Limiting Dilution Method Research Articles

T cell repopulation from functionally restricted splenic progenitors: 10,000-fold expansion documented by using limiting dilution analyses.

Mice depleted of T cells by thymectomy, lethal irradiation, and reconstitution with Thy-1-depleted syngeneic bone marrow were given graded doses of splenic T cells to see whether post-thymic cells had the ability to regenerate immune function in these hosts. Using limiting dilution methods to estimate the number of antigen- and mitogen-responsive cells in recipients 12 to 20 wk after reconstitution, we found that new helper and cytotoxic precursor cells were produced, but attained levels only 10 to 20% of normal. Because these repopulated mice were able to produce nearly normal levels of helper and cytotoxic activity in conventional, high density cultures, despite their relative paucity of precursors, we infer that their normal function in conventional assays may reflect a balanced deficiency of effector and regulatory cell types. Surface phenotyping of the progenitor cells responsible for repopulation showed that Lyt-2- cells were required for helper cell regeneration and that Lyt-2+ cells acted as progenitors only for the cytotoxic lineage, contrary to earlier speculation that the splenic Lyt-1+ 2+ (Ly-123) pool included cells antecedent to both effector lineages. Comparison of the number of injected progenitors needed to produce repopulation with the number of new precursor cells eventually produced suggests that the relevant progenitors are able to undergo 10,000-fold expansion in 12 to 20 wk. Numerical expansion in the periphery from thymic-processed cells could well be a major source of new lymphocytes in adult mice.

Monoclonal antibodies against the rat liver glucocorticoid receptor.

Splenic cells from one BALB/c mouse and one C57/BL mouse, immunized with purified rat liver glucocorticoid receptor (GR), were fused with the mouse myeloma cell line Sp 2/0-Ag 14. Screening for production of anti-GR-antibodies by the hybridomas was carried out with an enzyme-linked immunosorbent assay, using partially purified rat liver GR as antigen. Further screening was by a second-antibody immunoprecipitation assay using [3H]triamcinolone acetonide-GR complex from rat liver cytosol as tracer. Hybridomas from 10 different microplate wells, positive in both assays, were successfully cloned by the limiting dilution method to monoclonality. The different origins of the monoclonal antibodies were confirmed by their various isoelectric points when analyzed by isoelectric focusing. Four of the monoclonal hybridoma cell lines secreted IgM antibodies; two, IgG1; three, IgG2a; and one, IgG2b. The GR-antibody complex was identified in glycerol density gradients by a shift of the 4S GR to an 8.5S or 19S GR-antibody complex when incubated with monoclonal IgG or IgM antibody, respectively. The 10 monoclonal antibodies recognized different determinants on the GR, all situated on that domain of the receptor that is separate from the ligand and DNA-binding domains. Also, the cross-reactivity to the mouse liver GR varied among the monoclonal antibodies. No cross-reactivity was observed to the human lymphocytic GR. NaDodSO4 electrophoresis of a 0.5% pure GR preparation followed by immunoblotting using one of the monoclonal antibodies identified a single peptide with a molecular weight of 94,000, identical to the purified rat liver GR.

Infectivity to mosquitoes of Plasmodium falciparum clones grown in vitro from the same isolate

In an attempt to produce a line of cultured Plasmodium falciparum parasites consistently infective to mosquites, a Brazilian isolate, IMTM 22, was cloned by the limiting dilution method. Five of the resulting clones were examined in detail. The clones were found to differ in their ability to produce micro- and macrogametocytes, to exflagellate and to infect Anopheles freeborni mosquitoes. The stability of one clone in producing microgametocytes and in its ability to produce oocysts and sporozoites in mosquitoes has been documented through 15 subcultures. This clone should provide a reliable source of infectious gametocytes for genetic studies and vaccine development.

Age-associated decline in precursor frequency for different T cell-mediated reactions, with preservation of helper or cytotoxic effect per precursor cell.

We have begun to apply limiting dilution methods to address the cellular interactions that underlie age-associated changes in the immune response. By 18 mo of age, mice of the long-lived (BALB/c X C57BL/6)F1 strain are found to have a consistent, three to fivefold decline in precursor frequency for several different T cell-mediated responses: a) IL 2 response to T cell mitogens; b) antigen-nonspecific CTL production in mitogen-stimulated, IL 2-supplemented responses; and c) proliferation in response to concanavalin A. We also find a diminished precursor frequency for IL 2-secreting, KLH-specific helper T cells in the draining lymph nodes of KLH-immunized mice. Despite the decline in the fraction of responding precursor cells, the amount of "effect" (cytotoxicity, IL 2, or proliferation) generated per responsive cell is not altered by age. We speculate that the decline in all of these assays may reflect a common, underlying defect, one that reduces the proportion of T cells that can generate a sizeable clone of proliferating cells. This defect seems to act in several different T cell subsets, and in responses to several activating stimuli: alloantigens, soluble proteins, or mitogens.

Establishment of a myoid cell clone from rat thymus

In the course of rat thymus cultures, cloning of myoid cells (myoblasts) was successfully achieved. Myoblasts first appeared in the primary culture of the rat thymus in very limited number, but proliferated rapidly, started to fuse, and formed multinucleated myotubes contracting spontaneously. In the 15th month of culture, myoblasts were detached from the flasks to establish clones by the limiting dilution method. The cloned cells of IT-45R92 were identified as myoid cells; the small spindle-shaped cells proliferated rapidly and fused with each other forming large cross-striated myotubes. Ultrastructurally, thick and thin filaments with A, I, and Z bands as well as H and M bands occupied the cytoplasm of a large myotube. No epithelial origin of IT-45R92 cells was considered, and pluripotent stem cells were regarded as the most likely cells of origin. A difference is suggested between the factor stimulating fusion and the factor stimulating formation of fully differentiated myotubes. The significance of myoid cells is discussed in relation to acetylcholine receptors of IT-45R92 and the possible causes of myasthenia gravis.

Presence of mast cell precursors in the yolk sac of mice

Concentration of mast-cell precursors in hematopoietic tissues of mouse embryos was evaluated by a limiting dilution method. Cells from yolk sacs, livers, and bodies of (WB × C57 BL/6)F 1 (hereafter called WBB6F 1)-+/+ embryos were injected directly into the skin of adult WBB6F 1- W W v mice which were genetically depleted of tissue mast cells. Concentration of mast-cell precursors was calculated from the proportion of injection sites at which mast cells did not appear. Since the concentration of mast-cell precursors in the yolk sac was about 30 times as great as that of embryonic body at Day 9.5 of the pregnancy, the mast-cell precursors seemed to be generated within the yolk sac. The concentration in the yolk sac reached the maximum level at Day 11, and then dropped markedly at Day 13. In contrast, mast-cell precursors increased from Day 11 to Day 15 in the fetal liver. As a result, the concentration of 11-day yolk sacs was comparable to that of 15-day fetal liver. Although intravenous injection of 15-day fetal liver cells (2 × 10 6) rescued the general mast-cell depletion of WBB6F 1- W W v mice, the intravenous injection of the same number of 11-day yolk sac cells did not rescue it. In contrast with fetal livers, yolk sacs scarcely contained hematopoietic stem cells which were measured by spleen colony formation. Therefore, the mast-cell precursors of the yolk sac may not originate from such stem cells.

Preparation, characterization, and use of monoclonal antibodies to vitamin B6.

Monoclonal antibodies exhibiting various specificities for B6 vitamer forms have been prepared. The antigen preparation employed was a partially purified mixture of human placental proteins that had been derivatized by reaction with pyridoxal 5'-phosphate and sodium borohydride. Spleen cells obtained from mice immunized with the phosphopyridoxyl protein preparation were fused with the mouse myeloma cell line designated X63-Ag8.653. The resulting hybridomas were screened for production of antibodies to the haptenic phosphopyridoxyl group using an enzyme-linked immunosorbent assay. Clones producing such antibodies were isolated by limiting dilution methods. The monoclonal antibodies obtained in this fashion have been characterized with respect to their ability to interact with various forms of vitamin B6. In addition, these antibodies have been shown to be useful in the detection of cellular pyridoxal phosphate binding components using immunoblot techniques. Monoclonal antibodies to vitamin B6 derivatives are potentially powerful tools in the assessment of vitamin B6 nutritional status and in the study of the roles of pyridoxal phosphate binding components in relation to growth, differentiation, carcinogenesis, and steroid hormone action.

Analysis by limiting dilution of interleukin 2-producing T cells in murine ontogeny.

The ontogeny of interleukin 2 (IL 2) production in young CBA/HT6T6J mice was studied using both bulk culture and limiting dilution methods. The ability of spleen cells in bulk culture to produce IL 2 in response to concanavalin A (Con A) was found to rise through the first 2 weeks of life, from no production at day 1, through 20 units/ml at day 6, to 80-100 units/ml in adults. No evidence for suppression of IL 2 production by young spleen was found. Limiting dilution analysis of both young spleen and young lymph node (LN) shows that young spleen has a much lower complement of cells producing IL 2 in response to Con A or allogeneic stimulation than does adult spleen. The frequency of 6-day spleen cells producing IL 2 in response to Con A is 1/1000, while the adult frequency is approximately 1/50. Young LN, in contrast, has levels of IL 2-producing cells close to those of adult LN, with a frequency of responders to Con A of 1/20. No evidence was found for a deficiency in IL 2 production on a per cell basis, in either 6-day spleen or LN. In examining allogeneic reactivity, a high frequency of cells reacting to strong Mls stimulation was found in both young and adult spleen and LN.

Monoclonal antibody to soluble guanylate cyclase of rat brain

Guanylate cyclase (GTP pyrophosphate-lyase, cyclizing, EC 4.6 .1.2), the enzyme responsible for the synthesis of cyclic GMP, is detected mostly in soluble fraction and partly associated with particulate fraction of mammalian tissues [ l-61. In rat brain, guanylate cyclase is found mainly in synaptosomal soluble fraction suggesting its involvement in the neural transmission [6]. We have purified the soluble guanyiate cyclase from bovine brain to an apparent homogeneity and produced the antibody to the enzyme by immunizing a rabbit [7]. The antibody effectively inhibited the soluble guanylate cyclase activity from various mammalian tissues, but not the Triton-dispersed particulate guanylate cyclase from tissues. A method for preparing a monoclonal antibody has been established [8]. Because of its high specificity, a monoclonal antibody would offer a method for studying the different properties of enzyme molecules and for histochemically demonstrating the cellular as well as subcellular localization of guanylate cyclase. We report here production and properties of the monoclonal antibody to the soluble guanylate cyclase of rat brain. The purified guanylate cyclase (64OMg) was injected into BALB/c mouse intraperitoneally with 2 X lo9 heat-inactivated pertussis organisms. The booster injections were carried out twice at a 3-week interval without pertussis organisms. Spleen cells were taken from immunized mouse 3 days after the last booster injection and fused with mouse myeloma cells P3-NS I/l-Ag4-1 (NS I) as in [9]. Spleen cells (1.6 X 108) were fused with 4 X 10’ myeloma cells by the addition of 1 ml 50% polyethylene glycol4000. The cells were suspended in growth medium (RPM1 1640 supplemented with 15% fetal bovine serum, 2 mM L-glutamine and 1 mM pyruvate) and inoculated in two 96-well Nunc Microtest Plates. Two days later, 0.1 ml of HAT selective medium (growth medium supplemented with 100 PM hypoxanthine, 0.4 PM aminopterin and 16 PM thymidine) was added and half of the medium was replaced 3 times during the subsequent 10 days. Fourteen days after cell fusion, culture fluid (60 fl) was assayed for anti-guanylate cyclase antibody by measuring enzyme inhibition as described below. Monoclonal hybridoma cell lines were obtainec’ by a limiting dilution method repeated twice.

Establishment of Autoantibody‐Producing Cell Lines from Peripheral Blood Lymphocytes of Patients with Systemic Lupus Erythematosus

Autoantibody-producing B cell lines were established from peripheral blood lymphocytes of patients with systemic lupus erythematosus. Peripheral blood lymphocytes obtained from five of seven patients were successfully transformed by Epstein-Barr virus. Two of four established B lymphoblastoid cell lines examined in this study produced anti-nuclear factor antibodies and one of them produced anti-single-stranded DNA and anti-double-stranded DNA antibodies. These results indicate that B cell clones committed to self antigens are transformed by Epstein-Barr virus and continue to produce autoantibodies. In order to establish a monoclonal autoantibody-producing B cell line, the cells were cloned by a limiting dilution method. The data suggest that it is possible to establish a monoclonal autoantibody-producing B cell line by the combination of transformation of B cells by Epstein-Barr virus and extensive cloning.

The role of antigen in the immune response: Analysis by limiting dilution methods

The effect of antigen was examined in vitro under conditions where precursors (B cells) were limiting compared to accessory cells, e.g., carrier specific, thymus-derived cells (T cells), and adherent cells (A cells). Selective initiation of precursors was found at very low concentrations of antigen, further antigen was found to have an effect on clone size or “burst” size over a wide-range of antigen concentrations. Inhibition of the in vitro primary anti-hapten immune response by polyvalent, non-immunogenic, TNP-HSA seemed to occur by selective prevention of the initiation of precursors to plaque forming cells.

Kinetics of Cell Proliferation of Murine Bone Marrow Cells Cultured in Diffusion Chambers: Effect of Hypoxia, Bleeding, Erythropoietin Injections, Polycythemia, and Irradiation of the Host

Abstract Mouse bone marrow cells were cultured in diffusion chambers implanted in the abdominal cavity of host mice that were subjected to different kinds of treatment. Preirradiation of the chamber host enhanced the cell growth in the chambers as follows: the number of cells capable of proliferation and differentiation in the chambers (diffusion chamber progenitor cells, DCPC) as determined by a limiting dilution method increased significantly. Similarly, the spleen colony-forming units (CFU’s) in the chambers proliferated more rapidly in irradiated than in normal hosts. The total number of cells in the granulocytic series harvested after culture periods of 3-7 days was also significantly increased. The number of cells in the granulocytic series harvested per DCPC in irradiated animals was twice normal. These findings indicate that the progenitor cells probably proceed through multiplicative mitoses before differentiation in the irradiated animals. A higher cloning efficiency or a shortened generation time of stem cells are other possibilities. The increased number of differentiating granulocytes may simply be a consequence of the events taking place in the stem cell compartment, but a stimulating effect of the irradiation environment on proliferating cells in the granulocytic series cannot be excluded. Preirradiation also stimulated the growth of macrophages but less consistently. When normal mice carrying chambers with normal marrow were subjected to intermittent hypoxia during the culture period, the number of DCPC’s and of cells in the granulocytic series and macrophages were reduced approximately to the same extent. Hypoxic treatment of donor animals for 3-5 days resulted in a similar reduction in number of DCPC’s and CFU’s at both intervals. These findings may suggest competition for a common pool of stem cells for erythrocytes, granulocytes, and macrophages. However, a toxic effect cannot be excluded. Erythropoiesis, which is usually absent, was significantly increased when hypoxia and irradiation of the chamber hosts were combined. Bleeding and erythropoietin injections also enhanced erythropoiesis to some extent.

T. T. C. Test with Resistant Strains to Antibiotics of Streptococcus thermophlus for the Detection of Germ-Inhibitory Substances (Antibiotics) in Milk

A method detecting and identifying the variety of antibiotics in milk was completed by T. T. C. test. After the cultivation of resistant strains to various antibiotics (penicillin, streptomycin, chloramphenicol and tetracycline) of Streptococcus thermophilus antibiotics in milk were identified by the crossed T. T. C. test using those in comparison with the sensitive normal strain.Results of basic experiments carried out are as follows:1) Each resistant strain to penicillin, streptomycin, chloromycetin, achromycin or terramycin respectively was obtained by means of raising streptococcus thermophilus in succession in each of skim milk culture substrates which were added severally with the gradually increasing amount of antibiotics. These resistant strains gave a coloration at the higher levels of antibiotics content compared with the normal strain.2) Streptcoccus thermophilus showed different resistances according to the kind of antibiotics, but the resistant strain to terramycin and the one to achromycin showed crossed resistance each other.3) The differences of resistance caused by the variety of antibiotics were stabilized by making use of the limiting dilution method.4) When samples were heated at 85°C for 5 minutes in the T. T. C. test, no antibiotics were affected.Judging from the results of our basic experiments as mentioned above, we think this method is practically applicable to the identification of antibiotics in milk.