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PubMed1979

Selective affinity fractionation of murine cytotoxic T lymphocytes (CTL). Unique lectin specific binding of the CTL associated surface glycoprotein, T 145.

We have analyzed the lectin binding characteristics of cytotoxic T lymphocyte (CTL)-derived surface labeled glycoproteins by affinity chromatography of the labeled glycoproteins on a panel of immobilized lectin adsorbents. Evidence is presented for the specific interaction of the CTL-associated glycoprotein T 145 with a lectin derived from Vicia villosa seeds. Conditions are described for the preparation and use of lectin affinity adsorbents for the rapid isolation of T 145 bearing cytotoxic T lymphocytes. Direct proof is given to show that T 145-positive cells arising from a variety of T-cell activations constitute the only subpopulation of cells with ability to perform cell-mediated T-cell cytotoxicity. Specific depletion of the CTLs by adherence to V. villosa adsorbents is shown by their depletion in the nonbound cell fraction and correspondingly enriched recovery in the sugar eluted cell fraction. Specific affinity fractionation of CTLs has occurred in every strain combination tested and irrespective of the actual antigen specificity of the effector cell population.

Animals↗
PubMed1993

T cell receptor (TCR) structure of autologous melanoma-reactive cytotoxic T lymphocyte (CTL) clones: tumor-infiltrating lymphocytes overexpress in vivo the TCR beta chain sequence used by an HLA-A2-restricted and melanocyte-lineage-specific CTL clone.

HLA-A2+ melanomas express common melanoma-associated antigens (Ags) recognized in vitro by autologous cytotoxic T lymphocytes (CTL). However, it is not known whether tumor Ags can drive in vivo a selective accumulation/expansion of Ag-specific, tumor-infiltrating T lymphocytes (TIL). Therefore, to evaluate this possibility, 39 CTL clones isolated from several independent mixed lymphocyte tumor cultures (MLTC) of TIL and peripheral blood lymphocytes (PBL) of an HLA-A2+ melanoma patient and selected for T cell receptor (TCR)-dependent, HLA-restricted tumor lysis, were used for analysis of TCR alpha and beta chain structure by the cDNA polymerase chain reaction (PCR) technique with variable gene-specific primers followed by sequencing. Despite absence of oligoclonality in fresh TIL and PBL, as well as in T cells of day 28 MLTC (day of cloning), sequence analysis of TCR alpha and beta chains of TIL clones revealed a dominance of a major category of melanoma-specific, HLA-A2-restricted T cells expressing a V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1 TCR. The same TCR was also found in 2 out of 14 PBL clones. The other PBL clones employed a V alpha 2.1 gene segment associated with either V beta 13.2, 14, or w22. Clones A81 (V alpha 2.1/J alpha IGRJ alpha 04/C alpha and V beta 14/D beta 1/J beta 1.2/C beta 1) and A21 (V alpha 8.2/J alpha AP511/C alpha and V beta 2.1/D beta 1/J beta 1.1/C beta 1), representative of the two most frequent TCR of PBL and TIL, respectively, expressed different lytic patterns, but both were HLA-A2 restricted and lysed only HLA-A2+ melanomas and normal melanocytes, thus indicating recognition of two distinct HLA-A2-associated and tissue-related Ags. Finally, by the inverse PCR technique, the specific TCR beta chain (V beta 2.1/D beta 1/J beta 1.1/C beta 1) expressed by the dominant TIL clone was found to represent 19 and 18.4% of all V beta 2 sequences expressed in the fresh tumor sample and in the purified TIL, respectively, but < 0.19% of V beta 2+ sequences expressed in PBL. These results are consistent with the hypothesis that a clonal expansion/accumulation of a melanocyte-lineage-specific and HLA-A2-restricted T cell clone occurred in vivo at the site of tumor growth.

Amino Acid Sequence↗
PubMed1990

The predominance of CD8+ T cells after infection with measles virus suggests a role for CD8+ class I MHC-restricted cytotoxic T lymphocytes (CTL) in recovery from measles. Clonal analyses of human CD8+ class I MHC-restricted CTL.

Stimulation of PBMC, in children recovering from acute measles, with autologous EBV-transformed and measles virus (MV)-infected lymphoblastoid cell lines (B-LCL) expanded primarily MV-specific CD8+ T cells. A large number of CD8+ T cell clones were obtained either by passaging of bulk cultures at limiting dilutions or by direct cloning of PBMC without previous stimulation in bulk culture. The MV-specific CD8+ T cell clones responding in a proliferative and a CTL assay were found to be class I MHC restricted. In contrast, CD4+ MV-specific T cell clones, which were generated by the same protocol, recognized MV in association with class II MHC molecules. Analysis of processing requirements for Ag presentation to CD8+ and CD4+ T cell clones, measured by the effect of chloroquine in a proliferative T cell response, revealed that both types of T cells recognized MV Ag processed via the endogenous/cytoplasmic pathway. Thus, these studies indicate that, as in most other viral infections and in contrast to previous suggestions, the class I MHC-restricted CTL response by CD8+ T cells may be an important factor in the control and elimination of MV infection. Therefore, the role proposed for CD4+ class II-restricted T cells in recovery from measles needs to be reevaluated.

Antigen-Presenting Cells↗
PubMed1990

Cytotoxic activity and lymphokine production of T cell receptor (TCR)-alpha beta+ and TCR-gamma delta+ cytotoxic T lymphocyte (CTL) clones recognizing HLA-A2 and HLA-A2 mutants. Recognition of TCR-gamma delta+ CTL clones is affected by mutations at positions 152 and 156.

TCR-gamma delta+ CTL clones were generated from CD4-CD8- T cells that were stimulated twice with the cell line JY. Either IL-2 or IL-4 was used as growth factor. A number of TCR-gamma delta+ clones were found to lyse the stimulator cell line JY. Two of these clones secreted N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase activity after stimulation with JY cells. The cytotoxic activity of these two clones was blocked by a mAb specific for HLA-A2. Moreover, these two TCR-gamma delta+ clones selectively lysed human fibroblast line M1 and murine P815 cells transfected with DNA fragments encoding HLA-A2 but not those transfected with HLA-B7 encoding DNA, indicating that these clones recognize HLA-A2. Analysis of the recognition of HLA-A2 by using target cells transfected with mutated HLA-A2 encoding genes revealed that the nature of the amino acid at position 152 of the molecule is critical for recognition of the TCR-alpha beta+ as well as the TCR-gamma delta+ CTL clones since replacement of Val for Ala at that position resulted in abrogation of recognition of one TCR-gamma delta+ and one TCR-alpha beta+ clone and substitution of Val for Glu affected recognition of all clones. Substitution of Leu for Trp at position 156 abrogated recognition by one TCR-gamma delta+ and one TCR-alpha beta+ T cell clone, but recognition by the other clones was not changed. All clones were able to secrete IL-2, IFN-gamma, and GM-CSF but not IL-4 after activation.

Clone Cells↗
PubMed1995

CTL responses of HLA-A2.1-transgenic mice specific for hepatitis C viral peptides predict epitopes for CTL of humans carrying HLA-A2.1.

Vaccine development in animal models depends on ability to recognize epitopes seen by human T cells. In this work, we show that CTL responses in transgenic mice expressing human HLA-A2.1 prospectively predict the same four of 11 hepatitis C virus (HCV) structural protein-derived peptides, expressing a sequence motif for HLA-A2.1 binding, that are actually recognized by human A2.1-restricted CTLs. The CTLs also recognized targets endogenously expressing these proteins. Human CTLs from HCV-infected patients, tested by using the same peptides, revealed a virtually identical response repertoire. A highly conserved HCV core peptide was the most immunogenic, and may be a valuable component of a vaccine against a broad range of HCV isolates in HLA-A2-positive patients. These results suggest that, in spite of species differences, the T cell repertoire is plastic enough to allow a similar response when the same class I MHC molecule is presenting the peptide. Thus, the HLA molecule plays the primary role in determining which peptides are recognized by CTLs. This transgenic mouse model is important for the study of HLA-restricted CTL determinants and for an approach to design a potential HCV vaccine.

Adult↗
PubMed1978

Secondary in vitro generation of cytolytic T-lymphocytes (CTL's) in the murine sarcoma virus system. Virus-specific CTL induction across the H-2 barrier.

Following in vitro stimulation of murine sarcoma virus Moloney isolate (M-MuSV)-immune spleen cells with syngeneic antigenically related Moloney leukemia cells, highly efficient cytotoxic T-lymphocytes (CTL's) were generated. The cytotoxic effect was directed only against H-2-compatible target cells bearing M-MuSV tumor-associated antigens (TAA). However, in a cold target competition assay a weak but detectable capacity to block CTL activity was also obtained when allogeneic Moloney leukemia cells were added. Moreover, when M-MuSV-immune spleen cells from mice inoculated with virus 14 days previously were stimulated by allogeneic Moloney leukemia cells, a strong cytotoxic effect toward syngeneic and allogeneic tumor cells bearing M-MuSV TAA was elicited.

Animals↗
PubMed1994

MHC class I and non-MHC-linked capacity for generating an anti-viral CTL response determines susceptibility to CTL exhaustion and establishment of virus persistence in mice.

The influence of the murine MHC gene complex H-2 class I alleles or of the genetic background and the role of virus variants on establishment of a persistent infection with lymphocytic choriomeningitis virus (LCMV) was analyzed in immunocompetent mice of H-2d haplotype by evaluation of cytotoxic T cell responses. Susceptibility to establishment of a virus carrier state increased in various H-2d mice with lower CTL response and slower CTL kinetics. Low responder BALB/c-H-2dm2, lacking H-2Ld molecules to present the major T cell epitope amino acids 118-126 of the LCMV nucleoprotein or DBA/2 mice possessing relatively few CD8+ T cells, were more susceptible than high responder BALB/c mice expressing H-2Ld. Additional critical factors were LCMV isolate and dose of infection. The rapidly replicating LCMV-DOCILE and Cl 13 Armstrong induced viral persistence readily, whereas the slowly replicating parental virus strains WE or Armstrong did not. The presented findings illustrate a model of MHC-linked or MHC-unlinked susceptibility for virus persistence and may help to explain pathogeneses of chronic virus infections in humans that are often associated with slowly progressing immunopathology.

Animals↗
PubMed2001

Immunization with a tumor-associated CTL epitope plus a tumor-related or unrelated Th1 helper peptide elicits protective CTL immunity.

Immunization with cytotoxic T cell epitope SPSYVYHQF (AH1), derived from MuLV gp70 envelope protein expressed by CT26 tumor cells, does not protect BALB/c mice against challenge with CT26 tumor cells. By contrast, immunization with AH1 plus T helper peptides OVA(323-337) or SWM(106-118) eliciting Th1 and Th0 profiles, protected 83% and 33% of mice, respectively. Interestingly, immunization with AH1 plus both helper peptides reverted the efficacy to 33%. We identified the endogenous T helper peptide p(320-333) from gp70 which elicits a Th1 profile and is naturally processed. As for OVA(323-337), immunization with p(320-333) alone did not protect against tumor challenge. However, p(320-333) plus AH1 protected 89% of mice at day 10 after vaccination. Only 20% of mice vaccinated with AH1 + OVA(323-337) or AH1 + p(320-333) were protected when challenged 80 days after immunization. Treatment with OVA(323-337) or with p(320-333) around established tumors delayed tumor growth. Our results show that tumor-related as well as tumor-unrelated but strong Th1 peptides may be useful for inducing CTL responses in tumor immunotherapy.

Adjuvants, Immunologic↗
PubMed1985

A comparison of lysis mediated by Lyt 2+ TNP-specific cytotoxic-T-lymphocyte (CTL) lines with that mediated by rapidly internalized lymphotoxin-containing supernatant fluids: evidence for a role of soluble mediators in CTL-mediated killing.

A series of Lyt 2+, trinitrophenyl (TNP)-specific T-cell lines are shown to lyse 51Cr-labeled target cells in an antigen-specific, H-2-restricted fashion in a 4-hr assay. These cells also produce lymphotoxin, in addition to other factors, upon stimulation with TNP-haptenated syngeneic splenocytes. A technique for introducing macromolecules into the cytoplasm of fibroblasts by inducing the cells to pinocytose the molecule in hypertonic medium, and then lysing the newly formed pinocytic vesicles with a mild hypotonic shock was used to assess the role of soluble mediators in the cytotoxic T lymphocyte (CTL)-mediated lytic process. The technique itself has little effect on cell growth rate or viability. A minimum of 24 hr, and more frequently 48-72 hr is required for lymphotoxin to manifest it's lethal effect when it is merely included in the culture medium of growing fibroblasts. In contrast, supernatant fluids from the Lyt 2+ cells kill 51Cr-labeled fibroblasts in a dose-dependent fashion during a 4-hr assay when they are rapidly internalized via the osmotic procedure. The data serve as preliminary evidence of a role for soluble mediators such as lymphotoxin in T-cell-mediated lysis, and suggest that the cytotoxic-T-cell lethal hit may include a mechanism for rapidly internalizing a toxin into appropriate target cells.

Animals↗
PubMed1994

Acidification is essential for maintaining the structure and function of lytic granules of CTL. Effect of concanamycin A, an inhibitor of vacuolar type H(+)-ATPase, on CTL-mediated cytotoxicity.

An inhibitor of vacuolar type H(+)-ATPase, concanamycin A (CMA), inhibited the specific cytolytic activity of a CD8+ CTL clone, OE4. The inhibitory effect was observed when the effector cells, and not the target cells, were pretreated with CMA. CMA did not seem to inhibit early events, inasmuch as effector/target conjugate formation remained unaffected. Although CMA treatment of OE4 resulted in a slight decrease in the efficiency of granule exocytosis in response to anti-CD3 stimulation, the most prominent effect was a marked reduction of perforin activity and DNA degradation activity in lytic granules. Western blotting analysis indicated a drastic decrease in the amount of perforin in CMA-treated cells. Fluorescent microscopic observation of OE4 stained with acridine orange indicated that CMA raised the pH of the lytic granules. Under transmission electron microscopy, striking morphologic changes in cytoplasmic granular structures were observed after CMA treatment of OE4. The lytic granules of OE4 had homogeneously stained large cores and numerous small vesicles that filled peripheral areas. In contrast, the lytic granules of CMA-treated OE4 showed irregular shapes with no small vesicles, but with cores that became rough and loose. Vacuoles with no structure in them were seen occasionally. These results suggest that acidification through vacuolar type H(+)-ATPase is essential to maintain the structure and function of lytic granules.

Acid-Base Equilibrium↗
PubMed1990

Tumor-selective cytolysis is executed exclusively by CD45R+ CTL whereas allo-specific cytotoxicity can be executed also by CD45R- CTL.

Naive and memory CD4+ T helper cells can be distinguished on the basis of expression of the CD45R molecule. Whether this dichotomy applies also to CD8+ T cells has not yet been established. In the present investigation the cytolytic activity of peritoneal CD8+CD45R+ and CD8+CD45R- T cells from tumor- and allo-immunized rats has been studied. More than 90% of the CD8+ peripheral blood T lymphocytes expressed the CD45R molecule, whereas in the peritoneal cavity about 60% of the CD8+ T cells displayed the CD45R+ phenotype. Analysis of cytotoxicity of sorted peritoneal cells of W439 tumor-immunized donors demonstrated selective cytolytic activity of the CD5+CD4-CD8+CD45R+ subpopulation to W439 lymphoma target cells but no effect of CD5+CD4-CD8+CD45R- lymphocytes. None of these lymphocyte populations exhibited cytolytic activity to the NK-sensitive cell line YAC-1, whereas the CD5-CD45R+ population showed strong cytotoxicity to YAC-1 cells. In allo-immunized rats both CD5+CD4- CD8+CD45R+ and CD5+CD4-CD8+CD45R- peritoneal cells exhibited strong allo-specific cytolytic activity, but no activity to YAC-1 cells. Both CD5+CD4-CD8+CD45R+ and CD5+CD4-CD8+CD45R- cells from tumor-immunized rats proliferated in response to Con A and rIL-2. This is the first study demonstrating that tumor-selective cytolytic CD8+ T cells express the CD45R molecule and that allo-specific cytolytic CD8+ T cells are found in both the CD45R+ and CD45R- populations.

Animals↗
PubMed1988

CTL recognition of adenovirus-transformed cells infected with influenza virus: lysis by anti-influenza CTL parallels adenovirus-12-induced suppression of class I MHC molecules.

We examined the ability of influenza-specific cytotoxic T lymphocytes to lyse adenovirus-transformed cells infected with influenza virus. Cytotoxic T lymphocyte lysis of Ad12-transformed cells was greatly reduced relative to that of Ad5-transformed cells. Lysability of adenovirus-12-transformed cells was restored in parallel with interferon-gamma induced increases in major histocompatibility complex class I gene products. These findings establish that recognition of foreign molecules by self-restricted cytotoxic T lymphocytes is reduced in adenovirus-12-transformed cells. This provides further evidence that Ad12 tumorigenicity is related to its ability to suppress class I major histocompatability complex molecule expression thereby avoiding recognition by the host immune system.

Adenoviridae Infections↗
PubMed2003

Comparable in vivo efficacy of CD28/B7, ICOS/GL50, and ICOS/GL50B costimulatory pathways in murine tumor models: IFNgamma-dependent enhancement of CTL priming, effector functions, and tumor specific memory CTL.

Increasing evidence suggests that B7/CD28 interactions are important in clonal expansion and effector function of nai;ve CD4(+) T cells, whereas ICOS/GL50 interactions may optimize the responses of recently activated T(H) cells. In tumor models, it has been shown that engagement of ICOS, like CD28, by its ligands can be effective in enhancing tumor immunity. In this report, we have directly compared the in vivo efficacy of CD28 vs ICOS activation in the MethA fibrosarcoma and B16F1 melanoma tumor models. We studied the efficacy of systemic treatment of tumors with murine B7.2-IgG or GL50-IgG fusion proteins, and the therapeutic potential of B7.1 or GL50 vaccines given during various phases of the antitumor responses. In addition, we compare the efficacy of ICOS-ligand splice variants GL50 and GL50B in promoting tumor immunity. We find that each of these pathways is equally effective in promoting tumor immunity and that the efficacy of both GL50 and B7.1 vaccines is IFN-gamma but not IL-10 dependent. Our results suggest that CD28 or ICOS costimulation-based strategies may be equally efficacious as adjuvants to conventional cancer treatment.

Animals↗
PubMed2003

Derivation of HLA-B*0702 transgenic mice: functional CTL repertoire and recognition of human B*0702-restricted CTL epitopes.

Transgenic mice expressing chimeric human leukocyte antigen (HLA)-B*0702 and murine H-2K(b) class I molecules were evaluated as a model system to study the immunogenicity of human cytotoxic T lymphocyte epitopes. Immunization of these mice with six known HLA-B*0702-restricted cytotoxic T lymphocyte epitopes emulsified in incomplete Freund's adjuvant induced significant immune responses specific for all six epitopes. A comparison of the immune responses between HLA-B*0702/K(b) and HLA-A*0201/K(b) transgenic mice demonstrated that the HLA-B*0702/K(b) mice possess a T-cell receptor repertoire capable of recognizing human B*0702 epitopes. However, the magnitude of B*0702-specific responses induced in B*0702/K(b) mice were approximately tenfold lower than A*0201-specific responses induced in HLA-A*0201/K(b) transgenic mice. A panel of 24 B*0702 motif-bearing peptides was used to examine the relationship between immunogenicity and HLA-B*0702 binding capacity. All seven peptides with high binding affinities of 50% inhibitory concentration < or =50 NM (IC(50) 50 nM or less) were immunogenic. Similarly, 75% (9 of 12) of the intermediate binders (IC(50) nM of 50-500) were also immunogenic. Finally, only two of five peptides with binding capacity > 500 nM were found to have marginal immunogenicity, whereas the other three were completely negative. HLA-B*0702/K(b) transgenic mice were found to induce B*0702-specific responses after immunization with whole DNA genes or minigenes, suggesting that, at least to some degree, B*0702 epitopes were generated as a result of natural in vivo processing and presentation.

Amino Acid Sequence↗