Dendritic Cell Cross-Priming Is Essential for Immune Responses to Listeria monocytogenes

Cross-presentation is now recognized as a major mechanism for initiating CD8 T cell responses to virus and tumor antigens in vivo. It provides an elegant mechanism that allows relatively few Dendritic cells (DCs) to initiate primary immune responses while avoiding the consumptive nature of pathogenic infection. CD8 T cells play a major role in anti-bacterial immune responses; however, the contribution of cross-presentation for priming CD8 T cell responses to bacteria, in vivo, is not well established. Listeria monocytogenes (Listeria) is the causative agent of Listeriosis, an opportunistic food-borne bacterial infection that poses a significant public health risk. Here, we employ a transgenic mouse model in which cross-presentation is uniquely inactivated, to investigate cross-priming during primary Listeria infection. We show that cross-priming deficient mice are severely compromised in their ability to generate antigen-specific T cells to stimulate MHC I-restricted CTL responses following Listeria infection. The defect in generation of Listeria-elicited CD8 T cell responses is also apparent in vitro. However, in this setting, the endogenous route of processing Listeria-derived antigens is predominant. This reveals a new experimental dichotomy whereby functional sampling of Listeria-derived antigens in vivo but not in vitro is dependent on cross-presentation of exogenously derived antigen. Thus, under normal physiological circumstances, cross-presentation is demonstrated to play an essential role in priming CD8 T cell responses to bacteria

MHC Class I Endosomal and Lysosomal Trafficking Coincides with Exogenous Antigen Loading in Dendritic Cells

BACKGROUND: Cross-presentation by dendritic cells (DCs) is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I) from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing. CONCLUSIONS/SIGNIFICANCE: We conclude that DCs have 'hijacked' and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place

Exchange of H-2K^bOVA peptide is affected by MHC I tyrosine mutation on ΔY APCs.

<p>Splenocytes from K^bWT and ΔY mice were infected with <i>Listeria</i>-OVA between 0.001–0.1 MOI. Following 4 h infection, extracellular bacteria were removed and infected cells were incubated overnight. A. APCs were surface stained with a monoclonal antibody (mAb 25.D1.16) that specifically recognizes H-2K^b-OVA_(257–264) complexes and analysed by flow cytometry. Uninfected APCs or infected APCs pulsed with 1 µM OVA_(257–264) peptide for 1 h were used as negative and positive controls, respectively. B. Data is represented as a bar graph of the mean fluorescence intensity (MFI) of surface H-2K^bOVA_(257–264) complexes. Experiments were performed in triplicate and values are presented as average MFI plus SD with Student's <i>t</i> tests * <i>p</i><0.05.</p

Depletion of CD8 T cells in ΔY spleen following <i>Listeria</i>-OVA infection.

<p>K^bWT and ΔY mice were orally infected with 5e⁷ cfu <i>Listeria</i>-OVA or mock infected with Hepes buffered PBS. Infected spleens were harvested 9 days post infection and OVA-specific T cells expanded in culture for 5 days by incubation with specific OVA_(257–264) peptide. Cells were then counted by trypan blue exclusion and CD8 and CD4 T cells were stained and analysed by flow cytometry. Numbers of T cells were calculated and are presented as a bar graph plus SD of 2 mice per group infected; student's <i>t</i> test *, <i>p</i><0.05.</p

Proposed mechanisms of <i>Listeria</i>-derived antigen sampling following <i>in vitro</i> or <i>in vivo</i> infection.

<p>Schematic representation of the mechanisms involved in functional sampling of <i>Listeria</i>-derived antigens depending on the route of infection. A. <i>Listeria</i> directs its own phagocytic uptake into the cell. Within the phagosome, protease-mediated degradation of the bacterium generates peptides capable of binding MHC I molecules recycling from the surface, a process dependant on an intact tyrosine (Y) residue in the cytoplasmic tail of the MHC I molecule. Peptide exchange occurs allowing bacterially-derived antigens to be loaded on MHC I molecules and trafficked to the surface for cross-presentation. Predominantly, toxin-induced pore-forming of the phagosome occurs to allow the escape of the bacterium into the cytosol. Within the cytosol, <i>Listeria</i> is targeted for proteosome –mediated degradation and antigen transport to the ER where bacterial peptides are loaded on newly synthesized MHC I molecules. Loaded MHC I molecules follow the secretory pathway through the trans golgi network (TGN) to the surface for presentation. B. Following infection <i>in vivo</i>, directly infected cells undergo apoptosis, providing antigenic material for uptake by bystander DCs. In this scenario, apoptotic material containing bacterially-derived antigens is phagocytosed into a compartment competent for cross-presentation, named the endolysosomal compartment (ELC). MHC I molecules recycle from the surface into this compartment, peptide exchange occurs and bacterially-derived peptide antigens are loaded and recycled to the cell surface for cross-presentation.</p

ΔY APCs have decreased ability to stimulate naive T cell proliferation following <i>in vitro</i> infection with <i>Listeria</i>-OVA.

<p>A. Splenocytes from K^bWT and ΔY mice were infected with increasing doses of <i>Listeria</i>-OVA. Following 4 h infection, extracellular bacteria were removed and infected cells were incubated overnight. APCs were then cultured with OT-I T cells for 48 h. [³H]-Thymidine was then added to the cultures and 24 h later, proliferation was measured by [³H]-Thy incorporation. B. Data is presented as fold increases in T cell proliferation. Values are calculated by dividing [3H]-Thy count values by background. Experiments were performed in triplicate and values are presented as means plus SD with Student's <i>t</i> tests * <i>p</i><0.05; ** <i>p</i><0.005; ***<i>p</i><0.0005.</p

Antigen specific CD8 T cell responses in C57BL/6 mice differ following <i>Listeria</i>-OVA oral and i.v. routes of infection.

<p>C57BL/6 mice were infected with 1e⁹ cfu or 1e⁴ cfu <i>Listeria</i>-OVA by oral gavage or i.v. injection, respectively. Mock oral infections were performed with 0.1 M HEPES-PBS (Mock) while PBS alone was used for mock i.v. infections. At the peak of the cellular response, 9 days post infection, spleens were harvested and the cytolytic capacity of CD8 T cells was analysed in a standard ⁵¹-Cr release assay. T cells were expanded in culture with OVA_(257–264)-specific peptide for 6 days. Effector T cells were then incubated with OVA_(257–264) peptide pulsed, sodium chromate-labeled target cells and their ability to lyse targets was measured at indicated ratios. Experiments were performed in triplicate with 3 mice per group.</p

ΔY mice are deficient in generating specific CD8 T cell responses following <i>Listeria</i>-OVA infection.

<p>K^bWT and ΔY mice were orally infected with 5e⁷ or 1e⁶ cfu <i>Listeria</i>-OVA or mock infected with 0.1 M HEPES-PBS. Spleens and MLN were harvested 9 days post infection and analysed directly <i>ex vivo</i> or specific T cells were expanded in culture by incubation with OVA_(257–264) peptide A, Cytotoxicity assays were performed whereby OVA_(257–264) peptide pulsed, sodium chromate-labeled target cells were incubated with restimulated effector T cells from spleens of infected mice. The ability of effector T cells to lyse targets was measured as chromium release at indicated ratios. 2 mice per group were analysed for each dose of infection tested. Cytotoxicity experiments were performed in triplicate. B, C, <i>Listeria</i>-derived OVA_(257–264)-specific CD8 T cells from spleens or MLN of 2 mice per group were quantified by tetramer staining following oral infection with 5e⁷ cfu <i>Listeria</i>-OVA and restimulation of the T cells <i>in vitro</i> (B) or analysed directly <i>ex vivo</i> (C). <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007210#s2" target="_blank">Results</a> are presented as bar graphs of fold increases in % tetramer/CD8 T cells stained plus SD; Student's <i>t</i> test *, <i>p</i><0.05.</p

ΔY DCs have decreased ability to activate T cells following <i>in vitro</i> infection with <i>Listeria</i>-OVA.

<p>Bone marrow preparations from K^bWT and ΔY mice were cultured <i>in vitro</i> with GM-CSF to develop bmDCs. After 10 days in culture, cells were labeled with CD11c and H-2K^b and analysed by flow cytometry. A, Day 10 bmDCs from K^bWT, ΔY or TAP1^−/− mice were infected with increasing doses of <i>Listeria</i>-OVA for 4 h. After 4 h infection time, extracellular bacteria were removed and DCs were subsequently incubated overnight to allow processing of internalized bacteria. DCs were then incubated with the B3Z T cell hybridoma for 18 h and T cell activation was measured using a CPRG chemiluminescent assay. Uninfected bmDCs from of K^bWT, ΔY or TAP1^−/− mice served as negative controls. B, Data is presented as a fold increase in T cell activation by dividing CPRG absorbance values by background C. uninfected bmDCs from K^bWT, ΔY or TAP1^−/− mice were incubated with indicated doses of soluble whole OVA. Processing of internalized OVA was allowed to take place overnight followed by incubation with B3Z T cell hybridoma for 18 h before measuring T cell activation. BmDCs were also pulsed for 1 h with 1 µM OVA_(257–264)-specific peptide as positive control. Experiments were performed in triplicate and values are presented as means plus SD with Student's <i>t</i> tests * <i>p</i><0.05; ** <i>p</i><0.005; ***<i>p</i><0.0005.</p