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R V Blanden

Publications and source records attributed to R V Blanden.

At least 19 recordsLinked to original sources
PubMed1996

Granzyme A is critical for recovery of mice from infection with the natural cytopathic viral pathogen, ectromelia.

Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.

Animals↗
PubMed1995

Polymorphic peptide transporters in MHC class I monomorphic Syrian hamster.

We have already shown that in species with highly polymorphic major histocompatibility complex (MHC) class I molecules (human, mouse) no functional polymorphism of the peptide transporters TAP1 and TAP2 is detectable (Lobigs and Müllbacher 1993). Investigating the antigen-presentation machinery of the class I MHC monomorphic Syrian hamster using mouse MHC class I expression via recombinant vaccinia viruses (VV) we found that six hamster cell lines fall into two phenotypic classes. four cell lines (HaK, FF, MF-2, and HT-1) showed no defect in expressing four different H2 class I molecules (Kk, Kd, Kb, Dd) and the appropriate VV peptide recognized by mouse VV-immune cytotoxic T (Tc) cells on the cell surface. Two cell lines (BHK-21 and NIL-2) expressed Dd and Kb in association with VV peptides as recognized by VV-immune, H2-restricted Tc cells but not Kk and Kd. However, Kd was expressed on the cell surface, as shown by fluorescence-activated cell sorter (FACS) analysis and alloreactive Tc-cell recognition. Kk is only surface-expressed in these two cell lines when superinfected with two VV recombinants encoding rat TAP1 (VV-mtp1) and TAP2 (VV-mtp2). Superinfection with VV-mtp1 and VV-mtp2 rendered both cell lines, after infection with either VV-Kk and VV-Kd, susceptible to lysis by either Kk- or Kd-restricted VV-immune Tc cells. Thus Syrian hamster cell lines express functionally polymorphic peptide transporters. The TAP2 gene from FF cells was cloned and sequenced; comparison with human, mouse, and rat TAP2 sequences show 78%, 88% and 87% similarity, respectively.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Evolution of V genes: DNA sequence structure of functional germline genes and pseudogenes.

In this review we have examined the features of germline sequences of IgV genes from a number of species in an attempt to identify the "signature" of molecular mechanisms responsible for generating and maintaining diversity in the germline repertoire (after gene duplication by meiotic unequal crossover). We now summarize the relevant features point by point: 1. Codon analysis reveals a significant deficit of stop codons below the numbers that would be expected under random point mutational change. This implies that the majority of individual V genes have each been selected for the possession of open reading frames able to encode a functional Ig molecule. There is an extraordinarily high rate of apparent rescue of potential stop codons in both V genes and pseudogenes. Other (non-Ig) pseudogene sequences studied thus far do not show this high rate of rescue of stop codons. 2. The distribution of changes is concentrated in most cases in the 5' half of CDR2 (CDR2a), and coincides with the patterns of antigen-selected mutations in B lymphocytes. It does not coincide with expected non-antigen-selected (random) changes, as exemplified by hypermutated but unexpressed passenger V transgenes in B cells in Peyer's patches of unimmunized mice (Gonzalez-Fernandez and Milstein 1993). 3. In germline V genes of mice, there is no evidence of triplet codon insertion (or multiples thereof) as a mechanism generating germline diversity. This parallels a known absence of gene conversion as a mechanism generating somatic diversity in mice. In contrast, in germline chicken pseudogenes which are known to contribute to somatic generation of diversity by gene conversion, frequent examples of triplet codon insertions and deletions in CDRs are present. 4. The pattern of unique insertions and deletions in all species with sufficient sequence data available is consistent with hyper-recombination events targeting the transcription and/or coding unit. The distribution of these events does not correlate with known inducers of gene conversion, for example, inverted or direct repeats and palindromes. Furthermore, the 5' boundaries of somatic hypermutation and the 5' peak of germline nucleotide insertions and deletions coincide in IghV (Rothenfluh et al. 1993, 1994; Rogerson 1994) and in IgkV (Rogerson 1994; Rada et al. 1994, and analyses herein). It will be interesting to see how these features relate to each other in other gene sets as data become available.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1995

Why do class I MHC molecules bind smaller peptides than class II MHC molecules?

This article attempts to assemble theoretical-teleological argument to explore possible answers to the question of why class I MHC molecules bind smaller peptides than class II MHC molecules and the associated question of why the size of peptides binding to class I molecules is approaching the limit of the self-non-self discrimination. I propose that the small size of most class I-binding peptides precludes the production of 'MHC-restricted' antibodies. Such a strategy avoids the possibility of antibodies binding to the epitopes recognized by CD8+ T cells, thus blocking effector function required for clearance of potentially lethal infections.

Animals↗
PubMed1994

Analysis of patterns of DNA sequence variation in flanking and coding regions of murine germ-line immunoglobulin heavy-chain variable genes: evolutionary implications.

We analyzed the DNA sequence structure of the 5' flanking and coding regions of 52 VH186.2-related germ-line genes isolated by PCR from C57BL/6J and BALB/c mice. The aligned coding regions display hypervariable and conserved regions corresponding to some of the complementarity-determining regions (CDRs) and framework regions (FRs) found in somatically mutated rearranged immunoglobulin variable genes. Most of the coding regions (88.5%) display open reading frames, strongly suggesting positive selection by antigen. Phylogenetic comparisons of putative transcribed regions versus 5' nontranscribed regions show that they have evolved very differently. Inspection of the 52 murine VH186.2-related DNA sequences (as well as other vertebrate germ-line V sequences reported in the literature) reveals clusters of insertion and deletion events bracketing the transcription/coding unit. These data strongly suggest hyperrecombination events targeting the putative transcription/coding sequence. Given that the DNA of unrearranged germ-line V elements cannot be the direct target for "natural-selection" antigen-binding forces (since V elements are only expressed somatically when rearranged in a mature lymphocyte), it is difficult to explain how these nonrandom sequence variations appear in the germ-line DNA. A number of molecular genetic processes are considered, including antigen-driven soma-to-germ-line gene feedback operative during vertebrate germ-line V gene evolution.

Amino Acid Sequence↗
PubMed1994

The flavivirus nonstructural protein NS3 is a dominant source of cytotoxic T cell peptide determinants.

Vaccinia virus recombinants encoding regions of the Murray Valley encephalitis virus (MVE) genome, which together cover the entire viral coding region, were employed to identify the MVE protein which is the dominant source of CD8+, cytotoxic, T cell antigenic determinant(s) presented by the mouse H-2Kk major histocompatibility antigen. MVE and West Nile virus-immune, H-2k-restricted, effector cells recognized peptides derived from the MVE nonstructural polyprotein segment, and in this region the immunodominant determinant mapped to protein NS3. Interestingly, mapping of cytotoxic T cell antigenic determinants of other flaviviruses also identified the NS3 protein as the dominant source of antigenic peptides (A. B. Hill, A. Müllbacher, C. Parrish, G. Coia, E. G. Westaway, and R. V. Blanden, 1992, J. Gen. Virol. 73, 1115-1123; A. L. Rothman, I. Kurane, C.-J. Lai, M. Bray, B. Falgout, R. Men, and F. A. Ennis, 1993, J. Virol. 67, 801-806). Using an allele-specific peptide motiff for H-2Kk, we predicted 12 peptides in the MVE NS3 protein as ligands for the restriction element and identified three peptides which were recognized in association with H-2Kk by MVE-immune cytotoxic T cells. We also examined the effect of proteolytic processing in the MVE nonstructural polyprotein segment mediated by the viral proteinase NS3 on antigen processing and presentation of the MVE H-2Kk-restricted T cell determinant. Processing of the MVE polyprotein by the viral proteinase did not markedly influence the availability of this peptide determinant.

Alleles↗
PubMed1993

Affinity maturation of lymphocyte receptors and positive selection of T cells in the thymus.

In this review we have re-evaluated the dominant paradigm that TcR V genes do not somatically mutate. We highlight the many structural and functional similarities between Ig and TcR antigen-specific receptors on B and T cells. We have reviewed the factors influencing the somatic and germline evolution of IgV regions in B cells, have evaluated in detail various models which could be invoked to explain the pattern of variation in both transcribed and non-transcribed segments of germline IgV-gene DNA sequences, and applied this perspective to the TcR V beta and V alpha genes. Whilst specific TcRs recognize a complex of a short antigenic peptide bound to MHC Class I or II glycoprotein, and Ig receptors can recognize both oligopeptides and conformational determinants on undegraded polypeptides, they both employ heterodimer variable regions (Fabs) utilizing all three CDRs in epitope binding. We conclude that a plausible case can be made for the possibility that rearranged TcR V genes may undergo some type of somatic hypermutation process during T-cell development in the thymus (concurrent with or after the positive selection phase) thus allowing a repertoire of TvR alpha beta heterodimers to be both positively and negatively selected by the same set of ligands (self MHC + self peptide) in the thymus.

Animals↗
PubMed1993

Ir1 genes, peripheral cross-tolerance and immunodominance in MHC class I-restricted T-cell responses: an old quagmire revisited.

Previous studies have shown that peripheral tolerance of one MHC molecule may influence the ability of an animal to respond to a particular antigenic determinant in the context of another MHC molecule (cross-tolerance). We describe here an investigation of the extent to which the presence of MHC molecules other than the one involved in Tc cell recognition affected whether or not a particular "antigen" (MHC/peptide) was recognized during the response of mice to infection with the flavivirus, West Nile (WNV). WNV-immune Tc cells from intra-H-2 recombinant mouse strains and F1 hybrid animals were used. In general, the antigens identified as immunogenic for five prototype mouse strains were immunogenic wherever the F1 or recombinant animal possessed the required MHC gene, but there were several notable exceptions. Firstly, while no responses were associated with Kd and Dk in BALB/c (H-2d) and CBA/H (H-2k) mice, respectively, in C3H.OH mice (Kd, Dk) both of these MHC molecules were associated with detectable responses. Secondly, in B10.A(2R) (Kk, Db) mice responses restricted by Db were not found whereas they were present in B6 mice (Kb, Db). This phenomenon was similar to previously reported phenomena with H-Y, Sendai, influenza and VV. However, it differed in two important ways. Firstly, we have no definitive evidence as yet that peripheral cross-tolerance to Kk influences responses against Db plus flavivirus as it does with H-Y and VV. Secondly, in addition to Kk, genes in the H-2s, H-2q and H-2d haplotypes influence responses to Db plus flavivirus but not responses to the other "antigens" listed above. Alternatively, the data are compatible with the concept of "immunodominance" based on a hierarchy of affinities of TCR's. the responding T-cell population appeared to focus the immune response on a limited number of "antigens"; in the presence of certain more strongly immunogenic "antigens", responses to other apparently more weakly immunogenic "antigens" were not seen.

Animals↗
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↗