Showing papers on "Antigen published in 1968"

Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs.

Abstract: The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label (51Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the graft recipients, as well as its 19S and 7S fractions, protected target cells against the toxic effect of the spleen cells, but a lipoprotein antigen isolated from the tumour cells failed to inhibit the cytotoxic reaction. Target cell lysis as measured by specific release of 51Cr was partially inhibited by actinomycin-D and by cycloheximide at concentrations which effectively blocked DNA-dependent RNA and protein synthesis.

An antigen detected in the blood during the incubation period of serum hepatitis

Abstract: The aim of the following experiments was to provide an objective immunologic criterion for the diagnosis of serum hepatitis, as well as a possible means of screening for carriers of the agent of this disease. An antigen that reacted in the immunodiffusion test with serum from multiply transfused patients was detected in the blood during the incubation period prior to the onset of major chemical or clinical abnormalities. Double blind experiments suggest that this antigen is specific for serum hepatitis virus. Materials and Methods.-Clinical specimens: Sera from cases of transfusion-induced viral hepatitis, which we have collected, were obtained as part of a long-term study involving biweekly follow-up of transfused patients at The New York Hospital. Patients volunteering to participate in this study provided blood samples prior to transfusion and at least biweekly for a period of 6 months or more following transfusion. Test serum: The reference \"antiserum\" used in the majority of the studies to be described, hereinafter referred to as serum S, was obtained from a 24-year-old male patient with hemophilia who has received more than 10,000 units of blood, fresh-frozen plasma, and cryoprecipitate during the course of treatment for bleeding episodes. He has had no episodes of icteric hepatitis, but it was presumed that he had been multiply exposed to the virus or viruses of serum hepatitis. Serum S was chosen for these studies because the patient's multiple exposure was thought to ensure a hyperimmune status. Subsequently, four other sera from multiply transfused patients have been found to react in a manner similar to serum S. For some experiments, the serum was concentrated by ethanol fractionation. To each milliliter of serum to be concentrated, 8 ml of 30% ethanol in 0.1 Ml NaCl, 0.01 M tris(hydroxymethyl)aminomethane (Tris), 0.001 Ml ethylenediaminetetraacetate (EDTA), (pH 7.0 at -7oC) were added. This mixture was held at -70C and lyophilized. The dried globulin fraction was then rehydrated with distilled water to 0.1 the original volume of serum employed. Immunodiffusion technique: Double diffusion in agar gel was done by a micro-Ouchterlony technique.1 Nonspecific precipitation reactions between adjacent wells were eliminated by the use of 0.9% agarose dissolved in a buffer composed of 0.1 M NaCl, 0.01 M Tris (pH 7.6 at 250C), and 0.001 M EDTA containing 1 mg/ml protamine sulfate. Protamine sulfate has been recently suggested as a means of decreasing virus-agar interaction.2 Plates were incubated in a humid atmosphere at room temperature and read daily for 7 days. Strong reactions were evident after overnight incubation, while weaker reactions required 2 or 3 days' incubation and intensified for several days. Clinical chemical methods: Serum glutamic pyruvic transaminase (SGPT) was assayed by a kinetic spectrophotometric method with the Gilford multiple method sample recording spectrophotometer.3 Serum lactic dehydrogenase (LDH) enzymes were assayed by the method of Amador et al.4 with the same instrument. Serum LDH isoenzymes were separated by thin agar gel electrophoresis and quantitated fluorometrically.5 Results.-Demonstration of an antigen appearing in the blood during the incubation period of serum hepatitis: Failure in the past to isolate a causative virus from serum hepatitis could possibly be attributed to the fact that most isolation attempts have been carried out with specimens obtained early in the clinical course of the disease, at what is actually a late stage of the infection due to the

Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat.

Abstract: The peroxidase-labeled antibody method was modified to localize multiple tissue antigens in a single histologic section. The first antigen was localized by the indirect method and the antisera were removed from the section by elution, leaving the colored reaction products identifying the antigenic sites. The second and subsequent antigens were localized similarly, using substrates that develop reaction products of different colors. For the demonstration of the method, anti-human growth hormone (GH), anti-human thyrotropic hormone (TSH) and anti-human chorionic gonadotropin, which cross-reacts with rat luteinizing hormone (LH), were used with sections of pituitary gland of the rat. AntiGH reacted with small acidophilic cells. Anti-LH was localized in oval shaped cells, and anti-TSH was localized in angulated basophilic cells. Some cells were devoid of these three antigens and no cell contained more than one of them. A convenient method which would identify more than one amitigen in the same section of tissue would be of considerable value to cellular biologists. In the past, two tissue antigens have been localized simultaneously with the immunofluorescent method using antibodies labeled with fluorescent coml)ounds of different color (4, 5, 1012). This technique has obvious limitations, including the tendency of the fluorescence of one label to mask that of the other, and the tendency for one color to fade more rapidly than the other

Regulatory effect of antibody on the immune response.

Abstract: Publisher Summary This chapter deals with the regulatory effect of antibody on antibody formation. It is possible to analyze those factors that influence the process of antibody synthesis. Certainly antibody, the end product of the process, is among the most potent and specific of inhibitors of antibody synthesis. That this inhibition results from the interaction of antibody with antigen neutralizing the immunogenicity of the latter seems likely, and the evidence for this is critically presented. The potential use of this mechanism is suggested by its effectiveness in the enhancement of tissue grafts, its use in the therapeutic prevention of anti-D antibody responses in mothers of Rh-incompatible fetuses, and its possible role in the induction of some types of immunological tolerance. The immune response represents a predictable series of events, characterized by the sequential appearance of several classes of γ-globulin antibody molecules and the expression of various cell-mediated immune reactions. One mechanism is described for regulating the concentration of immunoglobulin (IgG) in the circulation. The mechanism operates by increasing the catabolic rate of IgG when the serum concentration is abnormally increased as, for example, in multiple myeloma. Two mechanisms are described both of which regulate the immune response. These are alteration of antigenic stimulation and suppression of the immune response by passive transfer of specific antibodies prior to or shortly after administration of antigen.

Cell to cell interaction in the immune response. II. The source of hemolysin-forming cells in irradiated mice given bone marrow and thymus or thoracic duct lymphocytes.

Abstract: 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.

Cell to cell interaction in the immune response : i. hemolysin-forming cells in neonatally thymectomized mice reconstituted with thymus or thoracic duct lymphocytes

Abstract: 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.

Antigens in immunity. XV. Ultrastructural features of antigen capture in primary and secondary lymphoid follicles.

Abstract: 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.

Studies on human antibodies : vi. selective variations in subgroup composition and genetic markers

Abstract: The composition of various isolated antibodies was determined by quantitative analyses for heavy chain subgroups and light chain types Certain antibodies such as anti-tetanus toxoid and anti-A isoagglutinins were predominantly of the major γG1-type However, a high preponderance of molecules of the minor γG2-subgroup was found for antibodies to dextran, levan, and teichoic acid These findings explain some unusual features previously noted for anti-dextrans such as weak PCA reactions and lack of Gm antigens Studies of several isolated antibodies from single heterozygous individuals showed a selective absence of genetic markers in certain antibodies and their presence in others The "allelic exclusion" principle was clearly evident in the isolated antibodies of two different individuals Large differences in the ratio of kappa to lambda light chains were observed for the same type of antibody from different individuals Subfractionation of dextran antibodies by affinity for specific glycosidic linkage or combining site size produced marked changes in the ratios The isomaltohexaose eluates of the dextran antibodies from two subjects were primarily kappa and the isomaltotriose eluates were predominantly lambda The one anti-levan antibody studied was uniquely homogeneous, consisting exclusively of γG2-heavy chains and kappa light chains By these criteria as well as others, it closely resembled myeloma proteins

Particles associated with Australia Antigen in the Sera of Patients with Leukaemia, Down's Syndrome and Hepatitis

Abstract: AUSTRALIA antigen was first identified using an antiserum produced in a transfused patient1,2. The antiserum gave a clear precipitin line in a double diffusion experiment when placed adjacent to the serum from an Australian aborigine. Pending further identification of the antigen, the geographic name “Australian antigen” was given to the reacting material found in the aborigine's serum. Specific antisera against this antigen can be produced by immunizing rabbits with serum containing Australia antigen, and subsequent absorption with serum which does not contain Australia antigen3. The precipitin band which forms between the haemophilia antiserum and the serum containing Australia antigen stains faintly with sudan black, indicating that the antigen contains lipid. It has a specific gravity of less than 1.21 and appears in the first peak in ‘Sephadex G-200’ column chromatography (indicating a high molecular weight)4.

The mechanism of immunological paralysis

Abstract: Publisher Summary This chapter presents the mechanism of immunological paralysis. The paralysis is the result of a central failure of responsiveness brought about by exposure to antigen that prevents potentially competent cells from initiating a specific antibody response. In cellular terms, paralysis results from an adverse effect of antigen directly on lymphocytes, and the recovery from paralysis appears to involve the recruitment of new competent cells, not a recovery of responsiveness by once paralyzed cells. The quantitative factors involved in this antigenic exposure are determined, and these data not only shed light on the process of immunological paralysis but also on those conditions essential to the initiation of an antibody response. The barrier that the homograft reaction imposes to tissue transplantation can be overcome by paralyzing the host with donor antigens. This form of treatment that is already a routine procedure in animals provides the best hope of suppressing a homograft reaction without interfering with other immunological defenses. The chapter focuses on protein antigens, particularly heterologous serum proteins. These are of special interest because of their application in exploring the induction of paralysis in adult animals by low doses. The competent cell that is affected appears to be the lymphocyte. Macrophages in or from paralyzed animals function normally, but lymphocytes display altered reactivity. There is some evidence to suggest that antigen acts directly upon lymphocytes in the induction of paralysis.

Antigenic modulation loss of tl antigen from cells exposed to tl antibody. study of the phenomenon in vitro

Abstract: Antigenic modulation (the loss of TL antigens from TL+ cells exposed to TL antibody in the absence of lytic complement) has been demonstrated in vitro An ascites leukemia, phenotype TL1,2,3, which modulates rapidly and completely when incubated with TL antiserum in vitro, was selected for further study of the phenomenon Over a wide range of TL antibody concentrations modulation at 37°C was detectable within 10 min and was complete within approximately 1 hr The cells were initially sensitized to C' by their contact with antibody, thereafter losing this sensitivity to C' lysis together with their sensitivity to TL antibody and C' in the cytotoxic test The capacity of the cells to undergo modulation was abolished by actinomycin D and by iodoacetamide, and by reducing the temperature of incubation to 0°C Thus modulation apparently is an active cellular process Antigens TL 1,2, and 3 are all modulated by anti-TL1,3 serum and by anti-TL3 serum This modulation affects all three TL components together, even when antibody to one or two of them is lacking aAnti-TL2 serum does not induce modulation and in fact impairs modulation by the other TL antibodies The influence of the TL phenotype of cells upon the demonstrable content of H-2 (D region) isoantigen, first shown in cells modulated in vivo, has been observed with cells modulated in vitro Cells undergoing modulation show a progressive increase in H-2 (D region) antigen over a period of 4 hr, with no change in H-2 antigens of the K region Restoration of the TL+ phenotype of modulated cells after removal of antibody is less rapid than TL+ → TL- modulation and may require several cell divisions

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity : iii. analysis of mechanism

Abstract: The cytopathic effect of lymph node cells from tuberculin-sensitized rats on rat embryo fibroblasts in the presence of PPD was not enhanced by admixture of normal (nonsensitized) lymph node cells. Preincubation studies showed that this in vitro response is initiated by the reaction of lymphocytes with specific antigen, beginning within 30 min, rather than uptake of antigen by the fibroblasts. The supernatant fluids from suspensions of sensitized cells incubated with PPD for 17 hr or more possessed cytotoxic activity. The target fibroblasts showed a marked increase in acid phosphatase content within 48 hr after the addition of sensitized lymph node cells and antigen.

Autologous immune complex nephritis induced with renal tubular antigen. I. Identification and isolation of the pathogenetic antigen.

Abstract: The nephritogenic antigen, responsible for the immunogenic stimulus in experimental allergic glomerulonephritis induced with tubular antigen, has been identified as a renal tubular epithelial (RTE)-specific antigen and has been isolated in a relatively purified form. This antigen, RTE-α5, is a distinct and antigenically specific lipoprotein of high density which is derived primarily from the brush border of proximal convoluted tubular epthelium of the rat kidney. It has been suggested that this molecule may be a plasma membrane subunit. Immunization of rats with as little as 3 µg N of RTE-α5 in complete Freund's adjuvant has effectively induced this form of membranous glomerulonephritis. RTE-α5 is not a constituent of normal rat glomeruli; however, with the onset of autologous immune complex nephritis it is deposited in a granular fashion along glomerular capillary walls indistinguishable from the deposits of γ-globulin and complement. The antigenic specificity of this antigen and its tissue derivation has been explored, and the observations support the autologous immune complex pathogenesis of the glomerulonephritis induced in rats by immunization with renal tubular antigen.

Ly-A and Ly-B: two systems of lymphocyte isoantigens in the mouse.

Abstract: Two systems of isoantigens confined to thymocytes and lymphocytes have been defined in the mouse by cytotoxic isoantisera. The two genetic loci have been designated Ly-A and Ly-B. Typing of 25 mouse strains and sublines indicates that each system comprises only 2 alleles, determining alternative isoantigens Ly-A. 1 or Ly-A. 2, and Ly-B. 1 or Ly-B. 2, respectively. There is no evidence of multiple alleles or that either locus is compound. Tests for genetic linkage were performed by serological typing of mice from segregating generations. None of the four loci H-2 (group IX), $\theta $, Ly-A and Ly-B is closely linked to any other of the four. Ly antigens are found in high concentration on thymocytes and in lesser amounts on lymphocytes. The small absorptive capacity of bone marrow cell suspensions is probably due to the presence of mature lymphocytes rather than thymocyte-lymphocyte precursors, which according to preliminary evidence lack Ly antigens. Ly-B. 1 is exceptional in that the disparity between thymocytes and lymphocytes in content of antigen is greater than that of the three other antigens. When Ly antiserum is injected into mice of the relevant Ly type, under conditions which give complete absorption of H-2 antibody in vivo, Ly antibody is not extensively absorbed; this is to be expected from the limited tissue distribution of Ly antigens. Absorption of Ly and $\theta $ iosantibodies in vivo is reduced by treatment of the recipients with cortisone or lethal total-body irradiation. The reduction in titre of Ly and $\theta $ antibodies in such mice may be no more than that resulting from dilution. Absorption of injected H-2 antibody also may be reduced by treatment of the recipients with cortisone but in this case the effect of cortisone does not approach the virtual abolition of absorption that is seen with Ly and $\theta $ antibodies. The thymocytes and lymphocytes of chimeras formed by restoring lethally irradiated mice with allogeneic bone marrow have the Ly-A type, Ly-B type and $\theta $ type of the donor. The content of Ly antigens on cells of different leukaemias varies widely and shows no correlation with presence or absence of TL (thymus-leukaemia) antigen; TL+ leukaemias may be Ly- or Ly+ and TL- leukaemias may be Ly- or Ly+. Five systems of isoantigens are now demonstrable on mouse thymocytes by means of cytotoxic isoantisera. Of these, TL is an exclusively thymic antigen. Ly-A and Ly-B antigens occur also on lymphocytes. $\theta $ occurs on thymocytes, on lymphocytes, and in brain. The fifth antigen, H-2, is still more widely distributed and differs from the other four in being represented less strongly on thymocytes than on lymphocytes.

Quantitative studies on the mixed lymphocyte interaction in rats iii. kinetics of the response

Abstract: The proliferative interaction of cultured rat lymphocytes of immunogenetically disparate origin—the mixed lymphocyte interaction—was employed as an experimental model to examine the initial stages of the immune response mechanism Using mixed cultures of cells derived from parental strain and F1 hybrid rats, in which only the parental lymphocytes respond, the following observations were made on the magnitude and kinetics of the reaction After initiation of the cultures, there was a latent period of approximately 40 hours during which time no mitotic activity was detected This inactive phase was followed by a period of proliferation in which previously nondividing cells entered the mitotic cycle for the first time Activity in the cultures, as detected by incorporation of radioactive thymidine and measured by radioautography or scintillation spectrometry, increased exponentially with a doubling time (T2) of 9–10 hr In this exponential proliferative phase, lasting approximately 100 hr, the dividing cells underwent a series of rapid sequential divisions with a generation time (Tc) of 8 hr, and few, if any, dropped out of the mitotic cycle In addition to the cells which first entered mitosis at the beginning of the proliferative phase and then proceeded through multiple divisions, significant numbers of new, previously nondividing cells continued to enter the mitotic cycle during the entire exponential growth phase The total number of these newly responsive, first division cells throughout the total culture period amounted to 1–3% of the original parental cell inoculum This is a surprisingly large proportion of peripheral blood lymphocytes with demonstrable reactivity to a particular antigen system, if it is assumed that these first division cells in vitro are functionally related to the hypothetical antigen-sensitive cells which proliferate and differentiate into immunological effector cells At present there is no entirely satisfactory explanation for this large number of reactive cells in the mixed lymphocyte interaction

Ultrastructural localization of antibody in differentiating plasma cells

Abstract: Antibody was localized by electron microscopy within differentiating and mature plasma cells of the spleens of hyperimmunized rabbits. Horseradish peroxidase was used as antigen. Intracellular antibody to peroxidase was revealed in glutaraldehyde-fixed tissue by coupling it with its antigen and then revealing the sites of peroxidase activity cytochemically. Antibody first appears in the perinuclear space of hemocytoblasts where it persists through differentiation into immature plasma cells, but it disappears from this site in mature plasma cells. Concomitant with the development of the ergastoplasm, antibody accumulates in many but not all of its cisternae. Antibody is present in the lamellar portion of the Golgi apparatus in all phases of plasmacytic differentiation. Mature plasma cells exhibit two types of antibody distribution, a concentration into large spherical intracisternal granules or an overflowing into all parts of the cytoplasm.

Macrophage-Lymphocyte Interaction in the Antigen-Induced Blastogenic Response of Human Peripheral Blood Leukocytes

Abstract: Glass bead column purified lymphocyte suspensions have reduced blastogenic responses to antigen when compared to unseparated leukocyte suspensions in vitro. Culture of these purified lymphocytes on macrophage monolayers generally restored the cells response to antigen. The response to phytohemagglutinin (PHA) was unaffected by column purification. The antigens used were streptolysin O (SLO), streptokinase-streptodornase (SK-SD), purified protein derivative (PPD) and small pox vaccine (POX). The response of the unseparated leukocytes, separated lymphocytes and separated lymphocytes on monolayers were 76%, 74% and 74% blasts generated in response to PHA; 18%, 0.0% and 10.0% blasts generated in response to SLO; 14.0%, 0.5% and 8.5% in response to SK-SD; 6%, 0% and 6% in response to PPD; and 7.5%, 0% and 1% in response to POX. The response of separated and unseparated cells to antigen but not to PHA was found to be dependent on the density of cells in culture. For Leighton tubes the optimal cell concentrations were 3 to 4 × 106 lymphocytes and 0.2 to 0.6 × 106 macrophages per culture. Separation of the lymphocytes and macrophages by a semi-permeable membrane prevented the restoration of blastogenesis. These results are consistent with the view that macrophage uptake of antigen and macrophage-lymphocyte interaction are necessary for the blastogenic response of lymphocytes to antigen in vitro.

Observations on Australia antigen in Japanese.

Abstract: Summary. Frequency of Australia antigen in blood donors of the Tokyo area was estimated nearly 1%. A higher frequency in male donors was observed. The antigen was not an invariable serum antigen in patients who received blood transfusion and in patients with viral hepatitis. The antigen was found often only in the early phase of viral hepatitis. The appearance of Australia antigen did not always accompany clinical nor biochemical hepatitis. The antibodies against Australia antigen were found not only in the recipients of Australia antigen positive but also of Australia antigen negative blood. The nature of the antigen and its significance in blood transfusion were discussed.

The Transformation of Column-Purified Lymphocytes with Nonspecific and Specific Antigenic Stimuli

Abstract: Summary Phytohemagglutinin (PHA)-stimulated cultures of column-separated “purified” human peripheral blood lymphocytes incorporated tritiated thymidine to the same extent as cultures of unseparated leukocytes. However, they incorporated less tritiated thymidine when stimulated with the nonspecific staphylococcal filtrate (SF), specific antigens or with low doses of PHA. Furthermore, SF, antigens, and homologous leukocytes stimulated a median of 10, 5 and 5% respectively of the small lymphocytes in unseparated leukocyte cultures to transform, whereas this decreased to 2, 0.5 and 0.5% respectively in the cultures of “purified” lymphocytes. Prior incubation of the leukocytes with antigens followed by column separation resulted in an intermediate proportion of transforming lymphocytes. The addition of autologous phagocyte-rich, lymphocyte-poor leukocytes to the purified lymphocytes also partially restored the response to antigens. Restoration was also achieved by growing the “purified” lymphocytes at a higher cell density. However, growing the purified lymphocytes in conjunction with WI-38 human embryonic lung monolayers failed to restore their antigen-stimulated transformation. The data suggest that the phagocytic cells facilitate the in vitro transformation of lymphocytes with antigenic as well as “nonspecific” stimuli.

Genetic control of the antibody response in inbred mice transfer of response by spleen cells and linkage to the major histocompatibility (h-2) locus

Abstract: The transfer of spleen cells from (C3H x C57Bl/6) F(1) mice, capable of responding to (T,G)-A--L, into irradiated C3H parental recipients, normally incapable of responding to (T,G)-A--L, transfers the ability to make either a primary or secondary immune response to this synthetic polypeptide antigen. This localizes the genetic control of the ability to respond to the spleen cell population and indicates that the genetic control is exerted upon a process directly related to antibody formation. Studies with congenic strains of mice and linkage studies in segregating backcross populations show that the ability to respond to (T,G)-A--L and (H,G)-A--L is linked to the H-2 locus and can thus be localized to the IXth mouse linkage group. Note Added in Proof: Of the three possible recombinant animals noted in Tables IV and V, two were infertile. The third animal was not a recombinant, since progeny testing and reimmunization showed that this animal was an H-2(2)/H-2(k) heterozygote capable of responding well to (T,G)-A--L.

Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. I. Characterization of the phenomenon.

Abstract: In the presence of specific antigen, lymph node cells from inbred rats with delayed hypersensitivity to tuberculoprotein, bovine gammaglobulin, and egg albumin produced progressive destruction of monolayers of rat embryo fibroblasts in tissue culture, first apparent at 48 hr and maximal at 72 hr. The effect was specific and did not depend on a genetic difference between the lymph node cells and target cells. It required antigen concentrations equal to or greater than 1.25 µg/ml and lymphocyte: target cell ratios of approximately 10 or 20:1. It could be evaluated both by a plaquing technique and by cell enumeration with an electronic particle counter.

A Modification of the Hemolytic Plaque Assay for Use with Protein Antigens

Abstract: Summary A method is presented which allows the in vitro enumeration of cells producing antibody against a variety of protein antigens. The proteins are covalently linked to red blood cells by means of a carbodiimide reagent. The concentrations of protein required for the plaque assay are greater than those required for passive agglutination. The method is simple and sensitive and the results mimic the kinetics of the response that is seen in in vivo assays of serum antibody. Both direct (probably 19 S) and indirect (probably 7 S) plaque-producing cells are detected.

A quantitative in vitro study of the chromatographic distribution and immunoglobulin characteristics of human blocking antibody.

Abstract: This report describes the immunoglobulin and chromatographic characteristics of human blocking antibody. Studies were carried out on a pool of serum from 20 allergic donors who had been immunized with ragweed antigen E as a form of therapy for ragweed hay fever. The serum and serum fractions were subjected to chromatography on DEAE-Sephadex, CM-cellulose, DEAE-cellulose and Sephadex G 200. Blocking activity was assayed by an in vitro technique: the inhibition of antigen E induced histamine release from isolated human leukocytes. This technique is more than 10 times as precise as the usual skin assay. Immunoglobulins were determined qualitatively by immunodiffusion against specific antisera and quantitatively by the Preer technique. Recovery of blocking activity was about 70%. In each fractionation procedure blocking antibody and IgG eluted together. Purified IgA had less than 1% the activity of IgG preparations and IgM antibody contributed less than 1% to the activity of the whole serum. Absorption of the serum with anti-IgA and treatment with 2-mercaptoethanol did not decrease its blocking titer, while absorption with anti-IgG caused a decrement of more than 97%. Circulating reaginic antibody appeared to account for less than 1% of the total serum activity. It was concluded that blocking activity is essentially a function of IgG molecules.

Reactions in vivo and in vitro produced by a soluble substance associated with delayed-type hypersensitivity*

Abstract: peritoneal exudate cells from sensitized guinea pigs is inhibited by the presence of specific antigen. In this system, it has been found that the immunological information for this response is possessed by the lymphocytes, while the macrophages serve as indicator cells which migrate.6 When such sensitized lymphocytes are cultured in the presence of specific antigen, they elaborate into the medium a substance, probably a protein, capable of inhibiting the migration in vitro of normal macrophages.6-10 This substance, termed migration inhibitory factor (MIF), is produced by lymphocytes from guinea pigs exhibiting delayedtype hypersensitivity but not from animals producing only circulating antibodies, 3 6 and is produced only after the sensitized lymphocytes interact with the specific antigen.11

Cell to cell interaction in the immune response iii. chromosomal marker analysis of single antibody-forming cells in reconstituted, irradiated, or thymectomized mice

Abstract: 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.

Autologous immune complex nephritis induced with renal tubular antigen. II. The pathogenetic mechanism.

Abstract: The pathogenetic mechanism involved in a form of experimental allergic glomerulonephritis induced by immunization of rats with renal tubular antigen has been investigated. A single immunization with less than a milligram of a crude renal tubular preparation, probably containing less than 25 µg of the specific nephritogenic antigen, is effective in the induction of this form of chronic membranous glomerulonephritis. In the nephritic kidney autologous nephritogenic tubular antigen is found in the glomerular deposits along with γ-globulin and complement. When large amounts of antigen are injected during induction of the disease the exogenous immunizing antigen can also be detected in the glomerular deposits. It appears that this disease results from the formation of circulating antibodies capable of reacting with autologous renal tubular antigen(s) and the deposition of these antibodies and antigen(s) plus complement apparently as immune complexes in the glomeruli. This pathogenetic system has been termed an autologous immune complex disease and the resultant glomerulonephritis has been similarly designated.