Cross-reaction of anti-simian immunodeficiency virus envelope protein antibodies with human immunoglobulins.

It has been recently established that retroviral envelope proteins (REPs) have structural features similar to those of immunoglobulins (Igs). In this study, we asked whether anti-REP antibodies cross-react with human Igs (hIgs). To this end, murine monoclonal antibodies (mMoAbs) that had been raised against a simian immunodeficiency virus (SIV) envelope protein, SIVMac251gp120, were screened for their ability to react with human monoclonal Igs (HMIgs). We show that two HMIgs, RFSJ2 (a rheumatoid factor) and PAMLN6 (a human anti-hIg V region antibody), but not a number of other HMIgs, could be weakly, but consistently, bound by anti-SIVMac251gp120 mMoAbs KK17 and KK46, as judged by indirect enzyme-linked immunosorbent assay and a liquid-phase inhibition immunoassay. Both mMoAbs are specific to amino acid residues in the V3 loop of the SIVMac251gp120. The RFSJ2 Ig heavy-chain V region (VH) is coded in part by a human VH gene, VH3-30.3 and includes the idiotope 7B4 (NKYY), which was previously shown to be present in the gp120 protein of a number of HIV-2 and SIV strains. However, an entirely different VH gene codes the PAMLN6 VH region, opening the possibility that epitope(s) shared between SIVMac251gp120 and hIgs may not be limited to the 7B4 idiotope.

Human B cells accumulate immunoglobulin V gene somatic mutations in a cell contact-dependent manner in cultures supported by activated T cells but not in cultures supported by CD40 ligand.

The acquisition of somatic mutations in the rearranged immunoglobulin V regions in B cells occurs within the tightly regulated microenvironment of a germinal centre. The precise mechanism responsible for turning on the mutational process is unknown. To dissect the role of different components of the germinal centre in this mechanism, we have used in vitro cultures of normal human IgD+ peripheral blood B lymphocytes co-cultured with activated CD4+ T cells, or with resting CD4+ T cells, or with CD40 ligand and IL-4. We observed that if the cultures included activated CD4+ T cells, then up to 100% of VH transcripts on day 14 were somatically mutated. Transcripts were found to carry from one to 36 substitutions (median five). In contrast, in the absence of activated T cells, transcripts contained only background levels of somatic mutation irrespective of the presence of resting T cells or CD40 ligand and IL-4. Cell-cell contact was required for mutation because mutations were not detected when B cells were separated from activated T cells by a membrane.

VH repertoire in human B lymphocytes stimulated by CD40 ligand and IL-4: evidence for positive and negative selection mechanisms coupled to CD40 activation.

In the normal immune system, B cells are thought to be negatively or positively selected at various checkpoints during their maturation; a process that maintains a broad immunoglobulin repertoire while eliminating non-functional or potentially harmful autoreactive antibodies. This study tested the hypothesis that utilization of certain immunoglobulin heavy chain variable region (VH) genes, possibly as a consequence of intrinsic affinity for various ligands, directs positive or negative B cell selection coupled to B cell activation in the periphery during the immune response. The specific prediction that the VH repertoire of CD40-activated B cells would differ from the repertoire of unstimulated cells from the same donor, was tested by assessing VH utilization among human B cell clones grown in vitro, following stimulation with CD40 ligand (CD40L) and IL-4. The results showed that, although utilization of the known VH families and of individual VH3 genes was similar to that found in unstimulated B lymphocytes of the same donor, utilization of individual VH4 genes in CD40-activated B cells displayed a pattern that was markedly different from that of the unstimulated B cells. An allele of V4-61, V4-61b, was over-represented among the activated cells and, in contrast, the V4-34 gene (known to encode cold agglutinins with strong autoreactive properties) was modestly represented among the VH4 activated B cells, although V4-34 was overwhelmingly predominant in the repertoire of resting B cells. These results point to the existence of selection mechanisms that operate during B cell activation in the periphery. These mechanisms may favor B cells utilizing certain VH genes and disfavor the cells that utilize other genes, possibly because utilization of the latter confers autoreactivity.

Functional CD40 ligand expressed by human blood dendritic cells is up-regulated by CD40 ligation.

We examined whether human blood dendritic cells (DCs) express a functional ligand for CD40 (CD40L). Human blood DCs expressed significant amounts of cell surface CD40L identical to that expressed on activated T cells, as detected by mAb to CD40L or a chimeric CD40.Ig fusion protein (CD40.Ig). Stimulation through CD40 up-regulated protein and mRNA CD40L expression in DCs, B cells, and B cell lines. CD40-mediated CD40L expression was inhibited by a protein tyrosine kinase inhibitor, herbimycin, in a dose-dependent manner, suggesting that the induction of CD40L expression via CD40 requires protein tyrosine kinase activity. CD40L surface expression correlated with constitutive or inducible levels of CD40L-specific mRNA, as determined by reverse transcribed PCR analysis (RT-PCR) using CD40L-homologous primers. Furthermore, CD40L on DCs was functional, since CD40L+ DCs, unlike CD40L- DCs, induced B cell IgG and IgA production, and this induction could be inhibited by blocking CD40L-CD40 interactions with mAb to CD40L. Thus, CD40L on DCs and CD40L induced by crosslinking CD40 may regulate B cell activation and maturation. The importance of DC CD40L expression on B cell function is discussed.

Predominant V-region gene configurations in the human antibody response to Haemophilus influenzae capsule polysaccharide.

The antibody response to Haemophilus influenzae type b polysaccharide (Hib PS) is known to be encoded by a few V-region genes. We have obtained four human monoclonal Hib PS antibodies from four healthy adult subjects immunized with diphtheria toxin-conjugated Hib PS vaccine. The VH gene segments that encode for these antibodies belong to the VH3 gene family, of which two are related to the V3-23 gene and two to the VH3b subfamily. Both hybridomas that express a V3-23-related gene use short D-segments (3 bp), the JH6 gene segment and a V kappa gene derived from the A2 germline gene. The two hybridomas that express VH3b genes use D-segments of conventional length (24-33 bp), the JH4 gene segment and a non-A2 V kappa gene. Comparison of our sequences with those reported by others suggests that the above patterns of V-region gene segment association exemplify two V-region gene configurations that are predominant in the Hib PS antibody response. The first configuration is reminiscent of antibodies produced by B-1 B cells while the second is more characteristic of antibodies produced by conventional B cells. The possibility that these two configurations, in fact, represent the products of two different B cell lineages remains to be elucidated.

Calcium channel ligand binding to intact, concanavalin A and cyclic AMP-treated cells of the immune system.

To examine whether the cells of immune system express calcium channel-forming proteins, we studied the binding of calcium channel ligands, known to detect certain types of the above channels in excitable tissues, to murine splenic and human peripheral blood mononuclear cells. Specific (i.e., displaceable by excess cold ligand) binding of the 3H-labelled dihydropyridine drugs PN200-110 and nitrendipine, was not detected in these cells. Specific binding of a phenylalkylamine drug, [3H]verapamil, was detected, but cannot be attributed to the existence of certain specialized receptors, since multiple [3H]verapamil binding sites (about 10(6) per cell) appeared to be occupied. [3H]Verapamil binding to murine splenic mononuclear cells was inhibited following exposure to either the polyclonal T-cell activator, concanavalin A, or a cell-permeable analogue of the second messenger, cyclic AMP, suggesting that processes of lymphocyte activation and/or intracellular signalling may down-modulate at least some of calcium channel ligand binding sites.

[The effect of the binding of the antigenic determinant of tetrodotoxin-sensitive protein on the phytohemagglutinin-induced proliferative response of the mononuclear cells in mouse spleen]. / Izuchenie vliianie sviazyvaniia antigennoi determinanty tetrodotoksinchuvstvitel'nogo belka na proliferativnyi otvet mononuklearnykh kletok selezenki myshi, indutsiruemyi fitogemaggliutininom.

Mammalian brain-derived "tetrodotoxin-sensitive protein" has been shown to share an epitope with as yet unidentified structure of the human and rodent lymphocyte surface. Previously obtained observations that a monoclonal antibody to this epitope induces a proliferative response of murine splenic mononuclear cells are confirmed. However, this antibody fails to modify the phytohemagglutinin-induced response. Moreover, lectin with submitogenic concentration inhibited the antibody-induced response provided that monocytes were present in the culture. The antibody-induced proliferation appeared to be less monocyte-dependent than the lectin-induced one. Taken together, these findings argue against hypothesis that a lymphocyte structure with epitope of the "tetrodotoxin-sensitive protein" is associated with either T cell receptor for antigen or interleukin-2 receptor.

Second messenger-induced modulation of Thy-1 antigen expression: an ELISA study using murine thymic and neuroblastoma cells.

Freshly isolated cells of murine thymuses and in vitro cultured murine neuroblastoma cells were seeded into microplates, treated with either phorbol myristate acetate (PMA) or dibutyryl-cyclic adenosine monophosphate (DBcAMP), and then processed for enzyme immunoassay to analyze with G4 monoclonal antibody the expression of Thy-1.2 antigen epitope. Pretreatment of thymic cells with PMA resulted in little, if any, decrease of Thy-1 expression, while treatment of these cells with DBcAMP caused a significant down-modulation of the epitope. DBcAMP did not affect binding of another murine IgG antibody to the thymic cells. Modulation of the epitope on thymic cells caused by DBcAMP was dose-dependent with maximal effect seen at the drug concentration of 10(-4) M. However, at various doses of DBcAMP (10(-6) to 10(-3) M) we were unable to detect any significant shift of Thy-1 expression on neuroblastoma cells. Though mechanisms of the above phenomena need further elucidation, we conclude that cellular ELISA may provide a useful alternative to more commonly used cytofluorimetric studies for the analysis of immune cell-surface antigen expression and its pharmacological and physiological modulation.

Monoclonal antibody to brain cytoplasmic tetrodotoxin-sensitive protein detects an epitope associated with lymphocytes and involved in lymphocyte activation.

We have previously derived a monoclonal antibody, BIP-4, which is specific to a mammalian brain protein representing a type of sodium channel. Here we show that this antibody detects an epitope associated with lymphocytes and that it triggers a proliferative response of the cells. BIP-4 epitope can be detected on both human peripheral blood and murine splenic mononuclear cells. Surface immuno-globulin-negative (i.e. resting T) lymphocytes are neither bound by the antibody nor proliferate to it. Proliferative response exerted in 7-day cultures of murine splenic mononuclear cells by recombinant interleukin-2 was blocked by BIP-4 antibody. We conclude that the epitope shared by a type of brain sodium channel protein and lymphocyte surface is involved in some, yet unrecognized, step of immune cell activation.

Possible relationship of brain cytoplasmic tetrodotoxin-sensitive protein to voltage-gated sodium channel shown by monoclonal antibody.

Biochemical events leading to the formation of mature membrane-associated sodium channel proteins are not completely understood. We have recently purified a protein from the cytoplasm of brain cells, which is able to become incorporated into liposomes and induce neurotoxin-dependent sodium permeability. Here we report data on a monoclonal antibody derived against this protein. This antibody crossreacts with cell membrane preparations. The antibody binding to viable neuroblastoma cells is inhibited by veratrine, indicating that membrane molecules antigenically related to the cytoplasmic protein may also be related to the voltage-gated sodium channel.

[Binding of monoclonal antibodies to cerebral cytoplasmic tetrodotoxin-sensitive protein to human lymphocytes]. / Sviazyvanie monoklonal'nykh antitel k tsitoplazmaticheskomu tetrodotoksinchuvstvitel'nomu belku golovnogo mozga s limfotsitami cheloveka.

To examine whether lymphocytes express antigenic determinants of brain cytoplasmic tetrodotoxin-sensitive protein (CTSP), anti-CTSP monoclonal antibody (Bab) binding to human peripheral blood lymphocytes was studied using ELISA assay. It was shown that the Mab bound human lymphocytes in a dose-dependent fashion. Halph-maximal binding of this Mab was significantly enhanced if test-cells were pretreated with concanavalin A or with mixed lymphocyte culture supernatant. Results are discussed from the point of view of the hypothesis that CTSP are metabolic precursors of membrane sodium channels.

[Effect of helium and argon on the oxygen uptake by lymphocytes]. / Vliianie geliia i argona na potreblenie kisloroda limfotsitami.

The effect of inert helium and argon gases on the tissue respiration has been studied on lymphocyte suspensions of white rats. It is shown that normoxic helium-oxygen mixture induces almost a two-fold increase of the O2 uptake by lymphocytes as compared with the control (air). No deviations in the value of the studied parameter are revealed in case of replacement of nitrogen from air by argon. Significance of the membrane structure in realization of effects of inert gases is under discussion.

[Monoclonal antibodies to cytoplasmic tetrodotoxin-sensitive brain protein. The effect of veratrine on antibody binding to neuroblastoma cells]. / Monoklonal'nye antitela k tsitoplazmaticheskomu tetrodotoksinchuvstvitel'nomu belku golovnogo mozga. Vliianie veratrina na sviazyvanie antitel s kletkmai neiroblastomy.

Recently a glycoprotein capable to induce tetrodotoxin-sensitive sodium permeability being incorporated to liposomes was purified from the cytoplasm of the bovine brain. It is shown that a monoclonal antibody derived against this protein binds intact murine neuroblastoma cells. Veratrine, neurotoxin referred to modulate the activity of voltage-gated sodium channels, is shown to compete with the antibody for the neuroblastoma surface epitope. It is postulated that molecular moiety bound with the antibody is either identical or spatially related to veratrine (veratridine) binding site.

[Antibody adsorption to tetrodotoxin-sensitive cytoplasmic proteins by murine neuroblastoma cells]. / Adsorbtsiia antitel k tsitoplazmaticheskim tetrodotoksinchuvstvitel'nym belkam kletkami myshinoi neiroblastomy.

The study was aimed to examine properties of polyclonal antibodies elicited after immunization by cytoplasmic nerve cell glycoproteins forming sodium channels in liposomes. It was shown that intact murine neuroblastoma cells can absorb these antibodies. Absorbing cell dose-effect curves were found to have a characteristic form able to shift when the cell culture time or serum concentration in the growth medium varied.

[Common features of the antigenic determinants presented on the membranes of mammalian nervous system cells and cytoplasmic tetrodotoxin-sensitive proteins]. / Obshchnost' antigennykh determinant, predstavlennykh na membranakh kletok nervnoi sistemy mlekopitaiushchikh i na tsitoplazmaticheskikh tetrodotoksinchuvstvitel'nykh belkakh.

Hypothetical antigenic similarities between nerve cell membrane structures and cytoplasmic tetrodotoxin-sensitive proteins have been studied. Indirect ELISA binding assay combined with inhibition assay has been used. The results obtained indicate that cytoplasmic tetrodotoxin-sensitive proteins do share antigenic determinants with nerve cell membrane structures. This is consistent with the speculation that cytoplasmic tetrodotoxin-sensitive proteins are relatives of membrane sodium channels.

[Characteristics of micromethods for the analysis of the in vitro synthesis of antibodies against xenogenic erythrocytes]. / Kharakteristika mikrometodov issledovaniia sinteza in vitro antitel k ksenogennym éritrotsitam.

In order to choose an optimal assay for screening cell microcultures containing anti-xenogenic erythrocyte antibodies in their supernatants, two microassays were compared: microhemagglutination and hemolytic patch assay. Samples of murine sera, each in both assays, were examined for the antibody titer. It is shown that the sensitivity of the assays is approximately at the same level, as the statistically significant difference in the amount of antibodies evaluated either by hemagglutination or by patch assay was not found. Hence one should not prefer any of the assays from the standpoint of sensitivity, when choosing the screening procedure for hybridomas of lymphoid cell microcultures secreting antibodies against xenogenic red blood cells.

[Production of antibodies by hybridomas in a serum-free culture medium]. / Produktsiia antitel gibridomami v pitatel'noi srede, lishennoi syvorotki.

Evidence is presented that hybridomas maintained in serum-free medium continue to secrete antibodies for some time. The dynamics of this secretion appears to be close to that in analogous cultures kept in the serum medium but apparently differs from that in analogous cultures grown in the balanced salt solution. The possibility of hybridomas to synthesize antibodies in protein free medium and not only to release those formerly synthesized is under discussion.