T-cell regulation of pokeweed-mitogen-induced polyclonal immunoglobulin production in mice. III. Role of Lyt 1+2- non-helper T cells in the suppression.

A large number of cells which can replicate vesicular stomatitis virus (VSV) were generated in pokeweed mitogen (PWM)-stimulated cultures of normal mouse spleen cells. The optimal dose of PWM for the generation of VSV-replicating cells was within the range 25 and 50 micrograms/ml, which had previously been shown to be optimal for the induction of suppressor cells. The VSV-replicating cells in this system were shown to be Thy 1+ and Lyt 1+2-. These cells, however, were unlikely to be helper T cells, but may be a subset of T cells involved in suppression. Thus, inoculation of VSV into a PWM-stimulated culture of spleen cells resulted in marked augmentation of polyclonal immunoglobulin production. Further, it was shown that the suppressive activity of T cells which had been precultured with a high dose of PWM was abolished by the infection with this virus.     

Cellular quiescence modulates and replication of L15 mutants of vesicular stomatitis virus

Infectious particle production by temperature-sensitive (ts) mutants of vesicular stomatitis virus (VSV) was measured in a variety of different host cell types maintained in a state of quiescence or stimulated to proliferate. At permissive temperatures, all ts mutants and the wild-type virus replicated equally well and with the same kinetics in both quiescent and proliferating cells. At semi-permissive temperatures, however, Lts mutants, with temperature-sensitive virion polymerases, showed a delay of about 6 h in infectious particle production relative to wild-type virus in proliferating cells and greater than 16 h in quiescent cells. The effect was specific for the Lts class of mutants and was not seen for representative mutants in any of the four other complementation groups of VSV. Regarding cellular determinants, the effect was correlated only with the growth phase and not with the species of origin, interferon inducibility or with malignant transformation.     

T cell hybridomas define the class II MHC-restricted response to vesicular stomatitis virus infection

T cell hybridomas with specificity for VSV (vesicular stomatitis virus)-infected cells were generated in an attempt to better define the la-restricted helper T cell response to VSV. The hybridomas were created by fusing BALB/c (H-2d) anti-VSV immune spleen cells to the murine thymoma BW 5147. These hybridomas produce IL-2 when stimulated with VSV-infected spleen cells. They were found to recognize viral antigens in association with I-Ad and, in addition, could also be stimulated by VSV-infected A20 cells (an Ia-positive B cell lymphoma of H-2d origin). The purified viral membrane glycoprotein, G protein, and Gs (secreted G protein that lacks the hydrophobic and intracytoplasmic domains) both stimulated IL-2 production when added to cultures of A20 and the hybridomas. These hybridomas therefore recognize a viral antigenic determinant on G protein. Since chemically-fixed antigen-presenting cells fail to stimulate the hybridomas after exogenous addition of purified G protein we can conclude that these T cell hybridomas recognize a processed form of the G protein. Stimulator cells created by expression in A20 of a transfected cDNA encoding G protein were also recognized. Recognition in this case was I-Ad-restricted, as anti-I-Ad monoclonal antibodies blocked stimulation, and an Ia-negative cell (P815) expressing a transfected G protein gene failed to stimulate the hybridomas. Even after paraformaldehyde fixation, G gene-transfected, Ia-positive cells could stimulate the hybridomas, suggesting that processing of this endogenously-synthesized antigen has occurred.     

Host gene influence on interferon action in adult mouse hepatocytes: specificity for influenza virus

Primary monolayer cultures of hepatocytes isolated from adult mice either genetically resistant (bearing the autosomal dominant allele Mx) or genetically susceptible toward influenza virus infection were tested for susceptibility to three viruses: a hepatotropic influenza A virus, vesicular stomatitis virus, and herpes simplex type I virus. Hepatocytes of both genotypes spontaneously released equal amounts of interferon during the first day of culture. Spontaneous interferon was sufficient to protect the cells markedly against vesicular stomatitis virus and marginally against herpesvirus infection. With respect to influenza virus, only Mx-bearing hepatocytes lost their initial susceptibility. Treatment with anti-interferon antibodies during the first 24 hr of culture rendered Mx-bearing cells as permissive for all three viruses as non-Mx-bearing cells. In such anti-interferon-treated cultures the antiviral resistance could be restored with highly purified mouse type I interferon. The antiviral state induced by exogenous interferon displayed the same characteristics as that induced by spontaneous interferon: it was highly efficient in inhibiting influenza A virus replication in Mx-bearing cells and inefficient in non-Mx-bearing cells. Inhibition of vesicular stomatitis virus or herpesvirus was independent of the Mx genotype.     

T helper cell unresponsiveness: Rapid induction in antigen-transgenic and reversion in non-transgenic mice

T cell tolerance is usually established by clonal deletion of self-specific T cells in the thymus, or some times, in the periphery. Alternatively, tolerance may also be achieved by induction of clonal T cell unresponsiveness by a poorly understood mechanism called “anergy”. We found that transgenic mice expressing a soluble form of vesicular stomatitis virus (VSV) glycoprotein (G) predominantly in liver and kidney exhibited normal B cell responses. VSV-G-specific T help-independent neutralizing IgM responses were within normal ranges, but no T help-dependent neutralizing IgG antibodies were generated upon immunization with recombinant VSV-G protein and recombinant vaccinia virus expressing VSV-G. This demonstrated absence of B cell tolerance but presence of T helper cell unresponsiveness. After adoptive transfer of transgenic spleen cells into thymectomized immuno-incompetent hosts, the unresponsive T helper cells regained function and switched the neutralizing IgM response to IgG, comparably to control T helper cells, within 7 days. Conversely, when naive non-transgenic spleen cells were transferred into transgenic mice, VSV-G-specific T helper cells became unresponsive within 3–4 days. These results suggest that VSV-G-specific T helper cells are rendered unresponsive within a few days in the VSV-G transgenic host also outside of the thymus and that this unresponsiveness was reversed by transfer into antigen-free recipients.     

Lysis by natural killer cells requires viral replication and glycoprotein expression

Several lines of evidence suggest that natural killer cells (NK) have an important role in antiviral defense. To be thus effective, NK cells have to recognize and cause lysis of virus-infected cells. The mechanism underlying this interaction was investigated in a murine system using vesicular stomatitis virus (VSV). A large number of cell lines was screened to identify a permissive target for VSV infection and the B16 murine melanoma cells were chosen since VSV replicates in these cells without producing cytopathic effects 24 h after infection. Spontaneous lysis of B16 melanoma cells by spleen cells occurred only if the target cells were previously infected with VSV. Treatment of spleen cells with anti NK 1.1 or anti Thy 1.2 plus complement decreased the specific lysis by 50%, therefore, the phenotype of the effector cells in this system corresponds to the NK cell subset. Immunofluorescent staining with polyclonal and monoclonal anti-VSV antibodies demonstrated that the viral glycoprotein G is abundantly expressed on the target cell surface. Treatment of the VSV-infected targets with tunicamycin prevented glycosylation of newly synthesized VSV glycoprotein G on the cell membrane. This treatment abrogated completely NK-mediated killing of the infected B16 melanoma cells. These results indicate that virus replication and membrane expression of glycosylated protein G are essential for recognition and lysis of VSV-infected targets by NK cells.     

Vesicular stomatitis virus plaque production in monolayer cultures with liquid overlay medium: description and adaptation to a one-day, human interferon-plaque.

Vesicular stomatitis virus forms discrete, microscopic plaques in stationary cultures of the WISH amnion cell line. Microplaque formation is rapid, reproducible, and easily quantitated, occurs at temperatures ranging from 33 to 40 degrees C, and does not require a semisolid overlay. WISH cells, however, are less sensitive to vesicular stomatitis virus than are chicken embryo, 3T6, or Vero cells. WISH amnion cells also are highly sensitive to the antiviral effects of human interferon, and a quantitative human interferon assay, based on vesicular stomatitis virus plaque reduction in WISH cells, is described. This interferon assay can be performed within 1 day, uses a liquid overlay medium, does not require a vital stain, is as sensitive as other methods that use diploid cell strains, and is performed in a microtiter system.     

Effect of cellular inhibitors on the infection of various susceptible cells with vesicular stomatitis virus

The effect of some cellular function inhibitors on adsorption and successive events of penetration of vesicular stomatitis virus to phylogenetically unrelated permissive cells was investigated. Treatment of HeLa, CER, EPC and Aedes albopictus cells with colcemide and cytochalasin D which affect cytoskeleton organization indicated that microfilaments but not microtubules were involved in the early events of VSV infection. Inhibitors of oxidative phosphorylation such as dinitrophenol and sodium azide, and the glycolysis inhibitor 2-deoxy-D-glucose, did not allow multiplication of prebound virions, demonstrating that VSV replication is largely energy dependent in either host cells examined.     

TAP1-independent loading of class I molecules by exogenous viral proteins

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1?/?) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glycoand nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1 ?/? cells as by control cells. Peptide regurgitation was not involved. Since particulate, nonreplicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.     

Interferon induction by vesicular stomatitis virus and its role in virus replication.

Antibody against human fibroblast interferon increased the yields of vesicular stomatitis virus in human cells. The results show that endogenous interferon produced in the course of multicycle vesicular stomatitis virus infection depresses virus yields.     

Replication of vesicular stomatitis virus in mouse spleen cells.

Mouse spleen cells which normally cannot support the in vivo replication of vesicular stomatitis virus (VSV) became susceptible to VSV infection after the intraperitoneal growth of certain syngeneic and allogeneic tumors. After 3 days' growth of P815 tumor cells in syngeneic DBA/2 mice, the viral-permissive state for VSV replication had been established. By 7 days after tumor in inoculation, up to 18% of the spleen cells were producing virus yielding greater than 10(8) plaque-forming units per spleen. Similarly, P815 cells induced the viral-permissive state in allogeneic C3H/HeN mice. Tumors other than P815 were also effective in permitting VSV growth in the spleen. The presence of tumor cells themselves was not sufficient for VSV growth, yet cell-free ascitic fluid from mice bearing syngeneic tumors inoculated 3 h before infection allowed for VSV replication. Cell-free supernatant from a T-cell hybridoma synthesizing interleukin-2 was also effective in permitting virus growth when inoculated 3 h before infection. The virus-permissive cell has been characterized as a nylon wool-adherent and plastic dish-nonadherent spleen cell.     

Direct priming of antiviral CD8+ T cells in the peripheral interfollicular region of lymph nodes

It is uncertain how antiviral lymphocytes are activated in draining lymph nodes, the site where adaptive immune responses are initiated. Here, using intravital microscopy we show that after infection of mice with vaccinia virus (a large DNA virus) or vesicular stomatitis virus (a small RNA virus), virions drained to the lymph node and infected cells residing just beneath the subcapsular sinus. Naive CD8+ T cells rapidly migrated to infected cells in the peripheral interfollicular region and then formed tight interactions with dendritic cells, leading to complete T cell activation. Thus, antigen presentation at the lymph node periphery, not at lymphocyte exit sites in deeper lymph node venules, as dogma dictates, has a dominant function in antiviral CD8+ T cell activation.     

Homologous interference induced by a temperature-sensitive mutant derived from an HVJ (Sendai virus) carrier culture.

Homologous interference between a temperature-sensitive small plaque mutant (HVJ-pB) derived from an HVJ (haemagglutinating virus of Japan - the Sendai strain of parainfluenza I virus) carrier culture of BHK cells and the original wild-type virus (HVJ-W) has been investigated. Prior infection of LLCMK2, HeLa, BHK or mouse L cells with HVJ-pB, both at permissive and non-permissive temperatures, for 24 h resulted in a reduced yield of superinfecting HVJ-W, reflecting a smaller number of cells capable of producing the superinfecting virus. However, HVJ-pB did not interfere with the replication of vesicular stomatitis virus, Sindbis virus or Newcastle disease virus. Interference in this system seems to be due to inhibition of the attachment of superinfecting HVJ-W as a result of intracellular mechanisms operating at a late stage in the replication of the interfering virus. There is also blocking or destruction of cellular receptors by extra-cellular particles of the interfering virus. Protein synthesis coded for by the complete virus genome is required to establish and maintain the interference, and treatment with actinomycin D has no effect on the interference phenomenon.     

Lymphocyte activation: IV. The ultrastructural pattern of the response of mouse T and B cells to mitogenic stimulation in vitro

Thymocytes from cortisone-treated mice (`T' cells), `B' spleen cells (B lymphocytes from thymectomized, irradiated, marrow reconstituted mice) and normal spleen (T + B) cells were examined by electron microscopy after 60 hours stimulation by Concanavalin A (a T cell specific mitogen), endotoxin (B cell specific mitogen), and pokeweed mitogen (which stimulates both T and B cells). Stimulation of T cells by Con A or PWM induced the appearance of lymphoblasts (Type I) and only PWM or endotoxin stimulated B cells developed `plasmablast' features (dilated, vesicular rough endoplasmic reticulum; Type II). A few stimulated B cells also had lymphoblast morphology. Large cells from normal (T + B) spleen stimulated by PWM were heterogeneous consisting of 55–60 per cent plasmablasts and 40–45 per cent lymphoblasts. It was concluded that the ultrastructure of stimulated lymphocytes depended on whether T or B cells were stimulated and not primarily on the mitogen used. In general, the response evoked by mitogens paralleled at the ultrastructural level that induced by antigens. It was also found that multivesicular bodies and glycogen particles occurred predominantly in the cytoplasm of stimulated T cells (lymphoblasts).     

Vaccinia virus stimulates the growth of vesicular stomatitis virus at the level of protein synthesis in mouse L cells

Coinfection with vaccinia virus increases the growth of vesicular stomatitis virus (VSV) in mouse L cells by 10- to 20-fold. Although vaccinia has no significant effect on RNA synthesis by VSV, VSV protein synthesis is dramatically stimulated by double infection. The enhancement of VSV growth is correlated with the ability of vaccinia to inhibit the VSV-mediated damage to the host translational machinery. Coinfection with vaccinia fails to stimulate the growth of a VSV mutant which is deficient in its ability to shut off protein synthesis during infection.     

Detection of mitogen-activated T and non-T lymphocytes by virus plaque assay. Virus plaque assay on the cells fractionated by unit gravity sedimentation.

Virus plaque assay (VPA) was utilized for the quantitative evaluation of activated lymphocytes. We examined what types of cells, especially which of activated T and non-T lymphocytes, were detected as infective centres after infection with vesicular stomatitis virus. Marked increases in DNA synthesis and in virus-plaque forming cells (V-PFC) were observed not only during the activation of T lymphocytes with Con A, but also, though to a lesser extent, during the activation with lipopolysaccharide (LPS) of non-T lymphocyte preparations of nude spleen from which theta-positive lymphocytes and macrophages were completely depleted. The latter observation was further confirmed by the VPA on the populations enriched in LPS-activated non-T lymphocytes fractionated by the unit gravity sedimentation method. Fast sedimenting cells were found to be more active in DNA synthesis and contained more infective centres after infection than those sedimenting slowly and original unfractionated cells. Both the capacity for DNA synthesis and virus-replication were considered to be general properties accompanying lymphocyte activation.     

Measles virus-induced promotion of dendritic cell maturation by soluble mediators does not overcome the immunosuppressive activity of viral glycoproteins on the cell surface

Measles virus (MV) infection promotes maturation of dendritic cells (DC), but also interferes with DC functions, and MV renders the DC inhibitory for T cell proliferation. We now describe that MV infection triggers the release of type I IFN from monocyte-derived DC (Mo-DC) which contributes to DC maturation. There is no evidence that soluble mediators are released interfering with the stimulatory activity of uninfected DC. Since inhibition of allogeneic T cell proliferation was unaffected by a fusion inhibitory peptide (Z-fFG), MV infection of T cells did not contribute to inhibition. Allogeneic T cell proliferation depended on the percentage of DC expressing MV F/H glycoproteins within the DC population and their surface expression levels, was induced upon addition of UV-inactivated MV to a mixed lymphocyte reaction stimulated by lipopolysaccharide-matured DC, and was not induced by DC infected with a recombinant MV encoding the ectodomain of vesicular stomatitis virus G protein (MG/FV) instead of the MV glycoproteins. Similarly, DC infected with MV, but not with MG/FV inhibited mitogen-induced proliferation of T cells. Thus, a dominant inhibitory signal is delivered to T cells by the MV glycoproteins on the surface of DC overcoming positive signals by co-stimulatory molecules promoted by maturation factors released from infected DC.