies were stabilized by back-fusion with a mouse x human heterohybrid myeloma partner, SBC-H20. One of the antibodies so produced, confers total protection of mice from tetanus, unlike a few recent reports where only partial protection (delay in the onset of tetanus) was achieved with single HuMAbs. Experiments to localize the neutralizing epitope(s) of the toxin using the protective monoclonal antibodies revealed that the antibody recognizes a conformational determinant that is destroyed on SDS-treatment. Preliminary studies show that Fab preparations of the protective antibody are capable of neutralizing tetanus toxin, suggesting that it might be possible to clone and express the Fab in a stable vector for large scale production.

Immunosuppressive effects of fish oil in normal human volunteers: Correlation with the in vitro effects of eicosapentanoic acid on human lymphocytes

We have studied the effects of dietary supplementation with fish oil on immunological parameters in a group of six normal volunteers, four of whom received a fish oil extract (total EPA dose of 2.4 g/day, which is on the lower range of clinically effective doses) for 6 weeks and two of which received a placebo (olive oil) for an identical period of time. Each volunteer was followed up for a period of 23 weeks after the dietary intervention was ended. All volunteers were boosted with tetanus toxoid (TT) at the onset of the trial. Several immune parameters were followed longitudinally, including NBT reduction and lysozyme release to test neutrophil function; lymphocyte subpopulations; mitogenic responses to phytohemagglutinin (PHA), concanavalin A (Con A) and anti-CD3; IL-2 release after PHA and pokeweed mitogen (PWM) stimulation; immunoglobulin and anti-TT antibody (ATT) synthesis by stimulated lymphocytes; and serum levels of immunoglobulins and of ATT. No consistent changes were observed in neutrophil function tests, mitogenic responses to PHA and Con A, and lymphocyte subsets. The mitogenic response to anti-CD3 and the release of IL-2 after stimulation with PHA and PWM appeared reduced as a consequence of fish oil ingestion, and levels of serum immunoglobulins decreased in three of the volunteers receiving fish oil supplementation. The systemic humoral response after the TT booster appeared not to be influenced by the ingestion of fish oil. However, in those subjects who were given fish oil supplementation, the specific in vitro response of their peripheral blood lymphocytes to TT appeared to be compromised at Week 3. This could reflect the need for progressive accumulation of EPA in lymphocyte membranes for the suppressive effect to be detectable, but it could also reflect a differential sensitivity to the effects of fish oil of circulating B lymphocytes vs. bone marrow B lymphocytes. All the parameters apparently affected by fish oil ingestion were also affected by the incubation of normal lymphocytes with EPA in vitro. In conclusion, low doses of fish oil may have a mild immunosuppressive effect affecting both T and B cell functions. These observations stress the need for more extensive trials designed to determine whether immunosuppressive effects can be consistently elicited and for studies aimed at determining the mechanisms by which omega-3 fatty acids affect the immune system.

The Immune Response to Anti-Idiotype Antibodies Bearing an Internal Image Epitope of Tetanus Toxin/Toxoid

Primary immune responses to tetanus toxoid (TT) and primary and secondary immune responses to a rabbit TT internal image bearing anti-idiotype antibody (Ab2 beta 1) inoculated in Freund's complete adjuvant (FCA), saline (SAL) or syntex adjuvant formulation vehicle (SAF) via the intraperitoneal or subcutaneous route, were examined in mice. High anti-TT antibody (Ab3) titres are reported although the titre and persistence of the antibody response varied according to the adjuvant used in the priming and challenge inocula of Ab2 beta 1. Mouse Ab3 antibodies were elicited in mice inoculated with rabbit Ab2 beta 1 antibodies which in turn were elicited by an inoculum of mouse monoclonal anti-TT Ab1 antibody. Ab3 was shown to be identical to Ab1 by immunoblot analysis. Primary and secondary immune responses elicited by rabbit Ab2 beta 1 antibody protected mice against a lethal dose of tetanus toxin.

Quantitation of anti‐tetanus and anti‐diphtheria antibodies by enzymoimmunoassay: Methodology and applications

We have developed enzymoimmunoassays (EIA) for the quantitation of antibodies (Ab) to tetanus and diphtheria toxoids (TT, DT) using Immulon I plates coated with the appropriate toxoid. A preparation of human tetanus immunoglobulin with a known concentration of anti-TT Ab was used as calibrator of the anti-TT antibody assay. The assay of anti-DT Ab is calibrated with a pool of human sera whose anti-DT Ab concentration was determined by quantitative immunoelectrophoresis, using a horse anti-DT with known Ab concentration as calibrator. A peroxidase-conjugated anti-human IgG was used in both assays. ABTS was used as substrate, and the reaction was stopped after 1 min incubation with citric acid and the OD measured at 414 nm on a Vmax reader. The assays have been applied to a variety of clinical situations. In patients suspected of having tetanus, the quantitation of antibodies has been helpful in establishing a diagnosis. In patients with a history of hypersensitivity to tetanus toxoid, verification of the levels of anti-TT antibody may prevent unnecessary and potentially harmful immunizations. The assays have also been used for the diagnostic evaluation of the humoral immune response to TT and DT, both in pediatric patients and in immunosuppressed patients. Several non-responders have been detected, and we have recently used the assay to monitor the effects of fish oil administration on the humoral immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

Decreased specific antibody synthesis in old adults: Decreased potency of antigen-specific B cells with aging

The rise in rates of infection in adults over the age of 60 is accompanied by a decreased ability of older adults to make specific immune responses after immunization with a variety of specific antigens (Ag). This investigation delineates age-related changes in Ag-specific humoral immunity, comparing adults over age 60 to young adults aged 18–40, using tetanus toxoid (TT) as an immunologic probe. A culture system which does not require TT booster immunizations of study subjects was used to induce in vitro specific antibody responses. The amount of anti-TT antibody (Ab) produced in serum and in culture was measured by a TT-specific enzyme-linked immunosorbent assay (ELISA). The numbers of anti-TT Ab-secreting B cells were measured by a TT-specific ELISA-plaque assay. The TT-specific responses of old subjects were significantly less than that seen for young control subjects in the following measures: (1) serum anti-TT Ab titers (mean ± S.E. log 2 titer = 3.3 ± 1.1 vs. 9.5 ± 1.4, P < 0.01); (2) anti-TT Ab produced by peripheral blood lymphocytes (PBL) in cultures stimulated with TT (6 ± 2.1 ng/ml vs. 22 ± 8.4 ng/ml, P < 0.01); (3) numbers of anti-TT Ab secreting B cells per million cells culured (6.7 ± 3.4 vs. 26.6 ± 7.6, P < 0.001) and (4) mean ng Ab secreted per anti-TT Ab-secreting B cell (0.6 ± 0.4 ng vs. 12.7 ± 7.8 ng, P < 0.01). This study shows that both decreased numbers of Ag-specific immune B cells and decreased potency on a per cell basis contribute to the impaired immune responses to immunizations in older adults.

The Stimulation of T Cells with Anti‐CD2 Monoclonal Antibodies Facilitates the Induction of Polyclonal B‐Cell Responses but does not Enhance the Activation of Antigen‐Specific B Cells

In this report we compare the effect of stimulation of peripheral mononuclear cells (PBMC) by using two monoclonal antibodies (MoAb) directed against the CD2 receptor on T cells or by using autologous erythrocytes (E) which express on their surface lymphocyte function-associated antigen 3 (LFA3), a natural ligand for CD2. The addition of autologous erythrocytes to pokeweed mitogen (PWM)-stimulated PBMC results in the enhancement of polyclonal immunoglobulin synthesis and of antigen-specific B-cell responses. Because B cells lack the CD2 molecule, it can be concluded that their enhanced activity is a consequence of the delivery of activating signals by activated T lymphocytes. When PBMC cultures were stimulated with a pair of anti-CD2 MoAb (Leu5b and VIT13) we were able to induce polyclonal immunoglobulin synthesis, particularly IgM, in cultures supplemented with interleukin 2(IL-2). Specific responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) were also enhanced by the addition of autologous E to PWM-stimulated PBMC. Significant anti-TT responses were observed in cultures stimulated with E + TT + IL-2. In contrast, stimulation of PBMC with VIT13 + Leu5b + IL-2 + antigen was not effective in inducing anti-TT antibody and only weakly effective in inducing anti-KLH antibodies. Replacing Leu5b by anti-CD3 had no effect on the induction of specific antibody responses; in contrast, replacement of Leu5b by E enhanced anti-TT antibody production while the effect on polyclonal production of IgM was minimal. Therefore, it appears that the signal delivered by the association of CD2 with LFA3 is a better potentiating signal for specific B-cell responses than the signal delivered by pairs of MoAb to different epitopes of CD2 or to CD2 and CD3 epitopes.

HC‐IgA antibodies of different specificities are normally present in serum: quantitation by ELISA and relationship to the major Ig classes

Enzyme-linked immunosorbent assays (ELISA) were developed for direct measurement of protein HC-IgA complexes (HC-IgA) in serum with antibody specificity for rabbit IgG (rheumatoid factor (RF) activity), lipopolysaccharide from Yersinia enterocolitica serotype O:3 (Y3) and tetanus toxoid (TT). About 80% of patients with rheumatoid arthritis had increased concentrations of HC-IgA-RF. The values were correlated with the concentrations of IgA-RF and IgM-RF. HC-IgA anti-Y3 was measured in 45 sera with anti-Y3 antibodies of IgM, IgG and IgA class. The HC-IgA anti-Y3 levels were correlated with those of anti-Y3 of IgG and IgM class, but not of IgA class. For HC-IgA anti-Y3, the closest correlation was that with the specific IgM antibody concentration, rs = 0.63 (p less than 0.001). In 25 normal sera, significant correlations were observed between HC-IgA anti-TT and specific antibodies of IgG and IgA class, but not of IgM class. In 107 sera containing IgA M-components, the total concentration of HC-IgA correlated poorly with both protein HC and with IgA concentrations. It was concluded that specific HC-IgA antibodies are normal constituents of serum, and that their concentrations are not directly related to the serum content of specific IgA antibodies.

Expression of Human Anti-Tetanus Toxoid Antibody in Transfected Murine Myeloma Cells

Mouse-human heterohybridoma cell line, GF4/1.1, secretes a human anti-tetanus toxoid (anti-TT) antibody of the γ3 isotype. This line was used as a source of mRNA for the construction of cDNA libraries and the subsequent isolation of the sequences encoding the human heavy and light chains. Expression vectors containing the cDNAs were constructed and introduced into a non-Ig producing, murine hybridoma cell line, together with a dihy-drofolate reductase (dhfr) selectable marker gene. Transfected cells producing human anti-TT antibody were grown in medium with increasing concentrations of the selecting drug (methotrexate). The level of antibody secretion was tested in cells that had adapted to the higher drug concentration and was found to increase in parallel with increases in the steady-state level of Ig mRNA. This increased productivity was not associated with detectable levels of gene amplification. Stable clones were obtained that secrete levels of human antibody in either open suspension cultures or within microcapsules that compare favorably with those of murine hybridomas.

Development of an antigen-specific CD8 suppressor effector clone in man.

A long-term cultured IL-2-dependent keyhole limpet hemocyanin (KLH)-specific CD8 (T8) suppressor clone (5B9) was generated from a healthy donor hyperimmunized with KLH. The 5B9 clonal population suppressed in vitro anti-KLH antibody response but did not suppress anti-TT antibody response or PWM-driven IgG synthesis. Moreover, 5B9 cells could not suppress anti-TT antibody response even in the presence of free KLH. 5B9 cloned cells suppressed the anti-KLH antibody response of B cells cultured with CD4+4B4+ cells without requiring the presence of CD8+ cells. This KLH-specific CD8 suppressor clone is an effector type rather than an inducer type of suppressor T cell. The cloned cells expressed alpha- and beta-TCR proteins (defined by WT-31 antibody) on their cell surface. More importantly, the CD3:TCR complex was functionally important in the suppression induced by this clone, because after CD3 antigen modulation from its cell surface, the suppressor effector function was abolished.