Ochre Suppression in SALMONELLA TYPHIMURIUM

A bacterial strain was constructed which permitted positive selection for ochre suppressor mutations as well as for the loss of suppressor function. A derivative bearing an ochre suppressor mutation was selected following mutagenesis with N-methyl-N-nitroso-N'-nitroguanidine. The suppressor-bearing strain was treated with nitrous acid to eliminate suppressor function by mutation, and a strain lacking suppressor activity was selected. The selected strain which had lost suppressor function was then subjected to mutagenesis to induce a second suppressor mutation. The alternating sequence (induction of an ochre suppressor mutation → induction of a mutation eliminating ochre suppressor activity) was repeated 29 and one-half times in a single strain. Some of the suppressor mutations were tentatively mapped at four locations on the chromosome. The first suppressor mutation selected maps at about minute 30 on the chromosome. The second suppressor selected maps at approximately minute 60, while the third suppressor maps nearby, possibly as far as minute 72. Among the subsequently selected suppressor mutations, all eleven which were mapped were cotransducible with the gal and nic loci near minute 36 on the chromosome and may represent more than one suppressor gene. Deletions were selected which inactivate two of the ochre suppressor alleles mapping near the gal-nic region, suggesting that one or more such genes are dispensable. Some evidence also suggests that the occurrence of either deletion mutations or transduction-mediated recombination events in the gal-nic region can cause instability of nearby suppressor alleles.     

Proliferative and cytotoxic immune functions in aging mice. IV. Effects of suppressor cell populations from aged and young mice

The changes with age in three splenic suppressor cell populations were studied in C57BL/6 mice. Allospecific Ts cells and nonspecific non-T suppressor cells were both generated in vitro in allogeneic MLC. The presence of "pre-existing" suppressor cells in fresh spleen cells from normal mice was examined. Suppressor cell activities were assayed for their ability to suppress proliferation in a fresh allogeneic MLC after treatment to prevent their own proliferation. The ability to generate both specific and nonspecific suppressor cells decreased with age, whereas pre-existing suppressor cells were detected in spleens from the majority of the aged animals but not in spleens from young animals. The decrease in suppressor cell activity was not due to any requirement for age matching between donors of suppressor and target cells. The specific and nonspecific MLC-generated suppressor cells inhibited both the proliferative response in the assay MLC and the generation of cytotoxic cells. The pre-existing suppressor cells only suppressed the proliferative response and not the generation of cytotoxic cells. The changes seen with age in these suppressor cell populations suggest that the ability to generate suppression (both allospecific and nonspecific) to newly encountered Ag declines with age, whereas a resident splenic suppressor cell population accumulates over the lifetime of the animals.     

Regulatory function of T lymphocytes in the immune response to polyvinyl pyrrolidone. II. Characterization of the intrinsic suppressor and the induced suppressor.

Anti-PVP antibody² response is regulated by two categories of suppressor cells, the intrinsic suppressor and the induced suppressor. The intrinsic suppressor activity was dependent on the thymus-derived cells, which were relatively short-lived, insensitive to the treatment with a small dose of ATS (T₁), resistant to HC, and may be adherent to NW. On the other hand, precursor of the induced suppressor was long-lived, susceptible to ATS (T₂), and insensitive to HC. Induced suppressor itself was sensitive to HC, radiosensitive, and nonadherent to NW.These results suggest that the intrinsic suppressor and the antigen-induced suppressor belong to different subsets of T cells.     

Screening system for orthogonal suppressor tRNAs based on the species-specific toxicity of suppressor tRNAs

Incorporation of unnatural amino acids into proteins in vivo, known as expanding the genetic code, is a useful technology in the pharmaceutical and biotechnology industries. This procedure requires an orthogonal suppressor tRNA that is uniquely acylated with the desired unnatural amino acid by an orthogonal aminoacyl-tRNA synthetase. In order to enhance the numbers and types of suppressor tRNAs available for engineering genetic codes, we have developed a convenient screening system to generate suppressor tRNAs with good orthogonality from the available library of suppressor tRNA mutants. While developing an amber suppressor tRNA, we discovered that amber suppressor tRNA with poor orthogonality inhibited the growth rate of the host, indicating that suppressor tRNA demonstrates a species-specific toxicity to host cells. We verified this species-specific toxicity using amber suppressor tRNA mutants from prokaryotes, eukaryotes, and archaea. We also confirmed that adding terminal CCA to Methanococcus jannaschii tRNA^Tyr mutant is important to its toxicity against Escherichia coli. Further, we compared the toxicity of the suppressor tRNA toward the host with differing copy numbers. Using the combined toxicity of suppressor tRNA toward the host with blue–white selection, we developed a convenient screening system for orthogonal suppressor tRNA that could serve as a general platform for generating tRNA/aaRS pairs and thereby obtained three suppressor tRNA mutants with high orthogonality from the tRNA library derived from Mj tRNA^Tyr.     

Regulation of the immune response to tumor antigen. IV. Tumor antigen-specific suppressor factor(s) bear I-J determinants and induce suppressor T cells in vivo.

The nature and function of suppressor factor(s) elaborated by suppressor T cells in response to certain chemically induced tumors have been further defined. Thus, suppressor factor(s) specific for the S1509a methylchol-anthrene-induced fibrosarcoma have been shown to bear determinants encoded by the I-J subregion of the murine MHC since suppressive activity is removed by passage of the factor through an immunoadsorbent composed of anti-I-Jk coupled to Sepharose. No loss of activity was observed after passage of factor through control columns composed of normal mouse globulin. Furthermore, activity could be recovered from the relevant immunoadsorbent by elution with high salt. The administration of crude suppressor factor(s) to normal animals for 4 days resulted in the development of a population of suppressor cells that act in a manner analogous to the suppressor cell population used for production of factor. These factor-induced suppressor cells are T cells and exhibit an antigen specificity similar to that displayed by the tumor-induced suppressor cells. Thus, tumor-specific suppressor factor(s) bear I-J determinants and are capable of inducing the appearance of suppressor T cells in the nontumor-bearing host, which may then act in a specific manner to limit host responsiveness to tumor antigen.     

Network regulation of the immune response: modulation of suppressor lymphocytes by alternative signals including contrasuppression

In the preceding paper we demonstrated that comparison of alternative designs for the immune network can be used to examine the functional significance of specified interactions in normal immune responses. In this paper we examine mathematically the functional significance of three interactions affecting the production of suppressor lymphocytes involved in regulation of normal immune responses. The interactions examined in detail are 1) antigenic stimulation of the production of suppressor lymphocytes, 2) idiotypic stimulation of the production of suppressor lymphocytes, and 3) antigenic inhibition of the production of suppressor lymphocytes (i.e., contrasuppression). The results of our analysis suggest that an immune system with only antigenic stimulation of suppressor production is less effective than a system with both antigenic and idiotypic stimulation of suppressor production on the basis of all of the criteria examined in this study. In turn, the latter system is less effective than a system with only idiotypic stimulation of suppressor production. Furthermore, a system with both idiotypic stimulation and antigenic inhibition of suppressor production can be equal or superior to a system with only idiotypic stimulation of suppressor production on the basis of the same criteria. Similar conclusions hold for the comparison of systems in which regulation by the suppressor lymphocytes of interest is exerted upon production of effector molecules rather than upon production of effector lymphocytes, and also for the comparison of systems in which interactions affecting the production of suppressor factors are of interest.     

Regulatory mechanisms in cytotoxic T lymphocyte development. I. A suppressor T cell subset that regulates the proliferative stage of CTL development

The alloantigen-induced suppressor function of cells from 3-day mixed leukocyte culture (MLC) was studied. These cells, when co-cultured with normal syngeneic lymphocytes and cells of the same haplotype as the original inducing alloantigen, inhibited the generation of cytotoxic T lymphocytes (CTL). Suppression was mediated by a radiation-resistant Lyt-2+ T cell. The suppressor T cells appeared to act by inhibiting the clonal expansion of CTL precursors in the responder cell population, determined by limiting dilution analysis. Levels of endogenous interleukin 2 (IL 2) in co-cultures with suppressor T cells were diminished, and the addition of exogenous IL 2 to co-cultures cancelled the suppressor T cell effects. The suppressor cell population was shown to be capable of absorbing IL 2 from lymphokine preparations, and in contrast to mitogen-induced suppressor T cells, after exposure to IL 2 the allostimulated suppressor T cell remains active. The results are discussed in terms of possible modes of action of the suppressor T cell.     

Mechanisms of corticosteroid action on lymphocyte subpopulations. IV. Effects of in vitro hydrocortisone on naturally occuring and mitogen-induced suppressor cells in man.

The effects of in vitro hydrocortisone (OHC) on human peripheral blood (PB) suppressor cell function were investigated. Two types of suppressor cells were studied: (i) the naturally occurring PB suppressor cell seen in 10% of normal people whose lymphocytes do not respond to in vitro PWM stimulation with direct anti-SRBC PFC responses, and (ii) Con A-generated suppressor cells. The addition of OHC to PWM-stimulated cultures from nonresponders reconstituted the PFC response in two of three individuals. The addition of OHC to allogenic cocultures of nonresponder and responder lymphocytes completely inhibited the ability of the naturally occurring suppressor cell of the nonresponder cultures to inhibit the PFC responses of normal responders. Preincubating the nonresponder cultures in 10^?5M OHC for 30 min followed by washing did not inhibit suppressor function, whereas readdition of OHC to cocultures did inhibit nonresponder suppressor cell function. The addition of up to 10^?4M OHC to previously generated Con A-activated suppressor cell-fresh cell cocultures in vitro did not prevent or inhibit mitogen-activated suppressor cell function. However, preincubation of PB cells for 6 hr prior to the addition of Con A prevented the generation of suppressor cells and in two of eight experiments generated a population of cells which were in and of themselves mitogenic for autologous fresh PB. Thus, the function of naturally occurring suppressor cells as well as the induction but not the function of Con A-activated suppressor cells is sensitive to pharmacologic levels of OHC. The effect of OHC on naturally occurring suppressor cell function or on the generation of suppressor cells by Con A did not involve cell lysis, but rather was a reversible phenomenon requiring the continued presence of OHC in culture.     

A suppressor of mutations in the region adjacent to iterons of pSC101 ori.

Some single-base changes in a 14-bp region (the downstream region) adjacent to three repeated sequences (iterons) in pSC101 ori are very deleterious for replication. We isolated a host suppressor mutation for one of these mutations and found that the suppressor suppressed all the mutations tested in the downstream region. The nucleotide sequence of the suppressor revealed that the suppressor gene was identical to dksA, which encodes a multicopy suppressor of the heat shock gene dnaK.     

Endometrial suppressor cells in beef cattle

Endometrial cells were recovered post mortem from cyclic and pregnant crossbred beef cattle (n = 5 each) on Days 16 to 18 after estrus, and were evaluated for their ability to suppress lymphocyte responses and release suppressor factor into the culture medium. The suppressor factor was assessed for transforming growth factor-beta (TGF-beta) activity. In addition, Percoll was used to fractionate endometrial cells from Angus cows (n = 4) on Days 16 to 18 of pregnancy to determine the density of the suppressor cells. Endometrial cells from cyclic and pregnant cows suppressed lymphocyte proliferative responses and released suppressor factor into the culture medium. The suppressor factor exhibited TGF-beta activity. Suppressor activities tended to be greatest for fractionated cells with densities of 1.01 and 1.095 g/mL. In conclusion, the bovine endometrium contains low- and high-density suppressor cells capable of releasing suppressor factor. The factor seems to be associated with TGF-beta.     

Identification and initial characterization of concanavalin A- and interferon-induced human suppressor factors: evidence for a human equivalent of murine soluble immune response suppressor (SIRS)

Human suppressor T cells activated by leukocyte interferon have properties similar to murine suppressor cells activated by interferon or by concanavalin A. Murine suppressor cells release a soluble mediator, soluble immune response suppressor (SIRS), which accounts, at least in part, for suppressive activity in murine systems. To compare and contrast murine and human suppressor pathways, we evaluated the suppression of human polyclonal plaque-forming cell responses by concanavalin A, by leukocyte interferon, and by immune interferon, or by suppressor cells activated by these agents. In each instance, suppressive activity was prevented by levamisole, ascorbic acid, catalase, or 2-mercaptoethanol, agents known to interfere with murine SIRS activity. Furthermore, concanavalin A, immune interferon, and leukocyte interferon induced T lymphocytes to release 110,000 to 150,000 m.w. proteins which suppressed responses only when added early in the culture period. As with murine SIRS, suppression by each of these human factors was inhibited by 2-mercaptoethanol, ascorbic acid, catalase, or levamisole. The reaction of human suppressor factors with H2O2 (10(-6) M) activated suppressor factors so that they suppress responses when added late in the culture period. Human suppressor factors were protease- and acid (pH 2)-sensitive. The similarities between these human suppressor factors and murine SIRS show the existence of a human SIRS pathway.     

The effect of indomethacin on the activation and effector function of suppressor cells from tumor-bearing mice

Summary The spleens of mice with large M-1 fibrosarcomas contain two populations of suppressor cells with the properties of macrophages and T cells. In this study, we tested the effect of indomethacin on suppressor cell activation and effector function. Neither the activation nor the effector function of the suppressor macrophages was inhibited by indomethacin, and the activity of suppressor macrophages correlated with the tumor size. In contrast, the treatment of tumor-bearing mice with indomethacin from the day of injection of tumor cells completely blocked the in vivo activation of suppressor T cells. Indomethacin did not, however, depress suppressor T cell activity if mice were treated only during the third week of tumor growth. The effector function of the suppressor T cells, as assessed in mixing assays, was partially blocked by indomethacin, while selective suppression by low-molecular-weight factors was completely blocked if indomethacin was present in the cultures. Furthermore, the in vitro activation of suppressor cells by soluble factors secreted by tumor-bearer spleen cells was completely blocked by indomethacin, and this inhibition was reversed by prostaglandin E₁. These data are consistent with the hypothesis that prostaglandins are involved in the activation, but not the effector function, of tumor-activated suppressor T cells.     

Splicing of a yeast proline tRNA containing a novel suppressor mutation in the anticodon stem

The intron-containing proline tRNAUGG genes in Saccharomyces cerevisiae can mutate to suppress +1 frameshift mutations in proline codons via a G to U base substitution mutation at position 39. The mutation alters the 3' splice junction and disrupts the bottom base-pair of the anticodon stem which presumably allows the tRNA to read a four-base codon. In order to understand the mechanism of suppression and to study the splicing of suppressor pre-tRNA, we determined the sequences of the mature wild-type and mutant suppressor gene products in vivo and analyzed splicing of the corresponding pre-tRNAs in vitro. We show that a novel tRNA isolated from suppressor strains is the product of frameshift suppressor genes. Sequence analysis indicated that suppressor pre-tRNA is spliced at the same sites as wild-type pre-tRNA. The tRNA therefore contains a four-base anticodon stem and nine-base anticodon loop. Analysis of suppressor pre-tRNA in vitro revealed that endonuclease cleavage at the 3' splice junction occurred with reduced efficiency compared to wild-type. In addition, reduced accumulation of mature suppressor tRNA was observed in a combined cleavage and ligation reaction. These results suggest that cleavage at the 3' splice junction is inefficient but not abolished. The novel tRNA from suppressor strains was shown to be the functional agent of suppression by deleting the intron from a suppressor gene. The tRNA produced in vivo from this gene is identical to that of the product of an intron+ gene, indicating that the intron is not required for proper base modification. The product of the intron- gene is a more efficient suppressor than the product of an intron+ gene. One interpretation of this result is that inefficient splicing in vivo may be limiting the steady-state level of mature suppressor tRNA.     

Suppressor T cell activation by human leukocyte interferon

Murine fibroblast interferon (IFN beta) activates murine suppressor T lymphocytes in vitro, which suppress plaque-forming cell responses by spleen cells. Suppression of human in vitro immune responses by IFN was investigated to determine whether human IFN also activates suppressor T cells. Human leukocyte IFN (IFN alpha) suppressed pokeweed mitogen-induced polyclonal immunoglobulin production by human peripheral blood mononuclear cells (PBMC) by 80 to 90% at doses of 200 to 350 U/ml. Responses by IFN alpha-treated PBMC were suppressed in a dose-dependent manner; control cultures had maximal responses on day 7. PBMC incubated with 10,000 U/ml of IFN alpha contained activated suppressor cells that decreased pokeweed mitogen-stimulated, polyclonal immunoglobulin production by autologous cells by 70 to 80%. Suppression mediated by these cells was prevented by catalase, ascorbic acid, and 2-mercaptoethanol (2-ME). In murine systems, these reagents interfere with expression of suppressor T cell activity by preventing activation of soluble immune response suppressor. Selection procedures with monoclonal antibodies identified the suppressor cell as an OKT8+ (suppressor/cytotoxic) T lymphocyte. Selected OKT8+ cells required less IFN alpha (1000 U/ml) for activation and were effective in smaller numbers than unfractionated activated PBMC. IFN alpha-activated suppressor cells also inhibited proliferation in mixed lymphocyte and mitogen-stimulated PBMC cultures; again, catalase and 2-ME blocked suppression. These results indicate that IFN alpha activates suppressor T cells in human PBMC cultures; the ability of catalase, 2-ME, and ascorbic acid to block suppression suggests that these suppressor T cells have certain similarities to IFN beta or to concanavalin A-activated murine suppressor T cells.     

Interaction of UAG suppressors and omnipotent suppressors in Saccharomyces cerevisiae

Haploids bearing the dominant UAG suppressor, SUP7-a, and various alleles of the omnipotent suppressor sup35 were examined. The presence of the UAG suppressor reduced the efficiency of some alleles of sup35, and caused other sup35 alleles to be lethal. A nonclassical interaction of the dominant suppressor tRNA and the ribosome is proposed to explain these observations.     

High efficiency temperature-sensitive amber suppressor strains of Escherichia coli K12

Summary A set of eight strains combining the supD43,74 ts¹ suppressor gene with alleles of three suppressor-enhancing (sue) genes have been constructed and characterized. The sue mutations work cooperatively to raise suppressor activity and together raise the activity of the supD43, 74-encoded suppressor 40-fold. These strains further expand the utility of the ts suppressor system by providing as much as 100% suppressor activity at temperatures at or below 20°C to as little as 0.015% suppressor activity at 43°C.     

Genetic control of effector cell characteristics: con A-induced suppressor cells carry H-2 I region encoded determinants.

Activation of splenic lymphocytes with Con A leads to the formation of suppressor cells capable of interfering with the activity of several polyclonal B-cell-activating substances. Thus, these suppressor cells, or their products, most probably act directly on B cells. Suppressor cells could be recovered from the effluent cell population of nylon wool columns, and they were absent from the spleens of athymic nude mice. Furthermore, they were absent from the thymus of normal as well as cortison-treated mice. Cortisone treatment did not abolish the formation of Con A-induced suppressor cells in the spleen. Treatment of activated suppressor cells with antisera specific for distinct products of the H-2 I region revealed that they carried I-J cell surface antigens. We conclude that the suppressor cells in our test system, which unlike other Con A-induced suppressor cell populations have a direct effect on B cells, had antigenic characteristics similar to those previously described for I-J carrying suppressor cells.     

Characterization and isolation of IgE class-specific suppressor factor (IgE-TsF) I. The presence of the binding site(s) for IgE and of the H-2 gene products in IgE-TsF

IgE-specific reverse plaque assay for the direct comparison of the IgE and IgG antibody responses was established and the method was employed for the assessment of the activity of IgE class-specific suppressor factor (IgE-TsF). In the in vitro culture system, the addition of IgE-TsF to DNP-KLH-primed spleen cells inhibited an antigen-induced increase of IgE-producing cells but did not show any effect on the IgG or IgM responses. Absorption of IgE-TsF with IgE-producing hybridoma cells removed the suppressor activity but IgM-producing hybridoma cells did not absorb the suppressor activity. The suppressor activity of IgE-TsF was removed by murine IgE-conjugated Sepharose column but not by IgM-, IgG-, or human IgE-conjugated column. The suppressor activity was eluted from IgE-column with glycine-HCI buffer, pH 3.2, or acetate buffer, pH 4.0, and the suppressor factor eluted from IgE-column was reabsorbed by anti-H-2d conjugated column. The results showed that IgE-specific suppressor factor was composed of the binding sites for IgE molecules and the H-2 gene products.     

Regulatory action of immune B cells on delayed-type hypersensitivity in mice

Suppressor cells in delayed-type hypersensitivity (DTH) to soluble protein antigens were induced in vitro from BALB/c spleen cells. Transfer of these cells into syngeneic recipients resulted in suppression of the hosts' DTH responses in an antigen-specific manner. These suppressor cells were characterized as B cells by their adherence to nylon-wool columns, resistance to treatment of anti-Thy 1, -Ly 1, and -Ly 2 antibodies plus complement, adherence to anti-mouse immunoglobulin-coated dishes, and nonadherence to uncoated plastic dishes. In addition to being radiation sensitive, these suppressor B cells showed the capability of binding to the primed antigen. Thus, it was demonstrated that our in vitro-induced suppressor cells were antigen-specific B cells. When these suppressor B cells were transferred into the recipients, serum titers of specific antibodies were elevated and effector phase suppressor T cells were induced in the recipients. These results suggest that suppressor B cells exert their suppressor activity through the idiotype-anti-idiotype network.