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PubMedDate not supplied

Difluoromethylornithine (DFMO) arrests murine CTL development in the late, pre-effector stage.

DL-alpha-Difluoromethylornithine (DFMO) is a specific inhibitor of the rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC). DFMO (1 mM) added to C57BL/6 anti-DBA/2 murine mixed lymphocyte cultures (MLC) inhibited cytolytic T lymphocyte (CTL) activity on days 3 and 5 by 88% and 96%. Putrescine (PUT; 1 mM) and spermidine (SPD; 0.01 mM) reversed DFMO inhibition, indicating that DFMO inhibition was caused by ODC antagonism. T helper (Th) cell and accessory cell functions were not affected since DFMO did not inhibit MLC proliferation or lymphokine production. Furthermore, exogenous IL-1, IL-2, IL-4, interferon-gamma, or a rat Con A supernatant failed to abrogate DFMO inhibition. Inhibition was reversible within 48 h of removing cells from DFMO; moreover, subsequent development of DFMO-blocked CTL did not require CD4+ cells. Clonal expansion of CTL treated with 1 mM DFMO for three days in MLC, determined by subsequent analysis in limiting dilution microcultures, was only approx. 1 cell division less than control. These results indicate DFMO inhibition is exerted directly on the CTL, and that the process of differentiation was more affected by a reduction in polyamine biosynthesis than proliferation. This may be a useful model to the study stages and events of CTL development, and the roles played by polyamines in supporting these processes.

Animals↗
PubMed1996

Three regions of HIV-1 gp160 contain clusters of immunodominant CTL epitopes.

HIV-1 infection stimulates a strong CTL response that coincides with resolution of viremia in acute infection and declines with development of opportunistic infections. Recognition of HIV gp160 by PBMC-derived T cell lines from 20 HIV-infected subjects is dominated by the response to a small number of peptide epitopes. Overlapping CTL epitopes restricted by multiple MHC Class I elements were identified in 3 relatively conserved regions of gp160 (amino acids 49-68, 591-600 and 844-863). CTL from five of 20 subjects recognized three overlapping immunodominant epitopes in the 49-68 a.a. region restricted by A24, B38, and B55. CTL from four subjects recognized at least three distinct epitopes in a.a 591-600 in the context of A24, B8, B14, and B27. CTL from seven subjects recognized epitopes within a.a. 844-863 restricted by A30, B7, B8 and B35.

Cell Line↗
PubMed1986

Enhancement of murine lymphoma cell lysability by CTL and by LAK cells, after treatments with mitomycin C and with adriamycin.

Murine lymphoma cells (RDM4) were treated, in culture, with adriamycin (ADM) and with mitomycin C (Mit C) at various concentrations for 20 h. They were then used as 51Cr-labelled target cells and tested for their sensitivity to the killing action both of specifically allosensitized cytotoxic T lymphocytes (CTL) and of lymphokine-activated killer (LAK) cells. They were also used as "cold target" inhibitors, in cytotoxicity assays involving CTL or LAK and 51Cr-labelled target cells. The results showed that treatments with different concentrations of Mit C enhanced the sensitivity of RDM4 cells to the killing actions of both CTL and LAK cells. ADM slightly enhanced sensitivity to the killing action by CTL, but to a much lesser extent than Mit C did. However, ADM proved to be almost as efficient as Mit C in its sensitizing action, in the case of lysis by LAK cells. Treatments with both drugs did not seem to modify the capacity of RDM4 cells to act as "cold target competitors" in cytotoxicity assays involving CTL killers, but did so in experiments involving the lytic action exerted by LAK cells.

Animals↗
PubMed1988

In vivo mechanisms of alloreactivity--IV. Cyclosporine differentially impairs accumulation of donor-reactive CTL but not donor-reactive alloantibody in murine sponge matrix allografts.

We have studied the influence of Cyclosporine on accumulation of donor-reactive CTL, donor-reactive cytotoxic alloantibody and as an immunosuppressive drug in individual murine sponge matrix allografts. During these studies, we observed that: (1) levels of CsA in sponge matrix allografts rapidly reach levels that are immunosuppressive in vitro, i.e. greater than 100 ng/ml, following daily i.m. or subsponge CsA injections of 30 mg/kg; (2) donor-reactive CTL continue to accumulate and develop cytolytic activity in sponge allografts from mice treated i.m., but not subsponge with CsA; (3) delay or interruption of subsponge CsA therapy decreases the ability of CsA to block CTL accumulation in sponge matrix allografts; (4) relatively high local concentrations of CsA appeared to be necessary to influence CTL behavior in sponge matrix allografts; (5) CsA therapy had a different effect on the development of donor-reactive cytotoxic alloantibody. Intramuscular CsA therapy potentiated alloantibody production whereas subsponge CsA therapy temporarily delayed alloantibody production. These studies demonstrate that CsA can differentially impair the accumulation of donor-reactive CTL, but not donor-reactive alloantibody in sponge matrix allografts. These results have several theoretical and clinical implications. They also illustrate the utility of sponge matrix allografts from concomitant in vivo immunologic and pharmacologic studies at the site of a localized immune response.

Animals↗
PubMed1984

A cloned cytotoxic T-lymphocyte (CTL) line recognizing a subtype of HLA B27.

The lymphoblastoid cell-line JY (HLA-A2,2;B7,7;C-; DR4,w6) was used to stimulate T cells from donor HG (HLA-A2,w23; B40,w44; Cw4; DRw6,7). Cloned CTL line were obtained by limiting dilution after tertiary stimulation. Strong cytotoxic activity on stimulator cells was found with all CTL clones obtained. One of the clones (HG-31 recognized a subtype of the HLA-B7 antigen. In this paper, we describe another long-term cloned CTL line (HG-61). This line, when tested on a panel of 107 target cells from unrelated individuals, recognized a subtype of HLA-B27 (B27 "K"). There was no significant association with any other HLA antigen. The cloned CTLs were T8+ and their cytotoxic activity could be blocked by the monoclonal antibody W6/32 which recognizes a framework determinant on HLA-A, -B, and -C molecules. In families, reactivity with cells of the CTL line (HG-61) segregated with HLA. It is concluded that the CTL line interacts with an antigenic determinant shared between the HLA-B7 antigen and JY and the subtype of HLA-B27 (B27 "K"), or detects products of a gene closely linked to HLA-B, not revealed by present-day serology.

Antibodies, Monoclonal↗
PubMed2003

Induction of anti-melanoma CTL response using DC transfected with mutated mRNA encoding full-length Melan-A/MART-1 antigen with an A27L amino acid substitution.

Modification of the parental immunodominant Melan-A/MART-1 peptide (MART-1(26-35)) by replacing the alanine with leucine (A27L) enhances its immunogenicity. Because of the reported advantages of RNA over peptides in DC vaccines, we sought to mutate the MART-1 gene to encode a full-length MART-1 antigen with an A27L amino acid substitution. Human DC were transfected with A27L-mutated MART-1 RNA (A27L RNA) or native MART-1 RNA, and then used to stimulate autologous T cells from a series of 8 HLA-A2+ volunteers. After three stimulations, all CTL induced with DC/A27L RNA exhibited more tetramer+ cells, and demonstrated stronger antigen-specific IFNgamma-secreting activity compared to CTL induced with DC/native RNA. A potent MART-1-specific, and predominantly class-I-restricted lysis was detected in most CTL induced with DC/A27L RNA, while native RNA-induced CTL showed minimal and non-specific lysis. HLA-A2+ DC and MART-1 negative/A2+ melanoma cells transfected with the A27L RNA were recognized and killed by MART-1-specific CTL, suggesting that these APC efficiently processed the A27L RNA and presented correct MART-1-specific epitope(s). In summary, introducing an A27L mutation into the MART-1 full-length mRNA sequence enhanced the immunogenicity of the encoded MART-1 Ag. The ease with which such a mutation can be made in RNA presents another potential advantage of using RNA for immunotherapy. Our results support considering this strategy for enhancing the immunogenicity of DC-based RNA vaccines.

Amino Acid Substitution↗
PubMed2004

Macrophages exposed to lymphotropic and monocytotropic HIV induce similar CTL responses despite differences in productive infection.

Macrophages are accessory cells that are vulnerable to infection by HIV-1. HTLV-IIIB, a lymphotropic strain of HIV, infects macrophages poorly resulting in either no or low levels of virus expression compared to high levels of productive infection after exposure of macrophages to the monocytotropic HIV strain Ada-M. Whether this results in an impaired ability of HTLV-IIIB-exposed macrophages to initiate protective cytotoxic T lymphocyte (CTL) immune responses against these strains is not well defined. We investigated the ability of monocyte-derived macrophages (MDM) exposed to lymphotropic and monocytotropic HIV strains to initiate primary CTL responses in vitro. MDM exposed to HTLV-IIIB induced a specific primary CTL response that was comparable to MDM exposed to the monocytotropic strain Ada-M despite marked differences in productive HIV infection in MDM between the two strains. CTL generated in this model were MHC-restricted, strain-specific, and CD8+. These data demonstrate that high levels of productive HIV infection in accessory cells are not a prerequisite for the generation of a primary CTL response, suggesting a novel immunologic interaction between MDM and lymphotropic HIV strains.

CD4-Positive T-Lymphocytes↗
PubMed2006

Dominant CTL-inducing epitopes on GAD65 are adjacent to or overlap with dominant Th-inducing epitopes.

Immune responses to GAD65 are associated with progression to T1D in NOD mice and humans. Our previous data suggested that dominant CTL-inducing and Th-inducing determinants might preferentially occur in proximal GAD65 sequences. Using a panel of 192 GAD65 peptides we discovered that four of the eight CTL-inducing peptides, including those most biologically relevant, were proximal to previously described I-A(g7)-restricted determinants that characterize natural islet autoimmunity in NOD mice. The CTL determinants 546-554 and 88-98 were presented by GAD65-expressing cells and were displayed on pancreatic LNC, along with 268-278, following beta cell damage. p546-554-specific CTL were detectable in young naive mice and transferred significant islet inflammation into NOD.scid mice. These findings demonstrate that unique regions of GAD65 may be favored during antigen processing, such that diverse dominant epitopes are produced from overlapping sequences, which can engage distinct T cell subsets. Additionally, cross-presentation may enhance GAD65-specific CTL responses in T1D.

Amino Acid Sequence↗
PubMed2004

Analysis of HLA-A24-restricted CMVpp65 peptide-specific CTL with HLA-A*2402-CMVpp65 tetramer.

Developing precise and efficient methods of monitoring immune responses against cytomegalovirus (CMV) infection in immunocompromised transplantation patients is important. With the aim of optimizing the monitoring strategy, an HLA-A24-CMVpp65 tetramer-based analysis of CMVpp65 peptide-specific CTL lines was performed. Previously, the HLA-A24-restricted CTL epitope of CMVpp65 matrix protein was identified (QYDPVAALF aa 341-349). In the present study, CMVpp65 (aa 341-349) peptide-specific CTL lines were obtained from PBLs of 12 HLA-A24+ healthy donors by two stimulations with peptide-pulsed dendritic cells (DC). Among 12 CTL lines, 9 showed HLA-A*2402-CMVpp65 tetramer staining, which was found to be strongly co-related to the amount of IFN-gamma produced by CMVpp65 peptide-restimulated CTL lines (r=0.943, P<0.001). These results suggested that HLA-A*2402-CMVpp65 tetramer staining was an efficient way to monitor immune responses against CMV infection in HLA-A24+ immunocompromised hosts.

Apoptosis↗
PubMed2006

Improved preparation of class I HLA tetramers and their use in detecting CMV-specific CTL.

Different methods were used to prepare HLA tetramers and the yields of each method were compared. Our results indicate that preliminary refolding of the heavy chain (Hc) and light chain (beta 2m) yields more monomer than the typical conventional method with urea-solubilized Hc and beta 2m. We then used the corresponding tetramers to detect cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL). Increasing data suggest that the adoptive transfer of CMV-specific CTL constitutes an effective strategy against CMV infections. We designed a method that efficiently induces CMV-specific CTL to a higher frequency in vitro than is currently achieved. This method increased the percentage of CMV-specific CTL from below 1% to 20% of PBL, accounting for more than 40% of CD8+ T cells. Successful HLA tetramer preparation provides the basis for the subsequent detection of CMV-specific CTL in clinical applications.

Cytomegalovirus↗
PubMed2004

Influenza virosomes enhance class I restricted CTL induction through CD4+ T cell activation.

Immunopotentiating reconstituted influenza virosomes (IRIV) are one of the few adjuvants currently licensed for human use. While their adjuvant capacity in the induction of humoral responses is clearly documented, few data exist on their effects on T cell immune response. Here we addressed IRIV adjuvance in the induction of HLA class I restricted cytotoxic T lymphocytes (CTL) in vitro. Lymphocyte stimulation with IM(58-66) and IRIV resulted in marked expansion of specific CTL as compared to cultures performed in the presence of either antigen alone or antigen and control liposomes (L). Studies addressing underlying adjuvant mechanisms demonstrated that IRIV activated CD4/CD45RO+ T cells, induced a cytokine profile consistent with T helper 1 (Th1) stimulation and increased the percentage of CD4+ T cells expressing CXCR3. Furthermore, supernatants from IRIV stimulated PBMC cultures promoted dendritic cell maturation. Most importantly, IRIV mediated CTL adjuvance required the presence of live CD4+ T cells. Powerful adjuvant effects of IRIV were also observed in the induction of CTL specific for the melanoma associated Melan-A/MART-1(27-35), HLA-A0201 restricted epitope. Taken together these findings indicate that IRIV are endowed with a high adjuvant capacity for HLA class I restricted CTL induction, largely attributable to their ability to antigenically stimulate CD4+ T cells.

Adjuvants, Immunologic↗
PubMed2004

Prediction of CTL epitopes using QM, SVM and ANN techniques.

Cytotoxic T lymphocyte (CTL) epitopes are potential candidates for subunit vaccine design for various diseases. Most of the existing T cell epitope prediction methods are indirect methods that predict MHC class I binders instead of CTL epitopes. In this study, a systematic attempt has been made to develop a direct method for predicting CTL epitopes from an antigenic sequence. This method is based on quantitative matrix (QM) and machine learning techniques such as Support Vector Machine (SVM) and Artificial Neural Network (ANN). This method has been trained and tested on non-redundant dataset of T cell epitopes and non-epitopes that includes 1137 experimentally proven MHC class I restricted T cell epitopes. The accuracy of QM-, ANN- and SVM-based methods was 70.0, 72.2 and 75.2%, respectively. The performance of these methods has been evaluated through Leave One Out Cross-Validation (LOOCV) at a cutoff score where sensitivity and specificity was nearly equal. Finally, both machine-learning methods were used for consensus and combined prediction of CTL epitopes. The performances of these methods were evaluated on blind dataset where machine learning-based methods perform better than QM-based method. We also demonstrated through subgroup analysis that our methods can discriminate between T-cell epitopes and MHC binders (non-epitopes). In brief this method allows prediction of CTL epitopes using QM, SVM, ANN approaches. The method also facilitates prediction of MHC restriction in predicted T cell epitopes.

Algorithms↗
PubMed2005

Co-immunization of BALB/c mice with recombinant immunogens containing G protein fragment and chimeric CTL epitope of respiratory syncytial virus induces enhanced cellular immunity and high level of antibody response.

With the goal to develop effective immunogens against infection of respiratory syncytial virus (RSV), vectors co-expressing chimeric CTL epitope or G protein fragment of RSV with carrier protein DsbA (disulfide bond isomerase) were constructed. The capacity of the expressed recombinant immunogens to induce cellular and humoral immunities were evaluated. It was demonstrated that the presence of G protein fragment was able to enhance the CTL activities induced by the chimeric CTL epitope, though G protein fragment alone had no effect on induction of CTL response. In contrast, the level of antibody response to RSV and neutralization titer in co-immunization with G protein fragment plus chimeric CTL epitope was lower than that in immunization with G protein fragment alone. The challenge experiments indicated that co-immunization further reduced RSV titers both in lung tissue and nasal track, indicating the combination of humoral and cellular immunities is more effective. This data imply that the combination of the two protein immunogens would be a viable strategy for a RSV vaccine.

Animals↗
PubMed2005

Endoplasmic reticulum targeting sequence enhances HBV-specific cytotoxic T lymphocytes induced by a CTL epitope-based DNA vaccine.

CD8(+) T cells play a critical role in protective immunity against Hepatitis B Virus (HBV). Epitope-based DNA vaccines expressing HBV-dominant CTL epitopes can be used as candidate vaccines capable of inducing cytotoxic T Lymphocytes (CTL) responses. A plasmid DNA encoding a CTL epitope of HBV core antigen, HBc(18-27), was constructed. Intramuscular immunization of C57BL/6 mice with this DNA vaccine resulted in successful induction of HBV-specific CTL responses. In order to promote transportation of the peptide into endoplasmic reticulum (ER) to bind to MHC class I molecules for optimal class I antigen presentation, an ER targeting sequence (ERTS) was fused with the C(18-27) encoding gene. ERTS fusion significantly enhanced specific CD8(+) T cell responses in terms of CTL cytolysis as well as IFN-gamma secretion. This enhancement was correlated with promoted epitope presentation on target cell surface. We report here an enhanced immunogenicity of an epitope-based DNA vaccine using an ER targeting signal sequence, which has significant implications for future design of therapeutic HBV vaccine.

Animals↗
PubMed2003

Mucosal immunization with PLGA-microencapsulated DNA primes a SIV-specific CTL response revealed by boosting with cognate recombinant modified vaccinia virus Ankara.

Systemically administered DNA encoding a recombinant human immunodeficiency virus (HIV) derived immunogen effectively primes a cytotoxic T lymphocyte (CTL) response in macaques. In this further pilot study we have evaluated mucosal delivery of DNA as an alternative priming strategy. Plasmid DNA, pTH.HW, encoding a multi-CTL epitope gene, was incorporated into poly(D,L-lactic-co-glycolic acid) microparticles of less than 10 microm in diameter. Five intrarectal immunizations failed to stimulate a circulating vaccine-specific CTL response in 2 Mamu-A*01(+) rhesus macaques. However, 1 week after intradermal immunization with a cognate modified vaccinia virus Ankara vaccine MVA.HW, CTL responses were detected in both animals that persisted until analysis postmortem, 12 weeks after the final boost. In contrast, a weaker and less durable response was seen in an animal vaccinated with the MVA construct alone. Analysis of lymphoid tissues revealed a disseminated CTL response in peripheral and regional lymph nodes but not the spleen of both mucosally primed animals.

AIDS Vaccines↗
PubMed1998

A dominant V beta bias in the CTL response after HSV-1 infection is determined by peptide residues predicted to also interact with the TCR beta-chain CDR3.

Many T cell responses are dominated by restricted TCR expression and can range from repeated usage of particular TCR Vbeta- and/or Valpha-elements, to the preferential usage of both V- and J-elements, often in conjunction with conserved V-D-J or V-J junctional sequences. Cytotoxic T lymphocytes specific for a Kb-restricted determinant from the herpes simplex virus glycoprotein B (gB) preferentially express a dominant TCRBV10 beta-chain with sequence conservation of a tryptophan-glycine located in the V-D junction. Here we have examined whether immunisation of C57BL/6 mice with the gB-peptide can mimic the CTL response seen after HSV-1 infection. Immunisation with the gB-peptide resulted in the generation of gB-specific CTL that showed a similar TCRBV10 bias to that observed after HSV-1 infection. When the gB-determinant was expressed as a part of a fusion protein, immunised mice again exhibited the TCRBV10 bias with the junctional sequence conservation in the responding CTL. C57BL/6 mice were then immunised with variants of the gB-peptide that contained amino acid substitutions at positions previously predicted to contact the TCR beta-chain CDR3. Analysis of the TCRBV usage of variant specific CTL lines showed that substitutions at the TCR-contact positions 4, 6 and 7 of the gB-peptide resulted in a loss of the TCRBV10 bias. These results suggest that the TCRBV10 bias seen in gB-specific CTL after HSV-1 infection is due to antigenic selection by the minimal peptide and is determined by residues proposed to contact the TCR beta-chain CDR3.

Amino Acid Sequence↗
PubMed2001

Mother-to-child transmission of HIV infection and CTL escape through HLA-A2-SLYNTVATL epitope sequence variation.

Cytotoxic T lymphocytes (CTL) play a central role in containment of HIV infection. Evasion of the immune response by CTL escape is associated with progression to disease. It is therefore hypothesised that transmitted viruses encode escape mutations within epitopes that are required for successful control of viraemia. In order to test this hypothesis, escape through the dominant HLA-A2-restricted CTL epitope SLYNTVATL (p17 Gag residues 77-85 SL9) in the setting of mother-to-child-transmission (MTCT) was investigated. Initial data from two families in which the HIV-infected mother expressed HLA-A*0201 and had transmitted the virus to other family members were consistent with this hypothesis. In addition, analysis of the gag sequence phylogeny in one family demonstrated that CTL escape variants can be successfully transmitted both horizontally and vertically. To test the hypothesis further, a larger cohort of transmitting mothers (n=8) and non-transmitters (n=14) were studied. Variation within the SL9 epitope was associated with expression of HLA-A2 (P=0.04) but overall no clear link between variation from the SL9 consensus sequence and MTCT was established. However, the high level of background diversity within p17 Gag served to obscure any possible association between escape and MTCT. In conclusion, these studies highlighted the obstacles to demonstrating CTL escape arising at this particular epitope. Alternative strategies likely to be more definitive are discussed.

Amino Acid Sequence↗
PubMed2003

Comparison of CTL reactivity in the spleen and draining lymph nodes after immunization with peptides pulsed on dendritic cells or mixed with Freund's incomplete adjuvant.

OBJECTIVE: To compare CTL reactivity in the spleen and the draining lymph nodes (LN) from C57BL/6 mice after immunization with self and non-self peptides pulsed on autologous dendritic cells (DC) or mixed with Freund's incomplete adjuvant (FIA). METHODS: Peptides showing high to low binding affinities for H-2 Kb/Db were emulsified in FIA or pulsed on bone marrow (BM)-derived DC and injected subcutaneously into C57BL/6 mice. Eight days later, the mice were sacrificed and cell suspensions were prepared from the spleen and draining LN. Splenocytes or LN cells were cultured for 5 days with irradiated syngeneic spleen cells (as APCs) pulsed with the appropriate peptide in vitro. 51Cr-release assay using peptide pulsed target cells was used to detect CTL reactivity. RESULTS: Both self and non-self peptides can induce specific CTL responses with the adjuvant FIA and DC. Peptide pulsed DC were found to be more effective than peptides mixed with FIA to induce specific CTL responses towards non-self peptides and can induce much stronger responses in the spleen than in the draining LN both for non-self and self peptides. Self peptides emulsified in FIA generated the strongest responses in the draining LN, whereas non-self peptides mixed with FIA generated the strongest response in the spleen. CONCLUSIONS: DC-based immunization with non-self and self peptides is more efficient than immunization based on peptides mixed with FIA. DC-based immunization focuses the CTL response towards the spleen. Immunization based on FIA focuses the response against self peptides towards the draining LN and non-self peptides towards the spleen.

Amino Acid Sequence↗