Bases for the Recognition of Antigenicity1

[CANCER RESEARCH 28, 1423-1429,July 19Â«8] Bases for the Recognition of Antigenicity1

G. J. V. Nossal and A. Abbot The Walter and Eliza Hall Institute of Medical Research, Melbourne 3060, Australia

Summary immune studies, and cancer research. In this brief review, I The recognition of antigenicity is a problem that can be will attempt to outline our current concepts of how mammalian broken into two main portions. First, there is the response of species actually do recognize antigenic molecules and what re the immune system to antigens and second, the recognition of actions may follow such recognition. Broadly speaking, the foreignness at the local tissue level. The former is best separated problem may be broken into two major portions. First, there operationally into two parts, the response to soluble or par- is the general response of the lymphoid and reticuloendothelial ticulate-injected antigens, and the response to antigens of systems to the entry or emergence of antigens. This is the prov grafted cells. ince of students of inflammation and immunology. Second, The present paper deals mainly with the basis for recogni there is the problem of local tissue reactions which may occur tion of injected antigens. Evidence is presented that a specific following the appearance of cells with changed surface antigens in an organ. This is a much less well-defined area of study, but antibody, either natural or immune, is an important steering mechanism for immune responses. It influences not only the one which may turn out to have great significance in cancer extent of antigen capture, but, just as important, the internal research. The first area, which will be the major subject of this distribution of antigen between certain well-defined compart paper, can again be broken into two parts, purely for didactic ments of the reticuloendothelial system of lymph nodes. Spe purposes. These are the response to soluble or paniculate cifically, if antibody or natural opsonin to an injected antigen antigens introduced by infection or injection, and the reaction is present in an animal, localization of antigen in primary lym- to cellular antigens such as grafts. phoid follicles and in germinal centers is an inevitable conse Recognition of Foreignness of Injected Materials quence. In these locations electron microscopic autoradiography shows that antigen in follicles is held in an extracellular, mem The most precise information that we have on recognition brane-associated location without being subjected to phagocy of foreignness comes from direct examination of what actually tosis. The immunologie consequences of follicle antigen-trapping happens to foreign materials when they are injected into the are germinal center formation and, in all probability, the de body of an adult animal. Even here, definition of the problem velopment of memory cells. Whether membrane-associated an is not easy, because in each case we must compare the way tigen is important in primary immune responses is not known. that a foreign material is distributed with the way in which Some key papers in the literature concerning early recogni an autologous constituent of similar physical and chemical tion steps in antibody formation are briefly reviewed. The characteristics would have been handled in the body. Obviously, if we had a window into an animal's own bloodstream and conclusion is reached that there may well be factors other than circulating or cell-bound antibodies concerned, some of which could determine the fate of its red cells, serum albumin, and are under genetic control. The chemical nature of these is not circulating insulin molecules, we would get widely differing known. answers. Therefore, it is reasonable to expect significant dif Present knowledge in the fields of cellular recognition of ferences in distribution pattern also among foreign constituents grafts, allogeneic inhibition, and tissue surveillance is briefly which enter the body. Nevertheless, there is one common fea surveyed. ture which applies to all but the smallest foreign molecules and that is their tendency to be taken up by the reticuloendo Introduction thelial system. Autologous constituents are not entirely exempt The current immunology research explosion has been moti from phagocytosis, as witness the disposal of effete red blood vated by many considerations far removed from the field of cells and the autophagocytosis of lymphocytes In germinal defense against microbial invasion. Among the most important centers. This has speculatively been ascribed to some antigenic factors has been the realization that recognition of foreignness change in the cells concerned, but concrete evidence is lacking. is a concept of great importance in organ transplantation, auto- In general, however, foreign red cells are phagocytozed much more rapidly and extensively than autologous red cells, and 1 This work was supported by grants and contracts from the the same is true of smaller foreign particles and molecules. This National Health and Medical Research Council, Canberra, much will have been familiar to all readers, but there are Australia; the National Institute of Allergy and Infectious Dis eases (AI-0-3958) ; and the United States Atomic Energy Com subtleties to the role of the reticuloendothelial system in anti mission (AT-30-D-3695). This is Publication No. 1213 from The gen handling that are not as widely known and which we must Walter and Eliza Hall Institute of Medical Research. now discuss.

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The Role of Antibody in Antigen Trapping tion) has made a detailed study of what portion of the immuno- One of the most important factors in the recognition of globulin molecule is instrumental in bringing antigen into soluble or particulate antigens is antibody. Much attention follicles and holding it there. Most of the evidence indicates has been given to the role of a specific antibody in accelerating that there are one or more sites on the various immunoglobulin the rate of uptake of materials by the reticuloendothelial sys heavy chains which tend to bind to follicle reticular cells. A tem, but far less to the equally important question of the large number of different antigens have now been tested for effect of antibody on the internal distribution of antigens in follicular localization. In every case where there is antibody the different compartments of the reticuloendothelial system in the serum, follicular localization is a major feature of lym which exist in lymphoid tissue. We will deal first with evidence phoid antigen trapping. In fact, injection of labeled antigen into an animal and observation of follicular localization 4-24 which comes from use of specific, immune antisera and will then examine the role of natural antibodies or opsonins. hours later can be an exquisitely sensitive test for the presence A convenient way of illustrating the point is to compare the of antibody. This is illustrated by studies in tolerant animals distribution of an antigen in normal adult rats in the presence (2, 12) where a trace of antibody in an animal exhibiting sub and absence of antibody. If human scrum albumin (HSA) stantial partial tolerance can be picked up by the tendency labeled with 1-5I is injected into the hind footpad of a rat, for enhanced follicular localization even when sensitive serum- antibody-titration methods fail to reach threshold levels. only around 0.02% of it will be stably trapped in the popliteal nodes (15). Autoradiographic analysis shows that virtually all Specific antibody in immunized animals is thus a clear and of this is in macrophages in the lymph node medulla, chiefly obvious example of one basis for recognition of antigenicity. We now come to the more vexing question of how virgin, non- within the sinuses but also scattered through the cords. On the other hand, if HSA is complcxod with a specific antibody immunized animals recognize antigens. In an earlier study (1), either in vitro or in vivo, the amount trapped is increased by we noted that in unimmunized animals there was a broad a factor of about 10, and the internal distribution in the lymph correlation between the degree of immunogenicity of a material node is quite different. Some early medullary localization is and its tendency for follicular localization. Highly foreign ma present, but the most prominent stable antigen depots are in terials such as bacterial flagellar antigens showed excellent the cortical lymph node follicles. The antibody has little, if follicular localization; moderately immunogenic products such any, effect on the extent of medullary macrophage-trapping of as horse ferritin showed less; heterologous albumin showed the antigen, but it has a dramatic effect on retention in lym- less still; and minimally antigenic proteins such as gelatin showed no follicular localization at all. We now recognize that phoid follicles. The question immediately comes up as to other factors besides "degree of foreignness" influence follicular whether the size of the foreign particle is a major factor in antigen handling. This is not a likely complete explanation, as localization in virgin rats, such as particle size and other USA aggrega tcd by heat to a particle size comparable with physicochemical characteristics. For example, carbon particles antigen-antibody complexes can be shown to be trapped much (6, 18) and saccharated iron oxide (6) show some follicular better in the medulla of the nodes of normal rats than native localization though they are not generally considered to be HSA; but in the absence of antibody, heat-denatured HSA antigenic. Still, in all probability the reason why some smaller shows no tendency for follicular localization (10). molecules show follicular localization in virgin animals is the Further insights into the differences between follicular and presence in their serum of natural antibodies or opsonins fre medullary HSA localization can be gained by electronmiero- quently in amounts undetectable by standard serologie tech scopic autoradiography of lymph nodes. Our technics for this niques. Interestingly, the broad generalization seems to hold procedure have been fully described elsewhere.- In the medulla that molecules to which animals exhibit natural antibody are the antigen is rapidly sequestered into membrane-bound inclu more likely to be good immunogens than those to which none sions (phagolysosomes) and subjected to the action of digestive is present. enzymes (Fig. 1). In the cortical follicles, the antigen maintains An example of the role of opsonins in antigen trapping in unimmunized rats is the work of Williams (27). Using lar'I- a largely extracellular location. Much of it is attached to the plasma membranes of processes of specialized follicular reticular labeled polymerized flagellili as antigen, Williams was able to cells (Fig. 2). These interdigitale extensively and form a com show that after 5 days of chronic thoracic duct drainage, an plex antigen-retaining web which permeates both primary and tigen localization in rat lymph node follicles was markedly secondar}' lymphoid follicles. In many cases where antigen is impaired. Significant restoration of localization followed the stably trapped in the spleen, virtually all of it is in this injection of normal rat serum. The hypothesis was proposed labyrinthine follicular location. The follicle reticular cells show that a proportion of recirculating lymphocytes of unimmunized no tendency to phagocytozc antigens; they cannot be correctly rats manufactured opsonins important for correct antigen described as macrophages and appear to be quite different handling. It seems possible that their absence could be one from the "tingible body" macrophages of lymphoid follicles, factor involved in the diminished immune responsiveness of which phagocytozc dead lymphocytes. Lummus (in prepara- the drained rats.

Positive Feedback Mechanisms Involving IgM Antibody 2 Nossal, G. J. V., Abbot, A., and Mitchell, J. Antigens in Im munity, XIV. Electron Microscopio Autoradiographic Studies of Many indirect pieces of evidence point to the probability Ani Â¡nonCapture in the Lymph Node Medulla. J. Exptl. Mcd., that uptake of antigen by the reticuloendothelial system is a 127: 263-276, 1968. necessary prelude to antibody production. For example, par-

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1968 American Association for Cancer Research. Antigenicity Recognition ticulate antigens tend to be more highly immunogemc than immunoglobulins or component chains on their surfaces before soluble molecules of the same chemical composition. An ele any detectable antibodies are made. The thought that antibody gant experiment by Frei et al. (9) showed that if bovine is the only basis for the recognition of antigenicity has the serum albumin is injected into a rabbit and the most avidly appeal of simplicity, especially as far as humoral antibody phagocytozed portion is removed by the reticuloendothelial formation is concerned. However, the view that the relevant system, the material which remains in the circulation causes receptors exist solely on the surface of clonally individuated tolerance rather than antibody formation upon reinjection into lymphocytes cannot be correct because of two important types another rabbit. Perhaps the most direct evidence for the value of experiments (4, 19) which we will shortly examine. of prÃ©existentnatural antibody in ensuring maximal antibody From an operational point of view, there are thus two ways production comes from the recent work of Jerne (13). This in which opsonins are recognition factors important in deter showed that the injection of purified IgM antibody could mining quantitative features of antibody production. First, markedly increase the immune response of mice to sheep red they facilitate correct antigen handling; second, inasmuch as blood cells. IgG antibody, as had been shown previously (26), they are caused by prior antigenic stimulation, they may re acted as a negative feedback, with a dose-response curve of flect the existence of memory cells for a cross-reacting antigen, steeper slope. From a teleological point of view, this makes which could be fired off even by an antigen somewhat different good sense. In most immune responses, IgM is synthesized from the one which caused their development. It still remains first and IgG considerably later. Thus, the system has an to be determined whether antigens of bacterial or viral origin inbuilt positive feedback early on when maximal acceleration are so powerfully immunogenic because of some as yet un of synthesis could conquer an infection, and a later negative determined physical or chemical property, or because of the feedback to prevent wastefully large amounts of antibody fact that all normal animals possess opsonins for them. If being made. While Jerne did not seek to determine the mech anything, work on germ-free animals favors the former view, anism of the positive IgM feedback, it seems likely to be due because, in general, their immune responses to such antigens to some aspect of antigen handling by the reticuloendothelial are just as good as those of conventional animals. On the other system rather than to any direct effect on the lymphoid cells. hand, most studies of germ-free animals have examined only the ceiling levels of antibody formed, which depend on activa Nature of Opsonins tion of negative feedback. Quantitative studies on the numbers It seems likely that all opsonins are immunoglobulins. The of antigen-sensitive cells and on rates of antibody synthesis question remains whether these natural antibodies which play early after immunization would be revealing. such an important role in the recognition of antigens are syn thesized regardlegs of antigenic stimulation. Studies which Genetic Aspects of Recognition of Antigens employ antigens of bacterial or viral origin are frequently Important insights into the mechanism of recognition of criticized because of the possibility that they actually induce antigenicity have been gained by the experiments of Benacerraf secondary responses. It is argued that animals used in putative (4). He has found that guinea pigs can be divided into two primary responses may have encountered cross-reacting an categories with respect to the capacity to form antibodies tigens in their previous life. Certainly it is true that many against 2,4-dinitrophenyl-poly-L-lysine (DNP-PLL). There are natural antibodies are present in reduced amounts in the responders which, when injected with the antigen in complete serum of germ-free animals, and further, that cross-reactions adjuvants, manufacture up to 7 mg/ml of antibody; there among antigens are very common. Moreover, it is clear that are also nonresponders that fail to form detectable antibody, an animal's prior antigenic experience can exert a profound as judged by a test with a threshold of <1 /ig/ml. Mating ex effect on its response to a first encounter with an antigen (8). periments suggest that the ability to respond is the expression In some ways the argument is a rather academic one. Even of a single, dominant, autosomal gene. The gene seems to con germ-free animals are by no means antigen-free, and the use trol a recognition step because if DNP-PLL is attached to an of a synthetic antigen or a haptene is no guarantee against immunogenic carrier such as aggregated bovine serum albumin, the presence of natural antibodies. Also, many of the antigens genetic DNP-PLL nonresponders make excellent antibodies commonly praised as suitable for studies of theoretical im to the DNP-PLL now acting as a haptene. Thus, the capacity munity must be given together with an adjuvant, and/or by to form immunoglobulin molecules with combining sites of the multiple injections. This militates severely against a detailed necessary specificity is not lacking in the nonresponders. Their study of early inductive events, and indeed of most of the deficiency is not due to the absence of pre-aclapted cells with biologically important problems in antibody formation. The the correct potential. Rather, they lack the ability to recognize question which we would like to be able to pose is the fol the immunogenic nature of DNP-PLL, a defect which can be lowing: how does an animal that has never been stimulated overcome by the trick of chemical conjugation with a readily with an antigen of any sort recognize foreignness? Is it through phagocytozed material. In some respects these animals are the the presence of natural antibodies either in the serum or on the converse of animals rendered immunologically tolerant. We surface of lymphocytes? In selective theories of antibody for do not know what the nature of the initial recognition step mation (5), cells possess the genetic potential to synthesize may be, or whether it involves cytophilic or other natural antibodies and are postulated to do so at a minimal rate even antibody. However, the experiment does provide evidence for before antigenic stimulation. The work of Sell and Gell (24) the view that recognition potential and synthetic potential are suggests that the lymphocytes of young rabbits may have not synonymous.

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While the initial recognition step is a necessary prerequisite stem cells of the whole lymphoid system reside in the bone for antibody formation, it is not a sufficient condition. There marrow and are seeded out to the thymus and peripheral lym is now substantial evidence to indicate that animals in which phoid compartment via the circulation, probably throughout immunologie tolerance has been induced can recognize antigens life. At least as far as cells capable of forming antibody to sheep just as well as normal animals (2), and that their macrophages red blood cells are concerned, the bone marrow stem cells need can produce factors capable of inducing antibody formation to come under a thymic influence, probably humoral, to mature in normal, nontolerant lymphoid cells (20). This is consistent into cells capable of reacting to antigen. Knowledge of this with a two-step view of antibody formation. The initial recog maturation step so far comes from experiments on the recon nition step involves the reticuloendothelial system. The syn stitution of immunologie competence following X-irradiation. thetic step involves the existence of appropriate lymphoid cells. We do not know to what extent, if any, this pathway of dif ferentiation is dependent, directly or indirectly, on antigenic The Nature of Receptors for Antigen as Determined by in stimulation. It would be most helpful to be able to generate Vitro Induction of Antibody Formation antigen-sensitive cells in vitro from the 2 organ sources in Mitchison (19) has reached much the same conclusion as volved. Finally, analysis of recognition mechanisms would cer Benacerraf, starting from a completely different viewpoint. tainly be facilitated if the whole induction and production could He addressed himself to the question of whether the stimula take place under defined in vitro conditions. Recent progress in tion of antibody formation was simply the result of union of this area has been rapid (7, 23). It is now possible to induce antigen and antibody-receptors on the surface of lymphocytes. good, apparently primary immune responses in free cultured He used as test antigens various haptenes linked to protein suspensions of spleen cells. However, to date no reports have carriers as stimulators of secondary responses in vitro. Mice come forward showing antibody formation in vitro with mixed were primed with haptene-protein adjuvant mixtures. After cultures of macrophages and lymphocytes reconstituted from periods of around 100 days, they were killed and lymphoid purified cell suspensions. As soon as this becomes feasible, many cells were restimulated in vitro and injected into syngeneic, new experimental approaches will emerge. irradiated recipients. These were subsequently assayed for serum antibody levels. It was found that pure suspensions of Basis of the Recognition of Allogeneic Cells by Lymphocytes lymphocytes could not be stimulated; the presence of some There are by now a number of model systems available for other cell type, presumably a macrophage, was necessary. the study of the behavior of lymphocytes placed in contact Stimulation in vitro took a definite time, 2 hours incubation with allogeneic cells, both in vivo (10) and in vitro (11, 21, 22). being necessary for a maximal effect. Induction could be readily There is no dispute about the fundamental observations com inhibited by cross-reacting haptenes. Most important for our mon to all systems. These include blast cell transformation of present discussions, alterations to the carrier dramatically af a proportion of small lymphocytes and eventual damage to the fected the response to the haptene. Experiments using inert allogeneic cells. The initiating mechanism is still speculative. In carbon-chain "spacers" lent no support to the idea that the the in vivo situation, it appears that under some circumstances, postulated site on the lymphocyte was large enough to embrace e.g., a kidney allograft, direct contact between lymphocytes both haptene and carrier. Rather, the results suggested that and allogeneic cells is sufficient to cause sensitization (25), two or more receptors were jointly involved in the inductive whereas under others, e.g., a skin allograft (3), antigenic ma phenomenon, one recognizing the carrier and the other the terial must first pass from the graft to the draining lymph node haptene. It is quite possible that these were on two different before effective sensitization occurs. In both cases the pre cells, the former on a macrophage, the latter on a lymphocyte. ferred site for the actual proliferation of the transformed cells is in lymphatic tissue. This results in the genesis of progeny Proposals for the Future Analysis of the Basis of Recognition small lymphocytes which migrate to the allograft and cause its of Antigens as a Prelude to Antibody Formation destruction. Studies of target cell destruction in vitro have sug It is clear that we have only limited insights into the initial gested that the damage may be inherently nonimmunologic in steps of recognition as yet. Future studies in this field should nature, reflecting the existence of a phenomenon akin to allo center around 3 themes: the use of antigen-free animals, the geneic inhibition. According to this notion, when two cells of creation of antigen-sensitive cells in vitro by thymus:bone mar allogeneic character are placed into close approximation, some row interactions, and the use of total in vitro systems of anti surface interaction between the opposed, antigenically differing body formation. Substantial progress is being made on the de plasma membranes leads to mutual inhibition. MÃ¶ller(22) has velopment of mice that are not only germ-free but that have postulated that lymphocytes may be specifically differentiated been reared on macromolecule-free diets. In such animals un to be stimulated rather than destroyed by such surface inter resolved problems include vertically transmitted viruses and actions. The resultant blast cell seems to be a particularly possible antigens arising from somatic mutation in body cells. potent inflicter of target cell damage (11). The way in which Nevertheless, they ought to be much cleaner tools for an analy specific immunization is thought to help is by providing anti sis of the problem of stimulation of virgin cells than either body on the surface of certain of the lymphoid cells which germ-free or conventional animals. A different way of achieving acts as a "glue" ensuring close contact between aggressive a population of truly virgin antigen-sensitive cells is embodied lymphocyte and target cell. In many in vitro experiments, in the notion of bone marrow:thymus interactions. It has re target cell damage cannot be mediated by lymphocytes from cently become apparent (17) that the true self-perpetuating nonimmunized animals unless some extraneous "glue" such as

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1968 American Association for Cancer Research. Antigenicity Recognition phytohaemagglutinin or heterologous antiserum is used to is good evidence (a) for the existence of at least two recogni cause tight clumping of lymphocyte and target cell. In the case tion sites (19) possibly in different cells and (6) for a recog of specifically preimmunized animals, such additional factors nition step which can be under direct genetic control (4), the are not necessary, possibly because of the presence of cell chemical nature of which is quite obscure. surface-associated anti-target cell antibodies on many lympho Evidence is mounting that the recognition of antigenicity, cytes. The biochemical steps involved in target cell damage, i.e., of the immediate proximity of a nonidentical cell, may be or indeed of any of the phenomena related to allogeneic in a general phenomenon of which most somatic cells are capable. hibition, are still unknown. Inhibitor studies (22, 11) suggest Delineation of the nature of this recognition will probably that DNA, UNA, or protein synthesis are not involved, but turn out to be a problem in membrane physiology and bio that active cell respiration is essential. chemistry rather than conventional immunology. Allogeneic inhibition is one manifestation of this basis of recognition; Extension of Allogeneic Inhibition Concept to the Problem cellular immunity may be no more than an extreme example of Tissue Surveillance of allogeneic inhibition. The inadequacies of present meth If the line of reasoning pioneered by the Stockholm group odologies constitute a formidable barrier to progress and render and summarized all too briefly above is basically correct, then detailed interpretations quite speculative. Nevertheless, it the ability to recognize foreignness is a fundamental charac seems certain that the concept of allogeneic inhibition will be teristic of living cells, and immune reactions represent only a of prime importance for the future development of cancer special and perhaps extreme example of a general biologic immunology. phenomenon. If so, as Klein (14) has forcefully pointed out, allogeneic inhibition could represent a particularly efficient ACKNOWLEDGMENTS mechanism of tissue surveillance against antigenic variants arising through somatic mutation, viral transformation, or We wish to thank Dr. G. L. Ada for help with iodinations and other means. Normally, any cell exhibiting foreign antigens on Mrs. J. M. C. Mitchell for participation in autoradiographic its surface would be rapidly eliminated, thus avoiding the need experiments. for the activation of a cellular immune response with the quantitative constraints imposed by the need to consider its REFERENCES afferent, central, and efferent components. However, the exist ence of chimeric and mosaic animals with perfectly healthy 1. Ada, G. L., Nossal, G. J. V., and Austin, C. M. Antigens in tissues poses same problems for the universality of allogeneic Immunity, V: The Ability of Cells in Lymphoid Follicles to inhibition. This can be disposed of albeit rather vaguely, by Recognize Foreignness. Australian J. Exptl. Biol., /t2: 331-346, postulating a "tolerance induction" phase which would share 1964. some features with immunologie tolerance. The vital importance 2. Ada, G. L., Nossal, G. J. V., and Pye, J. Antigens in Immunity, of this research area to cancer immunology needs no further XI: The Uptake of Antigen in Animals Previously Rendered annotation. We await with interest the development of meth Immunologically Tolerant. J. Exptl. Biol. Med. Sci., 43: 337- odologies which will allow more precise analysis of allogeneic 344, 1965. inhibition in local tissue situations. 3. Barker, C. F., and Billingham, R. E. An Artificial Immuno logically Privileged Site. In: J. Dausset, J. Hamburger and G. Conclusions MathÃ© (eds.), Advances in Transplantation, p. 25. Copen The only basis for the recognition of antigenicity that we hagen: Munksgaard, 1968. understand from a chemical point of view is antibody. Spe 4. Benacerraf, B. The Immune Response of Guinea Pigs to cific antibody, either natural or immune, is an important Hapten-Poly-L-Lysine Conjugates as an Example of the factor in promoting correct handling of antigens by the Genetic Control of the Recognition of Antigenicity. Cold reticuloendothelial system as a prelude to antibody formation. Spring Harbor Symp. Quant. Biol., SS: 1967, in press. In all probability, natural antibodies reflect mainly previous 5. Burnet, F. M. The Clonal Selection Theory of Acquired Im stimulation of the animal by antigens cross-reacting with some munity. Cambridge: Cambridge University Press, 1959. thing that the animal has encountered previously. Thus there 6. Cohen, S., Vassalli, P., Benacerraf, B., and McCluskey, R. T. The Distribution of Antigenic and Nonantigenic Compounds is a second factor tending to encourage brisk antibody forma Within Draining Lymph Nodes. Lab. Investigation, 15: 1143- tion following the injection of antigens to which opsonins exist 1155, 1966. in the serum. This is the presence, in the animals concerned, 7. Dutton, R. W., and Mishejl, R. I. Cellular Events in the of cells with memory for the cross-reacting antigen. However, results from germ-free animals suggest that there must be Immune Response. The in vitro Response of Normal Spleen Cells to Erythrocyte Antigens. Cold Spring Harbor Symp. additional factors involved in the inherent immunogenicity of Quant. Biol., SÃ©:1967,in press. many viral and bacterial antigens. 8. Fazekas de St. Groth, S. Cross-Recognition and Cross- While the clonal selection hypothesis of antibody formation Reactivity. Cold Spring Harbor Symp. Quant. Biol., 32: 1967, (5) provides a useful framework in which to think about anti in press. genic stimulation of lymphoid cells, it appears that the nature 9. Frei, P. C., Benacerraf, B., and Thorbecke, G. J. Phagocytosis of antibody-like receptor sites on lymphocytes is not the sole of Antigen, a Crucial Step in the Induction of the Primary factor in determining the results of antigenic stimulation. There Response. Proc. Nati. Acad. Sei. U. S., 53: 20, 1965.

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10. Gowans, J. L. and McGregor, D. D. The Immunological Ac VI: The Phagocytic Reticulum of Lymph Node Follicles. J. tivities of Lymphocytes. Progr. Allergy, 9: 1-78, 1965. Exptl. Biol. Med. Sci., 40: 445-448, 1967. 11. Holm, G., and Perlmann, P. Phytohemagglutinin Induced 19. Mitchison, N. A. Receptors for Antigen. Cold Spring Harbor Cytotoxic Action of Unsensitized Immunologically Competent Symp. Quant. Biol., SÃ©:1967, in press. Cells on Allogeneic and Xenogeneic Tissue Culture Cells. 20. Mitchison, N. A. Immunological Tolerance. In: B. Cinader Nature, Â«07:818, 1965. (ed.), Proceedings of Symposium on Regulation of the Im 12. Humphrey, J. H., and Frank, M. M. The Localization of Non- mune Response, in press. Microbial Antigens in the Draining Lymph Nodes of Tolerant, 21. MÃ¶ller, E., and MÃ¶ller, G. Inhibition of Tumor Growth by Normal and Primed Rabbits. Immunol., 13: 87-100, 1967. Confrontation with Incompatible Cells. Cancer, 20: 871-879, 13. Jerne, N. K. The Depressive Effects of 7 S Antibody and the 1967. Enhancing Effects of 19 S Antibody in the Regulation of the 22. MÃ¶ller, G., Zukoski, C., Lundgren, G., Beckman, V., and Primary Immune Response. In: J. Killander (ed.), Nobel MÃ¶ller, E. In Vitro Cytotoxicity by Non-immune Lymphoid Symposium III, Gamma Globulins, p. 421. Stockholm: Alm- Cells: Differential Mechanism of Action between Various qvist and Wiksell, 1967. Cell Types. In: 3. Dausset, J. Hamburger, and G. MathÃ© 14. Klein, G. Lymphocytes and Antibodies in Relation to Malig (eds.), p. 67. Copenhagen: Munksgaard, 1968. nant Disease. In: G. E. W. Wolstenholme and R. Porter (eds.), 23. Robinson, W. R., Marbrook, J., and Diener, E. Primary Anti- Ciba Foundation Symposium on The Thymus, pp. 348-359. genie Stimulation and Measurement of Antibody to Sheep London: J. & A. Churchill, Ltd., 1966. Red Blood Cells in vitro. J. Exptl. Med., 126: 347-356, 1967. 15. Lang, P. G. and Ada, G. L. Antigen in Tissues. IV. The Effect 24. Sell, S., and Gell, P. G. H. Studies on Rabbit Lymphocytes in of Antibody on the Retention and Localization of Antigen in Vitro. J. Exptl. Med., m: 423, 1965. Rat Lymph Nodes. Immunology, 13: 523, 1967. 25. Strober, S. and Gowans, J. L. The Role of Lymphocytes in the 16. Lang, P. G. and Ada, G. L. The Localization of Heat De Sensitization of Rats to Renal Homografts. J. Exptl. Med., natured Serum Albumin in Rat Lymph Nodes. Australian J. 1SS: 347-360, 1965. Exptl. Biol. Med. Sci., 45: 445-448, 1967. 26. Wigzell, H. Studies on the Regulation of Antibody Synthesis. 17. Miller, J. F. A. P., Mitchell, G. F., and Weiss, N. S. Cellular Cold Spring Harbor Symp. Quant. Biol., 32: 1967, in press. Basis of the Immunological Defects in Thymectomized Mice. 27. Williams, G. M. Antigen Localization in Lymphopenic States. Nature, SI4: 992-997, 1967. I. Localization Pattern Following Chronic Thoracic Duct 18. Miller, J. J. Ill, and Nossal, G. J. V. Antigens in Immunity, Drainage. Immunology, 11: 467-474, 1966.

Fig. 1. Electron microscopic autoradiograph of a medullary macrophage from a rat and popliteal lymph node 1 day after the injec tion of a complex of HSA-125I and anti-HSA antibody into the hind foot-pad. Note that nearly all the grains are within membrane- bound inclusions (phagolysosomes; see Ref. 23). HSA, human serum albumin. Fig. 2. Antigen capture in a primary lymphoid follicle; same antigen and time as Fig. 1. In follicles, there exists a profusion of fine cytoplasmic processes, some of which emanate from reticular cells and others from lymphocytes. Antigen preserves a largely extracellu lar, membrane-associated localization.

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G. J. V. Nossal and A. Abbot

Cancer Res 1968;28:1423-1429.

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