An inter-laboratory retrospective analysis of immunotoxicological endpoints in non-human primates: T-cell-dependent antibody responses

The Immunotoxicology Technical Committee of HESI sponsored a retrospective analysis of T-cell-dependent antibody responses in non-human primates (NHP). Antibody responses to keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), and/or sheep red blood cells (SRBC) in 178 NHP (from 8 sponsors, 13 testing sites, 30 studies) were statistically analyzed. Rates of positive or negative anti-KLH, -TT, and -SRBC primary and secondary IgM and IgG responses were compared. The influence of gender, country of origin, and previous immunization with a different antigen on response rate and kinetics of anti-KLH and anti-TT responses were analyzed. In addition, the magnitude of the antibody responses and the impact of the above-mentioned factors were analyzed. In addition, based upon the inter-individual variability of the peak response values, power calculations were conducted. The analysis demonstrated that the rates of positive responses were similar between the two genders, were high for KLH, SRBC, and TT challenges by 21 days following immunization (87, 100, and 84%, respectively, for IgGs) and did not include statistically significant differences based on NHP country of origin. Mean peak secondary responses were greater than peak primary responses; the magnitude of the response to KLH was increased by incomplete Freund's adjuvant (IFA). Gender had little effect on the magnitude and variability of these responses. KLH and TT were associated with similar inter-animal variability, whereas in some situations KLH responses were less variable than responses to SRBC. The data suggested that inter-animal variability with KLH was similar with or without IFA. Power analysis illustrated that animal group sizes of typical standard toxicology studies (generally ≤ 4/sex) are likely to detect only fairly large treatment effects. However, combining males and females, when appropriate, will improve the power: an N of 8 to 12 could detect ≤ 3.1-fold differences in anti-KLH IgG responses.     

T-cell-dependent antibody response: assay development in cynomolgus monkeys

The current study was designed to develop and test a T-cell dependent antibody response to keyhole limpet hemocyanin (KLH) in cynomolgus monkeys. In an optimization experiment, monkeys (3/sex) were given a single intramuscular injection of KLH at 10 mg/animal to evaluate the kinetics of the antibody response. Serum samples were collected pretest, and on Days 4, 6, 8, 11, 15 and 22 for measurement of anti-KLH IgM and IgG endpoint titers. In a subsequent experiment, female monkeys (3/group) were treated once daily by gavage with the immunosuppressive agent cyclosporine (Neoral) at 0, 10 and 50 mg/kg for 21 days, and the effects of drug treatment on anti-KLH IgM and IgG responses were determined. The effects of cyclosporine on hematology, biochemistry, bone marrow, organ weights, gross and histopathology, and peripheral lymphocyte subsets also were evaluated. Robust anti-KLH IgM and IgG responses were seen in monkeys given a single intramuscular injection of KLH at 10 mg/animal, with peak antibody responses at approximately 10-14 days post-immunization for anti-KLH IgM, and 14-21 days for anti-KLH IgG. Decreases in anti-KLH IgG endpoint titers were seen in 1 monkey given cyclosporine at 10 mg/kg, and 1 monkey dosed at 50 mg/kg. Relative to vehicle control animals, mild lymphoid depletion was evident in lymph nodes and tonsil of monkeys with suppressed anti-KLH IgG titers. Collectively, these findings in individual animals provided evidence of cyclosporine-induced immunosuppression. Cyclosporine at 10 and 50 mg/kg did not alter anti-KLH IgM production, hematology, biochemistry, bone marrow, organ weights, or peripheral lymphocyte subsets. Lastly, the results of this study demonstrated that KLH immunization at 10 mg/animal did not alter the standard toxicity endpoints evaluated in control animals.     

Evaluation of primary and secondary responses to a T-cell-dependent antigen,keyhole limpet hemocyanin,in rats

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.     

Nasal challenge with diesel exhaust particles can induce sensitization to a neoallergen in the human mucosa

BACKGROUND: Diesel exhaust particles (DEPs) increase in vivo IgE and cytokine production at the human upper respiratory mucosa, exacerbating allergic inflammation. OBJECTIVE: We examined the ability of DEP exposure to lead to primary sensitization of humans by driving a de novo mucosal IgE response to a neoantigen, keyhole limpet hemocyanin (KLH). METHODS: Ten atopic subjects were given an initial nasal immunization with 1 mg of KLH followed by 2 biweekly nasal challenges with 100 microg of KLH. Identical nasal KLH immunization was then performed on 15 different atopic subjects, but DEPs were administered 24 hours before each KLH exposure. RESULTS: Exposure to KLH alone led to the generation of an anti-KLH IgG and IgA humoral response, which was detected in nasal fluid samples. No anti-KLH IgE appeared in any subjects. In contrast, when challenged with KLH preceded by DEPs, 9 of the 15 subjects produced anti-KLH-specific IgE. KLH-specific IgG and IgA at levels similar to that seen with KLH alone could also be detected. Subjects who received DEPs and KLH had significantly increased IL-4, but not IFN-gamma, levels in nasal lavage fluid, whereas these levels were unchanged in subjects receiving KLH alone. CONCLUSION: These studies demonstrate that DEPs can act as mucosal adjuvants to a de novo IgE response and may increase allergic sensitization.     

Distinctive development of IgG4 subclass antibodies in the primary and secondary responses to keyhole limpet haemocyanin in man

The human primary and secondary IgG subclass antibody responses to keyhole limpet haemocyanin (KLH) have been measured by ELISA using IgG subclass-specific monoclonal antibodies. KLH-specific IgG1 and IgG2 antibodies were detected 3 weeks after primary immunization, and IgG1, IgG2 and IgG4 antibodies after secondary immunization. IgG3 antibodies were observed less frequently in both primary and secondary responses. Unlike the other subclasses, IgG4 antibodies developed very slowly during the primary response, with no antibody detected at 3 weeks and often with only low titres 1 year after immunization. In one individual, this IgG4 primary response peaked around 10 months, but there was considerable variation between individuals. Comparing primary and secondary responses, the greatest increase in KLH antibody was for the IgG4 subclass (45-fold rise), followed by IgG1 (7.3-fold rise), whilst IgG2 and IgG3 KLH-specific antibodies did not show a significantly increased secondary response. There was no detectable IgG4 antibody response when secondary immunization was performed 1 month after the primary, even though IgG1, IgG2 and IgG3 antibodies were present. Reasons for the different time-course of IgG4 anti-KLH development and the isotype-related differences in 'memory' responses are discussed.     

Abnormal immunoglobulin G subclass production in response to keyhole limpet haemocyanin in atopic patients.

A proportion of patients with atopic dermatitis have elevated serum levels of IgG4. In order to investigate further this abnormality of IgG subclass production, atopic patients were immunized with the protein antigen keyhole limpet haemocyanin (KLH), and IgG subclass responses following primary and secondary immunization were analysed. In the primary response, titres of IgG1, 2 and 3 antibodies were lower in the atopic patients than in the controls. In contrast, titres of IgG4 were much higher for the patient group. In both patients and controls, the kinetics of IgG4 antibody production following the initial immunization with KLH showed a slow rise reaching a peak at 30 weeks. This time course indicated that the high IgG4 response was unlikely to be due to previous exposure of the patients to a cross-reacting antigen. A higher proportion of IgG4 was also seen in the atopic patients following secondary immunization; indeed, IgG4 was the major subclass in the secondary response in the patient group. In the controls, but not in the patients, titres of IgG4 anti-KLH correlated with total serum levels of IgG4, and some of the highest IgG4 antibody responses were detected in atopic patients whose serum IgG4 concentration was in the normal range. The results suggest that raised serum levels of IgG4 in atopy may reflect abnormal isotype regulation in response to protein antigens.     

Murine lung immunity to a soluble antigen

Although it is known that soluble antigen is immunogenic when deposited in the respiratory tract, less is known about lung immunity to soluble antigen than is known about lung immunity to particulate antigen. To test the hypothesis that soluble antigen triggers antigen-specific immunity in the respiratory tract in a fashion similar to that reported for particulate antigen, we examined the development of local and systemic immunity in C57BL/6 mice after intratracheal (i.t.) instillation of a soluble, large molecular weight protein neoantigen, keyhole limpet hemocyanin (KLH). Specific anti-KLH IgG and IgM first appeared in the sera of mice on day 7 after primary immunization by i.t. instillation of KLH, with specific serum antibody concentrations remaining elevated at day 11. Cell populations prepared from lung-associated lymph nodes of immunized mice released specific anti-KLH IgG and IgM in vitro; peak levels were obtained from cells isolated 7 days after antigen instillation, with levels of specific antibody released by cells isolated on days 9 and 11 decreasing markedly. Cultured spleen cells obtained from mice after primary immunization released only low levels of specific IgM, and no specific IgG. No specific antibody was released by cell populations derived from the lungs of animals undergoing primary immunization. When presensitized mice were given an i.t. challenge with KLH, responses differed markedly from those following primary immunization. Lung-associated lymph node cell populations from challenged mice released greater amounts of specific antibody earlier than did cell populations from mice undergoing primary immunization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunoglobulin response to intrapulmonary immunization of asthmatics.

Atopic asthmatics, compared to non-atopic individuals, exhibit an increased amount of serum antigen-specific IgE and IgG4 antibody directed toward many aeroallergens. We tested the hypothesis that this difference between atopics and non-atopics extends to the response to intrapulmonary deposition of a neoantigen, keyhole limpets haemocyanin (KLH). We immunized nine atopic asthmatics and nine non-atopic controls with 500 micrograms KLH instilled into a subsegment of the lingula and examined serum anti-KLH, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, and IgM and specific antibody production by peripheral blood mononuclear cells for 25 days. We also determined specific antibody in bronchoalveolar lavage fluid (BALF) in both the immunized and a non-immunized lobe 11 days after immunization. We found specific serum antibody in all immunized subjects with no difference between atopics and normals in the amount or kinetics of anti-KLH IgG1, IgG2, IgG3, IgA1, IgA2 and IgM. However, the atopics exhibited more anti-KLH IgG4 than the normal controls. Specific anti-KLH antibody-producing cells were detected in peripheral blood in most subjects at day 8 to 12 after immunization with no difference between atopics and normals. Specific IgA1, IgA2, IgG1 and IgM antibodies were detected in BALF from the immunized lobes but not from the non-immunized lobes of both groups of subjects with no difference between atopics and normals. We conclude that atopic asthmatics respond to intrapulmonary KLH with more serum anti-KLH IgG4 than normal controls, consistent with a bias toward a Th2 response to intrapulmonary exposure to antigen.     

Characterization of anti-Fab'' antibodies in human sera: identification of soluble immune complexes that contain hidden anti-KLH and blocking anti-immunoglobulins following immunization with keyhole limpet haemocyanin

After immunization with keyhole limpet haemocyanin (KLH), increased concentrations of anti-KLH and anti-Fab' antibodies (Abs) were demonstrated in sera from 18 of 20 volunteers. In many cases, post-immunization sera contained soluble immune complexes that incorporated both anti-Fab' and 'hidden' or 'blocked' anti-KLH antibodies. The complexes containing hidden anti-KLH and blocking anti-Fab' Abs were not found in pre-immunization sera. The hidden Abs to KLH were revealed by demonstrating increases in anti-KLH activity in sera incubated previously with Fab' fragments, immobilized on plastic microtitre tray wells. Incubation with insoluble Fab' did not influence the quantity of anti-tetanus toxoid (TT) that was detected in these sera. Addition of affinity purified anti-Fab' Abs to samples, previously 'unblocked' by adsorption with immobilized Fab', depressed their anti-KLH activity to levels present before adsorption, but did not change the quantity of Abs to Dermatophytin, Trychophyton, or TT therein. These results suggest that some autoantibodies generically recognized as 'Fab' specific' have properties that are usually considered to be characteristic of autoanti-idiotypes.     

Involvement of antigen-presenting cells in the enhancement of the in vitro antibody responses by cholera toxin B subunit.

The enhancing effect of cholera toxin B subunit (CTB) on primary antibody responses to keyhole limpet haemocyanin (KLH) and the cellular basis of the effect were investigated, using in vitro cultures of mouse spleen cells. CTB (1-100 ng/ml) enhanced anti-KLH IgM, IgG and IgA antibody responses in a dose-dependent manner, when added to the cultures with KLH. This immunoenhancement was antigen specific, but not due to either polyclonal activation of the spleen cells or antigenic cross-reactivity between CTB and KLH. CTB did not affect the kinetics of the anti-KLH antibody responses. Early (Days 0-1) addition of CTB to the cultures enhanced the anti-KLH antibody production, whereas late (Days 5-7) addition of CTB did not. Addition of CTB with KLH to splenic adherent cells (SAC) resulted in a dose-dependent enhancement of the anti-KLH antibody responses, when the SAC were reconstituted with unimmunized non-adherent cells. Moreover, CTB enhanced IL-1 secretion from SAC incubated with KLH. These results suggest that CTB enhances the primary anti-KLH antibody responses in vitro by acting on early events in the responses, and that antigen-presenting cells play a major role in the enhancement.     

No impairment of local intestinal immune response to keyhole limpet haemocyanin in the absence of Peyer's patches.

The role of Peyer's patches in the local intestinal and serum antibody responses to keyhole limpet haemocyanin (KLH) was studied in rabbits with chronically isolated ileal loops. Four weekly doses of 400 microgram KLH were administered into loops prepared with and without Peyer's patches. Isotype-specific IgA and IgG anti-KLH in loop secretions collected twice each week and in sera collected weekly were assessed by enzyme-linked immunosorbent assay. Fluid IgA anti-KLH in loops without Peyer's patches first showed a statistically significant increase on day 25, 1 week later than control loops with Peyer's patches. However, some animals in the group without Peyer's patches showed a rise as early as day 7, and the differences from controls were not statistically significant at any time. No statistically significant rise in fluid or serum IgG anti-KLH occurred in either group. Thus, Peyer's patches were not essential for local intestinal antibody response to KLH, a soluble macromolecular antigen. The findings suggest that the innumerable small lymphoid nodules in the gastrointestinal tract, or other mechanisms of antigen processing, play an important role in local intestinal immune responses.     

Cholera toxin as a mucosal adjuvant: effects of H-2 major histocompatibility complex and lps genes.

In previous studies we found that cholera toxin (CT) can act as a mucosal adjuvant; i.e., it can stimulate an intestinal secretory immunoglobulin A (S-IgA) response to an unrelated protein antigen when both are fed together to mice. The purpose of this study was to determine whether the mucosal adjuvanticity of CT is restricted by either H-2 major histocompatibility complex or lps genes by using congenic inbred strains that differ at only a single genetic locus. Groups of five mice each were fed saline, CT (10 micrograms), keyhole limpet hemocyanin (KLH) (5 mg), or both CT and KLH on four different days, and samples of intestinal secretions and plasma were obtained 1 week after the last feeding. In the mice fed both CT and KLH, the intestinal S-IgA anti-KLH response was higher in H-2b congenic strains than in H-2k congenic strains, and in addition there was a highly significant positive correlation between the intestinal S-IgA anti-KLH and S-IgA anti-CT responses in the intestinal secretions of individual mice. Similarly, in the lps congenic strains, mice of the endotoxin-responsive strain that were fed both CT and KLH had substantially higher S-IgA and plasma IgG responses to KLH than did mice of the endotoxin-unresponsive strain. The effect of CT on the induction of oral tolerance to KLH in the H-2 congenic strains was also examined. In contrast to the results above, the abrogation of oral tolerance to KLH by CT occurred in all strains regardless of H-2 haplotype. Similarly, the adjuvant effect of CT on plasma IgG anti-KLH responses after both were given together intraperitoneally was not restricted by H-2. I conclude that the mucosal adjuvanticity of CT is influenced by both the H-2 and lps genetic loci and that it appears to depend on a vigorous mucosal immune response to CT itself.     

Autologous Red Blood Cells Potentiate Antibody Synthesis by Unfractionated Human Mononuclear Cell Cultures

We have tried to determine the most favourable conditions for the in vitro induction of specific antibody (Ab) responses to tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH). Human peripheral blood mononuclear eells (PBMNC) were obtained from normal volunteers and stimulated with PWM, TT, KLH. and mixtures of PWM and antigens in the presence or absence of autologous red blood cells (RBC) (I:50 ratio of PBMNC/RBC). The cultures were harvested on day 11: immunogiobulins were determined immunonephelometrically and Ab levels by ELISA with human antibodies used for calibration. While anti-TT responses were easy to induce with PBMNC from recently boosted individuals, the production of anti-IT from PBMNC obtained from non-recently boosted individuals was only possible when PBMNC were stimulated with TT and PWM in ihe presence of autologous RBC Similarly, anti-KLH responses were easier lo induce with PBMNC frt)m an immune donor; maximal response was observed after slimuiation wiih PWM + KLH in the presence of autologous RBC. Stimulation of primary anti-KLH responses with PBMNC from non-immune donors was only successful when the cells were stimulated with KLH + PWM in the presence of autologous RBC. The potentiation of human B-cell responses with autologous RBC can be abrogated by pretreatment of PBMNC with anti-CD2 antibodies and is associated with increased expression of IL-2 receptors and increased production of gamma interferon (IFN-γ). However, addition of IFN-γ in different doses and at different times to PWM-slimulaled PBMNC cultures was not as effective as addition of RBC in enhancing the production of immunoglobulin and antibody.     

Restraint stress augments antibody production in cyclophosphamide-treated mice

These studies evaluated the effects of a psychological stressor (restraint, RST) on antibody production in male BALB/cByJ mice. In Experiment 1, mice were immunized with keyhole limpet hemocyanin (KLH, 100 microg i.p.) 8 h prior to 15 h of RST or food and water deprivation (FWD). RST mice exhibited higher serum anti-KLH IgM and IgG antibodies than FWD mice. In Experiment 2, mice were given either cyclophosphamide (CY, 15 mg/kg) or saline (SAL) prior to immunization with KLH and RST or FWD. ANOVA revealed serum anti-KLH IgG antibody titers in CY+RST animals to be significantly higher than in CY+FWD, SAL+FWD, and SAL+RST mice. Anti-KLH IgM titers of CY+RST mice were higher than those of other groups before and after a second immunization with KLH. In Experiment 3, we show that these changes in antibody production are not likely to be mediated via CY-induced alterations in the reactivity of the hypothalamo-pituitary-adrenal axis to RST. Together, these results indicate two potentially immunomodulatory parameters (RST and CY) can interact to alter a humoral immune response. In addition, these data support the hypothesis that humoral immune response of mice can be more reactive to stress when the mice are given a low dose of an immunomodulatory drug prior to stressor exposure.     

Human immune responses in vivo to protein (KLH) and polysaccharide (DNP-Ficoll) neoantigens: normal subjects compared with bone marrow transplant patients on cyclosporine

Thymus-independent (TI) and thymus-dependent (TD) primary immune responses were measured in 67 controls and 13 bone marrow transplant (BMT) recipients treated with cyclosporine (CSP) by immunizing with a synthetic antigen (DNP-Ficoll) and keyhole limpet haemocyanin (KLH). DNP-Ficoll induced similar TI antibody responses in controls and BMT recipients except that antibody levels declined much more rapidly in BMT recipients. The IgM and IgG antibodies induced by DNP-Ficoll only recognized the DNP epitope and not the Ficoll carrier. Both IgM and IgG classes of antibody showed similar TI behaviour upon immunization and re-immunization. The antibodies to DNP-Ficoll were overwhelmingly of the IgG1 subclass. The TD response to KLH evoked both delayed hypersensitivity (DH) and antibody production. DH developed at the site of immunization in 68% of controls and in 88% upon subsequent challenge with KLH. None of the BMT recipients on CSP developed DH. KLH antibody arose in 88% of controls but in only one BMT recipient on CSP. Eight BMT recipients were re-immunized with KLH 2-6 weeks after stopping CSP and only one made primary DH and antibody responses, arguing that CSP inhibited priming as well as any detectable response to KLH. The immunization procedure described has proved a sensitive and comprehensive method of quantitating human immune responses in vivo and is readily adaptable for in vitro studies.     

Fc-mediated immune precipitation. III. Visualization by electron microscopy.

Fc-mediated interactions between immune complexes are of major importance for the precipitin reaction. In the present study these interactions were investigated by means of electron microscopy. Keyhole limpet haemocyanin (KLH) was adsorbed to a thin glow charged carbon supporting film and reacted with either rabbit anti-KLH IgG or anti-KLH F(ab')2 fragments. The Fc-Fc interactions were investigated by reacting these surface-adsorbed antibody-rich KLH immune complexes with soluble, antigen-rich ferritin-anti-ferritin complexes using either rabbit anti-ferritin IgG or the corresponding isomolar F(ab')2 fragments as antibody. Fc-Fc interactions were indicated by the formation of clusters or ring structures of ferritin molecules, which were only seen when using KLH anti-KLH IgG and ferritin-anti-ferritin IgG complexes. When F(ab')2 fragments were used as antibody, no reaction between KLH anti-KLH complexes and ferritin-anti-ferritin complexes could be demonstrated.     

Lack of specific antibody response in common variable immunodeficiency (CVID) associated with failure in production of antigen-specific memory T cells

Several T cell defects have been described in the antibody deficiency disease, CVID, but there have been few data on the generation of responses of specific T cell populations to primary neoantigens. We have now used immunization with the neoantigens, keyhole limpet haemocyanin (KLH) and DNP-Ficoll, to evaluate immune responses in CVID patients and normal donors. B and T cell responses were examined 2 and 4 weeks post-immunization. Sera were examined for IgM and IgG anti-KLH responses by ELISA and for anti-DNP-Ficoll activity by haemagglutination. The frequency of KLH-responsive T cells was measured by DNA synthesis in a limiting dilution culture system. Low density cells enriched for dendritic cells were pulsed with KLH and cultured with different numbers of autologous T cells. T cells from normal donors and from patients showed a low frequency of antigen-specific precursor T cells (≤1:200 000). After KLH immunization the frequency increased in normal donors (1:60 000 and 1:30 000 at 2 and 4 weeks, respectively), while in CVID patients it did not change from the pre-immunization level. The defect may extend to a dysfunction of antigen-specific cells, rather than being solely due to the reduced numbers of cells, since mean responses of ‘positive’ wells were also reduced. The serum-specific antibody response paralleled the T cell data, in that all normal donors but none of the CVID patients generated IgG KLH-specific antibodies. CVID patients did produce IgM antibodies against the T-independent DNP-Ficoll, but at a lower level than normal controls. These data show that both T and B cells from CVID patients have defective responses to specific antigen, implicating both lineages in the antibody deficiency.     

The induction and migration of antigen-specific helper cells for IgA responses in the intestine.

The distribution of keyhole limpet haemocyanin (KLH)-specific helper cells for antibody responses of IgA, IgM and IgG isotypes in Peyer's patch (PP), mesenteric lymph node (MLN) and peripheral lymph node (PLN) was examined following oral, intraduodenal (ID), intraperitoneal (IP), intra-Peyer's patch (IPP) or subcutaneous (SC) immunization with KLH. Oral or ID immunization gave little or no response in any tissue studied. IP immunization with or without a subsequent ID challenge gave rise to a modest IgA and IgM helper response in MLN but a small IgA and IgM helper response in PP and PLN. IP immunization alone did not stimulate IgG-specific help in any tissues studied, but a small IgG helper response occurred in MLN and PLN after subsequent ID challenge. IPP was the most effective route of immunization, giving rise to a large helper response for IgA, IgM and IgG isotypes in PP, a smaller response in MLN and no response in PLN. The helper response following IPP immunization was not augmented by subsequent ID challenge. SC immunization gave a small but significant helper response for all isotypes in PLN but no response in PP or MLN. The kinetics of the helper response were examined in PP, MLN, PLN and thoracic duct lymph (TDL) following IPP immunization. The helper response for all isotypes in PP was maximal at 2 weeks and then declined. Similar kinetics but of lower magnitude were observed in MLN and TDL. The presence of IgA-specific helper cells in TDL demonstrates that these cells migrate, presumably from GALT, and may constitute an important component of mucosal responses at extraintestinal sites.     

The CD4+ T-cell response to protein immunization is independent of accompanying IFN-gamma-producing CD8+ T cells.

By virtue of their strong bias towards production of interferon-gamma (IFN-gamma), CD8+ T cells have the potential to promote the development of type 1 immune responses. We have previously shown that the CD4+ T-cell response to immunization with the protein antigen keyhole limpet haemocyanin (KLH) has a mixed interleukin-4 (IL-4)/IFN-gamma production profile. Here we show that this immunization regimen also stimulates accumulation in the draining lymph nodes of CD8+ T cells, which preferentially contain IFN-gamma mRNA ex vivo and secrete IFN-gamma protein in vitro. This provides a model to test whether CD8+ cell-derived IFN-gamma participates in the normal control of the immune response to a non-viable exogenous antigen. To investigate regulation of the anti-KLH response by the CD8+ population or IFN-gamma produced by this or other cell types, mice were administered depleting antibodies. Depletion of CD8+ cells had no effect on the frequency of clonogenic KLH-specific CD4+ T cells, the IL-4/IFN-gamma profiles of their progeny, or the isotype profiles of the serum antibody response to KLH. In contrast, IFN-gamma neutralization diminished cell accumulation in the lymph nodes and reduced both the frequency of KLH-specific CD4+ T cells that gave rise to IFN-gamma-producing clones and serum titres of KLH-specific IgG2a and IgG3. Therefore, despite the potential for cross-regulation, the CD4+ T-cell response to this immunogen is independent of the IFN-gamma-skewed CD8+ response.     

Genetically Detoxified Mutants of Heat-Labile Toxin from Escherichia coli Are Able To Act as Oral Adjuvants

Detoxified mutants of the Escherichia coli heat-labile toxin (LT) act as mucosal adjuvants to intranasally presented coadministered antigens. Here, we compare the adjuvant activity of a panel of detoxified derivatives of LT, using both intranasal (i.n.) and oral (p.o.) routes of administration. The mutants used as adjuvants varied in sensitivity to proteases and toxicity. With keyhole limpet hemocyanin (KLH) as the bystander antigen, the immune responses to i. n. immunizations were consistently higher than the equivalent p.o. -delivered proteins. LT-G192, a mutant which demonstrates a 10-fold reduction in toxicity in vitro, demonstrated wild-type adjuvant activity both i.n. and p.o., inducing similar titers of KLH specific antibody in the sera and immunoglobulin A in local mucosal secretions as wild-type LT. In line with previous data, the nontoxic holotoxoid LT-K63 induced intermediate immune responses in both the serum and mucosal secretions which were lower than those achieved with wild-type LT but at least 10-fold higher than those measured when the antigen was administered with LT-B. Although significant levels of local and systemic anti-KLH antibodies were induced following p.o. immunization with LT-K63, cellular proliferative responses to KLH was poor or undetectable. In contrast, LT and LT-G192 induced significant T-cell responses to KLH following p.o. immunization. These proliferating cells secreted both gamma interferon and interleukin-5, suggesting that the type of immune response induced following p.o. coimmunization with LT and purified protein is a mixed Th1/Th2 response.