Sendai virus stimulates chemiluminescence in mouse T and B lymphocytes

Mouse spleen cells emit a burst of chemiluminescence (CL) starting within a few seconds on infection with Sendai virus. We have analyzed the cell types responsible for the generation of CL in the spleen cell population. Cell suspensions, treated with iron carbonyl powder to remove adherent cells, showed similar CL compared to untreated spleen cells suggesting that lymphocytes, rather than phagocytic cells, were responsible for CL generation. Furthermore, killing of T lymphocytes with anti- Thy-1.2 antibody and complement, and removal of Ig⁺ cells from the cell population, respectively, showed that both T and B lymphocytes are active in CL generation. The results suggest that changes in lymphocyte metabolism occur within the first few minutes of cell-virus contact.     

Carbohydrate composition of the envelope glycoproteins of Sendai virus

The carbohydrate composition of the two envelope glycoproteins and glycolipids of Sendai virus was determined by gas chromatographic analysis. The larger glycoprotein (HANA), which bears neuraminidase and hemagglutinin, was found to contain approximately 9% carbohydrate by weight, and the smaller glycoprotein (F) approximately 15%. Fucose, mannose, galactose, and N-acetylglucosamine were found in both the glycoproteins, but less fucose and N-acetylglucosamine were present in the HANA glycoprotein than in the F glycoprotein.The main carbohydrate components of the glycolipids are galactose and glucose. Sialic acids have been found to constitute less than 0.01% of the virion.Each radiolabeled glycoprotein was digested with pronase and then subjected to gel filtration using a Bio-Gel P-6 column. Oligosaccharides from the HANA glycoprotein were found to be composed of two distinct types of carbohydrate chains, whereas those from the F glycoprotein were found to be composed of a single type of carbohydrate chain. Possible structures of the oligosaccharides from each glycoprotein are proposed.The amino acid composition of each glycoprotein was also analyzed.     

Involvement of GANP in B cell activation in T cell-dependent antigen response

Adaptive immunity is dependent on proliferation of antigen-driven B cells for clonal expansion in germinal centers (GCs) against T cell-dependent antigens (TD-Ag), accompanied with somatic hypermutation of variable-region gene and class switching of B cell antigen receptors. To study molecular mechanisms for B cell differentiation in GCs, we have identified and studied a 210kDa GANP protein expressed in GC-B cells. GANP has domains for MCM3-binding and RNA-primase activities and is selectively up-regulated in centrocytes surrounded with follicular dendritic cells (FDCs) upon immunization with TD-Ag in vivo and in B cells stimulated with anti-CD40 monoclonal antibody in vitro, which suggested that GANP plays a certain important role in the maturation of immunoglobulin or selection of B cells in GC during the immune response to TD-Ag. Since this up-regulation has not been detected in T cells in GCs and in Concanavalin A-stimulated T cells in vitro, selective function of GANP molecule on B cell proliferation and differentiation might exist.     

Inhibition of T cell-dependent human B cell proliferation and B cell differentiation by polyspecific monomeric IgG.

A commercially available polyspecific, monomeric IgG preparation suitable for intravenous administration (IgSRK; Sandoglobulin) can inhibit pokeweed mitogen (PWM)-induced proliferation of peripheral blood mononuclear cells (PBMC) by a small, but statistically significant, amount compared to control cultures. Such inhibition could not be demonstrated when PBMC were stimulated with the T cell mitogen phytohaemagglutinin. Surface phenotype analysis of the PWM-stimulated cells indicated that in IgSRK-containing cultures, the proportion of B cells was decreased and the proportion of T cells was increased compared to control cultures. This alteration in T:B ratio was not due to antigenic modulation of B or T cell markers from their surfaces. In addition, IgSRK inhibited the proliferation of T cell-depleted PBMC cultures stimulated by B cell proliferation factors (BCPF) but not by fixed protein A-bearing Staphylococcus aureus strain Cowan I. The capacity to inhibit B cell proliferation was independent of and distinct from its capacity to inhibit B cell differentiation, since IgSRK inhibited the differentiation of a B cell differentiation factor (BCDF)-sensitive line by BCDF (which contains no BCPF activity). IgSRK inhibited PWM-induced generation of cytoplasmic Ig+ cells but had no effect on Ig secretion from mature Ig-secreting cells. Taken together, these findings suggest that IgSRK (which contains the IgG fraction from pooled plasma from 2,000 healthy donors) can inhibit T cell-dependent or T cell factor-dependent B cell proliferation and B cell differentiation.     

T helper cell-dependent induction of resting B cell differentiation need not require cognate cell interactions

We have analyzed the role of cognate interaction with helper T cells (Th) in support of resting B cell differentiation to plaque formation. Co-culture of histoincompatible resting B cells and resting Th cells resulted in the induction of plaque-forming cells when dimeric but not monomeric fragments of anti-T cell receptor (TcR) antibody were added to culture. The efficiency of B cell activation was comparable to that supported by lipopolysaccharide and lectin-mediated Th-B cell conjugate formation. Further, if resting Th cells were preactivated with antigen and histocompatible antigen-presenting cells, the requirement for addition of anti-TcR to mixtures of histoincompatible Th and B cells was obviated. These results demonstrate that TcR-mediated Th recognition of major histoincompatibility complex class II/antigen composites on the resting B cell membrane does not provide obligate signals for B cell differentiation to plaque formation. We are left with two possibilities. Either the entire process of Th cell-dependent induction of resting B cell differentiation is mediated by soluble lymphokines or if Th-B cell contact is mandatory, it is mediated through nonpolymorphic cell surface determinants.     

Costimulation through the inducible costimulator ligand is essential for both T helper and B cell functions in T cell-dependent B cell responses

Costimulation through the inducible costimulator (ICOS) and its ligand (ICOSL) is essential for T cell-dependent B cell responses, but the cellular and temporal dynamics underlying its in vivo effects are poorly defined. Here we have shown that Icosl(-/-) and Icos(-/-) mice had similar phenotypes and that ICOS-ICOSL costimulation modulated the early but not late phases of IgG1 affinity maturation. Exploiting the adoptive transfer of T or B cells from primed Icosl(-/-) mice, we provided genetic evidence that costimulation through ICOSL was essential for primary but not secondary helper T cell responses and for the control of both T and B cell activities, resulting in T cell-dependent IgG1 production.     

Modulation of suppression of mitogen-induced T cell-dependent B cell responses by natural killer cells

We examined the ability of human natural killer (NK) cells to modualte pokeweed mitogen (PWM)-induced polyclonal antibody production. Highly purified NK cells inhibited plaque-forming cell (PFC) responses and this suppression could be substantially increased by preincubation of NK cells with the known enhancers of NK cell lytic activity, interferon (IFN)-alpha, IFN-gamma, and interleukin-2. Additionally, costimulation of NK cells with two anti-CD2 antibodies (9-1 and 9.6), which recognize different epitopes on the CD2 molecule, also augmented the inhibitory effect. When subpopulations of NK cells were assayed for suppressor cell activity, this activity was primarily mediated by NK cells bearing Leu-7 but not Leu-2 (CD8) antigens. Thus, alteration of NK cell lytic activity may have significant effects on the immunoregulatory functions of these cells, which may have important implications for the in vivo manipulation of cytotoxic responses.     

Separation of Sendai virus glycoproteins by using glutaraldehyde-treated erythrocytes and preparation of monospecific antisera against the glycoproteins

Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) glycoproteins, F and HN, were separated from Triton X-100- or Nonidet P-40-solubilized envelopes as unadsorbed and eluted fractions, respectively, by using glutaralde-hyde-treated chicken erythrocytes. These separated glycoproteins were biologically active. Monospecific antisera (in terms of monoreactivity to virus glycoproteins in gel diffusion precipitation patterns) were prepared by using these fractions as immunogens. Anti-HN rabbit serum inhibited all of the viral activities tested (infectivity, neuraminidase, hemagglutinating, and viral hemolysis), whereas anti-F serum definitely inhibited viral hemolysis only, although the two antisera enhanced neutralization in the presence of complement. The advantages and disadvantages of this separation method were discussed.     

Unique B cell responses in B cell-dependent and B cell-independent EAE

Previous studies characterized B cell-dependent and B cell-independent models of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. To further characterize the B cell response generated in these two models, the serum antibody response and the B cell surface immunoglobulin (Ig) repertoire were analyzed following immunization of wild-type C57BL/6 mice with either recombinant myelin oligodendrocyte glycoprotein (MOG; B cell-dependent EAE) or the encephalitogenic MOG(35-55) peptide (B cell-independent EAE). Plasma ELISA revealed responses to unique linear epitopes of MOG following immunization with recombinant MOG that were absent in MOG(35-55)-immunized animals. B cell repertoire analysis by RT-PCR identified a unique response restricted to 7183 Ig heavy chain variable gene family in mice immunized with recombinant MOG that was not observed in MOG(35-55)-immunized mice. These insights could aid in the identification of the relevant B cell populations important to the pathogenesis of B cell-dependent EAE and in the mechanisms by which these B cell populations contribute to disease.     

Sendai virus-specific T cell clones II. Induction of interferon production by Sendai virus-specific T helper cell clones

Interferon (IFN) was detected upon co-culture of cloned Sendai virus (SV)-specific T lymphocyte with SV-presenting syngeneic stimulator cells. The antiviral activity was defined as IFN-alpha, beta. The T cell clones, upon contact with antigen-presenting stimulator cells, stimulated adherent cells present in the stimulator cell population to secrete IFN. Induction of IFN production was independent from interleukin 2 production by the T cell clones.     

Host cell-dependent lateral mobility of viral glycoproteins

The lateral mobility of viral envelope proteins on the plasma membranes of infected cells is an important factor in both virus assembly and pathogenesis. The envelope glycoproteins of measles and human parainfluenza virus are mobile on the surfaces of infected HeLa cells and undergo lateral redistribution in the presence of specific antibody, forming unipolar caps. In contrast, no such redistribution was observed with influenza virus hemagglutinin (HA) or vesicular stomatitis virus (VSV) G glycoproteins on infected HeLa cell surfaces. However, the HA and G glycoproteins were both found to be mobile in the plasma membrane of CV-1 cells, or human or murine peritoneal macrophages. These results indicate that host cell-dependent as well as virus-specific factors are involved in determining viral glycoprotein mobility. No significant differences in the patterns of synthesis of influenza or VSV viral proteins were found in the various cell types examined. The HA and G proteins, when expressed from vaccinia virus recombinants, were each found to be immobile in HeLa cells and mobile in CV-1 cells, thus indicating that the host cell-dependent differences in mobility are an intrinsic property of each viral glycoprotein molecule and not the result of interaction with other viral components. It is suggested that the association of viral glycoproteins with either the cytoskeleton or membrane-associated cellular proteins may be related to the observed differences in lateral mobility.     

In vitro induction of T cell-dependent B cell differentiation in patients with common varied immunodeficiency

Patients with common varied immunodeficiency (CVI) are characterized by hypogammaglobulinemia. We investigated in vitro T cell-dependent B cell differentiation in CVI peripheral blood mononuclear cells by stimulating the T cells with an anti-CD3 (T3) monoclonal antibody. In cultures from CVI patients with no detectable circulating B cells, little immunoglobulin (Ig) was produced following anti-CD3 stimulation. In cultures from CVI patients with near-normal numbers of circulating B cells, anti-CD3 stimulation induced a normal percentage increase in Ig-secreting cells and appreciable (albeit subnormal) increases in IgG and IgM secretion. Cell-mixing experiments pointed to a quantitative, rather than qualitative, defect in B cell function in most of these CVI patients. Nevertheless, CVI T cells can induce substantial differentiation of autologous (and normal) B cells following anti-CD3 stimulation (which may mimic physiologic stimulation). This raises the possibility of correcting the hypogammaglobulinemia of CVI by in vivo or ex vivo administration of appropriate T cell stimuli.     

Selective triggering of B cell subpopulations by mitogens

We studied the pattern of responsiveness of normal spleen cells to three different B cell mitogens: dextran sulfate (DS), lipopolysaccharide (LPS) and purified protein derivative of tuberculin (PPD). Activation by DS results in extensive proliferation of cells, most of which are not high-rate antibody-secreting cells and can be reactivated. PPD activates high-rate antibody synthesis in cells which are more restricted in mitotic capacity. LPS seems to activate both of these subsets of B cells. LPS exhibits strong mitogenicity in vivo and a large fraction of LPS-activated cells are high-rate antibody-secreting end cells. It is suggested that these distinct subsets of B cells include cells at different stages of differentiation.     

Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens.

Techniques which identify hapten-specific B cells in tissues have been used to determine the sites of B cell activation in rat spleens in response to T cell-dependent (TD) antigens and T cell-independent type-1 (TI-1) antigens. Surface-associated hapten binding by specific memory B cells and B blasts was distinguished from the strong cytoplasmic hapten binding by specific plasma cells and plasmablasts. Blast cells in S phase were identified in tissue sections by staining cells which had been pulse labeled in vivo with 5-bromo-2'-deoxyuridine. Hapten-specific B blast cells are found in three sites: (a) around interdigitating cells in the T cell-rich zones; (b) in the follicular dendritic cell network and (c) in association with macrophages in the red pulp. Hapten-binding memory B cells, which are not in cell cycle, accumulate in the marginal zones and to a lesser extent the follicular mantles in response to TD and TI-1 antigens. The hapten-specific blast response in T zones is confined to the first few days after antigen is given and is low for primary responses to TD antigens, but massive on secondary challenge, when marginal zone memory B cells migrate to the T zones. Both the primary and secondary T zone responses to TI-1 antigens are impressive and in these responses hapten-specific B blasts are also found in the splenic red pulp. The follicular response to TD antigens starts with a small number of B blasts (fewer than five) entering each follicle. These increase in number exponentially so that by the 4th day after immunization they fill the follicle. The oligoclonality of the response is shown in simultaneous responses to two haptens where 6%-31% of the follicles on day 3 after immunization contain blasts specific for only one of the two haptens. During the 4th day classical zonal pattern of germinal centers develops. The surface immunoglobulin-positive B blasts are lost from the follicle center, while one pole of the follicular dendritic cell network fills with surface immunoglobulin-negative centroblasts. Centroblasts do not increase in numbers but divide to give rise to centrocytes, which re-express sIg and migrate into the follicular dendritic cell network. Cell kinetic studies indicate that the centrocyte population is renewed from centroblasts every 7 h. Centrocytes either leave the germinal center within this time or die in situ.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neutralizing activity of the antibodies against two kinds of envelope glycoproteins of Sendai virus

Summary Murine monoclonal antibodies against the fusion (F) and hemagglutininneuraminidase (HN) proteins of Sendai virus (SV) were prepared and studied on their antiviral activities, particularly on the neutralization of infectivity. On the analysis with solid phase competitive ELISA, 26 anti-HN antibodies were divided into at least four groups (HN-I, -II, -III and -IV). Antigenic sites recognized by the HN-I, -II, and -III group antibodies topographically separate from each other. Sites recognized by the HN-IV group antibodies overlaps partially with ones recognized by the HN-I, HN-II and -III group antibodies. The antibodies belonging to the HN-III group highly neutralize the infectivity of SV and weakly or not at all inhibit the hemagglutination (HA). In contrast, the HN-IV group antibodies strongly inhibit HA, but weakly neutralize the infectivity. Adsorption of SV to chicken red blood cells or L cells is inhibited by the HN-IV antibodies, but scarcely by the HN-III antibodies. On the other hand, incubation with HN-III antibodies of HeLa cells that have been preadsorbed with SV at 4° C, followed by culture at 37° C, causes inhibition of infection, but the HN-IV antibodies do not effectively interfere with such infection.The competitive ELISA showed that 17 anti-F antibodies were divided into two groups (F-I and -II). Two antigenic sites recognized by the antibodies, however, seem to be near to each other because a certain competition is observed between the antibodies of both groups. Two of the seven antibodies belonging to the F-II group inhibit the hemolysis activity and also neutralize the infectivity of SV, but the other five F-II antibodies do not. One of the anti-F antibodies has a low HI activity, and, in competition tests, competes with one of the anti-HN antibodies (HN-IV).With 2 Figures     

Interleukin-23 restrains regulatory T cell activity to drive T cell-dependent colitis

Interleukin-23 (IL-23) is an inflammatory cytokine that plays a key role in the pathogenesis of several autoimmune and inflammatory diseases. It orchestrates innate and T cell-mediated inflammatory pathways and can promote T helper 17 (Th17) cell responses. Utilizing a T cell transfer model, we showed that IL-23-dependent colitis did not require IL-17 secretion by T cells. Furthermore, IL-23-independent intestinal inflammation could develop if immunosuppressive pathways were reduced. The frequency of naive T cell-derived Foxp3+ cells in the colon increased in the absence of IL-23, indicating a role for IL-23 in controlling regulatory T cell induction. Foxp3-deficient T cells induced colitis when transferred into recipients lacking IL-23p19, showing that IL-23 was not essential for intestinal inflammation in the absence of Foxp3. Taken together, our data indicate that overriding immunosuppressive pathways is an important function of IL-23 in the intestine and could influence not only Th17 cell activity but also other types of immune responses.     

Correlation of rotational mobility and flexibility of Sendai virus spike glycoproteins with fusion activity

The rotational mobility of Sendai virus glycoprotein spikes was measured by flash-induced transient dichroism of eosin triplet probes. The possible importance of this molecular motion for function was investigated by parallel assays of hemagglutination and fusion with erythrocytes. For mobility measurements, the glycoproteins were labeled on amino groups with eosin-5-isothiocyanate and on the galactose residues of the oligosaccharide chains with eosin-5-thiosemicarbazide. The decay of the absorption anisotropy of both probes, which has a time constant of about 100-200 musec at 37 degrees is attributed to the rotation of the proteins about an axis normal to the plane of the membrane. This motion was inhibited by crosslinking of the spike proteins with glutaraldehyde or by the specific binding of human erythrocyte glycophorin (a virus receptor) to the HN glycoprotein. Low values of the initial anisotropy for both probes indicate the existence of a second, faster motion. This is attributed to segmental motion of the glycoproteins. Segmental motion is inhibited by crosslinking with glutaraldehyde but appears to be little affected by interaction with glycophorin. The temperature dependence of the segmental and rotational motion of the proteins revealed a pronounced increase in mobility in the range of 30-35 degrees which was not paralleled by the lipid motion of the Sendai virus envelope membrane. Since the temperature dependence of virus-induced hemolysis has a similar characteristic, the mobility of glycoproteins appears to be correlated with the fusion activity. The hemagglutination activity, however, is not dependent on the mobility of the glycoprotein spikes.     

Interaction between Sendai virus and the cell membrane II. The role of Sendai virus neuraminidase in haemolysis

When chicken erythrocytes (CRBC) were pretreated with non-haemolytic Sendai virions or isolated HANA spikes, they acquired a resistance to the haemolytic action of "second challenge" viruses. This resistance was dependent on the quantity of N-acetylneuraminic acid liberated from the surface of the CRBC by the initial virus, and not on the use of different viral sources. When exposed to Sendai virus, CRBC were more difficult to be lysed and easier liberated N-acetylneuraminic acid than human 0 erythrocytes. The restricted number of virions able to fuse CRBC was explained by such neuraminidase function of the HANA spike of the virion.     

Priming of helper T cell-dependent antibody responses by hemagglutinin-transgenic B cells

Mice expressing the hemagglutinin (HA) gene of influenza virus PR8 (H1 subtype) under the control of x light chain promoter and enhancer have been generated. They express HA in and on B cells, and are tolerant to HA. In vitro, only lipopolysaccharide (LPS) blasts but not resting B cells of transgenic mice can stimulate HA-specific helper T cells of HA-specific α/β T cell receptor (TCR)-transgenic mice. Transfer of HA-transgenic LPS blasts into syngeneic, non-transgenic recipients primes HA-specific antibody responses. Resting, small HA-transgenic B cells, which were purified by fluorescence-activated cell sorting, prime lower antibody responses. Host B cells produce the HA-specific antibody response. The donor HA-transgenic B cells need to express major histocompatibility complex (MHC) class II molecules and need to be alive to induce the antibody response in the host. Most notably, the host antibody response never produces detectable levels of IgM, but only of switched IgG isotypes. Neither resting nor activated HA-transgenic B cells induce tolerance in antibody responses. These results suggest that HA-transgenic B cells, presenting both the intact antigen on the cell surface and peptides of the antigen on MHC class II, are effective inducers of helper T cell responses, and as judged by the Ig-isotype response pattern, which is mainly IgG1, of Th2 type.     

Interleukin 2 induces T cell-dependent IgM production in human B cells

Conditioned medium, obtained from mononuclear cells after activation with concanavalin A and phorbol myristate, strongly stimulated the in vitro production of IgM by human mononuclear cells. Partial purification by gel filtration showed that this activity co-eluted with interleukin 2 (IL 2). Via removal by adsorption and further purification by chromatofocusing techniques, it was demonstrated that the factor in this conditioned medium, responsible for the IgM production, was in fact IL 2. Experiments with purified IL 2 from the human IL 2-producing Jurkat cell line as well as recombinant IL 2 confirmed its capacity to induce IgM production. In the absence of T cells, IL 2 could not activate Ig synthesis, suggesting an indirect effect of IL 2 in the induction of the helper signals for B cells. Evidence is presented that partially purified conditioned medium contained another factor, distinct from IL 2, with the capacity to differentiate human B lymphocytes in the absence of T cells into IgM-producing cells.