Search

Loading of most endogenous peptides on major histocompatibility complex class I molecules is conditional on their transport into the endoplasmic reticulum (ER) by the peptide transporter TAP. We describe an HSV-2/1 cross-reactive cytotoxic T-cell (CTL) epitope that is processed in a TAP1-independent manner in vivo following immunization of TAP1-/- mice with naked DNA or a recombinant vaccinia virus. These data indicated that TAP1-independent processing of endogenous proteins is sufficient to prime CTLs in vivo. TAP1-independent processing of this epitope was not due to ER targeting by signal sequences and exogenous loading of MHC-I molecules and was not influenced by the amino acids flanking this epitope. In contrast, TAP1-/- mice infected with HSV-2 or HSV-2 mutants did not mount a CTL response against this epitope., (Copyright 2001 Academic Press.)

The immunology of hepatitis C virus (HCV) infection should be studied in the context of HCV antigen expression in the liver, because HCV primarily infects this organ. Indeed, the nature, function, and fate of T cells primed after antigen expression in the liver might differ from those primed when antigens are expressed systemically or in other organs, because the nature of the antigen-presenting cells (APCs) involved may be different. In addition, the normal liver contains a resident population of lymphocytes that differ from those present at other sites. Thus, we investigated whether HCV-specific CD8(+) cytotoxic T cells (CTLs) could be elicited following portal vein (PV) injection of plasmid DNA in mice whose hepatic veins were transiently occluded. We show that PV injection of mice with "naked" DNA expressing the HCV-NS5a protein, under the control of a liver-specific enhancer/promoter, resulted in NS5a expression in the liver and the priming of HCV-specific CTLs. These results suggested that such a model might be relevant to the study of HCV-specific immune responses primed during natural infection.

Priming of strong cellular immune responses to hepatitis C (HCV) is thought to be important for eradication of infection. Although productive infection of HCV occurs only reproducibly in humans and chimpanzees, definition of HCV-specific T cell epitopes in mice is necessary to screen efficiently HCV vaccine strategies for their ability to prime cellular immune responses. Out of seven strains of mice screened for immunodominant CTL epitopes against HCV-1a Core, E2, NS5a and NS5b, only one epitope (p214K9) in only one mouse strain was identified. Enumeration of p214K9-specific CD8+ cells by flow cytometry revealed that the number of epitope specific CTL primed by 'naked' DNA immunization was lower than that reported during viral infection. The p214K9 epitope described here, combined with analysis of CTL responses by flow cytometry, should be instrumental in ranking various HCV vaccine strategies for their ability to prime CTL responses.

Despite advances in the clinical management of HIV infection, using combinations of antiretroviral pharmaceuticals, a safe and efficacious vaccine is needed to limit the AIDS pandemic. It is now thought that an effective HIV-1 vaccine should prime both cross-neutralizing antibodies and long-lasting cytotoxic CD8+ T lymphocytes (CTLs) recognizing multiple codominant HIV-1 epitopes. To that end, many novel vaccine strategies have been tested. However, only a few of these strategies, beside those relying on live-attenuated viruses, are able to prime strong CTL responses in nonhuman primates and humans. In this study, three rhesus macaques were immunized with HIV-1 p55gag virus-like particles (VLPs) in the absence of adjuvant to assess the potential of such a vaccine to prime CTL responses. After intramuscular injection of p55gag VLP, all three animals mounted CTL responses against HIV-1 p55gag. Notably, these CTLs primed by vaccination recognized naturally processed peptides and were long lived (>8.5 months) both in the peripheral blood and draining lymph node. Furthermore, these CTLs were directed against multiple HIV-1 p55gag epitopes. This indicated that immunization with p55gag VLP primes strong MHC class I-restricted, CD8+ cell-mediated immune responses and suggested that HIV-1 p55gag VLPs should be a reasonable vaccine candidate, when combined with strategies priming cross-neutralizing antibodies.

Vaccination can elicit CD8(+) cytotoxic T lymphocytes (CTL) that recognize peptides presented by class I MHC molecules. Relatively little is known, however, about the genetic factors that shape the repertoire of T cell clonotypes responding to any given epitope. We report here that H-2(b) mice immunized with a plasmid DNA vaccine or vaccinia virus encoding for HIV-1SF2p55gag elicit CD8(+) CTL against the H-2Db-restricted immunodominant epitope (pgagb). This response involved three different T cell populations based on their recognition of alloantigens: one that cross-reacted with the alloantigen H-2Ld, one that cross-reacted with H-2Kd, and one that did not cross-react with either H-2(d) or H-2(k) molecules. Using the TAP-deficient cell line T2-Ld, we showed that pgagb-specific CTL cross-react with H-2Ld and a yet unidentified self-peptide. In mice expressing H-2(b) and H-2(d) allotypes, we investigated whether tolerance to H-2(d) influenced the HIVp55gag-specific CTL repertoire as a consequence of thymic deletion of the cross-reactive CTL repertoire. In (H-2(dxb))F1 mice heterogygosity at the MHC-I level prevented maturation of some but not all TCR combinations specific for H-2Db+pgagb, illustrating the concept that self-tolerance can influence the repertoire of antiviral T cells., (Copyright 1998 Academic Press.)

Intramuscular injection of mice with an adeno-associated virus (AAV) vector expressing herpes simplex virus type 2 glycoprotein B led to the generation of both gB-specific major histocompatibility complex class I-restricted cytotoxic T lymphocytes and anti-gB antibody. AAV-mediated immunization was more potent than plasmid DNA or protein in generating antibody responses.

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.

Analysis of Ag specificity of TRC-gamma delta+ T cells in humans has been hampered by the fact that cloned lines of these cells expanded in IL-2 generally display high NK-like cytotoxic activity. A TCR-gamma delta+ CTL clone, isolated in IL-4, strongly lysed a specific stimulator cell, the EBV-transformed cell line JY, but failed to lyse K562 and other target cells sensitive for NK cell activity. Subsequent culture of this clone (CD124) in IL-2 induced high cytotoxic activity against the NK sensitive target cells. K562 cells were unable to induce the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester [(BLT)-serine esterase] or influx of Ca2+ ions in clone CD124 cultured in either IL-4 or IL-2. In contrast, JY cells induced high BLT-serine esterase secretion and an increase of cytosolic Ca2+ levels. By using a combination of a 51Cr-release assay and a BLT-serine esterase secretion assay, the reactivity of clone CD124 against a limited number of target cells was analyzed. CD124 which expresses HLA-A2 and -B7, recognized an Ag shared by JY (HLA-A2; B7; C blank; DR4,6) and one haplotype expressed by the cell line SPS (HLA-A1; B14; Cw6; DR4). The only specificity shared by SPS and JY was HLA-DR4. However, clone CD124 failed to lyse 5 other HLA-DR4+ target cells. The cytotoxic activity of clone CD124 was inhibited by the class I MHC specific mAb W6/32 and the anti-beta 2m mAb A88, but not, or only marginally, by the anti HLA-DQ mAb SPV-L3 or the anti-HLA-DR mAb 135. These data strongly suggest that clone CD124 recognizes a class I MHC Ag different from HLA-A, -B, or -C.

IL-4 has been shown to act as a growth factor for human T cells. In addition, IL-4 can enhance CTL activity in MLC, but blocks IL-2 induced lymphokine activated killer cell activity in PBL. In our study, the cloning efficiencies, Ag-specific CTL activity and non-MHC-restricted cytotoxicity of CTL clones generated in IL-2 were compared to those generated in IL-4. In a first experiment, T cells were stimulated with the EBV-transformed B cell line JY and cloned 7 days later with feeder cells and either IL-2 or IL-4. In a second experiment, stimulation of the T cells was carried out in the presence of IL-2 plus anti-IL-4 antibodies or IL-4 plus anti-IL-2 antibodies in order to block the effects of IL-4 and IL-2, respectively, produced by the feeder cells. Although the cloning efficiencies in the second experiment were lower than those obtained in the first experiment, the cloning efficiencies obtained with IL-2 or IL-4 were similar in both experiments. The overall proportion of TCR alpha beta+ T cell clones cytotoxic for the stimulator cell JY established in IL-2 or IL-4 were comparable. A striking difference between the clones obtained in IL-2 or IL-4 was that a large proportion of the clones obtained in IL-4 expressed CD4 and CD8 simultaneously, whereas none of the clones isolated in IL-2 were double positive. Also gamma delta+ T cell clones could be established with IL-4 as a growth factor. TCR gamma delta+ T cell clones isolated in either IL-2 or IL-4 were CD4-CD8- or CD4-CD8+, but the proportion of CD4-CD8+ clones isolated in IL-4 was higher. Interestingly, one TCR gamma delta+ clone isolated in IL-2 was CD4+CD8-. Most of the TCR alpha beta+ and TCR gamma delta+ CTL-clones isolated in IL-2 lysed the NK cell sensitive target cell K562. In contrast, only a small proportion of the TCR alpha beta+ or TCR gamma delta+ CTL clones isolated in IL-4, lysed K562. One TCR gamma delta+ T cell clone (CD-124) isolated in IL-4 and subsequently incubated in IL-2 acquired lytic activity against K562.(ABSTRACT TRUNCATED AT 400 WORDS)

Human rIL-4 was studied for its capacity to induce lymphokine-activated killer (LAK) cell activity. In contrast to IL-2, IL-4 was not able to induce LAK cell activity in cell cultures derived from peripheral blood. IL-4 added simultaneously with IL-2 to such cultures suppressed IL-2-induced LAK cell activity measured against Daudi and the melanoma cell line MEWO in a dose-dependent way. IL-4 also inhibited the induction of LAK cell activity in CD2+, CD3-, CD4-, CD8- cells, suggesting that IL-4 acts directly on LAK precursor cells. IL-4 added 24 h after the addition of IL-2 failed to inhibit the generation of LAK cell activity. Cytotoxic activity of various types of NK cell clones was not affected after incubation in IL-4 for 3 days, indicating that IL-4 does not affect the activity of already committed killer cells. No significant differences were observed in the percentages of Tac+, NKH-1+ and CD16+ cells after culturing PBL in IL-2, IL-4 or combinations of IL-2 and IL-4 for 3 days. IL-4 also inhibited the activation of non-specific cytotoxic activity in MLC, as measured against K-562 and MEWO cells. In contrast, the Ag-specific CTL activity against the stimulator cells was augmented by IL-4. Collectively, these data indicate that IL-4 prevents the activation of LAK cell precursors by IL-2, but does not inhibit the generation of Ag-specific CTL.