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At least 973 records · Page 54Linked to original sources
PubMed1994

Resistance of CTL to perforin-mediated lysis. Evidence for a lymphocyte membrane protein interacting with perforin.

Cytotoxic T lymphocytes are highly resistant to killing by their own cytolytic protein perforin. We have investigated the molecular basis of this self-protection mechanism. CTL withstood high dosages of perforin, when complete lysis was obtained with various tumor target cell lines. A peptide-specific CTL clone readily lysed tumor targets presenting the peptide, but was unable to kill the peptide-presenting CTL in spite of equal degranulation. With streptolysin O, a pore-forming lytic protein of bacterial origin, no difference in susceptibility between the various targets was detected. Perforin was radioactively labeled by using the Bolton and Hunter method without any loss of its lytic activity. Experiments performed at 4 degrees C revealed that, on all of the cells studied, binding of the labeled perforin to the membrane is reversible and strictly Ca(2+)-dependent. After a short exposure to 37 degrees C, perforin was no longer dissociated from cell membranes by EDTA. Although no correlation between susceptibility and the extent of perforin binding was detected, differences in the conformation of membrane-bound perforin were observed. Perforin adsorbed to resistant cells was cleaved by trypsin into a 55-kDa (C-terminal) and a 10- to 15-kDa (N-terminal) fragment, whereas this cleavage was not found on tumor cell-bound perforin. A portion of perforin was included in the Triton X-114 detergent phase at 4 degrees C on resistant CTLs only. Our results are compatible with the notion that a protective molecule, specifically expressed on CTLs, interacts with perforin, thereby rendering it lytically inactive.

Animals↗
PubMed1995

A MHC class I B locus allele-restricted simian immunodeficiency virus envelope CTL epitope in rhesus monkeys.

In light of the importance of virus-specific CTL in the control of the spread of the AIDS virus, it will be important to assess the generation of these effector cell responses in trials of novel vaccine strategies for the prevention of AIDS virus infections. To facilitate such studies in the simian immunodeficiency virus (SIV)/macaque model for AIDS, we have defined a rhesus monkey SIVmac CTL epitope carboxy terminus to both the CD4-binding and V4 regions of the envelope glycoprotein. We also used one-dimensional isoelectric focusing to characterize the MHC class I molecule of the rhesus monkey that binds this 9-amino-acid SIVmac envelope fragment. Cloning and sequencing of the cDNA encoding this rhesus monkey MHC class I molecule demonstrated that it is a newly described HLA-B homologue, Mamu-B*01. The definition of this viral CTL epitope and its restricting MHC class I molecule will facilitate the use of the SIVmac/rhesus monkey model for studies of envelope-based vaccine strategies for the prevention of AIDS.

Alleles↗
PubMed1993

Abrogation of H-2-restricted CTL responses and efficient recognition of HLA-A3 molecules in DBA/2 HLA/A24 responder mice.

DBA/2 (H-2d) HLA-B7 x human beta 2-microglobulin transgenic and DBA/2 nontransgenic mice stimulated by DBA/2 HLA-A3 x human beta 2-microglobulin transgenic mouse spleen cells developed potent H-2Kd restricted cytolytic responses with recognition of a peptide from the second domain of the HLA-A3 H chain. These H-2Kd-restricted responses obliterated, as a rule, cytolytic responses with direct recognition of the HLA-A3 molecules, even in HLA-B7 transgenic mice. These immunodominant H-2Kd-restricted responses could be abrogated in DBA/2 HLA-A24 mice because of cross-tolerance, the HLA-A3 derived-H-2Kd presented peptide being shared by several (including A24) HLA class I H chain allelic variants. Under such experimental circumstances, strong CTL responses with exclusive direct recognition of HLA-A3 molecules constantly developed. Further analysis of these responses in six DBA/2 HLA-A24 responder mice indicated that a large fraction of the mouse V beta and V alpha genes could be used to mount such CTL responses. Thus, by combining classical HLA class I transgenesis and selective destruction of H-2K and H-2D genes, it should be possible to derive useful strains of mice for the study of HLA class I-restricted CTL responses.

Animals↗
PubMed1995

Protective CTL-dependent immunity and enhanced immunopathology in mice immunized by particle bombardment with DNA encoding an internal virion protein.

Anti-viral CTL were induced in vitro using a particle bombardment device or "gene-gun" to deliver plasmid DNA encoding the nucleoprotein of the lymphocytic choriomeningitis virus (LCMV). Using this plasmid we were able to study T cell-mediated immunity in the absence of a neutralizing Ab response. Upon a single DNA immunization, a nearly 2 log10 reduction in splenic viral titers was observed 3 days after LCMV infection. After two or three immunizations a greater than 3 log10 inhibition of viral titers in the spleen was observed, with most animals having no detectable virus. C57BL/6 mice immunized with DNA encoding the nucleoprotein gene were also challenged with LCMV intracranially. Upon intracranial challenge, vaccinated animals displayed either protection or enhanced immunopathology leading to accelerated kinetics of death. Using limiting dilution analysis it was possible to detect LCMV-specific CTL precursors in both the spleen and lymph nodes of vaccinated animals. C57BL/6 mice inoculated with DNA demonstrated an anamnestic CTL response detectable at day 4 after LCMV challenge. Thus DNA vaccines are capable of inducing an anti-viral T cell response that can inhibit viral replication and mediate either protective immunity or immunopathology. Vaccination with DNA may therefore provide a useful alternative to current viral or subunit vaccines once the efficacy of immunization with DNA is optimized.

Amino Acid Sequence↗
PubMed1995

IL-10 converts mouse lymphoma cells to a CTL-resistant, NK-sensitive phenotype with low but peptide-inducible MHC class I expression.

IL-10 has a variety of effects including: inhibition of monocyte MHC class II-dependent Ag presentation, Th1 cytokine production, and inhibition of T cell proliferation. Recently we have shown that IL-10 inhibits Ag presentation to human tumor-specific and allospecific CTL. In the present study we showed that transfection of the mouse lymphoma RMA (H-2b) with the IL-10 gene induced conversion to a RMA-S-like phenotype. The changes included an inhibition of lysis by minor histocompatibility or tumor Ag-specific CTLs and, conversely, a dramatic increase in susceptibility to lysis by NK cells. The RMA-10 transfectants showed levels of H-2 expression as low or even lower than those found on RMA-S. The levels of tested adhesion molecules were unaltered. Treatment of RMA with rIL-10 gave a less pronounced change in phenotype. In addition, relative to untreated target cells, IL-10 pretreated cells or IL-10 transfectants were unaltered in their capacity to affect cytotoxicity by cold target inhibition, arguing against the possibility that the observed effect could be a direct effect of IL-10 on the CTL. The expression of H-2 was partially restored by coculturing RMA-10 transfectants with class I-binding peptides. Taken together, these results indicate that IL-10 exerts a post-transcriptional effect on H-2 expression, compatible with an induced decrease in the access of peptides to the MHC class I complex. IL-10 is the first cytokine reported to have this effect and also the first factor shown to induce NK sensitivity and reduced sensitivity to CTL, an effect that may be of physiologic relevance.

Animals↗
PubMed1994

The effect of mutant beta 2-microglobulins on the conformation of HLA-B27 detected by antibody and by CTL.

The arthritis-predisposing HLA-B27 consists of a heavy chain, a small peptide, and the monomorphic beta 2-microglobulin (beta 2-m). CTLs and a mAb, Ye-2, which recognize the complex with specificities both for the heavy chain and for the peptide, are available. The beta 2-m is in noncovalent association with the heavy chain at multiple points and is exchangeable with free beta 2-m outside of the complex. The purpose of our experiments was to test whether mutant beta 2-m capable of modulating HLA-B27 activity could be created. Eighteen recombinant mutants of the human beta 2-m were experimentally generated. In 14 of these, mutations were at or near residues that are either contact residues or interface residues with the heavy chain. Relative to the parent beta 2-m, two-thirds of the mutants showed reduced ability to exchange into HLA-B27 complexes. However, at least four of them induced more than 80% decrease in Ye-2 Ab reactivity. Two mutants were able to induce a minor decrease in susceptibility to lysis by four CTL clones. One of the CTL clones was autoreactive. Two of the CTL clones were specific for HLA-B27 cells experimentally infected with arthritis-causing Yersinia enterocolitica. These results indicate that certain beta 2-m residues play an indirect role in peptide presentation, although they are not directly associated with the peptide residues.

Amino Acid Sequence↗
PubMed1994

CTL access to tissue antigen is restricted in vivo.

MHC class I-restricted, hepatitis B surface Ag (HBsAg)-specific, CD8+ polyclonal CTL lines and clones cause a severe necroinflammatory liver disease when they are injected i.v. into transgenic mice that display widespread tissue expression of HBsAg. Surprisingly, the same CTLs fail to cause disease in any other HBsAg-positive tissue in these animals. However, the CTLs are highly cytopathic for HBsAg-positive renal tubules and choroid plexus epithelial cells when they are injected extravascularly, either beneath the kidney capsule or intracerebrally. Analysis of the microvascular anatomy of these tissues reveals that the hepatic sinusoid is characterized by a discontinuous endothelium and the absence of a basement membrane. In contrast, the vasculature of most other tissues displays a continuous endothelium and basement membrane; and the epithelial cells of many of these tissues are also surrounded by a separate basement membrane. These results suggest that viral, tumor, and normal self Ags expressed in the liver are readily accessible to class I-restricted T cells, whereas the vascular endothelium and/or the vascular and cellular basement membranes constitute an extremely effective barrier that precludes CTL access to tissue Ag at many other sites. Because class I-restricted Ag recognition occurs throughout the body in many natural diseases and disease models, the vascular barrier must be breached by other events before CTL access to endogenously synthesized epithelial Ag can occur.

Animals↗
PubMed1994

CTL responses to the gag polyprotein encoded by the murine AIDS defective retrovirus are strain dependent.

Murine AIDS (MAIDS) is induced by a mixture of retroviruses, of which a replication defective virus is the proximal agent of disease. This defective virus harbors a single intact gene that encodes an aberrant gag polyprotein. Certain mouse strains are genetically resistant to MAIDS, with several genes, including H-2Dd, contributing to this resistance. Because MHC class I gene products present intracellular Ags to CTL, recombinant viruses were used to determine whether gag-specific CTLs mediate the genetic linkage between H-2Dd and resistance. Interestingly, while genetically resistant BALB/cByJ and C57BL/KsJ mice (H-2d) generated gag-specific CD8+ CTLs, a similar response was not detected in susceptible BALB.B and C57BL/6J mice (H-2b). However, this CTL response does not appear to be responsible for genetic resistance because: 1) a vigorous CTL response could also be generated by susceptible (C57BL/6 x BALB/cBy) F1 mice and 2) the relevant epitope is H-2Kd restricted.

Animals↗
PubMed1996

Generation of primary tumor-specific CTL in vitro to immunogenic and poorly immunogenic mouse tumors.

This study investigated the generation of primary tumor-specific CTL activity in vitro to several mouse tumors. We report that the development of optimal primary tumor-specific CTL to the P815 mastocytoma, the EL4 thymoma, and the Lewis lung carcinoma is dependent on tumor Ags, on enhancement of T cell costimulation by B7.1, and on exogenous T helper activity in the form of IL-2 and IL-4. A relatively low concentration of IL-2 and IL-4 was required to limit the induction of lymphokine-activated killer cells. In the case of P815, the CTL were directed toward molecularly defined tumor rejection Ags. These primary cultures yielded long term T cell lines that were heterogeneous in fine tumor Ag specificity and in cytokine production.

Animals↗
PubMed1996

[Analysis of tumor rejection antigen peptides recognized by specific CTL].

Tumor rejection antigens, recognized by cytotoxic T lymphocytes (CTL), have been identified in several tumors. In malignant melanoma MAGE-1 and -3 antigen peptides, recognized by specific CTL, were defined. Tyrosinase, gp100 and Melan A/MART-1, normally expressed in the melanosome, were also shown to be recognized by specific CTL. In murine tumors, three antigenic peptides were identified. These are P1A in mastocytoma P815, MUT1 in murine lung carcinoma (3LL) derived from connexin 37, and pRL1 in murine leukemia RL male 1 derived from c-akt proto-oncogene. Analysis of the tumor rejection antigen peptides will elucidate the molecular nature of the tumor rejection antigen and facilitate their therapeutic use as tumor vaccine.

Animals↗
PubMed1996

Identification of a population of CD4+ CTL that utilizes a perforin- rather than a Fas ligand-dependent cytotoxic mechanism.

Although cytotoxic activity has generally been shown to reside in the CD8+ population of T lymphocytes, there are numerous examples of cytotoxic CD4+ T cells. In the present study, we found that after depletion of CD8+ T cells, CD4+ T cells cultured in short-term in vitro mixed lymphocyte cultures developed strong Ag-specific cytotoxic activity. Such CD4+ CTL lysed both Fas+ and Fas- target cells and developed in Fas ligand-deficient gld mice. Thus, in contrast to several recent reports involving long-term T cell clones or short-term mitogen-activated splenocytes, the cytotoxic activity of these CD4+ CTL is not dependent on Fas-Fas ligand interactions. However, CD4+ cells derived in a similar manner from perforin knockout mice showed greatly decreased target cell lysis, indicating that their cytotoxic activity is primarily dependent on a perforin-based mechanism. The Fas-Fas ligand pathway has been shown to be important in the maintenance of peripheral tolerance, but to have a minimal role in protection against viral and bacterial pathogens. Thus, the existence of a population of CD4+ CTL that utilizes perforin re-emphasizes the likely importance of these cells in a classical defensive role against foreign pathogens, in addition to their already implied role as modulators of the immune response.

Animals↗
PubMed1996

Induction of heat-stable antigen expression by phagocytosis is involved in in vitro activation of unprimed CTL by macrophages.

Costimulation is required for activation of unprimed CTL. While the costimulatory molecules B7 and heat stable Ag (HSA) play a role in CTL response induction generated by dendritic cells, HSA but not B7 contributes to primary CTL response induced by macrophages (M phi), but only if particulate material has been ingested. This is a finding that correlates well with the observation that soon after phagocytosis of latex beads by M phi, cell surface expression of HSA rapidly increases. This increase could not be prevented by addition of drugs that blocked the synthesis or intracellular transport of newly synthesized HSA. However, inhibitors of protein kinase C did significantly down-regulate HSA expression. Other proteins appear to be regulated by a similar mechanism, because the surface expression of the CD45 isoform B220, of IL-2R, and of CD26 also increased immediately following ingestion of beads by M phi. These data suggest that there exists a sequestered pool of proteins that can be exposed coordinately at the cell surface via a protein kinase signaling mechanism that detects phagocytic events. In the case of HSA, we suggest that the ability to rapidly modulate the cell surface level of costimulatory molecules is a useful mechanism by which M phi are able to quickly up-regulate their T cell stimulatory capabilities during the time when, most likely, they are presenting foreign Ag.

Amino Acid Sequence↗
PubMed1996

Regulation of CTL recognition by receptors for HLA-DR molecules.

NK cells and some T cells have receptors for MHC class I molecules that negatively regulate the recognition of these cells. In the present study, we show alpha beta CTL clones carrying the receptors for HLA-DR molecules that inhibit this cytotoxicity. The cytotoxicity of two CTL clones possessing NK activity was inhibited by the surface expression of HLA-DR and HLA class I molecules on the target cells. The inhibitory effect of DR molecules on the cytotoxicity of the CTL clones was confirmed by using T2 transfectants expressing HLA-DR3. These results suggest that receptors for both HLA-DR and HLA class I molecules negatively regulate the recognition of T cells.

Antigen Presentation↗
PubMed1996

Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified at HLA-A*0201-binding residues.

Recognition of the melanoma Ag gp100 by tumor-infiltrating lymphocytes (TIL) in vitro has been correlated with tumor regression in patients with metastatic melanoma treated with the adoptive transfer of TIL plus IL-2. Three common gp100 epitopes have been identified that are recognized in the context of HLA-A2 by TIL from different patients: G9154 (KTWGQYWQV), G9209 (ITDQVPFSV), and G9280 (YLEPGPVTA). Upon stimulation with these peptides, melanoma-reactive CTL could be induced in vitro from PBL of some HLA-A2+ melanoma patients. However, numerous restimulations were required, and specific reactivity could not be generated in many patients. Therefore, to enhance the immunogenicity of gp100 peptides, amino acid substitutions were introduced into G9154, G9209, and G9280 at HLA-A*0201-binding anchor positions, but not at TCR contact residues, to increase peptide class I MHC-binding affinity. Several modified gp100 peptides bound with greater affinity to HLA-A*0201 than unmodified peptides and were recognized by TIL specific for the natural epitopes. These peptides were used to sensitize PBL from HLA-A2+ melanoma patients in vitro using peptide-pulsed autologous PBMC as stimulators. After five weekly restimulations with either the native G9209 or G9280 peptide, melanoma-reactive CTL could only be induced from two of seven patients. However, amino acid substitutions in these peptides enabled the induction of melanoma-reactive CTL from all seven patients. These results suggest that modified gp100 peptides may be more immunogenic than the native epitopes, and may be useful in immunotherapy protocols for patients with melanoma.

Amino Acid Sequence↗
PubMed1995

Association of cytotoxic T lymphocyte (CTL) escape mutations with persistent hepatitis C virus (HCV) infection.

Mechanisms by which HCV evades the cellular immune response in persistently infected humans and chimpanzees are poorly defined, but could involve mutations in epitopes recognized by class I MHC restricted CTLs. To investigate this possibility, we identified an epitope in the NS3 protein of HCV that was recognized by intrahepatic CTLs from a chimpanzee that developed persistent HCV infection after experimental challenge with the virus. Fine mapping studies with truncated synthetic peptides revealed that the epitope was 9 amino acids in length, encompassing residues 1445 to 1454 (GDFDSVIDC) of NS3. This sequence was completely conserved in all full-length NS3 genomes described to date. In view of the fact that the major genotypes of HCV may differ by up to -30% in overall amino acid homology, it appears in contrast that this epitope is highly conserved. The role of CTL escape mutations in HCV persistence was assessed in the virus inoculum used to infect this chimpanzee and in post-inoculation plasma samples. Sequencing of 6-10 M13 clones containing a 232-nucleotide fragment amplified with NS3-specific primers revealed that the epitope in the challenge inoculum and a post-inoculum plasma sample obtained at week 4 were identical to the published sequence of HCV-I. In contrast, all molecular clones sequenced from week 16, 25 and 28 plasma samples contained a single Asp--> Glu (D-->E) amino acid substitution at residue 1449. Significantly, four independently derived CTL clones established from the liver of this chimpanzee at various times up to two years after infection recognized target cells pulsed with a nonameric peptide representing the wild-type HCV-I sequence, but not those pulsed with a peptide containing the D-->E mutation. These data suggest that CTL escape mutations may play a role in viral persistence.

Amino Acid Sequence↗
PubMed1997

CD3+ CD8+ CTL activity within the human female reproductive tract: influence of stage of the menstrual cycle and menopause.

The human female reproductive tract (RT) has been analyzed by others with respect to NK cell cytolytic activity, but not CD3+ T cell (CTL) cytolytic activity. Here, we describe the cytolytic capacity of mucosal CD3+ T cells both longitudinally within the RT (Fallopian tube, uterine endometrium, endocervix, ectocervix, and vaginal mucosa) and temporally throughout the menstrual cycle, using a redirected lysis assay system. Cytolysis by CD3+ CD8+ T cells is found throughout the RT and appears to be hormonally regulated, since in the uterine endometrium, the capacity for CD3+ T cell cytolytic activity is present during the proliferative phase of the menstrual cycle and absent during the subsequent secretory (postovulatory) phase. In contrast, in postmenopausal women the entire RT, including the uterus, retains the capacity for strong CD3+ T cell cytolytic activity. These findings suggest that the high levels of estradiol and progesterone present during days 14 to 28 of the menstrual cycle down-regulate CTL activity in the uterus. As a consequence, the absence of this activity may allow implantation of a semiallogeneic embryo that would otherwise be rejected. Further, these studies indicate that CTL activity is regulated differentially in different regions of the RT, persisting in the cervix and vagina throughout the menstrual cycle.

Adult↗
PubMed1997

Cytolytically active memory CTL present in lymphocytic choriomeningitis virus-immune mice after clearance of virus infection.

Generally, it has been assumed that memory T cells are dormant and inactive cells in the absence of their specific Ag. Recent work has challenged this assumption by showing that a portion of the CD8+ memory T cell pool is in cycle. In this study, we demonstrate that a significant number of blast-size memory CD8+ T cells in mice, long after lymphocytic choriomeningitis virus (LCMV) infection, mediate cytolysis against highly sensitive targets without any in vivo or in vitro restimulation and expansion with Ag. Peptide-coated RMA-S targets were sufficiently sensitive to detect low but significant cytolytic activity in bulk 51Cr release assays in nonstimulated LCMV-specific splenic memory CTL populations. Most of the directly cytotoxic activity was against the GP33 epitope, and this persisted throughout the lifetime of the mouse following infection. The cytotoxic activity was not inhibited by cyclosporin A, indicating that these cells were already in an active state and not dependent on further stimulation in vitro. It was formally shown that the cytotoxic activity was mediated by the CD8+ CTL by sorting for the blast-size CD8+ population and by blocking target cell lysis with anti-CD8 Ab. Thus, at any time after the original infection some portion of the memory CD8+ T cell pool is cycling, and it remains cytolytically active long after resolution of the original infection. These CTL may provide a rapidly acting defense mechanism against reinfection.

Animals↗
PubMed1997

Peptides derived from a wild-type murine proto-oncogene c-erbB-2/HER2/neu can induce CTL and tumor suppression in syngeneic hosts.

In this analysis, we examined whether peptides derived from a wild-type murine proto-oncogene, c-erbB-2, function as tumor rejection Ags. Expression of murine c-erbB-2 examined by means of reverse transcription-PCR was observed in several normal adult tissues, such as intestine, kidney, and testis. We then transduced human and murine c-erbB-2 cDNA into two mutually noncross-reactive fibrosarcoma lines of BALB/c origin, CMS7 and CMS17. In BALB/c mice immunized with CMS17HE (CMS17 transduced with human c-erbB-2 cDNA), the growth of subsequently challenged CMS7HE (CMS7 transduced with human c-erbB-2 cDNA) was significantly suppressed. CTL against human c-erbB-2-expressing cells were generated from BALB/c spleen cells in vivo and in vitro sensitized by CMS17HE. The CTL activity was also directed against murine c-erbB-2-expressing cells, CMS7ME and CMS17ME, and was blocked by anti-CD8 or anti-Kd mAbs. A series of peptides of human or murine c-erbB-2 compatible with the Kd binding motif was synthesized. The CTL were reactive with P1.HTR (H-2d) pulsed with three of these peptides, p63-71 (human c-erbB-2 derived), p63-71(A) (murine c-erbB-2 derived), and p780-788 (common for human and murine c-erbB-2). Spleen cells immunized in vivo and in vitro with syngeneic spleen cells pulsed with these peptides became cytotoxic for CMS17HE and/or CMS17ME, but not CMS17neo (CMS17 transduced with control vector). The growth of CMS7ME was suppressed in mice immunized with the murine c-erbB-2-derived peptide, p63-71(A) or p780-788. There was no apparent pathologic change in mice that rejected CMS7ME after vaccination with these peptides.

3T3 Cells↗