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S A Safley

Publications and source records attributed to S A Safley.

5 recordsLinked to original sources
PubMed1996

The presentation of class I and class II epitopes of listeriolysin O is regulated by intracellular localization and by intercellular spread of Listeria monocytogenes.

The hemolysin, listeriolysin 0 (LLO), produced by Listeria monocytogenes is both a virulence factor and an immunodominant Ag. In this study, we investigated how the lytic activity of LLO effects the context of presentation of two known LLO epitopes by either class I or class II MHC molecules. T cell hybridomas were used to monitor each peptide/MHC ligand. APCs infected with strains of Listeria expressing hemolytic LLO strongly presented the class I MHC epitope; however, this ligand was not well presented by cells infected with nonhemolytic Listeria. In contrast, there was almost no presentation of the class II-binding LLO epitope in cells infected with fully hemolytic Listeria. Only hemolysin-deficient Listeria were presented by class II MHC. Listeria expressing wild-type LLO but deficient in other virulence factors showed a presentation pattern equivalent to that of hemolytic Listeria. To address further the divergence of presentation, we used an intercellular spread assay to detect Ag presentation by cells neighboring the primarily infected one. We found that hemolytic Listeria were presented by both class I and II MHC on cells adjacent to the initially infected one(s). Finally, our kinetic analysis of presentation revealed that the class II ligand is presented over 4 h before the class I ligand. We have demonstrated that LLO's lytic activity potentiates presentation of listerial Ags by class I MHC and inhibits presentation via class II MHC. LLO-mediated intracellular localization (cytoplasmic vs endosomal) of bacteria corresponds to the operative presentation pathway.

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PubMed1995

Mechanisms of T cell epitope immunodominance analyzed in murine listeriosis.

We demonstrate conclusively that the bacterial exotoxin listeriolysin O (LLO) is a target Ag for eliciting CD4+ T cell responses following infection with Listeria monocytogenes. The minimal I-Ek-restricted immunodominant CD4+ T cell epitope was identified as peptide 215-226 (p215-226). Most LLO-specific T cell hybridomas recognized p203-226, p208-226, p215-226, and p215-234, although each exhibited a characteristic pattern of preferential reactivity. One hybridoma (IIIC5) reacted to p203-226 but not to p208-226 or any other LLO peptide tested. With APCs from B10 congenic mice and cells transfected with either I-Ak or I-Ek, IIIC5 recognized p203-216 with I-Ak, while a different hybridoma (IB5) recognized p215-226 with I-Ek. Competitive binding studies demonstrated that of 15 LLO peptides tested, only p203-226, p215-226, and p215-234 had high affinity for I-Ek, while p203-226 could also bind to isolated I-Ak. Of nine LLO peptides tested, only p215-234 bound multiple class II MHC alleles. These findings suggest that the immunodominance of p203-226 may be due in part to the presence of multiple T cell epitopes with I-Ek- and I-Ak-binding capability. Many of the rules of immunodominance observed with model Ags are also operative in our murine model of bacterial infectious disease. Furthermore, a novel mechanism of immunodominance based on newly defined structural features of MHC molecules is implicated. This information is crucial for rational vaccine development.

Amino Acid Sequence↗
PubMed1995

Human T-cell recognition of Listeria monocytogenes: recognition of listeriolysin O by TcR alpha beta + and TcR gamma delta + T cells.

The cell-mediated immune response to Listeria monocytogenes has been well characterized in the mouse. Listeriolysin O (LLO) is a major antigen in murine T-cell recognition of L. monocytogenes. In this study, we show that LLO is also recognized by human TcR alpha beta T cells and TcR gamma delta T cells. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with live listeriae and then expanded with interleukin 2 were shown to respond to purified LLO. The generation of LLO-responsive T cells was dependent on the use of live bacteria during the initial in vitro challenge. LLO-induced proliferation of T cells expanded by exposure of PBMC to live listeriae was major histocompatibility complex restricted. PBMC cultured with formalin-fixed listeriae and subsequently expanded by interleukin 2 gave high proliferative responses to fixed bacteria but failed to respond to LLO. PBMC stimulated in vitro with fixed listeriae contained predominantly TcR alpha beta + T cells. In contrast, PBMC obtained from 85% of the donors studied generated high numbers of TcR gamma delta + T cells following in vitro culture with live listeriae. Using a panel of synthetic amphipathic LLO peptides, we found that LLO-specific T cells from different individuals recognized both common and unique peptides. LLO 470-508 was recognized by three of five individuals, while LLO 203-226 and LLO 107-126 were recognized by two of six individuals. A TcR gamma delta + T-cell line was established from PBMC stimulated with live listeriae and was shown to recognize LLO 470-508. Proliferative responses could be induced in this cell line by peptide-pulsed autologous PBMC but not by peptide-pulsed allogeneic PBMC. Our results establish the importance of LLO in human T-cell recognition of listeriae and show that both TcR alpha beta + T cells and TcR gamma delta + T cells recognize this antigen. Finally, since LLO 470-508 has a high degree of homology with other gram-positive bacterial toxins, the recognition of this peptide by TcR gamma delta + T cells suggests that an important role of these T cells in host defense is the recognition of bacterium-derived toxins.

Amino Acid Sequence↗
PubMed1991

Role of listeriolysin-O (LLO) in the T lymphocyte response to infection with Listeria monocytogenes. Identification of T cell epitopes of LLO.

Using a murine model, we investigated the role of the bacterial exotoxin listeriolysin O (LLO) in cellular immunity to Listeria monocytogenes. A correlation between LLO production by infecting bacteria and generation of protective immunity to virulent LLO-producing bacteria was noted. Using isogeneic hemolysin (Hly+ or Hly-) strains of L. monocytogenes, we demonstrated that LLO production by infecting bacteria is required to elicit T cells reactive both to bacteria-associated Ag and to the secreted LLO molecule as measured by IL-2 production in vitro. Distinct sets of T cells specific for largely nonoverlapping pools of antigenic determinants represented by LLO and cell-associated Ag (heat-killed L. monocytogenes) are generated after infection. We have used models for prediction of T cell epitopes based on primary structure of LLO, and synthetic amphipathic LLO peptides were evaluated as Ag in vitro or as immunogenes in vivo. Infection of several strains of mice (H-2k and H-2d) with LLO-producing L. monocytogenes resulted in the generation of T cells that could respond consistently to two peptides, LLO 215-234 and LLO 354-371. Mouse strains lacking expression of I-E molecules (e.g., B10.A(4R) and C57BL/6) responded to LLO but not to the peptides tested. With C3HeB/FeJ mice, antibodies to I-Ek blocked the presentation of LLO 215-234. The importance of the N-terminal portion of LLO 215-234 was evidenced by the drastic reduction in antigenic activity of truncated peptides (e.g., LLO 221-234 and LLO 224-234). LLO 215-234, the strongest and most consistent activator of T cells from L. monocytogenes-immune mice, fit well some models for antigenic peptides in several ways. It could be predicted to form an amphipathic alpha-helix, it contained multiple "Rothbard motifs" (charged residue or glycine, two or three hydrophobic amino acids and then a glycine or polar residue), it had a net charge of +2, and it contained the correct spacing of amino acids (five to six residues between a hydrophobic and basic amino acid) that is characteristic of I-Ek-binding peptides. Immunization with 8 of 10 synthetic LLO peptides generated T cells that recognized the immunizing peptide in vitro, but such T cells were only poorly reactive with LLO. Our results indicate that LLO is an important target Ag for stimulation of CD4+ L. monocytogenes-specific T cells, and that LLO 215-234 is antigenically dominant in C3HeB/FeJ mice.

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