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At least 865 records · Page 48Linked to original sources
PubMed2000

Epitope affinity for MHC class I determines helper requirement for CTL priming.

We show here that priming and memory generation of antigen-specific CD8+ cytotoxic T lymphocytes (CTL) does not require help if the immunogen binds major histocompatibility complex (MHC) class I molecules with high affinity. This conclusion was based on the study of three chemically distinct optimal length CTL epitopes with high affinity for the restriction element Kb. In contrast, when two subdominant epitopes with intermediate MHC binding affinity were studied, either a class II MHC-restricted T helper cell epitope or administration of antibody to CD40 was required to obtain significant CTL priming. Depending on the epitope, one source of help was much more efficient than the other.

Animals↗
PubMed2004

HIV evolution: CTL escape mutation and reversion after transmission.

Within-patient HIV evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. Whether this intrapatient accumulation of escape mutations translates into HIV evolution at the population level has not been evaluated. We studied over 300 patients drawn from the B- and C-clade epidemics, focusing on human leukocyte antigen (HLA) alleles HLA-B57 and HLA-B5801, which are associated with long-term HIV control and are therefore likely to exert strong selection pressure on the virus. The CTL response dominating acute infection in HLA-B57/5801-positive subjects drove positive selection of an escape mutation that reverted to wild-type after transmission to HLA-B57/5801-negative individuals. A second escape mutation within the epitope, by contrast, was maintained after transmission. These data show that the process of accumulation of escape mutations within HIV is not inevitable. Complex epitope- and residue-specific selection forces, including CTL-mediated positive selection pressure and virus-mediated purifying selection, operate in tandem to shape HIV evolution at the population level.

Adult↗
PubMed2003

Dendritic cells transfected with tumor RNA for the induction of antitumor CTL in colorectal cancer.

Dendritic cells (DC) are the most potent antigen-presenting cells known, currently tested for vaccination studies in cancer patients. The use of tumor-derived RNA to load DC overcomes the requirement of defined HLA types and the identification of tumor antigens expressed by the tumors. Here, we show that human monocyte-derived DC generated under serum-free conditions by GM-CSF, IL-4 and TNF-alpha acquire a mature phenotype and expression of the chemokine receptor CCR-7, which plays a pivotal role in DC migration to the afferent lymph nodes. We demonstrate the feasibility of total RNA transfection into such DC using the renal cell carcinoma (RCC) cell line N43-EGFP, which was stably transfected with an EGFP-encoding vector. Moreover, we show that DC transfected with RNA from colorectal cancer cells present HLA class I-restricted antigenic epitopes to induce a primary antitumor CTL response in vitro. Interestingly, the CTL induced by SW480 RNA also recognized another colon cancer line, HCT116, and the RCC line A498. Our results confirm the feasibility of total RNA transfection of serum-free generated DC for the induction of CTL against colon cancer and RCC cells, and support the relevance of shared tumor rejection epitopes between colorectal cancer and RCC.

Carcinoma, Renal Cell↗
PubMed1996

The adjuvant effect of a non-toxic mutant of heat-labile enterotoxin of Escherichia coli for the induction of measles virus-specific CTL responses after intranasal co-immunization with a synthetic peptide.

The intranasal route has been shown to be effective for immunization. However, immunization via this route may require the use of potent and safe adjuvant. The construction of non-toxic mutants of heat labile enterotoxin of Escherichia coli (LT), which is a potent mucosal adjuvant, is a major breakthrough for the development of mucosal vaccines. In this study we have assessed the ability of an LT mutant (LTK63) to act as an adjuvant following intranasal co-immunization with a peptide corresponding to a measles virus cytotoxic T lymphocyte (CTL) epitope. LTK63 was more effective at potentiating the in vivo induction of peptide-specific and measles virus-specific CTL responses than was administration of the peptide in saline. A concentration of 10 micrograms/dose of LTK63 was found to be the most effective in potentiating the in vivo priming of peptide-specific and measles virus-specific CTL responses. These findings highlight the potential of the non-toxic mutant of LT as a safe mucosal adjuvant for use in humans.

Adjuvants, Immunologic↗
PubMed2002

Human CD8+ CTL recognition and in vitro lysis of herpes simplex virus-infected cells by a non-MHC restricted mechanism.

Cytotoxic T lymphocytes (CTL) are important for the recognition and lysis of virally infected cells, but their effectiveness can be limited by viral immune evasion mechanisms. We investigated the immunophenotype and function of human CD8+ T cells raised in response to herpes simplex virus (HSV). The expanded population contained cells of an activated and mature phenotype, as determined by the expressions of CD25, CD45RO, CD57, CD95 and HLA-DR. Cultured cells also expressed CD45RA. These cells lysed autologous and allogeneic HSV-infected lymphoblastoid cell line (LCL) targets via a non-major histocompatibility complex (MHC) restricted recognition pathway. Inhibition assays showed the mechanism of cytotoxicity to be calcium-dependent, granule exocytosis pathway, rather than the internal disintegration pathway. Cold target competition assays indicated that a common CTL population contributed to the recognition of autologous and allogeneic-infected targets. These effectors showed recognition of infected targets which was distinct from that of K562 cells. Non-MHC restricted lysis-associated molecule 2B4 (CD244) was upregulated on culturing and made a significant contribution to lysis of FcgammaR-bearing targets in a redirected killing assay. These findings suggest that CTL can recognize virally infected cells through a combination of non-MHC restricted mechanisms and may result in more efficient lysis than classical CD8+ T cells.

Antigens, CD↗
PubMed2003

Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16.

Many specific antiviral and antitumour immune responses have been attributed to the protective effects of antigen-specific CD8+ cytotoxic T lymphocytes (CTL). Recognition of virus infected or tumour cells by CTL requires presentation of at least one peptide epitope from a virus or tumour-specific antigen by the relevant MHC Class I molecule. Viral genes with mutations which remove CTL epitopes may thus be favoured for survival. Human cervical cancers are caused by papillomavirus infection, and these cancers consistently express the E7 protein of the oncogenic papillomavirus. We therefore investigated the MHC Class I restricted T cell epitopes of the human papillomavirus type 16 E7 oncoprotein using mice of five different genetic backgrounds, and an IFN-gamma ELISPOT assay, to determine the frequency with which MHC Class I epitopes might be expected in this small oncoprotein (98 amino acids). No MHC Class I restricted responses were detected in E7 immunized BALB/c (H-2d), CBA/CaH (H-2 k), FVB/N (H-2q) or A2KbH2b human HLA2.1 transgenic mice. In C57BL/6 J (H-2b) mice, a previously identified single antigenic epitope was detected. Therefore, we conclude that there is a paucity of MHC Class I restricted T cell epitopes in HPV16 E7 protein because of its small size. This might be advantageous to the virus. Furthermore here we present a quick and easy method to exhaustively determine CD8 T cell epitopes in proteins using a unique set of overlapping 8, 9 and 10 mer synthetic peptides.

Animals↗
PubMed2000

Sensitization of tumour cells to lysis by virus-specific CTL using antibody-targeted MHC class I/peptide complexes.

A number of cell surface molecules with specificity to tumour cells have been identified and monoclonal antibodies (mAb) to some of these antigens have been used for targeting tumour cells in vivo. We have sought to link the powerful effector mechanisms of cytotoxic T-cells with the specificity of mAb, by targeting recombinant HLA class I molecules to tumour cells using an antibody delivery system. Soluble recombinant MHC class I/peptide complexes including HLA-A2.1 refolded around an immunodominant peptide from the HIV gag protein (HLA-A2/gag) were synthesized, and the stability of these complexes at 37 degrees C was confirmed by enzyme-linked immunosorbent assay using a conformation-specific antibody. MHC class I-negative lymphoma cells (Daudi) were labelled with a biotinylated mAb specific for a cell surface protein (anti-CD20) then linked to soluble biotinylated HLA-A2/gag complexes using an avidin bridge. Flow cytometry revealed strong labelling of lymphoma cells with HLA-A2/gag complexes (80-fold increase in mean channel fluorescence). CTL specific for HLA-A2/gag efficiently lysed complex-targeted cells, while control CTL (specific for an HLA-A2.1-restricted epitope of melan-A) did not. Similarly, SK-mel-29 melanoma cells were also efficiently lysed by HLA-A2/gag-specific CTL when HLA-A2/gag complexes were linked to their surface via the HMW-MAA specific anti-melanoma antibody 225.28s. With further consideration to the in vivo stability of the MHC class I/peptide complexes, this system could prove a new strategy for the immunological therapy of cancer.

Antibodies, Monoclonal↗
PubMed2006

Generation of in vitro B-CLL specific HLA class I restricted CTL responses using autologous dendritic cells pulsed with necrotic tumor lysate.

New approaches in the treatment of chronic B lymphocytic leukemia (B-CLL) have led to improved clinical response rates. In this setting there is a need to evaluate novel therapeutic approaches that aim to eradicate minimal residual B-CLL cells following an initial favorable response. The use of tumor lysate-pulsed dendritic cells (DC) represents a potentially important development in the field of cancer vaccination. B-CLL is ideally suited for DC-based vaccination since tumor cells are readily available (peripheral blood) and both known (tumor idiotype) and unknown antigens can be exploited to stimulate immune responses. In the current study we have evaluated the ability to stimulate in vitro autologous immune reactivity against target B-CLL cells using autologous DCs pulsed with B-CLL tumor lysate. Enhanced specific T cell IFN-gamma expression was detected in 9 of 14 patients evaluated. These responses were specific with increased levels of IFN-gamma mRNA measurable in T-cells stimulated with NC-DCs and not unpulsed DCs or DCs pulsed with normal B cell lysate. CTLs demonstrating increased levels of IFN-gamma mRNA also lysed autologous B-CLL targets cells in an MHC class 1-restricted manner by (51)chromium release assay. Priming target leukemic cells with CD40 ligand and IL-4 enhanced CTL killing. The effector CTL displayed negligible toxicity against NK susceptible target cells K-562 and spared CD19(+)CD5(-) normal B cells in cytotoxicity assays. The specificity of the CTL response was confirmed by blocking HLA class I molecules and cold target inhibition assays.

Cell Differentiation↗
PubMed1996

Ribosomal scanning past the primary initiation codon as a mechanism for expression of CTL epitopes encoded in alternative reading frames.

An increasing amount of evidence has shown that epitopes restricted to MHC class I molecules and recognized by CTL need not be encoded in a primary open reading frame (ORF). Such epitopes have been demonstrated after stop codons, in alternative reading frames (RF) and within introns. We have used a series of frameshifts (FS) introduced into the Influenza A/PR/8 /34 nucleoprotein (NP) gene to confirm the previous in vitro observations of cryptic epitope expression, and show that they are sufficiently expressed to prime immune responses in vivo. This presentation is not due to sub-dominant epitopes, transcription from cryptic promoters beyond the point of the FS, or internal initiation of translation. By introducing additional mutations to the construct exhibiting the most potent presentation, we have identified initiation codon readthrough (termed scanthrough here, where the scanning ribosome bypasses the conventional initiation codon, initiating translation further downstream) as the likely mechanism of epitope production. Further mutational analysis demonstrated that, while it should operate during the expression of wild-type (WT) protein, scanthrough does not provide a major source of processing substrate in our system. These findings suggest (i) that the full array of self- and pathogen-derived epitopes available during thymic selection and infection has not been fully appreciated and (ii) that cryptic epitope expression should be considered when the specificity of a CTL response cannot be identified or in therapeutic situations when conventional CTL targets are limited, as may be the case with latent viral infections and transformed cells. Finally, initiation codon readthrough provides a plausible explanation for the presentation of exocytic proteins by MHC class I molecules.

Animals↗
PubMed1998

Translation of a retained intron in tyrosinase-related protein (TRP) 2 mRNA generates a new cytotoxic T lymphocyte (CTL)-defined and shared human melanoma antigen not expressed in normal cells of the melanocytic lineage.

We report here the identification of a new shared human melanoma antigen recognized by a human leukocyte antigen (HLA)-A*68011-restricted cytotoxic T lymphocyte clone (CTL 128). The cDNA encoding this antigen is composed of a partially spliced form of the melanocyte differentiation antigen tyrosinase-related protein (TRP)-2, containing exons 1-4 with retention of intron 2 and part of intron 4 (TRP-2-INT2). The sequence coding for the antigenic epitope is located at the 5' end of intron 2 and is available for translation in the same open reading frame of the fully spliced TRP-2 mRNA. This peptide is also recognized by CTL 128 when presented by the HLA-A*3301, a member of the HLA-A3-like supertype that includes the HLA-A*68011. Quantitative reverse transcription PCR analysis carried out on total and/or cytoplasmic mRNA demonstrated that, in contrast to the fully spliced TRP-2 mRNA expressed in melanomas, normal skin melanocytes, and retina, the TRP-2-INT2 mRNA could be detected at significant levels in melanomas but not in normal cells of the melanocytic lineage. Instead, in these normal samples, both the spliced and the unspliced transcript of gp100 were expressed at high levels. Absence of endogenous TRP-2-INT2 expression in melanocytes was also confirmed by lack of recognition of HLA-A*68011-transduced, TRP-2(+) melanocyte lines by CTL 128. These results indicate that a partially spliced form of a differentiation antigen mRNA, present in the cytoplasmic compartment of neoplastic but not normal cells of the melanocytic lineage, can be the source of a melanoma-restricted T cell epitope.

Alleles↗
PubMed2004

Limited T cell receptor diversity of HCV-specific T cell responses is associated with CTL escape.

Escape mutations are believed to be important contributors to immune evasion by rapidly evolving viruses such as hepatitis C virus (HCV). We show that the majority of HCV-specific cytotoxic T lymphocyte (CTL) responses directed against viral epitopes that escaped immune recognition in HCV-infected chimpanzees displayed a reduced CDR3 amino acid diversity when compared with responses in which no CTL epitope variation was detected during chronic infection or with those associated with protective immunity. Decreased T cell receptor (TCR) CDR3 amino acid diversity in chronic infection could be detected long before the appearance of viral escape mutations in the plasma. In both chronic and resolved infection, identical T cell receptor clonotypes were present in liver and peripheral blood. These findings provide a deeper understanding of the evolution of CTL epitope variations in chronic viral infections and highlight the importance of the generation and maintenance of a diverse TCR repertoire directed against individual epitopes.

Amino Acid Sequence↗
PubMed2006

Maintenance of viral suppression in HIV-1-infected HLA-B*57+ elite suppressors despite CTL escape mutations.

Rare human immunodeficiency virus 1-infected individuals, termed elite suppressors (ES), maintain plasma virus levels of <50 copies/ml and normal CD4 counts without therapy. The major histocompatibility complex class I allele group human histocompatibility leukocyte antigen (HLA)-B*57 is overrepresented in this population. Mutations in HLA-B*57-restricted epitopes have been observed in ES, but their significance has remained unclear. Here we investigate the extent and impact of cytotoxic T lymphocyte (CTL) escape mutations in HLA-B*57+ ES. We provide the first direct evidence that most ES experience chronic low level viremia. Sequencing revealed a striking discordance between the genotypes of plasma virus and archived provirus in resting CD4+ T cells. Mutations in HLA-B*57-restricted Gag epitopes were present in all viruses from plasma but were rare in proviruses, suggesting powerful selective pressure acting at these epitopes. Surprisingly, strong CD8+ T cell interferon-gamma responses were detected against some mutant epitopes found in plasma virus, suggesting the development of de novo responses to viral variants. In some individuals, relative CD8+ T cell interleukin-2 responses showed better correlation with the selection observed in vivo. Thus, analysis of low level viremia reveals an unexpectedly high level of CTL escape mutations reflecting selective pressure acting at HLA-B*57-restricted epitopes in ES. Continued viral suppression probably reflects CTL responses against unmutated epitopes and residual or de novo responses against epitopes with escape mutations.

CD4 Lymphocyte Count↗
PubMed1990

Inhibition of the in vitro generation of class II-restricted, HSV-1-specific, CD4+ CTL by HIV-1.

Previously, we demonstrated that cultures of human peripheral blood lymphocytes (PBL) stimulated with herpes simplex virus type 1 (HSV-1) generate antigen-specific, major histocompatibility complex (MHC) restricted cytotoxic T lymphocytes (CTL) that tend to be CD4+ and restricted to HLA-DR antigens. In this study, we present evidence that when HSV-1 stimulated human peripheral blood lymphocytes (PBL) are cocultured with human immunodeficiency virus type 1 (HIV-1), the generation of CD4+, DR-restricted CTL during the 5-day culture period is inhibited. In contrast, HIV-1 had no effect on either natural killer (NK) activity, or on the unrestricted NK-like killers which are often detected in HSV-1-stimulated cultures after the depletion of CD16+ cells. HIV-1 also failed to inhibit the generation of CTL against Epstein-Barr virus (EBV), a response that principally involves CD8+, CD4-, class I-restricted killers.

Animals↗
PubMed1991

Induction of HIV-specific CTL and antibody responses in mice using retroviral vector-transduced cells.

Recombinant retroviral vectors can efficiently transduce and express foreign genes in mammalian cells. We have examined the utility of retroviral vector-mediated gene transfer to deliver genes which encode human immunodeficiency virus type I (HIV) antigens capable of stimulating specific immune responses. Murine fibroblast cell lines were transduced with a nonreplicating murine retroviral vector carrying the gene encoding the HIV-IIIB envelope protein and were shown to express the gp160/120 protein. Mice immunized with syngeneic vector-transduced cells developed CD8+, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) specific for targets expressing the HIV envelope protein. The CTL also exhibited lytic activity on target cells coated with synthetic peptides derived from the gp120 V3 hypervariable region of both the HIV-IIIB and HIV(MN) isolates. Following adoptive transfer in a murine tumor model, these CTL were shown to be effective in vivo by their ability to eliminate established tumor cells expressing the HIV protein. Vector-transduced syngeneic cells were also capable of eliciting HIV envelope-specific antibody responses in immunized mice. Sera obtained from these mice were found to bind to the HIV-IIIB gp160 protein as well as a peptide-defined neutralizing antibody epitope contained within the V3 domain of gp120. These sera exhibited virus-neutralizing activity in that they markedly reduced the ability of HIV to infect and form syncytia of a human T-cell line. This is the first demonstration that cells transduced with a retroviral vector encoding the HIV-IIIB envelope protein are capable of inducing effective HIV-specific cellular and humoral immune responses in mice.

Amino Acid Sequence↗
PubMed1981

CTL helper factor(s) produced by peritoneal cells of mice immunized to Listeria monocytogenes: relationship to interferon.

We have studied the relationship of interferons (IFN) to soluble helper factor(s) produced in vitro by activated peritoneal exudate cells of mice immunized to Listeria monocytogenes. This helper factor(s) can assist the generation of cytotoxic lymphocytes (CTLs) in response to allogeneic thymocytes that have been heat-treated to remove functional antigens that would normally stimulate CTLs in the absence of added exogenous help. In addition to IFN and CTL helper activity, a thymocyte proliferative activity also was detected. There appeared to be collaboration between activated nonadherent T cells and macrophages (MO) in the production of all three activities. The IFN produced appeared to be primarily IFN-gamma, however, some IFN-alpha/beta probably was present as well. Although CTL helper activity and IFN were both contained in fractions comprising the major peak of thymocyte proliferative activity when supernatant fluids containing all three were chromatographed, functional IFN could be deleted with pH-2 and anti-IFN treatment without appreciably altering the other two activities. Furthermore, when fluids containing helper activity, but depleted of functional IFN, were added to cultures of responder lymphocytes and heat-treated thymocytes, IFN was produced during the course of the five day culture as were CTLs. In parallel cultures to which fluids containing helper factor(s) were not added, no IFN was produced, and no CTLs were generated. Thus, although fluids having activities required for the generation of CTLs may be depleted of IFN at the time they are added to cultures, a role for IFN in some phase of CTL development cannot be excluded.

Animals↗
PubMed2002

Important contribution of p15 Gag-specific responses to the total Gag-specific CTL responses.

OBJECTIVES: HIV-1 p15 Gag and its protease cleavage products, NCp7 and p6, are believed to play a major role in viral infectivity and assembly during the early and late stages of the retroviral life cycle. However, the extent to which p15 Gag is targeted by the host immune system in natural infection as well as precise cytotoxic T lymphocyte (CTL) epitopes within this protein remains to be defined. METHODS: In this study, 57 HIV-1 infected individuals and 10 HIV-1 negative controls were screened for CD8 and CD4 T-cell responses using overlapping peptides spanning the entire p15 Gag protein as well as the p17 Gag and p24 Gag proteins. Peptide-specific interferon-gamma production was measured by Elispot assay and flow-based intracellular cytokine quantification, and cytotoxic activity was confirmed after isolation of peptide-specific CD8 T-cell lines. RESULTS: CD8 T lymphocytes specific to p15 Gag were found in 46% (26/57) of HIV-1 infected individuals studied and contributed on average 17% (range, 0-100%) to the total Gag-specific T-cell responses. Responses were clustered within three immunodominant regions of p15 Gag, mapping to important functional sites. These studies also include the description of the first three optimally defined CTL epitopes within p15 Gag. CONCLUSIONS: These results indicate that p15 Gag is frequently recognized by HIV-1-specific CD8 T cells in HIV-1 infection and will be important in the comprehensive assessments of CTL responses in infected persons, as well as the design and testing of future HIV-1 vaccines and immunotherapeutic interventions.

Amino Acid Sequence↗
PubMed1996

Analytical study of the clinical response to two distinct adoptive immunotherapies for advanced hepatocellular carcinoma: comparison between LAK cell and CTL therapy.

To evaluate the effect of two distinct adoptive immunotherapies, tumor-specific cytotoxic T-cell (CTL) therapy and lymphokine-activated killer (LAK) cell therapy, the clinical responses of patients with stage IV primary hepatocellular carcinoma (HCC) treated with these therapies were studied. Of 18 patients treated with CTL, 3 had complete regression (CR), 2 had partial regression, and 3 had minor regression (MR). Their median survival was 21 months after the end of therapy, and 1 CR patient survived for > 6 years. On the other hand, in the LAK-cell-treated group of eight patients, four had MR and their median survival was only 2 months. No survival was observed 27 months after the end of LAK cell therapy. These results indicate that tumor-specific CTL therapy is more effective than LAK cell therapy and that it might be a promising therapeutic tool for advanced HCC patients.

Adult↗
PubMed1998

Dendritic cells pulsed with CEA peptide induce CEA-specific CTL with restricted TCR repertoire.

The human carcinoembryonic antigen (CEA), which is expressed in several cancer types is a potential target for antigen-specific immunotherapy. In this study, we show that dendritic cells (DC) pulsed with an HLA class I restricted CEA cytotoxic T lymphocyte (CTL) peptide epitope can stimulate T cells to kill CEA peptide loaded T2 target cells as well as CEA expressing tumor lines in the presence of interleukin-7 (IL-7) in an HLA-restricted manner. This has been demonstrated for carcinoma patients as well as healthy donors. The DC-CEA + IL-7 stimulated cultures contained predominantly CD3+CD8+CD56- cells indicative of MHC class I restricted CTL. In addition, DC-CEA + IL-7 stimulated cells showed higher levels of CD69 expression compared with cells stimulated with IL-7 alone, implying an activated phenotype. When the T-cell receptor (TCR) from CTL cultures stimulated with DC-CEA + IL-7 was analyzed, an oligoclonal pattern of expression was found for certain V beta subfamilies compared with the polyclonal patterns shown by IL-7 or phytohemagglutinin stimulated T cells from the same donors. This TCR restriction appeared to be maintained and enhanced after additional rounds of restimulation with DC-CEA + IL-7. The association between cytotoxicity and TCR restriction suggests that TCR analysis may be useful as an in vitro indicator to monitor alterations in the T-cell population in response to antigen-specific immunotherapies.

Amino Acid Sequence↗