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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.

Non-lineage section studied in total 90 mAb samples, including 23 submitted as known CD specificities. Thirty four samples submitted as unknown and potentially novel specificities recognized actually well known molecules (HLA class I, CD7, 11b, 14, 18, 44, 45, 45RB, 47, 59, 62L, 71, 82, 147). Seven samples reacted with newly defined CD molecules (CD281, 282, 284, 298, 315, 316, 321) and specificities of 12 samples remained unresolved.

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N- and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.

Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4+ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptide-induced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the beta subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change.

Lymphocyte membrane rafts contain molecules critical for immunoreceptor signaling. Here, we report identification of a new raft-associated adaptor protein LIME (Lck-interacting molecule) expressed predominantly in T lymphocytes. LIME becomes tyrosine phosphorylated after cross-linking of the CD4 or CD8 coreceptors. Phospho-LIME associates with the Src family kinase Lck and its negative regulator, Csk. Ectopic expression of LIME in Jurkat T cells results in an increase of Csk in lipid rafts, increased phosphorylation of Lck and higher Ca2+ response to CD3 stimulation. Thus, LIME appears to be involved in regulation of T cell activation by coreceptors.

A key molecule necessary for activation of T lymphocytes through their antigen-specific T cell receptor (TCR) is the transmembrane adaptor protein LAT (linker for activation of T cells). Upon TCR engagement, LAT becomes rapidly tyrosine phosphorylated and then serves as a scaffold organizing a multicomponent complex that is indispensable for induction of further downstream steps of the signaling cascade. Here we describe the identification and preliminary characterization of a novel transmembrane adaptor protein that is structurally and evolutionarily related to LAT and is expressed in B lymphocytes, natural killer (NK) cells, monocytes, and mast cells but not in resting T lymphocytes. This novel transmembrane adaptor protein, termed NTAL (non–T cell activation linker) is the product of a previously identified WBSCR5 gene of so far unknown function. NTAL becomes rapidly tyrosine-phosphorylated upon cross-linking of the B cell receptor (BCR) or of high-affinity Fcγ- and Fcε-receptors of myeloid cells and then associates with the cytoplasmic signaling molecules Grb2, Sos1, Gab1, and c-Cbl. NTAL expressed in the LAT-deficient T cell line J.CaM2.5 becomes tyrosine phosphorylated and rescues activation of Erk1/2 and minimal transient elevation of cytoplasmic calcium level upon TCR/CD3 cross-linking. Thus, NTAL appears to be a structural and possibly also functional homologue of LAT in non–T cells.

Circulating human natural killer (NK) lymphocytes have been functionally defined by their ability to exert cytotoxic activity against MHC class I-negative target cell lines, including K562. Therefore, it was proposed that NK cells recognized the “missing self.” We show here that the Ig-like CD160 receptor expressed by circulating CD56dim+NK cells or IL-2-deprived NK cell lines is mainly involved in their cytotoxic activity against K562 target cells. Further, we report that HLA-C molecules that are constitutively expressed by K562 trigger NK cell lysis through CD160 receptor engagement. In addition, we demonstrate, with recombinant soluble HLA-Cw3 and CD160 proteins, direct interaction of these molecules. We also find that CD158b inhibitory receptors partially interfere with CD160-mediated cytotoxicity, whereas CD94/CD159a and CD85j have no effect on engagement with their respective ligands. Thus, CD160/HLA-C interaction constitutes a unique pathway to trigger NK cell cytotoxic activity.

A highly attractive approach to investigate the influence and hierarchical organization of viral proteins on cellular immune responses is to employ mutant viruses carrying deletions of various virus-encoded, immune-modulating genes. Here, we introduce a novel set of deletion mutants of the human CMV (HCMV) lacking the UL40 region either alone or on the background of a deletion mutant devoid of the entire US2–11 region. Deletion of UL40 had no significant effect on lysis of infected cells by NK cells, indicating that the expected enhancement of HLA-E expression by specific peptides derived from HCMV-encoded gpUL40 leader sequences was insufficient to confer target cell protection. Moreover, the kinetics of MHC class I down-regulation by US2–11 genes observed at early and late phases postinfection with wild-type virus correlated with increased susceptibility to NK lysis. Thus, the influence of HCMV genes on NK reactivity follows a hierarchy dominated by the US2–11 region, which encodes all viral genes capable of down-modulating expression of classical and non-classical MHC class I molecules. The insights gained from studies of such virus mutants may impact on future therapeutic strategies and vaccine development and incorporate NK cells in the line of defense mechanisms against HCMV infection.

A broadly used pan-HLA class I-reactive monoclonal antibody W6/32 is believed to recognize a conformational epitope dependent on association between heavy chains and beta2-microglobulin (beta2m). However, in the present study we report that W6/32 does recognize at least some free HLA class I heavy chains under the partially denaturating conditions of nonreducing Western blotting, namely nearly all HLA-B allelic products. Furthermore, we confirm and largely extend our previous observation that complexes of beta2m with heavy chains of a few HLA class I allelic forms (most notably HLA-B27) exhibit unusual resistance to dissociation by SDS, which is reminiscent of MHC class II molecules. In addition, our data indicate the existence of covalent (disulfide-linked) heterodimers of certain HLA class I heavy chains (namely Cw1 and Cw4) and beta2m.

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.

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 Mg 2+ /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 imegrins.

One of the recently described antigens broadly expressed on human leukocytes is CDw149, which was defined at the 6th Human Leukocyte Differentiation Antigen (HLDA) Workshop by means of 2 monoclonal antibodies (mAbs). Molecular characterization of this antigen has been lacking. In the present study we demonstrate that these anti-CDw149 mAbs actually recognize a clustered subset of a well-defined membrane protein, CD47, also known as integrin-associated protein (IAP). This clustered subset is present on leukocytes but not erythrocytes. The anti-CDw149 mAbs bind with only low affinity to a monomeric (unclustered) subset of CD47 but with high avidity to the CD47 clusters. A fraction of CD47 is associated with large complexes containing cytoplasmic signaling molecules (Src family kinases and heterotrimeric G-proteins) similar to glycosphingolipid-enriched microdomains (GEMs), which may explain the previously described signaling capacity of CD47. The low-affinity anti-CD47 mAbs may be useful tools targeting specific receptor complexes involved in cell activation. Specific reactivity of low-affinity mAbs with clustered subsets of cell surface antigens may more generally explain the nature of poorly defined “activation forms” or activation neoepitopes described previously for several cell surface 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, melanotransf errin (gp97).

Mice were immunized i.p. with soluble or heat-denatured protein antigens [ovalbumin, beta-galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat-denatured (100 degrees C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30-fold higher doses. If the heat-treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat-denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA-A2/H-2K(b) hybrid gene with heat-denatured, recombinant HPV 16-E7 protein induced D(b)-restricted CTL specific for the peptide 49-57 of E7, indicating that this epitope is immunodominant over any A2-restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100 degrees C or even autoclaved was still able to induce virus-specific CTL and BALB/c spleen cells heated to 100 degrees C could still cross-prime minor H-specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.

The T-lymphocyte co-receptors of MHC glycoproteins CD4 and CD8 are known to be associated with the protein tyrosine kinase Lck via cysteine-containing sequences in the cytoplasmic domains of CD4 and CD8 and in the N-terminal domain of Lck. Here we demonstrate that a fraction of CD4 and CD8 molecules are associated with very large, detergent-resistant complexes containing several glycosylphosphatidylinositol-anchored proteins, (glyco)lipids, and protein tyrosine kinases Lck and Fyn but apparently no other major transmembrane proteins. Association of Lck and Fyn with these large complexes is, in contrast to simple CD4/CD8-Lck complexes, not sensitive to alkylation with iodoacetamide. These large complexes therefore represent an alternative way of association of CD4 and CD8 with the protein tyrosine kinases, which may play a role in signaling through these receptors.

The glycosyl phosphatidylinositol (GPI)-linked antigen recognized by monoclonal antibody (mAb) MEM-102 is expressed on all peripheral blood lymphocytes, both resting and activated. Its properties are very similar to a previously described activation antigen, Blast-1. The amino acid sequence deduced from the structure of cloned cDNA is identical to that of the Blast-1 antigen except for a single amino acid residue. There are several other minor differences in the nucleotide sequence of the Blast-1 and MEM-102 cDNAs that do not affect the predicted structure of the polypeptide product. The amino acid sequence of the first 15 N-terminal residues of the antigen purified from Raji cells is found in the deduced sequence close to the presumed boundary between the leader peptide and mature polypeptide. Properties of the recombinant product expressed in COS cells are similar to the antigen isolated from peripheral blood mononuclear cells (PBMNCs) or B-and T-cells lines. The antigen purified on immobilized mAb MEM-102 is recognized by all six known CD48 mAbs under western blotting conditions. COS cells transfected with MEM-102 cDNA react with all the CD48 mAbs. It is concluded that mAb MEM-102 is directed against the as yet poorly characterized antigen CD48, which is therefore structurally closely related to Blast-1. Several possibilities are discussed that might account for the apparent discrepancy between the broad pan-leucocyte expression of the between the broad pan-leucocyte expression of the MEM-102/CD48 antigen and much more restricted expression of the epitope recognized by the previously described mAb defining the Blast-1 antigen.

Human glutamate carboxypeptidase II (GCPII) is a co-catalytic metallopeptidase and its putative catalytic domain is homologous to the aminopeptidases from Vibrio proteolyticus and Streptomyces griseus. In humans, the enzyme is expressed predominantly in the nervous system and the prostate. The prostate form, termed prostate-specific membrane antigen, is overexpressed in prostate cancer and is used as a diagnostic marker of the disease. Inhibition of the form of GCPII expressed in the central nervous system has been shown to protect against ischemic injury in experimental animal models. Human GCPII consists of 750 amino acids, and six individual domains were predicted to constitute the protein structure. Here, we report the analysis of the contribution of these putative domains to the structure/function of recombinant human GCPII. We cloned 13 mutants of human GCPII that are truncated or extended at one or both the N- and C-termini of the GCPII sequence. The clones were used to generate stably transfected Drosophila Schneider's cells, and the expression and carboxypeptidase activities of the individual protein products were determined. The extreme C-terminal region of human GCPII was found to be critical for the hydrolytic activity of the enzyme. The deletion of as few as 15 amino acids from the C-terminus was shown to completely abolish the enzymatic activity of GCPII. Furthermore, the GCPII carboxypeptidase activity was abrogated upon removal of more than 60 amino acid residues from the N-terminus of the protein. Overall, these results clearly show that amino acid segments at the N- and C-termini of the ectodomain of GCPII are essential for its carboxypeptidase activity and/or proper folding.

Monoclonal antibodies to specific families of TCR variable domains serve as highly useful immunochemical tools for basic research in T-cell biology and diagnosis of autoimmune diseases. Monoclonal antibody MEM-262 characterized in this communication recognizes beta chains of the TCR expressed by HPB-ALL cell line (carrying Vbeta5.3) and a small subset of peripheral blood T cells. This subset is larger than that recognized by a previously described Vbeta5.3-specific mAb. MEM-262 potently stimulates selective expansion of the T-cell subset, efficiently immunoisolates native TCR complexes as well as free beta chains and uniquely recognizes denatured TCRbeta chains under the conditions of Western blotting.

Nonclassical major histocompatibility complex (MHC) class I human leukocyte antigen E (HLA-E) and HLA-G molecules differ from classical ones by specific patterns of transcription, protein expression, and immunotolerant functions. The HLA-G molecule can be expressed as four membrane-bound (HLA-G1 to -G4) and three soluble (HLA-G5 to -G7) proteins upon alternative splicing of its primary transcript. In this study, we describe a new set of monoclonal antibodies (mAbs) called MEM-G/01, -G/04, -G/09, -G/13, MEM-E/02, and -E/06 recognizing HLA-G or HLA-E. The pattern of reactivity of these mAbs were analyzed on transfected cells by flow cytometry, Western blotting, and immunochemistry. MEM-G/09 and -G/13 mAbs react exclusively with native HLA-G1 molecules, as the 87G mAb. MEM-G/01 recognizes (similar to the 4H84 mAb) the denatured HLA-G heavy chain of all isoforms, whereas MEM-G/04 recognizes selectively denatured HLA-G1, -G2, and -G5 isoforms. MEM-E/02 and -E/06 mAbs bind the denatured and cell surface HLA-E molecules, respectively. These mAbs were then used to analyze the expression of HLA-G and HLA-E on freshly isolated cytotrophoblast cells, on the JEG-3 placental tumor cell line, and on cryopreserved and paraffin-embedded serial sections of trophoblast tissue. These new mAbs represent valuable tools to study the expression of HLA-G and HLA-E molecules in cells and tissues under normal and pathologic conditions.

An unusual CD18 monoclonal antibody (mAb) MEM-148 binds, in contrast to standard CD18 mAbs, specifically to peripheral blood monocytes and neutrophils activated by various stimuli such as phorbol myristate acetate, opsonized zymosan, heat-aggregated immunoglobulin, and (after priming with lipopolysaccharide, tumor necrosis factor, or granulocyte-macrophage colony-stimulating factor) also by formyl-methionyl-leucyl-phenylalanine. In addition, in vivo activated neutrophils obtained from urine of patients following recent prostatectomy were also strongly positive for MEM-148. On the activated myeloid cells the mAb recognized a 65- to 70-kd protein identified immunochemically and by mass spectrometric peptide sequencing as a membrane-anchored fragment of CD18 (the common chain of leukocyte integrins) produced by proteolytic cleavage. The CD18 fragment originated mainly from integrin molecules stored intracellularly in resting cells, it was unassociated with CD11 chains, and its formation was inhibited by several types of protease inhibitors. Thus, the 65- to 70-kd CD18 fragment represents a novel abundant activation marker of myeloid cells of so far unknown function but possibly involved in conformational changes in leukocyte integrin molecules resulting in increased affinity to their ligands.

SIT (SHP2-interacting transmembrane adaptor protein) is a recently identified transmembrane adaptor protein, which is expressed in lymphocytes. Its structural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR-mediated recruitment of SH2 domain-containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly interacts with the SH2-containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibition motif (ITIM). Moreover, SIT is capable to inhibit TCR-mediated signals proximal of activation of protein kinase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize the molecular interaction between SIT and intracellular effector molecules and to identify the protein(s) mediating its inhibitory function. We demonstrate that SIT not only interacts with SHP2 but also with the adaptor protein Grb2 via two consensus YxN motifs. However, mutation of both Grb2-binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine-based signaling motif Y(168) ASV completely abrogates the ability of SIT to inhibit T cell activation. Co-precipitation experiments revealed that the tyrosine kinase p50(csk) could represent the negative regulatory effector molecule that binds to this motif.

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 α/β 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 α/β 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.

A CDw78 mAb FN1 was shown to recognize DP and/or DR molecules under the conditions of Western blotting. DP molecules were specifically retarded on a column of the FN1 immunosorbent; binding of FITC-labeled FN1 to B cell lines was completely blocked by excess of mAb to DR/DP beta chains, partially by several mAb to DP and weakly by some mAb to DR. The binding of two other CDw78 mAb, FN4 and MR11, to the B cell surface was most strongly inhibited by excess of different mAb to DR. Kinetics of stable binding of the CDw78 mAb indicated that their monovalent binding is of low affinity and that the stable binding to the surface is due to bivalent binding to two spatially close MHC class II molecules. FN1-based immunosorbent effectively immunoisolated complexes of MHC class II proteins with several tetraspanin molecules from a mild detergent lysate of a B cell line. It is concluded that FN1 and most likely also the other two CDw78 mAb recognize with low affinity determinants on MHC class II molecules (DP or DR) and preferentially bind in a stable fashion to dimerized or aggregated MHC class II molecules. Such dimers or aggregates may either exist as preformed on the cell surface or may be gradually formed and stabilized by bivalent interaction with mAb. These structures may be related to the previously described 'superdimers' of MHC class II and/or 'MHC-tetraspanin complexes'. CDw78 mAb may be valuable tools targeting such aggregated fraction of MHC class II molecules which can exhibit important signaling and antigen-presenting properties.

Cathepsin D, a lysosomal aspartic proteinase, is secreted in the form of enzymatically inactive proenzyme by many types of human breast cancer tissue and exerts mitogenic activity toward these tissues. Flow cytometry was used to test the binding of procathepsin D purified from the secretion of the breast cancer cell line ZR-75-1 to human breast cancer cells. No previously known surface antigens or soluble M6P-R or anti-M6P-R antibodies were found to inhibit the specific binding of procathepsin D-FITC. Similarly, none of these potential inhibitors was found to inhibit growth factor activity of procathepsin D. Our results indicate that procathepsin D growth factor activity is mediated by a new, previously unknown receptor moiety and that the binding activity can be localized in position 27-44 of the activation peptide of procathepsin D. Furthermore, in vivo experiments indicate that treatment with anti-procathepsin D antibodies can reverse the growth of human breast tumors in athymic nude mice.

Four of the tetraspans family antigens expressed in B cells, CD37, CD53, TAPA-1, and R2/C33, as well as at least two other molecules, CD19 and CD21, coprecipitate with DR antigens from mild detergent lysates of human B-cell lines and tonsillar B cells. Coprecipitation and preclearing experiments indicate the existence of large multicomponent complexes containing jointly the seven components, although some "incomplete" complexes lacking some of the components may also exist. The complexes contain only a relatively small fraction of the total cellular pool of relevant molecules. The existence of these "tetraspans-DR complexes" may be related to the previously reported antiproliferative and signaling effects of antibodies against most of their components.

Monoclonal antibodies (MAbs) to plum pox virus (PPV) were obtained after immunization of BALB/c mice with purified PPV-W isolate. Spleen cells from a mouse showing a high serum titer were used for fusion with Sp2/0-Ag14 myeloma cells. Culture supernatants were screened for specific antibody production against PPV-W isolate using indirect ELISA. A total of six stable hybridoma lines producing MAbs of IgG class were obtained. All four PPV isolates tested (W, A, D and M) can be distinguished by these MAbs. Two highly efficient antibodies were chosen for practical purposes, and their applicability in PPV diagnostics has been studied since 1989 in parallel with polyclonal antibodies and commercial kits (Boehringer, Bioreba). The experiments have proven comparable sensitivity with a detection limit ranging between 10 and 50 ng of virus per ml sample. In routine diagnostics of plums, peaches and apricots our MAbs ranked at least as high as the commercial kits.

BALB/c, (BALB/c x B10.A)F1 and (BALB/c x B10)F1 hybrid mice were immunized with C-peptide of human proinsulin. The (BALB/c x B10.A)F1 hybrids were the best responders and yielded 3 hybridomas secreting specific monoclonal antibodies. One of them, C-PEP-01, bound the C-peptide with high affinity (Kas = 1.1 x 10(9) l/mol), cross-reacted fully with human proinsulin but not with insulin, glucagon or somatostatin and apparently recognized the regions of C-peptide comprising amino acid residues 8-13 and 25-31. A RIA system could be set up employing this monoclonal antibody suitable for estimation of C-peptide concentrations in a diagnostically useful range (1-50 ng/ml).

The effect of an H-21J disparity on skin graft survival was studied in 18 mouse donor-recipient strain combinations, in which the recipients were treated with an efficient immunosuppressant, lentil lectin (LCA). The simultaneous I-J disparity essentially had no (or a slightly adverse) effect on the graft survival times in strain combinations differing at the K and I-A loci or in the entire H-2 complex. In two strain combinations incompatible at the D locus, the simultaneous I-J disparity promoted graft survival. The disparity at the I-J locus therefore seems to have only a marginal effect on the survival of allografts in most of the LCA-treated recipients, but it may promote graft survival in some animals. A similar tolerance-promoting effect was also observed with D disparity.

The previously demonstrated role of H-2 gene products in nonimmune cell interactions was further analyzed. As model of such interactions served the erythropoietic performance (in terms of 59Fe uptake) of bone marrow cell (BMC) inocula in the cellular environment of the spleen of massively irradiated hosts. When the performance in syngeneic hosts is taken as standard, the performance of cells from the same pool in H-2 disparate (congenic) hosts is subnormal. The inhibition was also observed in two congenic strain combinations where the H-2 disparity did not involve the I region. The capacity of BMC to distinguish H-2 -identical and H-2 -disparate hosts could be greatly reduced or virtually abolished by enzymatic digestion of certain cell surface carbohydrates. An essential effect of neuraminidase was further increased by emulsin. When the digested cells were left for 6 h in an enzyme-free medium, their capacity to dinstinguish H-2 disparities recovered. A pretreatment with papain, which cleaves a large fragment of the H-2 protein carrying both the antigenic site and the carbohydrate chain, had an effect comparable with but no greater than the sugar-removing enzymes. The enzymatic effect was largely due to the impairment of the performance of BMC in H-2 -identical hosts rather than to an improvement in H-2 -disparate ones. The neuraminidase- and emulsin-sensitive groupings thus seem to be essential for the cells to recognize “self” rather than “non-self” and to interact with H-2 -identical cells in physiological processes. A hypothesis is proposed that in H-2 glycoproteins, the antigenically and physiologically active sites are distinct, being located in the protein and carbohydrate parts of the molecules, respectively. It is further suggested that the normal function of the H-2 products might consist in providing the cell surface with structures which are complementary to similar structures on other cells; the carbohydrate moiety seems to be essential for this type of physiological interaction in contrast to the key and lock-type of complementarity in immune interactions which is based on protein structures.

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.

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.

The carboxy-terminal amino acid sequence of the soluble form of the 53,000 mol. wt monocyte surface antigen, CD 14, 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. Science239, 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 CD 14 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-CD 14 antibody, MEM-18, and polyclonal rabbit anti-CD 14 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 CD 14 antigen in human sera.

Two monoclonal antibodies, MEM-15 and MEM-18, were prepared which recognize a monocyte 53-kDa antigen. A soluble form of this antigen was found in most normal sera and in urine of some nephrotic patients. Milligram amounts of this glycoprotein were isolated by immunoaffinity chromatography from urine and used for quantitative amino acid and carbohydrate analysis and N-terminal amino acid sequencing. The reactivity of the isolated antigen with several previously described monoclonal antibodies indicates that it is the antigen previously called MY-4 or MY-23. A significant homology exists between the sequenced N-terminal portion of the antigen and sequences found in the kinase-related transforming protein src and in the precursor of epidermal growth factor.

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.

A convenient simple polyacrylamide gel electrophoresis system is described, suitable for semiquantitative analysis of the content of monoclonal antibodies in ascitic fluids of hybridoma-bearing mice.

The interaction of lectins with carbohydrate receptors on the plasma membrane of eukaryotic cells results in a wide variety of biological effects1. One effect which has been extensively studied is the stimulation of lymphocytes to blastogenesis and proliferation. High doses of concanavalin A (Con A) and phytohaemagglutinin (PHA) have been shown to activate in vitro regulatory cells capable of suppressing proliferation of other cells in the culture2,3 . However, Con A and PHA treatment were used effectively to prolong the survival of skin and heart allografts4–8 before it was recognised that some lectins have an activatory effect on suppressor cells in vitro. A possible explanation of these tolerogenic effects is the activation of specific suppressor cells. In this letter we have compared systematically various lectins differing in their carbohydrate specificity and mitogenicity in relation to their ability to induce prolonged skin allograft survival in mice with the aim of selecting the most effective lectin treatment schedule. Some preliminary results of this study have been mentioned in a recent review9.

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: (a) ‘depletion’ of helper T cells and (b) a relative increase in the regulatory (suppressor) cell ratio, which may support allograft survival.

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.

EXAMINATION of the chemical nature of histocompatibility antigens is not easy, because of the rapid inactivation of cell-free extracts and the difficulty in obtaining these antigens in a soluble form.