Serum and urinary levels of CD222 in cancer: origin and diagnostic value.

The mannose 6-phosphate/insulin-like growth factor 2 receptor (CD222, M6P/IGF2R) is a multifunctional transmembrane type I receptor, mostly localized intracellularly, less on the surface of all types of mammalian cells. It is known both to transport lysosomal enzymes through their mannose 6-phosphate moieties and to internalize extracellular ligands like insulin-like growth factor 2 or plasminogen. CD222 is involved in regulation of cell proliferation, migration, T cell activation, and apoptosis. Soluble CD222 has been found in higher concentrations in sera of liver disease patients. In this study, we analysed the level of CD222 present in body fluids, namely in serum and urine, of cancer patients. We found significantly elevated levels of soluble CD222 in sera of cancer patients compared to healthy controls irrespective of the type of disease. The urine CD222 levels were increased specifically in breast cancer and multiple myeloma. In contrast to serum, CD222 was present within CD222-positive exosomes in urine pointing to different origins of CD222 present in various human body fluids. Based on this work, we propose serum soluble CD222 as a general biomarker for tumorigenesis.

Arbuscular mycorrhizal fungal community differences among European long-term observatories.

Arbuscular mycorrhizal fungal (AMF) communities have been demonstrated to respond to a variety of biotic and abiotic factors, including various aspects of land management. Numerous studies have specifically addressed the impact of land use on AMF communities, but usually have been confined to one or a few sites. In this study, soil AMF assemblages were described in four different long-term observatories (LTOs) across Europe, each of which included a site-specific high-intensity and a low-intensity land use. AMF communities were characterized on the basis of 454 sequencing of the internal transcribed spacer 2 (ITS2) rDNA region. The primary goals of this study were (i) to determine the main factors that shape AMF communities in differentially managed sites in Europe and (ii) to identify individual AMF taxa or combinations of taxa suitable for use as biomarkers of land use intensification. AMF communities were distinct among LTOs, and we detected significant effects of management type and soil properties within the sites, but not across all sites. Similarly, indicator species were identified for specific LTOs and land use types but not universally for high- or low-intensity land uses. Different subsets of soil properties, including several chemical and physical variables, were found to be able to explain an important fraction of AMF community variation alone or together with other examined factors in most sites. The important factors were different from those for other microorganisms studied in the same sites, highlighting particularities of AMF biology.

Arbuscular mycorrhizal fungal communities and Rhizophagus irregularis populations shift in response to short-term ploughing and fertilisation in a buffer strip.

Short-term effects of soil physical disturbance by ploughing and nitrogen and phosphate fertilisation on arbuscular mycorrhizal fungal (AMF) communities and on intraspecific populations of Rhizophagus irregularis in a buffer strip surrounded by arable fields were studied. Pre-grown Plantago lanceolata plantlets were transplanted into fertilised and/or ploughed experimental plots. After 3 months, the glomeromycotan communities in the roots of these trap plants were analysed using 454 pyrosequencing of a fragment of the RNA polymerase II gene (RPB1). Intraspecific populations of R. irregularis were studied by restriction fragment length polymorphism (RFLP) analysis of the mitochondrial large ribosomal subunit (mtLSU) gene. Soil disturbance significantly increased the diversity of species-level molecular taxa (MTs) and altered community structure, whilst fertilisation alone had no significant effect, unless coupled with ploughing. At the population level, the expected shift from genotypes of R. irregularis typically found in grasslands to those usually found in arable sites was only partially observed. In conclusion, in the short-term, physical soil disturbance, as well as nitrogen fertilisation when coupled with physical soil disturbance, affected AMF community and to a smaller extent population composition.

H-2 restriction as a consequence of intentional priming. Frequency analysis of alloantigen-restricted, trinitrophenyl-specific cytotoxic T lymphocyte precursors within thymocytes of normal mice.

An in vitro acute-depletion protocol was used to detect trinitrophenyl (TNP)-specific, allo-major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) within thymocytes of inbred mice. After removal of alloreactivity, the negatively selected cells could be sensitized to become TNP-specific, allo-MHC-restricted cytotoxic T cells. A precursors frequency analysis revealed a three- to ninefold lower frequency of allo-MHC-restricted CTL precursors (CTL-P) as compared to self-MHC-restricted CTL-P. The specificity analysis of clonally distributed allo-MHC-restricted CTL-P excluded cross-reactivity as an explanation of allo-MHC restriction. These results provide direct evidence that thymic T cells are composed of a mixture of self-MHC- and allo-MHC-restricted immunocompetent T cells and that antigen-driven selection of precommitted T cells dictates the H-2-restriction phenotype, i.e., H-2 restriction is a consequence of priming.

T cell stimulation via the erythrocyte receptor. Synergism between monoclonal antibodies and phorbol myristate acetate without changes of free cytoplasmic Ca++ levels.

We observed that certain E-receptor antibodies (CD2 antibodies) can induce proliferation of resting human T cells in the presence of PMA, while other CD2 antibodies fail to have such an effect. The same CD2 antibodies that were mitogenic in the presence of PMA (9.6, X11, VIT13), but not the nonreactive ones, were also able to induce T cell proliferation via the so-called alternative pathway of T cell activation, i.e., when added pairwise in certain combinations to T cells in the absence of PMA. While the simultaneous addition of two comitogenic CD2 antibodies (9.6 or X11 plus VIT13) or the addition of a single nonmitogenic CD3 antibody (VIT3) led to a clearcut elevation of intracellular Ca++ levels, no such effect could be observed after the addition of one CD2 antibody alone. Even in the presence of PMA, one comitogenic CD2 antibody alone was unable to trigger a significant Ca++ response, although this combination induced a proliferative response. These data indicate that, distinguishable by their influence on free cytoplasmic Ca++, there are two different mechanisms of T cell activation via CD2. While simultaneous triggering with two antibodies leads to cell proliferation preceded by an increase of Ca++ levels, stimulation with one antibody plus PMA results in proliferation without a measurable early Ca++ response. We conclude that T cells treated by certain CD2 antibodies alone already recognize an activation signal probably unrelated to Ca++ homeostasis, a signal that can further be developed by PMA to result in a completely developed proliferative response.

Frequency analysis of cytotoxic T lymphocyte precursors in chimeric mice. Evidence for intrathymic maturation of clonally distinct self-major histocompatibility complex- and allo-major histocompatiblilty complex-restricted virus-specific T cells.

To study whether the thymic major histocompatibility complex (MHC) imposes a constraint on the receptor repertoire of maturating cytotoxic T lymphocyte (CTL) precursors, the restriction phenotypes of virus-specific CTL of MHC-compatible and of MHC-incompatible thymus- and bone marrow-grafted (A X B)F1 chimeric mice were compared. Dependent on the mode of in vitro sensitization, thymocytes or splenocytes of both types of chimeric mice generated Sendai virus-specific, self-MHC-or allo-MHC-restricted CTL. By applying the limiting-dilution technique, the CTL-precursor (CTL-P) frequencies of self-MHC-restricted and allo-MHC-restricted virus-specific T cells as well as of alloreactive T cells were determined. The data obtained revealed that independent of MHC differences between thymus and bone marrow, the frequencies of self-MHC-restricted and allo-MHC-restricted CTL-P were comparable, and in the same older of magnitude as those previously determined in conventionally reared mice. Self-MHC-restricted, virus-specific CTL-P were in a three- to fivefold excess over allo-MHC-restricted CTL-P. A segregation analysis revealed that clonally distinct CTL-P give rise to either self-restricted or allo-MHC-restricted, virus-specific CTL. Both sets were found not only in the spleen, but also in the thymus of chimeric mice, formally demonstrating the intrathymic differentiation pathway of self-MHC as well of allo-MHC-restricted CTL-P. These data reveal no major constraint of the thymic MHC on the capacity of T cells to recognize viral antigens either in the context of self-MHC or of allogeneic MHC products.

Interaction of CD31 with a heterophilic counterreceptor involved in downregulation of human T cell responses.

CD31 is a 130-kD glycoprotein of the immunoglobulin (Ig) superfamily expressed on the surface of endothelial cells, platelets, and several leukocyte subsets. Previous reports indicated that CD31 can mediate intercellular adhesion via both homophilic and heterophilic interaction mechanisms. Using a soluble recombinant CD31-Ig fusion protein (CD31 receptor globulin [Rg]), we demonstrate here that human CD31- T lymphocytes and CD4+CD31- T cell clones express a heterophilic CD31 ligand that is upregulated 18 h after activation. Interaction of CD31Rg with CD31- T helper cell (Th) clones was divalent cation independent but could be blocked by heparin, thus indicating that the CD31 counterreceptor on T cells can be distinguished from the ligands identified on other cell types. Moreover, a single chain protein of 120 kD was precipitated by CD31Rg from the lysates of CD31- Th clones. CD31Rg completely downregulated the proliferative response and cytokine production (interleukin-4, interferon-gamma, and tumor necrosis factor-alpha) of CD31- Th clones when the cells were maximally stimulated via immobilized CD3 monoclonal antibody. These results suggest that interaction of CD31 with a heterophilic counterreceptor on T lymphocytes can interfere with a positive regulatory pathway of T cell activation, or directly signal T cells to downregulate immune function.

Urokinase plasminogen activator receptor, beta 2-integrins, and Src-kinases within a single receptor complex of human monocytes.

The glycosylphosphatidylinositol (GPI)-anchored membrane protein urokinase plasminogen activator-receptor (uPA-R; CD87) is one of the key molecules involved in migration of leukocytes and tumor cells. uPA bound to uPA-R provides the cell proteolytic potential used for degradation of extracellular matrix. uPA-R is also involved in induction of cell adhesion and chemotaxis. Here, we provide a molecular explanation for these uPA-R-related cellular events. By size fractionation of monocyte lysate and affinity isolation on its natural ligand uPA, we demonstrate uPA-R as a component of a receptor complex of relatively large size. Reprecipitation and immunoblotting techniques allowed us to detect the protein tyrosine kinases (PTKs) p60fyn, p53/56lyn, p58/64hck, and p59fgr as components of this "uPA-R complex". Activation of monocytes even with enzymatically inactivated uPA resulted in induction of tyrosine phosphorylation, suggesting modulation of uPA-R-associated PTKs upon ligand binding. In spite of their presence in large complexes, we did not find the GPI-linked proteins CD14, CD58, and CD59 in the uPA-R complex, which indicates the presence of different receptor domains containing GPI-linked proteins in monocytes. However, we identified the leukocyte integrins LFA-1 and CR3 as components of the uPA-R complex as indicated by coisolation of these molecules, as well as by cocapping and comodulation of uPA-R and leukocyte integrins on the monocyte surface. The assemblage of uPA-R, PTKs and membrane spanning beta 2-integrins in one receptor complex indicates functional cooperation. In regard to the involvement of these molecules in pericellular proteolysis, signal transduction, as well as adhesion and chemotactic movement, we suggest uPA-R complex as a potential cellular device for cell migration.

Activated human T lymphocytes express MHC class I heavy chains not associated with beta 2-microglobulin.

We present here the molecular characterization of a new activation-induced surface structure on human T lymphocytes, termed LA45, with high homology (93% at protein level) to MHC class I molecules. Antigen modulation and sequential immunoprecipitation experiments revealed that LA45 and HLA class I proteins do not crossreact with the corresponding antibodies. Furthermore, LA45 is not associated with beta 2-m. On the other hand, we could show that the separation of HLA-A,B,C and beta 2m molecules, induced by SDS-denaturation, leads to a conformational change in the heavy chain in such a way that it becomes reactive with LA45. The 90/45 kD LA45 proteins thus appear to be non-beta 2m-associated MHC class I alpha chains that are selectively expressed by activated but not by resting human T lymphocytes.

Polyunsaturated fatty acids inhibit T cell signal transduction by modification of detergent-insoluble membrane domains.

Polyunsaturated fatty acids (PUFAs) exert immunosuppressive effects, but the molecular alterations leading to T cell inhibition are not yet elucidated. Signal transduction seems to involve detergent-resistant membrane domains (DRMs) acting as functional rafts within the plasma membrane bilayer with Src family protein tyrosine kinases being attached to their cytoplasmic leaflet. Since DRMs include predominantly saturated fatty acyl moieties, we investigated whether PUFAs could affect T cell signaling by remodeling of DRMs. Jurkat T cells cultured in PUFA-supplemented medium showed a markedly diminished calcium response when stimulated via the transmembrane CD3 complex or glycosyl phosphatidylinositol (GPI)- anchored CD59. Immunofluorescence studies indicated that CD59 but not Src family protein tyrosine kinase Lck remained in a punctate pattern after PUFA enrichment. Analysis of DRMs revealed a marked displacement of Src family kinases (Lck, Fyn) from DRMs derived from PUFA-enriched T cells compared with controls, and the presence of Lck in DRMs strictly correlated with calcium signaling. In contrast, GPI-anchored proteins (CD59, CD48) and ganglioside GM1, both residing in the outer membrane leaflet, remained in the DRM fraction. In conclusion, PUFA enrichment selectively modifies the cytoplasmic layer of DRMs and this alteration could underlie the inhibition of T cell signal transduction by PUFAs.

GPI-anchored cell-surface molecules complexed to protein tyrosine kinases.

Binding of ligand or antibody to certain cell-surface proteins that are anchored to the membrane by glycophosphatidylinositol (GPI) can cause activation of leukocytes. However, it is not known how these molecules, which lack intracellular domains, can transduce signals. The GPI-linked human molecules CD59, CD55, CD48, CD24, and CD14 as well as the mouse molecules Thy-1 and Ly-6 were found to associate with protein tyrosine kinases, key regulators of cell activation and signal transduction. A protein tyrosine kinase associated with the GPI-linked proteins CD59, CD55, and CD48 in human T cells, and with Thy-1 in mouse T cells was identified as p56lck, a protein tyrosine kinase related to Src. This interaction of GPI-linked molecules with protein tyrosine kinases suggests a potential mechanism of signal transduction in cells.

Exposure by desialylation of myeloid antigens on acute lymphoblastic leukemia cells.

The 3-fucosyl-N-acetyllactosamine structure, a sugar sequence contained in the human milk oligosaccharide lacto-N-fucopentaose III, is recognized by most of the granulocyte-specific monoclonal antibodies (MoAb) reported in the literature, including the six MoAb from our laboratory. Blast cells from patients with acute myeloblastic leukemia (AML) displayed a heterogeneous reaction pattern when they were exposed to MoAb against this moiety, and the proportion of reactive cells in individual cell samples was highly variable. The intensity of the reaction was strongly enhanced by neuraminidase treatment of AML blasts, and reactive structures were exposed on previously negative AML blast cells. Surprisingly, this granulocyte-associated antigen was exposed by desialylation not only on malignant myeloid precursor cells but also on common acute lymphoblastic leukemia cells. No such effect was seen when normal peripheral blood lymphocytes, lymphocytes from patients with chronic lymphatic leukemia, or blast cells from patients with B-cell acute lymphoblastic leukemia, acute erythroid leukemia, and acute megakaryoblastic leukemia were treated with neuraminidase.

Terminal deoxynucleotidyl transferase and CD7 expression in acute myeloid leukemias are not associated with a high frequency of immunoglobulin and/or T cell receptor gene rearrangement.

Within normal hemopoiesis, the intranuclear DNA polymerase TdT seems to be exclusively expressed by T and B lymphoid precursor cells. Double staining experiments showed that TdT can also be expressed in blast cells of certain acute myeloid leukemias. Recent reports described a very strong association between TdT expression and rearrangements of IgH and TcR genes in such AML specimens, suggesting a predominant lymphoid commitment of these TdT positive AML blasts. When submitting 24 serologically and morphologically well-characterized TdT positive AML specimens for additional genotypic analysis to determine the IgH and TcR gene configuration, we observed that only four had clonally rearranged IgH and/or TcR genes, whereas 20 had germ line configuration. This frequency is clearly lower than previously reported and not necessarily different from rearrangement frequencies reported for TdT negative AML (4-40%). It would seem to us, therefore, that the expression of TdT in otherwise well-defined AML blasts is not necessarily associated with a higher frequency of immunoglobulin and/or T cell receptor gene rearrangement.

Monoclonal antibodies to the carbohydrate structure Lewis(x) stimulate the adhesive activity of leukocyte integrin CD11b/CD18 (CR3, Mac-1, alpha m beta 2) on human granulocytes.

Several carbohydrate structures on human granulocytes have been discussed as potential ligands for C-type lectins (selectins) on endothelial cells. Among them are the lacto-series type II chain antigens sialyl-Lewis(x) (SLe(x), Lewis(x) (Le(x)), and VIM2. We demonstrated in this study that monoclonal antibodies (mAbs) to Le(x) and to SLe(x), but not other anticarbohydrate mAbs (VIM2, CDw17, CD24), can stimulate granulocytes to form homotypic aggregates. This effect was particularly noticeable with three distinct anti-Le(x) mAbs (3C6, 4D1, 6C7). Much less impressive effects were also seen with nine other anti-Le(x) mAbs and with the anti-SLe(x) mAb CSLEX1. Aggregation was shown to be an active process. It is temperature and energy dependent, requires divalent cations, and is selective in terms of mAb specificity. Anti-Le(x)-induced homoaggregate formation could be inhibited with CD11b mAb JML-H11 and CD54 (ICAM1) mAb LB-2 and thus seems to be associated with activation of the beta 2-integrin cytoadhesion pathway.

Studies on differentiation of committed hemopoietic progenitor cells with monoclonal antibodies directed against myeloid differentiation antigens.

In the present study we evaluated the reactivity of monoclonal cytotoxic antibodies directed against myeloid differentiation antigens with hemopoietic precursor cells. VIM-D5 and VIM-2 inhibit the proliferation of clusters and colony formation after seven days of incubation. Day-14 CFU-GM are not affected by these antibodies. After complement-mediated cytolysis with VIM-2, the number of BFU-e was significantly reduced; however, this effect was largely abrogated by addition of leukocyte-conditioned medium to the cultures as an exogenous source of burst-promoting activity. Furthermore, the maturation of myeloid progenitor cells has been examined by delayed treatment with VIM-D5 and complement during the in vitro culture period. In these experiments a different maturation behavior of day-7 and day-14 CFU-GM was demonstrated. To study whether a cryptic carbohydrate structure is present on more immature CFU-GM, the effect of neuraminidase treatment of myeloid progenitor cells on reactivity with VIM-D5 was tested.

Acquisition of host cell-surface-derived molecules by HIV-1.

OBJECTIVE: To determine the acquisition of host cell-membrane-derived molecules by HIV-1 during the budding process, and to investigate whether the uptake of these molecules is cell-type-specific and selective. DESIGN: Virions, propagated by four different cell types were analysed for the presence of adhesion molecules, glycosylphosphatidylinositol (GPI)-anchored proteins and various cell-surface markers. The pattern was compared with the phenotype of the HIV-1-infected cell. METHODS: For phenotypic analysis of virions a two-step assay was used. In the first step, virus was captured with monoclonal antibodies (in some cases polyclonal sera) against different cell-membrane proteins. In a second step, the presence of virus was measured by determining the concentration of the virus-specific p24 core antigen. The expression of surface molecules on uninfected and HIV-1IIIB-infected cells was analysed by FACS. RESULTS: Depending on the cell type used for virus propagation, different cell-membrane molecules were found on the virus surface reflecting the corresponding cell type. The uptake of these molecules was selective to a certain degree. No CD4 and CD87 molecules were detectable on HIV-1, although both molecules were present on uninfected and HIV-1-infected cells. CR3 and CDw108 could not be seen on uninfected cells, but wre detectable on infected cells and virions. CONCLUSIONS: During the budding process HIV-1 acquires a variety of cell-type-specific cell-surface molecules. Certain cell-membrane molecules become upregulated during HIV-1-infection and are then found on virions, whereas other molecules remain on the cell surface and do not become incorporated.

Platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin cooperatively regulate fibroblast growth factor-induced modulations of adherens junction functions.

Cellular adherens junctions are formed by cadherins linked to proteins of the catenin family. In endothelial cells, not only vascular endothelial cadherin but also platelet endothelial cell adhesion molecule-1 localizes into junctions and associates with beta-catenin. To explore a putative cooperation of platelet endothelial cell adhesion molecule-1 and vascular endothelial cadherin, we analyzed transfectants expressing either platelet endothelial cell adhesion (CD31 cells) or vascular endothelial cadherin (CD144 cells) or both molecules (CD31/CD144 cells), and, for comparison, human umbilical vein endothelial cells. Basic fibroblast growth factor completely dissociated vascular endothelial cadherin/beta-catenin complexes and robustly moved beta-catenin into the nucleus in CD144 cells, whereas in CD31/CD144 cells as well as in human umbilical vein endothelial cells, fibroblast growth factor only partially dissociated the junctional complex followed by a significantly reduced nuclear translocation of beta-catenin. In contrast, in CD31 cells, the subcellular distribution of beta-catenin remained unaffected by fibroblast growth factor. As a functional consequence, fibroblast growth factor induced a complete collapse of the F-actin network in CD144 cells, a limited rearrangement of F-actin fibers in CD31/CD144 cells and no F-actin rearrangement in CD31 cells. We also analyzed the effect of fibroblast growth factor-induced rearrangement of junctions on junction permeability for leukocytes: in line with our observation that vascular endothelial cadherin was required for cells to respond to fibroblast growth factor, only in CD31/CD144 cells, but not in CD31 cells, leukocyte transmigration was significantly enhanced by fibroblast growth factor. In conclusion platelet endothelial cell adhesion molecule-1 cooperates with vascular endothelial cadherin in a mutual fashion; platelet endothelial cell adhesion molecule-1 reduces and temporarily limits fibroblast growth factor-induced dissociation of vascular endothelial cadherin/beta-catenin complexes, but requires vascular endothelial cadherin to control leukocyte transmigration in dependence of fibroblast growth factor.

Secretory group II phospholipase A2 in human atherosclerotic plaques.

Atherosclerotic plaques exhibit a series of features that are similar to those of chronic inflammation. Based on the fact that during inflammation several cell types synthesize and secrete a group II phospholipase A2 (PLA2), an immunohistochemical study was undertaken to explore whether this enzyme can be identified in human atherosclerotic lesions. Tissue specimens obtained from 13 patients who had undergone arteriectomy and three specimens with advanced atherosclerotic plaques obtained at autopsy were analyzed and compared to arteries free of atherosclerosis. The results showed that in all areas with atherosclerotic lesions, a staining with monoclonal antibodies raised against group II PLA2 was evident. In normal arteries without thickened intima, this immunostaining was completely negative. With the use of specific monoclonal antibodies against macrophages (anti-KP-1) and smooth muscle cells (anti-alpha-actin), PLA2-positive cells were identified as foam cells mainly derived from macrophages. In addition to these cells, other regions of the thickened intima gave a partially positive reaction with anti-PLA2 antibodies, but could not be stained with either anti-KP-1 or anti-alpha-actin. Some of these regions were localized on edges of calcification and cell necrosis. Other PLA2-positive regions seem to be associated with extracellular matrix structures. In summary, the findings of this study may be regarded as further evidence to support the link between atherosclerosis and chronic inflammatory processes. In view of the fact that the in vitro modification of lipoproteins by PLA2-treatment induces lipid deposition in macrophages, the results of this study suggest that group II PLA2 may actively be involved in the formation of foam cells in vivo.

A rapid and simple immunoperoxidase staining procedure for blood and bone marrow samples.

A fast and simple indirect immunoperoxidase staining technique is described, which can also be used for the characterization of cells with high endogenous peroxidase activity such as many haemopoietic cells. It is based on the combination of a newly developed glucose-oxidase plus glucose procedure for the inhibition of endogenous peroxidase activity with a standard 2 or 3 layer immunoperoxidase staining protocol. Glucose-oxidase plus glucose mixture completely inhibits endogenous peroxidase activity without having a detectable deleterious effect on any of the cellular antigens so far studied. In many instances this permits the use of only 1 layer of horseradish peroxidase (HRP)-labelled antibodies. The glucose-oxidase plus glucose mixture can also be added to cells together with the HRP-labelled antibody solution without losing its inhibitory effect for endogenous peroxidase activity and without leading to a visually detectable loss of the activity of the HRP conjugate. Consequently, the separate incubation step previously necessary for the inhibition of endogenous peroxidase activity becomes superfluous.

Induction of human complement activation without cytolysis by mouse monoclonal antibodies to human leukocyte antigens.

Ten monoclonal antibodies to human leukocyte subsets that had previously been shown to lyse their respective target cells in the presence of rabbit serum as complement source were evaluated for their cytolytic capacity with human complement. Four of the ten were lytic with human complement. All were of IgM type. Antibodies were also evaluated for their capacity to induce C3 binding to target cells. With this method we could demonstrate that, indeed, 3 of the 6 noncytolytic antibodies had the capacity to initiate the human complement activation process and to induce C3 binding. Two of these 3 antibodies were of IgM class (VIT3 and VIM13), one of IgG3 (562). From the practical point of view the most interesting of these 3 antibodies is the nonmitogenic anti-CD3 pan-T cell antibody VIT3. Therefore, this antibody was analyzed in more detail. VIT3 antibody concentrations needed to induce detectable C3 binding to human T cells are very low (down to 1 ng VIT3/ml). Human serum as complement source can also be considerably (100X) diluted before C3 binding becomes undetectable. Activation of C3 is a prerequesite for VIT3-induced C3 binding, and bound C3 seems to lack the C3a fragment. Bound C3, in contrast to the quickly occuring antigenic modulation of the CD3 complex and the simultaneous disappearance of the antibody coat, remains expressed also after prolonged incubation at 37 degrees C. C3 fragments bound to T cells after activation with VIT3 are also recognized by cells bearing C3 receptors of types CR1 and CR2.