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At least 505 records · Page 28Linked to original sources
PubMed1997

Potentiation of antitumor CTL response by GM-CSF involves a B7-dependent mechanism.

We have previously demonstrated the importance of endogenous GM-CSF production for the B7-2-dependent potentiating effect of exogenous TNF for CTL generation by stimulation cultures of splenic cells from mice bearing a large MOPC-315 tumor. Here we show that addition of GM-CSF to stimulation cultures of such tumor-bearer splenic cells also leads to the generation of enhanced anti-MOPC-315 CTL activity via a B7-dependent mechanism. However, while the potentiating effect of TNF was previously shown to be IL-2-independent, the potentiating effect of GM-CSF is shown here to be completely IL-2-dependent. Still, the potentiating activity of exogenous GM-CSF for the in vitro generation of CTL activity is shown to depend completely on endogenous TNF production. Finally, TNF and GM-CSF may cooperate in enhancing the in vivo generation of CTL activity in MOPC-315 tumor bearers because low-dose melphalan (L-phenylalanine mustard) therapy, which was previously shown to lead to the rapid up-regulation of TNF production at the tumor site and the subsequent TNF-dependent in vivo acquisition of potent CTL activity, is shown here to lead to the rapid up-regulation of GM-CSF production at the tumor site.

Animals↗
PubMed1999

Reduction in mitochondrial membrane potential is an early event in Fas-independent CTL-mediated apoptosis.

Cytotoxic T lymphocytes (CTL) can destroy target cells via the Fas-mediated pathway or the granule-mediated pathway. We used Fas-negative target cells to examine for target-cell reduction in mitochondrial membrane potential (DeltaPsi(m)) induced by intact CTL via the granule-mediated pathway. We find that reduction in DeltaPsi(m) is an early step in Fas-independent CTL killing of target cells that precedes phosphatidyl serine translocation, cytosolic protein release, or loss of plasma membrane integrity. Target-cell reduction in DeltaPsi(m) and cytoplasmic protein release in Fas-independent CTL killing were inhibited by N-carbobenzoxy-Ala-Pro-Phe chloromethyl ketone, but not by caspase inhibitors N-carbobenzoxy-Val-Ala-Asp fluoromethyl ketone (z-VAD-fmk) or N-carbobenzoxy-Asp-Glu-Val-Asp fluoromethyl ketone (z-DEVD-fmk). This contrasts with Fas-mediated apoptosis, in which the reduction in DeltaPsi(m) can be inhibited by z-VAD-fmk or z-DEVD-fmk. Assessing the changes in target-cell DeltaPsi(m) can provide for a sensitive and rapid means with which to monitor CTL activity.

Amino Acid Chloromethyl Ketones↗
PubMed1998

Modelling the dynamics of LCMV infection in mice: conventional and exhaustive CTL responses.

Lymphocytic choriomeningitis virus (LCMV) infection in mice provides an example of an extraordinarily dynamic process with an extreme sensitivity of phenotype of infection to parameters of virus/host interaction. A mathematical model is developed to examine the dynamics of virus-specific cytotoxic T lymphocyte (CTL) response for LCMV infection in mice. The model, formulated by a system of nonlinear delay-differential equations, considers the interacting populations of viruses, precursor CTLs, terminally differentiated effector CTLs and total virus antigen load. Clonal elimination of virus-specific cytotoxic T cells in high-dose LCMV-Docile infection represents an example of the classical phenomenon--high zone tolerance. To describe both conventional and exhaustive CTL responses in the acute phase of LCMV-D infection two mechanisms are invoked: the high virus antigen load inhibition of T-cells proliferation via energy induction and the activation-induced cell death by apoptosis. Parameters of the model, characterizing the rates of virus and CTL production and elimination in spleen, are estimated by assimilating with the model data on the LCMV-D infection in C57BL/6 mice for low-, moderate- and high-dose infections. It is suggested that not only the clonal expansions have to be described in mathematical models as being virus regulated but also the later phases of primary immune response. Down-regulation of the primary CTL response is controlled by a network of mechanisms inducing anergy and apoptosis in activated T cells. The model is used to investigate the effect of variations in virus and CTL response parameters on LCMV infection outcome and suggest predictions for experimental studies, in particular the phenotype of LCMV-WE infection in C57BL/6 as a function of initial virus doses.

Animals↗
PubMed1996

Identification of HTLV-1-specific CTL directed against synthetic and naturally processed peptides in HLA-B*3501 transgenic mice.

Previous studies of CTL responses to influenza peptides in HLA single transgenic mice resulted in the identification of at most one immunodominant epitope. Since HLA-B*3501 is known to present multiple HIV-1-specific T cell epitopes we tested the cellular immune response of HLA-B*3501 transgenic mice to synthetic HTLV-1 peptides mixed with the lipohexapeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)propyl]cysteinyl-seryl-lysyl-l ysyl- lysyl-lysine, which is a biocompatible, Th-epitopeindependent adjuvant. Eleven of 37 tested HLA-B*3501 binding peptides mounted a CTL response after three in vitro stimulations. The HLA-B*3501 affinity of peptides correlated with their ability to induce CTL in HLA-B*3501 transgenic mice. Seven peptides derived from env-gp46 (VPSPSSTPLL, VPSSSSTPL, YPSLALAPH, and YPSLALAPA), pol (QAFPQCTIL), gagp19 (YPGRVNEIL), and tax (GAFLTNVPY) proteins induced peptide-specific CTL Bulk CTL generated by four peptides derived from env-gp46 (SPPSTPLLY, VPSPSSTPLLY, and VPSPSSTPLL) and pol (QAFPQCTILQY) killed peptide-pulsed and recombinant vaccinia-infected target cells. The latter peptides therefore present T-cell epitopes and are vaccine candidates for our transgenic mouse model.

Animals↗
PubMed1997

The role of proximal and distal sequence variations in the presentation of an immunodominant CTL epitope encoded by the ecotropic AK7 MuLV.

An emv-14-derived, replication-competent ecotropic murine leukemia virus [MuLV], designated AK7, was previously cloned from the AKXL-5 recombinant inbred mouse strain and partially characterized. While genetically encoding for an envelope-derived immunodominant CTL epitope [KSPWFTTL] located in the transmembrane region of p15TM, this virus, unlike the emv-11-derived virus AKR623, fails to be efficiently recognized by AKR/Gross MuLV-specific cytotoxic T lymphocytes [CTL]. AK7 thus provides the opportunity to study the role of retroviral sequence variations that are located outside of the immunodominant epitope as a mechanism of escape from CTL-mediated immune surveillance. In an attempt to identify which region[s] of the AK7 genome could account for its ability to evade efficient recognition by AKR/Gross MuLV-specific CTL, we have constructed recombinant murine retroviruses. The direct influence of a sequence variation twelve amino acids N-terminal to KSPWFTTL was explored with the use of chimeric viruses and determined not to significantly impair the presentation of KSPWFTTL to AKR/Gross MuLV-specific CTL. The long terminal repeat [LTR] derived from the AK7 virus, which possesses only one copy of the 99-base pair transcriptional enhancer in the U3 region, in contrast to AKR623 that possesses two copies of the tandem direct repeat enhancers, was also analyzed for its influence on the presentation of KSPWFTTL. Interestingly, our data indicate that the enhancer region derived from AK7 negatively influences the presentation of KSPWFTTL in the context of a recombinant AKR623 virus.

AKR murine leukemia virus↗
PubMed1998

Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway.

To evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus of bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses.

Animals↗
PubMed1998

Viral evolution and CTL epitope stability during JHMV infection in the central nervous system.

The JHM strain of mouse hepatitis virus (JHMV) establishes a persistent infection in the murine central nervous system (CNS) associated with chronic ongoing demyelination in the absence of detectable virus. To distinguish between immune and replication associated mechanisms of persistence, brains from acutely and persistently infected mice were analyzed for viral RNA mutations in the encapsidation sequence (ECS) and regions encoding either the transmembrane domains of the matrix (M) protein or a protective CTL epitope in the nucleocapsid (N) protein. Detection of the ECS to 120 days post infection (p.i.) indicated low levels of replication. The ECS remained stable whereas the fragment encoding the CTL epitope revealed extensive diversity with mutation frequencies in the order of 2.0 per 1000 nts. The M gene also remained stable despite random mutations during the acute phase. Mutations in the N gene were random and not selected for during persistence, with the exception of a single prominent Pro363 to Ser substitution in a region not associated with any known regulatory function or immune response. Mutations within the CTL epitope affecting CTL recognition were found early in responder BALB/c mice (H-2d), but also in non-responder C57BL/6 (H-2b) mice, suggesting that CTL escape variants play no significant role in establishing persistence.

Animals↗
PubMed1985

Antigen/mitogen induced cytolytic activity and IL-2 secretion in memory-like CTL-hybridomas.

Memory-like monoclonal CTL hybridomas, derived from fusion of the AKR thymoma BW5147 with secondary CTL generated in vivo or in MLC cultures, have been used to study the mechanism whereby antigen/mitogen induces anamnestic CTL responses. Specifically, we have asked whether induction of cytolytic activity can be promoted by an antigenic/mitogenic signal without involvement of IL-2 receptors, IL-2, or other extrinsic factors. We have found that antigen/lectin alone can trigger the cytolytic potential of the hybridomas and induce IL-2 secretion. Pure IL-2 and conditioned medium were ineffective inducers of cytotoxicity. Moreover, IL-2 receptors were not detected on the hybrid cells before and after antigenic stimulation, demonstrating that expression of IL-2 receptors and induction of specific killing activity are not genetically linked. Non-activated and activated cells conjugated with target cells equally well, suggesting that induction of cytolytic activity involves a post target cell binding step. Close linkage between cytotoxicity and IL-2 secretion has been observed: induction of killing was consistently associated with IL-2 secretion and stimulation of both activities could be blocked by Cyclosporin A. IL-2 was secreted by the CTL hybrids as early as 3 h following stimulation. We propose that the immediate supply of IL-2 by such memory CTL enhances antigenic response of other, IL-2-dependent T cells.

Animals↗
PubMed1988

Genetic control of CTL responses to AKR/Gross virus: effect of inheritance of Akv proviruses.

Our earlier observations suggested that the AKR/Gross leukemia virus-specific C57BL/6 cytolytic T lymphocyte (CTL) response was directed to Akv-1, but not Akv-3 or Akv-4, provirus-associated determinants. Based on these data, the present experiments were performed with various AKXL RI mouse strains of the responder H-2b haplotype which had inherited different combinations of the Akv-1, -3, and -4 proviruses, to determine whether these strains were able to mount specific antiviral CTL responses. In a comparison with control responder C57BL/6 mice, a clear pattern emerged. Akv-negative mice of the AKXL-29 strain were fully responsive, but five other AKXL strains which had inherited the Akv-1 provirus failed to mount significant antiviral CTL responses (less than or equal to 10% of control). In contrast, an Akv-1-negative but Akv-4-positive strain (AKXL-5) was partially responsive (approximately 24% of the C57BL/6 control). These results were consistent with a direct relationship between the Akv-1 provirus and the nominal antigens recognized by antiviral CTL, and with an inverse correlation between in vivo expression of viral antigens by normal cells and the ability to generate antiviral CTL. The possible mechanisms accounting for this unresponsiveness are discussed along with the utility of this system for investigating the interactions of retroviruses with the immune system.

AKR murine leukemia virus↗
PubMed2005

Analysis of a rare melanoma patient with a spontaneous CTL response to a MAGE-A3 peptide presented by HLA-A1.

We describe an HLA-A1 melanoma patient who has mounted a spontaneous cytolytic T cell (CTL) response against an antigenic peptide encoded by gene MAGE-A3 and presented by HLA-A1. The frequency of anti-MAGE-3.A1 CTLp was 5 x 10(-7) of the blood CD8 cells, with a dominant clonotype which was present in six out of seven independent anti-MAGE-3.A1 CTL clones. After vaccination with a recombinant poxvirus coding for the MAGE-3.A1 antigen, the blood frequency of anti-MAGE-3.A1 CTLp increased tenfold. Twenty-two independent CTL clones were derived. Surprisingly, only one of them corresponded to the dominant clonotype present before vaccination. Two new clonotypes were repeated 12 and 7 times, respectively. Our interpretation of these results is that the spontaneous anti-MAGE-3.A1 CTL response pre-existing to vaccination was polyclonal, and that the vaccine restimulated only some of these clones. To estimate the incidence of spontaneous anti-MAGE-3.A1 CTL responses in melanoma patients with a tumor expressing gene MAGE-A3, we measured the blood frequency of anti-MAGE-3.A1 T cells in 45 patients, and found only two clear responses.

Aged↗
PubMed1985

Differential resistance to growth of a tumor expressing incompatible minor alloantigens reflects regulatory influences rather than differences in anti-minor-CTL-P frequencies.

In previous studies it was found that BALB/c (H-2d) was more susceptible than (BALB/c X A)F1 (H-2d X H-2a) to a tumor bearing multiple mismatched minor histocompatibility antigens, the DBA/2 (H-2d) mastocytoma P815, and that this resistance was H-2 linked. In the present studies the immunologic basis of this effect was examined by comparing the cytotoxic-T-lymphocyte (CTL) responses of BALB/c with those of (BALB/c X A)F1. Despite the BALB/c X A)F1's 34-fold greater resistance to P815 in vivo, the numbers of effector cell precursors were found to be similar in the two hosts as shown by (a) similar anti-P815 CTL responses in vitro with T-cell growth factor, (b) similar secondary anti-DBA/2 MiHA responses after in vivo priming with irradiated P815, and (c) similar frequencies of anti-DBA/2 CTL precursors by limiting-dilution analysis. However, priming with proliferating P815 in vivo revealed a defect in the BALB/c animals: Spleen cells from such animals were unable to control the growth of contaminating P815 cells in vitro or to mount strong secondary CTL responses to DBA/2 antigens. The defective priming of BALB/c could be corrected when DBA/2 spleen cells were added to the P815 inoculum. This impaired priming by living tumor cells was not seen in (BALB/c X A)F1. It is concluded that the use of living P815 tumor cells revealed a defect in immunoregulation in BALB/c mice, which rendered them susceptible to tumor growth in spite of apparently adequate numbers of anti-minor-CTL precursors. How the additional H-2 products expressed in the (BALB/c X A)F1 might correct this defect is discussed.

Animals↗
PubMed1987

Mouse embryonic cells become susceptible to CTL lysis after midgestation.

The susceptibility of murine embryonic cells to CTL lysis has been assessed. Zona-free blastocysts were not lysed during 6 hr incubation with CTL sensitized against the embryonic genotype and remained capable of implantation and normal development in vivo as well as outgrowth in vitro. Freshly dissociated cells from postimplantation embryos before the 13th day of gestation were not lysed in a 51Cr-release assay, but cells from 13- to 16-day embryos were susceptible to lysis. Cells from Day 12 embryos cultured for 40 hr in the presence or absence of interferon became susceptible to CTL lysis. Cultured cells from Day 10 embryos were only lysed following culture in medium containing interferon. Cell populations lysed in the CTL assay remained resistant to lysis by NK effector cells. These experiments indicate that the murine fetus is protected from the maternal immune system at least during the first half of pregnancy by an intrinsic ability of early embryonic cells to resist cell mediated lysis. Suppression of maternal CTL responses may not be essential for fetal survival until later stages of gestation.

Animals↗
PubMed1995

Nonresponsiveness of AKR.H-2b congenic mice for anti-AKR/Gross MuLV CTL responses: involvement of inhibitory cells as defined by adoptive transfer experiments.

AKR.H-2b mice are unable to elicit AKR/Gross murine leukemia virus (MuLV)-specific cytolytic T lymphocyte (CTL) responses. The participation of inhibitory cells was addressed through adoptive transfer experiments utilizing young AKR.H-2b:Fv-1b congenic responder mice as the recipients of AKR.H-2b donor cells. The adoptive transfer of unfractionated viable splenocytes led to inhibition of virus-specific CTL responsiveness without affecting minor histocompatibility or allogeneic (H-2d)-specific CTL responses. Negative cell selection studies indicated that of the donor AKR.H-2b spleen cells that mediate specific inhibition, B lymphocytes, CD4-CD8+ and CD4+CD8- T lymphocytes, but not macrophages, even though they are viral antigen positive (as are B and T lymphocytes), were the cells responsible for the diminution of the generation of AKR/Gross virus-specific CTL by AKR.H-2b:Fv-1b mice. To evoke maximal inhibition, the adoptive transfer of AKR.H-2b cells had to be performed prior to in vivo priming with viral antigen. Anti-AKR/Gross MuLV nonresponsiveness of AKR.H-2b mice could not be overcome through utilization of exogenous IL-2 at either the priming or in vitro restimulation phases of CTL generation. These results illustrate the complex interaction between retroviruses and lymphocytes and are relevant to understanding how retrovirus-infected cells may not only escape immune surveillance themselves, but also may inhibit the cytolytic T cell response directed at other infected cells, such as tumor cells.

AKR murine leukemia virus↗
PubMed1992

Kinetics of killing by monoclonal cytotoxic T lymphocytes. I. Colorimetric detection of cryptic CTL determinants on adherent target cells and survivorship analysis.

Some targets of cell-mediated cytolysis do not efficiently release 51Cr but manifestly are killed in direct viability assays. We characterize and validate an alternative and non-radioactive (colorimetric) method for measuring killing of adherent targets by monoclonal CTL. The method obviates concerns about the effects of trypsinization, is technically simple, quantitative and in some cases more sensitive than conventional 51Cr assays. Target loss obeyed first-order kinetics with respect both to [CTL] and time. These results are consistent with an exponential (Poisson) model of killing and support the use of a single kinetic parameter to describe the lytic activity of monoclonal CTL on adherent targets. When monoclonal CTL are used at appropriate effector:target ratios (less than or equal to 1:1), the residuals obtained after least squares linear regression are homoscedastic and normally distributed, justifying the use of commonly available statistical calculators or programs for the analysis of CTL data.

Animals↗
PubMed1992

In vivo primary induction of virus-specific CTL by immunization with 9-mer synthetic peptides.

A primary cytotoxic T lymphocyte (CTL) response in vivo requires antigen presentation by cytosolic processing and can not in general be obtained by vaccination with soluble proteins. In the present work we have found that vaccination of mice with pre-processed synthetic peptides, corresponding to endogenous 9-mers produced in influenza A virus-infected cells, resulted in strong primary CTL responses. The generated CTL efficiently killed virus-infected target cells with preference for viral strains having the identical amino acid sequences to the peptides used for immunization. The optimal conditions for a primary in vivo CTL response was obtained with 100 micrograms peptide dissolved in incomplete Freund's adjuvant and injected s.c. at the base of tail. Spleen cells which had been primed 7-10 days earlier were restimulated for 5 days in vitro, using an optimal low peptide concentration (0.05 microM) and tested against virus-infected and peptide-treated target cells. The peptide-induced CTL were major histocompatibility complex class I restricted and CD8 positive.

Amino Acid Sequence↗
PubMed1996

In vivo CTL immunity can be elicited by in vitro reconstituted MHC/peptide complex.

The use of peptides as a vaccine is a potentially powerful immunization strategy. We explored the possibility of inducing an efficient cytotoxic T lymphocyte (CTL) mediated immune response in mice, using in vitro reconstituted major histocompatibility complex (MHC) class I/peptide complexes as the immunogen. Recombinant derived H-2Kb and beta 2-microglobulin (beta 2m) were properly folded into an MHC class I complex using the vesicular stomatitis virus (VSV)-8mer from the natural nucleocapsid proteinN52-59 (RGYVYQGL), an immunodominant Kb epitope in C57BL/6 (B6) mice. After immunizing mice with the H-2Kb class I/VSV peptide complex and a subsequent in vitro stimulation with the VSV peptide alone, a specific CTL response was demonstrated. The method was also applicable to other peptides, for example, the Sendai virus (SV) peptideN324-332 (FAPGNYPAL). The CTL response was mediated by CD3+/CD8+ T cells and was shown to be allele specific, as only peptide loaded target cells expressing the H-2Kb allele could be recognized. It is of interest that extremely small amounts of injected MHC class I/peptide complex (i.e. 500 pg) could generate a measurable CTL response. The MHC class I/peptide complex had to be intact and properly folded to elicit an immune response, suggesting that the complex protected the peptide for internalization by antigen presenting cells (APCs) or for delivering to the proper site for peptide exchange on the cell surface of APCs. The described immunizing method can be routinely used to prime a CTL response by employing in vitro folded MHC class I/peptide complexes, without the use of adjuvants. It appears to be efficient, sensitive and specific. By using the recombinant protein system, unlimited amounts of MHC class I/peptide complex can be produced for immunization. Moreover, this protocol permits different in vitro combinations of allelic MHC class I molecules and peptide variants.

Amino Acid Sequence↗
PubMed1996

Mucosal immunization with a measles virus CTL epitope encapsulated in biodegradable PLG microparticles.

The immunogenicity of a cytotoxic T cell epitope (CTL) representing residues 52-60 from measles virus (MV) nucleoprotein, encapsulated in poly(lactide-co-glycolide) (PLG) microparticles was evaluated after mucosal immunization. After intranasal administration of the encapsulated CTL epitope linked at the carboxyl terminus of two copies of a T-helper epitope (TT-NP6), peptide-specific and MV-specific CTL responses were detected in splenocytes. However, these responses were lower than the responses observed when the TT-NP6 peptide was administered intranasally in saline or using CTB as an adjuvant. Intranasal coadministration of the encapsulated TT-NP6 peptide with CTB did not result in any significant potentiation of the CTL responses. The effectiveness of biodegradable PLG microparticles for mucosal delivery of CTL epitopes, combined with their excellent tissue compatibility and biodegradability suggests that they represent a valuable delivery system for synthetic immunogens. However, further work is needed to define the requirements for effective absorption by the nasal epithelium.

Animals↗
PubMed1995

In vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells stimulated with synthetic peptides.

A protocol for in vitro induction of primary, antigen-specific CTL from human peripheral blood mononuclear cells (PBMCs) was developed. Antigen presenting cells (APCs) consisted of Staphylococcus aureus Cowan-I (SAC-I) activated PBMCs treated with a citrate-phosphate buffer at pH 3 to release endogenous peptides bound to surface MHC. This treatment resulted in transient expression of empty class I molecules which could be subsequently stabilized with peptide and beta 2-microglobulin (beta 2m). SAC-I activated PBMCs from HLA-A2.1 normal donors loaded with HBV core 18-27 peptide following acid treatment were used to stimulate PBMCs depleted of CD4+ T cells, in the presence of recombinant interleukin-7 (rIL-7). After 12 days, cells were restimulated with autologous, peptide-pulsed, adherent cells and tested for CTL activity 7 days later. In 23 independent experiments from 13 different HLA-A2.1 donors, this protocol resulted in induction of primary CTL more than 90% of the time. As indicated by both the frequency and magnitude of the response against peptide-sensitized target cells, SAC-I activated PBMCs treated with acid were the most efficient stimulator APC. Thirteen per cent of the cultures generated were capable of lysing target cells transfected with the HBV core antigen and, in general, these CTL cultures exhibited high avidity for the HBV core peptide. This protocol is generally applicable to different antigens and class I alleles, and thus, may be utilized to screen large numbers of peptides to identify human CTL epitopes.

Antigen-Presenting Cells↗