anti-DNP Response Research Articles

Cellular Basis of Tolerance to Serum Albumin in Adult Mice

Abstract The surface markers and size of suppressor cells were determined in adult (BALB/c × C57BL/Ka)F1 mice which were tolerized with a single injection of deaggregated bovine serum albumin (BSA). Suppressor cells from the spleens of tolerized donors were assayed in a cell transfer system in which graded numbers of cells were injected into irradiated syngeneic mice along with limiting numbers of T cells primed to BSA and an excess of B cells primed to DNP-BSA. Adoptive hosts were challenged with DNP-BSA in saline, and the anti-DNP response was measured. Suppressor cells were antigen specific as shown by the inhibitory activity of BSA-tolerant spleen cells on the response to DNP-BSA, but not to DNP-BGG. Suppressor cells were eliminated by in vitro treatment with anti-Thy 1.2, anti-Ly-2.2, anti-I-J subregion antisera and C, but not with anti-Ly-1 or anti-I-A subregion antisera. Neither unprimed nor primed helper T cells were detected in the spleen of tolerized donors after in vitro treatment with anti-Ly-2.2 antisera. Both helper and suppressor T cells from the spleens of primed or tolerized donors, respectively, showed a rapid sedimentation velocity (S > 3.7 mm/hr).

Recovery of immune competence after tumour resection in mice: correlation with loss of suppressor elements

Changes in the immune competence and levels of suppressore elements were assessed by mitogen stimulation and in vitro antibody production, after resection of a transplantable sarcoma. Spleen cells from tumour-resected animals were found to have depressed responses to conA as well as to the antigens SRBC and DNP-LPS. This inability to respond was gradually overcome and, by Day 21 after resection, spleen cell competence had returned to normal levels. Suppressor cells isolated from the spleens of tumour-resected animals were capable of suppressing the conA response and PFC response of normal syngeneic spleen cells in vitro. The ability to suppress the conA response of normal cells disappeared by Day 1 after resection, while the ability to suppress the anti-SRBC and anti-DNP PFC response of normal cells disappeared by Day 8 and Day 14 respectively. Serum from tumour-resected mice was also found to be suppressive to the conA response of normal spleen cells. The inhibitory material responsible for suppression eluted with the Ig-containing fraction on Sephadex G-150. This inhibitory material gradually disappeared from the serum of tumour-resected mice and was no longer apparent by Day 14. Therefore, it appeared that the return of normal lymphocyte function after tumour-resection was concomitant with the disappearance of splenic suppressor cells and suppressive serum factor.

Analysis of the Restricted Dinitrophenyl (DNP) Antibody Response to the DNP Conjugate of Poly-(L-Glu56, L-Lys35, L-Phe9)

Abstract The anti-dinitrophenyl (DNP) response in B10 congenic mice to the DNP conjugate of the random linear terpolymer of L-Glu, L-Lys, L-Phe (GLØ) is restricted in the number of DNP-specific antibody-producing clones expressed as determined by the technique of isoelectric focusing. A limited strain survey indicated that a gene(s) outside the major histocompatibility complex may influence the degree of restriction in the DNP response to DNP-GLØ. Evaluation of sera run in juxtaposition established that a high percentage of B10 congenic mice express a common clone of anti-DNP antibody-producing cells in response to DNP-GLØ. The expression of this clone is independent of H-2 haplotype. These findings contrasted with the extremely heterogeneous populations of anti-DNP antibodies produced in these strains when immunized with DNP-ovalbumin (DNP-OVA). Adoptive transfer experiments revealed, however, that unprimed or GLØ-primed T cells can cooperate with syngeneic DNP-OVA-primed B cell clones to generate a heterogeneous anti-DNP response upon challenge with DNP-GLØ.

Ontogeny of B Lymphocyte Function

Abstract Lethally irradiated mice reconstituted with adult T cells and neonatal or fetal B cells produce an anti-DNP response of restricted heterogeneity of affinity when compared with the response of mice reconstituted with T and B cells from adult donors. The capacity to reconstitute adult mice to give a heterogeneous response matures between 7 and 10 days after birth. The maturation of B cells from day-15 fetal or neonatal donors to produce a heterogeneous response was followed in the adult, cell transfer recipient by immunizing them at different times after cell transfer. It was found that B cells both from day-15 fetal mice and from neonatal mice acquire the capacity to produce a heterogeneous response within 3 days in the adult, cell transfer recipient. Thus, the B cell population matures more rapidly in the cell transfer recipient than in the intact donor. The kinetics of maturation in the adult recipient is the same for B cells from day-15 fetal and neonatal donors. The data imply that all information required to produce a fully heterogeneous response is already present in the day-15 fetus. In addition, the data strongly support the hypothesis that a factor in the adult mouse acts to induce this step in the maturation of the B lymphocyte population. Thus, the data seem to be inconsistent with the view that the timing of the occurrence of this differentiation event is precoded in an internal cell clock in the B lymphocyte line. Clearly, B cells from day-15 fetal mice are already capable of differentiating in response to the inducing factor which is present in the adult animal.

Mechanisms of B cell tolerance: I. Dynamic nature of the induction of hapten-specific unresponsiveness by hapten-conjugated pneumococcal polysaccharide.

Exposure of 2,4-dinitrophenyl (DNP)-hemocyanin-primed spleen cells to DNP-conjugated pneumococcal polysaccharide (DNP-S3) in vivo or in vitro renders such cells unresponsive to DNP-hemocyanin challenge following adoptive transfer. The unresponsiveness is hapten-specific, independent of the presence of T cells and adherent cells, and not due to either toxic effect of S3 or carry-over of tolerogenic amounts of cell-bound DNP-S3, and thus presumably represents a model of B cell tolerance. The degree of suppression induced depends upon the dose of the tolerogen and most strikingly on the duration of exposure of cells to the tolerogen. Thus 2 to 6 h exposure to DNP-S3 has an insignificant effect on anti-DNP responses, while 24 and 48 h exposures in vitro and in vivo are highly suppressive. Such a dynamic process of tolerance induction suggests a "multiple hit" phenomenon, implying the generation of suppressive signals by repeated cycles of tolerogen-receptor interactions. The process can be interrupted by removal of extracellular DNP-S3, although this does not reverse the unresponsiveness that has already been induced.

Failure to demonstrate allotype-specific helper cells for antibody production in the rat

THE way in which the immune response is regulated is poorly understood but network theories, based on idiotype–anti-idiotype interactions have been widely discussed1. It has been suggested2,3 that in addition to the idiotype-based network an allotype network also exists. This hypothesis rests on the demonstration3 that in a particular mouse strain, carrier-primed T cells from Ig 1-a homozygotes collaborated in a secondary anti-DNP (dinitrophenol) response with Ig 1-a allotype, but not Ig 1-b allotype, B cells from DNP-primed 1-a/1-b heterozygotes. In attempting to understand the regulation of the immune response it is clearly essential to establish whether the heavy chain allotype specificity of helper T cells shown by these experiments is common to all allotypes and is not a particular feature of the system studied. Here we describe experiments in which we were unable to demonstrate allotype-specific helper cells for antibody production in the rat.

Mechanism of T-cell help in the immune response to soluble protein antigens. I. Evidence for in situ generation and action of T-cell-replacing factor during the anamnestic response to dinitrophenyl keyhole limpet hemocyanin in vitro.

The involvement of a nonantigen-specific T-helper factor in the anamnestic immune response to dinitrophenyl keyhole limpet hemocyanin is demonstrated employing cultures of unseparated spleen cell populations. In such cultures of primed and boosted spleen cells, a good IgG anti-DNP response could be obtained if the hapten was presented on a heterologous carrier, provided that the homologous carrier was added simultaneously. T-cell depletion and reconstitution experiments show that such a factor, presumably identical with T-cell-replacing factor is produced by primed helper cells upon rechallenge and helps primed B cells, stimulated by soluble heterologous carrier hapten conjugates, to become IgG-secreting cells.

Reaginic Antibody Production to <i>Ascaris suum</i> Allergen, ASC-1

Wistar/Furth rats were immunized with DNP-Asc-1 in various hapten-to-carrier ratios. The kinetics and intensity of the anti-DNP and anti-carrier IgE responses were dependent on the extent of carrier substitution. Lightly substituted conjugates induced excellent primary antihapten and anticarrier IgE antibodies while heavily substituted conjugates induced an early, short-lived and low antihapten response, but not anticarrier antibodies. The pattern of IgG antibody production was not dependent on the degree of substitution in the ranges tested. A single injection of carrier in CFA induced suppression of IgE antibodies following an injection with hapten-carrier conjugate, but enhanced the IgG antibody response to the hapten. Preimmunization or supplemental immunization with carrier in Al(OH)<sub>3</sub> suppressed the IgE anti-DNP response but did not prevent the appearance of anticarrier antibodies. The suppression of the antihapten IgE response was also demonstrated in an adoptive transfer system when donors of carrier-primed spleen cells were injected with carrier in CFA or with carrier in Al(OH)<sub>3</sub>. On the other hand, excellent collaboration between carrier-primed helper cells and hapten-primed antibody-forming cell precursors was demonstrated for both IgE and IgG antibody production when donors of either carrier-primed or of hapten-primed cells received <i>Bordetella pertussis</i> as adjuvant. The results are consistent with the concept that intrinsic differences between the induction patterns of the IgE and IgG antibodies exist and that various factors contribute each in a different fashion to the dissociation of these responses.

The antibody response during amphibian ontogeny

The maturation of the anti DNP-response in adult and larval amphibians has been compared in two species of anurans,Rana catesbeiana andXenopus laevis. IgM responses were very similar in larvae and adults of both species. Specific IgG antibody synthesis, studied inRana catesbeiana only, was not obvious in larval primary response but was easily detectable in adults. Although metamorphosis results in profound changes in the lymphoid system associated with the apparent acquisition of self tolerance to new antigens, immunological memory of antigens injected during larval life ofRana andXenopus persists after the metamorphosis period.

Maturation of B Lymphocytes in Rats

Abstract We examined the ability of large and small thoracic duct cells obtained from Lewis rats primed to DNP to restore the adoptive secondary anti-DNP response in irradiated syngeneic hosts given an excess of T helper cells. The large cells were four to five times more active on a per cell basis than were the small cells. However, the large cells constitute only 7 to 8% of the thoracic duct cells and, therefore, make a minor contribution to the restorative activity of the unfractionated cells. Pretreatment of thoracic duct cell donors with high specific activity 3H-TdR for 48 hr before cannulation markedly reduced the memory B cell activity of the large cells, but had little effect on that of small cells. In addition, the activity of the large cells diminished with time after primary immunization, but that of the small cells remained stable. Immunofluorescent staining of the large and small cells for surface IgM and IgG, and subsequent sorting on the fluorescence-activated cell sorter (FA CS), showed that surface IgM was present on large memory B cells, and that IgG was present on small memory B cells. The experimental results suggest that two subpopulations of memory B cells in the thoracic duct lymph differ by size, rate of turnover, persistence after primary immunization, and class of surface IgG.

Anti-DNP response in gibbons (Hylobates lar)

Anti-DNP response in gibbons (Hylobates lar)

Anti-DNP response in gibbons ( Hylobates lar)

The nature of antibody response in gibbon ( Hylobates lar) following immunization with dinitrophenylated bovine gammaglobulin (DNP 50-BGG) was investigated. Anti-DNP of the IgG class was purified by immunoadsorbent technique on a DNP-Sepharose column. The average intrinsic association constant ( K 0) was determined by the fluorescence quenching technique. The K 0 value of antibody formed during the course of the immune response varied from less than 10 3 to more than 10 81/mole. The biological activities of these antibody preparations, i.e. the ability to agglutinate passively sensitized sheep red blood cells, to activate the complement system, and to induce a PCA response, changed in the same general direction as the K 0 value. However, analysis of the data failed to disclose a direct proportional relationship between these parameters and the K 0 value since a large change in the average intrinsic association constant is associated with a smaller magnitude of the change in biological activity.

Effect of anti-carrier antibody on carrier-determined tolerance.

These experiments were originally designed to determine whether an anti-carrier antibody, e.g., anti-allotype could break hapten-specific tolerance in vivo. Tolerance to 2,4-dinitrophenyl (DNP) was induced in C57BL/6J mice using DNP-BALB/c IgG2a conjugate. When anti-allotype serum was injected in C57BL/6J mice one day after a single injection of DNP-IgG2a the mice were not tolerant. In contrast, when tolerance was induced by four weekly injections of tolerogen, the anti-allotype serum had no effect on the tolerant state. This effect was specific for tolerance-inducing carrier. Anti-carrier antibody injected in C57BL/6J mice one day after DNP-IgG2a produced a small but significant anti-DNP response without administration of the immunogen, whereas the tolerogen (DNP-IgG2a) by itself was not immunogenic. Similarly, despite multiple injections of DNP-IgG2a bearing the foreign allotype, only one out of 7 C57BL/6J mice showed a weak anti-carrier response. In contrast, a marked anti-carrier (IgG2a) response was obtained when the anti-allotype antibody was passively administered in C57BL/6J mice. In conclusion, these experiments suggest that tolerance to an antigenic determinant may be broken by an antibody directed not to this determinant, but to another on the same molecule. The significance of this finding in relationship to the mechanism of the carrier-determined tolerance and the breakdown of self-tolerance is discussed.

Ontogeny of B-lymphocyte function. II. Ability of endotoxin to increase the heterogeneity of affinity of the immune response of B lymphocytes from fetal mice.

The ontogeny of the functional capacity of B lymphocytes to generate a heterogeneous response to a haptenic determinant was studied by cell transfer techniques in LAF1 mice. Fetal liver, as a source of B lymphocytes, was transferred into adult, syngeneic, irradiated animals. All recipients received excess adult thymus cells so that T-cell activity did not limit the response and were immunized with DNP-BGG. The heterogeneity of avidity of their anti-DNP PFC response was assayed by hapten inhibition of plaque formation. Animals reconstituted with B lymphocytes from fetal donors produced a response that is highly restricted with respect to heterogeneity of affinity. Transfer studies using multiple fetal donors or mixtures of adult and neonatal cells for reconstitution suggest that the restriction in heterogeneity is not the consequence of suppressor T-lymphocyte activity. With animals reconstituted with B cells from day 16 or older fetal donors, injection of LPS together with antigen converted the response to a heterogeneous "adult-type" response. With animals reconstituted with B lymphocytes from day 14 fetal liver DxSO4, but not LPS, could convert the response to a highly heterogeneous one. Animals reconstituted with day 14 or 16 fetal liver as source of B lymphocytes were capable of producing a heterogeneous secondary response despite the fact that their primary response was of restricted heterogeneity. This implies the selection of high affinity B-memory cells, in the absence of high affinity PFC during the primary response with fetal B lymphocytes. Animals reconstituted with day 14 or 16 fetal liver produce only direct PFC, while animals reconstituted with day 18 fetal liver produce both direct and indirect PFC. Three differentiation events have therefore been defined in the functional development of B lymphocytes: (a) between day 14 and day 16 of fetal life they acquire responsiveness to LPS; (B) BETWEEN DAY 16 AND 18 OF FETAL DEVELOPMENT THEY ACQUire the capacity to produce indirect PFC; (C) between day 7 and 10 after birth they acquire the capacity to give a heterogeneous response after normal immunization. In addition, it was shown that LAF1 mice already have all of the information required to produce an "adult-type" heterogeneous anti-DNP response at day 14 of fetal life.

The immunological properties of haptens coupled to thymus-independent carrier molecules. IV. The IgG response to dinitrophenylated Ficoll.

Dinitrophenylated polysucrose (DNP-Ficoll) elicits T cell-independent IgM anti-DNP antibody formation in mice. This antigen also elicits a heterogeneous IgG1 and IgG2 anti-DNP response, which is operationally as T-independent as the IgM response. However, a concomitant graft-versus-host reaction markedly enhances the IgG response (allogeneic effect). These results confirm those of others, indicating that a certain proportion of the precursors of IgG-producing cells can be triggered by some T-independent antigens. However, our results suggest that even with such antigens optimal triggering of IgG precursors requires T cell help.

Ineffectiveness of Lipopolysaccharide for Preventing the Tolerance Induction in Bone Marrow-Derived Lymphoid Cells with Dinitrophenyl-Poly-l-(Glutamic Acid, Lysine)

The effect of endotoxin or lipopolysaccharide (LPS) on tolerance induction in bone marrow-derived lymphoid cells (B cells) was investigated. Dinitrophenylated amino acid copolymer-L-(glutamic acid, lysine) (DNP-GL) acts as a potent tolerogen on normal and DNP-primed B cells. LPS significantly enhanced the anti-sheep red blood cell plaque-forming cell (anti-SRBC PFC) response that occurred after the immunization with a low dose of SRBC. LPS did not induce the primary anti-DNP PFC response after the injection of DNP-GL, nor did it prevent the tolerance induction in normal and DNP-primed B cells that occured after the administration of DNP-GL.

The class of surface immunoglobulin on cells carrying IgG memory in rat thoracic duct lymph: the size of the subpopulation mediating IgG memory.

The fluorescence activated cell sorter was used to determine the class of immunoglobulin on the thoracic duct lymphocytes (TDL) which carried IgG memory. Although only about 3% of all TDL carried membrane IgG these cells accounted for most, if not all, of the adoptive IgG anti-DNP response. It is concluded that both CR+ and CR- cells mediating IgG memory in rat TDL bear the same class of membrane immunoglobulin as that secreted by their differentiated progeny. The class of membrane immunoglobulin on CR+ and CR- rat TDL was also examined. It was found that IgM+ cells, which made up over 80% of all Ig+ cells, were virtually all CR+. In contrast, the few percent of IgG+ and IgA+ cells present were to be found in both subpopulations. There was no evidence of a large population of B cells bearing exclusively heavy chains other than IgA, IgG, of IgM. The observation that some IgG+ cells as well as IgM+ cells possess a receptor for C3 appears to rule out the hypothesis that this receptor is involved in blocking a switch from IgM to IgG synthesis.

An improved autoradiographic technique for the detection of antibody-forming cells

An autoradiographic technique for the detection of antibody-forming cells has been developed for the assay of anti-DNP responses. The lymphoid cells suspension to be assayed was allowed to sediment on to a glass slide coated with DNP-conjugated gelatin to which the secreted antibody bound during subsequent incubation. The bound antibody and its Ig class was revealed by a second incubation using 125I-anti-immunoglobulin reagents followed by autoradiography. Studies on the sensitivity and specificity of the method are presented and its advantages over other techniques described. The technique should be readily applicable to other haptens.

Differences between virgin and memory IgM-antibody-forming-cell precursor B-cells, and correlations with the heterogeneity present in B-cell populations from unimmunized mice

Previous in vitro studies showed that a large proportion of DNP-reactive B-cells in the spleens of unimmunized mice, unlike B-cells reactive to fowl gamma globulin (FGG), did not adhere to glass-bead columns. B-cell reactivity was assessed by challenge with dinitrophenylated-polymerized flagellin (DNP-POL) or FGG in the presence of POL, both responses being thymus-independent. Now we have shown that when donor mice are immunized with FGG, a greater proportion of FGG-reactive B-cells becomes non-adherent and the column filtrates give an IgM anti-FGG response. This indicates then that the adherence properties of the IgM-producing antibody-forming-cell (AFC)-precursor differed in virgin and immunized B-cell populations. In vitro testing of the thoracic duct cell (TDC) population of normal and immunized mice revealed parallels between thoracic duct B-cells and the non-adherent splenic B-cell population. Thus while the in vitro anti-DNP response of thoracic duct lymphocytes from normal mice reached levels little below those of spleen cells, the anti-FGG response was much lower. However when TDC were taken from mice immunized with FGG, the anti-FGG responses were much higher. Thus a parallel was demonstrated both in terms of non-adherence to glass and presence in the thoracic duct, of a portion of the DNP-reactive B-cell population in unimmunized mice and the FGG-reactive population in FGG-immunized mice. This suggested that the non-adherent DNP-reactive B-cells in unimmunized mice may represent B-cells with experience of cross-reacting antigens. The significance of this heterogeneity of the B-cell populations reactive to certain antigens in unimmunized mice is discussed with particular reference to recent apparently contradictory findings in regard to B-cell activation.

Immunological properties of dinitrophenyl derivatives of beta-subunit of the human chorionic gonadotropin

Four different derivatives with varying proportions of dinitrophenyl (DNP) groups tagged covalently to β-subunit of HCG (DNP (1)-β-HCG, DNP (3.3)-β-HCG, DNP (6)-β-HCG and DNP (10)-β-HCG) have been prepared. Their immunogenicity as well as the reactivity of antibodies formed in mice, rabbit and rhesus monkey have been studied. The derivatives give rise to the formation of antibodies in all species tested which react with HCG and in some species with DNP moieties. The anti-DNP response increases with derivatives bearing more than 3–4 DNP groups per molecule of β-HCG. On the other hand the anti-HCG response diminishes with derivatives bearing higher than 3–4 DNP groups per β-HCG molecule. The antibody response in monkeys is of long duration (> 14 months). The association constants for binding with HCG are of the order of 4 × 10 9 to 6 × 10 10 L/mole. the affinity of the antibodies tends to increase with time.