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Biomedical subjects

B A Askonas

Publications and source records attributed to B A Askonas.

At least 19 recordsLinked to original sources
PubMed1990

Murine CD4+ T cell clones vary in function in vitro and in influenza infection in vivo.

Several CD4+ Th1 clones specific for influenza haemagglutinin or nucleoprotein were transferred into syngeneic mice after intranasal influenza infection to examine whether they accelerate viral clearance in vivo similarly to CD8+ cytotoxic T cells. We observed changes in functional properties of the CD4+ clones in vitro and variable effects on the course of infection in vivo. While some clones resulted in more rapid virus clearance, others had no protective effect, but rather exacerbated illness symptoms. Our results reflect problems in the in vivo use of CD4+ T cell clones maintained in long-term culture. Their IL-2 and IL-5 release and cytolytic activity varied, while IL-3 and gamma-IFN production as well as DTH induction were more stable. CD4+ T cells primed by infection became cytolytic only after prolonged culture. The data point to the fine balance between exacerbation of disease and protection by CD4+ T cells.

Animals↗
PubMed1990

The 22,000-kilodalton protein of respiratory syncytial virus is a major target for Kd-restricted cytotoxic T lymphocytes from mice primed by infection.

Recombinant vaccinia viruses containing the 22-kilodalton protein (matrixlike or 22K protein) or phosphoprotein gene from respiratory syncytial virus were constructed. These recombinant viruses expressed proteins which were immunoprecipitated by appropriate respiratory syncytial virus antibodies and comigrated with authentic proteins produced by respiratory syncytial virus infection. The new recombinant viruses (and others previously described containing the attachment glycoprotein, fusion, or nucleoprotein genes of respiratory syncytial virus) were used to infect target cells for cultured polyclonal cytotoxic T lymphocytes generated from the spleens of BALB/c or DBA/2 mice primed by intranasal infection with respiratory syncytial virus. Respiratory syncytial virus-specific cytotoxic T lymphocytes (CTL) showed strong Kd (but not Dd)-restricted recognition of the 22K protein. As previously reported, the fusion protein and nucleoprotein were both seen by CTL, but recognition of these proteins was comparatively weak. There was no detectable recognition of other respiratory syncytial virus proteins tested (including phosphoprotein). 22K protein-specific splenic memory CTL persisted for at least 11 months after infection of BALB/c mice. Priming BALB/c mice with recombinant vaccinia virus containing the 22K protein gene induced respiratory syncytial virus-specific memory CTL at lower levels than that previously reported following infection with a similar recombinant containing the fusion protein gene. These data identify the 22K protein as a major target antigen for respiratory syncytial virus-specific CTL from H-2d mice primed by respiratory syncytial virus infection.

Animals↗
PubMed1990

T-cell mediated cytolysis: evidence for target-cell suicide.

The mechanism by which cytotoxic T-lymphocytes (Tc) induce the death of specific target cells is still controversial. We have used quantitative cytochemical methods to distinguish the metabolic activities of the target cells from those of the Tc, even when they are attached to each other. Early events following Tc-P8(15) target cell interaction were first, increased glucose 6-phosphate dehydrogenase activity and second, labilization of the lysosomes within the target cell: these changes could be mimicked, in part, by polyamines and could be inhibited by inhibiting ornithine decarboxylase (ODC) activity. The crucial role of ODC in the chain of events that led to cytolysis in this particular experimental system was shown first, by measuring ODC activity directly and secondly, by the inhibition of cytolysis by the presence of a selective inhibitor of ODC activity.

Animals↗
PubMed1990

Influenza peptide-induced self-lysis and down-regulation of cloned cytotoxic T cells.

Virus-specific cytotoxic T-cell (Tc) clones can lyse target cells in vitro in the presence of their specific peptide epitopes. The lytic potency of murine influenza nucleoprotein (NP)-specific Tc clones was investigated after observing that target cell killing was reduced in the presence of high (greater than 0.2 microM) concentrations of specific NP peptide antigen. Following incubation of Tc for 16 hr in the presence of a range of peptide concentrations, two effects were observed; (i) a peptide dose-dependent mortality of Tc, which has been attributed to self-lysis by clonal Tc in the presence of specific peptide; (ii) and a reduced ability to specifically lyse NP-expressing target cells whilst retaining lectin-dependent lytic activity in the surviving Tc. This functional down-regulation was reversible after 24 hr incubation in the absence of peptide. Toxic effects were excluded, since inhibition of specific target lysis by Tc was mediated only be pretreatment with specifically recognized peptide.

Animals↗
PubMed1989

Primary stimulation by dendritic cells induces antiviral proliferative and cytotoxic T cell responses in vitro.

We used well-gassed hanging drop (20 microliters) cultures with high concentrations of purified T cells from normal BALB/c mice to examine whether dendritic cells (DC) can induce primary antiviral proliferative T cell responses and generate virus-specific CTL. We found that DC exposed to infectious influenza virus in vitro or in vivo in small numbers (0.1-1%) resulted in strong proliferation of responder T cells within 3 d, and this was strongly inhibited by antibodies to class II MHC molecules. In addition, in 5-d cultures, the influenza-treated DC generated CTL specifically able to lyse influenza-infected syngeneic target cells bearing MHC class I antigens. The most potent nucleoprotein (NP) epitope recognized by BALB/c CTL is peptide 147-158 (Arg156-) and influenza-infected DC in vitro stimulated CTL recognizing this peptide, thus mimicking the response in mice primed by intranasal influenza infection. We also induced T cell proliferation and virus-specific CTL in cultures of normal T cells by stimulating with DC pulsed with the natural NP sequence 147-158 or the potent peptide 147-158 (Arg156-). Small numbers of peritoneal exudate cells, after activation with Con A to produce class II MHC expression and after removal of DC with a specific mAb (33DI), did not lead to primary CTL generation but initiated secondary stimulation in vitro. Our results using the hanging drop culture method and DC as APC have implications for studying the T cell repertoire for viral components in humans without the necessity of previous immunization.

Animals↗
PubMed1989

Identification of residues necessary for clonally specific recognition of a cytotoxic T cell determinant.

The residues in an influenza nucleoprotein (NP) cytotoxic T cell determinant necessary for cytotoxic T cell (CTL) recognition, were identified by assaying the ability of hybrid peptides to sensitize a target cell to lysis. The hybrid peptides were formed by substituting amino acids from one determinant (influenza NP 147-158) for the corresponding residues of a second peptide (HLA CW3 171-182) capable of binding to a common class I protein (H-2Kd). Six amino acids resulted in partial recognition; however, the presence of a seventh improved the potency of the peptide. Five of the six amino acids were shown to be required for recognition. The spacing of the six amino acids was consistent with the peptide adopting a helical conformation when bound. The importance of each amino acid in CTL recognition and binding to the restriction element was investigated further by assaying the ability of peptides containing point substitutions either to sensitize target cells or to compete with the natural NP sequence for recognition by CTL. The T cell response was much more sensitive to substitution than the ability of the peptide to bind the restriction element. Collectively the separate strategies identified an approximate conformation and orientation of the peptide when part of the complex and permitted a potential location in the MHC binding site to be identified. The model provides a rationalization for analogues which have previously been shown to exhibit greater affinity for the class I molecule and suggests that the binding site in major histocompatibility complex (MHC) class I molecules might have greater steric constraints that the corresponding area of class II proteins.

Amino Acid Sequence↗
PubMed1989

Influenza virus-specific T cells lead to early interferon gamma in lungs of infected hosts: development of a sensitive radioimmunoassay.

A sensitive immunoradiometric assay for murine interferon gamma (MuIFN-gamma) has been developed and used reproducibly to measure low levels of MuIFN-gamma in lung lavage samples from influenza-infected mice. In control infected mice, IFN-gamma peaked on day 6, but transfer of virus-specific cytotoxic T cells or T helper cells, which reduced virus replication in vivo in infected hosts, resulted in an earlier peak on day 4.

Animals↗
PubMed1989

Influenza-specific cytotoxic T-cell recognition is inhibited by peptides unrelated in both sequence and MHC restriction.

Two related peptides from the nucleoprotein (NP) sequence 365-380, derived from influenza virus isolates A/PR/8/34 and A/NT/60/68, are recognized by mutually exclusive sets of Db (Class I)-restricted cytotoxic T-lymphocyte (CTL) clones. These peptides compete with each other for presentation on Db-bearing target cells in vitro. A Kk-restricted nucleoprotein epitope (NP 50-63), which is unrelated in sequence, competes more efficiently on H-2b target cells but is not itself recognized by virus-specific CTL from influenza-infected H-2b mice. A peptide sequence from the class I molecules Cw3 and Db can also compete, but additional unrelated peptides do not do so at equimolar concentrations. Our results show that competition is at the level of the target cell and imply that the binding specificity of the class I molecule Db is broader than indicated by the immune response phenotype of the C57BL (H-2b) mouse.

Animals↗
PubMed1989

Rapid recovery of lung histology correlates with clearance of influenza virus by specific CD8+ cytotoxic T cells.

Previous studies have shown that influenza nucleoprotein (NP)-specific cytotoxic T-cell clones do not prevent influenza infection of mice but lead to a more rapid viral clearance and recovery of the host. Here we examine the histology of the lung to see if viral clearance by cytotoxic T cells (Tc) correlates with recovery of pulmonary pathology or if it is in any way deleterious. Intransasal (i.n.) A/X31 virus infection of BALB/c mice produces lung tissue changes lasting 8-10 days in BALB/c mice, with the most severe abnormalities appearing between Days 4 and 6 (e.g. loss of epithelium, airway obliteration, peribronchiolar and perivascular cell accumulation). The transfer of Tc clone T9/13 into i.n.-infected BALB/c mice induces a transient enhanced loss of epithelium on Day 4, while by Day 6 epithelial abnormalities are much reduced in the lung compared to control infected mice. This correlates with a significant reduction in lung virus titres by Day 6; by Day 8 virus is cleared in Tc recipients and lung histology is normal. Another Tc clone (T5/5) with greater cytolytic activity resulted in significant recovery of the lung tissues by Day 4. Tc clones also resulted in enhanced perivascular infiltration of cells and variation in the infiltrating cell type. Quantification in our system required careful attention to the level of the airway assessed. These histological findings showing an enhanced tissue recovery support the previous assessment of reduced lung viral levels following the transfer of Tc cells, and show that a transient increase in lung pathology can occur.

Animals↗
PubMed1988

Competition between unrelated peptides recognized by H-2-Kd restricted T cells.

P815 (H-2d) target cells incubated with synthetic peptides corresponding to region 170-182 of HLA or to region 141-161 of influenza nucleoprotein (NP) are lysed by DBA/2 derived cytolytic T cells (CTL) specific for HLA or by BALB/c derived CTL-specific for NP, respectively. Both peptide Ag are recognized in the context of Kd. We show herein that these unrelated, nonhomologous peptides clearly compete reciprocally for recognition by the appropriate Kd restricted CTL. In contrast, different NP peptides that are recognized by other CTL restricted by HLA-B37, H-2-Db or KK, either failed to compete or were much less efficient as competitors than NP peptides recognized in the context of Kd. The efficiency of a peptide as a competitor correlated with its potency as an Ag. The most efficient competitor was a variant peptide of NP 147-158 with R156 deleted, which had been previously shown to be 1,000 times more efficient as an Ag than its natural homolog. Our results suggest that peptides recognized by CTL in the context of the same MHC class I restriction element may bind to the same or interdependent site(s) on the restriction molecule.

Amino Acid Sequence↗
PubMed1988

Cytotoxic T cells clear virus but augment lung pathology in mice infected with respiratory syncytial virus.

We have examined the function of class I MHC-restricted cytotoxic T cells in experimental respiratory syncytial virus (RSV) infection of BALB/c mice by transfer of T cell line MJC-A2 and CTL clone E8a into RSV-infected mice. The T cell line cleared pulmonary RSV infection within 5 d in persistently infected gamma-irradiated mice, but caused acute respiratory disease. This was only seen in infected mice and was often lethal after transfer of greater than 3 x 10(6) CTL. Lower numbers of CTL produced less severe disease but still cleared lung RSV, albeit over a longer time course (up to 10 d). Clearance of lung RSV in immunocompetent mice by the T cell line and CTL clone was again accompanied by acute and sometimes lethal respiratory disease. Bronchoalveolar lavage showed severe lung hemorrhage and frequent neutrophil efflux in mice with CTL-augmented disease.

Animals↗
PubMed1988

Enhanced recognition of a modified peptide antigen by cytotoxic T cells specific for influenza nucleoprotein.

A previously identified Kd restricted epitope of influenza A virus nucleoprotein (147-161) was modified, resulting in recognition by Kd restricted cytotoxic T cells at significantly lower concentrations than the natural peptide sequence. This was achieved by first refining the epitope to the minimum determinant 147-158. Deletion of arginine 156 resulted in a peptide that was shown to be greatly superior in both dose response titrations and in its rate of association with cells to form targets. Analog peptides were tested to determine the important amino acid changes. These data suggest that T cell epitopes can be modified to result in improved immunological recognition.

Amino Acid Sequence↗
PubMed1988

Rapid changes in target cell lysosomes induced by cytotoxic T cells: indication of target suicide?

Although many studies have attempted to elucidate how cytotoxic T (Tc) lymphocytes cause the death of target cells, the mechanism is still controversial. In the present study the effect on the integrity of the lysosomes of the target cell has been investigated. We show here that the specific recognition and attachment of cloned type A influenza-specific Tc cells to A/X31 influenza virus-infected target cells caused rapid change in the amount of lysosomal naphthylamidase activity that was bound within the lysosomes, indicating that the lysosomal membranes in the target cells had been totally labilized. Target cells infected with type B influenza virus served as controls. We therefore suggest that the viral specificity of Tc lymphocytes allows for recognition and intimate membrane contact with suitably infected targets. This intimate contact induces sufficient perturbation of the target cell plasma membrane so as to cause total labilization of the target cell lysosomes which could account for intracellular lysis.

Animals↗
PubMed1988

Helper T cell recognition of respiratory syncytial virus in mice.

In this study we aimed to define the protein and viral subtype specificities of helper Th cells to respiratory syncytial virus (RSV). BALB/c mice were primed by infection with RSV, or with vaccinia viruses (VV) containing genes encoding several individual RSV proteins. Priming for Th cell memory was assayed by stimulating spleen cells in vitro with different RSV isolates and measuring RSV-specific interleukin 2 (IL-2) release by T cells into supernatants using an IL-2-dependent CTLL cell line. Splenocytes from mice primed intranasally with RSV exhibited RSV-specific Th cell memory, whereas those from unprimed mice did not. Th cell recognition was in part specific to the strain of RSV used in priming and in part cross-reactive between RSV strains. Intraperitoneal priming with RSV fusion protein-expressing VV or nucleoprotein-expressing VV induced a stronger RSV-specific Th cell response than the attachment glycoprotein-expressing VV which produced only slight Th recognition. No Th cell recognition of two non-structural proteins (1A and 1B) could be demonstrated.

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