Tumor dormancy and cell signaling: anti-mu-induced apoptosis in human B-lymphoma cells is not caused by an APO-1-APO-1 ligand interaction.

Signal transduction initiated by crosslinking of antigen-specific receptors on T- and B-lymphoma cells induces apoptosis. In T-lymphoma cells, such crosslinking results in upregulation of the APO-1 ligand, which then interacts with induced or constitutively expressed APO-1, thereby triggering apoptosis. Here we show that crosslinking the membrane immunoglobulin on human lymphoma cells (Daudi) (that constitutively express APO-1) does not induce synthesis of APO-1 ligand. Further, a noncytotoxic fragment of anti-APO-1 antibody that blocks T-cell-receptor-mediated apoptosis in T-lymphoma cells does not block anti-mu-induced apoptosis. Hence, in B-lymphoma cells, apoptosis induced by signaling via membrane IgM is not mediated by the APO-1 ligand.

Gangliosides are neuronal ligands for myelin-associated glycoprotein.

Nerve cells depend on specific interactions with glial cells for proper function. Myelinating glial cells are thought to associate with neuronal axons, in part, via the cell-surface adhesion protein, myelin-associated glycoprotein (MAG). MAG is also thought to be a major inhibitor of neurite outgrowth (axon regeneration) in the adult central nervous system. Primary structure and in vitro function place MAG in an immunoglobulin-related family of sialic acid-binding lactins. We report that a limited set of structurally related gangliosides, known to be expressed on myelinated neurons in vivo, are ligands for MAG. When major brain gangliosides were adsorbed as artificial membranes on plastic microwells, only GT1b and GD1a supported cell adhesion of MAG-transfected COS-1 cells. Furthermore, a quantitatively minor ganglioside expressed on cholinergic neurons, GQ1b alpha (also known as Chol-1 alpha-b), was much more potent than GT1b or GD1a in supporting MAG-mediated cell adhesion. Adhesion to either GT1b or GQ1b alpha was abolished by pretreatment of the adsorbed gangliosides with neuraminidase. On the basis of structure-function studies of 19 test glycosphingolipids, an alpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of a gangliotetraose core is necessary for MAG binding, and additional sialic acid residues linked to the other neutral core saccharides [Gal(II) and GalNAc(III)] contribute significantly to binding affinity. MAG-mediated adhesion to gangliosides was blocked by pretreatment of the MAG-transfected COS-1 cells with anti-MAG monoclonal antibody 513, which is known to inhibit oligodendrocyte-neuron binding. These data are consistent with the conclusion that MAG-mediated cell-cell interactions involve MAG-ganglioside recognition and binding.

Cloning and comparative analysis of the human pre-T-cell receptor alpha-chain gene.

In immature T cells the T-cell receptor (TCR) beta-chain gene is rearranged and expressed before the TCR alpha-chain gene. At this stage TCR beta chain can form disulfide-linked heterodimers with the pre-T-cell receptor alpha chain (pTalpha). Using the recently isolated murine pTalpha cDNA as a probe, we have isolated the human pTalpha cDNA. The complete nucleotide sequence predicts a mature protein of 282 aa consisting of an extracellular immunoglobulin-like domain, a connecting peptide, a transmembrane region, and a long cytoplasmic tail. Amino acid sequence comparison of human pTalpha with the mouse pTalpha molecule reveals high sequence homology in the extracellular as well as the transmembrane region. In contrast, the cytoplasmic region differs in amino acid composition and in length from the murine homologue. The human pTalpha gene is expressed in immature but not mature T cells and is located at the p21.2-p12 region of the short arm of chromosome 6.

The BALB/c mouse B-cell response to pigeon cytochrome c initiates as a heteroclitic response specific for the self antigen mouse cytochrome c.

Direct evidence is presented in support of the longstanding but unproven hypothesis that B lymphocytes specific for self antigens (Ags) can be used in the immune response to foreign Ags. We show that the B cells in BALB/c mic responding early to pigeon cytochrome c (CYT) produce antibodies that recognize and bind the major antigenic site on mouse CYT with greater affinity than they bind pigeon CYT i.e., they are heteroclitic for the self Ag. Furthermore, these B cells express the same combination of immunoglobulin variable region (V) genes that are known to be used in B-cell recognition of mouse CYT. Over time, the response to pigeon CYT becomes more specific for the foreign Ag through the recruitment of B cells expressing different combinations of V genes and, possibly, somatic mutation of the mouse CYT specific B cells from early in the response. Cross-recognition of pigeon CYT by mouse CYT-specific B cells results from the sharing of critical amino acid residues by the two Ags. Although B-cell recognition of the self Ag, mouse CYT, is very specific, which limits the extent to which foreign Ags can cross-activate the autoreactive B cells, it is possible that polyreactive B cells to other self Ags may be used more frequently in response to foreign Ags.

Frequent somatic hypermutation of the 5' noncoding region of the BCL6 gene in B-cell lymphoma.

The BCL6 gene encodes a zinc-finger transcription factor and is altered by chromosomal arrangements in its 5' noncoding region in approximately 30% of diffuse large-cell lymphoma (DLCL). We report here that, in 22/30 (73%) DLCL and 7/15 (47%) follicular lymphoma (FL), but not in other tumor types, the BCL6 gene is also altered by multiple (1.4 x 10(-3) -1.6 x 10(-2) per bp), often biallelic, mutations clustering in its 5' noncoding region. These mutations are of somatic origin and are found in cases displaying either normal or rearranged BLC6 alleles indicating their independence from chromosomal rearrangements and linkage to immunoglobulin genes. These alterations identify a mechanism of genetic instability in malignant B cells and may have been selected during lymphomagenesis for their role in altering BCL6 expression.

The CD19 signal transduction molecule is a response regulator of B-lymphocyte differentiation.

The phenotypes of CD19-deficient (CD19-/-) mice, and human CD19-transgenic (hCD19TG) mice that overexpress CD19 indicate that CD19 is a response regulator of B-lymphocyte surface receptor signaling. To further characterize the function of CD19 during B-cell differentiation, humoral immune responses to a T-cell-independent type 1 [trinitrophenyl-lipopolysaccharide (TNP-LPS)], a T-cell-independent type 2 [dinitrophenyl (DNP)-Ficoll], and a T-cell-dependent [DNP-keyhole limpet hemocyanin (KLH)] antigen were assessed in CD19-/- and hCD19TG mice. B cells from CD19-/- mice differentiated and underwent immunoglobulin isotype switching in vitro in response to mitogens and cytokines. In vivo, CD19-/- mice generated humoral responses to TNP-LPS and DNP-KLH that were dramatically lower than those of wild-type littermates. Surprisingly, the humoral response to DNP-Ficoll was significantly greater in CD19-/- mice. In contrast, hCD19TG mice were hyperresponsive to TNP-LPS and DNP-KLH immunization but were hyporesponsive to DNP-Ficoll. These results demonstrate that CD19 is not required for B-cell differentiation and isotype switching but serves as a response regulator which modulates B-cell differentiation. Since humoral responses to both T-cell-dependent and T-cell-independent antigens were similarly affected by alterations in CD19 expression, these differences are most likely to result from intrinsic changes in B-cell function rather than from the selective disruption of B-cell interactions with T cells.

Characterization of a 23-kDa protein associated with CD40.

CD40 is a 45-kDa glycoprotein member of the tumor necrosis factor receptor (TNFR) family expressed on B cells, thymic epithelial cells, dendritic cells, and some carcinoma cells. The unique capacity of CD40 to trigger immunoglobulin isotype switching is dependent on the activation of protein-tyrosine kinases, yet CD40 possesses no kinase domain and no known consensus sequences for binding to protein-tyrosine kinases. Recently, an intracellular protein (CD40bp/LAP-1/CRAF-1) which belongs to the family of TNFR-associated proteins was reported to associate with CD40. We describe a 23-kDa cell surface protein (p23) which is specifically associated with CD40 on B cells and on urinary bladder transitional carcinoma cells. Protein microsequencing revealed that p23 shows no homology to any known protein. A rabbit antibody raised against a peptide derived from p23 recognized a 23-kDa protein in CD40 immunoprecipitates. In contrast to CD40bp/LAP-1/CRAF-1, p23 was not associated with TNFR p80 (CD120b). These findings suggest that p23 is a novel member of the CD40 receptor complex.

Expression studies of catalytic antibodies.

We have examined the positive influence of human constant regions on the folding and bacterial expression of active soluble mouse immunoglobulin variable domains derived from a number of catalytic antibodies. Expression yields of eight hybridoma- and myeloma-derived chimeric Fab fragments are compared in both shake flasks and high density fermentations. In addition the usefulness of this system for the generation of in vivo expression libraries is examined by constructing and expressing combinations of heavy and light chain variable regions that were not selected as a pair during an immune response. A mutagenesis study of one of the recombinant catalytic Fab fragments reveals that single amino acid substitutions can have dramatic effects on the expression yield. This system should be generally applicable to the production of Fab fragments of catalytic and other hybridoma-derived antibodies for crystallographic and structure-function studies.

Down-regulation of NF-kappa B protein levels in activated human lymphocytes by 1,25-dihydroxyvitamin D3.

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2)D3], a steroid hormone with immunomodulating properties, on nuclear factor kappa B (NF-kappa B) proteins was examined in in vitro activated normal human lymphocytes by Western blot analysis. Over a 72-hr period of activation, the expression of the 50-kDa NF-kappa B, p50, and its precursor, p105, was increased progressively. When cells were activated in the presence of 1,25(OH)2D3, the levels of the mature protein as well as its precursor were decreased. The effect of the hormone on the levels of p50 was demonstrable in the cytosolic and nuclear compartments; it required between 4 and 8 hr and was specific, as 25-hydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 were ineffective. Besides p50, 1,25(OH)2D3 decreased the levels of another NF-kappa B protein, namely c-rel. In addition, 1,25(OH)2D3 decreased the abundance of a specific DNA-protein complex formed upon incubation of nuclear extracts from activated lymphocytes with a labeled NF-kappa B DNA binding motif. Further, 1,25(OH)2D3 inhibited the transcriptional activity of NF-kappa B in Jurkat cells transiently transfected with a construct containing four tandem repeats of the NF-kappa B binding sequence of the immunoglobulin kappa light chain gene linked to the chloramphenicol acetyltransferase reporter gene. These observations demonstrate directly that there is de novo synthesis of NF-kappa B during human lymphocyte activation and suggest that this process is hormonally regulated.

An astrocytic binding site for neuronal Thy-1 and its effect on neurite outgrowth.

Thy-1, a member of the immunoglobulin superfamily, is one of the most abundant glycoproteins on mammalian neurons. Nevertheless, its role in the peripheral or central nervous system is poorly understood. Certain monoclonal antibodies to Thy-1 promote neurite outgrowth by rodent central nervous system neurons in vitro, suggesting that Thy-1 functions, in part, by modulating neurite outgrowth. We describe a binding site for Thy-1 on astrocytes. This Thy-1-binding protein has been characterized by immunofluroesence with specific anti-idiotype monoclonal antibodies and by three competitive binding assays using (i) anti-idiotype antibodies, (ii) purified Thy-1, and (iii) Thy-1-transfected cells. The Thy-1-binding protein may participate in axonal or dendritic development in the nervous system.

CD27-CD70 interactions regulate B-cell activation by T cells.

CD27, a member of the tumor necrosis factor (TNF) receptor family, binds to its ligand CD70, a member of the TNF family, and subsequently induces T-cell costimulation and B-cell activation. CD27 is expressed on resting T and B cells, whereas CD70 is expressed on activated T and B cells. Utilizing transfected murine pre-B-cell lines expressing human CD27 or CD70, we have examined the effect of such transfectant cells on human B-cell IgG production and B-cell proliferation. We show that the addition of CD27-transfected cells to a T-cell-dependent, pokeweed mitogen-driven B-cell IgG synthesis system resulted in marked inhibition of IgG production, whereas the addition of CD70-transfected cells enhanced IgG production. The inhibition and enhancement of pokeweed mitogen-driven IgG production by CD27 and CD70 transfectants were abrogated by pretreatment with anti-CD27 and anti-CD70 monoclonal antibodies, respectively. In contrast, little or no inhibition of IgG production and B-cell proliferation was noted with CD27-transfected cells or either anti-CD27 or CD70 monoclonal antibody in a T-cell-independent Staphylococcus aureus/interleukin 2-driven B-cell activation system. In this same system CD70-transfected cells enhanced B-cell IgG production and B-cell proliferation, and this enhancement could be gradually abrogated by addition of increasing numbers of CD27-transfected cells. These results clearly demonstrate that interactions among subsets of T cells expressing CD27 and CD70 play a key role in regulating B-cell activation and immunoglobulin synthesis.

Rabbit monoclonal antibodies: generating a fusion partner to produce rabbit-rabbit hybridomas.

During the last 15 years several laboratories have attempted to generate rabbit monoclonal antibodies, mainly because rabbits recognize antigens and epitopes that are not immunogenic in mice or rats, two species from which monoclonal antibodies are usually generated. Monoclonal antibodies from rabbits could not be generated, however, because a plasmacytoma fusion partner was not available. To obtain a rabbit plasmacytoma cell line that could be used as a fusion partner we generated transgenic rabbits carrying two transgenes, c-myc and v-abl. These rabbits developed plasmacytomas, and we obtained several plasmacytoma cell lines from which we isolated hypoxanthine/aminopterin/thymidine-sensitive clones. One of these clones, when fused with spleen cells of immunized rabbits, produced stable hybridomas that secreted antibodies specific for the immunogen. The hybridomas can be cloned and propagated in nude mice, and they can be frozen without change in their ability to secrete specific monoclonal antibodies. These rabbit-rabbit hybridomas will be useful not only for production of monoclonal antibodies but also for studies of immunoglobulin gene rearrangements and isotype switching.

One sequence, two folds: a metastable structure of CD2.

When expressed as part of a glutathione S-transferase fusion protein the NH2-terminal domain of the lymphocyte cell adhesion molecule CD2 is shown to adopt two different folds. The immunoglobulin superfamily structure of the major (85%) monomeric component has previously been determined by both x-ray crystallography and NMR spectroscopy. We now describe the structure of a second, dimeric, form present in about 15% of recombinant CD2 molecules. After denaturation and refolding in the absence of the fusion partner, dimeric CD2 is converted to monomer, illustrating that the dimeric form represents a metastable folded state. The crystal structure of this dimeric form, refined to 2.0-A resolution, reveals two domains with overall similarity to the IgSF fold found in the monomer. However, in the dimer each domain is formed by the intercalation of two polypeptide chains. Hence each domain represents a distinct folding unit that can assemble in two different ways. In the dimer the two domains fold around a hydrophilic interface believed to mimic the cell adhesion interaction at the cell surface, and the formation of dimer can be regulated by mutating single residues at this interface. This unusual misfolded form of the protein, which appears to result from inter- rather than intramolecular interactions being favored by an intermediate structure formed during the folding process, illustrates that evolution of protein oligomers is possible from the sequence for a single protein domain.

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases.

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.

Considerations on the folding topology and evolutionary origin of cadherin domains.

Cell-cell adhesion in zonula adherens and desmosomal junctions is mediated by cadherins, and recent crystal structures of the first domain from murine N-cadherin provide a plausible molecular basis for this adhesive action. A structure-based sequence analysis of this adhesive domain indicates that its fold is common to all extracellular cadherin domains. The cadherin folding topology is also shown to be similar to immunoglobulin-like domains and to other Greek-key beta-sandwich structures, as diverse as domains from plant cytochromes, bacterial cellulases, and eukaryotic transcription factors. Sequence similarities between cadherins and these other molecules are very low, however, and intron patterns are also different. On balance, independent origins for a favorable folding topology seem more likely than evolutionary divergence from an ancestor common to cadherins and immunoglobulins.