Distinct responses of splenic dendritic cell subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo.

To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α(+), CD4(+), and CD8α(-)CD4(-) DC and the B220(+) plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8(+) T cells at 24hpi. The CD8α(+) DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4(+) DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α(-)CD4(-) DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8(+) DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8(+) T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α(+) DC subset.

The mechanism of unresponsiveness to circulating tumor antigen MUC1 is a block in intracellular sorting and processing by dendritic cells.

Immunity to tumor Ags in patients is typically weak and not therapeutic. We have identified a new mechanism by which potentially immunogenic glycoprotein tumor Ags, such as MUC1, fail to stimulate strong immune responses. MUC1 is a heavily glycosylated membrane protein that is also present in soluble form in sera and ascites of cancer patients. We show that this soluble protein is readily taken up by dendritic cells (DC), but is not transported to late endosomes or MHC class II compartments for processing and binding to class II MHC. MUC1 uptake is mediated by the mannose receptor, and the protein is then retained long term in early endosomes without degradation. Long-term retention of MUC1 does not interfere with the ability of DC to process and present other Ags. We also demonstrate inhibited processing of another important glycoprotein tumor Ag, HER-2/neu. This may, therefore, be a frequent obstacle to presentation of tumor Ags and an important consideration in the design of cancer vaccines. It should be possible to overcome this obstacle by providing DC with a form of tumor Ag that can be better processed. For MUC1 we show that a 140-aa-long synthetic peptide is very efficiently processed by DC.

Concentration of MHC class II molecules in lipid rafts facilitates antigen presentation.

The plasma membranes of eukaryotic cells are not uniform and possess distinct cholesterol- and sphingolipid-rich raft microdomains that are enriched in proteins known to be essential for cellular function. Lipid raft microdomains are important for T cell receptor (TCR)-mediated activation of T cells. However, the importance of lipid rafts on antigen presenting cells (APCs) and their role in major histocompatibility (MHC) class II-restricted antigen presentation has not been examined. MHC class II molecules were found to be constitutively present in plasma membrane lipid rafts in B cells. Disruption of these microdomains dramatically inhibited antigen presentation at limiting concentrations of antigen. The inhibitory effect of raft disruption on antigen presentation could be overcome by loading the APCs with exceptionally high doses of antigen, showing that raft association concentrates MHC class II molecules into microdomains that allow efficient antigen presentation at low ligand densities.

Presentation of MUC1 tumor antigen by class I MHC and CTL function correlate with the glycosylation state of the protein taken Up by dendritic cells.

We previously reported that the glycosylated MUC1 tumor antigen circulating as soluble protein in patients' serum is not processed by dendritic cells and does not elicit MHC-Class II-restricted T helper responses in vitro. In contrast, a long synthetic peptide from the MUC1 tandem repeat region is presented by Class II molecules, resulting in the initiation of T helper cell responses. Here we addressed the ability of dendritic cells to present various glycosylated or not glycosylated forms of MUC1 by MHC Class I. We found that three different forms of MUC1, ranging from glycosylated and underglycosylated protein to unglycosylated synthetic peptide, were able to elicit MUC1-specific, Class-I-restricted CTL responses. The efficiency of processing and the resulting strength of CTL activity were inversely correlated with the degree of glycosylation of the antigen. Furthermore, the more efficiently processed 100mer peptide primed a broader repertoire of CTL than the glycosylated protein.

Naturally processed class II epitope from the tumor antigen MUC1 primes human CD4+ T cells.

Epithelial cell mucin MUC1 is expressed on adenocarcinomas in an underglycosylated form that serves as a tumor antigen in breast, pancreatic, ovarian, and other tumors. Two predominant MUC1-specific immune responses are found in patients: CD8+ CTLs, which recognize tandemly repeated epitopes on the MUC1 protein core, and IgM antibodies. There have been no reports to date of MUC1-specific CD4+ T-helper cells in cancer patients. We show here that MUC1-specific CD4+ T cells are neither deleted nor tolerized and that CD4+ T cell responses can be generated when an appropriate soluble form of MUC1 is used. Naive CD4+ T cells from healthy donors were primed in vitro to a synthetic MUC1 peptide of 100 amino acids, representing five unglycosylated tandem repeats, presented by dendritic cells. They produced IFN-gamma and had moderate cytolytic activity. We identified one core peptide sequence, PGSTAPPAHGVT, that elicits this response when it is presented by HLA-DR3.

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.

Interferon-gamma and interleukin-10 have cross-regulatory roles in modulating the class I and class II MHC-mediated presentation of epitopes of Listeria monocytogenes by infected macrophages.

IFN-gamma is an important cytokine in resistance to infection with Listeria monocytogenes, and interleukin 10 is known to exacerbate infection with Listeria and other intracellular pathogens. We examined the effects of these cytokines on antigen presentation by macrophages infected with live Listeria. Listeriolysin O, a hemolysin secreted by Listeria, is an immunodominant antigen presented by both class I and class II MHC on infected cells. Thioglycollate-elicited macrophages were pretreated with exogenous IFN-gamma, IL-10, or both cytokines overnight, infected with bacteria, and then fixed. Epitope-specific, MHC-restricted, T cell hybridomas were then added to detect the presentation of the class I or class II ligand. We found that IFN-gamma enhanced the presentation of both the class I and class II epitopes and IL-10 strongly inhibited the presentation of both ligands. The degree of inhibition of presentation caused by IL-10 was dose dependent. IL-10 was also able to inhibit the presentation of exogenously added class II-binding peptide but had a less dramatic effect on the presentation of the added class I-binding polypeptide epitope. Flow cytometric analysis of expression of class I and class II on treated macrophages demonstrated that the inhibitory effect of IL-10 on antigen presentation was not due to significant downregulation of MHC expression. This loss of antigen presentation was also not due to downregulation of the costimulatory molecule, B7-2. We have found that IFN-gamma and IL-10 have opposing immunoregulatory effects on the presentation of antigens derived from an intracellular pathogen and that the class I vs. class II-mediated presentation of antigens is differentially regulated by IL-10.

Mechanisms of processing and presentation of the antigens of Listeria monocytogenes.

Antigen processing is the series of events through which protein antigens become degraded and are sent to the cell surface for recognition by T cells. These events within the cell have been studied extensively using the model system of infection with Listeria monocytogenes. This bacteria resides primarily intracellularly; immunity to it is mediated by cellular responses. Upon phagocytosis by a macrophage, engulfed Listeria express a hemolytic molecule, listeriolysin O (LLO), and can disrupt the membrane of the endosome and escape into the cellular cytoplasm. This allows the organism to escape the hostile environment of the endosome and also gives it access to the processing machinery of the cytoplasm. Therefore, Listeria monocytogenes can be processed within an endosome and be presented by class II MHC, or can escape into the cytoplasm and be processed there and presented by class I MHC. This LLO molecule is not only an important virulence factor, it is also a dominant antigen in the cellular immune response to Listeria. The outcome of antigen processing and presentation can be influenced by the expression of LLO, by the state of activation of the macrophage, and by the cytokines involved in the immune response.