Article

THE REQUIREMENT OF MORE THAN ONE ANTIGENIC DETERMINANT FOR IMMUNOGENICITY

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Abstract

Rabbits primarily stimulated with a BSA (bovine serum albumin)-sulfanilic acid complex will produce a good secondary response to the sulfanilic acid hapten if the carrier used in the secondary stimulus is again BSA, and not if the secondary carrier is HGG (human gamma globulin). In the latter situation, a good secondary response is obtained, however, if the rabbits are pretreated a few weeks earlier with free HGG. We conclude that the immune stimulus involves the recognition of carrier determinants unrelated to the hapten. As the receptors for recognition of unrelated determinants are probably situated on different cells, we suggest that the immune stimulus leading to antibody formation requires the interaction of two antigen-bridged cells.

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... They are derived from internal proteins of tumor cells. The immunogenicity of proteins requires the recognition of more than one antigenic determinant, as discovered 50 years ago [6]. It is likely that this also holds true for the immunogenicity of TAs and tumor cells. ...
... Tumor vaccine design also has to consider the question of directing the response against one or several TAs. One lesson from basic immunological studies is that one antigenic determinant is not sufficient for protein immunogenicity [6]. Two antigenic determinants, a hapten that is by a hapten-carrier presenting B cell and a pMHC class II restricted carrier determinant recognized by a Th2 cell, were the minimal requirement. ...
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The topic is how to achieve long-term protective anti-tumor immunity by anti-cancer vaccination and what are its mechanisms. Cancer vaccines should instruct the immune system regarding relevant cancer targets and contain signals for innate immunity activation. Of central importance is T-cell mediated immunity and thus a detailed understanding of cognate interactions between tumor antigen (TA)-specific T cells and TA-presenting dendritic cells. Microbes and their associated molecular patterns initiate early inflammatory defense reactions that can contribute to the activation of antigen-presenting cells (APCs) and to costimulation of T cells. The concommitant stimulation of naive TA-specific CD4+ and CD8+ T cells with TAs and costimulatory signals occurs in T-APC clusters that generate effectors, such as cytotoxic T lymphocytes and T cell mediated immunological memory. Information about how such memory can be maintained over long times is updated. The role that the bone marrow with its specialized niches plays for the survival of memory T cells is emphasized. Examples are presented that demonstrate long-term protective anti-tumor immunity can be achieved by post-operative vaccination with autologous cancer vaccines that are modified by virus infection.
... As expected, if those mice were challenged with TNP-RGG, IgM and IgG anti-TNPsecreting B cells were detected (data not shown). For a recall antibody response, the booster immunization need not be the same haptencarrier conjugate (TNP-RGG) used for priming, so long as the T cells were separately primed using the secondary carrier[35]. Hence, we hypothesized that OVA-specific memory cells would provide the appropriate help for memory B cells if the booster immunization was TNP-OVA. ...
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... Studies have shown that it is possible to obtain an antitumor response when only achieving microchimerism (<1% of donor cells) (20)(21)(22)(23)(24)(25). It is speculated that T-cell and natural killer (NK)-cell alloreactivity could generate immediate anti-leukemic effects that awaken the anti-tumor immunity of the host, change the tolerance of the host to the tumor, and allow the host to undergo an immune response to the tumor (24,(26)(27)(28)(29)(30). ...
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... The articles of Rajewsky et al. (3) and Mitchison (4) described the involvement of the cellular cooperation of carrier-specific T cells and hapten-specific B cells for anti-hapten antibody response to hapten-carrier conjugates. Besides the carrier specificity of helper T cells, these cells also strictly recognized MHC class II molecules on antigen-presenting cells. ...
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Chapter
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Chapter
It was known for a long time that certain immune reactions, particularly some that manifested themselves as skin swellings, were elicited by cells rather than soluble antibodies. For example, classical experiments of Chase in 1945 had shown that immunity of guinea pigs to tubercle bacilli, as tested by a skin reaction to tuberculin, was transferable to non-immune animals by lymph node cells but not by serum of immune animals1. This type of immunity had been termed “cellular immunity”, not knowing what it really was, to distinguish it from antibody-mediated “humoral immunity”. In his first paper on the transfer of transplantation immunity by lymph node cells in 1953 Mitchison commented “transplantation immunity shares with ... immunity to tuberculin the property of being transferred with greater facility by cells than by serum”2. In the absence of a paradigm that included immune mechanisms without antibodies, these observations were not followed by any sort of interpretation.
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Autoantibodies against myelin oligodendrocyte glycoprotein (MOG) are associated with autoimmune central nervous system diseases like acute disseminated encephalomyelitis (ADEM). For ADEM, it is speculated that a preceding infection is the trigger of the autoimmune response, but the mechanism connecting the infection to the production of MOG antibodies remains a mystery. We reasoned that the ability of B cells to capture cognate antigen from cell membranes, along with small quantities of coexpressed "bystander" antigens, might enable B-cell escape from tolerance. We tested this hypothesis using influenza hemagglutinin as a model viral antigen and transgenic, MOG-specific B cells. Using flow cytometry and live and fixed cell microscopy, we show that MOG-specific B cells take up large amounts of MOG from cell membranes. Uptake of the antigen from the membrane leads to a strong activation of the capturing B cell. When influenza hemagglutinin is also present in the membrane of the target cell, it can be cocaptured with MOG by MOG-specific B cells via the B-cell receptor. Hemagglutinin and MOG are both presented to T cells, which in turn are activated and proliferate. As a consequence, MOG-specific B cells get help from hemagglutinin-specific T cells to produce anti-MOG antibodies. In vivo, the transfer of MOG-specific B cells into recipient mice after the cocapture of MOG and hemagglutinin leads to the production of class-switched anti-MOG antibodies, dependent on the presence of hemagglutinin-specific T cells. This mechanism offers a link between infection and autoimmunity.
Chapter
Antibody formation results from contact of the antigen with antigen-sensitive cells of the immune system. It occurs either under natural conditions during the development of the individual, or after an artificial antigen administration, i.e. vaccination and immunization. In man, vaccination is mainly performed to induce protection against infectious diseases, whereas immunization of animals, besides conferring protection, is primarily used to obtain antibodies for experimental purposes (immune sera, antisera).
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Antigen-bridging models of the interaction between T and B lymphocytes rose to prominence in the late 1960s, based principally on two different experimental approaches1–3. One involved the “carrier effect”, in which physical association between hapten and carrier is required for a secondary anti-hapten antibody response in animals primed separately with hapten and carrier, and the other involved thymus-marrow reconstitution of irradiated animals, demonstrating that hapten and carrier were recognized by different cell types in the anti-hapten antibody response.
Chapter
The term antigen was originally employed to describe any substance capable of stimulating a specific immune response. However, its usage has been broadened in recent years to include any substance which may not by itself be able to produce immunity but is capable of reacting specifically with antibodies or immune lymphocytes present naturally or stimulated by a closely related antigen. The term immunogen, on the other hand, characterizes materials able to cause specific immunity in an appropriate host. Since these definitions are operational and depend on the response of a lymphoid system which encounters an immunogen rather than on intrinsic molecular properties of the substance itself, it is not surprising that antigens share very few properties. In fact, the only truly common property may be that there exists in the DNA code of some host information capable of dictating the synthesis of a protein capable of combining specifically with each antigen. Most antigens are naturally occurring macromolecules, especially proteins and polysaccharides, although antibodies reacting with lipids and nucleic acids do occur. However, much of the work carried out during the past 15–20 years on the nature of the immune response has employed small snythetic molecules since the specificity of the immune reactions to such compounds can be studied more precisely. These small nonimmunogenic haptens are capable of stimulating the production of specific antibody if they are covalently linked to an immunogen.
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Chapter
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Chapter
When an immunogen is injected into a higher organism, antibodies reacting specifically with that immunogen are produced. Since a vast number of antibody specificities exist, and since cogent evidence indicates that antibody specificity is determined by the primary structure of the antibody polypeptide chains, the question has to be asked of how antigen induces the structural genes coding for the “right” antibodies, and only these, to initiate protein synthesis.
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Fish are the earliest vertebrates which have a well-developed immune system characterized by both cellular and humoral branches endowed with specificity and memory. More specifically, since invertebrates apparently lack a molecule resembling the vertebrate antibody molecule (Bang, 1973; Shapiro, 1975), fish are the first group of animals which clearly possess an immunoglobulin component. In addition, although there is evidence indicating cellular function in recognition of non-self in invertebrates (reviewed in Cooper, 1976), again fish are the first group of animals where T-like and B-like lymphocytes may actually attain a level of specialization.
Chapter
Certain immunological responses can occur in the absence of thymus influence and thymus-derived lymphocytes (Miller and Osoba, 1967). The purpose of this chapter is to examine the evidence for the existence of such responses and to review what is known of their mechanism and their biological significance.
Chapter
In the past few years the concept of cooperation between cells for the induction of an immune response has been well established. It is generally believed that three cells are involved in the process: a lymphoid cell derived from the bone marrow, another lymphoid cell also derived from the bone marrow but “influenced” by the thymus, and a phagocytic cell: the macrophage. The thymus-bone marrow cells interaction is discussed in detail elsewhere in this symposium. Briefly, the “thymus derived” cell plays a role in recognising the antigen but does not secrete antibody. The “bone marrow derived cell” secretes antibody but needs the help of the “thymus derived cell” to do so.
Chapter
The virtually limitless number of foreign antigenic determinants that the immune system must be able to distinguish among and respond to allows for only a relatively small number of cells to be committed to recognizing any one determinant. Clonal expansion is therefore necessary to achieve amplification of the appropriate recognition unit. This process must be carefully regulated because of the severe consequences of excess proliferation of a single clone responding to one foreign determinant, and of the development of clones responding to self-determinants. As a result of this requirement for both amplification and precise regulation, there are a myriad of interactions, both positive and negative, that occur among the cells of the system. The nature and number of these interactions have been extremely hard to determine, principally because insufficient markers have been found to separate the interacting individual components of the system from one another for detailed analysis. Further, even when one of the system’s components is isolated and studied, a Heisenberg-like principle of uncertainty exists: one can never be sure that the isolated component behaves in the same fashion in isolation as it does when being stimulated by signals from other parts of the interacting system.
Article
Priming mice with a protein (bovine serum albumin or ovalbumin) prepared them for a markedly enhanced anti-hapten response to the conjugate of a hapten NIP (4-hydroxy-3-iodo-5-nitrophenacetyl) and the protein. The enhancing effect of this preimmunization was carrier-specific and dependent on the priming dose of the carrier. Thus, on day 6, anti-NIP titers were 6 to 240 times (p<0.01-<0.001) higher in groups primed with 0.01 to 10 mg of BSA and challenged with NIP-BSA than in groups not primed with the carrier. The optimum dose (0.1 to 1 mg) of BSA for an enhanced anti-NIP response also resulted in the highest secondary anti-BSA titers. The enhancing effect of the carrier priming was observed only at the beginning of the anti-NIP response. Corresponding findings were obtained when ovalbumin was used for preimmunization and as the carrier in the challenge. This dose-response relationship in mice was different from that found earlier in chickens. Small doses of carrier induced optimal priming in chickens. Priming with higher doses which induced high titers of anti-carrier antibody had a suppressive effect on the anti-hapten response in these birds.
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Sheep antisera specific for the three major immunoglobulin groups of the rabbit, i.e. IgG (gamma-chain), IgA (alpha-chain), and IgM (micro-chain), are each able to induce blast transformation of the peripheral lymphocytes of the rabbit when added to in vitro cultures. The per cent of lymphocytes transformed with each antiserum indicate that one-fourth of the peripheral lymphocytes carry or have the capacity to synthesize molecules of all three of the major immunoglobulin groups, and that the remaining three-fourths carry or have the capacity to synthesize only two (IgG and IgM). The data do not permit direct conclusions concerning the ability of a single cell to produce molecules belonging to more than one immunoglobulin group at the same time, or the ability of a given cell to make a transition from the synthesis of molecules of one immunoglobulin group to those of another group.
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The addition of sheep antisera to rabbit IgG or to rabbit IgG subunits (L chain, H chain, Fab piece, Fc piece) to in vitro cultures of rabbit peripheral lymphocytes may induce up to 80-90% of the small lymphocytes to transform into immature "blast" cells. As many as 1 x 10(7) molecules of antibody may be required to stimulate each cell. It is concluded that each peripheral lymphocyte carries the antigenic specificities of the entire IgG molecule and thus could carry, and may produce specific antibody molecules. Such a conclusion supports the theory that the small lymphocyte may be the site of specific recognition of antigens in the primary response.
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The number of discrete hemolytic foci and of hemolysin-forming cells arising in the spleens of heavily irradiated mice given sheep erythrocytes and either syngeneic thymus or bone marrow was not significantly greater than that detected in controls given antigen alone. Thoracic duct cells injected with sheep erythrocytes significantly increased the number of hemolytic foci and 10 million cells gave rise to over 1000 hemolysin-forming cells per spleen. A synergistic effect was observed when syngeneic thoracic duct cells were mixed with syngeneic marrow cells: the number of hemolysin-forming cells produced in this case was far greater than could be accounted for by summating the activities of either cell population given alone. The number of hemolytic foci produced by the mixed population was not however greater than that produced by an equivalent number of thoracic duct cells given without bone marrow. Thymus cells given together with syngeneic bone marrow enabled irradiated mice to produce hemolysin-forming cells but were much less effective than the same number of thoracic duct cells. Likewise syngeneic thymus cells were not as effective as thoracic duct cells in enabling thymectomized irradiated bone marrow-protected hosts to produce hemolysin-forming cells in response to sheep erythrocytes. Irradiated recipients of semiallogeneic thoracic duct cells produced hemolysin-forming cells of donor-type as shown by the use of anti-H2 sera. The identity of the hemolysin-forming cells in the spleens of irradiated mice receiving a mixed inoculum of semiallogeneic thoracic duct cells and syngeneic marrow was not determined because no synergistic effect was obtained in these recipients in contrast to the results in the syngeneic situation. Thymectomized irradiated mice protected with bone marrow for a period of 2 wk and injected with semiallogeneic thoracic duct cells together with sheep erythrocytes did however produce a far greater number of hemolysin-forming cells than irradiated mice receiving the same number of thoracic duct cells without bone marrow. Anti-H2 sera revealed that the antibody-forming cells arising in the spleens of these thymectomized irradiated hosts were derived, not from the injected thoracic duct cells, but from bone marrow. It is concluded that thoracic duct lymph contains a mixture of cell types: some are hemolysin-forming cell precursors and others are antigen-reactive cells which can interact with antigen and initiate the differentiation of hemolysin-forming cell precursors to antibody-forming cells. Bone marrow contains only precursors of hemolysin-forming cells and thymus contains only antigen-reactive cells but in a proportion that is far less than in thoracic duct lymph.
Article
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An injection of viable thymus or thoracic duct lymphocytes was absolutely essential to enable a normal or near-normal 19S liemolysin-forming cell response in the spleens of neonatally thymectomized mice challenged with sheep erythrocytes. Syngeneic thymus lymphocytes were as effective as thoracic duct lymphocytes in this system and allogeneic or semiallogeneic cells could also reconstitute their hosts. No significant elevation of the response was achieved by giving either bone marrow cells, irradiated thymus or thoracic duct cells, thymus extracts or yeast. Spleen cells from reconstituted mice were exposed to anti-H2 sera directed against either the donor of the thymus or thoracic duct cells, or against the neonatally thymectomized host. Only isoantisera directed against the host could significantly reduce the number of hemolysin-forming cells present in the spleen cell suspensions. It is concluded that these antibody-forming cells are derived, not from the inoculated thymus or thoracic duct lymphocytes, but from the host. Thoracic duct cells from donors specifically immunologically tolerant of sheep erythrocytes had a markedly reduced restorative capacity in neonatally thymectomized recipients challenged with sheep erythrocytes. These results have suggested that there are cell types, in thymus or thoracic duct lymph, with capacities to react specifically with antigen and to induce the differentiation, to antibody-forming cells, of hemolysin-forming cell precursors derived from a separate cell line present in the neonatally thymectomized hosts.
Article
Full-text available
A suspension of mouse spleen cells can be separated into two populations on the basis of their ability or inability to adhere to plastic dishes. It was found that both adherent and nonadherent cells were necessary for the induction of antibody formation to sheep red blood cells in vitro. Exposure of adherent cells to antigen for brief periods of time was sufficient to initiate a maximal in vitro response.
Article
Full-text available
Rabbit lymphocytes may be stimulated in vitro with specific antiallotype sera to transform into "blast" cells and to synthesize DNA. This transformation only occurs when the donor cells are obtained from a rabbit having a given γ-globulin allotype (As4) and these cells are cultured in the presence of an antiserum prepared against the given allotype (As4). Heterologous (sheep, goat, and guinea pig) anti-rabbit γ-globulin sera also induce significant blast transformation and DNA synthesis in rabbit lymphocytes. Allotypic transformation and DNA synthesis are due to 7S antiallotype antibodies and do not require complement. The degree of transformation and rate of DNA synthesis is related to the concentration of antibody. Incubation of the appropriate cells with the antiallotype antibody for as short a time as 15 minutes results in a significant degree of "blast" transformation, indicating that the recognition of the antiallotype specificity in the cells and stimulation of the cellular changes leading to eventual transformation is rapid. The activity of the antiallotype sera as measured by transforming or haemagglutinating capacity, may be absorbed by lymphocytes of the appropriate allotype, but is not absorbed by lymphocytes from a donor rabbit not having the allotype to which the antiserum is directed. Transformation does not occur with mixtures of lymphocytes from different rabbits even if 1 donor is immunized against an allotype present in the other donor. Peripheral rabbit lymphocytes can also be induced to undergo "blast transformation" in vitro by phytohaemagglutinin and staphylococcal filtrate. The lack of demonstrable leucoagglutinins in staphylococcal filtrate and antiallotype serum indicates that agglutination is not a necessary prerequisite to the induction of blast transformation.
Chapter
Some 50 years ago Landsteiner showed the feasibility of the carrier method for eliciting hapten-specific antibodies in suitable hosts. Since then numerous investigators have successfully applied this methodology to obtain antibodies against a vast number of different haptens (Landsteiner, 1945). In every instance, until recently, the haptens were conjugated to the carrier by covalent bonds, and with but one exception the haptens were small molecules. Goebel and Avery (1931) reported that a pneumococcal capsular polysaccharide, which is not immunogenic in rabbits when injected in chemically pure form, became immunogenic once it was conjugated chemically to a carrier protein. Plescia et al. (1964) first showed that a carrier need not be conjugated chemically to a hapten, that a carrier need only form a stable complex with the hapten. Methylated bovine serum albumin (MBSA), complexed electrostatically with denatured DNA or type III pneumococcal capsular polysaccharide, proved to be an effective carrier. Rabbits, immunized with these complexes, produced DNA and polysaccharide-specific antibodies.
Article
Excerpt Studies conducted in our laboratory and elsewhere on the initiation of antibody formation in cultures of tissues or cells from “normal” animals have yielded information which suggests a model for antibody formation that involves the sequential action of two types of cells (Fishman, 1959). According to this model the antigen is ingested and processed by macrophages which then transfer immunogenic material to lymph node cells where the antibody is produced. With the aid of tritium labeled bacteriophage and electron microscopy it has been possible to trace the uptake of antigen by macrophages, its concentration in lysosomal vacuoles, and finally the partial breakdown of the phage particles within 30 minutes of incubation at 37 C (Fishman, Adler, van Rood, and Binet, 1965). It is thought that in organized tissues the transfer of material from macrophages to lymph node cells would most likely take place within the structural “islands” in which macrophages...
Article
Excerpt Numerous observations have demonstrated the importance of genetic factors in the recognition of immunogenicity and in the ability to identify certain antigenic determinants, even though the antibodies produced are a heterogeneous population of molecules. (1.) There is a statistically significant relation between the ability of parents and offspring to respond to some antigens (Carlinfanti, 1948; Fjord-Scheibel, 1943; Sang and Sobey, 1954) and pure strains of mice differ in the amounts of antibody which they produce to different antigens (Ibsen, 1959). (2.) More recently, Sobey, Magrath, and Reisner (1966) have observed that an occasional mouse from randomly bred stock is naturally unresponsive to bovine serum albumin (BSA) and that such mice, when bred, produce both responsive and unresponsive mice. The fraction of offspring which are unresponsive increases with successive generations, and after the 6th or 7th generation, approximately 90% of the offspring of such matings are unresponsive to BSA. These experiments...
Article
Upon immunization with LDH-III (subunit composition AABB) rabbits produce anti-A and anti-B antibodies in comparable amounts. These antibodies fit equally well to the hybrid enzyme and to LDH-V (AAAA) or LDH-I (BBBB) respectively, as tested by passive hemagglutination inhibition. No antibodies reacting with both LDH-I and LDH-V were detected. A minority of hybrid-specific antibodies was, however, present in the sera. Animals primed with LDH-III respond regularly to a boosting injection of LDH-V with the production of large amounts of anti-A (but not anti-B) antibodies. A similar injection of LDH-I stimulates (if it has any effect at all) the production of anti-B antibodies only. Stimulation with one of the pure types does not impair a subsequent response to the other. The majority of the animals primed with LDH-III responded not at all or weakly to a boosting injection of LDH-I, though antibodies to LDH-I were present in the sera at the time of stimulation. This effect can hardly be explained on the basis of serological sepcificity. Hyporesponsiveness to LDH-III can be induced by injection of LDH-V into the newborn. Both anti-A and anti-B titers are equally depressed. Within the dose range tested, LDH-I does not exert any tolerogenic action with respect to LDH-III. The carrier property of subunit A is evident in the induction of both immunity and tolerance to LDH-III. The early phase of the immune response to the hybrid enzyme may be carrier-specific, and receptors for the haptenic subunit B may not exist at that stage.
Article
Excerpt In order to understand the mechanism of immunological stimulation, it is worth starting with a minimum theory of antigen reception. We assume first that nothing except antibody recognizes antigen, and we must therefore assume that the receptor for antigen is antibody already present at a site, in or on the cell, prior to exposure to antigen. Presumably the receptor antibody is made by the cell, and represents an accurate sample of the antibodies that the progeny of the cell will produce if stimulation is successfully accomplished. The relationship between receptor and product may well be even simpler and more direct than this: an individual plasma cell makes a single amino acid sequence, and this sequence is present in the parent lymphocyte. The arguments in favor of this hypothesis can be found in a recent review (Lennox and Cohn, 1967), from which the present account is derived. It would be premature...
Article
1. Mild acetylation of lactate dehydrogenase-I (LDH-I, l-lactate: NAD⁺ oxidoreductase, EC 1.1.1.27) from pig heart changes the pH optimum of the enzyme as well as its electrophoretic mobility without affecting its catalytic activity under optimal conditions. The modified enzyme may be hybridized in vitro with native LDH-I or LDH-V, i.e. the association specificity of the LDH subunits remains unchanged. LDH-V can be acetylated in the same way with some loss of activity, and both enzymes have been obtained in crystalline state.
Article
Observations on the specificity of the secondary response of rabbits to a variety of protein antigens have been described. Previous immunization with one antigen will accelerate and magnify the immune response to the first injection of another antigen if the two antigens are related. Subsequent immunization with one antigen may increase the amount of circulating antibody exclusively oriented to another antigen administered earlier, if the two antigens are related. Neither of these phenomena can be elicited by unrelated antigens. Possible explanations for this degree of non-specificity of the secondary response are discussed.
Article
The capacities of cells of thymus and bone marrow origin taken from radiation chimaeras to produce antibody during a secondary immune response have been tested in an in vivo transfer system. Although it could be shown that thymus-derived cells respond vigorously by mitosis to antigenic stimulation they were not capable of antibody production. By contrast bone marrow-derived cells did not in the first 3 days respond mitotically to antigenic stimulation but they were capable of limited antibody production. Most antibody was found when both cell populations were allowed to react to antigenic stimulation. (C) Williams & Wilkins 1967. All Rights Reserved.
Article
In response to a second injection of rabbits with a dinitrophenylated antigen, given 2 months to 2 yr after the first injection, there was rapid synthesis of large amounts of antibody high in relative affinity for the dinitrophenyl (DNP) determinant. The antibodies formed 3 days after restimulation were already high in affinity. Amounts of antigen too small to elicit detectable antibody production may prime the animal for a partial secondary response characterized by the formation of antibody of intermediate affinity after a second antigenic stimulus. Investigations into the specificity requirements for the secondary response indicated that variation in the carrier protein and in the haptenic determinant could be tolerated. Thus, after immunization with DNP-bovine γ-globulin, DNP-hemocyanin elicited the vigorous production of high affinity anti-DNP antibodies. However, DNP serum albumin was much less effective: it elicited a secondary response in some animals primed with DNP-bovine γ-globulin only when the interval between injections was increased from 10 to 28 wk. A secondary response was also evoked when the haptenic determinent of the second immunogen differed slightly from that of the one injected initially (i.e., 2,4,6-trinitrophenyl versus 2,4-dinitrophenyl).
Article
Secondary responses late in the course of immunization may be obtained with DNP conjugated to proteins different from that used for primary immunization. These responses are characterized by a modest increase in serum antibody concentration and by a large increase in affinity for hapten. On the other hand, secondary responses to the immunizing antigen involve a large increase in serum antibody concentration with little or no change in affinity. Secondary responses in rabbits immunized with type III pneumococci may be achieved with purified capsular polysaccharide but only very late in the course of immunization. These results are discussed in terms of a thermodynamically driven selection of sub-populations of sensitive cells by antigen.
Article
Genetic nonresponder guinea pigs made tolerant to BSA and then immunized with DNP-PLL.BSA failed to make anti-DNP-PLL antibodies. Thus, tolerance to a carrier protein renders animals unresponsive to the hapten which it bears. The addition in vitro of DNP-PLL or DNP-GL to lymph node cell cultures derived from genetic responder animals immunized with these materials led to a significant stimulation of (3)H-thymidine incorporation into DNA. However, the addition of DNP-PLL or DNP-GL to lymph node cell cultures from nonresponder animals immunized with these materials failed to produce any stimulation of DNA synthesis. Furthermore, the addition of DNP-PLL to lymph node cell cultures from nonresponder animals immunized with DNP-PLL.BSA or DNP-PLL.OVA also failed to stimulate cell proliferation in spite of the fact that the lymph node cells of these animals were producing anti-DNP-PLL antibodies. The above facts suggest that the function of the PLL gene product is to act at an early crucial step in the immune mechanism to form an antigen-inducer complex. The specificity of this early step may be of a simple order and different than that of the antibody which is later produced in the immune response.
Article
Suspensions containing normal thymus, spleen, or marrow cells were injected into irradiated syngeneic mice which were subsequently given antigen. Normal spleen cells produced discrete areas of antibody production in recipient spleens, and the number of areas was proportional to the number of donor cells. Mice receiving both marrow and thymus cells produced more centers of hemolytic activity in their spleens than mice receiving cells of either type alone. Normal and immunized thymus cells produced little or no hemolytic activity, and normal marrow was also inactive.
Article
A single thymus lobe from a neonatal donor has the capacity to restore primary immunological competence to a thymectomized syngeneic radiation chimaera. If a cytologically marked thymus graft is used the fate of the introduced cells following antigenic stimulation may be studied. Thymus-derived cells can be found in the spleen and lymph-nodes of such mice and furthermore there is a transient but well-defined increase in the proportion of these cells dividing following an injection of sheep red blood cells or application of a skin homograft 30 days after irradiation and thymus grafting. It was found that an even greater proportion of thymus-derived cells divide in response to a second challenge with sheep cells. Similarly, a higher percentage of thymus-derived cells divide in the lymph-nodes draining a second skin homograft.
Article
Erythrocyte isoantigens determined by the B blood-group locus of chickens enhance the immune response to weak isoantigens. This adjuvant action occurs if the antigens are present on the same erythrocyte, and the recipients are capable of responding immunologically to the B antigens.
Article
Upon immunization with LDH-III (subunit composition AABB) rabbits produce anti-A and anti-B antibodies in comparable amounts. These antibodies fit equally well to the hybrid enzyme and to LDH-V (AAAA) or LDH-I (BBBB) respectively, as tested by passive hemagglutination inhibition. No antibodies reacting with both LDH-I and LDH-V were detected. A minority of hybrid-specific antibodies was, however, present in the sera. Animals primed with LDH-III respond regularly to a boosting injection of LDH-V with the production of large amounts of anti-A (but not anti-B) antibodies. A similar injection of LDH-I stimulates (if it has any effect at all) the production of anti-B antibodies only. Stimulation with one of the pure types does not impair a subsequent response to the other. The majority of the animals primed with LDH-III responded not at all or weakly to a boosting injection of LDH-I, though antibodies to LDH-I were present in the sera at the time of stimulation. This effect can hardly be explained on the basis of serological sepcificity. Hyporesponsiveness to LDH-III can be induced by injection of LDH-V into the newborn. Both anti-A and anti-B titers are equally depressed. Within the dose range tested, LDH-I does not exert any tolerogenic action with respect to LDH-III. The carrier property of subunit A is evident in the induction of both immunity and tolerance to LDH-III. The early phase of the immune response to the hybrid enzyme may be carrier-specific, and receptors for the haptenic subunit B may not exist at that stage.
Article
Genetic responder guinea pigs primed with either benzylpenicilloyl-poly-l-lysine (BPO13-PLL287) or dinitrophenyl-polylysine (DNP13-PLL287) and boosted with the double hapten conjugate BPO13-DNP14-PLL287 gave an anamnestic response to the priming hapten and a primary response to the new hapten. Primary and anamnestic responses were clearly differentiated on the basis of antibody titers, time of attainment of maximal titers, and mercaptoethanol sensitivity of the antibodies. Both haptens were shown to be present on the double conjugate molecule. The absence of a booster response to the new hapten did not appear to be due to antigenic competition. These findings indicate for the present system, that the specificity of the anamnestic response relates only to individual antigenic determinants of a complex antigen, and not to the entire antigen molecule. These results would tend to exclude an enhanced processing of the antigen molecule as the crucial mechanism of the anamnestic response. The relationship between these results and the apparently discordant results of others is discussed.
Article
The 2,4-dinitrophenyl group, combined with proteins through azo linkage or by substitution in free amino groups, has been studied as a determinant in the precipitin reaction. The precipitin curves obtained resemble those encountered with purified antigens of high molecular weight with the chief exception that in the region of antibody excess antigen is not completely precipitated. Dinitrophenyl-azo-ovalbumin was a less effective precipitant than the conjugated ovalbumin in which dinitrophenyl groups were substituted in free amino groups. The former, however, was relatively more effective in precipitating antibody from antisera to dinitrophenyl-azo-proteins than from antisera prepared against amino-substituted dinitrophenyl-proteins. The globulin fraction of pooled antisera to dinitrophenyl-bovine γ globulin contained at least three species of antibodies—viz: those precipitated by bovine γ globulin; those precipitated by dinitrophenyl-ovalbumin and those precipitated only by dinitrophenyl-bovine γ globulin. These were present in the ratio of 1:1:1.3, respectively. After maximal precipitation with each of these antigens the supernates' binding of ε-N-dinitrophenyllysine, a hapten in which dinitrophenyl is combined with lysine in the same way as in the immunizing conjugate, was determined by the method of equilibrium dialysis. The binding in the three cases, relative to the binding by the unabsorbed immune globulin, was in the ratio of 1:0.6:0.25, respectively. Hence, the antibodies precipitated by bovine γ globulin had no detectable specificity for the dinitrophenyl group, and the antibodies precipitated by the heterologous conjugate (dinitrophenyl-ovalbumin) had a greater affinity for the dinitrophenyl determinant than those antihapten antibodies that required for precipitation the fully homolgous conjugate (dinitrophenyl-bovine γ globulin). Antibodies with dual specificity, i.e., with one specificity for the hapten and a distinctly different one for the protein of the immunizing conjugate, were not detected.
Article
Injection of 15 μg of a haptenazoprotein, such as p-aminobenzoic acid diazotized to hen egg albumin, in Freund's adjuvant, into footpads of guinea pigs produces delayed hypersensitivity in 5 days and circulating antibody in 10-11 days after sensitization. Injection of 1 μg of such conjugate in saline produces delayed hypersensitivity but no detectable circulating antibody. Specificity of delayed hypersensitivity is oriented toward the protein; that of circulating antibody, toward the hapten. When a primary dose of 1 μg in saline of protein, azoprotein or heterologous hapten-homologous protein is followed by a secondary dose of hapten-homologous protein, antibody response to the hapten is accelerated. A primary sensitizing dose of hapten, such as p-aminobenzoic acid (PABA) + protein such as hen egg albumin (HEA), followed by a secondary dose of the same hapten attached to heterologous protein, does not result in accelerated response to the hapten. These phenomena are explained if delayed hypersensitivity is considered an early step in antibody synthesis.
Article
A hemagglutination system has been described which employs direct coupling of 1,3-difluoro 2,4-dinitrobenzene (FDNFB) to the red cell surface, obviating the need for a protein carrier of the hapten, which is required in passive hemagglutination systems now commonly being used. The system is suitable for detection of anti-dinitrophenyl antibodies and cross-reacting antipicryl antibodies; it is equal in sensitivity to the method of passive cutaneous anaphylaxis and is simple in execution. Under certain conditions of time and hapten-coupling concentration, FDNFB-coupled erythrocytes were found to be most sensitive for detection of homologous and cross-reacting anti-picryl antibody of both rabbit and guinea pig origins. The stability of the hapten-erythrocyte bond was not disturbed by extensive dialysis of the cells. Picryl chloride and DNCB could not be coupled to red cells successfully, presumably because of a lower order of reactivity of these compounds.
Article
Rabbits were immunized with bovine gamma globulin intravenously in saline and were reinjected with dinitrophenylated bovine gamma globulin intravenously. No secondary effect on anti-dinitrophenyl antibodies was observed by prior injection of bovine gamma globulin; on the contrary, no anti-dinitrophenyl antibody was produced in these animals whereas in controls, which had not received prior bovine gamma globulin injections, antibody production was detected, especially with highly derivated dinitrophenyl-bovine gamma globulin preparations. A secondary effect against bovine gamma globulin was observed, especially with the less derivated preparation.
Cellular co-operation in the antibody response
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