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At least 667 records · Page 37Linked to original sources
PubMed1995

Beta 2-microglobulin-deficient mice demonstrate class II MHC restricted anti-viral CD4+ but not CD8+ CTL against influenza-sensitized autologous splenocytes.

Mice transgenic for beta 2-microglobulin gene deletion (beta 2M-/-) can clear respiratory pathogens but at a slower rate than control mice. How these mice eliminate virus is not known, but the process is believed to involve CD4+ T cells. Recent studies from other laboratories have suggested a role for CD8+ cytotoxic T lymphocytes (CTLs) in both recognition of beta 2M deficient cells by allogeneic mice and rejection of MHC-incompatible tumor cells by beta 2M-/- mice. After influenza inoculation, we found no evidence for anti-influenza CD8 CTL activity from the lungs or spleens of beta 2M-/- mice. Anti-influenza CD4+, class-II restricted CTL activity was demonstrated from both the lungs and spleens. We next used mitogen-stimulated splenocytes from beta 2M-/- mice for targets in an in vitro CTL assay. This method for determining MHC class II CTL activity showed that the lungs and spleens of influenza-infected beta 2M-/- mice contained precursors to CD4+, but not CD8+, effector CTLs. The data indicate that CD8+ CTLs have no role in anti-viral activity in beta 2M-/- mice. Development of anti-tumor CTLs and anti-viral CTLs may arise via different mechanisms.

Animals↗
PubMed1984

Generation of CTL lines using cultured T cells (CTC) as feeder cells. I. Specificity for class I determinants.

Lines of cytotoxic T lymphocytes (CTL) may be grown in long-term culture in medium supplemented with Interleukin-2 (IL-2). In most cases a source of feeder cells is also necessary. Because they are available in large numbers, we tested whether cultured T cells (CTC) would serve as feeder cells. We confirmed the observation of others that CTL grown in medium supplemented with IL-2, but in the absence of feeder cells, grew poorly and became less cytotoxic with time. CTL lines directed class I HLA antigens and grown with either x-irradiated peripheral blood lymphocytes (PBL) or x-irradiated CTC proliferated well, were similarly cytotoxic, and demonstrated the same antigenic specificity. By using cells from members of a family in which one offspring had inherited a recombinant chromosome, it was possible to show that sharing of the Class I determinants between the original stimulating cells in the primary culture and the CTC feeder cells was sufficient to enable the maintenance of strongly cytotoxic CTL lines.

Cell Division↗
PubMed1996

Comparative efficiency of simple lipopeptide constructs for in vivo induction of virus-specific CTL.

We have previously shown that virus-specific CTL responses can be elicited in vivo by injecting, without adjuvant, 12-40 amino acid-long peptides, modified in C-terminal position by a simple lipidic amino acid. In this paper, we have studied the chemical accessibility, and the ability to induce in mice a CTL response, of a series of lipopeptides derived from the HIV-1 env (312-327) or (302-335) sequences. We showed that a single modification of these peptides by a lipidic amino acid, preferably in C-terminal position, results in the ability to reproducibly induce, without adjuvant, a relevant CTL response. No clear discrimination appeared concerning the nature of the lipidic modification. Our findings indicate that modification of a relatively long peptide by a N epsilon-palmitoyl-L-Lysylamide can be achieved by conventional methods of synthesis and characterization, offering the possibility to develop low-cost synthetic vaccines in models in which the CTL component is of importance.

Amino Acid Sequence↗
PubMed2003

Fewer CTL, not enhanced NK cells, are sufficient for viral clearance from the lungs of immunocompromised mice.

Activation of the aryl hydrocarbon receptor (AhR) causes numerous defects in anti-viral immunity, including suppressed CTL generation and impaired host resistance. However, despite a reduced CTL response, mice that survive infection clear the virus. Therefore, we examined the contribution of NK cells and pro-inflammatory cytokines to viral clearance in influenza virus-infected mice exposed to TCDD, the most potent AhR agonist. Infection caused transient increases in pulmonary TNFalpha, IL-1, and IFNalpha/beta levels, but neither the kinetics nor magnitude of this response was affected by AhR activation. No IL-18 was detected at any time point examined. Exposure to TCDD enhanced NK cell numbers in the lung but did not affect their IFNgamma production. Furthermore, depletion of NK cells did not alter anti-viral cytolytic activity. In contrast, removal of CD8+ T cells ablated virus-specific cytolytic activity. These results demonstrate that the pulmonary CTL response to influenza virus is robust and few CTL are necessary for viral clearance.

Animals↗
PubMed2006

Epitope mapping of full-length glycoprotein D from HSV-2 reveals a novel CD4+ CTL epitope located at the transmembrane-cytoplasmic junction.

The glycoprotein D of HSV-2 (gD2) is currently a leading candidate vaccine target for genital herpes vaccines as both cellular and humoral responses can be generated against it. However, little is known about how vaccine composition will affect T cell epitope selection. A panel of 15-mer peptides (with 11 amino acid overlap) spanning full-length gD2 was used to investigate the fine specificity of T cell responses to gD2 as well as the role of vaccine composition on epitope selection. Spleen cells from BALB/c mice (H-2(d)) immunized with gD2, formulated with or without AlPO(4) and/or IL-12, were stimulated in vitro with overlapping gD2 peptides. Cellular responses (lymphoproliferation and IFN-gamma expression) were mapped to four epitopes within the gD2 molecule: gD2(49-63), gD2(105-119), gD2(245-259), and gD2(333-347). CTL analysis of these four epitopes indicated that not all of them could serve as a CTL epitope. Mice immunized with gD2 expressed from a viral vector mounted CTL responses primarily to one epitope located in the extracellular domain of gD2 (gD2(245-259)). More importantly, mice immunized with gD2 co-administered with IL-12 mounted CTL responses to an additional epitope located at the transmembrane-cytoplasmic junction of gD2 (gD2(333-347)). The location of this novel epitope emphasizes the benefit of using full-length versions of glycoproteins when designing vaccine components.

Amino Acid Sequence↗
PubMed2006

Preparation of native and amplified tumour RNA for dendritic cell transfection and generation of in vitro anti-tumour CTL responses.

Recent research indicates that dendritic cells transfected with RNA-encoded tumour-associated antigens (TAA) can generate potent anti-tumour immune responses in vitro and in vivo. RNA is an important source of TAA, but its relatively unstable nature, in addition to often limited availability of tumour tissue, may represent a considerable obstacle for its use. Our first goal was to establish an efficient protocol for the preparation of high quality total RNA from tumour samples. This should then be used as such or be pre-amplified for DC transfection. Therefore native total RNA was prepared from stabilised tissue samples obtained from liver metastases of colon cancer using either solution- or silicagel-based protocols for RNA isolation. The first isolation protocol yielded higher amounts of total RNA, but with lower purity as compared to the second one. No degradation of RNA was observed regardless of the protocol used. Subsequently, we focused on the amplification of mRNA. The fidelity of the amplified mRNA was confirmed by RT-PCR for glyceraldehyde-3-phosphate-dehydrogenase (GADPH) and carcinoembryonic antigen (CEA) coding sequences. We found no differences in the induction of CEA-specific CTL responses between native and amplified RNA-transfected DCs. Additionally, we tested the induction of CTL responses and found that DCs transfected with amplified mRNA originating from either tumour tissue or a cell line were able to induce strong anti-tumour CTL responses in vitro. They were comparable to those induced by native total RNA-transfected DCs. Our results therefore indicate that the amplified mRNA is equivalent to the native one in the induction of anti-tumour CTL responses and can be used for generation of RNA-transfected DCs.

Cell Line, Tumor↗
PubMed2006

Migratory dendritic cells transfer antigen to a lymph node-resident dendritic cell population for efficient CTL priming.

Skin dendritic cells (DCs) are thought to act as key initiators of local T cell immunity. Here we show that after skin infection with herpes simplex virus (HSV), cytotoxic T lymphocyte (CTL) activation required MHC class I-restricted presentation by nonmigratory CD8(+) DCs rather than skin-derived DCs. Despite a lack of direct presentation by migratory DCs, blocking their egress from infected skin substantially inhibited class I-restricted presentation and HSV-specific CTL responses. These results support the argument for initial transport of antigen by migrating DCs, followed by its transfer to the lymphoid-resident DCs for presentation and CTL priming. Given that relatively robust CTL responses were seen with small numbers of skin-emigrant DCs, we propose that this inter-DC antigen transfer functions to amplify presentation across a larger network of lymphoid-resident DCs for efficient T cell activation.

Animals↗
PubMed2007

Effect of serum and CTL on focal growth of human cytomegalovirus.

BACKGROUND: In immunocompromised patients only cytotoxic T-lymphocytes (CTL) but not antiviral antibodies appear to be efficient in control of human cytomegalovirus (HCMV) infection. This is contrasted by the well-documented neutralising activity of patient sera against standard HCMV strains. OBJECTIVE: We tested the hypothesis that a cell-culture model based on a recent clinical HCMV isolate would more accurately approximate the clinical situation and provide an explanation for the failure of neutralising antibodies in efficient restriction of HCMV infection. METHODS: Sera from five bone marrow transplant recipients with or without prolonged HCMV replication were analysed by an enzyme-linked immunoassay for their capacity to neutralise cell-free HCMV preparations. The inhibitory effect of these sera on viral cell-to-cell-spread was then quantified by focus expansion assays using a recent clinical HCMV-isolate and was finally compared to the inhibitory effect of HCMV-specific CTL lines. RESULTS: Prolonged HCMV replication occurred in three patients despite high titres of neutralising antibodies. In contrast to the strong inhibitory effect on cell-free HCMV, their sera could not inhibit the focal growth of a recent cell-associated HCMV isolate, whereas CTL clones directed against pUL123 or pUL83 of HCMV effectively limited focal expansion of the clinical isolate in fibroblast culture. CONCLUSIONS: Focus expansion assays based on a cell-associated clinical HCMV isolate provide a model for the in vivo effectiveness of virus-specific CTL and neutralising antibodies. Our data support the assumption that due to their strict cell-association clinical HCMV strains are withdrawn from neutralising antibodies.

Antibodies, Viral↗
PubMed2004

Designing and engineering of DNA-vaccine construction encoding multiple CTL-epitopes of major HIV-1 antigens.

A synthetic T cell immunogen (TCI) has been designed as a candidate DNA-based vaccine against Human immunodeficiency virus (HIV)-1 using cytotoxic T lymphocytes (CD8(+) CTL) and T-helper lymphocytes (CD4(+) Th) epitopes retrieved from the Los Alamos HIV Molecular Immunology Database. The protein 392 amino acids in length contains about eighty CTL-epitopes, many of which are overlapping and are totally restricted by ten different HLA class I molecules. To be able to detect CTL responses induced by a DNA vaccine in experimental animals, additional epitopes, restricted by mouse and Macaque rhesus major histocompatibility complex (MHC) class I molecules, were included in the target immunogen. The gene encoding the TCI protein was assembled, cloned into vector plasmids and expressed in a prokaryotic and a eukaryotic system. The presence of HIV-1 protein fragments in the immunogen structure was ascertained by ELISA and immunoblotting using panels of HIV-1-positive sera and monoclonal antibodies to p24. It has been demonstrated that DNA vaccine can induce both specific T cell responses (CTL and blast transformation) and specific antibodies in mice immunized with pcDNA-TCI.

AIDS Vaccines↗
PubMed2004

Human airway epithelial cells present antigen to influenza virus-specific CD8+ CTL inefficiently after incubation with viral protein together with ISCOMATRIX.

In the present paper, an in vitro model was established in which the interaction between influenza virus-specific CD8+ T cells and human airway epithelial cells can be studied. To this end, the human lung epithelial cell line A549 was transduced with the HLA-A*0201 gene. This MHC class I allele is involved in the presentation of the immunodominant M158-66 cytotoxic T lymphocyte (CTL) epitope of the influenza A virus matrix protein. The A549-HLA-A2 cells and a CD8+ T cell clone specific for the M158-66 epitope were used to evaluate ISCOMATRIX (IMX), which is considered a potential mucosal adjuvant for influenza vaccines, for its capacity to activate virus-specific CTL after incubation with epithelial cells. It was found that virus infected epithelial cells activated virus-specific CTL efficiently. However, incubation of epithelial cells with ISCOMATRIX and recombinant M1 protein activated CD8+ T cells inefficiently, unlike the incubation of C1R cells expressing a HLA-A2 trans gene or HLA-A2+ B-lymphoblastoid cells with these reagents. It was concluded that this lack of antigen presentation by epithelial cells indicate that these cells are not subject to killing by virus-specific CTL upon instillation with ISCOMATRIX-based vaccines, which may be a favorable property of mucosal vaccines.

Adjuvants, Immunologic↗
PubMed2005

CTL escape and increased viremia irrespective of HIV-specific CD4+ T-helper responses in two HIV-infected individuals.

We investigated whether development of mutations leads to loss of CD8 T-cell recognition in HIV-1 infection and is possibly linked to alterations in HIV-1-specific CD4(+) T-cell responses in 2 HIV-infected individuals. In patient, H434 full genome sequencing of HIV-1 biological clones at early and late time points during disease progression showed development of fixed mutations in 16 predicted HIV-specific CTL epitopes. Loss of T-cell recognition and reactivity against wild-type and mutant epitopes was observed primarily for the HLA-B27-restricted KK10 epitope and HLA-A2-restricted SL9 epitope. Similarly, in patient H671, decreasing numbers of HLA-A3-restricted CD8(+) T cells specific for the wild-type RK9 epitope was observed after CTL escape. Only in patient H434 loss of CTL responses was paralleled by a decrease in HIV-specific IL-2(+) CD4(+) T-helper responses. This suggests that loss of T-cell reactivity may not be directly linked to HIV-specific CD4(+) T-cell responses but that increased viremia after CTL escape may influence CD4(+) T-helper responses.

Epitopes, T-Lymphocyte↗
PubMed2005

Spontaneous in vivo retrovirus-infected T and B cells, but not dendritic cells, mediate antigen-specific Fas ligand/Fas-dependent apoptosis of anti-retroviral CTL.

C57BL/6 (H-2b), but not spontaneous virus-expressing AKR.H-2b congenic, mice generate retrovirus-specific CD8+ CTL responses to the immunodominant Kb-restricted epitope, KSPWFTTL. AKR.H-2b non-responsiveness is mediated by a peripheral tolerance mechanism. When co-cultured with primed B6 antiviral pCTL, AKR.H-2b splenocytes are recognized by the antiviral TcR as "veto" cells, which inhibit by an exquisitely virus-specific, MHC-restricted, veto cell FasL/responder T cell Fas, mediated apoptotic mechanism. Here, AKR.H-2b thymus, lymph node, and bone marrow cells are also shown to inhibit antiviral CTL generation. Purified AKR.H-2b CD4+ and CD8+ T cells, and B cells, served effectively as FasL-dependent veto cells. In contrast, AKR.H-2b dendritic cells (DC) did not efficiently veto antiviral CTL responses, despite expressing sufficient MHC class I/viral peptide complexes for TcR recognition. AKR.H-2b DC also expressed FasL mRNA and cell surface protein, albeit at a lower level than AKR.H-2b T and B cells. These findings suggest a fail-safe escape mechanism by virus-infected cells for escape from CTL-mediated immunity.

AKR murine leukemia virus↗
PubMed1996

Identification of potential CTL epitopes of bovine RSV using allele-specific peptide motifs from bovine MHC class I molecules.

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young infants and housed calves. Depletion of CD8+ lymphocytes from calves inhibited their ability to clear the virus from the nasopharynx and lungs. To study these cells further, a cytotoxic T lymphocyte (CTL) assay was established. CTL could be demonstrated in the peripheral blood of gnotobiotic calves 7-10 days post infection (p.i.) with RSV and in lungs 10 days p.i. This response was both MHC-restricted and virus-specific. Following separation of the lung lymphocytes by magnetic activated cell sorting, it was shown that the cytolytic activity was mediated by cells of the CD8+ phenotype. To identify epitopes recognised by bovine CTL, the consensus motifs from MHC class I alleles found in the herd at Compton were identified. cDNA libraries were constructed and screened for full length class I sequences. The isolated cDNA clones were then transfected into mouse P815 cells and the expressed product immunoprecipitated and matched with a serological specificity. The bovine MHC class I molecules were isolated from lysed transfected cells by affinity chromatography, using a monoclonal antibody specific for bovine MHC class I, and bound peptides were separated by reverse-phase HPLC. Analysis of the protein sequences of bovine RSV for the defined motifs has identified potential CTL epitopes.

Alleles↗
PubMed1999

Detailed immunophenotype of CD8+ memory cytotoxic T-lymphocytes (CTL) against HIV-1 with respect to expression of CD45RA/RO, CD62L and CD28 antigens.

We have previously reported that circulating effector cytotoxic CD8+ T-lymphocytes (CTLs) against HIV-1 express CD38 and HLA-DR activation antigens. In this study, we performed two series of FACS sorts to phenotype and characterize precursors of CTL effectors. First we looked at memory CTL activity against HIV-1 stimulated by antigen as well as CTL activity stimulated by CD3 mAb with regard to whether the precursors expressed CD45RA and/or CD62L. We found that the precursor cells that could be stimulated with antigen to become effectors within 7 days predominated in the CD45RA CD62L subset. However. in donors with low levels of CD8+ T-cell activation as measured by CD38 antigen expression, memory cells could also be found in the CD45RA+ CD62L+ subset. Our data indicate that reversion of memory cells to the CD45RA+ CD62L+ phenotype can occur in humans, especially in donors with low levels of virus replication and minimal CD8 + T-cell activation. Next, we looked at CD28 expression with regard to antigen specific memory cells and again found that the level of virus replication and CD8+ T-cell activation influenced the subset that contained the memory cells. In donors with high levels of virus replication, our results indicated that CTL were being actively recruited from both CD28+ and CD28 subsets, while in donors with undetectable levels of viral replication, the memory cells were entirely in the CD28 compartment.

CD28 Antigens↗
PubMed2001

Identification of a peptide capable of inducing an HIV-1 Tat-specific CTL response.

Although Tat-specific CTL responses are elicited in HIV-infected patients and in non-human primate models, specific CTL epitopes within Tat have not been identified. In this study, we mucosally immunized mice with recombinant, full-length Tat protein or individual Tat-specific, overlapping peptides to map putative H-2d-restricted, Tat-specific CTL epitopes. Standard chromium release assays from splenocytes of immunized animals identified a peptide (QPKTACTNC) capable of inducing Tat-specific CTL responses. This newly-identified epitope lies within a region of low sequence variability among HIV-1 subtypes, suggesting its potential use in a multicomponent AIDS vaccine.

AIDS Vaccines↗
PubMed2003

Optimization of epicutaneous immunization for the induction of CTL.

The immune system of the skin has recently been exploited for the development of non-invasive vaccine technologies. However, one of the limitations of current vaccine protocols is the inefficient priming of cytotoxic T lymphocytes (CTL). In this study, we report that the application of either an immunodominant class I MHC restricted ovalbumin peptide or whole ovalbumin protein, to tape-stripped skin together with the co-application of the bacterial enterotoxin cholera toxin (CT) induces antigen-specific CTL. Tape-stripping (TS) was found to enhance the magnitude of antibody responses to co-administered protein and to promote the generation of antigen-specific IgG(2a) responses. As well, both cholera toxin and tape-stripping enhanced epidermal dendritic cell (DC) immigration into draining lymph nodes. The adjuvant effect of co-administered cholera toxin and tape-stripping in promoting CTL priming was not dependent on IL-12. Epicutaneous immunization has previously been shown to induce robust antibody responses to administered protein antigen. We now demonstrate the induction of robust and persistent CTL responses to epicutaneously administered protein antigen. Epicutaneous immunization is cheap, simple and effective. These findings suggest the potential use of the skin for the generation of protective immune responses to both viral and tumor challenge.

Administration, Cutaneous↗
PubMed2003

A novel multivalent human CTL peptide construct elicits robust cellular immune responses in HLA-A*0201 transgenic mice: implications for HTLV-1 vaccine design.

Cytotoxic T-lymphocytes are critical in the clearance of chronic viral infections such as HTLV-1. Peptide-based vaccines may have potential application in invoking antiviral CTL responses. In the development of vaccination strategies, it is becoming increasingly important to elicit a broad immune response against several epitopes simultaneously that may provide large population coverage. In the present study, we addressed this issue, namely the processing and presentation of multiple CTL epitopes simultaneously for the generation of multispecific CTL responses. We designed a novel multivalent peptide consisting of three HLA-A(*)0201 restricted CTL epitopes, with intervening double arginine residues in tandem. These epitopes were derived from the HTLV-1 regulatory protein Tax, which is an attractive target for vaccine development against HTLV-1. Arginine residues were included to provide cleavage sites for proteasomes, to generate the intended MHC Class I ligands. Proteasomal digestion studies and mass spectrometry analysis showed cleavage of the multivalent construct to generate the individual epitopes. Immunization of HLA-A(*)0201 transgenic mice with this construct efficiently elicited cellular responses to each intended epitope in vivo, further validating the applicability of this approach. These data may have potential in the development of immunotherapeutic strategies for the treatment of HTLV-1 disease and in the future design of multivalent subunit peptide vaccines.

Animals↗
PubMedDate not supplied

Induction of a cytotoxic T lymphocyte response by immunization with a malaria specific CTL peptide entrapped in biodegradable polymer microspheres.

We have previously reported that biodegradable polymer microspheres (MS) are capable of eliciting strong and long-lasting antibody and T cell proliferative responses for either natural protein antigens or synthetic peptides. In this study, we investigated the possibility of inducing antigen-specific cytotoxic T lymphocyte (CTL) responses in vivo with a short synthetic peptide from the circumsporozoite (CS) protein of Plasmodium berghei (Pb) 252-260 by using different MS formulations. We show that injection of mice with a short CTL epitope microencapsulated in MS or adsorbed on empty MS enhanced a specific CTL response comparable to that obtained with the incomplete Freund's adjuvant (IFA) formulation, indicating that MS are a potent antigen delivery system/immunostimulant for CTL response. These results might be of practical interest for MS preparation and development of subunit vaccines.

Animals↗