Production of monoclonal anti-idiotype antibodies which mimic an M-like protein of Streptococcus equi.

Rat monoclonal anti-idiotype antibodies (mAb2) were raised against two mouse monoclonal antibodies (mAb1), 1D10 and 2A6, with specificity for the M-like protein of Streptococcus equi. The capacity of the mAb2 to inhibit the binding between the corresponding mouse mAb1 against which the mAb2 were raised and the M-like protein was investigated in an inhibition EIA. One of the ten mAb2 examined, namely 5D1 (anti-mAb1 1D10), was able to inhibit this binding. The mAb2 5D1 bound to the mAb1 1D10 in such a way as to completely inhibit the subsequent binding of the M-like protein antigen to the paratope of the mAb1 1D10. The mAb2 5D1 is likely to represent a true image of the M-like protein antigen and may thus be described as an Ab2 beta anti-idiotype antibody.

Internal image (Ab2 beta) anti-idiotype vaccines. Theoretical and practical aspects.

Anti-idiotype antibodies that carry an internal image of an antigen epitope (Ab2 beta antibodies) can be used in vaccine preparations to favourably manipulate the immune network. These vaccines have been shown to induce protective immunity in animals that have not been intentionally exposed to the native antigen epitope. This review attempts to define certain theoretical and practical aspects of immunological network manipulations and their relevance to Ab2 beta internal image anti-idiotype antibody vaccine design.

Protection against Streptococcus equi infection by monoclonal antibodies against an M-like protein.

We have developed an in vivo passive transfer assay using mice to identify monoclonal antibodies (mAbs) which offer protection against Streptococcus equi infection. The assay was developed using serum antibodies collected from horses convalescing from strangles. In this study, we show that a preparation of M-like protein, acid-extracted from S. equi, affords 80% protection to mice immunized with it. A number of mouse mAbs directed against a preparation of M-like protein were then assessed for their ability to passively protect mice against challenge with a lethal dose of the bacteria. Two mAbs, 1D10 and 2A6, were shown to be highly protective. It was also demonstrated, by means of a competitive enzyme immunoassay, that these mAbs recognized different epitopes in the preparation. Examination of a dose-response curve for mAbs 1D10 and 2A6 revealed that optimal levels of protection were achieved using 1 mg of either 1D10 or 2A6, or 0.5 mg 1D10 and 0.5 mg 2A6 given together. Immunological reactivity of these mAbs with a preparation of M-like protein showed that the antigens they recognized were comparable in size to some of the antigens recognized by convalescent horse serum antibodies. The role of immunoglobulin isotype in conferring protection is discussed.

The immune response to anti-idiotype antibodies bearing an internal image epitope of tetanus toxin/toxoid. I. Induction of the humoral immune response.

Primary immune responses to tetanus toxoid (TT) and primary and secondary immune responses to a rabbit TT internal image bearing anti-idiotype antibody (Ab2 beta 1) inoculated in Freund's complete adjuvant (FCA), saline (SAL) or syntex adjuvant formulation vehicle (SAF) via the intraperitoneal or subcutaneous route, were examined in mice. High anti-TT antibody (Ab3) titres are reported although the titre and persistence of the antibody response varied according to the adjuvant used in the priming and challenge inocula of Ab2 beta 1. Mouse Ab3 antibodies were elicited in mice inoculated with rabbit Ab2 beta 1 antibodies which in turn were elicited by an inoculum of mouse monoclonal anti-TT Ab1 antibody. Ab3 was shown to be identical to Ab1 by immunoblot analysis. Primary and secondary immune responses elicited by rabbit Ab2 beta 1 antibody protected mice against a lethal dose of tetanus toxin.

The immune response to anti-idiotype antibodies bearing an internal image epitope of tetanus toxin/toxoid. II. Comparison of the primary humoral immune response to xenogeneic Ab2 beta 1 and Ab2 beta 2 internal image anti-idiotype antibodies.

Mouse monoclonal (Ab1) anti-tetanus toxin/toxoid antibodies were used to raise Ab2 beta (tetanus toxin/toxoid internal image bearing) anti-idiotype antibodies in rabbits. Those rabbit serum antibodies (Ab2 beta) that did not bind to mouse serum proteins on an affinity column gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response in mice. Rabbit serum antibodies that did bind to the affinity column, when eluted and used to inoculate mice also gave rise to an Ab3 anti-tetanus toxin/toxoid antibody response. It is suggested that one population of rabbit Ab2 beta anti-idiotype antibodies (unbound fraction) bears a partial or complete internal image of a tetanus epitope (Ab2 beta 1) while others (bound fraction) bear a complete or partial mirror image of a mouse immunoglobulin epitope as well (Ab2 beta 2).

The migration of lymphocytes into rat popliteal lymph nodes during antigenic promotion or competition between heterologous erythrocytes.

The injection of chicken and sheep red blood cells (CRBC and SRBC) into rat popliteal lymph nodes either together or sequentially 2, 4, 6, or 8 days apart resulted in an enhanced immune response when the second antigen was injected 2 or 4 days after the injection of the first antigen (antigenic promotion) or a suppressed immune response when the second antigen was injected 6 days after the injection of the first antigen (antigenic competition). The immune response to either antigen was dependent upon the time of administration of the second antigen with respect to the first antigen. Lymphocyte migration into antigenically stimulated lymph nodes was greater when the two antigens were injected sequentially rather than together. Further, the migration of lymphocytes into the lymph node was enhanced when the second antigen was injected during the inductive or suppressive phase of the immune response to the first antigen (CRBC) regardless of whether the same (CRBC) or an antigenically unrelated antigen (SRBC) was used as the second antigen. While antigenic promotion may in part be explained by the increased rate at which lymphocytes migrate into lymph nodes, lymphocyte migration is also enhanced during antigenic competition. This suggests that while suppressor cells/factors may regulate the effector phase of an immune response they do not directly modulate the migration of blood-borne lymphocytes into the lymph node.

A novel structure in the stomach and intestine of two species of Australian marsupial mice.

A tubular or sac-like structure with a well defined lumen which occasionally contained parasites was found in the outer muscle wall of the small and large intestine in 32 out of 40 male and 35 of 40 female marsupial mice of the two species, Antechinus swainsonii and Antechinus stuartii (Macleay). A similar structure was also found in the muscle layers of the stomach in 5 marsupial mice of both species. The cellular matrix of the structure appeared to be composed of randomly organized macrophage-like cells and in one case there was evidence of an inflammatory reaction to parasites within the lumen. The origin of this tube is either vestigial or the result of reactive changes.

Stress related involution of lymphoid tissues in Australian marsupial mice.

Involution of the thoracic thymus in two species of marsupial mouse, Antechinus swainsonii (Waterhouse) and Antechinus stuartii (Macleay) was shown to be unrelated to corticosteroid action and to be complete before puberty. A stress response in male marsupial mice is caused by an androgen related drop in the plasma corticosteroid binding globulin concentration which gives rise to an increase in the plasma free glucocorticoid concentration. The high concentrations of free glucocorticoids in the plasma just prior to the breeding season causes a rapid involution of the spleen and lymph nodes while the gut associated lymphoid tissues remain unaffected. The concentration of free glucocorticoids also rises in females, but it never attains the high concentrations observed in males. Nevertheless, the spleen and lymph nodes do involute to some extent in some females and the degree of involution appears to be related to the relative concentration of plasma free glucocorticoids. At the conclusion of the breeding season, there is a complete mortality in males of the population, due to a stress response in which the compromised immune system clearly plays a role.

The gut-associated lymphoid system of two species of Australian marsupial mice, Antechinus swainsonii and Antechinus stuartii. Distribution, frequency and structure of Peyer's patches and lymphoid follicles in the small and large intestine.

The distribution and number of Peyer's patches (5) in two species of marsupial mice, Antechinus swainsonii and Antechinus stuartii was found to be the same even though the length of the intestine in the latter species was half that of the former. Both species lack a caecum and appendix. The position of the Peyer's patches is unusual in that the first three Peyer's patches are on the right side of the small intestine whereas the penultimate and ultimate Peyer's patches are large, contain many lymphoid follicles and are in an anti-mesenteric position in the small intestine and sometimes in the large intestine (ultimate Peyer's patch). The number of Peyer's patches in eutherian mice of similar size, and intestinal length is greater (13) although the number of Peyer's patch lymphoid follicles per centimeter intestine is less (1.8) than in A. swainsonii (4) and A. stuartii (4.4). Marsupial mice have most of their lymphoid follicles confined to a few large Peyer's patches, whereas eutherian mice have fewer lymphoid follicles per unit intestinal length, more Peyer's patches (with fewer lymphoid follicles) evenly distributed along the intestine and more single lymphoid follicles interspersed between them.

Migration of blood-borne lymphocytes into antigen-stimulated lymph nodes. A study of the kinetics of lymphocyte recruitment throughout an immune response.

The kinetics of lymphocyte recruitment into rat popliteal lymph nodes stimulated with varying doses of allogeneic lymphocytes or purified protein derivative of tuberculin differed markedly. The number of blood-borne lymphocytes entering lymph nodes stimulated with allogeneic lymphocytes was greater and the duration of their entry more prolonged than in lymph nodes stimulated with the soluble antigen purified protein derivative of tuberculin. When individual antigenic doses were compared, lymphocyte recruitment into popliteal lymph nodes was shown to be dependent upon antigenic dose; higher doses of antigen resulting in increased levels of lymphocyte recruitment. In addition it was found that there is no linear relationship between lymph node weight changes and the extent of lymphocyte recruitment.

The migration of blood-borne lymphocytes into rat popliteal lymph nodes stimulated with xenogeneic red blood cells.

The recruitment of radiolabelled blood-borne lymphocytes into rat popliteal lymph nodes (PLN) was investigated following the injection of varying doses of chicken (CRBC) or sheep red blood cells (SRBC) into the hind foot pad. Within the dose range tested (10(5)-10(8) RBC) an increase in the dose of injected antigen resulted in elevated levels of lymphocyte recruitment into the draining popliteal lymph node. The kinetics of lymphocyte recruitment with the same dose of CRBC or SRBC was similar even though these red cells differ in size and are antigenically non-cross-reactive. While little is known of the mechanisms which control the rate of entry of blood-borne lymphocytes into antigen-stimulated lymph nodes, the extent of lymphocyte recruitment was shown to be directly related to the quantity of antigen injected.

The entry of T and B lymphocytes into rat popliteal lymph nodes undergoing a graft-versus-host reaction.

The entry of radiolabeled blood-borne T and B lymphocytes into resting popliteal lymph nodes and popliteal lymph nodes stimulated with semiallogeneic lymphocytes was investigated in rats. Thoracic duct lymphocytes separated into T- and B-lymphocyte populations on nylon-wool columns were radiolabeled with 51chromium and equal numbers of T or B lymphocytes were injected intravenously. While the ratio of T and B lymphocytes in the blood is approximately 3:1 it was found that the ratio of T to B lymphocytes migrating into lymph nodes was approximately 9 T to 1 B lymphocyte in both resting and antigenically stimulated lymph nodes. Since the ratio of T to B lymphocytes in thoracic duct lymph is similar to that of blood, there is a disparity between the number of T cells entering and leaving lymph nodes. These results suggest that some T lymphocytes may return to the blood directly and/or there is increased T lymphocyte death in lymph nodes.

Lymphocyte migration during the development of regional lymph node anergy in experimental tumor growth.

The development of lymph node anergy in Wistar rats to growing Walker carcinoma 256 was studied in vitro using the 51Cr-release cytotoxicity assay. Cell-mediated cytotoxicity to the tumor peaked in draining lymph nodes 11 days after tumor transplantation. By 14 days, the regional lymph node had become anergic to the tumor at a time when cell-mediated cytotoxicity was still increasing in the more distal contralateral lymph node. Lymphocyte migration into resting, cytotoxic, and anergic lymph nodes was analyzed to determine if altered cell migration into the regional lymph node was associated with the development of anergy. Lymphocyte migration was found to be enhanced in both cytotoxic and anergic regional lymph nodes of tumor-bearing animals. It is concluded that lymph node anergy in this experimental tumor system is not related to changes in lymphocyte migration patterns; rather, it is the result of alterations in the microenvironment of the lymph node which prevents the expression of cytotoxic effector cells.

Lymphocyte recirculation in the sheep fetus.

The numbers of circulating thymus-derived and surface Ig-bearing lymphocytes in the fetal lamb increase exponentially over the last third of gestation. Experiments in which [3H]thymidine was continuously infused into fetal lambs have established that these cells are long-lived in the fetus. The migration of 51Cr-labelled autologous lymphocytes from intestinal or prescapular lymph was compared in fetal lambs and adult sheep. A subpopulation of thymus-derived lymphocytes present in intestinal lymph of adults which migrated preferentially to the small intestine was not found in fetal intestinal lymph. There were marked differences in the migration of fetal and adult lymphocytes to the lungs and liver. In spite of the absence of circulating antibodies or immunoglobulins and of extrinsic antigen in the immunologically virgin sheep fetus, the circulation of lymphocytes through the spleen and lymph nodes of fetal lambs was more intense than in the adult, indicating that the pathways of recirculation and the capacity of cells to recirculate arise as a physiological process independently of antigenic stimulation.

Changing patterns of lymphocyte circulation during development.

Despite the absence of antibody, immunoglobulin, and extrinsic antigen, the output of T- and B-lymphocytes from single lymph nodes and the intestines in the sheeep fetus increases exponentially over the last third of gestation. The fetus possesses a huge pool of recirculating lymphocytes which have the same blood to lymph transit time through lymph nodes as adult sheep. A subpopulation of intestinal lymphocytes which migrates preferentially through the small intestines of adult sheep was not found in the fetus. The sheep fetus is immunologically virgin, so fetal recirculating lymphocytes cannot be memory cells nor can they be dependent on antigen for their development.

The migration of lymphocytes in the fetal lamb.

The migration of 51Cr-labeled autologous lymphocytes from intestinal or prescapular lymph was compared in fetal lambs and adult sheep. A subpopulation of lymphocytes present in intestinal lymph of adults which migrated to the small intestine was not found in fetal intestinal lymph. There were marked differences in the migration of fetal and adult lymphocytes to the lungs and liver. In spite of the absence of circulating antibodies or immunoglobulins and of extrinsic antigen in the immunologically virgin sheep fetus, the circulation of lymphocytes through the spleen and lymph nodes of fetal lambs was more intense than in the adult.

Collection of lymph from single lymph nodes and the intestines of fetal lambs in utero.

Techniques for establishing chronic lymphatic fistulae in the intestinal and prescapular lymphatic ducts of fetal lambs during the test third of gestation are described. Despite the absence of antibody, immunoglobulin and extrinsic antigen, the number of recirculating lymphocytes increases considerably through gestation. Both thymus-derived and surface Ig-bearing B lymphocytes are present in the fetal recirculating pool and they appear to increase in number at the same rate.

Behaviour of sheep-immunoglobulin-bearing and non-immunoglobulin-bearing lymphocytes isolated by nylon wool columns.

The technique of separating lymphocytes on nylon wool columns has been applied to sheep lymphocytes obtained from efferent lymph. The non-immunoglobulin-bearing (sIg-) lymphocytes which pass through the column were MLR-reactive, responsive only to T cell mitogens, and migrated in vivo like T lymphocytes described in rodents. Immunoglobulin-bearing (sIg+) lymphocytes were MLR-non-reactive, responsive to lipopolysaccharide and migrated in vivo like B lymphocytes in rodents. It is considered that the majority of sIg- lymphocytes obtained in this way are T lymphocytes and the majority of sIg+ lymphocytes are B lymphocytes.

The appearance of non-specific antibody-forming cells in the efferent lymph draining antigen-stimulated single lymph nodes.

Immunization of single lymph nodes with various antigens led to the appearance of cells in the efferent lymph that secreted antibody specific for the antigen which induced their formation and for a number of unrelated, non-crossreacting antigens. Immunization of single lymph nodes with mitogens led to the appearance of cells secreting antibodies specific for an even greater number of antigens, including one (TNP) that in all probability is not present in the animals' natural environment. When the node was primed with one antigen, a subsequent challenge with an unrelated antigen 12 weeks later led to the appearance of greater numbers of cells containing and secreting antibody against the previously experienced antigen, than was the case in unprimed lymph nodes. These findings indicate that the immune response to antigen provokes the maturation of lymphocytes of specificities unrelated to that of the injected immunogen. Such a mechanism may be important in maintaining immunological memory. Mitogens may directly activate lymphocytes into maturation and expression as antibody-secreting cells, whereas antigens appear to act indirectly.