Immediate-type hypersensitivity to keyhole limpet haemocyanin in the dog.

A model of immediate-type hypersensitivity (ITH) in the dog using keyhole limpet haemocyanin (KLH) as the antigen was studied. Long-lasting ITH was induced in fifteen of sixteen dogs by the i.v. (Kepron & Tse, 1975), intradermal (Rockey, Schwartzmann & Halliwell, 1971), inhalation (Patterson & Kelley, 1974) and i.d. plus inhalation (Rockey et al., 1971) routes. This ITH was demonstrated by direct skin reactivity, acute respiratory reactions and peripheral blood leucocyte and respiratory cell histamine release. The reaginic antibodies considered to mediate these reactions were larger than canine IgG, heat labile, 2-mercaptoethanol sensitive, and are probably canine IgE. In addition to cutaneous and respiratory reactions, these reagins were shown to mediate systemic reactions. In the course of these studies, an unexpected finding was the occurrence of ITH to KLH in three dogs which had had no prior experimental exposure to KLH. This ITH was of sufficient intensity to be associated with acute airway reactions to the inhaled antigen.     

Helper activity of T lymphocytes which have been stimulated by keyhole limpet haemocyanin in vitro.

Details are given of a system for keyhole limpet haemocyanin (KLH)-induced DNA synthesis by murine T lymphocytes in vitro. Lymph node T cells from mice primed with KLH and Bordetella pertussis were stimulated with KLH under the defined conditions, and it was found that such cultured cells exhibited substantial non-specific helper activity. In contrast similarly primed T cells which had not been cultured showed only antigen-specific help. It is concluded that proper account should be taken of non-specific effects when studying the activity of antigen-specific helper cells in in vitro.     

Suppression of the primary immune response to keyhole limpet haemocyanin by antimacrophage globulin.

Thioglycollate-stimulated, adherence and density gradient-enriched peritoneal exudate cells were used for the preparation of a specific, highly active antimacrophage reagent. After absorption with mouse lymphoid cells, an antimacrophage globulin (AMG) fraction was shown to be cytotoxic for peritoneal exudate macrophages but not spleen, lymph node or thymus cells. The AMG suppressed both the in vivo primary serum antibody and spleen PFC responses to KLH.     

Abortive ectromelia virus infection in peritoneal macrophages activated by Corynebacterium parvum

We have previously demonstrated that peritoneal macrophages (M phi S) from C3H mice were resistant to in vitro infection by ectromelia virus, following activation by intraperitoneal injection of the immunomodulator Corynebacterium parvum. In contrast, resident and mineral oil-elicited M phi S were fully susceptible to virus infection. This report analyzes the infectious cycle of ectromelia virus in C parvum-activated and mineral oil-elicited M phi S and demonstrates that an abortive infection occurred in the activated M phi S that blocked the infectious cycle prior to the release of DNA from the infecting virions. The kinetics of adsorption of radiolabeled virus were similar in both susceptible and resistant M phi cultures; however, viral-induced incorporation of uridine and thymidine occurred only in the mineral oil-elicited and not the C parvum-activated M phi S. In addition, the late protein hemagglutinin was only detected in infected cultures of susceptible mineral oil-elicited M phi S. An electron micrographic analysis of the infectious cycle indicated that the adsorption of virus to the plasma membrane, uptake into lysosomes, and the primary undercoating and release of viral cores into the M phi cytoplasm were identical in both M phi types. In contrast, secondary uncoating (release of genomic DNA from the viral cores into the cytoplasm) was never detected in infected C parvum M phi S. These data are consistent with our previous findings and with the hypothesis that activation of M phi S by C parvum induces an interferon-mediated resistance to ectromelia virus infection.     

Effect of rhTACI-Ig fusion protein on antigen-specific T cell responses from keyhole limpet haemocyanin challenged mice

In addition to modulate B cells function, B cell activating factor belonged to TNF family (BAFF) also regulates T cells response via BAFFR and transmembrane activator and calcium modulator and cyclophilin-ligand interactor (TACI) expressing on activated T cells. This study explored the effect of a recombinant fusion protein containing the extracellular ligand-binding portion of TACI and the Fc portion of human immunoglobulin G (TACI-Ig) on activated T cells that were obtained from antigen-specific T-cell responses mice model induced by keyhole limpet haemocyanin (KLH), the characteristics of KLH-challenged mice were observed simultaneously. KLH immunization leaded to a significant positive relationship between BAFF level in serum and the extent of spleen histopathology. Serum concentration of BAFF, APRIL, IgM and IgG antibodies to KLH, and IL-4 were increased under KLH immunization, but IL-2 synthesis was decreased, resulting in a downregulation of IL-2/IL-4 ratio. Antigen-specific T cells proliferation, IL-5 production, the percentage of Th and activation T cells were significantly upregulated, however, IL-2 secretion and the percentage of na?ve T cells were downregulated in vitro. RhBAFF co-stimulation further evoked T cells hyperplasy, IL-4 and IFN-γ expression, the subgroups of Th, early antigen activation and activation T cells were also further increased. On the contrary, na?ve T cells were further reduced under rhBAFF stimuli. Administration of rhTACI-Ig significantly inhibited T cells proliferation, cytokines production and T cells differentiation, and the inhibitory effects might be associated with its ability to neutralize both the exogenous and endogenetic BAFF and APRIL.     

The role of macrophages in the adjuvant effect on antibody production of Corynebacterium parvum.

Spleen cells from mice pre-treated with C. parvum gave an enhanced in vitro antibody response to SRBC, but not to DNP-POL. This enhancing activity was associated with the adherent, but not the non-adherent spleen cell population and was found to be radioresistant. It is concluded that macrophages are directly involved in the adjuvant effect of C. parvum and the possible mechanisms of action are discussed.     

Dose-response effects in immunizations with keyhole limpet haemocyanin and rabies vaccine: shift in some immunodeficiency states.

We investigated the primary antibody response to the antigens keyhole limpet haemocyanin (KLH) and rabies vaccine (RV). Eighty-one healthy volunteers were injected with nine doses of KLH (ranging from 10 to 2500 micrograms) and 66 volunteers with six doses of RV (ranging from 17 to 680 micrograms protein). Anti-KLH and anti-RV antibodies were determined by ELISA and immunofluorescence (IF) immediately before and 14 days after primary immunization. On the basis of the dose-response curves, optimal and supra-optimal antigen doses were chosen for the assessment of humoral immunocompetence in two groups of patients with uraemic disease, who were treated either by chronic intermittent (hospital) haemodialysis (HD) (n = 16), or continuous ambulatory peritoneal dialysis (CAPD) (n = 23). The patients were randomly immunized with 250 micrograms or 2.5 mg KLH and 170 micrograms or 680 micrograms RV and their antibody responses were compared with those obtained in healthy individuals. We found a definite deficiency in the primary response in haemodialysis patients after challenging with a suitable antigen dose. However, the differences in response rate between patients and controls tended to disappear upon stimulation with a supra-optimal antigen dose. This might indicate that the dose-response curve of a particular antigen is shifted towards higher doses of antigen in immunodeficiency states, which could have important consequences for the testing of immunocompromised patients.     

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.     

Synergistic effects of locally administered cytostatic drugs and a surfactant on the development of delayed-type hypersensitivity to keyhole limpet haemocyanin in mice

The immunomodulating effects of two locally administered cytostatic drugs, the active cyclophosphamide-derivative Z 7557 and the plant alkaloid VP-16, were compared with the effects of systemically administered cyclophosphamide and several established adjuvants: Freund's complete adjuvant, dextran sulphate, and dimethyl dioctadecyl ammonium bromide (DDA). All agents tested promoted the development of delayed-type hypersensitivity (DTH) to keyhole limpet haemocyanin (KLH) in mice. Locally administered cytostatic drugs were the most effective immunostimulatory compounds, whereas DDA was the least toxic agent tested. In order to increase the effectiveness and/ or reduce the toxicity of these agents we tested the efficacy of combinations of cytostatic drugs and DDA to enhance DTH. The results show that DDA and suboptimal amounts of locally administered cytostatic drugs act synergistically on DTH.     

Differences in antigen handling by peritoneal macrophages from the Biozzi high and low responder lines of mice

Peritoneal macrophages were isolated from high (Ab/H) and low (Ab/L) responder mice and their in vitro handling of three isotopically labeled antigens, ¹²⁵I-labeled keyhole limpet hemocyanin (¹²⁵I-KLH), ¹⁴C-labeled type 3 pneumococcal polysaccharide complexed with methylated bovine serum albumin (¹⁴C-SIII-MBSA) and (¹⁴C-LE), studied in relation to the immune responses which these compounds evoke in the corresponding animals. There was a striking morphological difference between cultured Ab/H and Ab/L macrophages and the latter showed higher lysosomal enzyme activities. All three antigens were taken up faster by Ab/L macrophages. The intracellular digestion of ¹²⁵I-KLH was also more rapid in these cells. Membrane-bound ¹²⁵I-KLH was persistent in Ab/H macrophages, but disappeared rapidly from the Ab/L cells. After the uptake of ¹⁴C-SIII-MBSA complexes, more was retained by Ab/L macrophages, although probably localized in the interior of the cell, not on the membrane. Following the exposure of the cells to ¹⁴C-LE, there seemed to be an equivalent degree of continuing association of the polysaccharide with Ab/H as well as Ab/L cells. Thus, KLH and SIII, which evoke maximal and minimal antibody production in the two mouse strains, were handled differently by Ab/H and Ab/L macrophages. On the other hand LE, which elicits equivalent IgM synthesis in both mouse strains, had a similar fate in the two kinds of macrophages. The results suggest that macrophages express genes which control the humoral immune responses in the high and low responder animals and that antigen presentation on the macrophage membrane is a major factor regulating antibody production in these mice.     

The in vitro production of cytokines by mucosal lymphocytes immunized by oral administration of keyhole limpet hemocyanin using cholera toxin as an adjuvant.

The in vitro production of a variety of cytokines by lymphocytes isolated from spleen mesenteric lymph node (MLN), Peyer's patches (PP) and lamina propria (LP) was measured, after oral immunization with keyhole limpet hemocyanin using cholera toxin as an adjuvant. LP responses were characterized by very high levels of interleukin (IL) 4, IL 5 and IL 6 with lower levels of IL 2 and interferon-gamma (IFN-gamma). The PP had lower levels of IL 4, IL 5 and IL 6 than LP but higher levels of IL 2, IFN-gamma was only present at very low levels in this organ. The MLN had a pattern of cytokine production similar to the PP but did produce IFN-gamma. The spleen produced all cytokines measured except IL 5. Antibody production was characterized by IgA in the LP and PP but IgG was the dominant isotype in the spleen, the MLN was a poor source of antibody-producing cells. We interpret the results to show that (a) the LP response to cholera toxin/keyhole limpet hemocyanin is dominated by Th2-type cytokines compared to a lower production of Th1 type and (b) that the PP has responses typical of an organ with a high proportion of resting lymphocytes which develop mainly into Th2-types cells. The spleen is less dominated by Th2-type cytokines than the mucosal sites and this difference is paralleled by IgA antibody production at the mucosal sites and IgG antibody dominance in the spleen.     

The human primary immune response to keyhole limpet haemocyanin: interrelationships of delayed hypersensitivity, antibody response and in vitro blast transformation

The immune response to a protein antigen, keyhole limpet haemocyanin, was studied in fourteen normal subjects and twenty-one patients with solid tumours. Immunological responsiveness was assessed by intracutaneous skin testing, by haemagglutinin titres and by in vitro blast transformation. No significant difference was found in the kinetics or magnitude of the immune response among subjects immunized with 0·01, 0·10, or 5·0 mg. Delayed hypersensitivity to keyhole limpet haemocyanin developed in thirty-two of thirty-four skin tested; a positive antibody titre occurred in all; and thirty-one of thirty-five had positive in vitro responses. Patients in good general condition (Group 1) had significantly greater delayed hypersensitivity and antibody responses than the normals but similar in vitro responses. All immunological parameters were depressed in the patients with advanced neoplastic disease (Group 2).

Although only skin test positive subjects had positive in vitro responses, no direct correlation was found between the degree of delayed hypersensitivity and the degree of in vitro blast transformation. Excellent correlation was demonstrated, however, between the in vitro response and the haemagglutinin titre (correlation coefficient +0·52, standard error ±0·09).

     

Protection of mice against mouse hepatitis virus by Corynebacterium parvum.

C57BL/6 mice that are highly susceptible to infection with mouse hepatitis virus type 3 were protected against intraperitoneal viral infection by simultaneous intraperitoneal injection of Corynebacterium parvum. No protection was observed when C. parvum was given intravenously or when it was injected intraperitoneally 3 days before viral infection. Protective effects were, however, consistently found when C. parvum was given 2 h before or 2 h after viral infection. Activity was seen only against 10 50% lethal doses and not against 100 50% lethal doses. C. parvum also caused a significant decrease of virus type 3. These data suggest a direct effect of C. parvum on virus-susceptible cells. Injection of C. parvum in mice caused activation of natural killer (NK) cells and of interferon production. However, these two effects were equally demonstrable at high and low doses of C. parvum, whereas protection against mouse hepatitis virus type 3 was not demonstrable at low doses of C. parvum. Thus, antiviral protection may be dissociated from activation of NK cells and induction of interferon.     

Duck antibody responses to keyhole limpet haemocyanin,human immunoglobulin G and the trinitrophenyl hapten. Evidence of affinity maturation

White Pekin ducks (three in each of four groups) received trinitrofluorobenzene (TNP) conjugated to keyhole limpet haemocyanin (KLH) or human immunoglobulin G (HIgG) either incorporated into adjuvants (complete Freund's adjuvant (CFA), then incomplete Freund's adjuvant (IFA) followed by intravenous (i.v.) injection) or by repeated i.v. injection. A rabbit received TNP-KLH with CFA, IFA, then i.v. Antibody (Ab) responses to the carrier proteins and to the TNP hapten were monitored by enzyme-linked immunosorbent assay (ELISA), optimized for use with duck Abs, and the response to TNP was also assessed in an ELISA designed to detect shifts in Ab affinity (Ka). The rabbit mounted good Ab responses to KLH and TNP, the Ka of the anti-TNP response increasing from 10^5.99 to 10^8.87 l/mol (758-fold) during the experiment. Ducks showed weak Ab responses to KLH and HIgG, with weakest responses among ducks receiving antigen by the i.v. route exclusively. The duck anti-TNP responses were vigorous in all cases. However, following i.v. administration of antigens (Ags), the anti-TNP responses were transient, being strong by 7 days but diminishing to background levels by 14 to 28 days after each inoculation. The duck Ab responses to TNP displayed affinity maturation. In ducks receiving Ags with adjuvants, Ka increased by levels comparable with or even exceeding those in the rabbit. Affinity increases were less, but nevertheless apparent, following i.v. administration of Ag. These results show that, contrary to expectation, the duck Ab response experiences affinity maturation. They also point to the transience of the duck Ab response to selected haptens/epitopes when Ag is not delivered in adjuvant. These findings have clinical implications for field responses to pathogens and vaccines.     

Measurement of the clearance function of macrophages with 125I-labelled polyvinyl pyrrolidone

The rate constant of the exponential decay in blood radioactivity between 18 and 48 hr after intravenous injection of 30–80 μg of ¹²⁵I-labelled polyvinyl pyrrolidone (PVP) in mice has the characteristics of a test of the clearance function of macrophages. It is reduced by carbon, PVP or hydrocortisone, and increased by oestrogen. Clearance is independent of dose over this range, and probably measures resting unstimulated phagocytic function; this is perhaps different from the function measured by existing techniques. Preliminary evidence suggests that it is applicable to man.     

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.     

Protection against herpes simplex virus infection in mice by Corynebacterium parvum.

Corynebacterium parvum administered in mice prior to herpes simplex virus (HSV) infection significantly protected them against lethal encephalitis. This was seen both with a mouse strain highly susceptible to HSV and with one relatively resistant to HSV. Mice immunosuppressed by cyclophosphamide and showing an increased mortality after HSV infection were also protected by C. parvum pretreatment. However, C. parvum given simultaneously with or after HSV infection did not exert a therapeutic effect.     

Cells binding the antigen keyhole limpet haemocyanin in the peripheral blood and in the lymphocyte cultures of non-immune and immunized human subjects

Cells binding the antigen Keyhole limpet haemocyanin (KLH) were detected among human peripheral blood leucocytes and antigen-stimulated cultured lymphocytes. They were detected by their formation of rosettes with antigen-coated human O red blood cells. In the peripheral blood 0·13% of the lymphocytes of non-immune and 0·80% of the lymphocytes of immune subjects were antigen binding. In KLH-stimulated cultures of the lymphocytes of non-immune subjects there were 0·1% antigen-binding cells and in those of immune subjects there were 8·5% antigen-binding cells. Binding was specific. Thus, lymphocytes from phytohaemagglutinin- and streptolysin O-stimulated cultures did not bind KLH-coated red blood cells and did not form rosettes with KLH-stimulated lymphocytes. Incubation of KLH-stimulated lymphocytes with heterologous anti-immunoglobulin serum revealed that the KLH receptor was related to IgM. Addition of metabolic blocking agents (puromycin, cycloheximide, actinomycin-D and arabinosyl cytosine) to KLH-stimulated lymphocyte cultures revealed that the generation of antigen-binding cells required both DNA and protein synthesis. After primary immunization of normal human subjects, antigen-binding cells were detected in their appropriate lymphocyte cultures at 7 days and reached their maximum level at 21 days, in five of the six kinetically followed individuals and at 14 days in the other. This data represents the first report of the generation of antigen-binding cells in lymphocyte cultures demonstrated by the rosette method. These methods should be useful in the study of a variety of immunological problems in man.