Surface-bound Antigen Research Articles

Maintenance of Vascular Endothelial Cell-Specific Properties after Immortalization with an Amphotrophic Replication-Deficient Retrovirus Containing Human Papilloma Virus 16 E6/E7 DNA

Primary human vascular endothelial cells were immortalized by the integration of a single DNA copy of an amphotrophic, replication-deficient retrovirus containing the E6/E7 genes of human papilloma virus. To date, the resulting cell lines, designated EC-RF7 and EC-RF24, have been cultured for more than 1 year. The cell lines have retained a diploid karyotype, display no abnormalities, and are able to grow in a polar mode. Analysis of the EC-RF cell lines by indirect immunofluorescence, using an extensive panel of monoclonal antibodies, showed expression of endothelial cell-specific soluble (von Willebrand factor) and surface-bound antigens (endoglin, PCAM-1) indistinguishable from that of primary cells. In addition, the expression of the markers CD9, 13, 14, 29, 36, 40, 51, and 55 that are not restricted to endothelial cells was also similar for the immortalized and the primary endothelial cells. Immortalization did not alter the expression of the surface adhesion molecules E-selectin, VCAM-1, and ICAM-1 nor transmigration of neutrophils. The regulation of extracellular proteolytic activity by EC-RF24 was established by measuring both the induction of functional tissue factor (promotion of Factor Xa generation) and the functional deposition of plasminogen activator inhibitor 1 in the subendothelial matrix (SDS-resistant complex formation with thrombin). Finally, the biosynthesis of the endothelial cell-specific von Willebrand factor was studied in detail in the EC-RF24 cell line and the results were compared with those of primary endothelial cells.

Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity.

Anthrax lethal toxin, which is composed of two proteins, i.e., protective antigen and lethal factor, is cytolytic to mouse peritoneal macrophages and the macrophage-like cell line J774A.1. After exposure of cells to lethal toxin, inhibition of protein synthesis occurred only slightly before the onset of cytolysis. Thus, cell death did not appear to be due to inhibition of protein synthesis. However, prior treatment of J774A.1 cells with cycloheximide or puromycin, which inhibited protein synthesis, protected them completely against lethal toxin-induced cytolysis, which suggested that continuous protein synthesis is required for the expression of lethal toxin activity. Inhibition of protein synthesis had no appreciable effect on the binding of protective antigen to the cell surface receptor or on proteolytic cleavage of surface-bound protective antigen. Furthermore, inhibition of protein synthesis did not alter the uptake of toxin, which suggested that protein synthesis is required at a later stage of the intoxication process. The protection provided by inhibition of protein synthesis was effective, even up to 1 h after exposure to anthrax lethal toxin. The increased uptake of calcium observed in cells exposed to lethal toxin did not occur when they were protected by blocking protein synthesis. Identifying the protein(s) synthesized during the intoxication process may help to understand the mechanism of cell death produced by anthrax lethal toxin.

A cell surface antigen (BAL) defined by a mouse monoclonal antibody inducing apoptosis in a human lymphocytic leukemia cell line.

The lack of apoptosis or programmed cell death in human tumor cells has been suggested to be one factor allowing uncontrolled growth of neoplasms. We have developed a mouse monoclonal antibody (MAb) that induces programmed cell death in a human acute leukemia cell line (KM-3) of the pre B-cell type. Stable, antibody-producing hybridomas were produced by fusing mouse myeloma cells to spleen cells from mice immunized with viable KM-3 cells. Incubation of KM-3 cells with the MAb (designated anti-BAL) resulted in growth inhibition and subsequent cell death within 2-3 days. Anti-BAL required cross-linking with a rabbit anti-mouse antibody to induce DNA fragmentation typical of apoptosis. Immunoblotting experiments with anti-BAL identified a 37-kDa protein, apparently different from any previously described apoptosis-related surface antigen. Strongest expression of the antigen was generally found on cells of lymphoid or myeloid origin. However, several other cell types such as fibroblasts and endothelial cells were also stained by anti-BAL in flow cytometry but less intensively. Despite the apparent presence of this cell surface-bound 37-kDa antigen on several normal and malignant cell types, anti-BAL induced cell death only in human malignant cell lines expressing a more immature phenotype.

Induction of immune response against a short synthetic peptide antigen coupled to small neutral liposomes containing monophosphoryl lipid a

We have investigated the parameters affecting the immunogenicity of a short synthetic hexapeptide associated with liposomes. The model peptide used had the sequence IRGERA which corresponds to the C-terminal hexapeptide region of histone H3. Immunogenicity was measured by the ability of anti-peptide antibodies to cross-react with the parent protein. By itself, the peptide was not able to induce significant antibody production. However, liposomes were shown to be able to render the peptide immunogenic, nevertheless a number of parameters were important: to be immunogenic the peptide had to be surface bound, rather than entrapped within the liposomes, and an adjuvant, monophosphoryl lipid A (MPLA), had to be present in the same population of liposomes. Additionally, the intensity and duration of the immune response were found to be dependent both on the charge of the liposomes; neutral liposomes yielding a longer lasting response than negatively charged liposomes, and on the immunisation schedule where a long time period between immunisation and boosting yielded a better result than a short time period. To account for these phenomena we propose a model in which surface-bound antigen targets liposomal MPLA to B lymphocytes specific for the antigen. These results demonstrate that liposomes containing the non-toxic adjuvant MPLA can act as carriers to induce a long-lasting IgG response against peptides, eliminating the need of protein carriers and conventional adjuvants. Such an approach may be useful for designing synthetic vaccines.

Cooperativity in the antibody binding to surface-adsorbed antigen.

The binding to surface-adsorbed antigen of monoclonal mouse IgG-antibodies (mAbs), with two different affinities to dinitrophenyl (DNP), was measured by a calibrated ELISA. The concentration-dependence of antibody binding to surface-bound antigen of different epitope densities was analysed using Scatchard plots. The dissociation of bound tritium-labelled antibodies was measured in the presence of unlabelled antibodies in the bulk. At low surface concentration of bound anti-DNP, both high-affinity mAb and low-affinity mAb show a positive cooperativity in the binding reaction to antigen of high epitope density. Using antigen of lower epitope densities, the positive cooperativity is more pronounced for low-affinity clones. At higher surface concentrations of bound anti-DNP, the Scatchard plots indicate a negative cooperativity of binding, which is also implied by the increased dissociation found in the presence of antibodies in solution. The study confirms previous findings that the binding of antibodies to surface-bound antigen not only depends on intrinsic antibody affinity measured in solution. Other factors, such as self-interaction, also affect the heterogeneous binding reaction.

Miniantibodies: use of amphipathic helixes to produce functional, flexibly linked dimeric FV fragments with high avidity in Escherichia coli

We have designed dimeric antibody fragments that assemble in Escherichia coli. They are based on single-chain FV fragments, with a flexible hinge region from mouse IgG3 and an amphiphilic helix fused to the C-terminus of the antibody fragment. The sequence of the helix was taken either from that of a previously reported four-helix bundle design or from a leucine zipper, optionally extended with a short cysteine-containing peptide. The bivalent fragments associate in vivo, either with covalent linkage or with a monomer-dimer equilibrium, and results from ultracentrifugation sedimentation studies and SDS-PAGE are consistent with dimers. All constructs are able to bind to surface-bound antigen under conditions in which only bivalent but not monovalent antibody fragments bind. The covalent bundle helix construct shows binding characteristics nearly identical to those of the much larger whole mouse antibody, resulting in substantially more stable immunoglobulin-antigen complexes than in the case of monovalent fragments. This modular design of natural and engineered protein domains directly leads to a boost of avidity, and it allows the construction of bispecific antibody fragments in functional form in E. coli.

Langerhans cells in HIV-1 infection

The skin-specific immune surveillance system protects against invading microorganisms and transformed cells expressing tumor-specific neoantigens. This system includes antigen-presenting Langerhans cells, dermal and epidermal T lymphocytes, cytokine-producing keratinocytes, and draining peripheral lymph nodes. In patients infected with human immunodeficiency virus-1 (HIV-1), this surveillance system appears to be compromised, as evidenced by a reduction in the epidermal Langerhans cell population. Because human epidermal Langerhans cell express surface-bound CD4 antigens, HLA-DR antigens, and Fc-IgG receptors, all of which are involved in HIV-1 binding to, or entry into, the target cell, the reduction in Langerhans cells in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) may be a direct consequence of HIV-1 infection and subsequent injury to Langerhans cells. Detailed ultrastructural studies have confirmed moderate to severe morphologic damage in some Langerhans cells of such patients and the presence of HIV-1-like particles on Langerhans cell surface membranes and in the extracellular spaces. The biologic consequences of Langerhans cell infection by HIV-1 could be either impaired antigen presentation function of viable Langerhans cells or possible transmission of the retrovirus to the T-cell compartment in skin or lymph nodes, with subsequent depletion of CD4+ T cells via widespread syncytia formation between HIV-1-infected and noninfected cells. The facts that herpes simplex virus, specific cytokines, and ultraviolet B radiation can activate signals for HIV-1 expression and that epidermal cells can elaborate large amounts of cytokines, particularly with enhanced ultraviolet B exposure, may have important clinical implications for HIV-1-infected patients.

Dendritic Epidermal T Cells: Activation Requirements and Phenotypic Characterization of Proliferating Cells.

Dendritic epidermal T cells (DETC) are CD45+, Thy-1+, CD5-, CD8-, CD4- murine lymphocytes that express surface-bound CD3 antigens associated with T cell receptor gamma/delta heterodimers. Using epidermal cells greatly enriched for DETC and depleted of Langerhans cells, we found that DETC have growth requirements quite different from those of accessory cell-depleted lymph node and splenic T cells. Although the latter cells strongly proliferate in response to phorbol myristate acetate (PMA) + ionomycin, DETC, when exposed to interleukin-1 (IL-1), interleukin-3 (IL-3), concanavalin A (ConA), PMA, and ionomycin used either alone or in combination, do not exhibit significant mitotic activity. Recombinant interleukin 2 (rIL-2), albeit ineffective by itself, leads to vigorous proliferation of DETC when used with either ConA or PMA + ionomycin + IL-1. In contrast, the combination of PMA and recombinant interleukin-4 (rIL-4), which triggers growth of lymph node T cells, does not induce proliferation of DETC. Although a portion of proliferating DETC expressed CD8 antigens, essentially none bore detectable amounts of surface-bound CD4 or CD5 antigens, or both. Continuing stimulation of primary DETC cultures with lectin/lymphokine-rich media results in the propagation of cells with the essential phenotypic features of resident DETC.

Dendritic epidermal T cells: Activation requirements and phenotypic characterization of proliferating cells

Dendritic epidermal T cells (DETC) are CD45+, Thy-1+, CD5−, CD8−, CD4− murine lymphocytes that express surface-bound CD3 antigens associated with T cell receptor γ/δ heterodimers. Using epidermal cells greatly enriched for DETC and depleted of Langerhans cells, we found that DETC have growth requirements quite different from those of accessory cell-depleted lymph node and splenic T cells. Although the latter cells strongly proliferate in response to phorbol myristate acetate (PMA) + ionomycin, DETC, when exposed to interleukin-1 (IL-1), interleukin-3 (IL-3), concanavalin A (ConA), PMA, and ionomycin used either alone or in combination, do not exhibit significant mitotic activity. Recombinant interleukin 2 (rIL-2), albeit ineffective by itself, leads to vigorous proliferation of DETC when used with either ConA or PMA + ionomycin + IL-1. In contrast, the combination of PMA and recombinant interleukin-4 (rIL-4), which triggers growth of lymph node T cells, does not induce proliferation of DETC. Although a portion of proliferating DETC expressed CD8 antigens, essentially none bore detectable amounts of surface-bound CD4 or CD5 antigens, or both. Continuing stimulation of primary DETC cultures with lectin/lymphokine-rich media results in the propagation of cells with the essential phenotypic features of resident DETC.

Identification and percentage frequency of isolated non-parenchymal liver cells (NPLC) in the mouse

The aim of the present study was to identify non-parenchymal liver cells (NPLC) in B10.D2 mice and to determine their percentage frequency. The isolation of NPLC was carried out using the collagenase/pronase technique. Using functional techniques (latex phagocytosis, immunocytochemical detection of surface-bound and intracytoplasmic antigens) and morphological methods (light and electron microscopy), the following cell types were identified, and their percentage frequency in the NPLC determined: endothelial cells (50%), macrophages (23%), desmin-positive cells (14%), immunocompetent cells (10%, including T-, B-cells, pit and large vacuolated cells-both immunopositive to the asialo-GM 1 antigen) and unidentified cells (3%). These results show that, apart from the more familiar varieties of NPLC, two groups of cells exist in the liver which have not yet been fully identified and in which the immunocompetent cells predominate numerically.

Expression of HLA-DR and OKT6 antigens on keratinocytes and dendritic cells in pemphigus

The expression of HLA-DR and OKT6 antigens by epidermal cells in pemphigus was examined by immunohistochemical methods using skin biopsy specimens from human patients with pemphigus vulgaris. Changes in the distribution and subpopulations of Langerhans cells were observed in lesional (involved) skin. HLA-DR-positive, OKT6-negative dendritic cells were numerous and generally confined to the margins of epidermal clefts. OKT6-positive dendritic cells were rare or absent in lesional skin. The HLA-DR-positive, OKT6-negative dendritic cells associated with intraepidermal clefts are most likely functionally active antigen-presenting cells, which may play a role in lesion development. Aberrant expression of HLA-DR molecules by keratinocytes was observed in both involved and uninvolved skin, and may function to facilitate the recognition of surface-bound pemphigus antigen(s) by immunocompetent cells.

Differential Expression of Class II Alloantigens by Keratinocytes in Disease

The purpose of this study was to determine whether keratinocytes in certain disease states such as cutaneous T-cell lymphoma and lichen planus, express HLA-DR antigens (corresponding to the murine I-E antigens) only or whether they are also capable of expressing HLA-DQ antigens (analogues of the murine I-A antigens). Cryostat sections from 11 biopsies from cutaneous T-cell lymphoma and from 11 lichen planus biopsy specimens were submitted to indirect immunofluorescence and a 4-step immunoperoxidase method. This consists of applying monoclonal antibodies recognizing HLA-DR and HLA-DQ molecules and the intracytoplasmic invariant chain of the class II molecules. In 8 of the 11 cutaneous T-cell lymphoma specimens and in 3 of the 11 lichen planus biopsies concomitant expression of HLA-DR and HLA-DQ molecules by keratinocytes was detectable with the immunoperoxidase method. However, with the indirect immunofluorescence technique HLA-DQ antigens on keratinocytes could not be detected. The simultaneous expression of surface-bound HLA-DR antigens and intracytoplasmic gamma-chains was demonstrable in all cases investigated and with both the immunohistologic methods applied.

DNP-phycobiliproteins, fluorescent antigens to study dynamic properties of antigen-IgE-receptor complexes on RBL-2H3 rat mast cells.

In RBL-2H3 rat mucosal mast cells, the crosslinking of cell-surface IgE-receptor complexes by multivalent antigens initiates a sequence of responses leading to degranulation. We have developed a family of dinitrophenol (DNP)-conjugated fluorescent antigens to study dynamic membrane events associated with these responses. Lysyl groups on the phycobiliproteins, B-phycoerythrin and C-phycocyanin, were labelled with DNP, yielding fluorescent conjugates that cause the release of [3H]serotonin from anti-DNP-IgE-primed RBL-2H3 cells. The binding of these antigens to IgE-receptor complexes was observed by fluorescence microscopy and quantified by flow cytometry. Incubation with 1 microgram/ml DNP42-B-phycoerythrin stimulates maximum degranulation from IgE-saturated cells. Under these conditions, approximately 26 X 10(3) molecules of DNP42-B-phycoerythrin are bound per cell at equilibrium. The rate and extent of antigen binding and of antigen-stimulated mediator release decrease in parallel as the concentration and DNP:protein ratio of the fluorescent conjugates is reduced. Secretion stops immediately when the nonfluorescent monovalent antigen, DNP-lysine, is added to degranulating cell suspensions. DNP-lysine also displaces surface-bound antigen when added during the first minutes after multivalent antigen. However, the ability of DNP-lysine to displace surface-bound DNP42-B-phycoerythrin from IgE-receptor complexes decreases progressively with time. Treatment with dihydrocytochalasin B and several analogs that prevent antigen-stimulated F-actin assembly enhances secretion and delays the transition of antigen to its DNP-lysine-resistant form. Cytochalasin treatment also permits the long-range movement of antigen into surface caps.(ABSTRACT TRUNCATED AT 250 WORDS)

T cell regulation of myelopoiesis: analysis at a clonal level.

Colony-stimulating factor (CSF) production by a series of cloned human T lymphocyte cell lines was examined by substituting cloned T cells for peripheral blood mononuclear cells in the feeder layer of a double-layer agar CFU-C assay system. Of 12 T cell lines tested, all produced CSF when stimulated by specific antigen, whereas CSF production in the absence of stimulation was generally negligible. In the case of soluble antigen-specific (ragweed or tetanus toxoid) clones, this required both nominal antigen and the appropriate MHC gene product on autologous antigen-presenting cells, whereas in the case of clones specific for EBV-transformed B cell lines (allogeneic or autologous), surface-bound EBV-related antigen and MHC was necessary. When tested in this manner, CSF production by different cloned T cells was heterogeneous in both amount and subclass. Thus, although most clones stimulated growth of granulocyte, monocyte, and eosinophil colonies, certain clones were identified which preferentially stimulated some colony types but not others. This heterogeneity was particularly evident with respect to eosinophil colony production. In addition, a soluble inhibitor of granulocyte colony growth was produced by one clone. These findings provide further support for the notion that antigen-specific T cells may, on activation, regulate myelopoiesis in a precise way, and provide a possible cellular basis for selective eosinophilia, monocytosis, or neutrophilia seen in certain disease states.

Release of Mycoplasma pneumoniae substances after phagocytosis by guinea pig alveolar macrophages.

Antibody-opsonized Mycoplasma pneumoniae cells with various radioactive markers were sedimented onto monolayers of guinea pig alveolar macrophages (AM). After 2 h of incubation, about 50% of the activity of [3H]palmitate-labeled mycoplasmas was associated with AM. Nonspecific attachment of the opsonized mycoplasmas to AM-free plastic surface areas was negligible. The occurrence of phagocytosis was proven by electron microscopy and monitoring of AM surface-bound antigen by 125I-labeled F(ab)2 fragments. The activity of [3H]palmitic acid-labeled mycoplasmas was only slowly released into the supernatant. About 55% of the activity remained AM-associated up to 70 h after phagocytosis. After phagocytosis of [3H]thymidine-labeled cells, about 70% of the radioactivity found non-precipitable by trichloracetic acid. 3H-amino acid-labeled protein was released to 50% within 8 h. Supernatants and AM were tested for M. pneumoniae antigen with enzyme-linked immunosorbent assay. Considerable amounts of antigenically active material could be found in the supernatant within 8 h. This antigen was totally inactivated by heat (80 degrees C). Trypsin treatment (1 mg/ml, 10 min) reduced the antigenicity by 80%. The results suggest a selective release of microbial material after phagocytosis.

Virally mediated changes in cellular permeability.

Conferences on membranes are now held as frequently as those on intermediary metabolism or proteins were some years ago; few discussions of membranes, however, deal with disease. And of those that do, the word “virus” hardly ever appears in the index (e.g., Trump and Arstila 1975; Bolis et al. 1976; Miller and Shamoo 1977; Andreoli et al. 1978). Yet it is obvious that viral diseases involve the surface membrane of cells: the initial interaction between viruses and susceptible cells is at the cell surface, membrane damage is a characteristic feature of infected cells (e.g., Thigpen 1971; Fenner et al. 1974), the interplay between infected cells and the immune system takes place at the cell surface, and the action of interferon in protecting against viral attack begins at the cell surface. The features of the cell surface that have been particularly studied to date (e.g., Poste and Nicholson 1977) are the nature of virus receptors (proteins and glycoproteins), the mechanism by which viruses enter cells (endocystosis or, in the case of certain enveloped viruses, virus-cell fusion), the appearance of viral antigens (proteins and glycoproteins) on the surface of infected cells, the mechanism of viral release, and the participation of surface-bound antigens in eliciting an immune (cell-mediated) response. What has been relatively less explored is the way in which the cytopathic effects of viruses come about. The aim of this chapter is to suggest that a change in cellular permeability is at the root of some of these effects and that such a change may underlie the pathological consequences, that is, the diseases, that result from viral infection in certain cases.

Fluorescent Antibody Analysis of Host Plasma Components on Bloodstream Forms of African Pathogenic Trypanosomes. I. Host Specificity and Time of Accretion in Trypanosoma congolense

The presence, host specificity, and time of accretion of rat plasma components on the surface of Trypanosoma congolense were determined. Parasites collected at peak parasitemia (6to 10-day rat infections) were separated from blood cells on a DEAE-cellulose column and exogenous plasma was removed by washing. For immunoelectrophoretic analysis, the trypanosomes were lysed, the lysate centrifuged, and the soluble antigen reacted with rabbit antiserum developed against normal rat plasma. At least two antigen-antibody complexes were observed. To determine the location, host specificity, and time of appearance of the antigens shared by the host and parasite, a quantitative indirect fluorescent antibody method was used. The washed trypanosomes were fixed in 2.5% or 10% formalin before being subjected to the sandwich method. Formalin at a 2.5% concentration was the most suitable for the preservation of antigens. It was established by titrations that the largest difference in the fluorescence levels of the experimental and experimental control preparations occurred when the nonconjugated (first layer) rabbit antisera were used at 1:16 dilutions, and the fluorescein isothiocyanateconjugated anti-rabbit IgG serum (second layer) at a 1:40 dilution. Fluorescence differences between ratand mouse-derived trypanosomes stained with anti-rat plasma serum indicated that most, if not all, of the plasma components were host-specific. In vivo and in vitro experiments were performed to determine the length of time needed for appearance of new-host plasma components on the trypanosome surface. The rat-derived bloodstream forms when transferred to mice for 48 hr or incubated for 5 hr in normal mouse plasma emitted fluorescence quantitatively identical to that of trypanosomes grown in mice by serial passages. Incubation of mouse-derived trypanosomes in normal mouse plasma for 5 hr did not change their fluorescence measurements, but such trypanosomes incubated for 3 to 5 hr in normal rat plasma had fluorescence levels similar to those of rat-derived trypanosomes. The speed of acquisition and host specificity of the surface-bound antigens suggested that they were host plasma components accreted on the parasites. The occurrence of host plasma proteins on the surfaces of trypanosomes was suggested originally by Desowitz and Watson (1953) with regard to a rodent-adapted strain of Trypanosoma vivax. Subsequently, antigens common to trypanosomes and their hosts were reported from bloodstream forms of stercorarian and salivarian species on the bases of the results obtained by agglutination, double diffusion, immunoelectrophoresis, and direct fluorescent antibody methods. Among Stercoraria, such antigens were observed in Trypanosoma lewisi by D'Alesandro (1972) and Dwyer (1976). Among Salivaria, they were found in a mouse-adapted T. vivax (Ketteridge, 1970), Received for publication 8 December 1976. * This investigation was supported by Research Grant AI 00742-21, from NIAID, U.S. Public Health Service. t Predoctoral Trainee, supported by Training Grant 5 T01 AI 00226-13, from NIAID, U.S. Public Health Service. rat-adapted Trypanosoma brucei gambiense (Seed, 1974), and Trypanosoma brucei brucei (Vickerman, 1972; Le Ray, 1975). The present study was undertaken to ascertain if antigens resembling those of the host plasma occur on Trypanosoma (Nannomonas) congolense Broden. Since such host-parasite shared components were found, in vivo and in vitro experiments were designed to determine the length of time needed for the appearance of new host plasma components on the surface of the parasites. MATERIALS AND METHODS

Use of antibody-coated red cells for the sensitive detection of antigen and in rosette tests for cells bearing surface immunoglobulins

Conditions for an improved chromic chloride method for the attachment of antibody to red cells are described. The method, which is applicable to whole Ig fractions as well as affinity-purified antibodies, is reproducible and has a sensitivity for detection of antigen in the nanogram range. The coated cells may be used in a rosette assay for the detection of cell surface-bound antigens.

Paraformaldehyde fixation in immunofluorescence and immunoelectron microscopy: Preservation of tissue and cell surface membrane antigens

By means of the immunofluorescence technique and immunoelectron microscopy it is demonstrated that fixation of tissues and cells with 1% paraformaldehyde does not seriously affect tissue and membrane-bound antigens. Higher concentrations (2% and 4%) of paraformaldehyde and addition of glutaraldehyde in a concentration of over 0.1% to 1% paraformaldehyde caused a rapid loss of antigenicity. Addition of glutaraldehyde in very low concentrations (0.01–0.05%) improved the morphological conditions required for immunoelectron microscopical studies. Surface-bound antigens were immobilized by the fixation procedure with 1% paraformaldehyde, which abolished or greatly reduced the capping process. Finally cells incubated with labelled antisera after fixation could be kept for about two weeks before screening.

Mechanisms at the cellular level during induction of high zone tolerance in vitro

The in vitro induction of high zone tolerance to polymerized flagellin of S. adelaide in CBA mouse spleen cells consists of two stages: (a) Attachment of tolerance inducing concentrations of antigen to the surface of immunocompetent cells. (b) Inactivation of the immunocompetent cells by the surface-bound antigen. The definition of these two stages is based on the finding that trypsin treatment of spleen cells within 16 hr after tolerance induction in vitro completely reverses the state of unresponsiveness. However, reversibility of the tolerant state could not be observed by trypsinization of cells after 3 days of culture with antigen. Trypsin treatment of spleen cells before exposure to antigen prevented the induction of tolerance in vitro. This suggests that antigen binding was abolished by removal of recognition sites by the proteolytic enzyme.