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At least 811 records · Page 45Linked to original sources
PubMed1982

Direct analysis of individual killer T cells: susceptibility of target cells to lysis and secretion of hydrolytic enzymes by CTL.

Direct identification, enumeration and biological characterization of cytolytic T lymphocytes (CTL) complementing the chromium release test had allowed us to propose a mechanism for T-cell mediated cytotoxicity (T-CMC). One CTL without accessory cells is able to lyse a specific target. Binding which allows cell contact (E-T doublets), should activate effector CTLs and render target cells susceptible to lysis. Secretion of hydrolytic enzymes localized at the CTL-target junction accounts for the lethal hit given by CTLs to susceptible targets. This hypothesis that the resulting cell lesion is identified with an alteration of the lipid bilayer membrane had been confirmed by single killer cell study. Furthermore, we have proposed that phospholipase enzymes are involved in the membrane alterations, since susceptible targets bound to effector cells (conjugates) were lysed in a much higher yield in the presence of phospholipase. Membrane cell lesion determined by hydrolytic enzyme (phospholipase) could represent a basic general mechanism for other cellular or molecular mediated processes.

Animals↗
PubMed1992

Clinical relevance of soluble HLA and interaction of papain derived class I molecules with alloreactive CTL.

MHC class I and class II molecules are expressed in soluble form in the serum of both healthy and diseased individuals. Our aim was to investigate whether soluble class I (sHLA) levels in allograft patients correlate with their clinical status. Altogether, 20 renal and 30 cardiac graft recipients were examined. High levels of sHLA were measured at least 5 days preceding acute rejection episodes. Immune complexes between anti-HLA antibodies and sHLA were detected in a patient who died of a severe vascular rejection. In another study the interaction of papain-derived sHLA on alloreactive CTL in vitro was investigated. In a chromium-51-release cytotoxicity assay, 1.25 microg/ml of papain-digested class I molecules reduced CTL cytotoxicity to background levels. On the contrary, immobilized molecules triggered the release of serine esterase allospecifically. These data showed that the MHC molecule alone was a sufficient ligand for the interaction with alloreactive CTL.

Biomarkers↗
PubMed2003

Her-2/ neu altered peptide ligand-induced CTL responses: implications for peptides with increased HLA affinity and T-cell-receptor interaction.

In this study, we developed two Her-2/ neu-derived E75 altered peptide ligands (APLs) that demonstrate increased affinities for the HLA-A*0201 allele compared with wild-type E75 peptide. The APLs contain amino acids from E75(369-377), an immunodominant Her-2/ neu-derived peptide, and preferred primary and auxiliary HLA-A*0201 molecule anchor residues previously identified from combinatorial peptide library screening with the recombinant molecule. CTL lines were generated against wild-type E75 peptide (KIFGSLAFL) and APLs by multiple rounds of peptide stimulation of peripheral blood mononuclear cells (PBMCs) from HLA-A2+ antigen normal individuals. CTL lines raised on wild-type E75 peptide cross-reacted with APLs and similarly, CTL lines raised on APLs cross-reacted with wild-type E75 peptide, as measured by IFN-gamma ELISpot and target cell lysis assays. One of five individuals demonstrated specificity for APL 2 (FLFGSLAFL), whereas APL 5 (FLFESLAFL)-specific responses were observed from all five individuals tested. Molecular models of the E75, APL 2, and APL 5/HLA-A2 complexes indicated that the substitution of glycine with glutamic acid at position four of APL 5 resulted in the presentation of a large, negatively charged side chain that interacts with the outer edge of the HLA-A2 antigen alpha helix and is freely available to interact with cognate T-cell receptors. The results of this study further substantiate the concept that rational design of T-cell epitopes may lead to stronger peptide immunogens than natural, wild-type peptides.

Antigens, Neoplasm↗
PubMed1999

Recombinant vaccinia viruses expressing an immunodominant epitope of HIV-1 envelope protein within an influenza hemagglutinin cassette predominantly prime epitope-specific CD8(+) CTL.

We constructed recombinant vaccinia viruses (RVV) expressing a 15-residue peptide (P18IIIB; RIQRGPGRAFVTIGK) of gp160 envelope protein from a human immunodeficiency virus type-1 (HIV-1) IIIB isolate using an H1 influenza virus hemagglutinin (HA) gene cassette. Immunofluorescent tests with antisera against both H1N1 influenza virus and P18IIIB localized chimeric HA molecules comprising influenza virus HA and P18IIIB peptide intracellularly, but the P18IIIB could not be seen on the outer surfaces of infected cells though weak fluorescence was detected regarding HA molecule. Consistent with these findings, Western blotting confirmed the expression of a polypeptide of about 74-kDa protein representing chimeric HA molecule in the infected cells. These recombinants markedly primed CD8(+) cytotoxic T lymphocytes (CTL) specific for P18IIIB as well as HA protein of the influenza virus, but failed to elicit P18IIIB-specific antibody despite stimulating production of HA-specific antibody. In addition, the P18IIIB-specific CTL could strongly lyse target cells expressing the whole HIV-1 envelope gene of IIIB strain. Thus, the influenza virus chimeric HA cassette vector system used in the present study appeared to be a useful tool for constructing vaccine candidates which will predominantly prime CD8(+) CTL specific for immunodominant determinants of various infectious agents.

Amino Acid Sequence↗
PubMed1989

Hierarchy of cytotoxic T cell clones. II. Intraclonal triggering of lysis by hapten-specific CTL.

Cells from clones of anti-hapten murine cytotoxic T lymphocytes (CTL) can act as both target and effector cells, but will not lyse members of the same clone. The effect of haptenation on the cytolytic activity of anti-fluorescein (FL) and anti-trinitrophenol (TNP) CTL clones was examined. Treatment of anti-FL clones with fluorescein isothiocyanate or anti-TNP clones with trinitrobenzene sulphonic acid induces these clones to kill in an antigen-independent fashion. Targets killed by the haptenated CTL included syngeneic and allogeneic B lymphocyte blast cells, P815, YAC-1 and in one case human GM 4072 tumor cells. The importance of CD8 and T cell receptor (TCR) occupancy is demonstrated by the ability to block autotriggering by antibody directed against Ly 2 and the TCR. The results demonstrate that effects other than antigen recognition of the target play a role in the final outcome of effector-target cell interactions and provide a mechanism which could lead to autodestruction and immunosuppression particularly in some types of viral infection.

Animals↗
PubMed1991

Increased tumor-specific CTL activity in human tumor-infiltrating lymphocytes stimulated with autologous tumor lines.

Two long-term tumor-infiltrating lymphocyte (TIL) lines and their autologous tumor lines have been established from solid tumors derived from different patients with metastatic melanoma. In 4-hr 51Cr release assays, each TIL culture lysed only the autologous cryopreserved fresh or established melanoma line, but failed to lyse other melanoma tumors or K562 cells. Repeated stimulation of TIL with the autologous melanoma lines resulted in significant increases in anti-tumor CTL activity with no apparent loss in specificity. Stimulated cells have retained cytotoxic activity for up to 5 months in culture. Tumor cell CTL activity for both long-term TIL lines is inhibited by several mAbs, including those against CD3, CD8, and class I MHC molecules, indicating that the effector cells are class I-restricted CD8+, CTL. Furthermore, recognition of Ag on one of the established melanoma lines by TIL is restricted by HLA A-2. The availability of autologous tumor lines may prove clinically useful for the selective stimulation and expansion of cells with anti-tumor activity within a heterogeneous TIL population.

Antibodies, Monoclonal↗
PubMed1984

A method for testing the specificity of influenza A virus-reactive memory cytotoxic T lymphocyte (CTL) clones in limiting dilution cultures.

This paper describes a system for determining the frequency and fine specificity of influenza A virus-immune memory cytotoxic T cell (CTL) clones from limiting dilution (LD) microcultures. We found that such experiments can only be performed (1) by analyzing the clonal response of CTL from wells of replica plates containing fractions of one original plate, (2) when it has been ascertained that splitting is possible at the clonal level, and that each fraction of a microculture well gives an identical response on target cells infected with the stimulating virus. With these requirements fulfilled we found that short term CTL clones from LD microcultures from influenza A virus (A/X-31)-immune mice (C57BL/6) occur at a frequency of f = 1:546 to f = 1:6303. The effector cells carry the Lyt-2.2 marker and are specific for target cells infected with the immunizing virus (influenza virus A/X-31). They do not lyse NDV (Newcastle disease virus) or HSV (herpes simplex virus)-infected or NK (YAC) target cells.

Animals↗
PubMed1985

Generation of antigen specific cytotoxic T lymphocytes (CTL) directed against class II molecules expressed by activated T cells.

Mitogen-activated T cells were used to provide a source of Class II antigens to CTL originally stimulated against mononuclear cells expressing both foreign Class I and Class II determinants. Our results indicated that 7-10-day-old activated T antigen-presenting cells, which shared only Class II antigens with the original priming cell, were able to stimulate the differentiation of CTL-recognizing Class II determinants. The use of 14-day-old activated T cells as target cells in the CML assay, compared with 72 h PHA blasts or 7-day-old activated T cells, enabled a more sensitive detection of the anti-Class II CTL.

Antigen-Presenting Cells↗
PubMed1990

Generation and characterization of peptide-specific, MHC-restricted cytotoxic T lymphocyte (CTL) and helper T cell lines from unprimed T cells under microculture conditions.

We describe a microculture system for the generation of CTL and T helper cells against peptides. Tryptic digest and cyanogen bromide fragments of chicken ovalbumin and synthetic peptides of ovalbumin (323-339) and influenza virus (NP 365-380) were used to generate CTL and T helper lines from unprimed T cells. These lines were both peptide-specific and MHC-restricted. The relative ease of generating peptide-specific, MHC-restricted CTL and helper T cell lines with as few as 10(6) unprimed lymphocytes can be an efficient method of detecting potential immunogenic determinants of an antigen.

Animals↗
PubMed1991

pH sensitive liposomes provide an efficient means of sensitizing target cells to class I restricted CTL recognition of a soluble protein.

Exogenous antigens are normally endocytosed and enter the class II pathway of processing and presentation. It had been shown earlier that soluble antigen could be introduced into the class I pathway of processing and presentation by osmotic loading. In this report, we have demonstrated that OVA containing liposomes that destabilize on exposure to low pH, referred to as pH sensitive liposomes, could sensitize target cells to lysis by class I MHC-restricted OVA-specific CTL. However, OVA-containing pH insensitive liposomes, native OVA, or OVA subjected to the same protocol as was used to make the liposomes, failed to sensitize targets to OVA-specific CTL lysis. The pH sensitive liposomal approach was less toxic and more efficient (about 20-fold) in delivering than the osmotic loading approach. The pH liposome approach may prove valuable to study CTL recognition characteristics of less available proteins such as viral proteins.

Animals↗
PubMed1992

Electroporation of CTL clones: a useful method to investigate signalling pathways leading to the expression of effector functions.

Signal transduction mechanisms leading to effector functions in mouse cytolytic T lymphocyte (CTL) clones were studied following the introduction of exogenous molecules by electroporation. Conditions were defined in which the application of an electric pulse permeabilized the CTL without affecting functions such as antigen-dependent or antibody-mediated cytotoxicity. When a non-permeant Ca2+ chelator such as EGTA was added in the external medium during the electric pulse, it inhibited subsequent target cell cytolysis carried out in the presence of external Ca2+, thereby indicating the efficiency of EGTA uptake. Results obtained in this system, using a 13 amino-acid protein kinase C (PKC) pseudo-substrate peptide, indicated that it selectively inhibited cytolysis, whereas a substrate peptide with one amino-acid substitution was not inhibitory. This suggests that the technique could be used to study the signal transduction mechanisms of CTL clones which lead to the expression of effector functions.

Amino Acid Sequence↗
PubMed1996

CTL lysis: there is a hyperbolic relation of killing rate to exocytosable granzyme A for highly cytotoxic murine cytotoxic T lymphocytes.

The lysis of susceptible targets by efficient cytotoxic T lymphocytes (CTL) increases both with time and with the ratio of CTL to target. Simple methods for calculating a killing rate constant from the time dependence of killing and for calculating the relation of the killing rate constant to the concentration of exocytosable granzyme A are given. Application of these methods to the killing of target cells by the highly efficient mouse CTL AR1 is presented. AR1 needed granzyme A for efficient killing. AR1 contained sufficient exocytosable granzyme A to kill at about 80% of the rate possible at infinite exocytosable granzyme A.

Animals↗
PubMed1995

Pretreatment with beta-funaltrexamine blocks morphine-mediated suppression of CTL activity in alloimmunized mice.

The effect of prolonged exposure to morphine on cytotoxic T lymphocytes (CTL) and splenic natural killer (NK) activity was investigated. Daily administration of morphine (50.0 mg/kg, s.c.) to alloimmunized mice for 11 days resulted in a significant decrease (25-50%) in peritoneal and splenic CTL activity but not splenic NK activity. To identify the effector cell population mediating cytolysis, cell enrichment studies were carried out. The results of these studies indicated the CTLs are CD8+ CD4-. Chronic morphine treatment increased the percentage (25-30%) of CD3+ CD4+ and CD8+, but not Ig+ cells in the spleen relative to saline-treated controls. Pretreatment of mice with the mu-selective antagonist, beta-funaltrexamine blocked morphine-mediated suppression of splenic and peritoneal CTL activity as well as the increase in CD3+ CD4+ and CD8+ splenic lymphocytes. These results indicate the generation of CTLs in vivo is sensitive to chronic morphine exposure implicating opiates as important co-factors through modulation of cell-mediated immunity.

Animals↗
PubMed1992

Novel LCMV-specific H-2k restricted CTL clones recognize internal viral gene products and cause CNS disease.

H-2k (C3H/Hej) cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were cloned. Three clones recognizing internal viral antigens were studied. One such CTL clone recognized neither the glycoprotein nor nucleoprotein encoded by the viral short RNA segment, but reacted with a protein encoded by the long RNA segment, either the viral polymerase, or the Z protein. This one clone, in addition to primary CTL harvested from immunized C3H mice, failed to lyse target cells expressing the Z protein, suggesting recognition was to the viral polymerase. Two other clones recognized the viral nucleoprotein, amino acids 93-100, as determined by protein deletion and peptide mapping studies. When introduced directly into the central nervous systems of LCMV-infected histocompatible mice, all clones were active in vivo and capable of causing immunopathologically mediated death.

Animals↗
PubMed1985

Inhibition of anti-class I cytotoxicity by anti-class II monoclonal antibodies (MoAb). II. Blocking of anti-class I CTL clones by anti-DR MoAb.

Monoclonal antibodies directed at monomorphic determinants on A,B,C (w6/32) or DR (L243, S4/24, S8/8) HLA antigens were used to inhibit the cytotoxic activity of class I- or class II-reactive CTL clones. Anti-class I (HLA-Bw62) cytotoxic T lymphocyte CTL clones were inhibited by w6/32, but not by L243, when tested on PHA blasts and LCL as targets. Interestingly, S4/24 and S8/8 demonstrated a differential blocking ability of class I reactive clones; these two monoclonal antibodies could not block Bw62-directed CTL clones when using PHA blasts as targets but strongly inhibited the cytotoxic activity of the same clones on LCLs as targets.

Antibodies, Monoclonal↗
PubMed1996

Induction of SIV capsid-specific CTL and mucosal sIgA in mice immunized with a recombinant S. typhimurium aroA mutant.

We have developed a new expression system based on the E. coli groEL promoter. The suicide vector constructed (called APC vector) allows simultaneous attenuation of a Salmonella strain by disruption of the coding sequence for aroA and stable integration of a gene into the bacterial chromosome. High-level expression of antigen is achieved after Salmonella is taken up by macrophages, a major antigen processing cell of the host. The chloramphenicol acetyltransferase (CAT) and the simian immunodeficiency virus capsid (p27gag) genes were cloned downstream of the groEL promoter and expressed within S. typhimurium. By measuring CAT activity, we showed that the groEL promoter was up-regulated during infection of the J774 macrophage line. The immune response to SIV capsid was assessed in Balb/c mice given one oral dose of vaccine. A local mucosal secretory IgA response against SIV capsid was detected but no systemic antibody response to the same antigen. A systemic CTL response was detected as early as 28 days to as late as 70 days post-immunization. CTL activity was MHC restricted (H-2d) and was mediated by CD3+, CD8+, CD4- T-lymphocytes. These results indicate that with only one oral dose of recombinant Salmonella using the APC vector, a systemic CTL response and a mucosal secretory response against the SIV capsid antigen are elicited in a mouse model.

Animals↗
PubMed1995

In vivo CTL induction with point-substituted ovalbumin peptides: immunogenicity correlates with peptide-induced MHC class I stability.

Class I molecules are conformationally sensitive to peptide binding, prolonging the complex's half-life on the surface of the cell. By making a series of H2-Kb anchor motif amino acid point substitutions in the ovalbumin 257-264 octamer, we were able to analyse subtle changes in peptide binding, Kb stabilization and in vivo immunogenicity. The cell line RMA-S was used to determine peptide-dependent Kb stabilization under equilibrium and non-equilibrium binding conditions. Sixteen conservative and non-conservative amino acid substitutions were made at positions 3, 5 or 8 of the peptide. At 37 degrees C, Kb stabilization was differentially affected by these substitutions, with several substitutions severely affecting Kb surface expression. When the substituted peptides were used as immunogens to prime cytotoxic T lymphocytes (CTL) in vivo, each peptide's ability to stabilize Kb directly correlated with the intensity of specific CTL activation. We conclude that peptide class I stabilization is an important influencing factor in determining cell surface steady-state expression of these peptides and thus the breadth of CTL recruitment. These concepts may relate the phenomenon of immunodominance to cell surface-presented peptide steady-state levels and may also aid in peptide vaccine design.

Amino Acid Sequence↗
PubMed2006

Comparative evaluation of CD8+CTL responses following gene gun immunization targeting the skin with intracutaneous injection of antigen-transduced dendritic cells.

The skin is an attractive target for antigen-specific vaccination. Particle bombardment of the epidermis with plasmid DNA using the gene gun results in antigen expression in keratinocytes of the epidermis leading to antigen presentation in the draining lymph nodes by migratory dendritic cells (DC). In order to better understand the role of the skin in stimulating antigen-specific CD8+cytotoxic T cells (CTL), we compared gene gun immunization with intracutaneous injections of antigen-transduced DC. A single intracutaneous injection of antigen-transduced DC was able to induce in vivo expansion of CD8+CTL specific for the model antigen chicken ovalbumin while four simultaneous shots with the gene gun were not effective. Antigen-transduced DC were much more efficient than particle bombardment of the epidermis in stimulating adoptively transferred TCR-transgenic CD8+CTL in the draining lymph nodes. Employing the novel technique of in vivo bioluminescence imaging, we demonstrated efficient gene transfer to the skin following gene gun bombardment and confirmed that a similar amount of antigen reached the lymph node when compared with injection of antigen-transduced DC. Our results suggest that direct transfection of the skin does not optimally reach and activate appropriate antigen-presenting DC. We believe that this reflects the immunological function of the epidermis which must balance immunity and tolerance to foreign antigens. Further investigations will have to address the role of Langerhans cells for the activation of cellular immunity in the skin.

Adoptive Transfer↗