Quantity and Quality of Anti-Hapten Antibodies in Normal and in T Cell-Deprived Mice Studied at the Cellular Level

The Immune response of normal mice as well as of thymus-deprived (TxB) mice against 2 different hapten-protein conjugates (NNP-BSA and NNP-CG) was tested at various times after immunization at the cellular level. Normal mice developed both direct and indirect plaque-forming cells against the hapten, as determined by a modified local haemolysis in gel assay for detection of anti-hapten antibody-secreting cells. However, TxB mice produced direct plaque-forming cells to the same extent as normal mice, whereas the indirect plaque-forming cell response was greatly impaired. In fact, significantly elevated levels of indirect plaque-forming cells were not detected in such mice. Studies on the affinity of secreted antibodies by means of hapten inhibition revealed that the indirect plaque-forming cells derived from normal immunized mice could be inhibited by decreasing concentrations of free hapten with time after immunization, indicating a gradual increase in the number of cells secreting antibodies of high affinity. No such change was detected in normal or TxB mice with regard to antibodies giving rise to direct lysis, presumably IgM antibodies.     

Hapten-Reactive T and B Mouse Lymphocytes

The kinetics of development of anti-hapten-Kactive T and B cells in groups of mice immunized against different dotes of two hapten-protein conjugates was measured at different times. after a primary immunization with a single batch of conjugated sheep erythrocytes. In addition, the affinity of the receptors on the specific antigen-reactive cells was determined. Immunogenic doses of NNP-HGG varied from 40_μg to 2mg, whereas 0.4_μg even with adjuvant were nonimmunogenic .The affinity of antibodies secreted by single cells was measured by a modification of the local hemolysis in-gel assay with different concentrations of free hapten incorporated in the .agar. which showed a marked shift in affinity for i.PFC in contrast to d.PFC with time The affinityof receptor bearing antigen-binding cells of both T and B origin was also Studied by a hapten inhibition assay With the aid of specific antisera against T and B spleen lymphoid cells. pure population of T or B cells could be obtained from spleens of mice immunizedwith different hapten-protein conjugates .The binding strength of antigen-binding B cells increased with time after immunization, whereas that of antigen-binding T cells did not. The results are discussed in the light of recent studies on the structure of the T-cell receptor     

T-cell-dependent oscillations of IgM antibody affinity during the immune response to DNP-Dextran to low or high epitope density.

Anti-hapten IgM antibody response and affinity were evaluated by haemolytic plaque inhibition assay on spleen cells from mice immunized with 2,4, dinitrophenyl (DNP)1.3-Dextran or DNP13-Dextran. Regardless of epitope density, affinity was found to mature with time after immunization and to be characterized by rapid oscillations independent of changes in anti-hapten plaque-forming cell (PFC) response and antibody secretion rate. Injection of the lower epitope density immunogen not only elicited higher PFC responses and higher affinity antibodies but also induced more pronounced affinity oscillations mainly confined to the higher affinity PFC subpopulation. Immunization of athymic nude mice with DNP1.3-Dextran elicited PFC responses comparable to those observed in similarly immunized euthymic mice. However, affinity oscillations were drastically reduced in athymic mice and the restricted variation of antibody affinity observed shortly after immunization was followed by no oscillations in any of the affinity PFC subpopulations. Athymic mice did not produce high affinity PFC which account to a large extent for the affinity oscillations observed in euthymic mice. These findings demonstrate the important role of T cells in the appearance of rapid changes in IgM antibody affinity. The generation of T-cell-dependent oscillations of high affinity antibody-producing cell subpopulations is discussed in terms of interactions among cells and soluble factors involved in regulatory circuits of the immune network.     

Anti-hapten response in vitro. Affinity differences in precursors of anti-hapten antibody-producing cells

To study affinity differences in precursors of anti-hapten antibody-forming cells, an anti-hapten response was induced in mouse spleen cell cultures. The response was measured as specific high and low affinity plaque-forming cells. Selective inhibition of high affinity response was achieved both by free hapten, and by removal of high affinity precursor cells as rosette-forming cells. The data suggest a clonal predetermination of specific precursor cells to different affinity classes.     

Specificity of Hapten-Reactive T and B Mouse Lymphocytes

Immunization of mice with hapten-protein conjugates leads to the appearance of increased numbers of hapten-specific antigen-binding cells of both T and B origin. as well as to the formation of anti hapten antibody-secreting cells. The affinity of cellular receptors of hapten-reactive T and B cells was studied after immunization with different antigen doses at different time intervals. It was found that the affinity of secreted antibodies of the IgG class as well as of B-cell receptors for hapten was dose- and time-dependent. The affinity of hapten-reactive T cells for the hapten itself was low, irrespective of dose and time. Binding strength of T-and B-cell receptors was also investigated with the aid of mono- and multivalent homologous and heterologous hapten-carner complexes. The affinity of T cells for the homologous monovalent ligand was considerably higher than for the hapten itself or a heterologous monovalent ligand. indicating carrier contribution to T-receptor affinity. The contribution of carrier to the affinity of 7S antibodies or B-cell receptors was relatively small The avidity of hapten-specific cells for multi valent ligands was high in both T and B cells, but the increment in avidity with such ligands compared to monovalent ligands was less pronounced in T cells     

Effect of the number of haptens coupled to each erythrocyte on haemolytic plaque formation

We varied the density of a hapten coupled to sheep erythrocytes (SRC), and used different hapten—erythrocyte conjugates to study single antibody forming cells from rabbits. Both direct and indirect (developed by anti-IgG) plaques were seen. Hapten density influenced both plaque number and size. With aliquots of the same lymphoid cell suspension heavily coupled SRC yielded more direct and more indirect plaques than lightly coupled SRC. This tendency decreased with continuing immunization. Free hapten inhibited the formation of plaques more easily in lightly than in heavily coupled erythrocytes. Direct plaques were more dependent on a dense epitope coat than indirect ones. Our data indicated, that cells producing IgG antibody could produce direct plaques.

To explain our data we suggest that the product of some anti-hapten producing cells needs the collaboration of several combining sites to bind efficiently onto the hapten—SRC. They are dependent on a dense coat. The product of other cells has a higher affinity per site and binds efficiently onto the lightly coupled SRC.

     

Dose- and time-dependent changes in the binding capacity of IgM antibody

Past experiments have revealed antigen dose-dependent changes in IgG but not in IgM affinity. This is of importance to the general validity of the receptor concept and led us to investigate IgM affinity as a function of antigen dose and of time after antigen administration. Rabbits were immunized with proteins conjugated with p-azobenzene arsonate and the hapten response was assessed by plaque formation. Dose- and time-dependent changes in binding properties of IgM were determined by inhibition with hapten. The quantity of hapten which led to inhibition of 50% of the plaques, I₅₀, was independent of the amount of hapten antibody in the system. All direct plaques could be inhibited by antibody directed against μ chains, but were not inhibited by antibody against γ chains. The amount of antibody against μ chain which inhibited 50 % of the plaques was the same in the early as in the late plaque-forming response. In the initial stages of antibody response, there was no change in I₅₀, over a considerable range of immunizing doses, including doses sufficient to trigger a plaque-forming response, but insufficient to maintain it. Dose related changes in IgM affinity were observed fifteen days after antigen injection. In general, the avidity of IgM plaque-forming antibody increased with time. However, at a very high dose of antigen, there were no changes in average affinity. After a second administration of antigen, plaque-forming cells were detected earlier than in the primary response, though not in greater numbers. The first plaque forming antibody to be detected was of relatively high affinity; antibody detected subsequently was of the same affinity as the antibody formed in the primary response. Results were discussed in terms of cooperation between receptor binding sites and in terms of interference with this cooperation when antigen antibody complexes trigger antibody formation.     

Studies on the control of antibody synthesis. VII. Change in affinity of direct and indirect plaque-forming cells with time after immunization in the mouse: loss of high affinity plaques late after immunization.

The change in avidity of anti-hapten antibody with time after immunization was studied in mice at the level of the antibody-forming cell. A progressive increase in avidity was seen in both direct and indirect plaque-forming cells. Late (38 days) after immunization with a large dose of antigen there was a preferential loss of high avidity plaque-forming cells and the average avidity decreased. High avidity memory cells were still present since boosting resulted in the prompt appearance of very high avidity plaque-forming cells. There was a strong positive correlation between the avidity of the direct and the indirect plaque-forming cells present in the same spleen. A pattern of change in avidity and heterogeneity of avidity similar to that observed with intact animals was seen in lethally irradiated mice reconstituted with normal spleen and thymus cells.     

Analysis of affinity of monoclonal antibody responses by inhibition of plaque-forming cells

It has been suggested that dominant monoclonal responses in mice after transfer of small numbers of spleen cells are the result of strong selection for cell clones producing antibody of high affinity. We have attempted to measure the affinity of anti-DNP (2,4? dinitrophenyl) and anti-NIP (4? hydroxy? 5? iodo? 3? nitrophenacetyl) antibodies produced in monoclonal responses by inhibition of plaque-forming cells (PFC) with free hapten. PFC from individual monoclonal spleen foci and from spleens after adoptive transfer of 5 × 10⁵ to 2 × 10⁶ primed spleen cells were generally inhibited at lower free antigen concentrations than the PFC found in a heterogeneous secondary response. It was possible to compare several individual cell clones, forming monoclonal antibody to DNP, with respect to the free antigen concentration needed for 50 % plaque inhibition. These comparisons did not correlate well with the relative affinities of serum antibody estimated by the Stupp-Farr technique. Published work analyzing PFC from plasma cell tumor MOPC 315 forming antibody to DNP might have predicted that the curve of PFC inhibition by free hapten for a monoclonal PFC population would be steep. Usually this was not the case, and 20–80 % PFC inhibition occurred over 100-fold concentration range of free hapten. Some of the anti-DNP clones formed large plaques and when these PFC were inhibited by free antigen, the plaque diameter decreased progressively as the hapten concentration increased. Large plaques consequently require more free antigen for inhibition than small plaques. The poor agreement between apparent affinity measured by plaque inhibition and that measured in serum antibody for monoclonal antibody responses may well be a result of the observed differences in plaque size between individual clones. We therefore have strong reservations about the use of plaque inhibition by free antigen to estimate antibody affinity at the cellular level.     

T cells specific to hapten carrier but not to carrier alone assist in the production of anti-hapten and anti-carrier antibodies

We examined the immune response of Balb/c mice to antigens prepared by conjugating 2-phenyloxazolone (phOx) to a foreign protein, ovalbumin (OVA), or a self-protein, mouse serum albumin (MSA), in order to study how these chemical modifications would affect immune recognition. We found that anti-OVA antibodies and CD4(+) T cells produced by OVA immunization reacted with OVA as well as with phOx-OVA. Anti-phOx antibodies were produced by phOx-OVA immunization and, interestingly, T cells from these mice reacted only with phOx-OVA but not with the intact OVA. These results suggested that the classical model of hapten-carrier immunization, in which B cells specific to hapten are activated with assistance from T cells specific to a carrier protein, might not be a major route for production of anti-hapten antibodies in hapten-carrier immunization. Furthermore, phOx-MSA immunization induced production of anti-phOx antibodies, which could not be accounted for in terms of the assistance of carrier-specific T cells because of the absence of MSA-specific T cells. Therefore, we proposed a new model in which anti-hapten B cells are assisted by T cells specific to the haptenated carrier.     

Affinity-immunoadsorbent fractionation of rat anti-streptococcal a carbohydrate antibodies of restricted heterogeneity

Antisera obtained from selectively bred Sprague-Dawley rats after a primary series of immunization with group A streptococcal vaccine exhibit specific anti-carbohydrate anti-bodies of restricted heterogeneity. Separation of anti-carbohydrate antibodies was achieved on the basis of differences in the relative binding affinities of the antibodies for an insoluble, hapten (N-acetyl-D-glucosamine) immunoadsorbent. Evaluation of several eluting reagents including hapten, thiocyanate, urea and acid-salt demonstrated the chaotropic ion thiocyanate to be most effective in separating anti-hapten populations of individual affinity characteristics. In general, there was no consistent relationship between the net electrical charge of an antibody and its relative binding affinity for the immunoadsorbent. Furthermore, non-precipitating rat anti-streptococcal A carbohydrate antibodies were characterized as antibodies of relatively low binding affinity for the hapten immunoadsorbent. From a practical point of view, thiocyanate is recommended for the routine elution of anti-carbohydrate antibodies from immunoadsorbents.     

Dynamics of lymphocyte receptor affinity for hapten during the primary immune response

Changes in affinity of antigen-binding receptors of lymphocytes for dinitrophenol (DNP) were studied during the primary immune response in CBA mice. The process was evaluated by the method of inhibition of rosette formation with DNP-ovalbumin-sheep's red cells complex by hapten (DNP-ε-lysine). An increase in affinity of immunoglobulin receptors on the lymphocyte surface for DNA was shown, starting from the 10th day after immunization. The question of the role of affinity and density of receptors on the surface of the immunocyte is discussed.     

Antibody production in mice: V. The suppressive effect of anti-carrier antibodies on cellular cooperation in the induction of secondary anti-hapten antibody responses

Cooperative induction of secondary anti-hapten antibody responses was studied by using non-cross-reactive carrier proteins, bacterial α-amylase (BαA), Taka-amylase A (TAA) and keyhole-limpet haemocyanin (KLH), and the 2,4-dinitrophenyl (DNP) and benzylpenicilloyl (BPO) groups as haptenic determinants.

Lymphoid cells obtained from mice primed with a hapten-carrier conjugate could be effectively stimulated with a hapten-heterologous carrier conjugate, provided that lymphoid cells primed to the heterologous carrier were also present. In the carrier-primed lymphoid cell population, helper activity of thymus-derived cells developed earlier following carrier immunization than did the capacity of antibody-forming cell precursors (AFCP) to produce an effective anti-carrier antibody response upon secondary stimulation. Attempts to generate hapten-specific helper cell activity were unsuccessful. Thus, cells primed to one haptenic determinant failed to exert a helper function with cells primed to a second hapten upon subsequent administration to the two haptens together on the same heterologous carrier molecule.

In order to distinguish among carrier determinant specificities which react with thymus-derived helper cells from those which react with bone marrow-derived AFCP, the capacity of various anti-carrier antibodies or antibody fragments to suppress either anti-carrier antibody production alone or together with helper cell function in adoptive secondary anti-hapten antibody responses was tested. In this system, it was found that 7S anti-carrier antibody suppressed the reaction of helper cells and carrier-specific AFCP such that both the anti-hapten and anti-carrier antibody responses were abrogated. By contrast, passively administered 3.5S fragments of anti-carrier antibodies selectively prevented the stimulation of carrier-specific AFCP to produce anti-carrier antibodies, but had no effect on the capacity of carrier-specific helper cells to facilitate the secondary anti-hapten antibody response. As expected, passively administered 7S anti-hapten antibodies selectively abrogated the production of anti-hapten, but not anti-carrier antibodies. These data are discussed in the context of suggesting that distinct determinant sites on carrier molecules are recognized independently by thymus-derived helper cells and by bone marrow-derived AFCP.

     

Thymus dependency in anti-trinitrophenyl (TNP) binding responses in the spleen of Ambystoma mexicanum effects of thymectomy and anti-thymocyte serum treatments

In Ambystoma mexicanum the production of a response to the hapten trinitrophenyl (TNP) requires specific carrier preimmunisation. Thymectomy or the administration of an anti-thymocyte serum abrogates the anti-TNP response. Two populations of lymphoid cells appear to interact in the production of anti-hapten antibodies in Ambystoma: one is specific for the carrier, helping the second to synthesize anti-hapten antibodies.     

Inhibition of plaque-forming cells with anti-idiotope or hapten: variation due to hapten density on indicator red cells

Phosphorylcholine (PC)-specific antibody plaque-forming cells (PFC) were enumerated in the spleen of BALB/c mice immunized with S. pneumoniae R36a bacterial vaccine. Two indicator red blood cells were compared: RBC coupled with the hapten, PC-RBC, and cells coupled with PC-bearing polysaccharide from the pneumococcus, PnC-RBC. Equal numbers of direct (IgM) PFC were detected with both types of indicator cells. However, a significant difference appeared in the attempt to inhibit the respective PFC either with hapten (monovalent PC chloride, PCCl) or with monoclonal antibodies against T15 idiotopes (anti-Id). At optimal coupling concentrations, inhibition of anti-PnC-RBC plaques required a 10-fold higher concentration of the hapten when compared to anti-PC-SRBC plaques. Also, the inhibition of anti-PnC-SRBC plaques with anti-Id required much higher concentration of the antibody. The phenomenon may be explained by a higher epitope density on PnC-RBC than on PC-RBC. Heavily coupled PC-RBC were more difficult to inhibit (much like the PnC-RBC) by either the hapten or the anti-Id than lightly coupled PC-RBC. A mathematical analysis of the experimental curves supports the notion that under the assay conditions used, inhibition by either the hapten or the anti-Id is influenced primarily by antibody secretion rate and epitope density on indicator cells. If the 2 variables are too high, a significant inhibition of PFC with a low affinity anti-Id may not be possible. The theory also predicted that addition of a small amount of hapten into the assay would be roughly equivalent to secretion rate reduction and would facilitate the plaque inhibition by anti-Id. Indeed, we show a synergism between a minute (non-inhibitory) amount of PCCl and anti-Id in the inhibition of PC-specific PFC. These results point out that the detection of an idiotype-bearing PFC by plaque-inhibition assay is greatly influenced by technical variation in the assay, in particular, the preparation of the indicator red cells.     

Selective inhibition of the secondary response of primed cells by incubation with hapten

Cells from a mouse immunized with a hapten-carrier conjugate were incubated in vitro with antigen. They gave a secondary antibody response when transferred to syngeneic irradiated mice. The response was partly inhibited if free hapten was added to the incubation mixture. The inhibition was greatest at low antigen concentrations, and in these circumstances production of IgG anti-hapten antibodies was inhibited more than that of IgM antibodies. At nearly all antigen concentrations studied antibodies to the immunizing hapten were inhibited more than those that reacted with a related hapten. With respect to hapten inhibition the characteristics of memory cells resemble those of free antibody.     

Studies on the control of antibody synthesis. XVII. Effect of specific suppressor cells on the affinity of the antibody response by naive or primed lymphocytes.

A comparison was made of the effects of carrier-specific helper T-cell tolerance and carrier-specific suppressor cells on the affinity of the anti-hapten PFC produced in both a primary and secondary anti-hapten response. Carrier-specific suppressor T cells caused a striking preferential loss of high affinity PFC in the primary response, but had only a slight effect on the affinity of the anti-hapten PFC formed in the secondary response. A carrier-specific state of tolerance, induced at a dosage which was shown not to generate significant suppressor cell activity, was associated with minimal alterations in the affinity of the primary anti-hapten PFC response. Carrier specific tolerance, induced in animals which had been previously primed to the hapten on a different carrier, had little or no effect on the affinity of the PFC response.     

Suppression of the anti-hapten IgE antibody response with hapten-modified spleen cells

Spleen cells of BALB/c mice were chemically modified with phosphorylcholine or benzylpenicilloyl hapten. The i.v. administration of such cells into syngeneic animals suppressed the formation of specific IgE antibodies against the respective hapten. The IgE antibody response against ovalbumin, which was used as an immunogenic carrier for the haptens, was not affected and the anti-hapten IgG or IgG1 response remained at the levels of the controls. The suppression could be transferred into X-irradiated mice by T cells from tolerized animals. Moreover, it was demonstrated that not only the induction of IgE, but also an established anti-hapten IgE antibody response is accessible to suppression by treatment with hapten-modified spleen cells from syngeneic animals. The results indicate that the i.v. administration of antigen coupled to syngeneic spleen cells induces T cells which suppress the formation of specific IgE antibodies in the primary and the secondary response without significantly affecting the formation of IgG antibodies.     

Polarity of immunogens: implications for vaccine design.

Peptide constructs have been engineered consisting of amino acid sequence determinant recognized by T cells (TD) co-linearly linked to haptenic peptides. It was found that high anti-hapten antibody titers were induced after immunization with those constructs which had the TD sequence in the N-terminal position with respect to the hapten. Low or zero titers were elicited when the TD was in C-terminal position. Also, a high anti-hapten antibody titer corresponded to a low or zero anti-TD antibody titer and vice versa. These results suggest that immunogens are polar and stress the relevance of searching the more adequate position of the TD within a peptide construct when designing immunogens or synthetic peptide vaccines.     

Immune Response in the Bovine Mammary Gland After Intestinal, Local, and Systemic Immunization

The immune response in mammary glands of cattle was measured after intestinal, local, and systemic immunization with T4 bacteriophage. Nonlactating pregnant cows were immunized by infusions into the intestine or mammary gland and by subcutaneous injections in the region of the prescapular or external inguinal lymph nodes. Titers of antibodies of different isotypes were measured in serum and in lacteal secretions by enzyme-linked immunosorbent assay, and numbers of cells producing antibodies of each isotype were determined in lacteal secretions by the Jerne plaque assay. Substantial increases in immunoglobulin G subclass 1 (IgG1) and IgG2 antibody titers were detected in serum and lacteal secretions of animals immunized through an intestinal fistula. IgM and IgA antibody responses were low or undetectable. Low numbers of IgA and IgG1 plaque-forming cells were occasionally detected. It is proposed on the basis of these data that migration of antigen-stimulated IgG lymphoblasts, and perhaps of antigen, to spleen and peripheral lymph nodes may be dominant events after intestinal immunization of ruminants. This is consistent with the predominance of serum-derived IgG antibodies in colostrum and milk. Intramammary infusion of antigen gave rise to increases in antibody titers in all classes which were greater not only in lacteal secretions but also in blood serum than with either systemic route used. There was clear evidence from relative antibody titers for local synthesis of antibodies, principally IgA and IgG1, in the immunized glands. Comparison of IgA titers in secretions from the immunized glands with those in serum also suggested that locally synthesized IgA antibodies may have contributed in some measure to serum titers. Local synthesis in both immunized and nonimmunized glands was also reflected by the presence of increased numbers of IgA and IgG1 plaque-forming cells. It is hypothesized that antibody-forming cells responsible for local synthesis originated in lymphoid tissue within the mammary gland or from peripheral lymph nodes, depending upon the route of immunization.