B cell lipid rafts regulate both peptide-dependent and peptide-independent APC-T cell interaction.

Formation of an immunological synapse (IS) between APCs and T CD4(+) lymphocytes is a key event in the initiation and the termination of the cognate immune response. We have analyzed the contribution of the APC to IS formation and report the implication of the actin cytoskeleton, the signaling proteins and the lipid rafts of B lymphocytes. Recruitment of MHC class II molecules to the IS is concomitant with actin cytoskeleton-dependent B cell raft recruitment. B cell actin cytoskeleton disruption abrogates both IS formation and T cell activation, whereas protein kinase C inhibition only impairs T cell activation. Pharmacological B cell lipid raft disruption inhibited peptide-dependent T lymphocyte activation and induced peptide-independent but HLA-DR-restricted APC-T cell conjugate formation. Such peptide-independent conjugates did not retain the ability to activate T cells. Thus, B cell lipid rafts are bifunctional by regulating T cell activation and imposing peptide stringency.

Ultrastructural and immunologic characteristics of mouse x cattle xenogeneic hybridomas originating from bovine leukemia virus-infected cattle.

Nine percent of xenogeneic hybridomas originating from a bovine leukemia virus (BLV)-infected cow secreted monoclonal IgM antibodies with multispecific reactivity. Similar reactivity was evident in some antibodies with an unusually long (> 50 amino acids) third complementarity-determining region of the heavy chain. Electron microscopy of hybridomas demonstrated the presence of c-type virus particles consistent with polymerase chain reaction detection of BLV env gene. Some hybridomas contained dilated rough endoplasmic reticulum and cisternae filled with moderately electron-dense granular substance compatible with plasma cells at presecretory stage. The number of chromosomes in xenogeneic hybridomas corresponded to the sum total of mouse and bovine chromosomes. None of the hybridomas showed polyploidy. The immunochemical and genetic analysis of stable bovine immunoglobulin-secreting xenogeneic hybridomas confirms that BLV infection causes polyclonal B cell activation regardless of antigen specificity. Presence of c-type particles in hybridomas suggests that T cell-derived cytokines are not required for sustained BLV expression.

Alterations of ionic membrane permeabilities in multidrug-resistant neuroblastoma x glioma hybrid cells.

A population of NG108-15 neuroblastoma cells resistant to doxorubicin (NG/DOXR) was established. The cells exhibited a multidrug resistance phenotype with cross-resistance to vinblastin and colchicine, overexpression of a 170 kDa membrane protein identified as P-glycoprotein and reversal of resistance by verapamil and quinine. Compared with NG108-15 cells, NG/DOXR cells showed an increase in Na+ current density and a decrease in cyclic-AMP-activated Cl- current density with no change in K+- and volume-sensitive Cl- current densities. As previously observed in NG108-15 cells, the vacuolar-type H+-ATPase inhibitors bafilomycin A1 and nitrate induced membrane depolarizations in NG/DOXR cells. The resting potentials of sensitive and resistant cells were not significantly different, but the depolarizations evoked by these agents were significantly larger in NG/DOXR than in NG108-15 cells. The resting membrane potential of NG/DOXR cells, but not that of NG108-15 cells, was depolarized by verapamil, and this effect was abolished by bafilomycin. The volume-sensitive Cl- currents of drug-sensitive and drug-resistant cells were inhibited by a decrease in intracellular pH from 7.3 to 6.8. Whereas bafilomycin prevents activation of Cl- currents in both drug-sensitive and drug-resistant cells, verapamil inhibited the Cl- current only in NG/DOXR cells. The results are discussed in terms of the roles of cytoplasmic pH and membrane potential in multidrug resistance.

Cell cluster formation during up-scaling of a human-mouse heterohybridoma producing a polyspecific human IgM antibody.

Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced. A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system. The process of fermentation has been carried out continuously over a period of 4 months. In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased. Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody. During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable. Furthermore, we detected formation of cell clusters in the fermentor system. These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies. Clusters were found to represent fusions of hybridoma cells using electron microscopy. Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas. We discuss these results in the content of the polyreactive binding properties of this particular antibody.

De novo formation of caveolae in lymphocytes by expression of VIP21-caveolin.

Caveolae are plasma membrane invaginations, which have been implicated in endothelial transcytosis, endocytosis, potocytosis, and signal transduction. In addition to their well-defined morphology, caveolae are characterized by the presence of an integral membrane protein termed VIP21-caveolin. We have recently observed that lymphocytes have no detectable VIP21-caveolin and lack plasma membrane invaginations resembling caveolae. Here we transiently express VIP21-caveolin in a lymphocyte cell line using the Semliki Forest virus expression system and show de novo formation of plasma membrane invaginations containing VIP21-caveolin. These invaginations appear homogeneous in size and morphologically indistinguishable from caveolae of nonlymphoid cells. Moreover, the glycosylphosphatidylinositol-anchored protein. Thy1, patched by antibodies, redistributes to the newly formed caveolae. Our results show that VIP21-caveolin is a key structural component required for caveolar biogenesis.

Functional requirement of the two TNF receptors for induction of apoptosis in PC60 cells and the role of mitochondria in TNF-induced cytotoxicity.

The rat/mouse T-cell hybridoma PC60 was transfected either with hTNF-R55 cDNA, hTNF-R75 cDNA, or both. Receptor-specific stimulation was achieved using agonistic monoclonal antibodies or receptor-specific muteins of hTNF. Either hTNF-R55 or hTNF-R75 could mediate the activation of NF-kappa B and the induction of GM-CSF, IL-6, and IFN-gamma. But only in cells carrying both hTNF-R55 and hTNF-R75, was TNF able to induce apoptosis. This apoptosis could be inhibited almost completely by cotransfection with human bcl-2 cDNA. Functional cooperation was observed between liganded and unliganded receptors for the induction of apoptosis. In vitro protein kinase activity was detected only in TNF-R75 immunoprecipitates from cells in which the receptor was signaling. Direct evidence was obtained for reactive oxygen intermediates of mitochondrial origin responsible for TNF-induced cytotoxicity in L929 cells.

Optimization of culture conditions for feline x murine heterohybridomas.

Feline splenocytes were fused to the murine myeloma lines NSO or Ag8. Autologous serum and taurine were used as media supplements for the cat x mouse heterohybridomas. The best results were obtained by the use of NSO as fusion line with taurine-supported media.

Changes at peptide residues buried in the major histocompatibility complex (MHC) class I binding cleft influence T cell recognition: a possible role for indirect conformational alterations in the MHC class I or bound peptide in determining T cell recognition.

Recent crystallographic studies on two peptide complexes with the mouse Kb molecule have shown that peptide binding appears to alter the conformation of the class I alpha-helical regions that flank the antigen binding cleft. Given that this study also showed that much of the foreign peptide is buried within the class I binding cleft with only a small portion accessible for direct interaction with the components of the T cell receptor, this finding suggests that at least some component of T cell specificity may arise as a consequence of peptide-induced conformational changes in the class I structure. To assess this possibility, we have made systematic substitutions at residues within the Kb-restricted determinant from ovalbumin (OVA257-264) that are thought to be buried on binding to the class I molecule. We have found that changes in this determinant at the completely buried second residue (P2) can influence T cell recognition without affecting binding to Kb, suggesting that the substitutions may indirectly determine T cell recognition by altering the conformation of the class I molecule or the bound peptide.

T hybridoma alpha/beta gene transfected in a murine T cell hybridoma: role of CD4 molecule in vitro and in vivo--engraftment in SCID mice induces T cell maturation.

In the present work, we tested in SCID and Balb/c mice the activity of T hybridoma transfected with T cell receptor (TCR) alpha/beta chain genes. A T cell hybridoma denoted D011107 was used as recipient for transfection of cytotoxic KB5C20 TCR alpha/beta heterodimer genes by protoplast fusion or electroporation. After transfection, the parental D011107 T cell line reexpressed CD5 and CD4 surface molecules. In vitro, we noted strong proliferation and unusual cytotoxic reactivities against H-2k target cells although the transfected cell line does not express the CD8 molecule. The fate of parental and transfected cells was examined in severe combined immunodeficient (SCID) and Balb/c mice at Day 16 after intravenous injection. Cells from bone marrow, thymus, and spleen tissues were analyzed by immunofluorescence. The transfected T cell hybridoma was CD3+ Desire 1+ CD4+ Thy1.2. The SCID mice grafted with the transfected T cell hybridoma presented a high percentage of CD3+ (15%), CD4+ (27%), Thy1.2+ (27.52%), and Desire 1+ (8.74%) cells in the spleen. The percentages of CD3+ (6.2%) and Thy1.2+ (5.06%) cells in the spleen from SCID mice grafted with parental T cell D011107 and from untreated SCID were similar and lower (CD3+, 3.52%; Thy1.2+, 4.34%). It seems that transfected T cells hybridoma grafted in the SCID mice induce significant expression of CD4+ Thy1.2+ Desire 1- cells (17%) in the spleen. These results indicate that transfected T cells graft may allow T cell differentiation. In Balb/c mice, the percentage of different T cell subsets in bone marrow, thymus, or spleen cells in mice injected with transfected T cells was similar to that in untreated mice. We did not observe any cytotoxic or significant allogeneic proliferation in vitro.

Effect of serum on the plasma membrane fluidity of hybridomas: an insight into its shear protective mechanism.

We have previously shown that decreasing the concentration of fetal bovine serum (FBS) increased the fragility of a mouse hybridoma (HB-32) during agitated batch cultivation and that increasing the plasma membrane fluidity (PMF) increased the shear sensitivity during exposure to laminar flow. In this study, the effect of FBS concentration on the PMF of HB-32 was investigated. PMF was evaluated by steady-state fluorescence anisotropy (rs) of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene. Increasing serum concentration increased the rs of hybridomas, indicating a decrease in their PMF. The effect of cholesterol modulation on the PMF and shear sensitivity was also evaluated. Hybridomas were exposed to turbulent fluid shear after modification of PMF by cholesterol modulation. Direct cholesterol enrichment of the plasma membranes caused a decrease in the PMF and shear sensitivity, while cholesterol depletion caused an increase in PMF and shear sensitivity. Low- and high-density lipoprotein supplementation to cultures in serum-free or complete medium decreased their shear sensitivity. Lipoprotein supplementation to serum-free cultures decreased the PMF. Altogether, these results suggest that the protective mechanism of serum against hydrodynamic damage relies, at least partially, on its ability to decrease the PMF of hybridomas possibly through the transfer of cholesterol from the serum lipoproteins into the plasma membrane.

Intracellular variation of rat intestinal mucin granules localized by monoclonal antibodies.

Monoclonal antibodies produced against rat small intestinal mucins were utilized to study variability of stored mucin granules within rat ileal goblet cells. Eleven antibody-secreting hybridoma cultures were produced; six of these uniformly labeled stored mucin granules in virtually all goblet cells, suggesting that some antigenic features are common to all granules. The other five stained goblet cells in the rat small intestinal epithelium nonuniformly. R803, R805, and R807 localized within almost all goblet cells but revealed differential labeling of centrally and peripherally located mucin granules. R804 uniformly labeled the mucin granules of most villous goblet cells; some of the crypt goblet cells were uniformly labeled, but the majority were only partially labeled, resulting in a mottled staining pattern. R808 stained only a small portion of crypt goblet cells; there is, however, an increase in both number of goblet cells labeled and in uniformity of staining of the stored granule mass from the base of the crypt to the surface, resulting in uniform labeling of virtually all goblet cells at the villus tip. This study demonstrates for the first time that rat small intestinal mucin granules are immunologically heterogeneous and nonuniformly distributed within the epithelium. Additionally, staining patterns within the stored granule mass suggest that structurally distinct subpopulations of mucin granules may exist within a single goblet cell.

Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A.

Cyclosporin A and FK506 inhibit T- and B-cell activation and other processes essential to an effective immune response. In T lymphocytes these drugs disrupt an unknown step in the transmission of signals from the T-cell antigen receptor to cytokine genes that coordinate the immune response. The putative intracellular receptors for FK506 and cyclosporin are cis-trans prolyl isomerases. Binding of the drug inhibits isomerase activity, but studies with other prolyl isomerase inhibitors and analysis of cyclosporin-resistant mutants in yeast suggest that the effects of the drug result from the formation of an inhibitory complex between the drug and isomerase, and not from inhibition of isomerase activity. A transcription factor, NF-AT, which is essential for early T-cell gene activation, seems to be a specific target of cyclosporin A and FK506 action because transcription directed by this protein is blocked in T cells treated with these drugs, with little or no effect on other transcription factors such as AP-1 and NF-kappa B. Here we demonstrate that NF-AT is formed when a signal from the antigen receptor induces a pre-existing cytoplasmic subunit to translocate to the nucleus and combine with a newly synthesized nuclear subunit of NF-AT. FK506 and cyclosporin A block translocation of the cytoplasmic component without affecting synthesis of the nuclear subunit.

Nucleosomes occurring in protein-free hybridoma cell culture. Evidence for programmed cell death.

In addition to monoclonal immunoglobulin, two kinds of nucleoproteins, NP1 and NP2, were isolated from the supernatants of hybridoma cultures set up in a protein-free medium. As shown by SDS-electrophoresis the two nucleoproteins shared a set of proteins (apparent Mr 11,000 to 15,000), and differed in the DNA moiety (approximately 150 bp in NP1, approximately 350 bp in NP2). The amino acid composition of the protein moiety confirmed the nucleosomal origin of NP1 and NP2. The findings support the view that in hybridoma cultures the cells undergo death by apoptosis, i.e. a programmed process characterized by initial fragmentation of chromatin.

Characterization of an established mouse-human heterohybridoma and its application for production of (mouse-human)-human triple hybridoma secreting human immunoglobulin.

We report the construction of a mouse-human (M-H) heterohybridoma by fusion of the murine myeloma cell line NS-1 and human spleen cells from a 17 week old fetus. The nonsecreting, cloned hybridoma cell line II was resistant to 8-azaguanine (8-AG) and sensitive to hypoxanthine, aminopterin and thymidine (HAT) medium. It grew rapidly in 8-AG containing medium (doubling time 20 hrs.), but did not grow in HAT medium or in non-serum medium. It had a high fusion frequency with human lymphocytes from regional lymph nodes. Five human chromosomes were retained stably for over 6 months by this cell line II. Nine (mouse-human)-human ((M-H)-H) triple hybridomas secreting human IgG 1 or IgM were established by the fusion of this parental cell line II and human lymphocytes from regional lymph nodes. Immunoglobulin secretion was stable and has been maintained for over 8-10 months without recloning in these hybridomas. Secretion of immunoglobulin varies from 2.1-3.0 micrograms/10(6) cells/day, and these hybridomas contain from 3 to 16 human chromosomes, including No. 14. So, this M-H heterohybridoma II is an excellent useful parental cell line for the production of hybridomas secreting human immunoglobulin.

Development of a cell surface reacting human monoclonal antibody recognizing ovarian and certain other malignancies.

A human monoclonal antibody designated AC6C3 was developed by fusing regional lymph node lymphocytes from a patient with epithelial ovarian carcinoma with cells of the hybrid myeloma SPAZ 4. This monoclonal antibody recognized a determinant expressed on the cell surface of ovarian tumor cell lines. The AC6C3 hybridoma has been maintained for more than 24 months by repeated cloning and secretes IgM at concentrations of 2-8 micrograms/10(6) cells/24h. The AC6C3 monoclonal antibody reacted with a cell surface component of ovarian tumor cell lines, as determined by cell surface immunofluorescence staining using the fluorescent activated cell sorter (FACS). In contrast, nylon wool nonadherent peripheral blood lymphocytes or red blood cells from normal donors were negative (less than 5% of the cells were stained). Immunoperoxidase staining with the AC6C3 monoclonal antibody of nonpermeabilized cryostat sections of freshly obtained or cryopreserved ovarian carcinoma specimens and human ovarian tumor xenografts demonstrated strong reactivity of these specimens. Most normal tissues including brain, liver, heart, kidney and peritoneum demonstrated negative or weak reactions with AC6C3. Other carcinomas including breast, colon and some malignancies of neuroectodermal origin were strongly reactive with AC6C3. AC6C3 mediated complement-dependent cytotoxicity and identified a 32 Kd band in Western blotting and immunoprecipitation experiments conducted on surface labelled SKOV3 cells. The association constant for AC6C3 was determined at 2.3 x 10(10) M-1.

Ultrastructural localization of gold-labeled anti-IgM-antibodies and type A retrovirus particles in endoplasmic cisternae and vesicles of IgM-producing human B cell hybridomas.

Simultaneous appearance of IgM and type A retro-virus particles in endoplasmic cisternae and vesicles of human hybridomas has been demonstrated for the first time by immunogold-labeling at ultrastructural level. A suspected link between type A particle and IgM gene-expression in hybridoma cells could not be substantiated in this study.

[In the dying cell secretory protein diffuses through the membranes into the cytosol]. / V gibnushchei kletke sekretornyi protein diffundiruet cherez membrany v tsitozol'.

Anti-horseradish peroxidase IgG (a-HRP) secreting hybridoma lymphoblasts grown subcutaneously in recipient mice have been studied light and electron microscopically 30-120 min following capitation of the animals. Conventional HRP-DAB immunocytochemical staining was performed for demonstration of a-HRP which in the living cells was restricted to the rough endoplasmic reticulum, the perinuclear cisterns, the Golgi apparatus and some microvesicles. 30 min after death in a number of the cells a-HRP began to invade the cytosol leaving, however, the nucleus and mitochondrial matrix free of the secretory marker. 30 to 90 min later staining intensity became similar in all cellular structures thereby making an impression of overall a-HRP spreading throughout the cell. In the light of these findings and the data obtained by other investigators a conclusion is made on the diffusion of macromolecules across intracellular membranes as a result of considerable post-mortem disturbances in membrane permeability.

[Defining the optimal conditions for immunoperoxidase cytochemistry at the submicroscopic level]. / Opredelenie optimal'nykh uslovii immunoperoksidaznoi tsitokhimii na submikroskopicheskom urovne.

The authors tested a number of experimental protocols and chemicals known to facilitate permeabilization of tissues to immunoperoxidase markers without ultrastructural alterations of the cells to be examined. Monoclonal antibodies producing hybridoma lymphomas served as a primary test object. None of the procedures employed (i.e., quenching of the fixative aldehydes by some reducing agents; cryopermeabilization; treatment by detergents) were shown either to intensify stainability or to increase the penetration of immunoreagents into the tissue depth. The diffusion efficiency depended only on the marker molecular mass and the thickness of the vibratome section incubated. The Elder and coworkers (1983) two-step technique has been found superior in the preservation of both immunoreactivity and fine structure of the cell.

Efficient hybridization of mouse-human cell lines by means of hypo-osmolar electrofusion.

The fusion of a mouse-human heteromyeloma with a mouse hybridoma is used as a model to define parameters to generate human hybridomas. Electrofusion of these cells in 300 mosM and 75 mosM solutions showed that strong hypo-osmolar conditions resulted in a dramatic increase in the efficiency of hybridoma formation. In contrast to iso-osmolar electrofusion, a high hybrid yield could be obtained by injection of only a single field pulse. The field strength was adjusted in proportion to the increased size of the cells in hypo-osmolar solutions. Hypo-osmolar electrofusion allowed the generation of approximately 0.45% hybrids at a suspension density of 1.75 X 10(5) mouse-human cells/ml corresponding to an input number of 3.5 X 10(4) mouse-human cells. A further increase in the efficiency of hybridoma formation to about 0.6% was achieved by cell alignment in an alternating field of modulated field strength. Experiments in which the total cell number per fusion chamber was decreased at constant optimum suspension density showed that a further increase in the efficiency of hybridoma formation in hypo-osmolar solution was not possible because of the increasing influence of the heterogeneity of the cell lines with decreasing cell number. The results allow the conclusion that hypo-osmolar electrofusion is a potential tool to enhance successful immortalisation of human B lymphocytes.

Electron microscopy of hybridoma cells with special regard to monoclonal antibody production.

Electron microscopy of mouse hybridoma cell lines shows that the major difference between non, low and high producer cell lines is the amount of endoplasmic reticulum. Vesicular-tubular or cavernous structures of endoplasmic reticulum, which can survive long after cell death, are particularly abundant in producer cell lines. Immunogold labelling with anti-mouse IgG reveals that antibodies are predominantly located in these structures. The cell membrane undergoes structural changes during the late stages of batch culture with the disappearance of microvilli and the appearance of blebs and deep indentations. Necrosis disrupts the cytoplasmic structures and the nucleus is last to degrade.