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ItemCell to cell interaction in the immune response. 3. Chromosomal marker analysis of single antibody-forming cells in reconstituted, irradiated, or thymectomized mice.(Rockefeller University Press, 1968-10-01)Two new methods are described for making chromosomal spreads of single antibody-forming cells. The first depends on the controlled rupture of cells in small microdroplets through the use of a mild detergent and application of a mechanical stress on the cell. The second is a microadaptation of the conventional Ford technique. Both methods have a success rate of over 50%, though the quality of chromosomal spreads obtained is generally not as good as with conventional methods. These techniques have been applied to an analysis of cell to cell interaction in adoptive immune responses, using the full syngeneic transfer system provided by the use of CBA and CBA/T6T6 donor-recipient combinations. When neonatally thymectomized mice were restored to adequate immune responsiveness to sheep erythrocytes by injections of either thymus cells or thoracic duct lymphocytes, it was shown that all the actual dividing antibody-forming cells were not of donor but of host origin. When lethally irradiated mice were injected with chromosomally marked but syngeneic mixtures of thymus and bone marrow cells, a rather feeble adoptive immune response ensued; all the antibody-forming cells identified were of bone marrow origin. When mixtures of bone marrow cells and thoracic duct lymphocytes were used, immune restoration was much more effective, and over three-quarters of the antibody-forming mitotic figures carried the bone marrow donor chromosomal marker. The results were deemed to be consistent with the conclusions derived in the previous paper of this series, namely that thymus contains some, but a small number only of antigen-reactive cells (ARC), bone marrow contains antibody-forming cell precursors (AFCP) but no ARC, and thoracic duct lymph contains both ARC and AFCP with a probable predominance of the former. A vigorous immune response to sheep erythrocytes probably requires a collaboration between the two cell lineages, involving proliferation first of the ARC and then of the AFCP. The results stressed that the use of large numbers of pure thoracic duct lymphocytes in adoptive transfer work could lead to good adoptive immune responses, but that such results should not be construed as evidence against cell collaboration hypotheses. Some possible further uses of single cell chromosome techniques were briefly discussed.
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ItemAutoradiographic studies on the immune response.I. The kinetics of plasma cell proliferation.(Rockefeller University Press, 1962-01-01)The origin and growth kinetics of plasma cells have been investigated using autoradiographic labeling techniques. Rats immunized once with Salmonella flagella were given a single pulse of H(3)-thymidine 4 or 40 weeks later. 2 hours after the tracer injection, they received a secondary antigenic stimulus. When animals were sacrificed immediately only certain cells from the resting primarily immunized lymph nodes, notably large and medium lymphocytes, were labeled. Subsequent to secondary stimulation, animals were killed at intervals; nearly all the plasma cells formed within the next 5 to 6 days were labeled. They must thus have been the progeny of cells already capable of synthesizing DNA in resting nodes, most probably of large lymphocytes. Plasmacytopoiesis began with little or no lag following secondary immunization, and the number of labeled plasma cells rose exponentially between the 2nd and 4th day, with a doubling time of about 12 hours. Studies of mean grain counts of primitive cells also suggested that the generation time of plasmablasts was 12 hours or less. The hypothesis was proposed that immunological memory depended on the persistence, following primary stimulation, of a continuously dividing stem line of primitive lymphocytes, reactive at all times to further antigenic stimulation.
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ItemAntigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles.(Rockefeller University Press, 1968-02-01)This paper describes the trapping of antigen in lymphoid follicles of rat popliteal lymph nodes as revealed by electron microscopic radioautographs following injection of (125)I-labeled Salmonella adelaide flagella and other materials. The antigen was taken up vigorously, and to an approximately equal extent, by both primary and secondary follicles. The rate of uptake was faster in preimmunized than in virgin adult rats. The bulk of the antigen in follicles was extracellular, and persisted in this location for at least 3 wk. Label was most frequently found at or near the surface of fine cell processes. Many of these were branches of dendritic follicular reticular cells. Such processes interdigitated with equally fine processes of lymphocytes, creating an elaborate meshwork. In some cases, antigen was found between lymphocytes which appeared to be in close apposition. Occasionally, a few grains appeared over lymphocyte nuclei and study of serial sections suggested that this probably represented true entry of small amounts of antigen into lymphocytes. The characteristic "tingible body" macrophages (TBM) of germinal centers appeared to play only a secondary role in follicular antigen retention. They showed degrees of labeling over their phagocytic inclusions varying from negligible to moderately heavy. Moreover, follicles lacking or poor in TBM retained antigen just as effectively as those containing numerous TBM. The hypothesis is advanced that TBM may be derived from monocytes that migrate down from the circular sinus. Follicular localization of three other materials was also studied, though not in such detail. These were (125)I-HSA complexed to anti-HSA: (125)I-labeled autologous IgG; and (125)I-monomeric flagellin. All of these showed the basic features of intercellular, membrane-associated deposition noted with (125)I-flagella. The role of follicular antigen depots in immune induction is discussed. The tentative conclusion is reached that follicular antigen in a primary follicle encounters natural antibody on the surface of certain antigen-reactive lymphocytes. The resultant reaction causes blast cell transformation and eventually the genesis of a germinal center.
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ItemANTIGENS IN IMMUNITY. VI. THE PHAGOCYTIC RETICULUM OF LYMPH NODE FOLLICLES.(Rockefeller University Press, 1964-12-01)The localization of antigen in primary follicles and germinal centers of rat popliteal lymph nodes described previously using I(125)- and I(125)-labeled antigen has been confirmed by direct staining with fluorescent antibodies. A fine web of phagocytic reticulum in primary follicles was found to be responsible for antigen localization in this area. The nature of this web was confirmed by studies of the localization of colloidal carbon. This unique feature of primary follicles is discussed in relation to its importance in the induction of immune responses, our belief being that the great surface area of antigen retaining cytoplasm in primary follicles is responsible for the appearance of germinal centers in these particular parts of the node.
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ItemANTIGENS IN IMMUNITY. IX. THE ANTIGEN CONTENT OF SINGLE ANTIBODY-FORMING CELLS.(Rockefeller University Press, 1965-06-01)Flagellar antigens from S. adelaide bacteria were labelled with carrier-free (125)I so as to achieve a substitution rate of 0.1 to 2.1 radioactive iodine atoms per flagellin molecule. Lymph nodes from rats injected with small amounts of these antigens were teased into a single cell suspension. Single antibody-forming cells were identified and submitted individually to autoradiography so as to measure their content of iodine (125)I. The study was confined to the 7S phase of the primary response. Grain counts over 216 single antibody-forming cells were no higher than counts over equivalent background areas in the emulsion. This finding suggested that the cells contained little or no macromolecular antigen, and it was considered very unlikely that there were sufficient macromolecules of antigen in a plasma cell to act as a direct template on polysomes for the formation of antibody. The question of the possible presence, in such cells, of fragments of antigen was considered. This possibility, while not supported by the present results, cannot be excluded at present.
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ItemSINGLE CELL STUDIES ON 19S ANTIBODY PRODUCTION.(Rockefeller University Press, 1964-03-01)Rats were immunized with Salmonella adelaide flagella. By zone centrifugation of serum samples in sucrose gradients, it was shown that, as in many other systems of antibody formation, the first response was the formation of 19S, mercaptoethanol (ME)-sensitive antibody. This was quickly replaced by 7S, ME-insensitive antibody. Popliteal lymph node cell suspensions were prepared, and cells with antibody on their surface were identified by the method of bacterial adherence. By micromanipulation such cells were washed, placed into microdroplets, examined under high-power phase contrast and broken to release intracellular antibody. These droplets were then studied in either of two ways. In the first method, each droplet was halved and one half treated with ME. Then both halves were titrated for immobilizing antibody through serial twofold dilution of the half microdroplets. Droplets showing destruction of antibody by ME were classified as 19S; those showing no reduction in titer as 7S; and those showing significant (>1 log(2)) reduction as double producers; i.e., cells containing both 7S and 19S antibodies. In the second method, droplets were divided into 4 equal quarters, for testing after treatment with either ME, or a specific rabbit anti-rat 7S globulin serum, or both. In these experiments, cells showing some remaining antibody after treatment with either reagent, but not after treatment with both reagents, were classified as double producers. Of 144 cells tested, 123 contained readily detectable amounts of antibody. These comprised 42 19S cells, 64 7S cells, and 17 double producers. The double producers were frequent at times when the switchover from 19S to 7S antibody production was occurring. All except 4 of the cells in the study could clearly be identified as members of the plasma cell series. Though 7S cells became more frequent as the cell population matured, no clear-cut correlation between cell immaturity and 19S production could be obtained. In the primary response many fully mature plasma cells contained only 19S antibody; conversely, in the secondary response many blasts contained 7S antibody. No morphological difference between 19S and 7S cells could be found. The results suggested that many cells or cell clones go through a sequence whereby each forms first 19S and later 7S antibody with identical combining sites.
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ItemAutoradiographic studies on the immune response. II. DNA synthesis amongst single antibody-producing cells.(Rockefeller University Press, 1962-01-01)The DNA-synthesizing capacity of single antibody-forming cells was tested by a combination of micromanipulatory and autoradiographic techniques. Rats were immunized with S. adelaide flagellin, a protein antigen known to contain significant contamination with somatic (O) antigen. Single cells from secondarily immunized rats were tested for production of anti-H and anti-O antibodies by previously described and newer techniques. Positive antibody producers were transferred onto clean dry slides by micromanipulation, and autoradiographs were performed. When rats had received tritiated thymidine 1 hour before killing, labeling of antibody-forming cells was taken to imply that the cell was preparing for further mitotic division. It was found that on the 2nd and 3rd day of a secondary response, many of the antibody-producing cells in the nodes (chiefly plasmablasts) were incorporating tritiated thymidine. At the height of the cellular response, however, at 4 and 5 days, the majority of active antibody producers (chiefly mature plasma cells) were incapable of DNA synthesis. There appeared to be an inverse relationship between the antibody-forming and DNA-synthesizing capacities of the cell population under study; as more of the cells studied formed detectable antibody, fewer of them incorporated the DNA precursor. The age of plasma cells was also studied. Animals were killed at the height of the cellular immune response, having previously received an injection of tritiated thymidine 1 to 48 hours before killing; i.e., at 63 to 110 hours after their secondary stimulus. As the interval between isotope injection and killing increased, the proportion of antibody-forming cells showing labeling increased. With an interval of 30 hours, about half the antibody-forming cells were labeled and of 48 hours, over 95 per cent were labeled. This was taken as evidence that, few, if any, antibody-forming cells found at the height of a secondary response were more than 48 hours old. On the basis of these experiments and those reported in the accompanying paper, a simplified scheme showing the development of an antibody-forming clone in the secondary response was proposed.
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ItemAntigens in immunity. XIV. Electron microscopic radioautographic studies of antigen capture in the lymph node medulla.(Rockefeller University Press, 1968-02-01)Details of antigen trapping and processing in the rat lymph node have been investigated by the technique of high resolution radioautography. A series of 24 adult rats was injected with 20 microg of (125)I-labeled Salmonella adelaide flagella, given as either a primary or a secondary stimulus into one hind foot-pad. At intervals ranging from 3 min to 3 wk, rats were killed and the popliteal nodes were processed for electron microscopic radioautography using Kodak NTE emulsion. The present paper deals with events in the lymph node medulla, and an accompanying report describes the radically different behavior of antigen in the cortical follicles. In the medulla, lightly labeled granulocytes were transiently encountered, but by far the greatest bulk of antigen was in macrophages. Antigen entered these cells in two ways: by direct penetration of the plasma membrane; and by pinocytosis. In either case, the antigen rapidly became surrounded by tiny vesicles which may have represented Golgi-derived "protolysosomes." Vacuolar fusion ensued and a series of progressively larger and more complex antigen-containing "phagolysosomes" was formed. Substantial amounts of antigen could be detected in such bodies for at least 3 wk. The antigen injection, as expected, caused extensive plasma-cytopoiesis. No evidence of label in plasma cells was obtained. No special anatomic relationship between plasma cells and antigen depot sites was discovered. These results are briefly discussed in relation to current theories of immune induction.
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ItemOntogeny of the immune response. I. The development of the follicular antigen-trapping mechanism.(Rockefeller University Press, 1966-07-01)Polymerized flagellin from Salmonella adelaide was labeled with I(125) and injected into rats varying in age from 0 to 42 days. Lymphoid organs were removed at various intervals and the progressive development of antigen-capturing structures was studied using autoradiography. The chief findings were as follows: 1. Newborn rats lack the follicular and medullary antigen-trapping structures characteristic of adult animals. 2. At the age of 10 to 14 days, the first signs of specific cortical antigen localization appear in lymph nodes. This initially takes the form of a continuous "cortical rim" of antigen localization. 3. Within a further 4 to 6 days, the Anlagen of true follicular antigen-capturing structures appear, the continuous rim being only a transitional mechanism. 4. The antigen-capturing part of the follicle appears before the lymphoid component; follicle Anlagen can be defined only on autoradiographs and cannot be seen on ordinary histological sections. 5. The system of medullary macrophages develops gradually over the period 2 to 6 weeks of age. 6. The ability of lymph nodes to retain antigen increases progressively, there being a fivefold increase in the amount of antigen retained per unit weight of lymphoid tissue between 2 and 6 wk of age.
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