Clinicopathological features of aggressive large granular lymphocyte leukaemia resemble Fas ligand transgenic mice.

Fas ligand triggers cell death after interaction with its receptor Fas. Altered expression of Fas has been associated with lymphoproliferation and autoimmune disorders in both mice and man. Apoptosis of lung and liver tissue is seen in Fas ligand transgenic mice. It is not known whether constitutive expression of Fas ligand can cause a similar human disease. Four patients with aggressive large granular lymphocyte (LGL) leukaemia involving lung and liver were studied. All four patients were severely ill with pulmonary involvement. Two patients presented with hypoxia and were oxygen dependent; the other two patients had severe pulmonary hypertension. Lung biopsies showed interstitial infiltration by leukaemic LGL. The infiltrating lymphocytes expressed both Fas and Fas ligand, whereas normal pneumocytes expressed only Fas. Similar findings were observed in liver biopsies from these patients. Features mimicking the pathological changes of graft-versus-host disease were observed, including pneumocyte apoptosis. All four patients had high levels of circulating Fas ligand. Successful treatment with oral methotrexate or 2-chlorodeoxyadenosine was associated with disappearance or marked reduction of circulating Fas ligand. These results suggest that dysregulated expression of Fas ligand can lead to human disease with pathological features resembling graft-versus-host disease.

Protein CVermont: symptomatic type II protein C deficiency associated with two GLA domain mutations.

This study investigates type II protein C deficiency in a family with manifestations of both arterial and venous thrombosis. Of 64 members of the kindred, 14 have been tested and 7 have PC deficiency. Among affected individuals (n = 7), mean protein C levels by different assays were as follows: enzyme-linked immunosorbent assay (ELISA), 3.8 micrograms/mL (2.1 to 4.3 micrograms/mL); amidolytic with venom activator, 115% (60% to 140%); clotting with venom activator, 42% (23% to 59%). The mean ratio of clotting to amidolytic assays for the affected individuals was 0.37 compared with a normal range of 0.8 to 1.2. Thus, the affected individuals have normal total protein C and their activated protein C has a normal active site assessed by chromogenic substrate; however, they have markedly diminished clotting activity. Immunoassay and chromatography data suggested an abnormality of carboxylation in the gamma carboxyglutamic acid (Gla) domain. Polymerase chain reaction amplification and direct DNA sequencing of exon 2 from genomic DNA of affected individuals showed two nucleotide substitutions. One of the mutations (A----C) results in Glu20----Ala, thereby eliminating a site for vitamin K-dependent gamma-carboxylation. The other substitution (G----A) results in a Val34----Met mutation. DNA sequencing of the other exons from affected individuals has shown no further difference from that of the wild-type gene. The former mutation also removes a Bgl II restriction endonuclease site, which has allowed us to confirm the mutation in affected individuals by direct digestion and Southern hybridization of genomic DNA from family members. This is the first reported family with documented Gla domain mutations in the protein C gene.

Bleeding diathesis due to decreased functional activity of type 1 plasminogen activator inhibitor.

We evaluated an elderly patient with a lifelong history of severe bleeding after surgery or trauma and with evidence of persistent hyperfibrinolysis. Routine coagulation studies were normal. Serum plasminogen (40%, normal 72-128%) and alpha 2-antiplasmin (55%, normal 70-145%) activities were decreased. Euglobulin clot lysis was abnormally shortened (50 min) and normalized in vitro with epsilon-aminocaproic acid (EACA). The patient was treated with EACA with prompt cessation of bleeding. Patient tissue-plasminogen activator (t-PA) levels in serum were normal (4.7 ng/ml, control 3.5-7.2) as detected by a two-site immunoradiometric assay (IRMA). Patient fibrinolytic inhibitor activities were assessed by incubating 125I-labeled t-PA with either whole blood or serum followed by SDS-PAGE and autoradiography to identify the resultant protease/protease inhibitor complexes. In comparison to blood samples obtained from normal donors, patient plasma and serum demonstrated reduced binding of a fast-acting plasminogen activator inhibitor to 125I-labeled t-PA. Immunoprecipitation experiments indicated diminished complex formation between type 1 plasminogen activator inhibitor (PAI-1) in patient serum and 125I-labeled t-PA. Low patient PAI-1 activity was confirmed in serum (0.36 U/ml, control 0.87-1.81; n = 3) and in platelet lysates using a functional IRMA to quantitate PAI-1 binding to immobilized t-PA. However, patient serum PAI-1 antigen was within the normal range when analyzed by IRMA (31.8 ng/ml, control 19.6-42.2); this result was confirmed in both serum and platelets by Western blot (n = 3). Mixing experiments using purified PAI-1 as well as patient and control sera did not show evidence for an inhibitor against PAI-1. We conclude that this patient's bleeding diathesis was due to hyperfibrinolysis and defective PAI-1. This patient provides the first demonstration of a link between decreased in vivo PAI-1 activity and disordered hemostasis, and supports a role for PAI-1 in control of vivo fibrinolysis.

In situ identification of idiotype-positive cells participating in the immune response to phosphorylcholine.

The phosphorylcholine idiotype (Id)/anti-Id system has been used to study the role of antigen-specific cells in antigen-induced microenvironmental changes. Anti-Id staining of lymph nodes following PC immunization shows the presence of Id on follicular dendritic cells at 12 h and in plasma cells beginning at day 3. Germinal centers began to form at day 3, peaking in size and number at days 8-10. Scattered Id-positive small lymphocytes are present in germinal centers but with rare exceptions over 98% of germinal center cells are Id-negative. Idiotype-positive small lymphocytes are depleted from primary follicles adjacent to germinal centers but not from distant, unstimulated nodes. These results extend previous studies showing architectural alterations in lymph nodes following antigenic stimulation and demonstrate antigen-specific cells are a prominent component of these antigen-induced microenvironmental changes.

Monoclonal antibody to the amino-terminal L sequence of murine leukemia virus glycosylated gag polyproteins demonstrates their unusual orientation in the cell membrane.

To analyze cell surface murine leukemia virus gag protein expression, we have prepared monoclonal antibodies against the spontaneous AKR T lymphoma KKT-2. One of these antibodies, 43-13, detects an AKR-specific viral p12 determinant. A second monoclonal antibody, 43-17, detects a novel murine leukemia virus-related antigen found on glycosylated gag polyproteins (gp95gag, gp85gag, and gp55gag) on the surface of cells infected with and producing ecotropic endogenous viruses, but does not detect antigens within these virions. The 43-17 antibody immunoprecipitates the precursor of the cell surface gag protein whether in its glycosylated or unglycosylated state, but does not detect the cytoplasmic precursor of the virion gag proteins (Pr65gag). Based on these findings, we have localized the 43-17 determinant to the unique amino-terminal part of the glycosylated gag polyprotein (the L domain). We have determined that gp95gag contains L-p15-p12-p30-p10 determinants, whereas gp85gag lacks the carboxyterminal p10 determinant, and gp55gag lacks both p30 and p10 carboxy terminal determinants. Analysis of cell surface gag expression with the 43-17 antibody leads us to propose that the L domain plays a crucial role in (i) the insertion and orientation of murine leukemia virus gag polyproteins in the cell membrane and (ii) the relative abundance of expression of AKR leukemia virus versus Moloney murine leukemia virus glycosylated gag polyproteins in infected cells.

Transformed lymphocytes from Abelson-diseased mice express levels of a B lineage transformation-associated antigen elevated from that found on normal lymphocytes.

Animals injected with Abelson murine leukemia virus (A-MuLV) rapidly develop fatal bone marrow-derived lymphosarcomas. In all such diseased animals tested, a subpopulation of bone marrow cells expressed a monoclonal antibody-defined, B lineage transformation-associated antigen (6C3 Ag) at levels increased from that detected on normal lymphocytes. Cells bearing a high level of this antigen were found to be transformed as measured by clonal growth in agar, and they expressed surface antigen markers characteristic of early pre-B cells. High-level antigen-expressing cells were found in the bone marrow, lymph nodes, and spleen, but never in the thymus of diseased animals. This distribution agrees with the published pathology of Abelson disease.

Transformation-associated proteins in murine B-cell lymphomas that are distinct from Abelson virus gene products.

In an effort to identify cellular proteins that may be involved in the Abelson murine leukemia virus (A-MuLV) transformation process, we have isolated a hybridoma antibody (6C3) that detects a tumor-associated antigen in all A-MuLV-induced pre-B-cell lymphomas. The 6C3 antibody immunoprecipitates two molecules of Mr 160,000 and Mr 125,000 from metabolically labeled A-MuLV tumors. The two proteins recognized by the 6C3 antibody are distinct from the A-MuLV-transforming protein in that they lack viral gag determinants and are neither phosphoproteins nor protein kinases. The 6C3 proteins can be detected in all A-MuLV pre-B-cell lymphomas and some nonviral B lymphomas but are not detected on any other tumor or normal cell, including A-MuLV-transformed fibroblast lines. Thus, the 6C3 proteins may represent the products of novel cellular genes whose expression is induced, stabilized, or amplified in B-cell tumors of both viral and nonviral origin. Further evidence in support of this hypothesis is provided by the finding that 6C3 antigen expression correlates with autonomous cell growth and the transformed phenotype in both normal bone marrow cultures and those infected with A-MuLV.

Isolation of molecules recognized by monoclonal antibodies and antisera: the solid phase immunoisolation technique.

A simple technique for the isolation of antigens recognized by antisera and monoclonal antibodies has been developed. This method, the solid-phase immunoisolation technique, employs the protein-binding properties of polyvinylchloride microtiter plates. Antibodies are adsorbed to the plates either directly or via an anti-immunoglobulin reagent. Antigen is then placed in the wells, and allowed to adsorb to the antibody. The well is washed, and the antigen is then eluted with a denaturing electrophoresis sample buffer for one- or two-dimensional analysis. The solid-phase immunoisolation technique has been used to isolate a variety of cell membrane antigens with high signals and low backgrounds. The ease of the procedure and the high signal-to-noise ratio make this method preferable to the use of a staphylococcal adsorbent for many applications.

Protection against syngeneic lymphoma by a long-lived cytotoxic T-cell clone.

The effect of a cloned T-cell line on the in vivo growth of syngeneic lymphoma cells was studied. 1E4 is an H-2-restricted cytotoxic T-cell clone that efficiently kills Abelson virus-induced lymphoma target cells (L1-2) at low effector/target ratios, as measured by in vitro cytotoxicity assays. In addition, it is long lived in vitro in the absence of stimulation and survives for more than 1 wk in vivo in the absence of exogenous antigen or growth factors. Mice injected intraperitoneally with lethal doses of L1-2 and then treated with 1E4 survived longer than animals treated with saline or with a control T-cell clone. Multiple weekly injections of effector cells, or a single injection in animals given a low dose of tumor cells, resulted in 50-80% long-term survivors. The observation that intravenous injection of killer cells was less effective than intraperitoneal treatment, coupled with the previous demonstration of markedly abnormal circulatory patterns for T-cell clones, suggests that those animals succumbing to progressively growing neoplasm die because the effector cells are unable to home into peripheral sites of tumor deposition. Thus, although this cytotoxic T-cell clone does have useful in vivo activity, its function may be partially limited by a generalized defect in migration.

Abnormal migration of T lymphocyte clones.

Several in vitro T cell clones were markedly deficient in their ability to home to peripheral lymphoid tissue. This was found for an alloreactive noncytolytic clone, a soluble antigen- (KLH)specific line, and cytotoxic clones specific for allogeneic cells and for Abelson virus-induced lymphoma cells. This abnormal circulation pattern was probably caused by the lack of the receptors of the lymphocytes for high endothelial venules (HEV), as implied by the lack of binding of these T cells to HEV in frozen sections of mouse lymph node and Peyer's patches. The loss of surface receptors that are necessary for normal lymphocyte migration may thereby alter the in vivo function of adoptively transferred T cells.

A monoclonal antibody that detects a V kappa-TEPC15 idiotypic determinant cross-reactive with a Thy-1 determinant.

To identify T lymphocyte antigens with immunoglobulin-like determinants, we prepared rat anti-mouse T cell monoclonal antibodies and screened them against a panel of purified mouse myeloma proteins representing all isotypes of immunoglobulin. One hybridoma, designated 42-21, was found to detect a novel antigenic determinant shared by V kappa-TEPC15 and the Thy-1 molecule on all T lymphocytes. Although several explanations for this unusual phenomenon exist, it may imply some role for the Thy-1 molecule in antigen and/or mitogen recognition. In any event, future studies of idiotypes on T lymphocytes must consider the possibility that anti-idiotypic sera detect cell surface molecules unrelated to classical immunoglobulin.

Effects of Lyt antibodies on T-cell functions: augmentation by anti-Lyt-1 as opposed to inhibition by anti-Lyt-2.

Monoclonal anti-Lyt-1 (alpha Lyt-1) and alpha Lyt-2 manifest inverse effects on allogeneic mixed lymphocyte reactions when added to the reaction mixtures without complement. alpha Lyt-1 augments cell proliferation and generation of cytotoxic cells but has no effect on cell-mediated cytolysis, whereas alpha Lyt-2 blocks cell proliferation, generation of killer cells, and cytolytic activity of killer cells. The augmenting effect of alpha Lyt-1 cannot be attributed to a direct mitogenic effect on T cells. Both inhibition by alpha Lyt-2 and potentiated by alpha Lyt-1 require interaction of responder cells with the antibodies during the first 24 hr of the mixed lymphocyte reaction, indicating that early stages of the reaction are sensitive to Lyt antibodies. The enhancing effect of alpha Lyt-1 on alloantigen-induced T-cell proliferation is associated with augmented production of T-cell growth factors. When alpha Lyt-1 is present in mixed lymphocyte cultures, the supernatant media collected after 24 or 48 hr of culture induce higher proliferation of activated T cells compared to media of mixed lymphocyte cultures incubated in the absence of antibodies or in the presence of alpha Lyt-2 which has no effect on secretion of growth factors. The differences in the effects of alpha Lyt-1 and alpha Lyt-2 could not be attributed to differences in heavy chain constant region functions because both were of the same lambda 2A immunoglobulin class and were used at the same concentration. The data suggest a possible role for Lyt-1 molecules in early activation and mitogenesis processes such as production of growth factors.

Blocking effect of lyt-2 antibodies on T cell functions.

Monoclonal anti-Lyt-2 antibodies blocked effector function of cytotoxic thymus-derived (T) cells in the absence of added complement. Cytolysis of both allogeneic cells and syngeneic lymphoma or sarcoma target cells was inhibited at the level of the effector lymphocytes. Anti-Lyt-1 and anti-Thy-1 antibodies did not block killer cells. Proliferation of T cells in mixed lymphocyte culture was also inhibited by anti-Lyt-2, but not affected by anti-Lyt-1 or anti-Thy-1 antibodies. Although Lyt-1+ lymphocytes were required in the mixed lymphocyte reaction as helper cells for proliferation of Lyt-2+ lymphocytes, their helper function was not affected by the presence of Lyt-1 antibodies. Thus, although anti-Lyt-1, anti-Lyt-2 and anti-Thy-1 were of the same gamma 2A immunoglobulin class, had high titers, and interacted with T cells to the same extent, only anti-Lyt-2 blocked T cell functions. Polyclonal activation of T lymphocytes by concanavalin A, in contrast to activation by alloantigens, was not inhibited by Lyt-2 antibodies, suggesting that Lyt-2 antibodies interfere with T cell function at the level of the T cell antigen-receptor. The role which Lyt-2 molecules may play in T cell function is discussed.

Murine leukaemogenesis: monoclonal antibodies to T-cell determinants arrest T-lymphoma cell proliferation.

We have proposed a receptor-mediated leukaemogenesis hypothesis wherein T lymphomas would be clones of T cells bearing mitogen-linked surface receptors specific for the envelope determinants of the inducing MuLV. A prediction of the hypothesis is that T-lymphoma proliferation is dependent on continued presentation of MuLV envelope determinants to these cell-surface receptors, and that substances which interfere with receptor-virus interactions should inhibit T-lymphoma proliferation. Rat monoclonal antibodies were raised to the AKR mouse T lymphoma KKT-2, and these antibodies were screened independently for blockade of virus-binding and for cytostatic activity on KKT-2 cells. We report here that those monoclonal antibodies which block virus binding inhibit growth of KKT-2 cells in vitro, whereas monoclonal antibodies which bind to these cells but do not block virus binding are not cytostatic. Three of the four cytostatic antibodies detect determinants on the Thy-1 molecule, while none of the other (noncytostatic) antibodies detect Thy-1. Antibody inhibition of KKT-2 cell growth is precluded by saturation of KKT-2 virus receptors with the inducing leukaemia virus.

Hooked trichomes: a physical plant barrier to a major agricultural pest.

Hooked epidermal appendages (trichomes) on leaves of field bean cultivars effectively capture nymph and adult leafhoppers. Frequency of capture and capture mortality are highly correlated with trichome density. Hooked trichomes inserted at angles less than 30 degrees are ineffective in capture.