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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
PubMed1997

Dendritic cells as targets for cytotoxic T lymphocytes.

Dendritic cells (DC) carry antigen into lymph nodes where they may cluster with CD4 and CD8+ lymphocytes and activate both subsets in the initiation of immune responses. Since DC do not leave the lymph nodes in the efferent lymph they may die within the lymph nodes. Another possibility is that they are targets for cytotoxic T cells (CTL) when expressing appropriate epitopes. This possibility was tested in vitro using human peripheral blood DC to stimulate the development of primary CTL in response to HIV-1 or one of its T-cell epitopes (e.g. env 111-126) and secondary CTL in response to type A influenza virus. Pooled CTL generated during six day cultures in 60 replicate 20 microliters hanging drops were tested in a conventional CTL assay. The HIV or HIV peptide stimulated CTL lysed HIV infected DC while the influenza-virus induced CTL killed DC targets infected with this virus. DC were not lysed significantly until they had been exposed to virus for 2-3 days and thus are not highly susceptible to lysis. However, killing of DC after 2-3 days infection with virus may be a feedback mechanism for removing antigen presenting cells after they have stimulated T cell responses. Removal of persistently infected DC by CD8+ CTL may also contribute to the reduction in DC numbers observed in blood and skin in HIV infection.

Antigens, Viral↗
PubMed1996

Regulation of house dust mite responses by intranasally administered peptide: transient activation of CD4+ T cells precedes the development of tolerance in vivo.

We have previously demonstrated that intranasal (i.n.) administration of an immunodominant peptide (p1-111-139) derived from the house dust mite (HDM) allergen Der p 1 inhibits antigen-specific CD4+ T cell responses in H-2b mice. Here we report that i.n. peptide induced a rapid but transient activation of MHC class II restricted CD4+ T cells that peaked 4 days after peptide treatment and was of similar magnitude to that induced by parenteral immunization with antigen in adjuvant. During the early phase of the response lymph node and splenic T cells secreted a range of lymphokines when re-stimulated in vitro with p1 111-139; however, by day 14 IL-2 and IFN-gamma secretion by T cells were down-regulated. Mice deficient in CD8+ T cells became tolerant by i.n. treatment with peptide, suggesting that CD8+ T cells are not involved in down-regulating the CD4+ T cell response. Rechallenging mice with a single dose of p1 111-139 21 days after the initial treatment elicited a further transient T cell response, which was subsequently down-regulated over time. Although the i.n. peptide induced a strong transient CD4+ T cell response, only low levels of peptide-specific antibodies were detected either after the initial or subsequent i.n. exposures to p1 111-139. Our findings address the mechanisms underlying peripheral T cell tolerance following i.n. administration of a high dose of immunogenic peptide and have implications for understanding the consequences of peptide immunothearapy.

Animals↗
PubMed1996

Human CD4+ T-cell recognition of influenza A virus hemagglutinin after subunit vaccination.

We have examined human CD4+ T-cell recognition of influenza A/Beijing/32/92 (H3N2) virus hemagglutinin following influenza virus HANA subunit vaccination. CD4+ T-cell repertoires were dominated by recognition of epitopes located in conserved regions of the molecule, in a major histocompatibility complex class II haplotype-dependent manner, analogous to that observed following natural infection.

Adult↗
PubMed1995

Human CD4+ T-cell repertoire of responses to influenza A virus hemagglutinin after recent natural infection.

The human CD4+ T-cell repertoire of responses to hemagglutinin (HA) of influenza virus A/Beijing/32/92 was examined 3 to 6 months after natural infection by using a panel of 16-mer peptides overlapping by 11 residues. Short-term CD4+ T-cell lines were derived by using full-length HAs of virus A/Beijing/32/92 from 12 unrelated, major histocompatibility complex (MHC) class I and II haplotyped adults with a history of influenza in November and December 1993 and from 6 adults with no history of influenza during the preceding 4 years but who responded to HA. In contrast to recent murine studies, the human CD4+ T-cell repertoire of responses was dominated by the recognition of highly conserved epitopes. The HA2 subunit, widely regarded as nonimmunogenic, induced strong responses in every donor. This resulted in functional cross-reactivity among influenza A viruses. Our study included one pair of unrelated donors expressing identical HLA DRB1 and DQB1 alleles and two pairs of donors sharing low-resolution MHC class II types. These pairs responded to identical peptides; furthermore, clearly identifiable patterns of response were seen in donors sharing single class II haplotypes, irrespective of the presence of other alleles and exposure history. Two conserved regions which induced responses in 17 of 18 donors were identified (residues 295 to 328 and 407 to 442). Possible implications for cross-reactive T-cell vaccines are discussed.

Adult↗
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↗