Search PubMed⌕ Search

Biomedical subjects

N H Ruddle

Publications and source records attributed to N H Ruddle.

At least 73 records · Page 4Linked to original sources
PubMed1987

Anti-tumor activity of class II MHC antigen-restricted cloned autoreactive T cells. II. Novel immunotherapy of B16 melanomas by local and systemic adoptive transfer.

Lyt-1+, L3T4a+ autoreactive cloned T cells, producing lymphotoxin (LT) and interferon-gamma (IFN-gamma) in response to self-class II major histocompatibility complex antigen in vitro were examined for their anti-tumor effect in vivo against B16 melanomas. Without the aid of exogenous interleukin 2, the autoreactive T cells, when injected immediately and at an equal cell number into the site of s.c. inoculated B16 melanoma cells inhibited tumor growth in sublethally irradiated and nonirradiated syngeneic mice. The autoreactive T cells also induced regression of tumors established 3 days earlier. Normal spleen cells or class II-restricted cloned T cells specific for chicken gamma-globulin (CGG) had no inhibitory effect on tumor growth. A single injection of autoreactive T cells delayed tumor growth and prolonged the survival of mice that had received a lethal dose of B16 melanoma cells. The autoreactive T cells caused extensive necrosis at the injection site. A treatment regime consisting of two successive injections of anti-I-Ab monoclonal antibody 3JP prevented the inhibition of tumor growth, supporting the hypothesis that the autoreactive T cells inhibited the growth of melanomas by releasing LT and IFN-gamma upon recognition of I-A antigen-bearing cells at the injection site. The CGG-specific control T cells did not cause necrosis and survived within the nests of uninhibited tumor cells. Autoreactive T cells administered i.v. immediately after i.v. injection of B16 melanoma cells markedly reduced pulmonary metastases, whereas CGG-specific T cells did not. These results indicate that autoreactive T cells can function in vivo as inhibitors of tumor growth.

Animals↗
PubMed1987

Lymphotoxin: cloning, regulation and mechanism of killing.

The gene for murine lymphotoxin (MuLT) has been cloned from a cDNA library prepared using poly(A)+ RNA from an activated murine IL-2-maintained cloned T cell line (21C11). This was accomplished with a MuLT BamHI fragment isolated from a murine genomic library by hybridization to a human LT cDNA probe. Northern blot analysis with RNA from 21C11, an L3T4+ (CD4+-equivalent) ovalbumin-specific class II-restricted T cell line, revealed a 15S band that hybridized to this MuLT fragment. A cDNA library prepared with poly(A)+ RNA from 21C11 cells contained 36 colonies that hybridized with the MuLT BamHI fragment. A full-length cDNA has been isolated, sequenced, expressed in COS-1 cells and used to map MuLT to mouse chromosome 17. The sequence and structure of the MuLT gene has been determined. MuLT cDNA has been used to analyse mRNA expression in several L3T4+ and Lyt-2+ (CD8+-equivalent) T cell clones activated with antigen, mitogen, or antibody to the T cell receptor. LT is expressed by both class I- and class II-restricted T cells. The mechanism of killing by both LT and the functionally related molecule TNF-alpha includes the induction of DNA fragmentation in the target cell.

Animals↗
PubMed1987

Suppressor cells induced by total lymphoid irradiation affect proliferation and lymphokine production of murine T helper cell clones.

A key response to antigen is the activation of helper T cells to release lymphokines which stimulate and effect the immune reaction. This T cell population can secrete many different factors with diverse, often multifunctional roles, such as amplifying T or B cell antigen responses or being effectors of cell mediated delayed type hypersensitivity. Among these lymphokines are gamma-interferon (gamma-IFN), interleukin-2 (IL-2), or T cell growth factor, and lymphotoxin (LT) which has cytotoxic activity against a variety of cells. Immune suppression in mice following total lymphoid irradiation (TLI) has been correlated with the presence in lympho-reticular tissues of an antigen non-specific, null suppressor cell. This study examined what effects radiation induced suppressor cells had upon the in vitro activation and lymphokine responses of the ovalbumin (OVA) specific T helper cell clone, 153E6, following antigen presentation. Splenocytes from TLI treated mice obtained early in the post-irradiation period exerted a pan-inhibitory effect upon OVA induced 153E6 proliferation and its concomitant release of gamma-IFN, LT, and IL-2. As the interval from irradiation increased, splenocytes from TLI treated mice showed persistent suppression of 153E6 proliferation and gamma-IFN release, but had rapidly diminishing effects on the T cell's capacity to produce LT and IL-2. These findings suggest that suppressor cells induced by TLI have a marked inhibitory effect in vivo upon T helper cell proliferative responses to antigen and the production of various T helper cell lymphokines necessary to mediate the immune response. Such processes could contribute to the immunosuppressive effects of extensive nodal irradiation.

Animals↗
PubMed1987

Target cell DNA fragmentation is mediated by lymphotoxin and tumor necrosis factor.

Supernatant fluids containing lymphotoxin (LT) activity were examined for their ability to mediate the fragmentation of cellular DNA in target L929 cells in the manner observed in cytotoxic T lymphocyte mediated killing. These supernatants were able to mediate L929 target cell DNA fragmentation, requiring a time course of 48 hours. In an effort to determine whether or not LT was directly involved in mediating this activity, recombinant-derived human lymphotoxin was tested for its ability to bring about the same effect. Both recombinant-derived LT and recombinant-derived tumor necrosis factor (TNF), a factor produced by macrophages and having some activities in common with LT, were able to cause DNA fragmentation in L929 over the same time course. Electrophoretic analysis of DNA isolated from LT-treated L929 target cells revealed that it is fragmented into low molecular weight fragments in a pattern similar to that produced in DNA isolated from target cells after attack by cytotoxic T cell lethal hit.

Animals↗
PubMed1986

DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T-cell lines, lymphotoxin-secreting helper T-cell clones, and cell-free lymphotoxin-containing supernatant.

A Lyt-2+, trinitrophenyl-specific, lymphotoxin-secreting, cytotoxic T-cell line, PCl 55, mediates the digestion of target cell DNA into discretely sized fragments. This phenomenon manifests itself within 30 min after effector cell encounter as measured by the release of 3H counts from target cells prelabeled with [3H]deoxythymidine and occurs even at very low effector to target cell ratios (0.25:1). A Lyt-1+, ovalbumin-specific, lymphotoxin-secreting T-helper cell clone, 5.9.24, is also able to mediate fragmentation of target cell DNA over a time course essentially indistinguishable from the cytotoxic T lymphocyte-mediated hit. Cell-free lymphotoxin-containing supernatants also cause release of DNA from targets, although they require a longer time course, on the order of 24 hr. In contrast, lysis of cells by antibody plus complement or Triton X-100 does not result in DNA release even after extended periods of incubation (24 hr). All three treatments that result in the release of DNA from cells cause fragmentation of that DNA into discretely sized pieces that are multiples of 200 base pairs. The results thus suggest that cytotoxic T cells, lymphotoxin-secreting helper clones with cytolytic activity, and lymphotoxin all effect target cell destruction by means of a similar mechanism and that observed differences in time course and the absence of target cell specificity in killing mediated by lymphotoxin may simply reflect differences in the mode of toxin delivery.

Animals↗
PubMed1985

A monoclonal antibody that recognizes the functional domain of Escherichia coli single-stranded DNA binding protein that includes the ssb-113 mutation.

We have isolated a monoclonal antibody against Escherichia coli single-stranded DNA binding protein (SSB) that recognizes the functional domain specified by the ssb-113 temperature-sensitive mutation, a domain which is distinct from the DNA-binding site. Although the ssb-113 and ssb-1 mutations result in many similar phenotypic defects, they differ significantly in others, indicating that they affect different functional domains of the protein. Whereas the SSB-1 mutant protein is clearly defective in tetramer formation and is also unable to bind single-stranded DNA at nonpermissive temperatures, no similar in vitro defects have yet been found in the SSB-113 mutant protein. In fact, the only reported in vitro effect of the ssb-113 mutation on the protein is a slight increase in its helix destabilizing ability. Competition radioimmunoassays using a monoclonal antibody demonstrated that SSB-113 mutant protein, containing a single amino acid substitution at position 176 (the penultimate residue), did not compete with SSB while SSB-1 protein (with a single change at position 55) did compete with SSB. This analysis was refined by studies with a proteolysis fragment and with peptides derived from both SSB and SSB-113. The results indicate that the antibody recognizes a determinant near the COOH-terminal end of the protein and that the SSB-113 mutation lies within or very close to this determinant.

Antibodies, Monoclonal↗
PubMed1985

A comparison of lysis mediated by Lyt 2+ TNP-specific cytotoxic-T-lymphocyte (CTL) lines with that mediated by rapidly internalized lymphotoxin-containing supernatant fluids: evidence for a role of soluble mediators in CTL-mediated killing.

A series of Lyt 2+, trinitrophenyl (TNP)-specific T-cell lines are shown to lyse 51Cr-labeled target cells in an antigen-specific, H-2-restricted fashion in a 4-hr assay. These cells also produce lymphotoxin, in addition to other factors, upon stimulation with TNP-haptenated syngeneic splenocytes. A technique for introducing macromolecules into the cytoplasm of fibroblasts by inducing the cells to pinocytose the molecule in hypertonic medium, and then lysing the newly formed pinocytic vesicles with a mild hypotonic shock was used to assess the role of soluble mediators in the cytotoxic T lymphocyte (CTL)-mediated lytic process. The technique itself has little effect on cell growth rate or viability. A minimum of 24 hr, and more frequently 48-72 hr is required for lymphotoxin to manifest it's lethal effect when it is merely included in the culture medium of growing fibroblasts. In contrast, supernatant fluids from the Lyt 2+ cells kill 51Cr-labeled fibroblasts in a dose-dependent fashion during a 4-hr assay when they are rapidly internalized via the osmotic procedure. The data serve as preliminary evidence of a role for soluble mediators such as lymphotoxin in T-cell-mediated lysis, and suggest that the cytotoxic-T-cell lethal hit may include a mechanism for rapidly internalizing a toxin into appropriate target cells.

Animals↗
PubMed1985

Activation of Lyt-1+ and Lyt-2+ T cell cloned lines: stimulation of proliferation, lymphokine production, and self-destruction.

Antigen-induced activation of a chicken gamma-globulin (CGG)-specific Lyt-1+ T cell clone measured both as a function of proliferation and immune interferon (IFN-gamma) production is restricted by a class II determinant of the major histocompatibility complex (MHC) mapped to the I-A subregion, as determined by studies with both recombinant inbred lines and monoclonal antibodies. Activation of Lyt-2+ picryl chloride (PC1)-specific cloned T cell lines by trinitrophenyl (TNP)-coupled spleen cells results in proliferation and the production of at least two lymphokines: lymphotoxin (LT) and IFN-gamma. This antigen-specific activation is restricted to a class I determinant of the MHC complex encoded in the K region. Thus, the common intracellular pathway leading to production of IFN-gamma by Lyt-1+ and Lyt-2+ T cells is mediated and restricted through different surface recognition units. The LT that is produced by antigen-specific activation of T cells not only kills fibroblasts, but it inhibits interleukin 2 (IL 2)-maintained T cells as well. Activation of T cells by concanavalin A (Con A) results in suicidal inhibition of proliferation and cell death by those clones that make LT, but not by those that produce only IFN-gamma under such induction conditions. These results indicate that it is neither Con A nor IFN-gamma that kills T cells, but LT. These results strongly suggest a self-regulatory role of LT in limiting continuing unrestricted T cell response to antigen activation.

Animals↗
PubMed1985

Protein-antigen specific Ia-restricted cytolytic T cells: analysis of frequency, target cell susceptibility, and mechanism of cytolysis.

We previously showed that cloned, antigen-specific, Ia-restricted L3T4a+ T cell lines can be cytolytic for antigen-pulsed B cell lymphoma targets. Such cells can also, under different experimental conditions, activate B cells to proliferate and secrete immunoglobulin. In the present experiments, we show that this functional phenotype is a common one among a panel of cloned T cell lines. In keeping with this finding, freshly isolated, antigen-activated lymph node T cells show similar functional properties. Such cytolytic L3T4a+ T cells differ from classical H-2K/D-restricted cytolytic T cells in two distinct ways. First, Ia-restricted cytolytic T cells can kill bystander targets, whereas H-2K/D-specific cytolytic T cells do not. Second, in testing a panel of target cells by using lectin-mediated cytolysis, Ia-restricted cytolytic clones reveal large differences in target cell susceptibility, whereas all targets are similarly susceptible to H-2K/D-specific killer cells. Finally, evidence is presented that both direct and bystander killing effected by L3T4a+ T cells are mediated by the same soluble factors, in that there is a strong positive correlation of these two activities for individual cloned lines. The relevant mediators appear to be lymphotoxin and IFN-gamma, although the latter molecule by itself is not cytolytic on our target lines.

Animals↗
PubMed1985

The differential inhibitory effect of lymphotoxin and immune interferon on normal and malignant lymphoid cells.

Supernatants containing lymphotoxin (LT) and immune interferon (IFN-gamma) or IFN-gamma alone were produced by antigen-stimulated murine T cell clones. These lymphokine preparations as well as cloned recombinant murine IFN-gamma (Genentech) were tested for antiproliferative activity on a variety of murine T, B, macrophage, and mastocytoma tumors and on T cell clones and LPS-stimulated B cells. The growth of every cell line tested was susceptible to the LT-containing supernatants. Cloned recombinant murine IFN-gamma inhibited the growth of A9 fibroblasts but not L929 cells. Neither the cloned murine IFN-gamma nor that produced by a T cell clone had any appreciable effect on the lymphoid cells except for one B cell lymphoma, A20. The sensitivity of nontransformed T and B cells to LT indicates that this lymphokine may play an immunoregulatory role. Indeed, the T cells that produce LT are sensitive to its cytotoxic activity and, therefore, regulate themselves. The differential inhibitory effects of LT and IFN-gamma on lymphoid target cells allow us to distinguish between preparations that contain IFN-gamma alone and those that contain LT and IFN-gamma. The susceptibility of lymphoid tumors to the antiproliferative activity of the LT-containing preparations indicates that LT either alone or together with IFN-gamma may be useful in tumor therapy.

Animals↗
PubMed1984

Messenger RNA for an antigen-specific binding molecule from an antigen-specific T-cell hybrid.

The T-cell hybridoma 51H7D specifically binds the hapten azobenzenearsonate (ABA). An antiserum (anti-PCIF) specific for antigenic determinants shared by antigen-binding molecules of T cells (TABM) was used to precipitate polyribosomes containing mRNA for TABM from this T-cell hybridoma. The resultant mRNA was translated in vitro. The translated product (TrP51H7D) bound specifically ABA and was bound by both anti-PCIF and anti-T-cell suppressor factor. The parent lymphoma BW5147 yielded a similar translated product with the same antiserum used to isolate specific mRNA containing polysomes. This product (TrPBW) was bound by the antiserum used but did not bind ABA. The specific translated protein from both cells had a pI of approximately equal to 5.0, and apparent molecular weight of 71,000 (reduced) or 145,000 (nonreduced). Both protein products, when treated with guanidine to break down all noncovalent bonds, revealed an elemental peptide of Mr 23,500. The cDNA made from the isolated mRNA had 600-900 bases. mRNA of this size is expected for a protein of Mr 25,000. Our data indicate that a TABM specific for ABA is composed of peptides of Mr 23,500.

Animals↗
PubMed1983

Analysis of normal and neoplastic lymphocyte surface-labeled proteins by two-dimensional polyacrylamide gel electrophoresis.

Normal and neoplastic murine and human lymphocytes were surface-labeled by lactoperoxidase-catalyzed radioiodination, and the cell lysates were subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analyses, combining isoelectric focusing in the first dimension and sodium dodecyl sulfate-PAGE (SDS-PAGE) in the second dimension. 2D-PAGE autoradiogram patterns were reproducible and reflected differences in cell types. A string of spots with a Mr of 100K was tentatively identified as a new T-cell marker (Tp100) which was present in all murine and human T cells examined including human T lymphomas. Murine and human B cells displayed markers characteristic to B cells of each species with some similarities between them. Human lymphomas and murine cell lines showed markers which were absent or only weakly visible in normal cells. Thus, 2D-PAGE analysis of lymphocyte surface proteins proved to be a method useful for searching for various markers.

Animals↗
PubMed1983

The delayed-type hypersensitivity response to (4-hydroxy-3-nitrophenyl) acetyl- (NP) coupled proteins is carrier-specific: in vivo and in vitro demonstrations.

In vivo and in vitro approaches for measuring DTH to NP and the cross-reactive hapten, NIP, were taken. Mice were immunized subcutaneously with NP-OVA, NP-BGG or NP-CGG in CFA or NP-spleen cells, challenged intradermally with NP or NIP-coupled to a heterologous carrier, and footpad or ear swelling determined 4, 24, and 48 h later. Alternatively, draining LNC were removed and challenged in vitro with either haptenated protein or haptenated, irradiated, syngeneic spleen cells to induce lymphotoxin (LT) production or proliferation. Our results show that although carrier-specific DTH responses are easily elicited both in vivo and in vitro, NP-specific DTH effector cells cannot be evoked by conventional immunization regimens. This failure to induce hapten-specific DTH is not due to suppressor mechanisms. Attempts to induce LT production and T cell proliferation by re-exposure to NP were unsuccessful. Immunization with NP-coupled protein in CFA does elicit an intense Arthus reaction when mice are challenged with the hapten 8 days later. The antibody-mediated nature of this hapten-specific response is indicated by the kinetics of the reaction, which peaks 4 hr after challenge, intensely positive ELISA of circulating anti-NP antibodies, sensitivity to pretreatment with a high dose of cyclophosphamide, and the ability to transfer the reaction to naive recipients with serum. This early response is highly cross-reactive with NIP and is not restricted to mice of the igh-1b allotype.

Animals↗
PubMed1983

Production of lymphotoxin, IFN-gamma and IFN-alpha, beta by murine T cell lines and clones.

The PCl-6 T cell line, derived from mice sensitized by skin painting with picryl chloride (PCl), shows antigen dependence for DNA synthesis and for lymphotoxin (LT) production. These cells produce LT, but not interferon (IFN), when exposed to 2,4,6-trinitrobenzene sulfonic acid- (TNBS) coupled syngeneic spleen cells. Concanavalin A (Con A) induces IFN production by PCl-6 cells, and IFN levels, but not LT titers, are increased by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). These results support the noncoordinate regulation of these two lymphokines. Line 32, a T cell growth factor- (TCGF) dependent T cell line and its Ly-1.2+, 2.2- derivative clone, 32H1, produce both antiviral and antiproliferative activity after exposure to several different mitogens. Tests for acid lability, sensitivity to anti-mouse IFN-alpha, beta antisera, and antiproliferative activity on non-mouse target cells indicates that an Ly-1+ clone, in the absence of both TCGF and accessory cells, can produce at least three separate lymphokine activities after Con A exposure: IFN-gamma (Type II), IFN-alpha, beta (Type I), and LT.

Animals↗
PubMed1982

A critical analysis of the T cell hybrid technique.

The T cell hybridization technique can be used to prepare continuous cell lines which express the antigen specificity and function of T cells within the milieu of a proliferating lymphoma. Technical details for the preparation and maintenance, selection and analysis of T cell hybrids are defined. Techniques for cloning of hybrid cells and production of hybrid-derived tumors are also presented. Parameters influencing the hybridization frequency and the production of functional hybrids are discussed. A variety of T cell subsets, including suppressor cells and delayed-type hypersensitivity effector cells as well as T cells maintained on TCGF, are excellent sources of primary parents for hybridization. When BW 5147 is used as the T lymphomas parent in these experiments, the resulting hybridization frequencies range between 10 and 434 X 10(-7). We have had moderate success using YAC-1; however, additional lines such as L5178Y, BALENTL 5, EL4 BU and S491TB.2 have proved ineffective as sources of T lymphoma parents. The technique of T cell hybridization is evaluation in terms of retention of differentiated functions and the stability and growth characteristics of the resultant hybrids.

Animals↗