Selective effects of anti-Ia serum and complement treatment upon polyclonal B lymphocyte responses to dextran sulfate and lipopolysaccharide.

Subpopulations of B lymphocytes have been shown to vary in their expression of Ia alloantigens and polyclonal responsiveness to thymic independent antigens. We have demonstrated that the polyclonal B cell antibody response to dextran sulfate is less sensitive to removal of Ia-positive cells than is the response to LPS. This is a consistent finding whether alloantibody and complement (C) pretreatment is directed toward cells bearing Ia antigens coded for by the entire I region or by the I-A or I-E subregions. Heterogeneity appears to exist within the dextran sulfate-sensitive population in that using high antibody; cell ratios during antibody and C-mediated cell selection results in an inhibition of the proliferative but not the antibody response. This result may indicate a differential expression of Ia antigens on dextran sulfate-sensitive B cells that respond by proliferation versus those cells that produce antibody. Alternatively, proliferative responses to dextran sulfate may be more dependent upon Ia-positive accessory cells than is the polyclonal antibody response.     

Chemotactic macrophage subpopulations defined by macrophage chemotactic factors from delayed hypersensitivity reaction sites

The chemotactic specificity of ia-positive and -negative macrophages was studied by using three macrophage chemotactic factors (MCF), -a, -b, and -c, isolated from delayed hypersensitivity reaction (DHR) skin sites in guinea pigs. Listeria-elicited macrophages migrated toward MCF-a, -b, and -c. The chemotactic responses suggested responsive subpopulations to MCF. The electronic programmable individual cell sorter (EPICS) was used to separate macrophages with anti-la monoclonal antibodies. Ia-positive subpopulations responded to MCF-c, although they did not migrate toward MCF-a and -b. In contrast, Ia-negative subpopulations migrated toward MCF-a and -b, but not toward MCF-c. Furthermore, MCF-c attracted Ia-positive macrophages, whereas MCF-a and -b were Ia-negative in vitro; MCF did not induce Ia-negative macrophages to express surface Ia-antigens in vitro. MCF-c was able to produce massive Ia-positive macrophage accumulations when injected i.p., whereas MCF-a accumulated Ia-negative macrophages. The data suggest that MCF-a and -b, which mediate initial macrophage reactions, attract Ia-negative macrophages, and that MCF-c, which mediates predominant macrophage reactions, attract Ia-positive macrophages in the DHR.     

Level of the A, B and H blood group glycosyltransferases in red cell membranes from patients with malignant hemopathies

Eight patients (4 suffering from acute myeloid leukemia) exhibiting a loss of ABO red cell antigens, as seen by a mixed-field reaction pattern in agglutination tests, were selected and examined for the level of the A, -B, -H blood group glycosyltransferases within membranes prepared from erythrocyte subpopulations (A or B positive and A or B negative red cells). A or B enzyme activities were largely decreased in membranes which had lost A or B antigens (A or B negative subpopulations) but were within normal level in membrane from cells which had not lost A or B antigens (A or B positive subpopulations). The H enzyme level which was frequently low in the serum was within normal limits in the membrane preparations examined. Since A or B negative subpopulations were normally glycosylated in vitro into A or B reactive structures, the results demonstrate that loss of A or B antigens is related to some alteration of the blood group gene products rather than to significant abnormalities of the membrane precursors.     

Expression of I-region gene products on mouse tail epidermis cells.

Mouse epidermal cells express Ia antigens. Epidermal cells from C3H and B10. A mice express I-A and I-E region gene products. Products associated with I-B and I-J were not detectable. A weak reaction was seen with anti-I-C sera. Products of the I-A region appear to be preferentially expressed when compared to I-E-region gene products. Ten percent of epidermal cells possess IgG-specific Fc receptors and 15% of epidermal cells can phagocytize latex particles. Our studies suggest that Ia-positive epidermal cells in mice are not necessarily limited to Langerhans cells.     

The growth of B cell receptor microcluster is a universal response of B cells encountering antigens with different motion features

B lymphocyte cell senses and acquires foreign antigens through clonal distributed B cell receptors (BCRs) expressed on the surface of plasma membrane. The presentation formats of antigens are quite diverse. Based on their Brownian diffusion mobility, there are three forms: free mobile soluble antigens, lateral mobile membrane bound antigens, and fixed immobile antigens. Here, using high resolution high speed live cell imaging approaches, we provide evidence that BCR microclusters are formed on the surface of B cells shortly after B cell’s encountering of antigens with each format of motion features. Through high speed live cell imaging, we determine that these BCR microclusters show dynamic growth feature and by doing so function as the basic platforms for B cells to acquire the antigens. We propose that the formation and dynamic growth of BCR microcluster is a universal mechanism for B cell to response to antigens with diverse motion features.     

Subcellular localization of glycoproteins encoded by the viral oncogene v-fms

The McDonough strain of feline sarcoma virus encodes a polyprotein that is cotranslationally glycosylated and proteolytically cleaved to yield transforming glycoproteins specified by the viral oncogene v-fms. The major form of the glycoprotein (gp120fms) contains endoglycosidase H-sensitive, N-linked oligosaccharide chains lacking fucose and sialic acid, characteristic of glycoproteins in the endoplasmic reticulum. Kinetic and steady-state measurements showed that most gp120fms molecules were not converted to mature forms containing complex carbohydrate moieties. Fixed-cell immunofluorescence confirmed that the majority of v-fms-coded antigens were internally sequestered in transformed cells. Dual-antibody fluorescence performed with antibodies to intermediate filaments (IFs) showed that the IFs of transformed cells were rearranged, and their distribution coincided with that of v-fms-coded antigens. No specific disruption of actin cables was observed. The v-fms gene products cofractionated with IFs isolated from virus-transformed cells and reassociated with IFs self-assembled in vitro. A minor population of v-fms-coded molecules (gp140fms) acquired endoglycosidase H-resistant, N-linked oligosaccharide chains containing fucose and sialic acid residues, characteristic of molecules processed in the Golgi complex. Some gp140fms molecules were detected at the plasma membrane and were radiolabeled by lactoperoxidase-catalyzed iodination of live transformed cells. We suggest that v-fms-coded molecules are translated as integral transmembrane glycoproteins, most of which are inhibited in transport through the Golgi complex to the plasma membrane.     

Antigens on human monocytes identified by monoclonal antibodies

Two antigens (Mo1 and Mo2) present on human peripheral blood monocytes have been defined by lytic IgM monoclonal antibodies. Both antigens are present on greater than 70% of adherent mononuclear cells (predominantly monocytes). Mo1 is expressed by monocytes, granulocytes, and Null cells, but is absent from T and B lymphocytes. Mo2, on the other hand, appears specific for peripheral blood monocytes. Neither antigen is present on Ia-positive B cell lines or on tumor cells from patients with B cell lymphoproliferative malignancies, further excluding the possibility that Mo1 and Mo2 are Ia antigens. Mo1 and Mo2 are, however, present on a significant number of blast cells from patients with monocytic leukemia (both myelomonocytic and pure monocytic variants), but relatively infrequently expressed by cells from patients with acute granulocytic leukemia. These results indicate that Mo1 and Mo2 are unique antigens that may represent distinct stages of late monocyte-granulocyte differentiation.     

Immunochemical characterization of synaptosomal membrane antigens from chicken brain

A set of synaptic membrane antigens has been investigated in a number of tissues by indirect immunofluorescence histochemistry, using an antiserum (SPM-I) raised against a purified synaptosomal plasma membrane fraction prepared from day-old chick forebrain. The antigens were found to be present in both the central and peripheral nervous systems and none of them were restricted to the forebrain. The antigens were not detectable in nonneural tissues except for the adrenal medulla. Since the antigens could not be detected in a number of clearly defined glial cell populations or a surgically induced gliosis of the optic nerve, the antigens appear to be nerve specific. The antigens were not present in all types of neurons, thus indicating that surface membrane differences exist between different classes of neurons. Within the plasma membrane of the nerve cell the antigens were not uniformly distributed: they were present in the synaptic region and, in some nerve cells, also in axonal region but were absent from perikaryal membranes and extended regions of dendritic membranes. In the sciatic nerve the antigens were transported at the fast rate of anterograde axonal transport as well as in the retrograde direction. These results have been compared with previous attempts to detect nerve-specific membrane components by immunological means.Part of this work has been presented at American Society for Neurochemistry Meeting, New Orleans, March 1974.     

Differentiation antigens on normal and Abelson virus transformed lymphocytes.

Rabbit antisera to Abelson leukemia virus (A-MuLV)-induced murine lymphomas have been analyzed by absorption with a variety of murine lymphoma lines. Antibody binding to a panel of cell lines and normal lymphocytes was visualized by using hapten-sandwich indirect membrane immunofluorescence. Novel membrane antigens thereby detected are shared between lymphosarcomas, B lymphomas, normal B lymphocytes, and normal membrane immunoglobulin negative (sIg-) bone marrow cells, but are not found on T cells, thymic lymphomas, plasmacytoid lymphomas, or myelomas. The existence of such shared differentiation antigens suggests that sIg- A-MuLV-induced lymphosarcomas may be transformed B cell precursors. Since differences in the expression of these antigens on individual plasma-cytoid lymphoma lines were found, this category of lymphomas may include cells at a variety of differentiation states.     

Accessibility of plasma membrane antigens

Serological techniques applied to intact cells register only those antigens of the plasma membrane that are exposed at the cell surface and are therefore accessible to antibody. Solubilization of the plasma membrane by detergent, used in the conventional surface-iodination immunoprecipitation technique, renders other plasma membrane antigens accessible. We have shown this by using a modified version of the technique in which lysis with detergent is postponed until after the cells have been reacted with antibody. Comparison of the conventional and modified methods confirms that the plasma membrane glycoprotein gp70 has antigen that is not exposed on the intact cells as well as accessible antigen, for example, G_IX. The modified surface-iodination immunoprecipitation method is useful for distinguishing cell-surface antigens from plasma membrane antigens that normally are not accessible. This is exemplified by the fact that standard anti-TL and anti-X.1 sera identify gp70 antigen in the plasma membrane that is registered by the conventional, but not by the modified method.Abbreviations used in this paper are anti - BALB BALB/c - gp70 MuLV envelope glycoprotein of molecular weight about 70,000 daltons, sometimes referred to as gp69/71 - gs group-specific - ¹²⁵I-imm-pptn surface labeling of viable cells with¹²⁵I followed by immunoprecipitation analysis - Ig immunoglobulin - MuLV murine leukemia virus - NMS normal mouse serum - PAGE polyacrylamide gel electrophoresis - PBS Dulbecco's phosphate-buffered saline, Ca⁺⁺- and Mg⁺⁺-free - SDS sodium dodecyl sulfate - TL thymus leukemia antigen     

The poised B cell: lymphokines induce an Ia-increase and antigen-presenting function in B cells

Individual murine B cells express a wide range of Ia densities on the plasma membrane. Here we demonstrate that a dramatic increase in B-cell Ia could be induced by overnight exposure to an uncharacterized lymphokine (LK). Membrane I-A and I-E molecules were both increased after LK treatment, whereas membrane IgM remained unchanged. Two subpopulations of B cells were identified, based on their requirements for expressing maximal Ia; one subpopulation required only LK, the other required both LK and T cells in the overnight culture. Functional changes accompanied the Ia increase. The functional capacity to present antigens to T cells was lacking in normal resting B cells, but was acquired following LK treatment. We suggest that the LK-treated B cell has achieved a new differentiation state, one of preparation for interaction with T cells. We term this state the "poised" B cell, and propose that B cells in the poised state may significantly contribute to T-cell activation as antigen-presenting cells. Moreover, poised B cells may themselves find an advantage over normal B cells in successfully acquiring T-cell help.     

Increased projection of MHC and tumor antigens in murine B16-BL6 melanoma induced by hydrostatic pressure and chemical crosslinking

The B16-BL6 melanoma, like most spontaneously arising tumors, is poorly immunogenic and expresses low levels of major histocompatibility complex (MHC) antigens. Treatment of cells of this tumor in vitro by hydrostatic pressure in the presence of adenosine 2,3-dialdehyde (oxAdo), a membrane-impermeant crosslinker, caused elevated projection of MHC and a specific tumor antigen as demonstrated by flow-cytometric analysis. Maximum projection of both the MHC and the tumor antigens could be reached by application of 1200 atm for 15 min in the presence of 20 mM oxAdo. It is not yet clear whether this passive increase in availability of antigens on the cell surface originated from a dormant pool of antigens in the plasma membrane or from pressure-induced fusion of antigen-rich intracellular organelles (e.g. the endoplasmic reticulum). The immunogenic properties of the antigen-enriched B16-BL6 cells are described in the following paper.     

Role of accessory cells in B cell activation. IV. Ia+ accessory cells are required for the in vitro generation of thymic independent type 2 antibody responses to polysaccharide antigens

It has been previously shown that the in vitro antibody response to TNP-Ficoll requires the presence of adherent accessory cells. In order to determine if this characteristic was unique to TNP-Ficoll or a general feature of the TI-2 antibody responses, responses to the polysaccharide antigens TNP-Levan and TNP-Dextran were studied. Also, it was determined if the functionally relevant accessory cell expresses Ia determinants. Passage of spleen cells over Sephadex G-10 abrogated the response to TNP-Levan and TNP-Dextran as well as to TNP-Ficoll. Addition of adherent accessory cells to the G-10 passed spleen cells reconstituted the response to all 3 antigens. Pretreatment of the adherent accessory cells with a specific anti-Ia serum plus complement abrogated the ability of these cells to provide accessory cell function in the responses to all 3 antigens. Thus, an Ia-positive adherent accessory cell is required for the generation of TI-2 antibody responses to these polysaccharide antigens. This raises the possibility that genetic restrictions may exist between the Ia-positive accessory cell and the lymphocytes involved in the responses to TNP-Ficoll, TNP-Dextran, and TNP-Levan.     

Immunogold localization of callose and other cell wall components in pea nodule transfer cells

Summary Transfer cells are located adjacent to xylem and phloem elements in pea nodule vascular tissues. The composition of the labyrinthine wall intrusions was investigated by immunogold labeling using specific antibody probes. Callose antigen was found at the base of newly formed cell wall intrusions and also in adjacent plasmodesmata. Sections through developed labyrinthine intrusions revealed that wall ingrowths had an internal structure with small domains of callose suggesting the presence of channels or vents. Xyloglucan and pectin antigens were uniformly distributed within the wall, but the distribution of extensin antigens was variable, with different antigens being detected in different regions of the wall ingrowth. A lectinlike glycoprotein, PsNLEC-1, was localized in intercellular spaces associated with nodule transfer cells. Previously, expression of this component was observed in other types of cells showing complex involution of the plasma membrane, namely root cortical cells harboring arbuscular mycorrhizae and nodule cells harboring nitrogen-fixing rhizobia.     

Enhanced interleukin 1 production by alveolar macrophages and increase in Ia-positive lung cells in silica-exposed rats

Inhalation exposure to silica dust enhanced interleukin 1 (IL-1) production by alveolar macrophages (AM), which is attributable to an increase in Ia-positive lung cells. While the proportion of Ia-positive cells in lavaged bronchoalveolar cells (BAC) was much lower (0-3%) in unexposed control rats, about a third of the rats that inhaled silica showed higher proportions (8.0-18.5%); these were designated "Ia-high" exposed animals. The number of total cells, Ia-positive cells and lymphocytes in BAC was significantly increased (P less than 0.05, P less than 0.001, and P less than 0.001, respectively) in these "Ia-high" exposed animals, compared to the control animals. Adherent AM populations obtained from BAC preparations also contained significantly higher (P less than 0.001) proportions of Ia-positive cells in the "Ia-high" exposed animals. When these adherent AM cultures were stimulated with lipopolysaccharide, IL-1 activity of the culture supernatants was enhanced and was significantly higher (P less than 0.001) in the "Ia-high" exposed rats, compared to the control animals. These results indicate that silica-exposure can induce populational changes in lung cells and also activation of AM associated with the increase in Ia-positive cells.     

Location and mobility of twin arginine translocase subunits in the Escherichia coli plasma membrane

The twin arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane. Two primary Tat complexes have been identified, comprising TatABC or TatA multimers, which may interact at the point of translocation. We have analyzed green/cyan/yellow fluorescent protein (XFP) fusions to each of the Tat subunits. We show that the TatB and TatC fusions are active and incorporated into purified TatABC complexes. Proteolytic clipping of the TatA-XFP fusion precludes a definitive conclusion regarding activity, but we do find that the full fusion protein is preferentially incorporated into the TatABC complex. A previous study has proposed that TatB and possibly TatC are localized at the cell poles, whereas TatA is distributed more uniformly throughout the plasma membrane. Here, we likewise show that TatA-XFP is primarily distributed around the periphery of the cell. However, whereas much of the TatB-XFP is found at the poles, quantitative imaging studies show that approximately half of the protein is uniformly distributed in the plasma membrane. Moreover, we show that the bulk of TatC-XFP is detected as a halo around the cells, in some cases as punctate areas that are much smaller than those occupied by TatB-green fluorescent protein (GFP), indicating a uniform distribution. No evidence for a polar localization of TatC-GFP was obtained. Although TatC-GFP is found correctly complexed with TatB, a high proportion of TatB-GFP is not linked to TatC, and we propose that this "free" TatB forms unphysiological assemblies, possibly because it is synthesized in excess. Since TatC is invariably complexed with TatB in wild-type complexes, the combined data demonstrate that TatABC complexes are uniformly distributed throughout the plasma membrane. The significance of the punctate TatA/B/C-GFP is unclear; fluorescence recovery after photobleaching measurements show that these pools of proteins are immobile, whereas nonaggregated proteins are highly mobile in the plasma membrane.     

Intraepithelial lymphocytes modulate Ia expression by intestinal epithelial cells

We have demonstrated that although intestinal epithelial cells in fetuses and young rats do not express Ia antigens, in adult rats intestinal epithelial cells do express Ia antigens, as indicated by immunoperoxidase staining with monoclonal antibodies. Ia expression by intestinal epithelial cells appeared to be related to an increase in the number of intraepithelial lymphocytes (IEL). Most of the IEL were T cells and expressed the phenotype associated with cytotoxic/suppressor T cells, and a large number contained cytoplasmic granules. To directly study a possible modulating effect of IEL on intestinal epithelium, an Ia-negative intestinal epithelial cell line (IEC 17) of rat origin was cultured in the presence of supernatants obtained from Con A- or PHA-stimulated lymphocytes. IEL, as well as spleen cells but not bone marrow cells, were able to secrete a factor(s) capable of inducing Ia antigens on IEC 17 cells, as judged by immunoperoxidase staining and radioimmunoassay. Ia-positive IEC 17 cells were detectable after 12 hr and maximum Ia expression was obtained by 48-hr incubation. Persistence of Ia expression by intestinal epithelial cells required the continued presence of Ia-inducing factor in the medium. Lymphocyte proliferation was not essential for the secretion of the Ia-inducing factor(s). The characteristics and the kinetics of secretion of the Ia-inducing factor were similar to that of an interferon-like activity, but not of interleukin 2. Con A-induced supernatants from IEL and spleen cells were also capable of suppressing the growth of IEC 17 cells. The results of this study indicate that IEL, because of their close association with intestinal epithelial cells, may be involved in modulating a variety of epithelial cell functions, including the expression of Ia antigens. This leads us to speculate that Ia-positive epithelial cells, like Ia-positive macrophages and dendritic cells, may be involved in antigen presentation to T lymphocytes.     

B cell activation. II. Receptor cross-linking by thymus-independent and thymus-dependent antigens induces a rapid decrease in the plasma membrane potential of antigen-binding B lymphocytes

We report the specific induction of B cell plasma membrane depolarization with the use of thymus-dependent and -independent antigens. We have utilized various trinitrophenol-carrier conjugates for the stimulation of isolated trinitrophenol-binding mouse B cells. Membrane depolarization was assessed by flow cytometric analysis of 3-3'-pentyloxacarbocyanine iodide (DiOC5[3])-stained cells. Entry into the cell cycle was determined by flow cytometric analysis of acridine orange-stained cells. The results indicate that polyvalent antigens, but not free hapten, induce B cell membrane depolarization by a large proportion of antigen-binding cells within 2 hr of stimulation. Although all polyvalent antigens induce membrane depolarization, only thymus-independent antigens induce the subsequent G0 to G1 transition, suggesting that the membrane Ig cross-linking signal alone, although sufficient to induce membrane depolarization and subsequent increased IA expression, is insufficient to drive the entry of B cells into the cell cycle. The G0 to G1 transition appears to be dependent on a second signal, perhaps mediated by the thymus-independent carrier or antigen-specific, Ia-restricted T cell helper.     

Antigenic phenotype of TdT-positive cells in human peripheral blood

Double immunofluorescence studies for terminal deoxynucleotidyl transferase (TdT) and leucocyte surface membrane antigens have been used to characterize the small subpopulation of TdT-positive cells in human peripheral blood. The predominant antigens demonstrated were those coded for by the major histocompatibility complex, namely HLA-A,B and Ia-like antigens. A small proportion of TdT+ cells expressed antigens restricted to B lymphocytes and their precursors (BA-1+ CALLA+). In contrast, antigens associated with T-lymphocyte differentiation were not detected using a panel of T-cell-specific monoclonal antibodies. These results preclude the possibility that circulating TdT+ cells are immature cortical thymocytes that have "leaked" into the bloodstream. Although bone marrow-derived prothymocytes, which have not yet acquired T-cell lineage markers, may be included amongst this subset, the expression of B-cell related antigens by some TdT+ cells indicates the likely existence of lineage heterogeneity amongst this population of lymphoid cells. The relevance of these findings to the monitoring of human acute lymphoblastic leukaemia is discussed.