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Biomedical subjects

R V Blanden

Publications and source records attributed to R V Blanden.

At least 19 recordsLinked to original sources
PubMed1992

Restimulated memory Tc cells have a higher apparent avidity of interaction with targets than primary virus-immune Tc cells as indicated by anti-CD8 blocking.

Previous experiments have shown that whereas a secondary in vitro Kunjin-immune cytotoxic T (Tc) cell population lysed equally well targets infected with either native flavivirus or a recombinant vaccinia virus expressing the immunodominant determinant, primary in vivo Kunjin-immune Tc cells were able to lyse only the recombinant vaccinia virus-infected targets. Using CD8 blockade to assess the avidity of T cell-target interaction, recombinant-infected targets express antigen more efficiently than native flavivirus infected targets and secondary in vitro Kunjin-immune Tc cells have a higher avidity for targets than do primary in vivo Kunjin-immune Tc cells. Secondary in vivo influenza-immune Tc cells are also of higher avidity than primary in vivo influenza-immune Tc cells. Thus, a restimulated memory Tc cell population interacts with targets with greater avidity than does a recently activated naive population.

Animals↗
PubMed1992

Broad cross-reactivity with marked fine specificity in the cytotoxic T cell response to flaviviruses.

Cytotoxic T (Tc) cells were generated in mice of five H-2 haplotypes against the flaviviruses Kunjin and West Nile (WNV). A panel of recombinant vaccinia viruses which between them expressed cDNA of the entire Kunjin virus genome were used to infect targets. Anti-Kunjin virus responses to determinants derived from non-structural proteins, especially NS3, NS4A and NS4B, were dominant in most mouse strains; usually only one class I major histocompatibility complex (MHC) restriction element was involved. WNV-immune Tc cells showed similar but not identical patterns of antigen recognition to Kunjin virus-immune Tc cells. The extent to which WNV-immune Tc cells recognized Kunjin virus-encoded determinants varied considerably between mice of different MHC haplotypes.

Animals↗
PubMed1992

Recombinant vaccine vector-induced protection of athymic, nude mice from influenza A virus infection. Analysis of protective mechanisms.

Athymic, nude mice, which normally succumb to virus infection, can resolve infection with recombinant vaccinia virus (rVV) engineered to express IL-2. We have demonstrated that interferon-gamma (IFN-gamma) produced by natural killer (NK) cells and other immunocytes in response to the virus-encoded interleukin-2 (IL-2) is crucial to recovery. Here, we extend this work to show that nude mice, when primed intravenously with rVV co-expressing both IL-2 and an influenza virus haemagglutinin (HA) gene, are also protected following challenge with a lethal dose of homologous influenza virus. A substantial increase in the number of influenza virus-reactive antibody-secreting cells producing antibody of the IgM isotype, but not of the IgG or IgA isotypes, was found in spleens and lungs of the protected mice. Treatment with monoclonal antibodies to IFN-gamma or to the NK marker, as GM1, at challenge and thereafter, led to their death however, though the specific IgM antibody response was unaffected. These data suggest that both specific antibody and non-specific antiviral reactivity are important elements of the protective response and show that this immunization strategy may be used to protect severely immunocompromised individuals.

Animals↗
PubMed1992

Analysis of murine major histocompatibility complex class II-restricted T-cell responses to the flavivirus Kunjin by using vaccinia virus expression.

The present paper analyzes the influence of major histocompatibility complex (MHC) class II (Ir) genes on MHC class II-restricted T-cell responses to West Nile virus (WNV) and recombinant vaccinia virus-derived Kunjin virus antigens and identifies the immunodominant Kunjin virus antigens. Generally, mice were primed by intravenous infection with WNV or Kunjin virus, and their CD4+ T cells were stimulated in vitro 14 days later with WNV or Kunjin virus antigens to pulse macrophage or B-cell antigen-presenting cells (APC). WNV-specific in vitro T-cell responses from H-2b mice were higher than those from H-2d, H-2k, and H-2q mice. When recombinant vaccinia virus-derived Kunjin virus antigen preparations were tested in vitro, Kunjin virus-immune T cells of H-2b haplotype responded most strongly to structural (prM, C, E) and membrane-associated nonstructural (NS1) proteins encoded by VKV 1031 and showed weaker responses to cytosolic nonstructural protein NS5 (VKV 1022), whereas the responders of H-2k haplotype responded most strongly to the antigens encoded by VKV 1022 and gave lesser responses to VKV 1031. H-2d T cells gave weaker responses than either H-2b or H-2k cells, with responses to VKV 1031 generally being higher than those to VKV 1022. Responses to VKV 1023 or VKV 1024 encoding all of the NS3 to NS5 gene sequence or to VKV 1023 encoding all of NS3 were weak or absent. Within a given inbred strain, B cells and macrophages differed in their abilities to present recombinant vaccinia virus-derived Kunjin virus antigens, both in terms of magnitude of T-cell responses induced and the particular Kunjin virus protein presented. T cells from different non-MHC genetic backgrounds varied in their requirements of macrophage numbers as APC for maximum reactivity, suggesting that the concentration of class II MHC antigens and other molecules affecting APC-T-cell interaction varied in mice with different genetic backgrounds. Regardless of MHC haplotype, responses to VKV 1024, which encompasses VKV 1023 and VKV 1022, were either absent or lower than those to VKV 1022, possibly reflecting differences in the processing requirements of these two proteins. When mice were primed intravenously with recombinant vaccinia virus and when their CD4+ T cells were stimulated in vitro with native Kunjin virus antigens, VKV 1031 primed more efficiently than Kunjin virus and VKV 1022 primed similarly to Kunjin virus.

Animals↗
PubMed1991

Immunobiology of infection with recombinant vaccinia virus encoding murine IL-2. Mechanisms of rapid viral clearance in immunocompetent mice.

CD8+ cytotoxic T (Tc) lymphocytes mediate recovery from vaccinia virus (VV) infection. In mice, anti-VV Tc cells are detectable on or after day 3 after infection, and cytolytic activity peaks between days 5 and 6. A rVV encoding murine IL-2, VV-hemagglutinin (HA)-IL-2, was cleared more rapidly, compared with a control rVV, VV-HA-thymidine kinase (TK), from tissues of infected euthymic normal mice. The mechanism of VV-HA-IL-2 clearance was operative early in infection and correlated with an elevated NK cell response, before the induction of anti-VV Tc cell response. We have investigated the roles of NK cells, T cells, and IFN-gamma in the rapid clearance of VV-HA-IL-2, by using specific mAb. Depletion of NK cells with mAb significantly enhanced VV-HA-IL-2 but not VV-HA-TK titers 3 days after infection. NK cells alone could not account for rapid viral clearance, because VV-HA-IL-2 titers in NK cell-depleted mice were not comparable to VV-HA-TK titers. Treatment with a mAb to IFN-gamma completely abrogated the IL-2-induced mechanism(s) of VV-HA-IL-2 clearance, and titers of the IL-2-encoding virus were comparable to control virus titers. In addition, the elimination of CD4+ but not CD8+ T cells resulted in significant increases in VV-HA-IL-2 titers.

Animals↗
PubMed1991

Alloreactive cytotoxic T cells recognize MHC class I antigen without peptide specificity.

In this report, experiments are described to differentiate between three potential models of class I MHC allorecognition, namely 1) recognition of peptide-free MHC, 2) peptide-MHC-specific recognition, and 3) peptide-MHC-nonspecific recognition. Using a nucleoprotein peptide (NPP) with a sequence derived from influenza virus nucleoprotein with high affinity for Kd class I MHC molecules, it is shown that target cells rapidly become lysable by Kd-NPP self-restricted cytotoxic T (Tc) cells, and retain sufficient Kd-NPP complexes for at least 72 h. Kd-specific alloreactive Tc cells at the clonal and polyclonal level do not show decreased lysis of Kd-bearing targets in the continuous long term (48 h) presence of NPP. Kd-stimulator cells modified with NPP are able to induce potent Kd-NPP-specific self-restricted Tc cells, however Kd-NPP stimulator cells do not generate Kd-NPP specific alloreactive Tc cells from CBA and B10.A (5R) mouse strains as tested by limiting dilution split clone experiments. Human cells infected with the vaccinia virus recombinant coding for the murine Kd class I MHC Ag can be lysed by murine Kd-specific alloreactive Tc cells. In addition the rate of reemergence of alloreactive and self-restricted Tc cell epitopes on virally infected target cells that had their cell-surface class I MHC Ag removed is identical. These results are consistent with model 3 namely that the majority of Tc precursor and effector cells recognize class I MHC Ag without peptide specificity.

Animals↗
PubMed1991

Effects of vaccinia virus-expressed interleukin 2 on the immune system of sublethally irradiated mice.

Vaccinia virus that expressed interleukin 2 (IL 2) was cleared from immunodeficient irradiated mice more efficiently than virus that did not express interleukin 2. These results extend the previously observed protection from nude mice to another model of immunodeficiency. No antibody or cytotoxic T lymphocyte response could be detected in sublethally irradiated mice that had been inoculated with IL 2-expressing vaccinia virus, but levels of splenic natural killer cell activity were elevated. Sublethally irradiated mice that had recovered from IL 2-plus hemagglutinin-expressing vaccinia virus were partially protected against both influenza virus and vaccinia virus. These results indicate that vaccinia virus-expressed IL 2 mediates clearance of primary viral infection via a mechanism that does not involve antibody or cytotoxic T lymphocytes. They also indicate that inclusion of lymphokine genes in live recombinant viral vaccine vectors may increase vaccine safety.

Administration, Intranasal↗
PubMed1991

Functional analysis of macrophages, B cells and splenic dendritic cells as antigen-presenting cells in West Nile virus-specific murine T lymphocyte proliferation.

In this paper, the relative efficacy of macrophages, B cells and splenic dendritic cells (SDC) in presenting West Nile virus (WNV) antigens to WNV memory CD4+ T cells is examined. The results indicate that, under appropriate conditions, all these cell types can function as antigen-presenting cells (APC). Listeria-induced peritoneal macrophages induced higher proliferative responses than SDC or B cells derived from naive or 14 day WNV-primed mice. The ability of Listeria-induced macrophage populations to present antigen was specifically inhibited by anti-Class II major histocompatibility complex (MHC) antibodies. On a cell population basis, B cells obtained from mice primed with WNV 14 days previously evoked higher responses than resting B cells. B cells from mice receiving weekly injections of WNV over a period of 4 weeks elicited optimal responses with lower doses of antigen than naive or 14 day WNV-primed B cells. When macrophages were used as APC, addition of specific antibodies to WNV resulted in increased efficiency of presentation, probably due to increased uptake of antigen by opsonization. In contrast, addition of anti-WNV antibodies to hyperimmune B cells reduced their efficacy presumably by reducing uptake of antigen by B cell surface immunoglobulin. When SDC from C57BL/6 mice were used as APC, WNV-specific proliferative responses were directly related to the number of stimulator cells used, and the background proliferation with mock antigen was two- to five-fold lower than specific responses. Higher levels of background proliferation were stimulated by SDC from CBA/H mice so that the antigen-specific responses were always less than two-fold higher than background.

Animals↗
PubMed1991

Effect of high ligand concentration on West Nile virus-specific T cell proliferation.

In this paper the phenomenon of suppression of proliferation in vitro of 14 day primed, West Nile Virus (WNV)-specific, murine CD4+ T cells by large numbers of antigen-presenting macrophages and B cells has been investigated. Suppression was apparently not mediated by prostaglandins, as the use of indomethacin in cultures at four times the usual concentration did not reverse suppression. Experiments were designed to evaluate the contribution of major histocompatibility complex (MHC) Class II and nominal WNV antigens in causing suppression of T cell proliferation. Listeria- or thioglycollate-induced macrophages from CBA/H (H-2k) mice, when treated with heat-killed Listeria in vitro for 1 h to maintain or increase, respectively, MHC Class II levels before the addition of alloreactive Iak-specific T cells caused inverse dose-responses; the highest T cell proliferation occurred at a stimulator to responder (S : R) ratio of 0.25 and profound suppression at a S : R ratio of 1 or 2. In contrast, untreated thioglycollate-induced macrophages, which express low MHC Class II levels, gave a direct dose-response with increasing T cell proliferation as antigen-presenting cell (APC) numbers increased. Addition of anti-Ia antibodies (or their Fab fragments) to cultures caused a significant reversal of suppression of anti-WNV T cells imposed by high numbers of Listeria-induced macrophages or 14 day WNV-primed B cell APC. Suppression was also reversed by reducing the concentration of WNV antigen. These observations support the notion that the suppression of T cell proliferation observed at high S : R ratios was due to high concentrations of ligand (WNV-derived peptide complexed with Class II MHC) on APC.

Animals↗
PubMed1991

In vitro T-cell proliferative response to the flavivirus, west Nile.

West Nile virus (WNV)-specific murine T-cell proliferation in vitro was investigated in terms of conditions that optimize antigen-specific responses and reduce background proliferation. The responder populations consisted of splenocytes from WNV-primed mice enriched for L3T4+ T cells. Ia+ antigen-presenting cells (APC) were derived from splenocytes of WNV-primed or naive mice. Antigen was a lysate prepared from WNV-infected Vero cells at 12 h postinfection. Strong virus-specific proliferative responses were observed when antigen-pulsed APC were cocultured with responders at a 1:1 ratio. Substantial nonspecific proliferation occurred when culture medium supplemented with 5% fetal bovine serum (FBS) was used, whereas with 1% normal mouse serum a higher degree of antigen specificity was evident, although the magnitude of the responses was lower. The best separation between antigen-specific and background proliferation was obtained by using an exogenous source of T-cell growth factors to amplify for 2 days the proliferation of L3T4+ cells triggered by an initial 3 days of culture with antigen-pulsed APC. This investigation has defined optimal conditions for investigating the stimulation of WNV-primed L3T4+ T-cell proliferation in response to the presentation of viral gene products by Ia+ APC. This assay should permit detailed analysis of the efficiency of various APC populations and identification of viral antigens that stimulate the proliferation of Class II MHC-restricted T cells.

Animals↗
PubMed1991

Preliminary analysis of murine cytotoxic T cell responses to the proteins of the flavivirus Kunjin using vaccinia virus expression.

A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide.

Animals↗
PubMed1990

Interferon gamma is involved in the recovery of athymic nude mice from recombinant vaccinia virus/interleukin 2 infection.

Athymic nude mice recover from an infection with recombinant vaccinia virus (VV) encoding murine interleukin 2 (IL-2), but treatment with a mAb to IL-2 accentuated infection. Administration of a mAb against interferon gamma (IFN-gamma) to mice infected with the IL-2-encoding virus completely prevented the IL-2-induced mechanisms of recovery. Both asialo-GM1+ (NK) and asialo-GM1- (non-NK) cells were participants in the IFN-gamma-mediated recovery of nude mice from infection with the IL-2-encoding VV recombinant. Depletion of asialo-GM1+ cells exacerbated infection, though not as much as anti-IFN-gamma mAb. In vitro, both asialo-GM1+ and asialo-GM1- nude mouse splenocytes produced IFN-gamma in response to IL-2.

Animals↗
PubMed1990

Selection of the T-cell repertoire in transgenic mice expressing a transplantation antigen in distinct thymus subsets.

Transgenic mice that expressed a transplantation antigen, H-2Kb, in an unusual tissue distribution have been developed. Gene-regulatory elements from the immunoglobulin heavy-chain locus (Emu enhancer and heavy chain promoter) were linked to the class I Kb gene and the construct microinjected into fertilized mouse eggs of a different haplotype. It was expected that such gene-regulatory elements would direct expression of the foreign class I molecules only to B and T lymphocytes. However, expression was also detected in a subset of thymus medullary epithelium. The Kb molecules expressed on this thymic subset were unable to positively select T cells for passage to the periphery. The mice were, however, tolerant of the cell types expressing the foreign Kb molecules and were also tolerant of Kb presented as skin grafts. These results suggest that not all components of thymic epithelium are involved in positive selection of T cells and that transplantation antigens expressed on non-dendritic cells can induce tolerance.

Animals↗
PubMed1990

Elevated natural killer cell responses in mice infected with recombinant vaccinia virus encoding murine IL-2.

The role of cytotoxic T cells and NK cells in the recovery of immunodeficient, athymic, nude mice infected with a recombinant vaccinia virus (VV) encoding murine IL-2 was investigated. Kinetic studies with the IL-2-encoding recombinant (VV-HA-IL2) and control (VV-HA-TK) viruses excluded a role for cytotoxic T cells but suggested the possible involvement of NK cells. In athymic nude mice given VV-HA-IL2, NK activity was at least threefold higher than mice infected with VV-HA-TK and this activity persisted for at least 6 days after infection. The effectors mediating the NK-like activity were asialo-GM1+ (as-GM1+), Thy1.2+/-, CD4- and CD8-, the phenotype of conventional NK cells. Elevated NK activity coincided with the rapid clearance of VV-HA-IL2 from ovaries of infected normal CBA/H mice but not from ovaries of CBA beige mice which had no detectable NK activity in spleens or ovaries. The expression of IL-2 in recombinant VV infection probably induces a cascade of immunologic effects of which elevated NK activity is one. We speculate that the chemoattractant and NK activity augmenting effects of IL-2 may contribute to recovery from VV-infection.

Animals↗
PubMed1990

Anti-asialo-GM1 inhibits vaccinia virus infection of murine ovaries: asialo-GM1 as an additional virus receptor?

The neurovirulent strain of vaccinia virus, VV-WR, or recombinants derived from VV-WR, cause highly productive infection of murine ovaries, but infection could be partially inhibited in vivo using an antiserum to asialo-GM1 (as-GM1). In vitro analysis by flow cytometry revealed that murine ovarian cells expressed a cell surface antigen identical to or cross-reactive with as-GM1. The capacity of VV-WR to infect murine ovaries appears to depend in part upon as-GM1 expression on ovarian cells.

Animals↗
PubMed1989

Induction of cell surface expression of HLA antigens by human IFN-gamma encoded by recombinant vaccinia virus.

A recombinant vaccinia virus (VV) encoding human IFN-gamma (VV-huIFN-gamma) was constructed and its effects on MHC Ag expression in human and murine cells in vitro analyzed by flow cytometry. At high multiplicities of infection (5 pfu/cell) the IFN-gamma expressed by vaccinia was not able to overcome the profound decrease of MHC concentration, normally associated with VV infection, in any of the cells tested. However, at successively decreasing multiplicities of infection, a gradual increase in MHC class I concentration above control levels was observed in human 143B cells but not in murine L929 cells, thus indicating that the species specificity of IFN-gamma is preserved in VV-huIFN-gamma-infected cells. We infer from these data that the IFN-gamma secreted by infected 143B cells is able to exert an MHC upregulating effect on uninfected cells in the vicinity. Antiviral activity of the IFN-gamma expressed by the virus was also assessed. Pretreatment for 24 to 48 h of human 143B cells with IFN-gamma containing supernatants had a significant antiviral effect comparable to rhuIFN-gamma. However, when added 1 h after virus infection, antiviral activity was much less evident. Also, the IFN-gamma secreted by infected 143B cells in monolayers infected at low multiplicity did not efficiently inhibit spread of infection to other cells in the vicinity.

Animals↗
PubMed1989

Flavivirus infection up-regulates the expression of class I and class II major histocompatibility antigens on and enhances T cell recognition of astrocytes in vitro.

West Nile virus (WNV) infection of astrocytes can up-regulate their expression of both class I and class II major histocompatibility complex (MHC) antigens as determined by flow cytometry with monoclonal antibodies specific for class I and class II MHC antigens. The up-regulation of class I MHC antigen expression could be partly caused by interferon secreted after WNV infection because the synthetic interferon inducer polyinosinic-polycytidylic acid (poly I:C) has similar effects. In contrast the up-regulation of class II MHC antigen expression was not induced by poly I:C. The increased MHC antigen expression by WNV infection had significant effects on T cell recognition. Thus, WNV and influenza virus A/WSN double-infected astrocytes but not astrocytes infected by A/WSN alone were lysed by influenza virus-immune cytotoxic T cells. Similarly, WNV-infected astrocytes were better stimulators than normal astrocytes for a class II MHC-reactive T cell line, both in terms of T cell proliferation and interleukin release.

Animals↗