Qat-4 and Qat-5, new murine T-cell antigens governed by the Tla region and identified by monoclonal antibodies.

Two new lymphocyte antigens, provisionally designated Qat-4 and Qat-5 have been identified with two different hybridoma-derived, monoclonal AKR antiC57BL/6 antibodies. These antigens are governed by genes located to the right (distal) end of the H-2 complex, within the Qa-2,3 region. Qat-4 and Qat-5 antigens which do not seem to be identical with Qa-2,3 or TL antigens are absent from Ig/ lymphocytes and thymocytes. They are only present on a fraction of peripheral T cells. Thus, Qat-4 is expressed on 70%, and Qat-5 on 30% of splenic and lymph node T cells, Qat-4 is also found on the majority of Ig- cells from athymic nude mice. These findings illustrate the complexity of the chromosome segment between the H-2D and Tla loci and they emphasize the role of major histocompatibility complex-associated genes for the differentiation of T cells into different subpopulations with possibly distinct immunologic functions.

Retinoids are important cofactors in T cell activation.

Murine thymic T cells depleted of antigen-presenting cells proliferate poorly in response to crosslinking anti-CD3 monoclonal antibodies or concanavalin A when cultured in conventional fetal calf serum-containing serum. However, in a serum-free medium formulated to contain, in addition to basic ingredients, insulin, transferrin, albumin, linoleic acid (ITLB), and retinol, proliferation is vigorous. The presence of retinol is critical, because when omitted, cells do not become activated. The subsets of T cells proliferating with the assistance of retinol cofactor are both CD4+ and CD8+ thymic T cells, and CD4+ peripheral T cells. Mature CD8+ T cells of lymph nodes can also be activated in ITLB medium plus retinol, provided that interleukin 2 (IL-2) is added. Retinol needs to be present at the time when T cell receptor triggering is initiated, suggesting that early activation events (G0 to G1 transition) are dependent on retinol. It is currently less clear whether or not subsequent events associated with G1 to S phase transition also require the presence of retinol. 14-hydroxy-retroretinol (14HRR) is a metabolic product of retinol in lymphocytes, and this retinoid effectively supports T cell activation in conjunction with a mitogen in lieu of retinol. Thus, while retinol and its intracellular product, 14HRR, are unable to activate T cells on their own, they are important cofactors. The requirement for retinol in CD3-mediated T cell activation cannot be satisfied by retinoic acid or ILs-1, 2, 4, and 6, and tumor necrosis factor-alpha whereas interferon gamma can substitute for retinol. Our experiments are compatible with the idea that retinol, in the course of cellular activation, is converted to 14HRR, which is needed as intracellular messenger. If substantiated by molecular studies now underway, our data should lead to the description of a new signal pathway distinct from the retinoic acid signal pathway observed in nonlymphoid cells, but perhaps functioning by a similar mechanism, i.e., ligand-assisted transcriptional regulation.

Growth control or terminal differentiation: endogenous production and differential activities of vitamin A metabolites in HL-60 cells.

Vitamin A (retinol) is a prohormone that exerts its pleiotropic biological effects after conversion into multiple metabolites. In this report we describe the identification of three endogenous, retinolderived effector molecules, 14-hydroxy-retro-retinol (14-HRR), anhydroretinol (AR), and retinoic acid (RA) and a putative storage form of retinol, retinylesters (RE) in the human promyelocytic leukemia cell line HL-60. Exogenous application of the retinol metabolites in retinol-depleted serum-free cultures of HL-60 allowed the identification of unique cellular functions for each metabolite: 14-HRR is a growth factor for HL-60. AR is a functional antagonist of 14-HRR with growth-inhibiting activity, and RA is a potent inducer of granulocyte differentiation accompanied by growth arrest. Finally, intracellular RE serves as storage form allowing continuous production of 14-HRR when no external retinol is available.

Scanning electron microscopy of tobacco mosaic virus-labeled lymphocyte surface antigens.

The study of surface antigen by immunoelectron microscopy has been hampered by the fact that thin sections of cells provide only a view of the cell perimeter in an essentially two- dimensional fashion. Although the reconstruction of the entire cell from serial sections has been accomplished (1), it remains too exacting a technique and will find only exceptional application. Carbon-platinum replicas (2) allow the inspection of larger surface areas and therefore are better suited for studying the distribution of antigens (3). But since only relatively smooth surfaces will yield stable replicas, cells with large numbers of microvilli are not amenable to this technique. Despire its limited resolution, scanning electron microscopy (SEM) seems to be the method of choice because it can provide a view of almost half of the surface of a cell close to its natural configuration, particularly after critical point or freeze drying (4, 5). Immunological-labeling methods have not yet been routinely applied to SEM although both latex spheres (6) and hemocyanin (7) have been used with some success. The optimal visual marker should possess the following properties: be of a distinctive shape, chemically stable, and have per se a low binding affinity for cell surfaces. Tobacco mosaic virus (TMV), a marker with which we are familiar in transmission electron microscopy (8), seems to meet these demands; it has rod-like shape and defined dimensions (15 x 300 nm) and in addition it can easily be distinguished from surface microvilli. As the hybrid antibody technique (9) is also applicable to TMV, we have attempted to combine such immunological labeling with SEM. We present evidence that surface antigens can indeed be visualized by SEM, using the TMV marker in conjunction with the hybrid antibody technique.

Tumor necrosis serum induces a serologically distinct population of NK cells.

Murine spleen cells generate nonspecific cytotoxic cells, referred to as natural killer (NK) cells, within 4 d of incubation in Mishell-Dutton cultures. This NK cell type does not arise in cultures of BALB/c.nu spleen cells or in cultures of T-cell depleted C57BL/6 spleen cells, indicating that its activation depends on T cells. Another type of NK cells is induced by tumor necrosis serum in murine spleen-cell cultures. It arises within 24 h and its activation does not depend on T cells. This cell type (and its precursor) expresses the recently discovered cell-surface marker Qa5 (controlled by the Q region of chromosome 17) that distinguishes this NK cell from the NK cell that depends for its activation on thymic function. Qa5+ NK cells are also induced by interferon.

Anhydroretinol: a naturally occurring inhibitor of lymphocyte physiology.

Vitamin A (retinol) is an essential cofactor for growth of B lymphocytes in culture and for activation of T lymphocytes by antigen receptor-mediated signals. 14-hydroxy-4,14-retro-retinol (14-HRR) a metabolite of retinol, has been implicated as the intracellular mediator of this effect. Anhydroretinol (AR) is a retinol derivative with retro structure produced in activated human B lymphocytes and the insect cell lines SF 21 and Schneider S2. AR reversibly inhibits retinol- and 14-HRR-dependent effects and blocks B lymphocyte proliferation as well as activation of resting T lymphocytes. The intracellular signaling pathway blocked by AR in T cell activation is distinct from the calcineurin/interleukin 2 pathway inhibitable by cyclosporine A or FK-506.

Use of hybrid antibody with anti-gamma-G and anti-ferritin specificities in locating cell surface antigens by electron microscopy.

Hybrid antibody [F(ab')(2)] with dual specificity for mouse gammaG and ferritin was prepared from the corresponding rabbit antisera, providing a precise reagent for locating mouse gammaG on cell surfaces. Viable cells were exposed successively to (a) mouse antibody to a cell surface antigen, (b) the rabbit hybrid antibody, and (c) ferritin, before preparation for electron microscopy. This method of See PDF for Structure. labeling is sensitive and specific and clearly lends itself to the introduction of visual markers other than ferritin. Other advantages are uniformity of labeling, ease of purification of the reagent, and circumvention of the many drawbacks arising from coupling ferritin to antibody chemically.

Antigenic structure of cell surfaces. An immunoferritin study of the occurrence and topography of H-2' theta, and TL alloantigens on mouse cells.

The representation of mouse alloantigens belonging to three systems, H-2, theta and TL, on the surface of cells from thymus, spleen, lymph nodes, and peritoneal cavity, was studied by electron microscopy with ferritin-labeled antibody. As expected from earlier serological data, TL was confined to thymocytes, theta was found on thymocytes and lymphocytes, and H-2 occurred to some extent on all cell types observed. On reticular cells, lymphocytes, plasma cells, and eosinophils, the majority of the cell surface was occupied by H-2; thymocytes had considerably less H-2, and erythrocytes and peritoneal macrophages least of all. In every instance the representation of antigen was discontinuous, the fraction of the cell surface covered being characteristic both of the antigen and of the type of cell. H-2 and theta provide a striking example of this; H-2 is present in far higher amounts on lymphocytes than on thymocytes, whereas the converse is true of theta. Within areas positive for H-2 or theta, protuberances of the surface membrane were often antigen-negative. A better definition of cell surface structure, gained from studies such as this, is necessary for further inquiry into how the cell surface is assembled, and into selective gene action in relation to cellular differentiation.

The cysteine-rich regions of the regulatory domains of Raf and protein kinase C as retinoid receptors.

Vitamin A and its biologically active derivatives, the retinoids, are recognized as key regulators of vertebrate development, cell growth, and differentiation. Although nuclear receptors have held the attention since their discovery a decade ago, we report here on serine/threonine kinases as a new class of retinoid receptors. The conserved cysteine-rich domain of the NH(2)-terminal regulatory domains of cRaf-1, as well as several select domains of the mammalian protein kinase C (PKC) isoforms alpha, delta, zeta, and mu, the Drosophila and yeast PKCs, were found to bind retinol with nanomolar affinity. The biological significance was revealed in the alternate redox activation pathway of these kinases. Retinol served as a cofactor to augment the activation of both cRaf and PKC alpha by reactive oxygen, whereas the classical receptor-mediated pathway was unaffected by the presence or absence of retinol. We propose that bound retinol, owing to its electron transfer capacity, functions as a tag to enable the efficient and directed redox activation of the cRaf and PKC families of kinases.

Retinol is essential for growth of activated human B cells.

When EBV-transformed human B cells are removed from conventional cell cultures, washed, and seeded at a low cell density in serum-free medium, their growth potential is greatly diminished. Fresh serum restores the growth of low density B cell cultures. We have traced this restorative effect to an essential factor present in the lipid fraction of serum and have identified it as all-trans retinol. The identification is based on the close similarities of the factor isolated from serum with authentic all-trans retinol as revealed by mass spectrometry, HPLC chromatography, and the ability to stimulate the growth of lymphoblastoid cells in the bioassay. Retinol is active at concentrations equal to its concentration in serum. Retinol is also a requirement for growth in suspension cultures at cell densities of 3 x 10(5)/ml. Cells removed at any time from such exponentially growing cultures and transferred to retinol-free medium cease to grow and consequently die, whereas in the continued presence of retinol, cell growth is unabated. All-trans retinal can substitute for retinol, but retinoic acid fails to stimulate the growth of lymphoblastoid cells at physiological concentrations. Normal human B lymphocytes also require retinol as a costimulator of proliferation after activation by anti-mu antibody or Staphylococcus aureus (Cowan strain) bacteria. In serum, retinol is bound to retinol-binding protein, which in turn forms a complex with prealbumin. Accordingly, we find that B cells respond to retinol bound to its physiological serum carrier, retinol-binding protein. In conclusion, human B cells are critically dependent for optimal growth in cell culture on an external supply of retinol.

Retro-retinoids in regulated cell growth and death.

Vitamin A serves as a prohormone from which three classes of active metabolites are derived: the aldehydes, the carboxylic acids, and the retro-retinoids. Although these three classes are united under the rubric of signal transduction, they act by different molecular mechanisms: the 11-cis-retinaldehydes combine with opsin to form the universal visual pigments and the retinoic acids form ligands for transcription factors, whereas the retro-retinoids, as shown here, intersect with signal transduction at a cytoplasmic or membrane site. The retro-retinoid, anhydroretinol (AR), has long been known to act as a growth inhibitor in lymphocytes, whereas 14-hydroxy-4,14-retro-retinol (14-HRR) is required for normal lymphocyte proliferation. A mutually reversible relationship exists between these two retro-retinoids as one can reverse the effects of the other when given in pharmacological doses. The common explanation for reversible inhibition is competition for a shared receptor. We now provide evidence that when AR is given to T cells unmitigated by 14-HRR, rapid cell death can occur. The circumstances are closely related to nonclassical forms of apoptosis: within 2 h of AR administration the T cells undergo widespread morphological changes, notably surface blebbing and ballooning and, inevitably, bursting. In contrast, nuclear changes are comparatively mild, as indicated by absence of chromatin condensation and overt DNA cleavage to discrete nucleosomal fragments, although DNA nicks are readily discernible by terminal deoxynucleotidyl transferase assay. What further distinguishes the AR-induced form of apoptosis from classical ones is a lack of requirements of messenger RNA and protein synthesis, suggesting that the events leading to cell death are primarily initiated and play themselves out in the cytoplasm. This view is further reinforced by the finding that herbimycin A can prevent the onset of programmed cell death. The importance of our findings is that they strongly suggest a second messenger role for vitamin A metabolites in the cytoplasmic realm that has not been seen previously. These findings are entirely compatible with a general notion that in a cell requiring multiple coordinated signals for survival, the provision of an unbalanced signal can initiate programmed cell death. Collectively, our data also challenge the paradigm that retinoids (outside vision) solely mediate their function via the steroid/ retinoic acid receptor family of nuclear transcription factors. Instead, a mode of action in the cytoplasmic realm akin to one attributed to other small lipophilic second messenger molecules, such as diacyl glycerol or ceramide, may apply to retro-retinoids.

Intracellular signaling by 14-hydroxy-4,14-retro-retinol.

In mammals, retinol is the precursor for retinoids, which affect various aspects of morphogenesis and development. However, B lymphocytes, although retinol-dependent, do not use retinoic acid as mediator. Retinol is metabolized by B lymphocytes and other cell lines to optically active 14-hydroxy-4,14-retro-retinol; it is this compound that mediates the growth control. Thus another second messenger molecule, in addition to retinoic acid and retinal, is derived from retinol.

Computational detection of allergenic proteins attains a new level of accuracy with in silico variable-length peptide extraction and machine learning.

The placing of novel or new-in-the-context proteins on the market, appearing in genetically modified foods, certain bio-pharmaceuticals and some household products leads to human exposure to proteins that may elicit allergic responses. Accurate methods to detect allergens are therefore necessary to ensure consumer/patient safety. We demonstrate that it is possible to reach a new level of accuracy in computational detection of allergenic proteins by presenting a novel detector, Detection based on Filtered Length-adjusted Allergen Peptides (DFLAP). The DFLAP algorithm extracts variable length allergen sequence fragments and employs modern machine learning techniques in the form of a support vector machine. In particular, this new detector shows hitherto unmatched specificity when challenged to the Swiss-Prot repository without appreciable loss of sensitivity. DFLAP is also the first reported detector that successfully discriminates between allergens and non-allergens occurring in protein families known to hold both categories. Allergenicity assessment for specific protein sequences of interest using DFLAP is possible via ulfh@slv.se.

Expression of c-src in cultured human neuroblastoma and small-cell lung carcinoma cell lines correlates with neurocrine differentiation.

Human cell lines with neuronal and neuroendocrine features were examined for their expression of pp60c-src, the cellular homolog of the transforming gene product pp60v-src of Rous sarcoma virus. Four neuroblastoma (LA-N-5, SH-SY5Y, Paju, and SK-N-MC) and three small-cell lung carcinoma (U-2020, U-1690, and U-1285) cell lines were selected on the basis of their stage of neurocrine differentiation, as determined by the expression of neuron-specific enolase. In an immune complex protein kinase assay, all seven cell lines displayed c-src kinase activity which was considerably higher than that found in nonneurocrine cells (human diploid fibroblasts, glioma, and non-small cell lung carcinoma cell lines). Furthermore, the c-src kinase activity, as determined by autophosphorylation or phosphorylation of an exogenous substrate, enolase, correlated with the stage of neurocrine differentiation. There was an approximately 30-fold difference in c-src kinase autophosphorylation activity between the cell lines representing the highest and lowest stages of neurocrine differentiation. A similar variation was found in the steady-state levels of the c-src protein of these cell lines. Highly differentiated neuroblastoma cells expressed two forms of the src protein. Digestion by Staphylococcus aureus V8 protease did reveal structural diversity in the amino-terminal ends of these c-src molecules. In summary, we found a clear correlation between c-src kinase activity and the stage of neuronal and neuroendocrine differentiation. Thus, the phenotypic similarity between neurons and neuroendocrine cells includes high c-src expression.

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.

Different regulation of N- and c-myc expression during phorbol ester-induced maturation of human SH-SY5Y neuroblastoma cells.

The mRNA expression of c-myc and N-myc in the human neuroblastoma cell line SH-SY5Y was found not to change appreciably during the cell cycle and was also unaffected by proliferative inhibition induced by serum starvation or polyamine depletion. However, an early (0.5-8.0 h post-induction) transient reduction of c- and N-myc transcripts were observed in these cells upon induction to differentiation with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Treatment of these neuroblastoma cells with TPA for longer periods (1-8 days), which induces morphological and functional differentiation and growth arrest, was followed by decreased expression of both myc genes. However, the rate of disappearance differed considerably. The N-myc mRNA level was slightly decreased after 4 days and was still detectable 8 days after induction, whereas the c-myc transcript was down-regulated much faster. In contrast, when the cells were exposed to retinoic acid, which results in a maturation along an alternative pathway, the inhibition of N-myc and c-myc expression was similar. The c-fos mRNA expression increased in TPA-treated SH-SY5Y cells and remained high during extended exposure to the drug. The highest c-fos transcript level in induced cells coincided in time with the transient reduction of N-myc and c-myc. Thus, the TPA-induced neuronal differentiation of SH-SY5Y cells was compatible with high c-fos and a substantial N-myc mRNA expression.

The role of glycosylation in secretion and membrane expression of immunoglobulins M and A.

The role of glycosylation in membrane expression and secretion of IgM and IgA was investigated in murine lymphoma and hybridoma cell lines, derived from I.29 tumor, which synthesize IgM or IgA with identical variable regions. Tunicamycin, a selective inhibitor of N-linked glycosylation, prevented the membrane expression of both isotypes, as demonstrated by immunofluorescence, radioiodination and endogenous labeling experiments. Selective immunoprecipitation and immunochemical analysis of membrane, intracellular and secreted molecules permitted us to determine the amount of membrane heavy chain externalized in the presence or absence of tunicamycin. Id 150 and Id 43, two I.29-derived hybridomas secreting IgA and IgM respectively, were differently affected by tunicamycin. While secretion of IgM was inhibited to greater than 95%, no inhibition of secretion of non-glycosylated IgA could be detected in Id 150 cells. These results indicate that different requirements for glycosylation exist in the biosynthetic pathways of immunoglobulin isotypes, and suggest that distinct intracellular transport systems may operate for membrane and secreted alpha-chains.

Monoclonal antibodies detect polymorphic determinants shared by I-A and I-E antigens.

Binding data on inbred mouse strains and immunochemical isolation of Ia antigens with subsequent separation on non-reduced/reduced two-dimensional gels provide evidence for the cross-reactivity of monoclonal antibodies with I-A and I-E products. Thus two monoclonal antibodies were found to react with A alpha A beta as well as E alpha E beta dimers. One of these mAbs, K22 -42, reacts with the precursor form of E beta chain of B10.GD mice which is associated with the invariant chain (Ii). This indicates that the respective determinant on E beta is formed prior to association of E beta with E alpha.