The transmembrane adaptor protein TRIM regulates T cell receptor (TCR) expression and TCR-mediated signaling via an association with the TCR zeta chain.

T cell receptor (TCR)-interacting molecule (TRIM) is a recently identified transmembrane adaptor protein, which is exclusively expressed in T cells. Here we demonstrate that in mature T cells, TRIM preferentially interacts with the TCR via the TCR-zeta chains and to a lesser extent via the CD3-straightepsilon/gamma heterodimer. Transient or stable overexpression of TRIM in Jurkat T cells results in enhancement of TCR expression on the cell surface and elevated induction of Ca(2+) mobilization after T cell activation. TRIM-mediated upregulation of TCR expression results from inhibition of spontaneous TCR internalization and stabilization of TCR complexes on the cell surface. Collectively, our data identify TRIM as a novel integral component of the TCR complex and suggest that one function of TRIM might be to modulate the strength of signals transduced through the TCR through regulation of TCR expression on the cell surface.

Phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG), a novel ubiquitously expressed transmembrane adaptor protein, binds the protein tyrosine kinase csk and is involved in regulation of T cell activation.

According to a recently proposed hypothesis, initiation of signal transduction via immunoreceptors depends on interactions of the engaged immunoreceptor with glycosphingolipid-enriched membrane microdomains (GEMs). In this study, we describe a novel GEM-associated transmembrane adaptor protein, termed phosphoprotein associated with GEMs (PAG). PAG comprises a short extracellular domain of 16 amino acids and a 397-amino acid cytoplasmic tail containing ten tyrosine residues that are likely phosphorylated by Src family kinases. In lymphoid cell lines and in resting peripheral blood alpha/beta T cells, PAG is expressed as a constitutively tyrosine-phosphorylated protein and binds the major negative regulator of Src kinases, the tyrosine kinase Csk. After activation of peripheral blood alpha/beta T cells, PAG becomes rapidly dephosphorylated and dissociates from Csk. Expression of PAG in COS cells results in recruitment of endogenous Csk, altered Src kinase activity, and impaired phosphorylation of Src-specific substrates. Moreover, overexpression of PAG in Jurkat cells downregulates T cell receptor-mediated activation of the transcription factor nuclear factor of activated T cells. These findings collectively suggest that in the absence of external stimuli, the PAG-Csk complex transmits negative regulatory signals and thus may help to keep resting T cells in a quiescent state.

Expression of glutamate carboxypeptidase II in human brain.

Glutamate carboxypeptidase II (GCPII) is a transmembrane glycoprotein expressed in various tissues. When expressed in the brain it cleaves the neurotransmitter N-acetylaspartylglutamate (NAAG), yielding free glutamate. In jejunum it hydrolyzes folylpoly-gamma-glutamate, thus facilitating folate absorption. The prostate form of GCPII, known as prostate specific membrane antigen (PSMA), is an established cancer marker. The NAAG-hydrolyzing activity of GCPII has been implicated in a number of pathological conditions in which glutamate is neurotoxic (e.g. amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, epilepsy, schizophrenia, and stroke). Inhibition of GCPII was shown to be neuroprotective in tissue culture and in animal models. GCPII is therefore an interesting putative therapeutic target. However, only very limited and controversial data on the expression and localization of GCPII in human brain are available. Therefore, we set out to analyze the activity and expression of GCPII in various compartments of the human brain using a radiolabeled substrate of the enzyme and the novel monoclonal antibody GCP-04, which recognizes an epitope on the extracellular portion of the enzyme and is more sensitive to GCPII than to the homologous GCPIII. We show that this antibody is more sensitive in immunoblots than the widely used antibody 7E11. By Western blot, we show that there are approximately 50-300 ng of GCPII/mg of total protein in human brain, depending on the specific area. Immunohistochemical analysis revealed that astrocytes specifically express GCPII in all parts of the brain. GCPII is enzymatically active and the level of activity follows the expression pattern. Using pure recombinant GCPII and homologous GCPIII, we conclude that GCPII is responsible for the majority of overall NAAG-hydrolyzing activity in the human brain.

Characterization of a broadly expressed human leucocyte surface antigen MEM-43 anchored in membrane through phosphatidylinositol.

A monoclonal antibody MEM-43 was prepared, which recognizes an antigen expressed on all peripheral blood leucocytes, on erythrocytes and several cell lines, but is absent from U937, Nalm-6, Daudi and Raji cell lines. The antigen isolated by immunoaffinity chromatography from several cell lines is an 18,000-25,000 mol. wt glycoprotein. An apparently identical antigen isolated from erythrocytes binds to several lectins and has a 14,000 mol. wt polypeptide backbone, modified by an endoglycosidase F-sensitive carbohydrate moiety. The epitope recognized is reduction-sensitive. The sequence of N-terminal 17 amino acid residues was determined; five out of six N-terminal amino acids are identical to those found at the N-terminus of the mouse lymphocyte surface antigen Ly-6C. The antigen is completely released from the cell surface after treatment with phosphatidylinositol-specific phospholipase C.

Structural relationship between the soluble and membrane-bound forms of human monocyte surface glycoprotein CD14.

The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD14, was determined by carboxypeptidase Y digestion and compared with the complete amino acid sequence of this protein as predicted from the structure of cloned cDNA [Goyert et al. Science 239, 497-500 (1988)]. The soluble antigen isolated from urine appears to lack eight C-terminal amino acid residues predicted for the full-size translation product, but possesses a major part of the C-terminal hydrophobic domain originally suggested as the membrane-spanning segment. The CD14 antigen can be removed from the monocyte surface by phosphatidylinositol-specific phospholipase C treatment, indicating that this glycoprotein is anchored in the membrane by a phospholipid and is not a transmembrane protein. The soluble form occurring in serum and in supernatants of cultured monocytes thus probably arises by phospholipase-mediated cleaving off the cell surface antigen. A sensitive sandwich ELISA was developed using a monoclonal anti-CD14 antibody, MEM-18, and polyclonal rabbit anti-CD14 antiserum for quantitation of the soluble antigen concns in sera and cell culture supernatants. Using this assay, the antigen present in the supernatant of phospholipase treated peripheral blood mononuclear cells could be estimated. The assay was also used for estimation of the concns of the soluble form of the CD14 antigen in human sera.

Nitrocellulose membrane as an antigen or antibody carrier for screening hybridoma cultures.

Nitrocellulose membranes were used as carriers of antigen for rapid screening of specific monoclonal antibodies or as carriers of the monoclonal antibody (in a crude form) for a simple and economic determination of isotype of the antibody by a modification of enzymoimmunoassay.

Monoclonal antibodies against human alpha-fetoprotein. Exploitation of an unusual calcium-dependent interaction with the antigen for analytical and preparative purposes.

Seven murine hybridomas were established producing IgG1 monoclonal antibodies (mAbs) against three different epitopes of human alpha-fetoprotein (alpha FP). The interaction of two of these mAbs (AFP-01 and AFP-02) with the antigen was strongly inhibited by calcium-chelating agents. The mAb AFP-11 could be used for estimation of alpha FP by RIA in the concentration range of 5-100 ng/ml. Several combinations of the mAbs could be used in a two-site sandwich-ELISA for similar purposes. Immunoaffinity chromatography on a column of immobilized mAb AFP-01 permitted the purification of alpha FP.

Uveal melanoma: no expression of HLA-G.

PURPOSE: To investigate whether uveal melanoma cells express HLA-G, a nonclassical HLA class I molecule that has been shown to be a critical mediator in the inhibition of natural killer (NK) cell-mediated cytolysis. METHODS: Eleven human uveal melanoma cell lines were analyzed for the expression of HLA-G by flow cytometry, immunocytochemistry, Western blot analysis, and RT-PCR followed by Southern blot analysis. Two HLA-G-specific monoclonal antibodies were used, 87G and MEM-G/1. In addition, HLA-G expression was determined on frozen tissue sections of 17 primary uveal melanomas. RESULTS: With all HLA-G detection methods, no evidence for HLA-G expression by uveal melanoma cells was found. In contrast, the trophoblast cell line JEG-3 clearly expressed HLA-G transcripts and protein in all cases. Furthermore, interferon-gamma did not induce HLA-G expression in the uveal melanoma cell lines. Notably, all cell lines expressed HLA-E, and this expression was significantly enhanced by interferon-gamma. CONCLUSIONS: Because none of the uveal melanoma cell lines nor any of the primary uveal melanomas displayed expression of HLA-G, it is unlikely that HLA-G plays a role, direct or indirect, in the modulation of cellular immunity against uveal melanoma tumors.

Murine hybridoma monoclonal antibodies against insulin: cross-reactivity with insulins of three species and blocking of insulin binding to its receptor.

Six hybridoma cell lines secreting monoclonal antibodies against pig insulin and cross-reacting with human and bovine insulins were obtained. Five of these monoclonal antibodies were IgG1, kappa, one IgG2b, kappa; their pI values were in the range of pH 6.3-7.4 and dissociation constants of the insulin-antibody complexes were 0.3-2 X 10(-8) mol/l, as determined by an immunoradiometric inhibition assay. All of these antibodies reacted with sterically closely related determinants and blocked the binding of 125I-pig insulin to the receptor on human MOLT-4 cell line.

Modification of suppressor/cytotoxic and helper subsets in lentil lectin-pretreated mice.

The effect of lentil lectin (LCA) on various lymphocyte subpopulations in the spleen, lymph nodes and thymus of CBA mice was studied. The most marked effect after five 1-mg LCA doses was observed in the spleen. LCA caused a drop in both the total and relative number of cells carrying all the markers examined, i.e., Thy 1, L3/T4a, Lyt 2, and sIg, so that large numbers of lymphocytes did not express any of them. A dramatic decrease in the Thy 1-positive cell number is mainly due to depletion of the L3/T4a (helper) T cell subpopulation, whereas the Lyt 2+ (suppressor/cytotoxic) cells are less affected. The decrease in the Thy 1+, Lyt 1+ and L3/T4a+ cell numbers in the thymus and lymph nodes is smaller, the percentage of Lyt 2+ cells is even increased after LCA treatment. Our results indicate that two mechanisms play a role in the induction of allotransplantation tolerance by LCA: 'depletion' of helper T cells and a relative increase in the regulatory (suppressor) cell ratio, which may support allograft survival.

Characterization of the human leukocyte GPI-anchored glycoprotein CDw108 and its relation to other similar molecules.

The CDw108 glycoprotein is expressed on the surface of some leukemic cell lines, erythrocytes and on activated lymphocytes. Its surface expression is rapidly upregulated following various activating stimuli (PHA, PWM, Con A, PMA, anti-CD3) and subsequently gradually decreases. The molecule is anchored in the membrane via glycosylphosphatidylinositol (GPI) moiety, it has molecular mass of 75-80 kDa and pI of 5.0-5.5. Endoglycosidase F and H reduce its apparent size as determined by SDS PAGE by approx. 15 and 22 kDa, respectively. It is a component of large, detergent-resistant GPI-complexes associated with protein kinases. In addition to the previously described identity of CDw108 with the JMH blood group antigen, we demonstrate here its identity to the previously described glycoprotein recognized by monoclonal antibodies H105 and KS.2, and exclude its identity with another GPI-anchored glycoprotein of similar size, melanotransferrin (gp97).

A novel anti-CD18 mAb recognizes an activation-related epitope and induces a high-affinity conformation in leukocyte integrins.

Monoclonal antibody MEM-148 was previously shown to recognize CD18 chains in a free form unassociated within leukocyte integrin heterodimers, but yet it is paradoxically able to induce a high-affinity conformation in the native, cell surface expressed LFA-1 molecules. Our results based on kinetics of binding, immunoprecipitation and cell-aggregation experiments demonstrate that the mAb does bind to and stabilizes a specific conformation of LFA-1 heterodimers apparently distinguished by an increased affinity to its cellular ligand(s). A similar high-affinity conformation of LFA-1, in which the MEM-148 epitope becomes exposed, is induced also by a Mg2+/EDTA or low pH (5.5-6.5) treatments which may mimic physiologically relevant situations in normal or inflamed tissues. Thus, mAb MEM-148 is a novel valuable tool for detection and induction of specific conformations of human leukocyte integrins.

Immunosuppressive activity of bovine seminal ribonuclease and its mode of action.

Two preparations of dimeric BS RNase-native and recombinant proteins caused identical immunosuppressive effects on MLC-stimulated human lymphocytes. The monomers of RNase A and BS RNase were ten times less active. The inhibitory effect on MLC-stimmulation was followed by 90% inhibition of cell-mediated lympholysis (CML) caused by BS RNase (10 micrograms/ml). This effect indicated that BS RNase suppressed the recognition phase of the cytotoxic reaction, resulting in inhibition of generation of cytotoxic effector cells. BS RNase exerted a similar effect on generation of cytotoxic LAK cells. Cytotoxic activity of LAK cells or CTLs against K562 target cells was abrogated only when BS RNase was added at the beginning of the sensitizing phase, but the cytotoxicity of effector cells in the destruction phase was not influenced. The effect of RNase A on the generation of cytotoxic cells was much less pronounced. To get more information about the site of action, the effect of BS RNase on early lymphocyte stimulation by PHA was investigated by using fluorescein cell probes. BS RNase (100 micrograms/ml) prevented a shift in fluorescein emission occurring within one hour of activation using fluorescein diacetate as a marker for changes in the cytoplasmic matrix. On the contrary, it did not block the shift in fluorescence emission when tested with diphenylhexatrien as a marker for changes in membrane fluidity. Furthermore the effect of BS RNase on expression of membrane antigens expressed on activated human lymphocytes was estimated. BS RNase significantly inhibited the expression of CD25, CD38 and CD71 antigens on PHA-, Con A- and MLC-stimulated human T and B lymphocytes. No substantial change in expression of these antigens was observed on IL-2-stimulated cells, but DNA synthesis was totally abrogated. These results indicate that the mode of action of BS RNase on activated T and B lymphocytes is based mainly on the suppressed expression of receptors for interleukin-2-alpha-chain and transferrin.

Augmentation of NK cell activity and proliferation in cultured lymphocytes of leukemic patients by monoclonal antibodies CD3 and interleukin-2.

The aim of the present study was to induce NK cell activity in peripheral mononuclear cells (PMNC) of normal individuals or of leukemic patients. For this purpose, monoclonal antibodies (mAbs) anti-CD3 (MEM 57, OKT 3 and MEM 92), anti-CD59 (MEM 43) and anti-CD43 (MEM 59) were tested. MEM 43, MEM 59, MEM 57 and OKT 3 stimulated markedly the NK activity in fresh isolated PMNC of normal individuals, whereas the cytotoxicity in 3-day cultivated PMNC was enhanced only by MEM 57 and OKT 3. In comparison to interleukin-2 (IL-2), MEM 57 and OKT 3 induced less PMNC cytotoxicity but more cell proliferation. MEM 92 (anti-CD3, IgM) compared to MEM 57 or OKT 3 (anti-CD3, IgG) did not show any effect on both reactions mentioned above. PMNC of untreated leukemic patients exerted very negligible NK cell activity. Following 3-day culture of leukemic PMNC with OKT 3 or IL-2 the anti-K 562 cytotoxicity was markedly enhanced. In some cases the stimulating effect was more pronounced by IL-2, in others by OKT 3. Nevertheless, the best effect was gained in 7 out of 12 patients using a combination of IL-2 and OKT 3. On the other hand, OKT 3 did not synergize with IL-2 in normal PMNC culture.

Association of GPI-anchored glycoproteins with other components of the leucocyte membrane.

The leucocyte surface glycosylphosphatidylinositol (GPI)-anchored membrane proteins are localized within specific membrane microdomains which also contain specific (glyco)lipids and intracellular proteins including protein kinases. These "GPI-domains" are devoid of most abundant transmembrane proteins, but in T-cells they appear to contain small amounts of CD4 and CD8 and in B-cell lines, small amounts of CD10. The existence of these relatively detergent-resistant membrane microdomains explains the signal-transducing ability of GPI-anchored receptors. In addition to the "GPI-microdomains", several other types of analogous very large detergent-resistant complexes/domains appear to exist, such as those containing T-cell receptor, others containing CD45R molecules associated with a protein kinase, and still others composed mainly of several proteins of the tetraspan family. Therefore, we suggest that the leucocyte surface is a mosaic of microdomains of unique composition associated with specific signal-transducing molecules.

Effects of silica pretreatment on allogeneic tumour growth in mice treated with lentil lectin, spleen extract or alloantisera.

The effect of silica treatment (5 mg intravenously) on tumour growth was studied with regard to three types of immunosuppression induced by: lentil lectin (LCA), spleen cell extract (SE) (active enhancement), and alloantiserum (AAS) (passive enhancement). Two experimental tumours were used, which differ in their sensitivity to humoral antibodies. CBA mice were challenged with Sa 1 (A) tumours and BALB/c mice with EL-4 (C57BL/6) lymphomas. The results indicate that silica treatment caused a time-dependent promotion of tumour growth. In passive enhancement, tumour growth promotion by silica treatment was obtained in both tumour systems. When silica was given along with LCA or SE, the two combined treatments had no enhancing effect on Sa 1 targets and enhancing effects on EL-4. The different behaviour of the two graft-recipient combinations is discussed in the light of the inherent properties of both experimental tumours and of the donor-recipient relationships.

Allograft tolerance as in immunologically active process: analysis in a restricted H-2 incompatibility due to mutation (M504) in mouse strain B10.D2.

Cell-free spleen and liver extracts were used for attempted induction of allograft tolerance across a restricted H-2 barrier due to a chemically induced mutation in the B10.D2 mouse strain. When using mice of the latter strain as donors and the new congenic strain carrying the mutant H-2 hyaplotype as recipients, and with long-term administration of the cell-free antigens in adult life up to 100% yields (according to the dose of antigen) of permanently tolerant animals (particularly males) was obtained with spleen extract, but not with liver extract. This contrasts with a higher tolerance-inducing efficiency of liver extracts in various non-H-2 incompatibilities. In the reversed donor-recipient combination, neither spleen nor liver extracts gave positive results. The immune status of the permanently tolerant animals was analyzed and it was shown that with their bone marrow normal responsiveness can be conferred upon lethally irradiated syngeneic hosts whereas the specific allograft tolerance can be adoptively transferred to sublethally irradiated recipients by means of spleen or lymph node cells. It is concluded that in the present system the state of allograft tolerance is maintained by an immunologically active process of peripheral lymphocytes.

Persistence of regulation mechanism responsible for inhibition of allotransplantation reactions in the absence of alloantigen.

The purpose of the present work was to examine the persistence of regulatory cells implicated in the inhibition of allotransplantation reactions in recipient mice in the absence of antigenic material. Two experimental systems were used: (1) When neonatally induced tolerance (obtained as a result of cell-free spleen extract administration in the B10.D2 versus M504 strain combination) was adoptively transferred, the persistence of suppressor cells was detected in secondary recipients receiving a second homograft as long as 30 days after removal of a first homograft to the tolerance inducing haplotype. The removal of the skin homograft presenting a source of antigen for 60 days, resulted in tolerance interruption when a second test graft was applied. (2) When tolerance was induced in CBA mice with spleen-cell antigenic extracts and CFA and spleen cells from such animals were transferred to secondary recipients the following results were observed: Secondary recipients grafted with SaI tumour cells derived from the tolerance inducing A strain 20 days after tolerance induction presented an enhanced tumour growth. The adoptive transfer of this effect was at least partly due to IJ+ lymphocytes, since their removal reduced adoptive enhancement. However, if the cell transfer was delayed for 56 days after tolerance induction, the depletion of IJ+ or Thy 1.2+ cells did not affect the capacity of spleen cells to cause an enhanced growth of the homografted SaI cells. A delay of 100 days following donor enhancement induction did not result any adoptive transfer of SaI enhancement.

Immunogenic capacity of mouse alloantigens from ischaemic liver.

Immunogenic versus tolerogenic capacity of mouse alloantigens from ischaemic liver was tested in two different congenic strain combinations (H-2 compatible, non-H-2 incompatible). The recipients of test skin allografts were pretreated with tissue homogenate or particulate fraction and the survival pattern of the skin allografts was used as the criterion of the induced status. The results showed a difference in the immunogenicity of antigenic extracts from normal (healthy) and ischaemic liver. Following a single dose, alloantigens from ischaemic liver had an increased immunogenicity (which could be reduced through perfusion) as compared to control (extracts from healthy liver). Following three larger doses of ischaemic liver extracts, the effect was tolerogenic. Possible mechanism of the altered immunogenicity of alloantigens due to ischaemization of the liver tissue and the effect of perfusion are discussed.

The use of murine monoclonal antibody B2M-01 for detection and purification of human beta 2-microglobulin.

A murine hybridoma cell line was obtained producing monoclonal antibody specific for human beta 2-microglobulin (beta 2m). The antibody reacts strongly with cell-associted human beta 2m, weakly with the cells of the monkey Cercopithecus aethiops, but not with any other non-primate cells tested. It is of IgG2a isotype, its pI is 6.9-7.1 and dissociation constant of its complex with soluble beta 2m is K = 1.5 X 10(-8) mol/l. The antibody also binds to beta 2m after electrophoretic separation under the reducing and denaturating conditions and blotting onto nitrocellulose. Immobilization of this antibody on Sepharose 4B yields an efficient immunosorbent for purification of beta 2m.