Enhanced in vivo targeting of an asymmetric bivalent hapten to double-antigen-positive mouse B cells with monoclonal antibody conjugate cocktails

In order to target specifically double-Ag-positive cells in vivo, we synthesized chemically two mAb conjugates with specificities for both an allelic murine B cell-surface Ag and for a synthetic hapten. One conjugate was designed for its specificities for I-Ek and for N-epsilon-(2,4-DNP)-amino-caproate, and the other one for its reactivity to Lyb-8.2 and to indium-diethylenetriamine pentaacetate. A radiolabeled tracer, containing both the N-epsilon-(2,4-DNP)-amino-caproate and the indium-diethylenetriamine pentaacetate haptens, was obtained by reacting diethylenetriamine pentaacetic acid dianhydride with mono-[N-epsilon-(2,4-DNP)-amino-caproyl]-tyrosyl-lysine and labeling with indium-111. Mice from various strains (CBA/N: I-Ek+, Lyb-8.2+; AKR/N: I-Ek+, Lyb-8.2-; BALB/c: I-Ek-, Lyb-8.2+; and DBA/2: I-Ek-, Lyb-8.2-) were given simultaneous i.v. injections of microgram amounts of less than anti-[N-epsilon-(2,4-DNP)-amino-caproate], anti-I-Ek greater than and of [anti-(indium-diethylene-triaminepentaacetate), anti-Lyb-8.2] antibody conjugates and picomole amounts of the tracer. As expected, specific uptake of the tracer by the spleen was observed in strains where spleen cells expressed at least one Ag (CBA/N, AKR/N, and BALB/c). Furthermore, spleen cells from the double-Ag-positive mouse strain (CBA/N), when compared with spleen cells from single-positive mouse strains, exhibited a significantly higher uptake of the bivalent hapten. This specificity for double-Ag-positive cells, it is suggested, occurs through the formation of stable complexes between both cell-surface Ag, both conjugates, and the asymmetric bivalent hapten. The use of such asymmetric bivalent haptens, together with matched (anti-hapten, anti-cell) antibody conjugates, is proposed as a general method for increasing the in vivo specificity of immunoimaging and radioimmunotherapy.     

Blocking of delayed hypersensitivity by humoral antibody in an in vivo mouse transfer assay using sensitized guinea pig cells

Humoral antibody was shown to interfere specifically with the expression of cell-mediated immunity (delayed hypersensitivity) in an in vivo system. Mice that received peritoneal exudate cells obtained from guinea pigs sensitized to 1-chloro-2,4dinitrobenzene (DNCB) exhibited delayed hypersensitivity reactions after challenge with the sensitizing agent. While control groups that received either normal sera, saline, or anti-BSA (bovine serum albumin) in addition to peritoneal exudate cells from sensitized guinea pigs exhibited positive delayed reactons to challenge with DNCB, mice that received anti-DNP (dinitrophenyl group) in addition to the senstized cells were prevented from exhibiting a delayed reaction to DNCB.     

Involvement of host Fc receptors in antibody-mediated cutaneous basophil hypersensitivity reactions.

Cutaneous basophil hypersensitivity (CBH) reactions are heterogeneous delayed time course basophil-rich responses that can be mediated by either T cells, B cells, or serum antibodies. The current study examined the mechanism by which antibodies mediate CBH in guinea pigs. Fc competition experiments were constructed by passively transferring mixtures of anti-KLH serum and normal heterologous gamma-globulins. It was found that rabbit IgG and its isolated and purified Fc fragment [but not the (Fab')2 fragment] inhibited the ability of guinea pig immune serum to transfer CBH. Concurrent inhibition of transferred KLH-specific CBH and systemic passive cutaneous anaphylaxis (PCA) reactions by rabbit IgG or its Fc fragment, and not by sheep or bovine gamma-globulins, indicated that Fc receptors on cutaneous mast cells were probably involved in both CBH and PCA. It was also found that the basophil aspect of delayed cutaneous responses elicited by PHA was inhibited by Fc competition maneuvers. This could mean that some forms of apparently T cell-mediated CBH may be T cell dependent, but via secretion of molecules that bind to Fc receptors, as seems required in antibody-mediated CBH.     

In vitro correlates of hapten-specific delayed hypersensitivity

Azobenzenearsonate conjugates of l and d-amino acid polymers were studied in respect to their ability to stimulate lymphocytes from guinea pigs sensitized to the hapten. Using the two in vitro techniques of thymidine incorporation and inhibition of macrophage migration, it was shown that only conjugates of the l-amino acid polymers were active.The results confirm previous in vivo studies and suggest as one possibility that a preliminary processing event by macrophages may be obligatory for all associated phenomena of hapten-specific delayed hypersensitivity.     

Potentiation of antibody responses by specific IgM: specificity and thymus dependency

Modulation of antibody responses induced by IgM directed against the immunogen was investigated. When IgM directed against ox erythrocytes (ORBC) was given together with trinitrophenyl (TNP)-ORBC, the subsequent antibody response to the carrier, ORBC, as well as the response to the hapten, TNP, was potentiated. In contrast, IgG with carrier specificity inhibited both responses. The hapten-specific potentiation was found in both direct and indirect plaques, and was antigen-dose dependent, i.e., no potentiation was found with the lowest antigen doses. The response to 2,4-dinitrophenyl (DNP)-labeled proteins was potentiated by a monoclonal IgM with specificity for the hapten. The effects were observed both in primary and secondary responses. One strict requirement for IgM potentiation to occur was observed. The determinant against which potentiation was achieved had to be physically linked to the determinant against which the IgM was directed, be it hapten or carrier determinants. Thus, irrelevant IgM-antigen complexes were incapable of potentiating the responses. Similar specificity requirements were found for IgG induced suppression of antibody responses. Experiments with nude mice and their euthymic littermates showed that IgM potentiation of antibody production is T-cell dependent. Furthermore, passive transfer of carrier-primed spleen cells together with antigen challenge suggests that IgM potentiation of secondary antibody responses is dependent on specific carrier-primed immune T cells.     

The role of B lymphocytes in cell-mediated immunity II. Delayed hypersensitivity induced by dinitrophenyl-ficoll in dinitrophenyl-keyhole limpet hemocyanin-immunized guinea pigs.

Dinitrophenyl (DNP)-Ficoll will elicit typical delayed hypersensitivity skin reactions in guinea pigs immunized with DNP-keyhole limpet hemocyanin (KLH). We observed that lymph node cells (LNC) from these animals produced the lymphokine, monocyte chemotactic factor (MNL CTX) when stimulated by DNP-Ficoll in vitro. This response was antigen and hapten specific since LNC from nonimmune guinea pigs or those immunized with nonDNP containing antigens were not stimulated by DNP-Ficoll. Lymph node cells were fractionated into T- and B-cell-enriched populations to determine the nature of the DNP-Ficoll-responsive cell. Only the B-lymphocyte-enriched population produced MNL CTX in response to DNP-Ficoll. The purity of the B-cell population was demonstrated by its failure to respond to PHA and by the fact that B cells derived from DNP-although they could no longer respond without T-cell help to the T-dependent antigen, DNP-OVA. These findings suggest that the hapten-specific response of guinea pigs to DNP-Ficoll may be a form of B-cell-mediated delayed hypersensitivity.     

Cell-mediated immune responses to poliovirus. I. Conditions for induction, characterization of effector cells, and cross-reactivity between serotypes for delayed hypersensitivity and T cell proliferative responses

Human polioviruses are categorized into three distinct serotypes (types 1, 2, and 3) based upon their reactivity with specific antibodies. Although a great deal of information has been amassed about the induction and characterization of poliovirus antibody responses, little is known about cell-mediated immunity to poliovirus and its role in protection. Here, we show that intracutaneous injection of ultraviolet light-inactivated poliovirus into the tailbase of BALB/c mice induces delayed hypersensitivity (DTH) and T-cell proliferative (Tprlf) responses. Both DTH and Tprlf responses to poliovirus are mediated by Ly-1high2-, L3T4-bearing T cells. Moreover, known serologic cross-reactivity (i.e., antibody-mediated) of poliovirus serotypes is not predictive of cross-reactivity between the cell-mediated immune responses.     

Contact hypersensitivity reactions to dinitrofluorobenzene mediated by monoclonal IgE anti-DNP antibodies

Several studies have suggested a possible role for IgE antibodies in the pathogenesis of cutaneous hypersensitivity reactions that reach maximum intensity 24 to 48 hr after antigen challenge. The recent availability of murine monoclonal IgE anti-hapten antibodies has made possible the direct examination of the range of cutaneous inflammatory reactions that can be mediated by such antibodies. We have examined the effects of passively sensitizing BALB/c mice with monoclonal IgE anti-dinitrophenyl (DNP) antibody 48 hr before antigen challenge. Inflammatory responses were assessed by measuring ear swelling in mice challenged on the ears with the reactive hapten 2,4-dinitrofluorobenzene (DNFB). Compared with unsensitized controls, the ears of mice passively sensitized with IgE anti-DNP displayed a biphasic pattern of ear swelling after DNFB challenge. An early, transient response (present within 15 to 30 min of challenge and returning to control levels within 4 to 9 hr) was followed by a second, more persistent increase in ear swelling that peaked 24 to 48 hr after challenge. This biphasic pattern of ear swelling seen in IgE-sensitized mice was temporally indistinguishable from that observed in mice conventionally sensitized for allergic contact dermatitis reactions by epicutaneous application of DNFB 5 days before DNFB ear challenge. Antigen specificity of the IgE-mediated contact hypersensitivity reactions was demonstrated by the failure of mice passively sensitized with IgE anti-DNP to display early or delayed ear swelling greater than unsensitized controls when challenged with either of two noncross-reacting haptens, fluorescein isothiocyanate or oxazolone. Mice passively sensitized with a monoclonal IgA anti-DNP antibody (MOPC 315) 48 hr before DNFB challenge failed to display early or delayed ear swelling greater than unsensitized controls. Heat inactivation of the IgE anti-DNP ascitic fluid at 56 degrees C for 30 min completely abolished its capacity to passively sensitize mice for contact hypersensitivity reactions after DNFB challenge. These results document the existence of an antigen-specific, IgE-mediated, delayed-in-time cutaneous hypersensitivity response that can be elicited by epicutaneous challenge (contract) with a reactive hapten.     

Role of antigen and,antibody in the regulation of the immune response

The enzyme dextranase could degrade antigenic dextran in vivo even when given 6-15 d after the antigen. Dextranase injected after the antigen suppressed the immune response when given 24 but not 48 h after the antigen, indicating that the antigen must interact with the immune system for 48 h to initiate a response. Thereafter, the B cells are independent of further antigen stimulation. To show whether antibody-mediated suppression of the immune response was determinant specific FITC-conjugated SRC were applied as immunogen and antibodies were raised both against the carrier (SRC) and the FITC hapten. When these antibodies were injected 1-3 h after the immunogen they only suppressed the immune response to the corresponding determinant. Anti-carrier antibodies usually enhanced the response to the hapten. Therefore, antibody-mediated suppression of the immune response is determinant-specific and cannot be mediated in vivo to a detectable extent by the Fc part of the antibodies.     

Characteristics of the immune response of inoculated and intact animals to the administration of toxigenic and nontoxigenic strains of Clostridium tetani

In experiments on guinea pigs the immune reactions of the animals immunized and not immunized against tetanus in response to the injection of C. tetani spores were studied. In the immunized animals an increase in the production of tetanus antitoxin, the development of delayed hypersensitivity and the activation of the mechanisms of cell-mediated immunity were observed. The nonimmunized animals showed specific changes in the T-system of immunity without the appearance of the clinical symptoms of tetanus, which is, probably, one of the mechanisms of natural immunity.     

Relationships between cell-mediated immunity and the IgE antibody response: II. Delayed hypersensitivity and antibody production to DNP-Ascaris conjugates

Rats of the W/F strain were immunized with DNP-Ascaris conjugates using complete Freund's adjuvant (CFA), Al(OH)₃ gel (alum), or B. pertussis vaccine as adjuvants. Cell-mediated immunity was assessed by lymphotoxin in vitro and by delayed hypersensitivity in vivo. IgE and IgG antibody determinations were made on serum pools obtained at various times during the primary and secondary responses. Although delayed hypersensitivity appeared earlier than lymphotoxin, these two parameters correlated during the primary but not during the secondary response. The discrepancies suggested that different cells may be responsible for these two phenomena. Antibody production was influenced by the adjuvant used. CFA led to IgG antibody responses to both hapten and carrier but not to IgE antibody production. The use of B. pertussis resulted in both IgE and IgG antibody production. In the case of alum, anti-hapten antibodies appeared during the primary response while anti-carrier antibodies of both IgE and IgG classes were detected after booster. The results indicated that cell-mediated immunity, IgE, and IgG antibodies appeared independently in an ordered, temporal sequence, and that these responses were not mutually exclusive but were under strong modulatory influences of the various adjuvants used.     

The Arabidopsis putative G protein-coupled receptor GCR1 interacts with the G protein alpha subunit GPA1 and regulates abscisic acid signaling

Heterotrimeric G proteins composed of alpha, beta, and gamma subunits link ligand perception by G protein-coupled receptors (GPCRs) with downstream effectors, providing a ubiquitous signaling mechanism in eukaryotes. The Arabidopsis thaliana genome encodes single prototypical Galpha (GPA1) and Gbeta (AGB1) subunits, and two probable Ggamma subunits (AGG1 and AGG2). One Arabidopsis gene, GCR1, encodes a protein with significant sequence similarity to nonplant GPCRs and a predicted 7-transmembrane domain structure characteristic of GPCRs. However, whether GCR1 actually interacts with GPA1 was unknown. We demonstrate by in vitro pull-down assays, by yeast split-ubiquitin assays, and by coimmunoprecipitation from plant tissue that GCR1 and GPA1 are indeed physically coupled. GCR1-GPA1 interaction depends on intracellular domains of GCR1. gcr1 T-DNA insertional mutants exhibit hypersensitivity to abscisic acid (ABA) in assays of root growth, gene regulation, and stomatal response. gcr1 guard cells are also hypersensitive to the lipid metabolite, sphingosine-1-phosphate (S1P), which is a transducer of the ABA signal upstream of GPA1. Because gpa1 mutants exhibit insensitivity in aspects of guard cell ABA and S1P responses, whereas gcr1 mutants exhibit hypersensitivity, GCR1 may act as a negative regulator of GPA1-mediated ABA responses in guard cells.     

Immunogenicity of liposomal model membranes sensitized with mono(p-azobenzenearsonic acid)tyrosylphosphatidylethanolamine derivatives. Antibody formation and delayed hypersensitivity reaction.

We have previously reported that hapten specific antibodies are produced in guinea pigs immunized with certain N-substituted phosphatidylethanolamine derivatives (either free or incorporated into liposomal membranes) in complete Freund's adjuvant. In this paper, we describe the synthesis of mono(p-azobenzenearsonic acid)tyrosylphosphatidylethanolamine (ABA-Tyr-PE). Immunication with this compound (either free or present in liposomes) not only results in the formation of anti-azobenzenearsonyl antibodies, but also confers cellular immunity as manifested by delayed hypersensitivity reactions elicited by challenge with either azobenzenearsonyl-bovine serum albumin or sensitized liposomes. Thus, ABA-Tyr-PE immunized guinea pigs differ from those immunized with azobenzenearsonyl-bovine serum albumin which produce anti-bodies but do not reveal a delayed reaction. Moreover, the ABA-Tyr-PE immunized animals differ from those immunized with mono(p-azobenzenearsonic acid)tyrosine; this substance has been shown by other investigators to confer cellular immunity without antibody formation in guinea pigs. However, the deacylated homolog of ABA-Tyr-PE (i.e., mono(p-azobenzenearsonic acid)tyrosylglycerophosphorylethanolamine) has the same immunological properties as mono(p-azobenzenearsonic acid)tyrosine. These observations justify the further exploitation of liposomal model membranes as novel immunogens that are able to elicit both cell and humoral mediated immune responses.     

Pushing antibody-based labeling systems to higher sensitivity by linker-assisted affinity enhancement

The sensitivity of antibody/hapten-based labeling systems is limited by the natural affinity ceiling of immunoglobulins. Breaking this limit by antibody engineering is difficult. We thus attempted a different approach and investigated if the so-called bridge effect, a corecognition of the linker present between hapten and carrier protein during antibody generation, can be utilized to improve the affinity of such labeling systems. The well-known haptens 2,4-dinitrophenol (2,4-DNP) and 2,4-dichlorophenoxyacetic acid (2,4-D) were equipped with various linkers, and the resulting affinity change of their cognate antibodies was analyzed by ELISA. Anti-2,4-DNP antibodies exhibited the best affinity to their hapten when it was combined with aminobutanoic acid or aminohexanoic acid. The affinity of anti-2,4-D antibodies could be enhanced even further with longer aliphatic spacers connected to the hapten. The affinity toward aminoundecanoic acid-2,4-D derivatives, for instance, was improved about 100-fold compared to 2,4-D alone and yielded detection limits as low as 100 amoles of analyte. As the effect occurred for all antibodies and haptens tested, it may be sensible to implement the bridge effect in future antibody/hapten-labeling systems in order to achieve the highest sensitivity possible.     

Requirements for inducing tolerance of hapten-specific delayed hypersensitivity: epitope density.

Tolerance to hapten-specific antibody formation and delayed hypersensitivity was examined in adult rabbits. The azobenzenearsonate (ABA) or sulfonate-specific antibody response to hapten-hemocyanin immunogens was suppressed by deaggregated hapten-rabbit IgG conjugates given 21 and 14 days before challenge. High affinity antibody was preferentially suppressed. Delayed hypersensitivity to ABA-tyrosine was suppressed by deaggregated ABA-rabbit IgG conjugates injected 17 and 10 days before challenge. Conjugates with a high hapten density, ABA15-23-rabbit IgG were effective tolerogens. Conjugates with four to six ABA groups per carrier molecule were very poor tolerogens. Increasing the amount of low substituted conjugate injected did not improve tolerogenicity. It appears that a high epitope density is required for effective induction of tolerance to ABA-specific delayed hypersensitivity in the rabbit.     

Effects of recombinant cholera toxin b subunit (rCTB) on cellular immune responses: enhancement of delayed-type hypersensitivity following intranasal co-administration of Mycobacterium bovis-BCG with rCTB

Recombinant cholera toxin B subunit (rCTB) is a safe and potent mucosal adjuvant. To gain insight into the mechanism underlying the adjuvant effect of rCTB, the effects of rCTB on cell-mediated immune responses of mice and guinea pigs were examined after intranasal administration of Mycobacterium bovis -bacillus Calmette-Guérin (BCG) with and without rCTB. Delayed-type hypersensitivity, for skin reactions in guinea pigs and for footpad swelling reactions in mice, to purified protein derivative (PPD) were enhanced by intranasal co-administration of BCG and rCTB, as compared to giving BCG alone to these animals. Moreover, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma production of spleen cells and antigen specific spleen cell proliferation, stimulated with PPD, were enhanced in the presence of rCTB. These results strongly suggest that rCTB enhances cellular as well as humoral immune responses.     

Immunological maturation in cell-mediated immunity in the rat: fine specificity discrimination by the cells responsible for peritoneal cell migration inhibition, and its increase with time after sensitization.

Two weeks or longer after sensitization of inbred Sprague-Dawley rats with ABA-Tyr in CFA, peritoneal cell migration in vitro was inhibited in the presence of the immunogen, which strengthened the concept that migration inhibition is a correlate of delayed hypersensitivity also in the rat. After sensitization with one of three strongly cross-reacting conjugates ABA-Tyr, ABA-His, or ABA-Try, the cells responsible for antigen recognition in migration inhibition could discriminate between the homologous and heterologous conjugates added to the culture medium. The degree of fine specificity discrimination increased significantly from 3 to 35 wk after sensitization, which showed that the average affinity of the recognition structures involved increased with time after sensitization.     

Purified Protoplasmic Peptides of Mycobacteria: In Vivo and In Vitro Comparison of the Species Specificity of Purified Protoplasmic Peptides and Purified Protein Derivatives of Mycobacterial Culture Filtrates

The specificity of purified protein derivatives (PPD) prepared from the culture filtrates of Mycobacterium tuberculosis (PPD), M. kansasii (PPD-Y), M. intracellulare (PPD-B), and M. scrofulaceum (PPD-G) were compared to comparable protoplasmic extracts (PPP) of the same organisms by gel diffusion and delayed hypersensitivity reactions in sensitized guinea pigs. PPD and, to a lesser degree, PPD-Y demonstrated specificities sufficient to enable identification of homologously sensitized guinea pigs in the above group of four mycobacteria. PPD-B and PPD-G did not always elicit the largest reaction in homologously sensitized animals. The PPP sensitins from M. tuberculosis and M. kansasii produced as good skin reactions at 24 and at 48 hr as did their PPD counterparts. The PPP from M. scrofulaceum and M. intracellulare were more specific and more reactive than corresponding PPD, regardless of the time of comparison. Although based on different immunological mechanisms, the specificity of these two groups of sensitins, as demonstrated by delayed hypersensitivity, correlated well with serological comparisons in the gel diffusion test. The low degree of specificity of PPD-B and PPD-G in contrast to that of corresponding PPP was reflected in the precipitin bands in agar gel.     

Cutaneous basophil hypersensitivity uncovered in the cell transfer of classical tuberculin hypersensitivity.

Cellular transfer of cutaneous basophil hypersensitivity (CBH) was studied. Guinea pigs immunized for CBH with incomplete Freund's adjuvant (IFA) provided cells which could transfer delayed and basophil-rich reactions in skin tests of recipients. Guinea pigs immunized with complete classical tuberculin-type delayed hypersensitivity reactions (DH), which are characteristically devoid of basophils. However, recipients of cells from donors with DH, surprisingly, were found to have delayed skin reactions containing large basophil infiltrates which were lacking in the donors. Thus, recipients of classical cell transfers of tuberculin-type DH had delayed reactions which resembled CBH. Control experiments verified that the cell transfer of CBH from donors with DH was due to passive transfer with live cells and not transfer of contaminating humoral factors or active sensitization of recipients. It was concluded that cutaneous basophil responses were suppressed in CFA-immunized donors and expressed in cell transfer recipients. Cells from donors immunized with CFA were enriched for nonadherent and nonimmunoglobulin-bearing lymphocytes by passage through nylon wool columns, and these cells transferred conjugate specific CBH reactions. It was concluded that cells mediating these transfers were probably T cells. The finding of basophils in cell transfers of DH and a variety of other findings suggesting complex regulation of basophil numbers in tissue lead to the conclusion that the term CBH be used to simply describe a basophil-containing skin reaction.     

Evanescent delayed-type hypersensitivity: mediation by effector cells with a short life span.

Four days after i.v. immunization of mice with optimal low doses of heterologous erythrocytes (2 x 10(5) RBC), strong delayed-type hypersensitivity (DTH) responses can be elicited in the footpad. At later intervals after immunization, DTH responsiveness is progressively diminished and replaced by 4-hr antibody-dependent reactions. These evanescent T cell-mediated DTH responses, which are progressively replaced by antibody-dependent reactions, resemble Jones-Mote type delayed hypersensitivity responses of humans and guinea pigs. Since higher doses of immunizing antigen activate suppressor mechanisms that inhibit DTH responses, we examined the possibility that the evanescence of DTH in mice immunized with an optimal low dose of antigen might also be due to suppression. Using techniques that could clearly demonstrate the suppression produced by high antigen doses, we failed to find evidence for either humoral or cellular suppression in optimally immunized mice with declining of DTH responses. Thus, it appears that the evanescence of produced by optimal low dose immunization with RBC may be due to an intrinsic short life span of the effector cells rather than to the activation of an identifiable shut-off mechanism.