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At least 649 records · Page 36Linked to original sources
PubMed2002

Investigation of antigen delivery route in vivo and imune-boosting effects mediated by pH-sensitive liposomes encapsulated with K(b)-restricted CTL epitope.

Using fluorescein isothiocyanate (FITC)-conjugated H-2K(b) CTL epitope (SIINFEKL) as a model system, we investigated the antigen delivery route by pH-sensitive liposomes in vivo. Fluorescence was initially detected in lymph nodes at 3 h after immunization, and its intensity reached a peak value in superticial inguinal lymph node at 9 h. No trace could be detected in spleen even with prolonged monitoring for up to 24 h. These results strongly suggest that the presentation of CTL-peptide antigen vehicled by pH-sensitive liposomes exclusively occurs in lymph nodes. In mice immunized with the H-2K(b) CTL epitope encapsulated pH-sensitive liposomes, peptide-specific CTL response was detected at day 3. The response was strongly augmented by the second immunization and persisted up to at least 45 days. These results suggest that pH-sensitive liposome formula functions as a potential adjuvant of peptide antigens and is useful for the induction of antigen specific CTLsv in vivo.

Animals↗
PubMed1994

Chronic morphine treatment suppresses CTL-mediated cytolysis, granulation, and cAMP responses to alloantigen.

Exposure to opioid drugs (e.g., morphine) in vivo has been shown to suppress natural killer cell activity. However, the effects of in vivo exposure to opioids on cytotoxic T lymphocyte (CTL) activity has not been investigated. The administration of morphine (50.0 mg/kg, sc) to alloimmunized mice for 11 days resulted in a significant decrease in peritoneal and splenic CTL activity. Moreover, the intracellular content of serine esterases and esterase release by CD8+ effector cells from chronic morphine-treated mice was reduced compared to that of effector cells from vehicle-treated controls. In addition, the CD8+ cAMP response to alloantigen was diminished compared to CD(8+)-enriched cells from vehicle-treated animals. However, conjugate formation between effector and target and subsequent killing of target by effector cells did not reveal significant differences between vehicle- and chronic morphine-treated animals. Serum corticosterone and dehydroepiandrosterone levels were significantly lower in the chronic morphine-treated animals while proopiomelanocortin gene expression (exon 3) in splenic lymphocytes did not correlate with morphine-mediated suppression of CTL activity. These results indicate that CTL activity is sensitive to chronic morphine exposure, implicating opioids as important cofactors during viral infections in suppressing cell-mediated immunity.

Animals↗
PubMed1994

Precursor CTL specific for retrovirus-induced T lymphoma are found at high frequency in unprimed syngeneic mice.

The retrovirus-induced RBL5 lymphoma can be rejected by adoptive transfer of noncytolytic CD4+ Th 1 lymphocytes in normal hosts, without a requirement for transfer of specific CD8+ CTL. Therefore, we hypothesized that host precursor CTL (pCTL) might cooperate with transferred CD4+ Th1 cells to mediate tumor rejection. To evaluate this hypothesis, lymphocytes from non-immunized mice were analyzed for cytolytic activity after short-term bulk lymphocyte tumor culture (BLTC) with rIL-2 (5U/ml). BLTC induced the differentiation of anti-RBL5 CTL distinct from non-MHC-restricted LAK. These effectors were CD8+, TCR alpha/beta+, and utilized the CD3-TCR complex for MHC class I-restricted lysis. The majority of pCTL were found within the CD44/PgP-1hi population of memory/activated lymphocytes. However, there was no serologic evidence for prior exposure to RBL5-related tumor or viral Ags. CTL activity was susceptible to partial blockade with mAbs directed against CD8 and MHC class I, suggesting a relatively low-affinity Ag-TCR interaction. These data are most consistent with the recruitment of a population of Ag-specific, but cross-reactive, pCTL during BLTC.

Animals↗
PubMed1995

Multiple CTL specificities against autologous HIV-1-infected BLCLs.

The cellular immune response to HIV-1 has been well studied but, in many respects, remains incompletely defined. Although CTL specificities against highly conserved HIV-1 determinants as dictated by vaccinia/HIV-1 vector constructs have been described, much less is known regarding patient cellular reactivities against autologous cells infected with HIV-1. One of the main obstacles in characterizing this cellular reactivity has been the absence of a targeting system which accurately represents the HIV infected cell in vivo and is, at the same time, adaptable for in vitro assays. Through the use of two separate strategies aimed at increasing cellular CD4 expression, we were able to infect B-lymphocyte cell lines (BLCLs) with multiple strains of HIV-1. HIV-1-infected BLCLs were recognized by autologous effector cells with cytolytic specificities against env, gag, or pol determinants. In addition, HIV-1-infected BLCLs were capable of eliciting in vitro CTL reactivities directed against env-, gag-, and pol-expressing targets. This cellular reactivity was mediated by CD8+ cells and was MHC Class I restricted, suggesting a classical CTL response. Since multiple antigens are recognized, an HIV-1-infected BLCL is a more natural representation of an in vivo cellular target than other available testing systems and should permit a more representative analysis of CTL responses during infection or following vaccination.

B-Lymphocytes↗
PubMed1996

Reversal of age-related deficient influenza virus-specific CTL responses and IFN-gamma production by monophosphoryl lipid A.

Old and young Balb/c mice, 24--26 and 2--4 months old, respectively, were infected with a 0.1 LD50 of influenza A/Taiwan/1/86 (A/H1N1) virus by small particle aerosol. Lung virus titers were determined 4, 6, 8, 12, and 17 days later. Old mice had significantly higher virus titers than young mice (P < 0.05-0.0001) and shed virus up to Day 17, while young mice were free of virus by Day 12. Splenic MHC class I CD8+ CTL activity (P < 0.08--0.001) and IFN-gamma production (0.1-0.008) measured on Days 8, 12, and 17 were significantly lower among old mice than among young mice. Coadministration of liposomal influenza vaccine with monophosphoryl lipid A (MPL) resulted in enhanced CD8+ CTL response and IFN-gamma production among old mice (35 and 12,000 times, respectively). These results demonstrate that MPL stimulates CTL and Th1 cytokines (IFN-gamma) in aged mice and may serve to reverse age-related CD8+ CTL deficiency and reduce severe influenza disease in elderly human populations.

Adjuvants, Immunologic↗
PubMed1998

Activation of donor-specific CTL in a tolerant recipient of cardiac allograft.

Permanent graft acceptance was induced with cyclosporine A in a low responder rat strain combination PVG (RT1(c)) to DA (RT1(av1)) of cardiac transplants. Challenge (alloimmunization) with donor and third-party WF (RT1(u)) cells was performed and followed by the analysis of alloreactive cytotoxicity. The cytotoxic T lymphocytes (CTL) from recipients with permanently accepted cardiac grafts manifested cytolytic activity against third-party target cells but not against donor target cells. The findings demonstrated unresponsiveness to be donor-specific, i.e., transplant tolerance was present. This was verified by retransplantation of the DA recipient with either a second PVG heart (which was accepted) or a third-party (WF) heart (which was rejected). Restoration of donor-specific CTL after injection of exogeneous interleukin(IL)-2 concomitant with challenge showed that the donor-specific CTL were not depleted, but unable to be activated as a consequence of IL-2 deficiency. Despite donor-specific CTL activation in vivo the recipients failed to reject their established grafts, implying an ongoing presence of suppression.

Animals↗
PubMed2000

HLA-A2.1/K(b) transgenic murine dendritic cells transduced with an adenovirus encoding human gp100 process the same A2.1-restricted peptide epitopes as human antigen-presenting cells and elicit A2.1-restricted peptide-specific CTL.

HLA-A2.1/K(b) transgenic mice (A2.1/K(b) mice) were used to investigate the processing of human gp100 melanoma antigen by murine antigen presenting cells (APC). Bone marrow-derived dendritic cells (DC) from A2.1/K(b) mice were transduced with adenovirus encoding human gp100 (Ad2/hugp100v2). The Ad2/hugp100v2-transduced DC express human gp100, as documented by immunoperoxidase staining. Flow cytometric analysis demonstrates that Ad vector transduction does not downregulate expression of several markers, including MHC class I. We show that Ad2/hugp100v2-transduced DC are recognized by peptide-specific, A2.1-restricted CTL, suggesting correct processing and presentation of the hugp100 antigen by murine DC. To assess dominance among the various A2.1-restricted epitopes encoded by hugp100, A2.1/K(b) transgenic mice were immunized with Ad2/hugp100v2-transduced DC. Resulting effector cytotoxic T lymphocytes (CTL) were assayed for peptide specificity using a panel of six synthetic peptides known to encode A2.1-restricted epitopes of human gp100 (denoted G154, G177, G209, G280, G457, G476). CTL obtained from Ad2/hugp100v2-transduced DC immunized A2.1/K(b) mouse lysed target cells presenting five of the six epitopes, supporting the observation that murine cells correctly process the hugp100 antigen. The immunogenicity of individual gp100 epitopes correlates with their binding affinity to A2.1. CTL generated from A2.1/K(b) mice immunized with Ad2/hugp100v2-transduced DC also specifically recognize A2.1(+)/gp100(+) human melanoma cells. These data suggest that murine APC process and present the same set of HLA-restricted peptides, similar to human APC. HLA transgenic mice serve as a useful model system to study class I-restricted epitopes of human tumor-associated antigens.

Adenoviridae↗
PubMed1996

Genomic organization of the murine CTL lipase gene.

Murine cytotoxic T-lymphocyte (CTL) lipase was originally identified as an IL-4-inducible gene in CD8-positive T cells. To further our understanding of both the function and the regulation of CTL lipase in T cells, we have cloned and characterized the murine gene. Two overlapping phage clones spanning 29 kb contain the entire CTL lipase gene. The exon structure is similar to those characterized for the human and canine pancreatic lipase-related protein 1 genes, with notable differences in the 5' end. Transcripts initiate from a site that matches a consensus for an initiator sequence. Potential cis-regulatory elements in the CTL lipase 5' regulatory region that would confer dual tissue specificity in exocrine pancreas and cytotoxic T lymphocytes are identified. The implications of this promoter organization are discussed.

Animals↗
PubMed1994

Mutations inside but not outside the peptide binding cleft of the H-2 Ld molecule affect CTL recognition and binding of the nucleoprotein peptide from the lymphocytic choriomeningitis virus.

In order to investigate the role of residues inside and outside the peptide binding cleft of the Ld molecule in peptide presentation to cytotoxic T lymphocytes (CTL), we constructed a series of point mutations in the Ld gene. We determined the effects of the mutations in the Ld molecule on the binding and recognition of an Ld-restricted CTL epitope derived from the nucleoprotein (NP) of the lymphocytic choriomeningitis virus (LCMV). Each of the mutations within the Ld peptide binding cleft resulted in a complete loss of CTL recognition. Addition of the LCMV NP peptide to cells expressing these mutants did not increase surface Ld expression, suggesting that the mutations altered peptide binding. Mutations involving pockets D and E within the cleft affected LCMV peptide binding and recognition as drastically as those in pocket B, which was predicted to interact with a main anchor residue of the peptide. In striking contrast, the mutations located outside the cleft did not change either recognition or binding. These results demonstrate that the Ld residues in the peptide binding cleft are the main determinants dictating LCMV NP peptide binding, and that the residues in each of the pockets within the cleft play a role in this interaction. Surprisingly, one mutation outside the peptide binding cleft, T92S, abrogated CTL lysis of target cells treated with the LCMV NP peptide, but not virus-infected cells. These data show that this mutation selectively altered the presentation of the LCMV NP peptide introduced to the cell exogenously, but not endogenously. This implies that the pathway by which peptides associate with class I molecules within the cell differs from that of exogenous peptide binding.

Amino Acid Sequence↗
PubMed1995

Intact antigen presentation for Epstein-Barr virus (EBV)-specific CTL by a lymphoblastoid cell line established from a patient with severe chronic active EBV infection.

Severe chronic active Epstein-Barr virus (EBV) infection is a lymphoproliferative disease characterized by extremely high antibody titers to EBV, fever, lymphadenopathy, hepatosplenomegaly, and pancytopenia, without any prior immunological abnormality. A spontaneous lymphoblastoid cell line was established from a 4-year-old boy with severe chronic active EBV infection. Immunofluorescence and Western blotting analyses showed that the cell line was of B cell origin and expressed Epstein-Barr nuclear antigens 1, 2 3a, 3b and 3c, and latent membrane protein 1, which are reported to be targets for EBV-specific cytotoxic T lymphocytes (CTL). The cytotoxicity of peripheral blood mononuclear cells derived from the patient and his HLA-identical sister was assayed against the cell line. The cell line was recognized and killed by anti-EBV CTL derived from the HLA-identical sister, but the patient's peripheral blood mononuclear cells had no cytotoxicity. We conclude that antigen presentation in the EBV-infected cells from the patient is intact and sufficient for generation of an EBV-specific CTL response. These observations suggest that severe chronic active EBV infection may not be caused by impaired EBV-antigen presentation of the infected cells but by impaired cellular immune responses to the virus. Our results also suggest the therapeutic possibility that this disease may be treated by adoptive transfer of EBV-specific CTL or bone marrow transplantation from an HLA-matched donor whose immune response to EBV is intact.

Animals↗
PubMed2004

Secretomers as a new tool for the monitoring of CTL responses.

Efforts to follow tumor-specific immune responses in patients are often thwarted by lack of knowledge of the appropriate tumor antigens and the CTL epitopes of those antigens. There is, therefore, a growing need for techniques to monitor tumor-specific immune responses in settings where tumor antigens, and antigenic epitopes, remain unidentified. Here we describe a novel system to follow tumor-specific CTL immune responses. A truncated, soluble murine class I MHC (H-2Db) molecule was fused with a rat IgG2a Fc, in order to allow secretion of the complex. Tumor-specific CTL could then be detected as a result of the complex fastening to specific T cell receptors (TCR). These constructs were inserted into the genome of a recombinant adenovirus vector. Infection of tumor cells with these adenovirus constructs results in the secretion of the complexes into the culture supernatant. These soluble divalent class I MHC molecules were used to detect and activate specific CTL populations.

Adenoviridae↗
PubMed2005

Vaccination with mRNAs encoding tumor-associated antigens and granulocyte-macrophage colony-stimulating factor efficiently primes CTL responses, but is insufficient to overcome tolerance to a model tumor/self antigen.

Immunization of mice with dendritic cells transfected ex vivo with tumor-associated antigen (TAA)-encoding mRNA primes cytotoxic T lymphocytes (CTL) that mediate tumor rejection. Here we investigated whether direct injection of TAA mRNA, encapsulated in cationic liposomes, could function similarly as cancer immunotherapy. Intradermal and intravenous injection of ovalbumin (OVA) mRNA generated specific CTL activity and inhibited the growth of OVA-expressing tumors. Vaccination studies with DNA have demonstrated that co-administration of antigen (Ag)- and cytokine-encoding plasmids potentiate the T cell response; in analogous fashion, the inclusion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA enhanced OVA-specific cytotoxicity. The ability of this GM-CSF-augmented mRNA vaccine to treat an established spontaneous tumor was evaluated in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) mouse, using the SV40 large T Ag (TAg) as a model tumor/self Ag. Repeated vaccination elicited vigorous TAg-specific CTL activity in nontransgenic mice, but tumor-bearing TRAMP mice remained tolerant. Adoptive transfer of naïve splenocytes into TRAMP mice prior to the first vaccination restored TAg reactivity, and slowed tumor progression. The data from this study suggests that vaccination with TAA mRNA is a simple and effective means of priming antitumor CTL, and that immunogenicity of the vaccine can be augmented by co-delivery of GM-CSF mRNA. Nonetheless, limitations of such vaccines in overcoming tolerance to tumor/self Ag may mandate prior or simultaneous reconstitution of the autoreactive T cell repertoire for this form of immunization to be effective.

Animals↗
PubMed2001

A TGF betaRII frameshift-mutation-derived CTL epitope recognised by HLA-A2-restricted CD8+ T cells.

Microsatellite instability (MSI) is recognised as genome-wide alterations in repetitive DNA sequences caused by defects in the DNA mismatch repair machinery. Such mutation patterns have been found in almost all analysed malignancies from patients with hereditary non-polyposis colorectal cancer, and in approximately 15% of sporadic colorectal cancers. In cancers with the MSI phenotype, microsatellite-like sequences in coding regions of various cancer-related genes, including transforming growth factor beta receptor type II (TGF betaRII), are targets for mutations. The TGF betaRII gene harbours a 10-bp polyadenine tract, and mutations within this region are found in 90% of colorectal cancers with MSI. The frameshift mutations result in new amino acid sequences in the C-terminal part of the proteins, prematurely terminating where a novel stop codon appears. In this study we have defined new cytotoxic T lymphocyte (CTL) epitope (RLSSCVPVA), carrying a good HLA-A*0201 binding motif, and resulting from the most common frameshift mutation in TGF betaRII. A CTL line and several CTL clones were generated from an HLA-A2+ normal donor by repeated stimulation of T cells with dendritic cells pulsed with the peptide. One of the CTL clones was able to kill an HLA-A2+ colon cancer cell line harbouring mutant TGF betaRII. This epitope may be a valuable component in cancer vaccines directed at MSI-positive carcinomas.

Amino Acid Sequence↗
PubMed1988

Recognition of influenza virus-infected B-cell lines by human influenza virus-specific CTL.

The cytotoxic activity on influenza virus-infected Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines (LCL-Flu) and influenza virus-infected phytohemagglutinin lymphoblasts (PHA-Flu) was compared with the use of influenza-A virus-specific cytotoxic T lymphocytes (CTL), generated in short-term bulk cultures. Cold-target inhibition experiments showed that the lysis of PHA-Flu was completely blocked by both cold LCL-Flu and cold PHA-Flu whereas the lysis of LCL-Flu was completely inhibited by cold LCL-Flu, but only partially by cold PHA-Flu, indicating that structures can be recognized on LCL-Flu which are absent from PHA-Flu. Monoclonal antibody (McAb) directed against a monomorphic determinant of major histocompatibility complex (MHC) class I molecules inhibited the lysis of PHA-Flu more strongly than the lysis of LCL-Flu. Since LCL have a high expression of MHC class II molecules compared to PHA lymphoblasts, we examined whether class II-restricted CTL activity was responsible for the (anti)class I McAb-resistant lysis of LCL-Flu. Neither anti-CD4 McAb nor anti-class II McAb inhibited the lysis of LCL-Flu which argues against a contribution of MHC class II-restricted CTL. Depletion of CD16+ cells, containing the majority of the nonspecific cytotoxic cells, did not affect the lysis of LCL-Flu, indicating that the remaining lysis on LCL-Flu was also not due to a nonspecific component. We suggest that cell-type-dependent variations exist in the nature of the immunogenic determinants to which CTL respond.

Antibodies, Monoclonal↗
PubMed1988

Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb and H-2Db class I antigens and H-2Kb- and H-2Db-restricted SV40-specific CTL-mediated lysis.

The E3/19-kDa glycoprotein (E3/19K) coded by adenovirus type 2 (Ad2) is known to inhibit the cell-surface expression of major histocompatibility complex (MHC) class I antigens by binding to the MHC antigens intracellularly, and thus reduces recognition of antigens by MHC-restricted cytotoxic T lymphocytes (CTL). We have studied the effect of the E3/19K expression in SV40-infected monkey cells, TC-7/H-2Kb and TC-7/H-2Db expressing transfected H-2Kb and H-2Db antigens, respectively, on the cell-surface H-2 class I antigens and on lysis of the cells by SV40 large tumor (T)-antigen-specific H-2Kb- and H-2Db-restricted CTL clones. H-2Db antigen expression on TC-7/H-2Db cells was drastically reduced by infection with Ad2 but not with an E3/19K-negative SV40-Ad2 hybrid virus, Ad2+ND1, as early as 12 hr postinfection. However, H-2Kb antigen expression on Ad2-infected TC7/H-2Kb cells remained unaltered, even at 24 hr postinfection. Specific lysis of SV40-infected TC-7/H-2Db cells by H-2Db-restricted SV40 T-antigen-specific CTL clones, Y-1 and Y-3, was strongly reduced by coinfection of the target cells with Ad2 but not with Ad2+ND1. Lysis of SV40-infected TC-7/H-2Kb cells by a H-2Kb-restricted SV40 T-antigen-specific CTL clone Y-4 was also reduced significantly by Ad2 infection, but not Ad2+ND1. These results indicate that the E3/19K protein affects cell-surface expression of H-2Db antigen but not H-2Kb antigen.

Adenovirus E3 Proteins↗
PubMedDate not supplied

CTX-B inhibits CTL cytotoxicity and cytoskeletal movements.

Binding of cytotoxic T lymphocytes (CTL) to specific targets induces cytoskeletal movements in the effector cell followed by delivery of the lethal hit which ultimately results in target cell lysis. The question whether movement of the cytoskeleton in CTL are obligatory for delivery of the lethal hit is not resolved. Here we report that the CTX-B subunit of cholera toxin which is devoid of the catalytic CTX-A subunit inhibits CTL function. Inhibition was found not to be due to interference with TCR expression, CTL-target conjugate formation, target induced transmembrane signalling or secretion of BLT-esterase. CTX-B however does interfere with F-actin patch formation at the effector target binding site and inhibits reorientation of the microtubule organizing center and Golgi apparatus towards the target binding site. It is concluded that interference with cytoskeletal movements is responsible for inhibition of cytolysis pointing to an important role of the cytoskeleton in the lytic reaction.

Actins↗
PubMed1989

Allosensitization in IL-2-containing limiting dilution cultures generates cytotoxic T lymphocytes (CTL) rather than LAK cells reactive with a syngeneic nonimmunogenic murine tumor.

We have previously demonstrated that high frequency (1/20) of potent cytotoxic cells reactive with the nonimmunogenic lymphoma PIR-2 of C57BL/6 (B6, H-2b) origin, can be obtained by allosensitization of syngeneic B6 splenocytes against BALB/c (H-2d) splenocytes in limiting dilution cultures (LDC). Since a high concentration (250 U/ml) of exogenous interleukin 2 (IL-2), sufficient for the elicitation of lymphokine-activated killer (LAK) cells, was used in the LDC, and because the LDC-derived cytotoxic cells were active against a wide spectrum of target cells, we investigated whether the anti PIR-2 effector cells are LAK cells or cytotoxic T lymphocytes (CTL). We found that depletion from the B6 responder cell population of Lyt2+ (CTL precursors), but not of asialo GM1+ (LAK cell precursors), prior to LDC, results in the ablation of anti PIR-2 activity. When B6 splenocytes were plated in LDC with IL-2, in the absence of allogeneic stimulating cells, the resulting anti PIR-2 activity was greater than 10- to 500-fold lower than that obtained in LDC in the presence of allogeneic stimulating cells and IL-2. These and other observations suggest that the cytotoxic response against syngeneic tumors elicited by alloantigens in LDC is mediated by CTL rather than LAK cells, and that allogeneic sensitization in LDC can provide a means for the generation of CTL against syngeneic, nonimmunogenic tumors.

Animals↗
PubMed1992

Perforin is expressed in CTL populations generated in vivo.

Immunization of C57BL/6 mice with syngeneic tumor cells, MBL-2, resulted in the generation of antitumor effector cells in vivo. The immunized C57BL/6 mice permanently rejected viable MBL-2 lymphoma cells, but not B16 melanoma cells. Cytotoxic T cells obtained from MBL-2-immunized mouse peritoneal cells (PEC) showed specific cytotoxicity against MBL-2, but not to YAC-1, RDM-4 and Meth A cells. By sorting with FACStar, the specific CTL were characterized as TCR alpha beta+ CD8+ T cells. Moreover, the cytoplasm of in vivo-induced CTL was stained with a monoclonal antibody against perforin. The localization of perforin in cytoplasmic granules of CTL was demonstrated by electron microscope analysis. This experiment presented the first evidence that in vivo-induced CD8+ CTL against syngeneic tumor cells expressed significant amounts of perforin.

Animals↗