Production of auto-anti-idiotypic antibody during the normal immune response. XII. Enhanced auto-anti-idiotypic antibody production as a mechanism for apparent B-cell tolerance in rabbits after feeding antigen

Rabbits fed trinitrophenylated bovine serum albumin (TNP-BSA) generated fewer anti-TNP plaque-forming cells but greater numbers of hapten (TNP)-augmentable IgM and IgG PFC following immunization with TNP-Ficoll or TNP-Brucella abortus than did animals not previously fed antigen. Spleen and mesenteric and bronchial lymph nodes were similarly affected. In addition more auto-anti-idiotype (Id) antibody (anti-anti-TNP) was eluted by hapten from spleen cells of antigen-fed rabbits than from spleen cells of control rabbits not prefed antigen. Gel filtration studies ruled out the possibility that the Id binding activity in the eluates was due to immune complexes. The isotype of the anti-Id was IgG except in one rabbit where it was IgM. The results are consistent with the interpretation that the production of auto-anti-Id antibody is one of the factors responsible for the specific depression of the IgM and IgG immune responses which follows antigen feeding. In contrast the antigen feeding resulted in priming for an IgA anti-TNP response without detectable hapten-augmentable IgA PFC.     

Production of auto-anti-idiotypic antibody during the normal immune response: IX. Characteristics of the auto-anti-idiotype antibody and its production

Using hapten-reversible inhibition of plaque formation as an assay for auto-anti-idiotype antibody (anti-Id) and as a means for following idiotype (Id) expression, we have obtained evidence that following immunization with trinitrophenyl (TNP) conjugates (a) there are differences in Id expression in the anti-TNP antibody response to different TNP conjugates although there is some overlap; (b) different strains, although showing some differences in Id expression, tend to produce cross-reactive Ids, thus no obvious allotype linked inheritance of Id expression is observed in this heterogeneous immune response; (c) the auto-anti-Id produced following immunization with TNP-Brucella abortus or TNP-Ficoll tends to be of the IgG_2a and IgG_2b isotypes.     

Evidence for histamine-induced auto-anti-idiotypic antibody immunoregulation in vivo

The in vivo effects of histamine injection in LAF₁ male mice on the immune reactivity to trinitrophenylated bovine γ-globulin was studied using plaque-forming cell (PFC) responses and their avidity distributions. Splenic anti-trinitrophenyl (anti-TNP) PFC responses of mice treated with histamine (5 × 10^?6 mol or 1 mg, intravenously) were significantly reduced in number and restricted in heterogeneity and characterized by a preferential loss of high-avidity IgG PFCs. The reduced PFC response in histamine-treated mice was dose and time dependent. No evidence of suppressor cell activity in the spleens from histamine-treated mice was demonstrable. Only histamine-treated mice produced a significantly high percentage of anti-idiotype-blocked, hapten-augmentable IgG PFCs, suggesting the presence of auto-anti-idiotypic activity. Immune sera taken from histamine-treated mice caused an inhibition of anti-TNP PFC in vitro. This PFC-inhibiting factor in immune sera of histamine-treated mice was an antibody of the IgG₁ and IgG_2a class, lacked anti-TNP antibody activity, but reacted with anti-TNP antibody of LAF₁ origin. Passive hemagglutination study of this sera showed anti-(anti-TNP F(ab′)₂-IgG) titer. Thus, the results of this study suggest that histamine in combination with antigen induces auto-anti-idiotypic antibody which, in turn, is involved in the normal regulation of the immune response to trinitrophenylated bovine γ-globulin in vivo.     

Selective suppression by auto-anti-idiotypic antibody of B-cell idiotype repertoires generated after stimulation with the same hapten on T-dependent and T-independent carriers

In the present study, we investigated whether auto-anti-idiotypic antibody in the immune sera from old mice could recognize antitrinitrophenyl (TNP) plaque-forming cells (PFC) generated after stimulation with the T-dependent and T-independent forms of the hapten, TNP. Young and old C57BL/6J male mice were immunized with a variety of T-dependent (TNP-bovine gamma-globulin, TNP-BGG; TNP-keyhole Limpet hemocyanin, TNP-KLH; ovalbumin, OVA; bovine serum albumin, BSA; BGG) and T-independent (TNP-Brucella abortus, TNP-BA; TBP-Ficoll; TNP-polyacrylamide beads, TNP-PAA) antigens either in complete Freund's adjuvant (CFA) or in soluble form. Splenic anti-TNP or antiprotein PFC responses were assayed for anti-idiotype-blocked, hapten- or protein-augmentable IgM, IgG and IgA PFC, 1-2 weeks after immunization. It was found that 8-month-old mice produced significantly a higher percentage of hapten augmentable (26-42%) IgM PFC response to T-independent antigens as compared with the 2-month-old mice (3-6% augmentation). Similarly, old mice produced a significantly higher percentage of hapten or protein augmentable (25-129%) IgG PFC response to T-dependent antigens as compared with the 2-month-old group (2-6% augmentation). The data support the view that age-related regulation of auto-anti-idiotypic antibody is a general phenomenon for immune responses to T-dependent and T-independent antigens. Hapten-reversible inhibition of plaque formation was used to determine whether anti-idiotypic antibody containing antisera from old mice could inhibit B-cell idiotype repertoires generated after stimulation with the same hapten, TNP, on T-dependent and T-independent carriers. Pools of immune sera from 8-month-old mice primed with T-dependent TNP-BGG or TNP-KLH antigens but not with T-independent TNP-PAA or TNP-BA antigens, or with the proteins OVA, BSA, or BGG selectively inhibited IgM, IgG, and IgA anti-TNP PFC from 2-month-old mice that were previously primed with either TNP-BGG or TNP-KLH. In contrast, immune sera from old mice primed with TNP on either T-dependent or T-independent carriers inhibited anti-TNP PFC from mice primed with T-independent TNP-PAA or TNP-BA antigens. Immune sera from old mice primed with OVA or BSA only inhibited the respective antiprotein PFC. The immune sera from young mice did not show any appreciable inhibition of PFC generated after stimulation by any of the antigens studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suppression of anti-hapten antibody responses by hapten-specific cytolytic T lymphocytes: role of I-A-associated antigen on target B lymphocytes

Cytolytic T lymphocytes (CTL) specific for 2,4,6-trinitrophenyl (TNP) determinants suppress the effector phase of a secondary anti-TNP antibody responses of murine syngeneic spleen cells in vitro. The cells mediating this suppression are hapten-specific, H-2-restricted, and possess properties typical of CTL. Moreover, the targets of the suppression appear to be antigen-primed B lymphocytes that are recognized by CTL via soluble antigen bound noncovalently to their Ig receptors. The effect of the CTL can be blocked by the addition of monoclonal antibodies directed against I-A molecules but not I-E or H-EK-encoded molecules on the target B cells, even in strain combinations in which the CTL-B cell interaction is restricted only by the H-2K and I regions of the MHC. This result suggests that B lymphocyte-bound antigen tends to associate preferentially with I-A rather than H-2K/D-encoded determinants, and that the suppressive effect of the CTL population is attributable to the minor subset that recognizes hapten-modified Ia antigens. These findings are also discussed in terms of the possible immunoregulatory function of Ia-restricted CTL.     

Nature of the antigenic complex recognized by T lymphocytes. VI. The effect of anti-TNP antibody on T cell responses to TNP-conjugated macrophages.

In this paper we examined the effect of anti-TNP antibody on guinea pig T cell proliferation in response to TNP-modified macrophages in vitro. The addition of anti-TNP to TNP-modified macrophages immediately after conjugation inhibited their ability to stimulate TNP-specific T cell proliferation. This inhibition appeared to be specific for the TNP response since anti-TNP had no effect on the ability of TNP-modified macrophages pulsed with either PPD or TNP-Ova to stimulate efficient PPD or Ova T cell responses. On the other hand, anti-TNP had no effect on the TNP-specific response to TNP-modified macrophages that had been cultured overnight before addition to primed T cells or to macrophages which had been pulsed with TNP-Ova. We also demonstrated that the same TNP-specific T cell subpopulation responds to both freshly TNP-modified macrophages and overnight cultured TNP-modified macrophages. These results suggest that the relevant TNP-determinants recognized by T cells are not exposed on the macrophage surface and raise the possibility that macrophages must process membrane-conjugated TNP to create the immunogen recognized by T cells.     

Immunodetection of nitrocellulose-adhesive proteins at the nanogram level after trinitrophenyl modification

A method for protein detection on nitrocellulose membranes based on modification with 2,4,6-trinitrobenzenesulfonic acid and reaction with anti-trinitrophenyl (TNP) serum as first antibody followed by peroxidase-conjugated second antibody is described. Protein quantities between 1 and 3 ng can be detected in the dot test. This method was used in a double immunodetection procedure after electrophoretic transfer of proteins localizing first a distinct antigen with its specific antiserum followed by visualization of the complete protein pattern on the same blot by the TNP/anti-TNP method as described above. As only water-soluble reagents are employed no shrinkage of the membrane occurs. Furthermore, the method can be used in a simultaneous immunodetection procedure visualizing the specific antigen together with TNP marker proteins using a mixture of the specific antiserum and the anti-TNP serum as first antibody.     

Restricted maturation of antibody-binding characteristics for hapten-specific IgM-plaque-forming cells in mice

Inhibition of plaque formation was used as a method for studying changes in binding characteristics of IgM antibody specific for the hapten, 2,4,6-trinitrophenyl (TNP). By incorporating successive concentrations of TNP ligand into specific hemolytic plaque assays, we were able to obtain inhibition curves and to determine relative ligand binding of anti-TNP antibodies secreted by plaque-forming cell (PFC) populations. The concentration of free hapten required to obtain 50% reduction in IgM PFC decreased with time after immunization, indicating that an increase in average binding affinity of their secreted antibodes occurred. The slopes of the inhibition curves also increased with time after immunization. Maturation with regard to the above two properties was virtually complete for IgM PFC 5–7 days after immunization. The manner by which the slopes of the inhibition curves changed suggested that maturation occurred as the result of a simple selective process, and it appeared to involve primarily precursor cells existing prior to administration of antigen.     

Immune response of guinea pig lymphoid cells in vitro: III. Induction by Concanavalin A of a secondary anti-TNP response in primed guinea pig cells

Spleen and lymph node cells of 2,4,6-trinitrophenyl-ovalbumin (TNP-OVA)-primed guinea pigs, show a secondary anti-TNP plaque-forming cell (PFC) response on culture with Concanavalin A which does not require the addition of TNP-OVA but this response may be modestly stimulated by soluble TNP-OVA. If TNP sheep red blood cells (SRBC) are added instead as antigen, the spontaneous anti-TNP response is suppressed but an anti-SRBC response is induced.     

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.     

Carrier-specific unresponsiveness in reaginic antibody formation. II. Suppression of hapten and carrier responsiveness in presensitized rats.

By inducing carrier-specific tolerance to sheep γ-globulin (SGG) in rats challenged with TNP-SGG in alum, it has been possible to study the effect of helper T-cell Unresponsiveness on IgE anti-TNP antibody formation. Rats primed to either the carrier (SGG) or the hapten (TNP as TNP-KLH) were treated with a single high dose (10 mg) of soluble SGG resulting in a suppression of both IgE anti-TNP and anti-SGG antibody which was maintained following a normally immunogenic secondary challenge with TNP-SGG in alum. This suppression was relatively long lasting, with no detectable IgE responsiveness to hapten or carrier observed for up to 8 weeks after tolerance induction. Suppressed animals were able to respond to the hapten when challenged with TNP-KLH, indicating that the induced effect did not directly involve the IgE antibody producing cells, but rather the carrier-specific helper cells. These results parallel our previous findings for IgM and IgG responses in a similar system. Such relatively long lasting and easily induced suppression in IgE antibody formation to specific protein antigens in primed animals may eventually provide a clinically useful means of allergic desensitization to large protein allergens.     

Primary in vitro antibody response from human peripheral blood lymphocytes.

A method for the induction of a primary in vitro antibody response from human peripheral blood lymphocytes is presented. Upon cultivation with trinitrophenyl conjugated polyacrylamide beads (TNP-PAA), an anti-TNP response can be obtained as indicated by the appearance of direct plaque-forming cells from day 5 of culture, with a reproducible peak on day 8. These plaques correspond to cells actively producing antibody of the IgM type, as shown by their inhibition by cycloheximide and by anti-human IgM serum, but not by anti-human Fc gamma serum. Their specificity for the TNP hapten can be demonstrated by the effector cell blockade phenomenon, with highly substituted TNP-human IgG. Although the anti-TNP response induced by TNP-PAA in mouse spleen cell cultures appears T independent the same response in human PBL may involve in addition the participation of T cells, since E-RFC depletion before culture led to a markedly decreased number of plaque-forming cells. A significant response could be obtained from the PBL of all of the 30 normal individuals tested. Importantly, the response was reproducible in its magnitude in the six individuals tested in at least three different experiments. Thus, the in vitro stimulation of human PBL by TNP-PAA can be proposed as a reliable test for the study of human B cell function in a specific primary antibody response.     

Production of auto-anti-idiotype antibody during the normal immune response. X. Response to TNP-Ficoll in the chicken

The spontaneous production of auto-anti-idiotype (Id) was demonstrated after injection of chickens with trinitrophenylated Ficoll (TNP-F) by: (a) the presence of hapten-augmentable plaque-forming cells (PFC), (b) the ability of serum and of hapten eluates from immune spleen cells to cause hapten-reversible inhibition of anti-TNP plaque formation, and (c) an enzyme-linked immunosorbent assay (ELISA). Tests for anti-Id using the ELISA and hapten-reversible inhibition of PFC correlated very well. As in the mouse, the incidence of hapten-augmentable PFC was reduced by thymectomy and increased by the transfer of TNP-F-immune spleen cells. Hapten-augmentable PFC were also observed during the immune response of chickens to p-azobenzene arsonate-conjugated Brucella abortus.     

Secretion rate independent evaluation of IgM antibody avidity at the level of single immunocytes.

The hemolytic plaque inhibition assay has been performed on spleen cells from mice immunized with TNP-HRBC to evaluate avidity of anti-TNP IgM antibodies. At different times after immunization direct plaques were inhibited by soluble TNP-EACA, TNP61-BGG, or anti-mu antiserum. Analysis of the inhibition data provided independent estimates of antibody avidity and secretion rate. Avidity was found to increase with time, to reach a maximum when the antibody response attained the peak value, and then to decline as the response was waning. There was a decrease followed by increase of the secretion rate concomitant with the rise and fall of the antibody response and avidity.     

Relationship between trinitrophenol and H-2 antigens on trinitrophenyl-modified spleen cells. III. Quantitative aspects of trinitrophenol binding on cells treated with trinitrobenzene sulfonic acid

Splenic lymphoblasts or normal spleen cells were treated with varying concentrations of TNBS in order to assess whether cell membrane H-2 molecules were derivatized with TNP. Cells treated with high concentrations of TNBS had their cell membrane H-2 molecules derivatized and functioned antigenically as inhibitors in a cold target TNP-CML competition assay. In contrast, cells derivatized with lower concentrations of TNBS had a significant proportion of their membrane proteins derivatized with TNP but did not have their H-2 molecules derivatized. These latter cells were unable to block anti-TNP cytotoxic effector cells in the competition assay. When cells were treated with 3H-TNBS, it was observed that TNP couples to cell membrane H-2, Ia and Ig molecules, and an estimate of the number of TNP molecules bound per cell at varying concentrations of TNBS was determined. The data obtained are consistent with there being a requirement for TNP to directly derivatize H-2 molecules on the cell membrane in order to create antigenic determinants that can be recognized by cytotoxic anti-TNP effector cells. As an alternative, there may be a requirement for the presence of a high density of TNP molecules per cell rather than direct H-2 derivatization by TNP in order to account for activity.     

Activation of human B lymphocytes by a particulate antigen

A specific IgM antibody response toward the trinitrophenol (TNP) hapten can be induced in mononuclear blood cell suspensions upon culture with a particulate antigen: polyacrylamide beads conjugated with the TNP hapten (TNP-PAA). The response, and its specificity, are demonstrated by an increase in the number of TNP binding B lymphocytes (specific rosette forming cells), by the appearance of cells producing anti-TNP antibody at a high rate (haemolytic plaques), (ELISA test). The anti-TNP response requires monocytes, the role of which is to produce interleukin-1 (IL-1) and T lymphocytes (belonging to the T4 helper subset) the role of which is to produce interleukins (the characterization of which is under study). We propose a model or B cell activation based on the following signals: an early specific signal, provided by the particulate antigen; several non specific signals, provided by T derived interleukins. The anti-TNP response is negatively regulated by monocytes, the functional states of which can be modified in certain situations (autoimmunity, aging) or influenced by glucocorticoids. Suppressor T lymphocytes of this response (not exclusively of the T8 phenotype) can be induced and this can allow the evaluation of T suppressor cell function. This was used in adult idiopathic thrombocytopenic purpura treated with high doses of intra-venous gammaglobulins.     

A trinitrophenyl(TNP)-poly-L-lysine-horseradish peroxidase conjugate for the detection of anti-TNP antibodies in vivo

A new conjugate for the detection of anti-trinitrophenyl(TNP) antibodies was developed to study the localization pattern of specific antibody containing cells and extracellular antibody in vivo. By means of a bridging molecule, poly-L-lysine, nine TNP groups and six horseradish peroxidase (HRP) groups were joined in one conjugate. Thus a higher specificity (more hapten) was united with a higher staining intensity (more enzyme) in the same conjugate. This conjugate made possible the simultaneous detection of anti-TNP antibody containing cells and establishment of their class (immunoglobulin M (IgM) and IgG). It was also used for the demonstration of anti-TNP antibodies in tissues where a TNP-alkaline phosphate (AP) conjugate could not be used due to high AP (endogenous) background staining. Thus we demonstrated anti-TNP antibody containing cells in gut associated lymphoid tissue and anti-TNP-(TNP-ovalbumin) immune complexes in the glomeruli of the kidney. We suggest that poly-L-lysine is a suitable bridging molecule for the preparation of hapten-HRP conjugates.     

Antibodies against neuroactive amino acids and neuropeptides. II. Simultaneous immunoenzymatic double staining with labeled primary antibodies of the same species and a combination of the ABC method and the hapten-anti-hapten bridge (HAB) technique.

In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.     

Enhanced transcription in a pre-B-like cell line transformed with cloned antibody genes after antigen-specific stimulation

The transducing vector, pSV2-neo, carrying the rearranged immunoglobulin (Ig) heavy (mu) and light (kappa) chain genes specific for the hapten 2,4,6-trinitrophenyl (TNP) was introduced into a pre-B cell line. The transformants expressed the TNP-specific IgM receptor on the surface. Furthermore, the addition of TNP-bovine serum albumin (hapten-carrier conjugate) to the culture media activated the expression of the transferred Ig genes and several endogenous genes such as v-abl and beta-tubulin. However, expression of the beta2-microglobulin gene was not affected. The results presented in this paper show that transfection of cloned Ig genes into B cells is a useful system for establishing monoclonal B cell lines expressing functional Ig receptor molecules with defined hapten specificity.     

Effect of bacterial lipopolysaccharides on anti-trinitrophenyl antibody-producing cells. Nonspecific modification of the affinity at the cellular level.

The effect of lipopolysaccharide (LPS) on anti-trinitrophenyl (TNP) direct plaque-forming cells (PFC) in the spleen of mice and the affinity of antibodies produced by these PFC were examined. Simultaneous injection of bacterial LPS and TNP-coupled sheep red blood cells(SRBC) induced an obvious increase in anti-TNP PFC numbers and heightened the antibody affinity at cellular levels. The higher the doses of LPS, the greater the effects. Concomitant injection of LPS in TNP-coupled homologous mouse red blood cells (MRBC) also elicited good anti-TNP PFC response and slightly heightened the affinity. Priming with LPS and SRBC together 7 days prior to immunization did not enhance the anti-TNP PFC response and it was difficult to alter the affinity. Preinjection with small amounts of TNP-MRBC or -rabbit red blood cells and LPS simultaneously did not induce any significant increase in anti-TNP PFC secondary response after reimmunization with TNP-SRBC, but obviously heightened the antibody affinity. Injection of LPS simultaneously with the secondary immunization was effective for both the anti-TNP PFC response and the alteration of antibody affinity. These results suggest that LPS affects the control mechanisms of anti-TNP antibody affinity via the non-thymus-derived helper cell function, and the adjuvant action and alteration of antibody affinity induced by LPS are regulated by different mechanisms.