Appearance Of Plaque-forming Cells Research Articles

Modulation of the antibody response to sheep red blood cells in normal and immunodeficient XID mice by myo-inositol.

Over the dose ranges tested, dietary vitamin A and myo-inositol induce similar changes in murine fetal growth and development. Because vitamin A is also known to affect the immune response, studies were conducted to determine if dietary myo-inositol might have an effect on antibody production. It was found that in vivo in inbred mice myo-inositol (4 mg/g of diet) accelerated the rate of appearance of plaqueforming cells (PFC) in a primary response to sheep red blood cells (SRBC). In vitro, myo-inositol accelerated the rate of appearance of colonies of anti-SRBC PFC (foci) and significantly increased the number of PFC per colony, but did not affect the number of foci per culture noted at the end of the culture period, myo-inositol had no effect on the PFC IgM:IgG ratio following a single exposure to the agent, but exposure to myo-inositol in vivo and in vitro resulted in a decrease in the number of IgM PFC per focus in a primary response and IgM and IgG PFC per focus in a secondary response. Based on studies suggesting that myo-inositol or a phosphorylated metabolite might act downstream from Bruton's tyrosine kinase (Btk) in a signal transduction pathway in B cells, immunodeficient CBA/CaHN-XID/J mice were fed a standard diet or the same diet supplemented with 0.4% myo-inositol. Mice given the supplemented diet produced significantly more IgM anti-SRBC antibody than did XID mice given the control diet (4.3 +/- 2.5 vs 1.7 +/- 2.8, 1/log2), and produced approximately the same amount as immunocompetent controls (2.9 +/- 0.9). When rechallenged with SRBC, XID mice given supplemental inositol produced significantly more IgM antibody than did the XID and immunocompetent controls (3.6 +/- 0.5 vs 1.8 +/- 1.1 and 1.5 +/- 0.7, respectively). Added dietary inositol did not have a significant effect on primary or secondary IgG responses to SRBC, which remained impaired. These results suggest that dietary myo-inositol or a derivative may be able to modulate B-cell IgM responses by interacting within the inositol second messenger system downstream from Bruton's tyrosine kinase.

Fungal metabolites. Part 11. A potent immunosuppressive activity found in Isaria sinclairii metabolite.

A potent immunosuppressive activity was found in the culture broth of the fungus Isaria sinclairii (ATCC 24400). The metabolite, ISP-I ((2S,3R,4R)-(E)-2-amino-3,4-dihydroxy-2- hydroxymethyl-14-oxoeicos-6-enoic acid, myriocin = thermozymocidin) suppressed the proliferation of lymphocytes in mouse allogeneic mixed lymphocyte reaction, but had no effect on the growth of human tumor cell lines. It also suppressed the appearance of plaque-forming cells in response to sheep red blood cells and the generation of allo-reactive cytotoxic T lymphocytes in mice after intraperitoneal or oral administration. The metabolite was 10- to 100-fold more potent than cyclosporin A as an immunosuppressive agent of the immune response in vitro and in vivo, and appears to be a candidate for clinical application as a powerful immunosuppressant.

Further evidence against random polyclonal antibody formation in mice with lupus-like graft-vs-host disease.

The pathologic symptoms in F1 mice with chronic graft-vs-host disease (GVHD) (GVH F1) strongly resemble those of systemic lupus erythematosus (SLE). Mice with SLE-like GVHD do not produce antibodies to a number of non-self and self antigens. This finding is inconsistent with the widely accepted view that the (auto)-antibody formation in SLE is polyclonal in the sense that B cells are triggered at random, i.e., irrespective of their specificity. In the present study, therefore, we performed a systematic study of the kinetics of total IgM- and IgG-secreting splenic B cells and tested their specificities. The total IgM-secreting B cell population was increased only in the first week after the initiation of SLE-like GVHD; it seemed to reflect a random, but self-limited, polyclonal B cell stimulation. In contrast, the total number of IgG-secreting cells in the GVH F1 mice was increased to a much higher extent than that of the IgM-secreting cells and remained increased. At no time during GVHD was there an increase in the number of plaque-forming cells (PFC) spontaneously secreting IgG antibodies to non-self antigens. The GVH reaction (GVHR) did, however, lead to the appearance of PFC that secreted IgG antibodies to DNA. Similarly, the GVH F1 mice showed high serum titers of antibodies to self antigens characteristic of SLE and to endogenous viruses, but during the entire observation period they failed to develop serum antibodies to non-self antigens and insulin. Hence, the enhanced production of Ig, especially that of IgG, that occurs in SLE-like GVHD is not a random process, because it requires the presence of antigen, or signal 1. The data support our hypothesis that only certain kinds of self antigen, such as DNA and cell membrane epitopes, can cross-link the Ig receptors on the corresponding B cells and thus provide an adequate signal 1. Given the increase in help, or signal 2, in chronic GVHD, only the B cell clones that simultaneously receive an adequate signal 1 seem to be driven into clonal proliferation and IgG secretion.

Polyclonal effect of HgCl2 in the rat, its possible role in an experimental autoimmune disease.

Mercuric chloride induces an autoimmune glomerulonephritis in Brown-Norway (BN) but not in Lewis (LEW) rats. Injection of HGCl2 into BN rats regularly produced a transient appearance of plaque-forming cells (PFC) of anti-2,4,6-trinitrophenyl and anti-sheep red blood cell specificity and circulating anti-single-stranded DNA antibodies. Addition of HgCl2 to spleen cell cultures from BN rats induced an increase in anti-trinitrophenyl PFC and reverse PFC. This effect was no longer observed when nylon wool column-depleted or anti-Thy-1 antiserum-treated spleen cells were cultured in the presence of HgCl1. These data suggest that HgCl2 acts as a polyclonal activator on spleen cells in BN rats, but not on isolated B lymphocytes. In contrast, no effect of HgCl2 on immunoglobulin production was observed in LEW rats. Since polyclonal activation and immune-type nephritis are both seen in BN but not in LEW rats, polyclonal activation may participate in the pathogenesis of the HgCl2-induced autoimmune disease of BN rats.

Splenic role in the regulation of immune responses

Evidence for a splenic role in regulating antibody production in other lymphoid tissue was obtained in a system in which cyclical fluctuations of splenic plaque-forming cells (PFC) occur following a single intravenous injection of aggregated human γ-globulin in rabbits. First, PFC arising simultaneously in the mesenteric nodes, peripheral blood, and spleen appear to be derived from the spleen since splenectomy prior to antigen injection abrogated these responses. Second, a noncyclical appearance of PFC in popliteal nodes of rabbits responding to subcutaneous injection of antigen was converted to a cyclical response by simultaneous intravenous injection of antigen, an effect which was abolished by splenectomy prior to antigen injection. It is suggested that, following an intravenous injection of antigen, both suppressor cells as well as antibody-forming precursors may be activated in the spleen and disseminated to other lymphoid tissue.

Antibody formation in mouse bone marrow: VII. Evidence against the migration of plaque-forming cells as the underlying cause for bone marrow plaque-forming cell activity: A study with parabiotic mice

After intravenous immunization of mice with Escherichia coli lipopolysaccharide (LPS) or sheep red blood cells (SRBC), the bone marrow can contain large numbers of plaque-forming cells (PFC). By means of parabiosis, it was studied whether or not this appearance of PFC in the bone marrow might be due to a migration of such cells from peripheral lymphoid organs into the marrow, as has been suggested in the literature. Using parabionts consisting of nonimmunized mice and mice immunized with LPS, only background numbers of PFC could be demonstrated in the bone marrow of the nonimmunized mice. In similar experiments, with SRBC as antigen, mice showing high anti-SRBC PFC activity in the bone marrow could only provide for minor numbers of anti-SRBC PFC in the bone marrow of affixed normal mice. These results suggest that migration of PFC can not be the main cause for bone marrow PFC activity in the mouse. This provides additional evidence for our view presented in previous papers of this series that the appearance of PFC activity in the bone marrow is dependent on local maturation of B cells into PFC rather than on immigration of PFC.

Formation of plaques by adherent spleen cells from nonimmunized mice, incubated in vitro with the nonadherent fraction of immune allogeneic cells.

Twenty-four-hour incubation of adherent spleen cells from normal C57BL mice with purified nonadherent spleen cells from CBA mice, in the peak of their primary response against sheep red blood cells, resulted in the appearance of plaque-forming cells in the adherent cell population. The immunological nature of these plaques was demonstrated by their complement dependency and the inhibitory effect of specific anti-mouse IgM serum. Direct, but not indirect plaque formation was found to be associated with the adherent cell population for which direct cell to cell contact is necessary. Experiments with cytotoxic specific anti-strain sera revealed that in this system, the adherent plaque-forming cells are the nonimmune adherent C57BL cells.

Regulation of the Antibody Response to Type III Pneumococcal Polysaccharide

Abstract The present study was conducted primarily to obtain information concerning the mechanism by which suppressor thymic-derived (T) cells influence the magnitude of the antibody response to type III pneumococcal polysaccharide (SSS-III). This was accomplished by examining the effects of treatment with antilymphocyte serum (ALS) and Velban, a mitotic inhibitor, on the kinetics for the appearance of plaque-forming cells (PFC) in mice immunized with SSS-III. Without ALS-treatment, the kinetics were essentially biphasic. First, there was a phase during which PFC appeared at a relatively rapid rate; this was followed by a phase during which the rate of appearance of PFC gradually declined until maximal numbers were attained. In contrast, similar rate changes were not noted for mice given ALS; here, PFC increased at a uniform exponential rate throughout the 1st 5 days of the antibody response. Velban completely arrested the development of additional PFC in ALS-treated but not non-ALS-treated mice. These findings indicate that suppressor T cells act primarily by limiting the extent to which antibody-forming bone marrow-derived cells proliferate following immunization.

Studies on Early Events in the Differentiation of Antibody-Producting Cells

Abstract The incorporation rates of tritium-labeled thymidine, uridine, and amino acids into macromolecules, and the appearance of plaque-forming cells (PFC) were studied at 4-hr intervals, in spleen cells obtained from C57BL mice after a secondary intraperitoneal injection of sheep red blood cells. The incorporation rates displayed, over the period studied, clear maxima and minima occurring in the following order; a peak of uridine incorporation at 40 hr after immunization, 4 hr later a peak of thymidine incorporation, and at 60 hr a peak of amino acid incorporation. The number of PFC increased stepwise, the first and the second increases occurring at 60 and 76 hr after immunization, respectively. A similar sequence of biosynthetic events was observed in mouse spleen cells restimulated with sheep red blood cells in vitro. These results were interpreted as reflecting an antigen-induced, synchronized metabolic activity of the cells. In an attempt to detect possible changes induced in the replication pattern of deoxyribonucleic acid (DNA) synthesized in response to antigenic stimulation, we compared the reassociation rate of DNA newly synthesized in spleen cells 47 to 48 hr after the antigenic stimulation with that of DNA from normal cells. The newly synthesized DNA displayed, between C0t 1000 and C0t 10,000 a faster reassociation rate than the bulk DNA obtained from the same cells or DNA obtained from normal cells. This suggests a temporary selective synthesis of that part of the genome during the pulse labeling. Further support for this assumption was provided by the changes in specific activity of single stranded “immune” DNA along the C0t curve.

Reconstitution of the Antibody Response in Vitro of T Cell-Deprived Spleen Cells by Supernatants from Spleen Cell Cultures

Abstract The antibody response of spleen cells against sheep red cells was tested in vitro with the Mishell-Dutton technique. Spleen cells deprived of thymus-derived (T) cells by anti-θ serum treatment gave a low response. Supernatants from cultures of either parental or syngeneic spleen cells could restore the response of T cell-deprived spleen cells against sheep red cells to a certain degree, but supernatants from mixed lymphocyte cultures between two H-2 incompatible parental strains were more efficient in this respect. These supernatants also could stimulate markedly the immune response of spleen cells from athymic “nude” mice. Addition of supernatants to cultures of normal or T cell-depleted cells caused an early appearance of plaque-forming cells by day 2 with a maximal number on day 3 for the T cell-depleted cells. Supernatants harvested on day 1 were more effective than supernatants from day 2 in restoring the immune response, whereas supernatants from days 3 and 4 were inhibitory. This was not due to an excessive production of the restorative factor. Both T cells and adherent cells were necessary for the production of the restorative factor. The active factor in the supernatants could be produced in serum-free medium and was capable of supporting a primary antibody response of spleen cells cultured in normal mouse serum. The findings suggest that T cells may stimulate the antibody response of bone marrow-derived cells by means of a soluble factor.

Cellular events during the induction of experimental thyroiditis in the rabbit.

With a modification of the Jerne plaque technique to enumerate plaque-forming cells (PFC) to bovine and rabbit thyroglobulin, the cellular kinetics of the antibody response were followed during two 5-day series of injections of an aqueous preparation of bovine thyroglobulin. The results support the suggestion that thyroiditis in the rabbit is mediated by antibody. The peak PFC appear in the spleen at the end of the second series of injections and are considerably greater for bovine than for rabbit thyroglobulin. PFC also appear in the thyroid gland; however, the numbers of PFC for bovine and rabbit thyroglobulin were similar, and they did not reach a peak until 7 days after the peak PFC in the spleen. There was an excellent correlation between the appearance of PFC in the thyroid gland and the appearance of thyroid lesions. The disappearance of antibody to rabbit thyroglobulin from the serum also correlated with the appearance of lesions. Migration inhibition factor (MIF) activity was not produced at any time throughout the study when rabbit thyroglobulin was added to peritoneal exudates of immunized rabbits containing circulating antibody to rabbit thyroglobulin. MIF activity was observed when bovine thyroglobulin was added to similar cells in the later stages of the study after lesions were present.

Cellular Formation of Vibriolytic Antibody by Mouse Immunocytes: Cytokinetics and Specificity

Abstract A vibriolytic plaque assay in agar gel was developed which enumerates immunocytes secreting antibody to the common and type-specific antigens of Vibrio cholerae. No antibody-forming cells were found in the spleen of random bred or inbred mice prior to immunization with V. cholerae. Following immunization there was a consistent latent period of approximately 2 days before the first appearance of plaqueforming cells (PFC), followed by a rapid increase that reached a peak approximately 10 to 12 days after immunization. PFC directed against both the common and the type-specific antigen could be readily enumerated by testing individual spleen cell suspensions against both the homologous and heterologous vibrio strains, which are known to share a common antigen. A good correlation was evident between the appearance of PFC and circulating vibriolytic and agglutinating antibody, except for the long delay between the appearance of serum antibody and attainment of peak titer after 28 days. Anti-mouse globulin serum inhibited PFC, attesting to the immunoglobulin nature of the reaction, and inhibition tests with vibrio antigens, in the form of intact vibrios or lipopolysaccharide extracts, evinced the specificity of the PFC response.

Immune Response to Alloantigens of Thymus Studied in Mice with Plaque Assay

Abstract A plaque assay in which allogeneic thymus cells were used as target antigen has been developed. Inbred mice of various strains were stimulated with an intravenous injection of 4 × 107 allogeneic thymus cells. Whenever host and donor differed in ϑ antigens, plaque-forming cells (PFC) were detected in the spleen of the host. When host and donor carried identical ϑ antigen, PFC were detected only in certain strain combinations and only at the higher immunizing dose of 4 × 108 donor thymus cells. Kinetics of the appearance of PFC was studied in the strain combinations differing in θ. In normal animals, the numbers of PFC were less than 10 per spleen. In the primary response, the peak numbers of PFC were observed in 4 to 7 days after the stimulations. In one strain combination, the mean number of PFC as high as 40,000 per spleen, was observed at the peak of the response. Marked differences in the magnitude of the PFC response to ϑ-AKR antigen were observed in various strains of mice which lack this antigen. In two strain combinations, the peak numbers of PFC were lower in the secondary than in the primary response. In one strain combination the reverse was true. Evidence was presented that PFC detected in this assay produce antibodies of IgM variety.

Immunoglobulin Classes of Antibody-Forming Cells in Mice

Abstract Facilitation of localized hemolysis-in-agar plaque-forming cells (PFC) using antisera specific for each of the five immunoglobulin classes of mice (IgM, IgA, γ1, γ2a and γ2b) reveals that the peak production of IgM (direct) PFC in the spleens of mice occurs 4 days after receiving an optimal dose of sheep erythrocytes and is followed in 24 hr by the appearance of PFC representing each of the 7S immunoglobulin classes (IgA, γ1, γ2a and γ2b). while the number of direct (IgM) PFC falls after reaching a peak on day 4, 7S immunoglobulin antibody producing cells (indirect plaques) appear, decline and reappear in phases. 7S immunoglobulin antibody-producing cells appear more rapidly and in greater numbers in the secondary response, while direct (IgM) PFC are found in fewer numbers in the secondary response than in the primary response. However, indirect IgM PFC, apparently due to the production of IgM hemolysin of “low efficiency,” are a prominent feature of the secondary response.

Depressed Cellular Formation of Antibody toShigellaAntigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.

Alloantibody production measured by plaque assay in relation to strong and weak histoincompatibility.

A plaque technique which provides a sensitive and quantitative determination of allohemolysin (isohemolysin) production by lymphoid cells of mice and Syrian hamsters is described. Much ongoing attention has been given to minimizing sources of spurious plaques while improving the reproducibility of the technique. Primary intravenous immunization with allogeneic blood cells led to the early appearance of plaque-forming spleen cells responding to both strong and weak histocompatibility antigens in inbred strains of mice. The strongest histoincompatibility (A/J --> BL/6), characterized by H-2 plus non-H-2 differences and a median skin allograft survival time of 8.2 +/- 0.5 days, gave a peak average plaque production of 29.6 per 10 x 10(6) viable spleen cells after 6 days. At the other extreme with an average allograft survival time of 91 days, C3H(H-1(a)) --> C3H.K(H-1(b)) showed a feeble production of plaque-forming cells with a peak response of 3.7 per 10 x 10(6) viable spleen cells at 9 days. Histocompatibility antigens determined by genes at the H-1, H-Y, and H-X loci were demonstrated to be erythrocyte antigens as well. Peak plaque production occurred earlier and reached higher levels across the stronger histocompatibility barriers (H-2 plus non-H-2 or H-2 alone) in temporal accord with the rapidity of skin allograft rejection. Weak H-Y or H-1 differences leading to prolonged allograft survival were associated with moderate, but not commensurate delays in the initial occurrence and recruitment of plaque-forming cells. With one exception (C57BL/10 --> B10.A), the average plaque counts found at the peak of the primary responses were inversely proportional to the median survival times of skin allografts in the same strain combinations. Nevertheless, in light of the disparity between the times of allograft rejection and the time course of appearance of plaque-forming cells with the "weaker" congenic combinations, the early 19S antibodies do not appear to be directly involved in allograft rejection. There appears to be no limitation in the weakness of histocompatibility antigens capable of evoking antibodies detectible by plaque assay. The extent to which molecular species of mouse antibodies other than 19S hemolysins are induced and detected under the conditions employed remains to be determined. The responses in strain combinations involving stronger histocompatibility barriers were characterized by exponential increases in plaque-forming cells, for 2 to 4 days until maximal levels were reached. Repeated immunization in several trials led to substantial increases over the maximum primary response in the number of demonstrable plaque-forming cells. Further preliminary results are reported concerning alloimmune plaque production in three strains of Syrian hamsters exhibiting intermediate to weak histocompatibility differences.