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At least 793 records · Page 44Linked to original sources
PubMed1997

Immunotherapy of cancer. Generation of CEA specific CTL using CEA peptide pulsed dendritic cells.

Antigen specific cytotoxic T lymphocytes (CTL) are being studied for their potential immunotherapeutic benefit in the treatment of cancer. Carcinoembryonic antigen (CEA) is an oncofetal protein best known for its overexpression in the majority of colorectal, gastric, pancreatic, non small cell lung, and breast carcinomas. We are using dendritic cells (DC) pulsed with the CEA CTL peptide epitope to generate CEA specific CTL. DC from HLA A2+ donors were isolated by culturing plastic adherent PBMC in GMCSF and IL4 for 7 days. As expected these DC expressed the relevant cell surface molecules including HLA DR, CD58, CD80, and CD86. The DC were stripped of their endogenous peptides, pulsed with the A2 restricted CEA peptide, irradiated and used to stimulate autologous CD8+ T cells in the presence of IL7. Using this approach we have been able to generate CEA specific CTL from the PBMC of breast and pancreatic carcinoma patients as well as HLA A2+ tumor cells expressing the CEA antigen. This data is being used to support a phase I active immunotherapy clinical protocol using DC pulsed with CEA peptide to treat patients with metastatic malignancies expressing CEA.

Antigen Presentation↗
PubMed1998

Amastigote surface proteins of Trypanosoma cruzi are targets for CD8+ CTL.

Amastigotes of Trypanosoma cruzi express surface proteins that, when released into the host cell cytoplasm, are processed and presented on the surface of infected cells in the context of MHC class I molecules to be recognized by CD8+ CTL. To further understand the role of CTL in T. cruzi infection, we used the available MHC class I peptide binding motifs to identify potential CTL target epitopes in two recently described T. cruzi amastigote surface proteins, ASP-1 and ASP-2. The predicted amino acid sequences of ASP-1 and ASP-2 were screened for H-2b allele-specific class I peptide motifs, and four peptides (PA11, PA12, PA13, and PA14) and six peptides (PA5, PA6, PA7, PA8, PA9, and PA10) were synthesized from ASP-1 and ASP-2, respectively. The majority of the peptides bound to some degree to H-2b class I MHC molecules, and six of 10 of the peptides stimulated spleen cells from T. cruzi-infected mice to lyse target cells sensitized with the homologous peptides. Short term T cell lines specific for three of these peptides also lysed T. cruzi-infected target cells. These results demonstrate that ASP-1 and ASP-2 are targets of in vivo generated CTLs and that this CTL response induced by T. cruzi infection is parasite and peptide specific, MHC restricted, and CD8 dependent.

Animals↗
PubMed1998

Enhancement of tumor outgrowth through CTL tolerization after peptide vaccination is avoided by peptide presentation on dendritic cells.

Synthetic peptide-based vaccines have been shown to induce potent protective and therapeutic T cell-mediated immunity in preclinical animal models and are now being evaluated in clinical phase I/II studies for their efficacy against tumors or infectious diseases. However, such vaccines might also specifically tolerize T cells causing enhanced tumor outgrowth, as shown by vaccination with two CTL epitopes derived from the adenovirus type 5 early region 1 (Ad5E1) oncogenes. We now report that modification of the Ad5E1 peptide vaccine either through incorporation of the peptides into liposomes or by ligation of the peptides to lipid tails, another vaccine formulation being tested in the clinic, fails to convert immunosuppression into effective antitumor vaccination. Inclusion of a helper T cell epitope into the vaccine likewise induces enhanced tumor outgrowth and thus does not diminish the capacity of the peptides to tolerize Ad5E1-specific CTL. In contrast, the Ad5E1-derived peptides evoke a strong tumor-protective CTL response when presented on dendritic cells (DC), indicating that the in vivo CTL-tolerizing potential of these peptides is converted to specific immunostimulation when presented on DC. These findings have important implications for the development of peptide-based immune intervention strategies and emphasize the superior nature of Ag-pulsed DC over other peptide-based vaccination protocols as well as the crucial importance of the mode of peptide-Ag delivery in setting the balance between T cell stimulation and tolerization.

Amino Acid Sequence↗
PubMed1998

A role for heat shock protein 27 in CTL-mediated cell death.

CTL exocytosis of granules containing perforin and granzyme proteases induces apoptotic cell death. Either granzyme A or B can act with perforin to trigger apoptosis. Granzyme B activates a ubiquitous apoptotic cascade induced by caspase cleavage, but the granzyme A pathway is largely unknown. Using affinity chromatography with recombinant mutant inactive granzyme A, we previously isolated two granzyme A-binding proteins, PHAP (putative HLA-associated protein) I and II. PHAP II, a substrate of granzyme A, is degraded within minutes of CTL attack. Two additional cytoplasmic proteins of 27 and 53 kDa bind strongly to the mutant granzyme A column, requiring 6 M urea to elute. Sequencing identified these as the monomer and dimer of hsp27, a small heat shock protein up-regulated by stress and cellular activation. Hsp27 coprecipitates with granzyme A from cytoplasmic lysates and is not a substrate of the enzyme. Hsp27 translocates to the detergent-insoluble fraction of target cells and relocalizes from diffuse cytoplasmic staining to long filamentous fibers, especially concentrated in a perinuclear region, within minutes of CTL attack. Hsp27 may participate in morphologic changes during granule-mediated lysis. Low or absent levels of hsp27 expression in T lymphocytes, even after heat shock, may play a role in CTL resistance to granule-mediated lysis.

Cell Death↗
PubMed1998

The clonal composition of a peptide-specific oligoclonal CTL repertoire selected in response to persistent EBV infection is stable over time.

The TCR repertoire of a peptide-specific HLA A11-restricted CTL response to persistent infection with EBV was followed for a period of 57 mo. Sequencing of TCR V alpha and V beta chains and alanine scanning mutagenesis analysis of 83 CTL clones isolated in five reactivation experiments demonstrated that this repertoire is composed of at least four distinct CTL clonotypes that are constantly reactivated from donor's blood and express structurally heterogeneous TCRs. Target cell recognition and CD8 blocking experiments indicate that the four clonotypes possess different avidity and TCR affinity for the specific Ag. This demonstrates that at least in some individuals a heterogeneous peptide-specific memory CTL repertoire selected by a persistent Ag can be remarkably stable in time and accommodate a range of TCR affinities and T cell avidities. Our results suggest that competition for the specific Ag may be not the major force driving the maintenance of memory CTLs and that the nature of the first antigenic challenge may largely determine the clonal composition of memory.

Amino Acid Sequence↗
PubMed1998

Peptide modification or blocking of CD8, resulting in weak TCR signaling, can activate CTL for Fas- but not perforin-dependent cytotoxicity or cytokine production.

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Animals↗
PubMed1999

Selective induction of protective MHC class I-restricted CTL in the intestinal lamina propria of rhesus monkeys by transient SIV infection of the colonic mucosa.

The identification of mucosal immune responses required for protection against sexual transmission of HIV is essential for the development of an efficacious vaccine. To gain a better understanding of these responses, we have characterized the immune responses in the lamina propria (LP) and epithelium of the jejunum, the mesenteric lymph nodes, and peripheral blood (PBMC) of 11 rhesus monkeys following colonic exposure to two molecular clones of SIV. Two monkeys had no signs of infection. Three monkeys became persistently infected. Transient infections, characterized by the sporadic detection of virus in the periphery and/or detection of SIV-specific immune responses in either the gut-associated tissues or PBMC, were induced in six of the monkeys. One persistently infected and three transiently infected monkeys had high levels of SIV env-specific MHC class I restricted CTL in the jejunal LP. Another transiently infected monkey had SIV-specific IgA secreting B cells in the LP. Three or six months postexposure, these animals and four naive controls were challenged intracolonically with the heterologous primary isolate, SIV/DeltaB670. All four monkeys with strong SIV env-specific MHC-restricted CTL in the LP were protected, whereas none of the naive controls or the remaining seven monkeys with little or no CTL in the LP were protected. These experiments provide the first direct evidence that transient mucosal infection can induce SIV-specific immunity that remains localized to the gut-associated tissues. Furthermore, a strong correlation between SIV env-specific MHC-restricted CTL in the LP and protection against colonic mucosal challenge was observed.

Animals↗
PubMed1989

Absence of homologous restriction factor does not affect CTL-mediated cytolysis.

Homologous restriction factor (HRF) is a membrane protein of erythrocytes and leukocytes that inhibits the complement (C5b-9)-mediated lysis in a species-restricting manner. HRF has also been reported to inhibit perforin-mediated cytolysis and postulated to play a role in cytotoxic T lymphocyte (CTL) self-protection. We show that paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes, lacking HRF, are no more sensitive to Ca2+-dependent human CTL-mediated lysis than normal erythrocytes. Furthermore, mouse and normal human erythrocytes, as well as PNH erythrocytes, are similarly lysed by isolated murine perforin-containing granules. We conclude that HRF does not inhibit perforin-mediated lysis and therefore is not likely to play a role in CTL self-protection.

Blood Proteins↗
PubMed2003

Identification of a sperm protein 17 CTL epitope restricted by HLA-A1.

Sperm protein 17 (Sp17) is a novel cancer-testis antigen. We previously reported the successful generation of Sp17-specific HLA-A1-restricted cytotoxic T-lymphocytes (CTLs) from the peripheral blood of a healthy donor using a recombinant Sp17 protein. These CTLs were able to kill not only target cells pulsed with the recombinant protein but also fresh Sp17+ tumor cells. In the present study, we have identified a nonapeptide sequence within the Sp17 protein that is predicted to have a high binding affinity for the HLA-A1 molecules. We generated the synthetic nonapeptide and successfully propagated a peptide-specific CTL line. We confirmed that peptide Sp17(103-111) (ILDSSEEDK) contains an Sp17 CTL epitope restricted by HLA-A1 and identified amino acid residues 104, 107, 109 and 110 as crucial for the CTL lysis. Our findings therefore provide the tool for the characterization of CD8 T-cell function in vivo and generation of epitope-specific treatment strategies.

Cytotoxicity, Immunologic↗
PubMed2006

B and CTL responses to the ALK protein in patients with ALK-positive ALCL.

Anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma (ALCL) has a good prognosis compared to ALK-negative ALCL, possibly as a result of the immune recognition of the ALK proteins. The aim of our study was to investigate the presence of both a B and cytotoxic T cell (CTL) response to ALK in ALK-positive ALCL. We confirmed the presence of an antibody response to ALK in all 9 ALK-positive ALCL patients investigated. An ELISpot assay was used to detect a gamma-interferon (IFN) T cell response after short term culture of mononuclear blood cells with 2 ALK-derived HLA-A*0201 restricted peptides: ALKa and ALKb. A significant gamma-IFN response was identified in all 7 HLA-A*0201-positive ALK-positive ALCL patients but not in ALK-negative ALCL patients (n = 2) or normal subjects (n = 6). CTL lines (>95% CD8-positive) raised from 2 ALK-positive ALCL patients lysed ALK-positive ALCL derived cell lines in a MHC-Class I restricted manner. This is the first report of both a B cell and CTL response to ALK in patients with ALK-positive ALCL. This response persisted during long-term remission. The use of modified vaccinia virus Ankara (MVA) to express ALK is also described. Our findings are of potential prognostic value and open up therapeutic options for those ALK-positive patients who do not respond to conventional treatment.

Adolescent↗
PubMed2006

Combined prophylactic and therapeutic cancer vaccine: enhancing CTL responses to HPV16 E2 using a chimeric VLP in HLA-A2 mice.

We identified the strategies to induce a CTL response to human papillomavirus (HPV) 16 E2 in HLA-A2 transgenic mice (AAD). A chimeric HPV16 virus-like particle (VLP) that includes full length HPV16 E7 and E2 (VLP-E7E2) was generated. The combination of E2 and E7 has the advantage that E2 is expressed in early dysplasia and neoplasia lesions, where E7 is expressed in more advance lesions. Since T cell response to E2 is less defined, we first evaluated the strategies to enhancing CD8(+) T cell responses to HPV E7, using different combinations of immune-modulators with VLP-E7E2. Data showed that the CTL response to E7 could be significantly enhanced by coinjection of GM-CSF and anti-CD40 antibodies with chimeric VLP-E7E2 without adjuvant. However, using the same combination, a low level of CD8(+) T cell response to E2 was detected. To enhance the CD8+ T cell response to E2, we analyzed T cell epitopes from E2 sequence. A heterogenous prime-boost with chimeric VLP-E7E2 and E2 peptides was performed. The data showed that the priming with chimeric VLP-E7E2, followed by boosting with E2 peptides, gave a better CTL response than 2 immunizations with E2 peptides. The enhanced immunity is due to the increase of CD11c(+) and CD11c(+) CD40(+) double positive dendritic cells in mice that received immune-modulators, GM-CSF and anti-CD40. Furthermore, the level of anti-L1 antibodies remains similar in mice immunized with chimeric VLP with/without immune-modulators. Thus, the data suggested that the chimeric VLP-E7E2 has a therapeutic potential for the treatment of HPV-associated CINs and cancer without diminishing VLPs potential as a prophylactic vaccine by inducing anti-L1 antibodies against free virus.

Animals↗
PubMed1994

Isolation of naturally processed peptides recognized by cytolytic T lymphocytes (CTL) on human melanoma cells in association with HLA-A2.1.

Cytolytic T lymphocyte (CTL) clones have previously been derived from peripheral blood of melanoma patient SK29(AV). They lyse autologous melanoma cells but not autologous Epstein-Barr virus (EBV)-transformed B lymphocytes. Immunoselection experiments indicate that these CTL clones recognize 4 different antigens (Aa, Ab, B, C) in association with a single HLA restriction element, HLA-A2.1. While the expression of antigens B and C appears to be confined to SK29-melanoma cells, antigens Aa and Ab are shared by a high proportion of allogeneic HLA-A2-positive melanoma lines. HLA-A2.1 and total HLA class I molecules have now been purified from SK29-melanoma cells using affinity chromatography and associated peptides have been eluted. Peptide pools eluted from HLA-A2.1 and total class I were separated by reversed phase high performance liquid chromatography (HPLC). Individual HPLC fractions were tested for their ability to sensitize target cells for recognition by SK29-CTL clones. The presence of antigens Aa, Ab, B and C was detected in distinct HPLC fractions that were identical for both peptide pools. As target for detection of peptide antigens in HPLC fractions, the use of the HLA-A2.1-positive antigen processing mutant cell line CEM x 721.174.T2 (T2), pre-incubated with anti-HLA-A2 monoclonal antibody (MAb) MA2.1, was shown to be essential. Single-peak target-sensitizing activity was found for antigens Ab and B, whereas multi-peak sensitizing activity was reproducibly detected for antigens Aa and C. We reason that at least some of these melanoma peptide antigens might occur in biochemically distinct isoforms.

Antigens, Neoplasm↗
PubMed1985

Appearance of cytolytic granules upon induction of cytolytic activity in CTL-hybrids.

Examining a CTL-line (B6.1) with specific cytolytic activity, we have confirmed the ability of CTL-lines to produce poly C9-like, tubular complexes of two sizes (polyperforin 1 and 2). Isolated cytoplasmic granules from this cell line can induce hemolysis and contain the lysosomal marker arylsulfatase. Electron microscopial studies show that granules contain precursor molecules which are assembled into polyperforin upon addition of Ca2+. Antibodies raised against granules predominantly react with a 27-kd protein. We compared the B6.1 cell with a hybrid, PC60, derived from a cross between B6.1 and a non-cytolytic rat-thymoma. This hybrid line exhibits inducible CTL-activity. In PC60 with induced cytolytic activity, polyperforins 1 and 2, cytolytic granules and granule-associated proteins are detected. Non-cytolytic PC60 cells lack cytolytic granules and polyperforin 1, but contain polyperforin 2. These results suggest that cytolytic granules appear in parallel with the expression of cytotoxicity during T-lymphocyte differentiation.

Animals↗
PubMed2005

Identification of an immunodominant H-2D(b)-restricted CTL epitope of human PSA.

BACKGROUND: Human prostate specific antigen (PSA) is expressed selectively in prostate epithelium and is a potential target for the immunotherapy against prostate cancer. Various PSA-based vaccines have been reported to induce cytotoxic T lymphocyte (CTL) responses in animal models. Here, we present the identification and validation of an immunodominant CTL epitope of PSA in C57Bl/6 mice (H-2(b)). METHODS: PSA-specific CTLs were induced by immunization with a plasmid expressing PSA. Epitope specificity of the CTLs was determined by their reactivity against a panel of C-terminus truncated or mutated PSA proteins and use of bioinformatical prediction with the SYFPEITHI algorithm. RESULTS: The majority of PSA-specific CTLs were directed against a single H-2D(b) restricted epitope corresponding to the amino acid residues 65-74 (HCIRNKSVIL) of the protein. The CTLs had similar functional avidity against two putative H-2D(b) binding peptides: a 9-aa-long psa65-73 (HCIRNKSVI) and a 10-aa-long psa65-74 (HCIRNKSVIL). CONCLUSIONS: We demonstrate that the psa65-73 peptide can be used for reactivation of PSA-specific CTLs in vitro and ex vivo, and H-2D(b) pentamers assembled with this peptide are an efficient tool for monitoring of PSA-specific CTL responses after DNA vaccination.

Amino Acid Sequence↗
PubMed1999

Presentation of MUC1 tumor antigen by class I MHC and CTL function correlate with the glycosylation state of the protein taken Up by dendritic cells.

We previously reported that the glycosylated MUC1 tumor antigen circulating as soluble protein in patients' serum is not processed by dendritic cells and does not elicit MHC-Class II-restricted T helper responses in vitro. In contrast, a long synthetic peptide from the MUC1 tandem repeat region is presented by Class II molecules, resulting in the initiation of T helper cell responses. Here we addressed the ability of dendritic cells to present various glycosylated or not glycosylated forms of MUC1 by MHC Class I. We found that three different forms of MUC1, ranging from glycosylated and underglycosylated protein to unglycosylated synthetic peptide, were able to elicit MUC1-specific, Class-I-restricted CTL responses. The efficiency of processing and the resulting strength of CTL activity were inversely correlated with the degree of glycosylation of the antigen. Furthermore, the more efficiently processed 100mer peptide primed a broader repertoire of CTL than the glycosylated protein.

Amino Acid Sequence↗
PubMed1997

Protein sorting and secretion during CTL killing.

Now that the key proteins involved in target cell destruction during the lethal hit from CTL have been identified, an understanding of the mechanisms which regulate their delivery becomes important for understanding their relative contributions during CTL killing. This review summarizes what is known about the packaging and delivery of the different effector proteins involved in target cell destruction. The mechanisms which regulate their delivery during killing contribute to some of the unusual features of CTL killing, and give some clues about the different contributions in target cell destruction.

Animals↗
PubMed1998

Reduction of respiratory syncytial virus titer in the lungs of mice after intranasal immunization with a chimeric peptide consisting of a single CTL epitope linked to a fusion peptide.

In the work described here, the effect of intranasal immunization of BALB/c mice with synthetic chimeric peptides consisting of a cytotoxic T-cell epitope (amino acids 81-95) from the M2 protein of respiratory syncytial virus (RSV) and a fusion peptide (amino acids 113-131) from the F1 protein of measles virus on response to challenge with RSV has been assessed. Three intranasal immunizations with the chimeric peptides without adjuvant induce peptide- and RSV-specific cytotoxic T-cell responses (CTL) at 1 or 3 weeks after the third immunization. The CTL responses significantly declined at 6 weeks after immunization. Furthermore, viral load in the lungs following challenge with RSV was significantly reduced in mice immunized with the F/M2:81-95 chimeric peptide compared to control animals at 1 or 3 weeks after immunization and no reduction of RSV titers was detectable 6 weeks after immunization. The CTL activity induced by F/M2:81-95 was therefore short-lived (less than 6 weeks) but was significantly correlated with the reduction in viral load in the lungs.

Animals↗
PubMed2001

Induction of adult-like antibody, Th1, and CTL responses to measles hemagglutinin by early life murine immunization with an attenuated vaccinia-derived NYVAC(K1L) viral vector.

Although initially developed in adult animals, novel viral vectors expressing recombinant measles antigens must eventually prove their success in the early life setting, where the efficacy of the currently used live-attenuated measles virus vaccine is limited. The immunological requirements for vaccine candidates include the generation of protective antibody responses as well as the induction of Th1 and cytotoxic T lymphocytes (CTL) responses, which is challenging in the neonatal setting. Here, we report that young BALB/c mice immunized with a single dose of a vaccinia-based NYVAC(K1L) vector generate adult-like antihemagglutinin (HA) antibody responses as well as adult-like Th1 and CTL responses. Despite this strong immunogenicity in early life, antibody responses (but not T-cell responses) to a single dose of NYVAC(K1L)-HA remained susceptible to inhibition by preexisting measles antibodies, calling for use of prime-boost strategies. NYVAC(K1L)-HA is the first attenuated live viral vector demonstrated as capable of inducing adult-like antibody, Th1, and CTL responses against measles in an early life murine immunization model, a capacity previously only reported for measles DNA vaccines.

Animals↗