Fine specificity and MHC restriction of trinitrophenyl-specific CTL.
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In this study, the fine specificity and MHC restriction of a CTL response specific to the trinitrophenyl (TNP) hapten was analyzed. Based on the structure of peptide/Kb complexes and ternary TCR/Ag/MHC complexes, four TNP peptides, two octamers, and two nonamers were chosen for eliciting anti-TNP CTL responses. Hapten was conjugated at position 4 in the octamers and at position 5 in the nonamers, positions which should allow engagement of the hapten by TCRs. Potent CTL activity for each of the TNP peptides was obtained that was highly hapten-specific; however, there were considerable differences in the extent of cross-reactivity with other TNP peptides, with the octamers generating more cross-reactive CTL than the nonamers. MHC restriction analysis suggested that anti-hapten responses were less dependent on MHC recognition than anti-peptide responses. This was evidenced by the relative ease of detecting cross-reactivity to haptenated peptides presented by allo-MHC and by the relative insensitivity of anti-hapten vs anti-peptide CTL to mutations in the Kb molecule at potential TCR interaction sites. One potential explanation for this insensitivity to MHC mutation was the finding that the anti-hapten response appeared to be of higher avidity, since a > 100-fold difference in the amount of Ag required to sensitize target cells was found between these two types of Ags. (+info)
CD86 (B7-2) can function to drive MHC-restricted antigen-specific CTL responses in vivo.
(42/8493)
Activation of T cells requires both TCR-specific ligation by direct contact with peptide Ag-MHC complexes and coligation of the B7 family of ligands through CD28/CTLA-4 on the T cell surface. We recently reported that coadministration of CD86 cDNA along with DNA encoding HIV-1 Ags i.m. dramatically increased Ag-specific CTL responses. We investigated whether the bone marrow-derived professional APCs or muscle cells were responsible for the enhancement of CTL responses following CD86 coadministration. Accordingly, we analyzed CTL induction in bone marrow chimeras. These chimeras are capable of generating functional viral-specific CTLs against vaccinia virus and therefore represent a useful model system to study APC/T cell function in vivo. In vaccinated chimeras, we observed that only CD86 + Ag + MHC class I results in 1) detectable CTLs following in vitro restimulation, 2) detectable direct CTLs, 3) enhanced IFN-gamma production in an Ag-specific manner, and 4) dramatic tissue invasion of T cells. These results support that CD86 plays a central role in CTL induction in vivo, enabling non-bone marrow-derived cells to prime CTLs, a property previously associated solely with bone marrow-derived APCs. (+info)
Identification of a second major tumor-specific antigen recognized by CTLs on mouse mastocytoma P815.
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Murine mastocytoma P815 induces CTL responses against at least four distinct Ags (AB, C, D, and E). Recent studies have shown that the main component of the CTL response against the P815 tumor is targeted against Ags P815AB and P815E. The gene P1A has been well characterized. It encodes the P815AB Ag in the form of a nonameric peptide containing two epitopes, P815A and P815B, which are recognized by different CTLs. Here, we report the identification of the P815E Ag. Using a cDNA library derived from tumor P815, we identified the gene coding for P815E. We also characterized the antigenic peptide that anti-P815E CTLs recognize on the MHC class I molecule H-2Kd. The P815E Ag results from a mutation within an ubiquitously expressed gene encoding methionine sulfoxide reductase, an enzyme that is believed to be important in the protection of proteins against the by-products of aerobic metabolism. Surprisingly, immunizing mice i.p. with syngeneic tumor cells (L1210) that were constructed to express B7-1 and P815E did not induce resistance against live P815, even though a strong anti-P815E CTL response was observed with splenocytes from immunized animals. (+info)
Assessment of immunogenicity of human Melan-A peptide analogues in HLA-A*0201/Kb transgenic mice.
(44/8493)
Previous studies have shown that substitution of single amino acid residues in human Melan-A immunodominant peptides Melan-A27-35 and Melan-A26-35 greatly improved their binding and the stability of peptide/HLA-A*0201 complexes. In particular, one Melan-A peptide analogue was more efficient in the generation of Melan-A peptide-specific and melanoma-reactive CTL than its parental peptide in vitro from human PBL. In this study, we analyzed the in vivo immunogenicity of Melan-A natural peptides and their analogues in HLA-A*0201/Kb transgenic mice. We found that two human Melan-A natural peptides, Melan-A26-35 and Melan-A27-35, were relatively weak immunogens, whereas several Melan-A peptide analogues were potent immunogens for in vivo CTL priming. In addition, induced Melan-A peptide-specific mouse CTL cross-recognized natural Melan-A peptides and their analogues. More interestingly, these mouse CTL were also able to lyse human melanoma cell lines in vitro in a HLA-A*0201-restricted, Melan-A-specific manner. Our results indicate that the HLA-A*0201/Kb transgenic mouse is a useful animal model to perform preclinical testing of potential cancer vaccines, and that Melan-A peptide analogues are attractive candidates for melanoma immunotherapy. (+info)
Molecular and functional analysis of a conserved CTL epitope in HIV-1 p24 recognized from a long-term nonprogressor: constraints on immune escape associated with targeting a sequence essential for viral replication.
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It has been hypothesized that sequence variation within CTL epitopes leading to immune escape plays a role in the progression of HIV-1 infection. Only very limited data exist that address the influence of biologic characteristics of CTL epitopes on the emergence of immune escape variants and the efficiency of suppression HIV-1 by CTL. In this report, we studied the effects of HIV-1 CTL epitope sequence variation on HIV-1 replication. The highly conserved HLA-B14-restricted CTL epitope DRFYKTLRAE in HIV-1 p24 was examined, which had been defined as the immunodominant CTL epitope in a long-term nonprogressing individual. We generated a set of viral mutants on an HX10 background differing by a single conservative or nonconservative amino acid substitution at each of the P1 to P9 amino acid residues of the epitope. All of the nonconservative amino acid substitutions abolished viral infectivity and only 5 of 10 conservative changes yielded replication-competent virus. Recognition of these epitope sequence variants by CTL was tested using synthetic peptides. All mutations that abrogated CTL recognition strongly impaired viral replication, and all replication-competent viral variants were recognized by CTL, although some variants with a lower efficiency. Our data indicate that this CTL epitope is located within a viral sequence essential for viral replication. Targeting CTL epitopes within functionally important regions of the HIV-1 genome could limit the chance of immune evasion. (+info)
Stimulation of CD40 on immunogenic human malignant melanomas augments their cytotoxic T lymphocyte-mediated lysis and induces apoptosis.
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Here, we report the functional expression of CD40 on human malignant melanomas (MMs). Comparison of tumor specimen from MM precursor lesions, primary tumors, and metastases revealed that CD40 surface expression is down-regulated during tumor progression. CD40 expression was confirmed in 7 human MM cell lines established from immunogenic primary tumors or metastases, whereas 11 cell lines established from advanced stages were CD40 negative. CD40 expression could be enhanced in CD40-positive MM by stimulation with IFN-gamma and tumor necrosis factor-alpha but not by interleukin (IL)-1beta or CD40 triggering. CD40 ligation on MM by CD40L-transfected murine L-cells or by a soluble CD40L fusion protein up-regulated their expression of intercellular adhesion molecule-1 and MHC class I and class II molecules and their secretion of IL-6, IL-8, tumor necrosis factor-a, and granulocyte macrophage colony-stimulating factor and also induced a rapid activation of the transcription factor nuclear factor kappaB. Furthermore, CD40 ligation of a HLA-A2+, MelanA/MART1+ MM cell line enhanced its susceptibility to specific lysis by a HLA-A2-restricted, MelanA/MART-1-specific CTL clone. Finally, CD40 ligation induced growth inhibition and apoptosis in MM. These results indicate that CD40-CD40L interactions may play an important role in augmenting antitumor immunity and inducing apoptosis in some CD40-positive immunogenic human MMs. (+info)
Mucosal immune system of the human genital tract.
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In contrast to the pronounced dominance of secretory IgA over other immunoglobulin isotypes in human saliva, tears, milk, and gastrointestinal fluids, secretions of both female and male genital tracts contain more IgG than secretory IgA. Both IgG and IgA are derived, to a variable degree, from the systemic immunoglobulin pool as well as from local synthesis. The origin of IgG- and IgA-plasma cell precursors destined for the genital tract is unknown, but indirect evidence suggests that mucosal inductive sites localized in the rectum, small intestine, and especially in the nasal cavity contribute such precursors to the female genital tract. Several studies indicated that intranasal immunization of various species, including humans, was efficient at inducing antigen-specific antibody responses in the female genital tract; however, whether this route is also effective in males has not been explored. (+info)
The identification of myriocin-binding proteins.
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BACKGROUND: Myriocin is a natural product that potently induces apoptosis of a murine cytotoxic T lymphocyte cell line (CTLL-2) and inhibits a serine palmitoyltransferase (SPT) activity that has been detected in cell extracts and is thought to initiate sphingolipid biosynthesis. Because SPT has never been biochemically purified and a comprehensive appraisal of myriocin-binding proteins has not been conducted, we isolated specific targets using myriocin-based affinity chromatography. RESULTS: Myriocin derivatives were synthesized and evaluated using CTLL-2 proliferation and SPT activity assays. Guided by these results, affinity chromatography matrices were prepared and two specific myriocin-binding proteins were isolated from CTLL-2 lysates. Analyses of these polypeptides establish conclusively that they are murine LCB1 and LCB2, mammalian homologs of two yeast proteins that have been genetically linked to sphingolipid biosynthesis. CONCLUSION: The ability of myriocin-containing matrices to bind factors that have SPT activity and the exclusive isolation of LCB1 and LCB2 as myriocin-binding proteins demonstrates that the two proteins are directly responsible for SPT activity and that myriocin acts directly upon these polypeptides. (+info)