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Cross-Presentation: How to Get there – or How to Get the ER

Christoph kreer.

1 Life and Medical Sciences Institute, University of Bonn, Bonn, Germany

Judith Rauen

Matthias zehner, sven burgdorf.

Antigen cross-presentation enables dendritic cells (DCs) to present extracellular antigens on major histocompatibility complex (MHC) I molecules, a process that plays an important role in the induction of immune responses against viruses and tumors and in the induction of peripheral tolerance. In order to allow intracellular processing for cross-presentation, internalized antigens are targeted by distinct endocytic receptors toward specific endosomal compartments, where they are protected from rapid lysosomal degradation. From these compartments, antigens are processed for loading onto MHC I molecules. Such processing generally includes antigen transport into the cytoplasm, a process that is regulated by members of the ER-associated degradation (ERAD) machinery. After proteasomal degradation in the cytoplasm, antigen-derived peptides have been shown to be re-imported into the same endosomal compartment by endosomal transporter associated with antigen processing, another ER protein, which is recruited toward the endosomes after DC maturation. In our review, we highlight the recent advances on the molecular mechanisms of cross-presentation. We focus on the necessity of such antigen storage compartments and point out important parallels to MHC I-restricted presentation of endogenous antigens. We discuss the composition of such endosomes and the targeting of extracellular antigens into this compartment by specific endocytic receptors. Finally, we highlight recent advances on the recruitment of the cross-presentation machinery, like the members of the MHC I loading complex and the ERAD machinery, from the ER toward these storage compartments, a process that can be induced by antigen encounter or by activation of the dendritic cell after contact with endotoxins.

Introduction

Adaptive immune responses are induced when dendritic cells (DCs) encounter antigens in the peripheral tissue. Upon antigen recognition, the DC migrates toward the draining lymph node, where it can activate antigen-specific T cells (Mellman and Steinman, 2001 ). Therefore, the corresponding antigen is internalized by the DC and processed in specialized intracellular compartments. The resulting antigen-derived peptides are subsequently loaded on major histocompatibility complex (MHC) molecules. Whereas antigen loading onto MHC II molecules can lead to the activation of antigen-specific CD4 + T helper cells, peptide loading onto MHC I can activate antigen-specific cytotoxic CD8 + T cells.

In classical antigen presentation, intracellular antigens are degraded by the cytosolic proteasome. The resulting peptides are subsequently transported through the transporter associated with antigen processing (TAP) complex into the ER, where they can be loaded onto MHC I molecules (Purcell and Elliott, 2008 ). Exogenous proteins are internalized into the dendritic cell by endocytosis and end up in a lysosomal compartment, where they are degraded by lysosomal proteases to be loaded onto MHC II molecules (Trombetta et al., 2003 ). Apart from these classical presentation pathways, a process termed cross-presentation allows the presentation of extracellular antigens also on MHC I molecules (Bevan, 1976 ; Kurts et al., 1996 ).

Cross-presentation has been demonstrated to play an important role in a variety of processes, including the induction of an immune response against viruses that do not infect antigen-presenting cells directly or against tumors of non-hematopoietic origin (Huang et al., 1994 ; Sigal et al., 1999 ; den Haan and Bevan, 2001 ; Heath and Carbone, 2001 ).

The molecular mechanisms of antigen cross-presentation, however, remain only partially understood. In this review, we highlight some of the recent advances on these underlying mechanisms. We will focus on antigen targeting into specialized storage compartments, on the composition of these compartments and on the recruitment of members of the MHC I loading machinery toward these compartments.

The Importance of Antigen Storage Compartments for Cross-Presentation

During cross-presentation, antigen-derived peptides are loaded onto MHC I molecules. Subsequently, these peptide–MHC I-complexes are transported toward the cell membrane, where they can be recognized by antigen-specific T cells. Whereas peptide-loaded MHC II molecules are stable at the cell membrane for several days (Cella et al., 1997 ), differing information on the half-life of peptide-loaded MHC I molecules can be found in literature (Eberl et al., 1996 ; Rescigno et al., 1998 ; Cella et al., 1999 ; Kukutsch et al., 2000 ). A direct comparison between the stability of loaded MHC I molecules and MHC II molecules, however, showed that the half-life of loaded MHC I molecules is markedly decreased compared to peptide-loaded MHC II molecules (van Montfoort et al., 2009 ). This shorter half-life of peptide-loaded MHC I molecules has important implications for antigen cross-presentation. After antigen internalization, the cross-presenting DC must migrate toward the draining lymph node to activate antigen-specific T cells. Since this process is estimated to take up to 48 h (Martin-Fontecha et al., 2003 ), a prolonged MHC I-restricted presentation is required for efficient T cell activation, implying that ongoing antigen processing and loading of antigen-derived peptides onto MHC I molecules is indispensible. To ensure continuous peptide loading, it is essential that internalized antigens are not degraded instantly within the endo/lysosomal compartments of the DC, since this would rapidly eliminate putative epitopes for cross-presentation. For these reasons, prolonged cross-presentation depends on antigen storage in endosomal compartments, where they are protected from lysosomal degradation.

Delamarre et al. ( 2005 ) demonstrated that DCs express less lysosomal proteases compared to macrophages, resulting in a limited capacity for lysosomal degradation and a slower degradation rate of internalized antigens in DCs. Additionally, antigen stability in DCs is increased by active inhibition of lysosomal acidification, a process that prevents the activation of lysosomal proteases and therefore increases cross-presentation (Hotta et al., 2006 ). Endosome acidification is mediated by vacuolar ATPase (V-ATPase), which transports protons from the cytosol into the endosome (Nishi and Forgac, 2002 ). In DCs, this process is antagonized by NOX2-mediated alkalization of the endosome. The NADPH oxidase NOX2 is recruited by Rab27a toward endosomal membranes (Savina et al., 2006 ), where it produces reactive oxygen species (ROS). Since the production of such ROS within endosomes consumes large amounts of protons, it causes a strong alkalization of the endosome lumen (Savina et al., 2006 ), which neutralizes V-ATPase-mediated acidification and a neutral endosomal pH can be maintained. As described above, such neutral pH prevents rapid antigen degradation, resulting in enhanced cross-presentation. NOX2-mediated alkalization has been shown to be involved in cross-presentation of particulate antigens in phagosomes (Savina et al., 2006 ) and of soluble antigens in endosomes (Mantegazza et al., 2008 ).

The decreased expression of lysosomal proteases in DCs and endosomal alkalization by NOX2 might also be responsible for the high stability of antigens that are internalized by DCs in form of immune complexes. Ferry Ossendorp and colleagues have demonstrated that OVA-containing immune complexes were cross-presented efficiently over a time period of several days (van Montfoort et al., 2009 ). Importantly, nearly full-length OVA was present for over 3 days in endosomal storage compartments, from where it was steadily processed for cross-presentation.

Taken together, prolonged MHC I-restricted presentation requires antigen deposition in specialized storage compartments, where they are protected from extensive proteasomal or lysosomal degradation and from where continuous processing for loading onto MHC I molecules can take place.

Antigen Targeting into Storage Compartments for Cross-Presentation by Distinct Endocytic Receptors

In many studies, efficient antigen cross-presentation was shown to be restricted to distinct subsets of DCs. In particular, the CD8α + splenic DCs were shown to be much better in cross-presentation under steady state conditions compared to their CD8α − counterpart in mice (den Haan et al., 2000 ; Pooley et al., 2001 ; Schnorrer et al., 2006 ). Accordingly, cross-presentation capacities in mice lacking CD8α + DCs were severely reduced (Hildner et al., 2008 ) and the human counterpart of murine CD8α + DCs was also demonstrated to have superior cross-presentation capacities (Bachem et al., 2010 ; Crozat et al., 2010 ; Jongbloed et al., 2010 ; Poulin et al., 2010 ).

Cross-presentation of antigens targeted toward DEC-205, an endocytic receptor that is predominantly expressed on CD8α + splenic DCs was demonstrated to be much more efficient than antigens targeted toward DCIR2, which is only expressed on CD8α − DCs (Dudziak et al., 2007 ). Additionally, specific targeting toward DEC-205 resulted in prolonged cross-presentation for over 2 weeks (Bonifaz et al., 2004 ). It was postulated that these differences in cross-presentation capacity are due to a reduced overall expression of the cross-presentation machinery in CD8α − DCs (Schnorrer et al., 2006 ; Dudziak et al., 2007 ). Indeed, NOX2-mediated alkalization of phagosomes was demonstrated to be more pronounced in CD8α + DCs (Savina et al., 2009 ). More recent studies demonstrate that also CD8α − DCs possess intrinsic cross-presentation capacities, but that the mechanism by which the DCs internalize the antigen is crucial for its cross-presentation (Kamphorst et al., 2010 ). This was demonstrated using transgenic mice expressing the human DEC-205 on both CD8α + and CD8α − DC subsets. Antigen targeting toward this receptor resulted in similar levels of cross-presentation in both CD8α + and CD8α − DCs, indicating that also CD8α − DCs are potent cross-presenters if the antigen is internalized via DEC-205 but not via DCIR2, demonstrating an important role for the endocytic receptor itself in cross-presentation.

In accordance to these findings, we previously demonstrated a clear correlation between the mechanism of antigen internalization and its presentation (Burgdorf et al., 2007 ). We could show that antigens internalized by DCs via fluid phase pinocytosis or scavenger receptor-mediated endocytosis were rapidly targeted toward lysosomal structures, where they were degraded instantly and processed for presentation on MHC II molecules. If, however, antigens were internalized via mannose receptor (MR)-mediated endocytosis, they were not targeted toward lysosomes but rather routed into a distinct endosomal subset, which maintained all characteristics of early endosomes for a prolonged time. Importantly, from these endosomes, MR-internalized antigens were processed exclusively for cross-presentation.

These observations emphasize the importance of the endocytic receptor for cross-presentation and point out that the endocytic receptor on the DC that makes contact to an antigen already determines its fate in terms of presentation. Additionally, targeting antigens intended for cross-presentation into a separate pool of endosomes might enable enhanced endosomal stability, which is essential for prolonged cross-presentation, without affecting overall lysosomal activity, which is essential for simultaneous MHC II-restricted presentation.

Interestingly, the MR has also been proposed to directly inhibit lysosomal maturation (Shimada et al., 2006 ; Sweet et al., 2010 ), because phagosomes containing MR-internalized glycopeptidolipids displayed impaired phagosome–lysosome fusion. Such alterations were only observed if the glycopeptidolipids were endocytosed by the MR, which then was present in the glycopeptidolipid-containing phagosomes. In both publications, it was postulated that these effects might be due to MR-dependent signaling inside the DC, resulting in an overall impairment of phagosome–lysosome fusion. Another possibility would be that, similar to the observations on the role of the MR in cross-presentation, the MR targets the glycopeptidolipids into a separate endosomal compartment, which does not undergo normal lysosomal maturation. Further studies will reveal whether signaling via the MR additionally alters endosomal trafficking within DCs.

As described above, the different cross-presentation capacities of CD8α + and CD8α − splenic DCs might to a large extend be due to the expression of different endocytic receptors. This notion might also explain observations demonstrating that certain yeast antigens and antigens targeted to the neonatal Fc receptor are cross-presented to a higher extend by the CD8α − subset (Backer et al., 2008 ; Baker et al., 2011 ). Future experiments will show whether CD8α − DCs bear specific receptors for these antigens, enabling their cross-presentation.

The importance of distinct endocytic receptors for cross-presentation is further supported by experiments of the group of Peter Cresswell. They demonstrated that expression of Fc receptors, whose engagement has been shown to lead to potent cross-presentation (van Montfoort et al., 2009 ), in the human 293 T embryonic kidney cell line enables this cell line to cross-present extracellular antigens (Giodini et al., 2009 ), pointing out the possibility that nearly every cell possesses intrinsic cross-presentation capacities if the cell expresses a suited receptor.

Although the decisive role of the endocytic receptor for cross-presentation is indubitable, this might only be one half of the story. Increasing evidence points out that also the nature of interaction between the endocytic receptor and the antigen has an important impact on antigen routing and presentation. First, it has been demonstrated that receptor cross-linking by multivalent antigens alters antigen targeting within the DC. It has been shown that dectin-1, an endocytic receptor associated with cross-presentation (Weck et al., 2008 ), targets monovalent β-glucans into non-lysosomal compartments. If, however, dectin-1 is cross-linked by the multivalent β-glucan zymosan, these antigens are targeted toward lysosomal structures (Herre et al., 2004 ), demonstrating that antigen valence can regulate antigen trafficking and degradation. Second, it has been shown that the region of the endocytic receptor that recognizes the antigen is of crucial importance. Antigen targeting using antibodies specific for the carbohydrate recognition domain of DC-SIGN has been shown to efficiently deliver such antigens to lysosomal compartments for MHC II presentation (Tacken et al., 2005 ). A recent study by the same group demonstrated however, that antigen targeting toward the neck region of DC-SIGN results in prolonged antigen retention in early endosomal compartments and in reduced lysosomal trafficking (Tacken et al., 2011 ). Importantly, these antigens were efficiently cross-presented, demonstrating that different regions of a single endocytic receptor can target antigens to different processing and presentation pathways.

These findings might also provide an explanation for the observation that antigen targeting toward the MR, which targets OVA specifically toward cross-presentation (Burgdorf et al., 2007 ) as described above, can induce antigen-specific CD4 + T cell responses (Dasgupta et al., 2007 ; He et al., 2007 ; McKenzie et al., 2007 ). In these studies, antigens were targeted toward the MR by conjugation to a MR-specific antibody, which might alter MR-mediated antigen targeting by receptor cross-linking. Alternatively, these antibodies might target other regions of the MR, resulting in different antigen processing and presentation.

In summary, efficient cross-presentation requires antigen recognition by distinct regions of specific endocytic receptors, which target the internalized antigens toward antigen storage compartments, from where they can be processed for loading onto MHC I molecules.

Molecular Mechanisms of Cross-Presentation

The vacuolar versus the phagosome-to-cytosol pathway.

Despite intensive investigations, the molecular mechanisms governing antigen processing and loading onto MHC I molecules for cross-presentation are not fully resolved yet. Importantly, the diversity of experimental evidence obtained by different research groups indicates that multiple pathways can lead to MHC I-restricted presentation of exogenous antigens, depending on the nature of the antigen, the nature of the antigen-presenting cell, and the immunological context of the cross-presentation process (Figure ​ (Figure1 1 ).

Overview of the molecular mechanisms of cross-presentation . In the vacuolar cross-presentation pathway, extracellular antigens are internalized and degraded in endosomal compartments by Cathepsin S. The resulting peptides are subsequently loaded onto MHC I molecules within the endosomal compartment. In the phagosome-to-cytosol pathway, internalized antigens are transported out of the endosomes into the cytosol for proteasomal degradation. The resulting peptides can be re-imported into the same endosomal compartment by endosomal TAP to be loaded onto MHC I molecules there. The transport of the cross-presentation machinery toward antigen-containing endosomes is induced after stimulation of TLRs. Alternatively, DCs can obtain peptides from neighboring cells via gap junctions. These peptides are thought to subsequently enter the endogenous MHC I-restricted presentation pathway in the ER.

In general, two major pathways are considered to be most relevant for antigen cross-presentation: the vacuolar pathway and the phagosome-to-cytosol pathway (Rock and Shen, 2005 ).

In the vacuolar pathway, which is also termed TAP-independent cross-presentation, internalized antigens are degraded in endosomal compartments by intra-endosomal proteases such as cathepsin S (Shen et al., 2004 ). After such degradation, antigenic peptides are loaded within the endosomes onto MHC I molecules, which reach the endosomes from the cell surface during endocytosis. The acid environment in these endosomes might allow already bound peptides to dissociate from the MHC I molecules, enabling the peptides generated within the endosomes to bind MHC I molecules.

Although several studies reported of cross-presentation via the vacuolar pathway (Shen et al., 2004 ; Bertholet et al., 2006 ), its physiological significance remains unclear. Therefore, the phagosome-to-cytosol pathway is considered to be the most relevant cross-presentation pathway in vivo (Rock and Shen, 2005 ).

In the phagosome-to-cytosol pathway, internalized antigens need to be transported from the endosomal lumen into the cytoplasm. Such antigen transport is required for consecutive degradation by the cytosolic proteasome, which is essential for cross-presentation by the phagosome-to-cytosol pathway (Kovacsovics-Bankowski and Rock, 1995 ; Ackerman et al., 2003 ; Palmowski et al., 2006 ).

Antigen transport from endosomal compartments into the cytosol and proteasomal degradation

One of the most intriguing questions concerning the molecular mechanisms of antigen cross-presentation is without any doubt how antigens pass the endosomal membrane to reach the cytosol. Although it has been demonstrated that phagosomes containing Cryptococcus neoformans lose membrane integrity (Tucker and Casadevall, 2002 ) and the presence of sphingosine within the phagosome might influence membrane stability and permeability (Werneburg et al., 2002 ), it is assumed that antigen transport into the cytoplasm is not due to disruption of the endosomal membrane. Increasing evidence points out that a pore complex spanning the endosomal membrane might rather mediate this process. Antigen translocation has been demonstrated to be size-selective. Although dextranes of 500 kDa and even 2,000 kDa can still be translocated into the cytosol, the efficiency is clearly lower than the transport of 40 kDa-sized dextranes (Rodriguez et al., 1999 ), which supports the notion that such antigen transport is not simply due to a simple disrupture of the endosomal membrane.

Increasing evidence points out that the ER-associated degradation (ERAD) machinery plays a very central role in this antigen transport into the cytoplasm (Imai et al., 2005 ; Ackerman et al., 2006 ; Giodini and Cresswell, 2008 ). The ERAD machinery has been studied extensively in the context of protein dislocation at the ER membrane. During dislocation, the ERAD machinery mediates the transport of misfolded proteins from the ER into the cytoplasm for proteasomal degradation, depicting an important function in preventing misfolded proteins from reaching the cell surface. One important member of the ERAD machinery in respect to cross-presentation is Sec61, which is thought to build the pore complex for dislocation of proteins trough the ER membrane. Similar to its role in dislocation, Sec61 has been postulated to build the pore complex through the endosomal membrane for cross-presentation (Ackerman et al., 2006 ). This hypothesis was based on observations, revealing that DC treatment with exotoxin A, which is assumed to be a specific inhibitor of Sec61, prevented antigen translocation into the cytoplasm and hence cross-presentation (Koopmann et al., 2000 ). Since such evidence for an involvement of Sec61 in antigen translocation was based on the effect of an inhibitor and therefore indirect, the role of Sec61 in cross-presentation has been questioned (Lin et al., 2008 ; Segura and Villadangos, 2011 ). In these studies, it was argued that the size of Sec61, which has been estimated to be about 5–8 Å (Van den Berg et al., 2004 ), might not be sufficient for antigen translocation. However, this size was calculated for closed or empty Sec61 and it has been postulated that the Sec61 pore complex during protein transport might encompass up to 40–60 Å (Hamman et al., 1997 ). Additionally, it has been demonstrated that proteins are unfolded during antigen translocation into the cytosol (Giodini and Cresswell, 2008 ), which might also enable them to transit also through a narrow pore complex.

More direct evidence for an involvement of Sec61 in antigen translocation was provided by experiments, in which Sec61 expression was down-regulated by siRNA (Imai et al., 2005 ). Such down-regulation prevented cytosolic translocation of the antigen and hence its degradation by the cytosolic proteasome, further supporting an important role of Sec61. However, since Sec61 also interferes with dislocation of MHC I molecules (Wiertz et al., 1996 ), it cannot be fully excluded that reduced cross-presentation observed in this study was due to an altered expression of MHC I molecules. Therefore, the exact role of Sec61 in antigen translocation for cross-presentation could not be unequivocally determined yet. Additionally, other members of the ERAD machinery, like derlin-1, have also been proposed as candidates to build the pore complex for antigen translocation across the endosomal membrane during cross-presentation (Lilley and Ploegh, 2004 ; Ye et al., 2004 ). But as for Sec61, future experiments are needed to reveal a decisive role of these proteins in intracellular antigen transport.

Another member of the ERAD machinery, which has been shown to play an important role in cross-presentation, is the soluble AAA ATPase p97 (Ackerman et al., 2006 ). P97, which is associated with both Sec61 and derlin-1, is recruited toward the endosomal membrane, where its ATPase activity provides the energy for antigen translocation. Expression of a dominant negative mutant of p97 has been demonstrated to abolish antigen translocation into the cytoplasm and hence cross-presentation (Ackerman et al., 2006 ; Zehner et al., 2011 ).

Recent evidence indicates that also Igtp, a protein involved in the generation of lipid bodies, influences antigen cross-presentation (Bougneres et al., 2009 ). Since Igtp deficiency abolishes cross-presentation but not MHC I-restricted presentation of antigens that were introduced directly into the cytoplasm, Igtp has been postulated as a putative regulator of antigen transport into the cytoplasm (Desjardins, 2009 ). Whether Igtp and/or lipid bodies indeed play a role in intracellular antigen translocation, however, remains to be elucidated.

After antigen translocation into the cytoplasm, it becomes ubiquitinated and processed by the cytoplasmic proteasome (Kovacsovics-Bankowski and Rock, 1995 ; Ackerman et al., 2003 ; Palmowski et al., 2006 ; Burgdorf et al., 2007 , 2008 ). Importantly, the proteasome constitution in DCs differs from most other cell types. Within DCs, the standard catalytic subunits β1, β2, and β5 are replaced by β1i/LMP2, β2i/MECL-1, and β5i/LMP7 to build the immunoproteasome (Macagno et al., 1999 ). Such immunoproteasomes display an altered protease activity and cleavage site preference, resulting in the more efficient generation of MHC I epitopes (Kloetzel and Ossendorp, 2004 ) and the more efficient degradation of poly-ubiquitinated proteins (Seifert et al., 2010 ). Recent studies additionally reported of proteasomes intermediate between constitutive proteasomes and immunoproteasomes, in which only one or two catalytic subunits were replaced and which displayed an additional cleavage specificity (Guillaume et al., 2010 ), even enlarging the repertoire of antigens presented on MHC I molecules. The constitutive expression of such immunoproteasomes provides DCs with a unique capacity to generate a broad spectrum of peptides for loading on MHC I molecules.

Since antigens intended for cross-presentation generally enter the DC as proteasome substrates (Norbury et al., 2004 ), the half-life of the antigen is of crucial importance and epitopes that are degraded shortly after their synthesis are cross-presented very poorly (Wolkers et al., 2004 ).

Loading of antigen-derived peptides on MHC I molecules

Subsequent to proteasomal degradation, cross-presentation requires functional TAP activity (Kovacsovics-Bankowski and Rock, 1995 ; Huang et al., 1996 ; Song and Harding, 1996 ; Norbury et al., 1997 ; Ackerman et al., 2003 , 2006 ), which led to the hypothesis that proteasome-derived peptides might enter the classical MHC I loading pathway in the ER (Kovacsovics-Bankowski and Rock, 1995 ). Although direct evidence supporting this hypothesis is missing, it was broadly accepted for years. First evidence that peptide loading for cross-presentation might occur in cellular compartments distinct from the ER was based on the observation that antigen-containing phagosomes contain members of the MHC I loading machinery such as calreticulin, ERp57, tapasin, β2-microglobulin, Sec61, MHC I itself, and functional TAP (Ackerman et al., 2003 ; Guermonprez et al., 2003 ; Houde et al., 2003 ). These observations lead to the assumption that proteasome-derived peptides might be re-imported into the same phagosomal compartment for loading onto MHC I molecules there. Indeed, after TAP-mediated peptide transport into these phagosomes, the generation of peptide-loaded MHC I molecules within these phagosomes could be detected (Guermonprez et al., 2003 ). Such intra-phagosomal peptide loading was further accomplished by the recruitment of proteasomes toward the phagosomal membrane (Houde et al., 2003 ), providing a spatial proximity of all components of the cross-presentation machinery, which might be essential to minimize rapid degradation of proteasome-derived peptides with very limited half-life (Reits et al., 2003 ) by cytosolic peptidases.

Formal evidence that peptide loading for cross-presentation indeed takes places in antigen-containing endosomes came from experiments that were aimed at inhibiting TAP activity in an endosome-specific fashion (Burgdorf et al., 2008 ). In this study, the soluble TAP inhibitor US6 (Ackerman et al., 2003 ), which inhibits TAP activity from its luminal side, was covalently linked to transferrin, resulting in its specific targeting to antigen-containing endosomes. Such endosome-specific targeting abolished TAP activity in endosomes without affecting TAP activity in the ER. By this approach, it was demonstrated that endosomal TAP was absolutely required for cross-presentation and that peptide loading for cross-presentation of MR-internalized antigens does not take place in the ER but occurs in antigen-containing endosomes. Such a spatial separation of endogenous MHC I-restricted antigen presentation and cross-presentation further supports the notion of a strong compartmentalization of MHC I-restricted antigen presentation (Lev et al., 2010 ), although peptide loading in the ER under certain circumstances cannot be excluded.

After proteasomal degradation and TAP-mediated transport into endosomal compartments, antigen-derived peptides must be trimmed into the suitable size for optimal binding to MHC I molecules, a function that is exerted in the endogenous MHC I presentation pathway by the ER-resident peptidase ERAP. Recent work by the group of Peter Van Endert identified IRAP as an endosome-specific peptidase required for such peptide-trimming in cross-presentation (Saveanu et al., 2009 ). IRAP, which is specifically targeted toward endosomes by its amino-terminal cytoplasmic tail (Hou et al., 2006 ), displays a broader pH optimum compared to ERAP, allowing IRAP activity at a slightly acidic endosomal pH (Georgiadou et al., 2010 ). IRAP activity might ensure that antigen-derived peptides, which were generated by proteasomal degradation in the cytoplasm and re-imported into the endosomes by endosomal TAP, are trimmed to their optimal size for loading on MHC I molecules, providing potent cross-presentation by the phagosome-to-cytosol pathway.

Cross-presentation via gap junctions-mediated peptide transfer

In addition to the vacuolar and the phagosome-to-cytosol cross-presentation pathway, it has been demonstrated that DCs can obtain peptides from other cells by gap junctions-mediated cell–cell contact (Neijssen et al., 2005 ; Mendoza-Naranjo et al., 2007 ). Such peptides are though to directly enter the endogenous MHC I-restricted presentation pathway via TAP-mediated transport into the ER. Saccheri et al. ( 2010 ) demonstrated that infection of melanoma cells with salmonella induced an upregulation of Cx43, which increased the formation of gap junctions with DCs. These gap junctions enabled peptide transfer from the melanoma cell toward the DC, which resulted in the induction of an anti-melanoma immune response (Neijssen et al., 2005 ; Mendoza-Naranjo et al., 2007 ; Saccheri et al., 2010 ). Whether cross-presentation via gap junctions-mediated antigen transfer has a broad physiological relevance, however, remains unclear, especially because cytoplasmic peptides are rapidly degraded by cytosolic peptidases and display a half-life of only a few seconds (Reits et al., 2003 ).

Cross-dressing of DCs with peptide–MHC I-complexes

Independent of cross-presentation of internalized and processed antigens, DCs can also acquire MHC I molecules that are already loaded with antigen-derived peptides from a donor cell, a process that has been termed cross-dressing (Dolan et al., 2006 ; Smyth et al., 2008 ). Within this process, the antigen-presenting cell can obtain peptide–MHC I-complexes from a large variety of living or apoptotic donor cells. Presentation of such complexes to antigen-specific T cells does not require further processing by the DC. Transfer of the loaded MHC I molecules has been shown to be mediated by direct cell contact between the DC and the donor cell rather than by transfer of secreted vesicles like exosomes (Dolan et al., 2006 ; Wakim and Bevan, 2011 ). Such transfer occurred even at limited antigen concentrations (Smyth et al., 2008 ) and allows a direct antigen transfer from infected cells to DCs also in vivo (Dolan et al., 2006 ; Wakim and Bevan, 2011 ). The relevance of cross-dressing compared to direct or cross-presentation by DCs in the control of an infection remains to be analyzed further and will be the topic of intensive future investigations.

Influence of DC Maturation on Cross-Presentation and on the Recruitment of MHC Loading Machinery from the ER Toward Endosomes

In the absence of inflammatory stimuli, cross-presentation of internalized antigens, which occurs at moderate efficiency in immature DCs (Burgdorf et al., 2008 ), leads to T cell tolerance. Once the DC becomes activated by the recognition of microbial substances, its cross-presentation capacities are enhanced. First, in maturing DCs, total antigen uptake is increased (Gil-Torregrosa et al., 2004 ). Additionally, the composition of the ubiquitin–proteasome system is altered during DC maturation (Ebstein et al., 2009 ) and overall proteasomal activity is increased (Gil-Torregrosa et al., 2004 ). Finally, also antigen translocation into the cytoplasm is increased in maturing DCs (Gil-Torregrosa et al., 2004 ). Since such antigen translocation is mediated by proteins derived from the ER, the transport of these proteins toward the endosomes is an important prerequisite for cross-presentation.

A process termed ER-mediated phagocytosis has been postulated to mediate the transport of ER membrane components to endosomal compartments (Gagnon et al., 2002 ). In this process, the ER serves as a membrane donor for the developing phagosome, which then contains fragments of both the ER and the plasma membrane. This model, however, has been discussed controversially (Touret et al., 2005 ) and its physiological significance remains unclear. The same is true for transient fusions between the ER and endosomes after internalization, which has also been proposed to be a putative mechanism for delivery of ER components to endosomal membranes. The existence of such fusion events, however, has never been clearly demonstrated.

Increasing evidence points out that the transport of ER components to endosomes is a process that is controlled very tightly and that is increased during DC maturation. Goldszmid et al. ( 2009 ) demonstrated that the transport of ER components to Toxoplasma gondii -containing phagosomes only occurred if living protozoa were present in the phagosome. Additionally, our group demonstrated that TAP is only transported toward the endosomal membrane upon DC stimulation with LPS and due to the activation of the TLR4–MyD88 signaling pathway (Burgdorf et al., 2008 ). Likewise, the group of Peter Van Endert demonstrated that in unstimulated DCs, TAP is not present in IRAP-containing early endosomes (Saveanu et al., 2009 ). After phagocytosis of yeast cells, however, a clear TAP translocation from the ER toward the IRAP-containing endosomes was observed. Importantly, ERAP was not recruited to the antigen-containing endosomes, but maintained in the ER. This demonstrates that not all ER components are transported toward the endosome, but a process regulated by microbial substances rather induces the transport of only selected ER proteins toward the endosomal membrane. The selectivity of this transport also implicates that these ER components might not be recruited by ER-mediated endocytosis or by transient fusion events between the ER and the endosomes, since such events would result in an equal transport of all ER components toward the antigen-containing endosomes. These results might rather imply that upon stimulation with microbial substances, selective members of the ER undergo a directed ER-to-endosome transport.

Such TLR ligand-mediated transport from the ER toward endosomes reminds very much of the transport of several TLRs themselves. It has been demonstrated that TLR3, TLR7, and TLR9 in unstimulated DCs are localized in the ER (Latz et al., 2004 ). Upon DC stimulation with TLR ligands, however, these receptors are rapidly translocated toward early endosomes, from where their signaling takes place (Kagan et al., 2008 ). It is thinkable that, upon DC stimulation with endotoxin, some ER components involved in antigen presentation are translocated along with the TLRs toward early endosomes, where they can exert their function in cross-presentation. This transport has been shown to occur without passing the golgi and is regulated by the polytopic membrane protein UNC93B1 (Kim et al., 2008 ). Interestingly, a loss of function mutation of UNC93B1 has a severe influence on antigen presentation and in particular cross-presentation (Tabeta et al., 2006 ). Whether this loss of cross-presentation capacity indeed is due to an impaired transport of the cross-presentation machinery toward endosomes upon TLR signaling, however, still needs to be investigated.

Additional to the recruitment of ER components by TLR ligands, the endocytic receptor seems to play an important role for the recruitment of soluble ERAD components. In a recent study, we demonstrated that the MR, which targets its antigens specifically toward cross-presentation as described above, plays an important role in the recruitment of p97 (Zehner et al., 2011 ). During the ERAD process, p97 is recruited toward the ER by binding to poly-ubiquitinated proteins at the ER membrane (Ye et al., 2003 ). The recruitment of p97 toward the endosomal membrane for cross-presentation seems to be regulated in a very similar way. P97 recruitment for cross-presentation of MR-internalized antigens was regulated by ubiquitination of the MR (Zehner et al., 2011 ). Ligand binding to the MR induced poly-ubiquitination of its cytoplasmic tail. Without receptor poly-ubiquitination, no p97 recruitment toward the endosomal membrane took place and antigen transport into the cytoplasm and cross-presentation were impaired. These data demonstrate that the endocytic receptor is not only required for antigen targeting into a suited endosomal compartment for cross-presentation as described above, but also is of decisive importance for the antigen to get out of the endosomal compartment to reach the cytosol for proteasomal degradation.

In conclusion, it is important to emphasize that there are many different roads to cross-presentation. Whether extracellular antigens are cross-presented via the cytosolic or via the phagosome-to-cytosol pathway might be determined by the physiological conditions of both the antigen-presenting cell and the antigen itself or might even vary for different epitopes of the same antigen.

Furthermore, future experiments are needed to fully understand the molecular mechanisms underlying cross-presentation. Of special interest will be the identification of the pore complex that mediated antigen translocation into the cytoplasm, which without any doubt is one of the most important remaining open questions regarding cross-presentation. In this context, it will be important to unequivocally determine the role of Sec61 and the other postulated candidates in building the transmembrane pore complex.

Another question that will be subject of intense research is the transport of members of the MHC I loading machinery from the ER toward antigen-containing endosomes. Future experiments will show whether such components might be transported along with the different TLRs as postulated in this review, providing an explanation for the dependency of efficient cross-presentation on DC activation by TLR ligands.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Acknowledgments

This work is supported by the German Research Foundation grant BU2441/1-1 and consortium SFB645 project C1.

• Ackerman A. L., Giodini A., Cresswell P. (2006). A role for the endoplasmic reticulum protein retrotranslocation machinery during cross-presentation by dendritic cells . Immunity 25 , 607–617 10.1016/j.immuni.2006.08.017 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ackerman A. L., Kyritsis C., Tampe R., Cresswell P. (2003). Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens . Proc. Natl. Acad. Sci. U.S.A. 100 , 12889–12894 10.1073/pnas.1735556100 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bachem A., Guttler S., Hartung E., Ebstein F., Schaefer M., Tannert A., Salama A., Movassaghi K., Opitz C., Mages H. W., Henn V., Kloetzel P. M., Gurka S., Kroczek R. A. (2010). Superior antigen cross-presentation and XCR1 expression define human CD11c+CD141+ cells as homologues of mouse CD8+ dendritic cells . J. Exp. Med. 207 , 1273–1281 10.1084/jem.20100348 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Backer R., van Leeuwen F., Kraal G., den Haan J. M. (2008). CD8- dendritic cells preferentially cross-present Saccharomyces cerevisiae antigens . Eur. J. Immunol. 38 , 370–380 10.1002/eji.200737647 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Baker K., Qiao S. W., Kuo T. T., Aveson V. G., Platzer B., Andersen J. T., Sandlie I., Chen Z., de Haar C., Lencer W. I., Fiebiger E., Blumberg R. S. (2011). Neonatal Fc receptor for IgG (FcRn) regulates cross-presentation of IgG immune complexes by CD8-CD11b+ dendritic cells . Proc. Natl. Acad. Sci. U.S.A. 108 , 9927–9932 10.1073/pnas.1019037108 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bertholet S., Goldszmid R., Morrot A., Debrabant A., Afrin F., Collazo-Custodio C., Houde M., Desjardins M., Sher A., Sacks D. (2006). Leishmania antigens are presented to CD8+T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo . J. Immunol. 177 , 3525–3533 [ PubMed ] [ Google Scholar ]
• Bevan M. J. (1976). Cross-priming for a secondary cytotoxic response to minor H antigens with H-2 congenic cells which do not cross-react in the cytotoxic assay . J. Exp. Med. 143 , 1283–1288 10.1084/jem.143.5.1283 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bonifaz L. C., Bonnyay D. P., Charalambous A., Darguste D. I., Fujii S., Soares H., Brimnes M. K., Moltedo B., Moran T. M., Steinman R. M. (2004). In vivo targeting of antigens to maturing dendritic cells via the DEC-205 receptor improves T cell vaccination . J. Exp. Med. 199 , 815–824 10.1084/jem.20032220 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Bougneres L., Helft J., Tiwari S., Vargas P., Chang B. H., Chan L., Campisi L., Lauvau G., Hugues S., Kumar P., Kamphorst A. O., Dumenil A. M., Nussenzweig M., MacMicking J. D., Amigorena S., Guermonprez P. (2009). A role for lipid bodies in the cross-presentation of phagocytosed antigens by MHC class I in dendritic cells . Immunity 31 , 232–244 10.1016/j.immuni.2009.06.022 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Burgdorf S., Kautz A., Bohnert V., Knolle P. A., Kurts C. (2007). Distinct pathways of antigen uptake and intracellular routing in CD4 and CD8 T cell activation . Science 316 , 612–616 10.1126/science.1137971 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Burgdorf S., Scholz C., Kautz A., Tampe R., Kurts C. (2008). Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation . Nat. Immunol. 9 , 558–566 10.1038/ni.1601 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Cella M., Engering A., Pinet V., Pieters J., Lanzavecchia A. (1997). Inflammatory stimuli induce accumulation of MHC class II complexes on dendritic cells . Nature 388 , 782–787 10.1038/42030 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Cella M., Salio M., Sakakibara Y., Langen H., Julkunen I., Lanzavecchia A. (1999). Maturation, activation, and protection of dendritic cells induced by double-stranded RNA . J. Exp. Med. 189 , 821–829 10.1084/jem.189.5.821 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Crozat K., Guiton R., Contreras V., Feuillet V., Dutertre C. A., Ventre E., Vu Manh T. P., Baranek T., Storset A. K., Marvel J., Boudinot P., Hosmalin A., Schwartz-Cornil I., Dalod M. (2010). The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8alpha+ dendritic cells . J. Exp. Med. 207 , 1283–1292 10.1084/jem.20100223 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dasgupta S., Navarrete A. M., Bayry J., Delignat S., Wootla B., Andre S., Christophe O., Nascimbeni M., Jacquemin M., Martinez-Pomares L., Geijtenbeek T. B., Moris A., Saint-Remy J. M., Kazatchkine M. D., Kaveri S. V., Lacroix-Desmazes S. (2007). A role for exposed mannosylations in presentation of human therapeutic self-proteins to CD4+ T lymphocytes . Proc. Natl. Acad. Sci. U.S.A. 104 , 8965–8970 10.1073/pnas.0702120104 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Delamarre L., Pack M., Chang H., Mellman I., Trombetta E. S. (2005). Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate . Science 307 , 1630–1634 10.1126/science.1108003 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• den Haan J. M., Bevan M. J. (2001). Antigen presentation to CD8+ T cells: cross-priming in infectious diseases . Curr. Opin. Immunol. 13 , 437–441 10.1016/S0952-7915(00)00238-7 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• den Haan J. M., Lehar S. M., Bevan M. J. (2000). CD8(+) but not CD8(-) dendritic cells cross-prime cytotoxic T cells in vivo . J. Exp. Med. 192 , 1685–1696 10.1084/jem.192.12.1685 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Desjardins M. (2009). The good fat: a link between lipid bodies and antigen cross-presentation . Immunity 31 , 176–178 10.1016/j.immuni.2009.08.003 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Dolan B. P., Gibbs K. D., Jr., Ostrand-Rosenberg S. (2006). Dendritic cells cross-dressed with peptide MHC class I complexes prime CD8+ T cells . J. Immunol. 177 , 6018–6024 [ PubMed ] [ Google Scholar ]
• Dudziak D., Kamphorst A. O., Heidkamp G. F., Buchholz V. R., Trumpfheller C., Yamazaki S., Cheong C., Liu K., Lee H. W., Park C. G., Steinman R. M., Nussenzweig M. C. (2007). Differential antigen processing by dendritic cell subsets in vivo . Science 315 , 107–111 10.1126/science.1136080 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Eberl G., Widmann C., Corradin G. (1996). The functional half-life of H-2Kd-restricted T cell epitopes on living cells . Eur. J. Immunol. 26 , 1993–1999 10.1002/eji.1830261124 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ebstein F., Lange N., Urban S., Seifert U., Kruger E., Kloetzel P. M. (2009). Maturation of human dendritic cells is accompanied by functional remodelling of the ubiquitin-proteasome system . Int. J. Biochem. Cell Biol. 41 , 1205–1215 10.1016/j.biocel.2008.10.023 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Gagnon E., Duclos S., Rondeau C., Chevet E., Cameron P. H., Steele-Mortimer O., Paiement J., Bergeron J. J., Desjardins M. (2002). Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages . Cell 110 , 119–131 10.1016/S0092-8674(02)00797-3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Georgiadou D., Hearn A., Evnouchidou I., Chroni A., Leondiadis L., York I. A., Rock K. L., Stratikos E. (2010). Placental leucine aminopeptidase efficiently generates mature antigenic peptides in vitro but in patterns distinct from endoplasmic reticulum aminopeptidase 1 . J. Immunol. 185 , 1584–1592 10.4049/jimmunol.0902502 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Gil-Torregrosa B. C., Lennon-Dumenil A. M., Kessler B., Guermonprez P., Ploegh H. L., Fruci D., van Endert P., Amigorena S. (2004). Control of cross-presentation during dendritic cell maturation . Eur. J. Immunol. 34 , 398–407 10.1002/eji.200324508 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Giodini A., Cresswell P. (2008). Hsp90-mediated cytosolic refolding of exogenous proteins internalized by dendritic cells . EMBO J. 27 , 201–211 10.1038/sj.emboj.7601941 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Giodini A., Rahner C., Cresswell P. (2009). Receptor-mediated phagocytosis elicits cross-presentation in nonprofessional antigen-presenting cells . Proc. Natl. Acad. Sci. U.S.A. 106 , 3324–3329 10.1073/pnas.0813305106 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Goldszmid R. S., Coppens I., Lev A., Caspar P., Mellman I., Sher A. (2009). Host ER-parasitophorous vacuole interaction provides a route of entry for antigen cross-presentation in Toxoplasma gondii -infected dendritic cells . J. Exp. Med. 206 , 399–410 10.1084/jem.20082108 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Guermonprez P., Saveanu L., Kleijmeer M., Davoust J., Van Endert P., Amigorena S. (2003). ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells . Nature 425 , 397–402 10.1038/nature01911 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Guillaume B., Chapiro J., Stroobant V., Colau D., Van Holle B., Parvizi G., Bousquet-Dubouch M. P., Theate I., Parmentier N., Van den Eynde B. J. (2010). Two abundant proteasome subtypes that uniquely process some antigens presented by HLA class I molecules . Proc. Natl. Acad. Sci. U.S.A. 107 , 18599–18604 10.1073/pnas.1009778107 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hamman B. D., Chen J. C., Johnson E. E., Johnson A. E. (1997). The aqueous pore through the translocon has a diameter of 40-60 A during cotranslational protein translocation at the ER membrane . Cell 89 , 535–544 10.1016/S0092-8674(00)80235-4 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• He L. Z., Crocker A., Lee J., Mendoza-Ramirez J., Wang X. T., Vitale L. A., O’Neill T., Petromilli C., Zhang H. F., Lopez J., Rohrer D., Keler T., Clynes R. (2007). Antigenic targeting of the human mannose receptor induces tumor immunity . J. Immunol. 178 , 6259–6267 [ PubMed ] [ Google Scholar ]
• Heath W. R., Carbone F. R. (2001). Cross-presentation in viral immunity and self-tolerance . Nat. Rev. Immunol. 1 , 126–134 10.1038/35100512 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Herre J., Marshall A. S., Caron E., Edwards A. D., Williams D. L., Schweighoffer E., Tybulewicz V., Reis e Sousa C., Gordon S., Brown G. D. (2004). Dectin-1 uses novel mechanisms for yeast phagocytosis in macrophages . Blood 104 , 4038–4045 10.1182/blood-2004-03-1140 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hildner K., Edelson B. T., Purtha W. E., Diamond M., Matsushita H., Kohyama M., Calderon B., Schraml B. U., Unanue E. R., Diamond M. S., Schreiber R. D., Murphy T. L., Murphy K. M. (2008). Batf3 deficiency reveals a critical role for CD8alpha+ dendritic cells in cytotoxic T cell immunity . Science 322 , 1097–1100 10.1126/science.1164206 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hotta C., Fujimaki H., Yoshinari M., Nakazawa M., Minami M. (2006). The delivery of an antigen from the endocytic compartment into the cytosol for cross-presentation is restricted to early immature dendritic cells . Immunology 117 , 97–107 10.1111/j.1365-2567.2005.02270.x [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Hou J. C., Suzuki N., Pessin J. E., Watson R. T. (2006). A specific dileucine motif is required for the GGA-dependent entry of newly synthesized insulin-responsive aminopeptidase into the insulin-responsive compartment . J. Biol. Chem. 281 , 33457–33466 10.1074/jbc.M608853200 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Houde M., Bertholet S., Gagnon E., Brunet S., Goyette G., Laplante A., Princiotta M. F., Thibault P., Sacks D., Desjardins M. (2003). Phagosomes are competent organelles for antigen cross-presentation . Nature 425 , 402–406 10.1038/nature01912 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Huang A. Y., Bruce A. T., Pardoll D. M., Levitsky H. I. (1996). In vivo cross-priming of MHC class I-restricted antigens requires the TAP transporter . Immunity 4 , 349–355 10.1016/S1074-7613(00)80248-4 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Huang A. Y., Golumbek P., Ahmadzadeh M., Jaffee E., Pardoll D., Levitsky H. (1994). Role of bone marrow-derived cells in presenting MHC class I-restricted tumor antigens . Science 264 , 961–965 10.1126/science.7513904 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Imai J., Hasegawa H., Maruya M., Koyasu S., Yahara I. (2005). Exogenous antigens are processed through the endoplasmic reticulum-associated degradation (ERAD) in cross-presentation by dendritic cells . Int. Immunol. 17 , 45–53 10.1093/intimm/dxh184 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Jongbloed S. L., Kassianos A. J., McDonald K. J., Clark G. J., Ju X., Angel C. E., Chen C. J., Dunbar P. R., Wadley R. B., Jeet V., Vulink A. J., Hart D. N., Radford K. J. (2010). Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens . J. Exp. Med. 207 , 1247–1260 10.1084/jem.20092140 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kagan J. C., Su T., Horng T., Chow A., Akira S., Medzhitov R. (2008). TRAM couples endocytosis of Toll-like receptor 4 to the induction of interferon-beta . Nat. Immunol. 9 , 361–368 10.1038/ni1569 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kamphorst A. O., Guermonprez P., Dudziak D., Nussenzweig M. C. (2010). Route of antigen uptake differentially impacts presentation by dendritic cells and activated monocytes . J. Immunol. 185 , 3426–3435 10.4049/jimmunol.1001205 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kim Y. M., Brinkmann M. M., Paquet M. E., Ploegh H. L. (2008). UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes . Nature 452 , 234–238 10.1038/nature06726 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kloetzel P. M., Ossendorp F. (2004). Proteasome and peptidase function in MHC-class-I-mediated antigen presentation . Curr. Opin. Immunol. 16 , 76–81 10.1016/j.coi.2003.11.004 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Koopmann J. O., Albring J., Huter E., Bulbuc N., Spee P., Neefjes J., Hammerling G. J., Momburg F. (2000). Export of antigenic peptides from the endoplasmic reticulum intersects with retrograde protein translocation through the Sec61p channel . Immunity 13 , 117–127 10.1016/S1074-7613(00)00013-3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kovacsovics-Bankowski M., Rock K. L. (1995). A phagosome-to-cytosol pathway for exogenous antigens presented on MHC class I molecules . Science 267 , 243–246 10.1126/science.7809629 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kukutsch N. A., Rossner S., Austyn J. M., Schuler G., Ltz M. B. (2000). Formation and kinetics of MHC class I-ovalbumin peptide complexes on immature and mature dendritic cells . J. Invest. Dermatol. 115 , 449–453 10.1046/j.1523-1747.2000.00084.x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Kurts C., Heath W. R., Carbone F. R., Allison J., Miller J. F., Kosaka H. (1996). Constitutive class I-restricted exogenous presentation of self antigens in vivo . J. Exp. Med. 184 , 923–930 10.1084/jem.184.3.923 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Latz E., Schoenemeyer A., Visintin A., Fitzgerald K. A., Monks B. G., Knetter C. F., Lien E., Nilsen N. J., Espevik T., Golenbock D. T. (2004). TLR9 signals after translocating from the ER to CpG DNA in the lysosome . Nat. Immunol. 5 , 190–198 10.1038/ni1028 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lev A., Princiotta M. F., Zanker D., Takeda K., Gibbs J. S., Kumagai C., Waffarn E., Dolan B. P., Burgevin A., Van Endert P., Chen W., Bennink J. R., Yewdell J. W. (2010). Compartmentalized MHC class I antigen processing enhances immunosurveillance by circumventing the law of mass action . Proc. Natl. Acad. Sci. U.S.A. 107 , 6964–6969 10.1073/pnas.0910997107 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lilley B. N., Ploegh H. L. (2004). A membrane protein required for dislocation of misfolded proteins from the ER . Nature 429 , 834–840 10.1038/nature02592 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Lin M. L., Zhan Y., Villadangos J. A., Lew A. M. (2008). The cell biology of cross-presentation and the role of dendritic cell subsets . Immunol. Cell Biol. 86 , 353–362 10.1038/icb.2008.3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Macagno A., Gilliet M., Sallusto F., Lanzavecchia A., Nestle F. O., Groettrup M. (1999). Dendritic cells up-regulate immunoproteasomes and the proteasome regulator PA28 during maturation . Eur. J. Immunol. 29 , 4037–4042 10.1002/(SICI)1521-4141(199912)29:12<4037::AID-IMMU4037>3.0.CO;2-T [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Mantegazza A. R., Savina A., Vermeulen M., Perez L., Geffner J., Hermine O., Rosenzweig S. D., Faure F., Amigorena S. (2008). NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells . Blood 112 , 4712–4722 10.1182/blood-2008-01-134791 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Martin-Fontecha A., Sebastiani S., Hopken U. E., Uguccioni M., Lipp M., Lanzavecchia A., Sallusto F. (2003). Regulation of dendritic cell migration to the draining lymph node: impact on T lymphocyte traffic and priming . J. Exp. Med. 198 , 615–621 10.1084/jem.20030448 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• McKenzie E. J., Taylor P. R., Stillion R. J., Lucas A. D., Harris J., Gordon S., Martinez-Pomares L. (2007). Mannose receptor expression and function define a new population of murine dendritic cells . J. Immunol. 178 , 4975–4983 [ PubMed ] [ Google Scholar ]
• Mellman I., Steinman R. M. (2001). Dendritic cells: specialized and regulated antigen processing machines . Cell 106 , 255–258 10.1016/S0092-8674(01)00449-4 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Mendoza-Naranjo A., Saez P. J., Johansson C. C., Ramirez M., Mandakovic D., Pereda C., Lopez M. N., Kiessling R., Saez J. C., Salazar-Onfray F. (2007). Functional gap junctions facilitate melanoma antigen transfer and cross-presentation between human dendritic cells . J. Immunol. 178 , 6949–6957 [ PubMed ] [ Google Scholar ]
• Neijssen J., Herberts C., Drijfhout J. W., Reits E., Janssen L., Neefjes J. (2005). Cross-presentation by intercellular peptide transfer through gap junctions . Nature 434 , 83–88 10.1038/nature03290 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Nishi T., Forgac M. (2002). The vacuolar (H+)-ATPases – nature’s most versatile proton pumps . Nat. Rev. Mol. Cell Biol. 3 , 94–103 10.1038/nrm729 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Norbury C. C., Basta S., Donohue K. B., Tscharke D. C., Princiotta M. F., Berglund P., Gibbs J., Bennink J. R., Yewdell J. W. (2004). CD8+ T cell cross-priming via transfer of proteasome substrates . Science 304 , 1318–1321 10.1126/science.1096378 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Norbury C. C., Chambers B. J., Prescott A. R., Ljunggren H. G., Watts C. (1997). Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells . Eur. J. Immunol. 27 , 280–288 10.1002/eji.1830270141 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Palmowski M. J., Gileadi U., Salio M., Gallimore A., Millrain M., James E., Addey C., Scott D., Dyson J., Simpson E., Cerundolo V. (2006). Role of immunoproteasomes in cross-presentation . J. Immunol. 177 , 983–990 [ PubMed ] [ Google Scholar ]
• Pooley J. L., Heath W. R., Shortman K. (2001). Cutting edge: intravenous soluble antigen is presented to CD4 T cells by CD8- dendritic cells, but cross-presented to CD8 T cells by CD8+ dendritic cells . J. Immunol. 166 , 5327–5330 [ PubMed ] [ Google Scholar ]
• Poulin L. F., Salio M., Griessinger E., Anjos-Afonso F., Craciun L., Chen J. L., Keller A. M., Joffre O., Zelenay S., Nye E., Le Moine A., Faure F., Donckier V., Sancho D., Cerundolo V., Bonnet D., Reis e Sousa C. (2010). Characterization of human DNGR-1+ BDCA3+ leukocytes as putative equivalents of mouse CD8alpha+ dendritic cells . J. Exp. Med. 207 , 1261–1271 10.1084/jem.20092618 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Purcell A. W., Elliott T. (2008). Molecular machinations of the MHC-I peptide loading complex . Curr. Opin. Immunol. 20 , 75–81 10.1016/j.coi.2007.12.005 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Reits E., Griekspoor A., Neijssen J., Groothuis T., Jalink K., van Veelen P., Janssen H., Calafat J., Drijfhout J. W., Neefjes J. (2003). Peptide diffusion, protection, and degradation in nuclear and cytoplasmic compartments before antigen presentation by MHC class I . Immunity 18 , 97–108 10.1016/S1074-7613(02)00511-3 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rescigno M., Citterio S., Thery C., Rittig M., Medaglini D., Pozzi G., Amigorena S., Ricciardi-Castagnoli P. (1998). Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells . Proc. Natl. Acad. Sci. U.S.A. 95 , 5229–5234 10.1073/pnas.95.9.5229 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rock K. L., Shen L. (2005). Cross-presentation: underlying mechanisms and role in immune surveillance . Immunol. Rev. 207 , 166–183 10.1111/j.0105-2896.2005.00301.x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Rodriguez A., Regnault A., Kleijmeer M., Ricciardi-Castagnoli P., Amigorena S. (1999). Selective transport of internalized antigens to the cytosol for MHC class I presentation in dendritic cells . Nat. Cell Biol. 1 , 362–368 10.1038/14058 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Saccheri F., Pozzi C., Avogadri F., Barozzi S., Faretta M., Fusi P., Rescigno M. (2010). Bacteria-induced gap junctions in tumors favor antigen cross-presentation and antitumor immunity . Sci. Transl. Med. 2 , 44–57 10.1126/scitranslmed.3000739 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Saveanu L., Carroll O., Weimershaus M., Guermonprez P., Firat E., Lindo V., Greer F., Davoust J., Kratzer R., Keller S. R., Niedermann G., van Endert P. (2009). IRAP identifies an endosomal compartment required for MHC class I cross-presentation . Science 325 , 213–217 10.1126/science.1172845 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Savina A., Jancic C., Hugues S., Guermonprez P., Vargas P., Moura I. C., Lennon-Dumenil A. M., Seabra M. C., Raposo G., Amigorena S. (2006). NOX2 controls phagosomal pH to regulate antigen processing during crosspresentation by dendritic cells . Cell 126 , 205–218 10.1016/j.cell.2006.05.035 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Savina A., Peres A., Cebrian I., Carmo N., Moita C., Hacohen N., Moita L. F., Amigorena S. (2009). The small GTPase Rac2 controls phagosomal alkalinization and antigen crosspresentation selectively in CD8(+) dendritic cells . Immunity 30 , 544–555 10.1016/j.immuni.2009.01.013 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Schnorrer P., Behrens G. M., Wilson N. S., Pooley J. L., Smith C. M., El-Sukkari D., Davey G., Kupresanin F., Li M., Maraskovsky E., Belz G. T., Carbone F. R., Shortman K., Heath W. R., Villadangos J. A. (2006). The dominant role of CD8+ dendritic cells in cross-presentation is not dictated by antigen capture . Proc. Natl. Acad. Sci. U.S.A. 103 , 10729–10734 10.1073/pnas.0601956103 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Segura E., Villadangos J. A. (2011). A modular and combinatorial view of the antigen cross-presentation pathway in dendritic cells . Traffic 12 , 1677–1685 10.1111/j.1600-0854.2011.01254.x [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Seifert U., Bialy L. P., Ebstein F., Bech-Otschir D., Voigt A., Schroter F., Prozorovski T., Lange N., Steffen J., Rieger M., Kuckelkorn U., Aktas O., Kloetzel P. M., Krüger E. (2010). Immunoproteasomes preserve protein homeostasis upon interferon-induced oxidative stress . Cell 142 , 613–624 10.1016/j.cell.2010.07.036 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Shen L., Sigal L. J., Boes M., Rock K. L. (2004). Important role of cathepsin S in generating peptides for TAP-independent MHC class I crosspresentation in vivo . Immunity 21 , 155–165 10.1016/j.immuni.2004.07.004 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Shimada K., Takimoto H., Yano I., Kumazawa Y. (2006). Involvement of mannose receptor in glycopeptidolipid-mediated inhibition of phagosome-lysosome fusion . Microbiol. Immunol. 50 , 243–251 [ PubMed ] [ Google Scholar ]
• Sigal L. J., Crotty S., Andino R., Rock K. L. (1999). Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen . Nature 398 , 77–80 10.1038/18038 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Smyth L. A., Harker N., Turnbull W., El-Doueik H., Klavinskis L., Kioussis D., Lombardi G., Lechler R. (2008). The relative efficiency of acquisition of MHC: peptide complexes and cross-presentation depends on dendritic cell type . J. Immunol. 181 , 3212–3220 [ PubMed ] [ Google Scholar ]
• Song R., Harding C. V. (1996). Roles of proteasomes, transporter for antigen presentation (TAP), and beta 2-microglobulin in the processing of bacterial or particulate antigens via an alternate class I MHC processing pathway . J. Immunol. 156 , 4182–4190 [ PubMed ] [ Google Scholar ]
• Sweet L., Singh P. P., Azad A. K., Rajaram M. V., Schlesinger L. S., Schorey J. S. (2010). Mannose receptor-dependent delay in phagosome maturation by Mycobacterium avium glycopeptidolipids . Infect. Immun. 78 , 518–526 10.1128/IAI.00257-09 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Tabeta K., Hoebe K., Janssen E. M., Du X., Georgel P., Crozat K., Mudd S., Mann N., Sovath S., Goode J., Shamel L., Herskovits A. A., Portnoy D. A., Cooke M., Tarantino L. M., Wiltshire T., Steinberg B. E., Grinstein S., Beutler B. (2006). The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9 . Nat. Immunol. 7 , 156–164 10.1038/ni1297 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Tacken P. J., de Vries I. J., Gijzen K., Joosten B., Wu D., Rother R. P., Faas S. J., Punt C. J., Torensma R., Adema G. J., Figdor C. G. (2005). Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody . Blood 106 , 1278–1285 10.1182/blood-2005-01-0318 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Tacken P. J., Ginter W., Berod L., Cruz L. J., Joosten B., Sparwasser T., Figdor C. G., Cambi A. (2011). Targeting DC-SIGN via its neck region leads to prolonged antigen residence in early endosomes, delayed lysosomal degradation, and cross-presentation . Blood 118 , 4111–4119 10.1182/blood-2011-07-367615 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Touret N., Paroutis P., Terebiznik M., Harrison R. E., Trombetta S., Pypaert M., Chow A., Jiang A., Shaw J., Yip C., Moore H. P., van der Wel N., Houben D., Peters P. J., de Chastellier C., Mellman I., Grinstein S. (2005). Quantitative and dynamic assessment of the contribution of the ER to phagosome formation . Cell 123 , 157–170 10.1016/j.cell.2005.08.018 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Trombetta E. S., Ebersold M., Garrett W., Pypaert M., Mellman I. (2003). Activation of lysosomal function during dendritic cell maturation . Science 299 , 1400–1403 10.1126/science.1080106 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Tucker S. C., Casadevall A. (2002). Replication of Cryptococcus neoformans in macrophages is accompanied by phagosomal permeabilization and accumulation of vesicles containing polysaccharide in the cytoplasm . Proc. Natl. Acad. Sci. U.S.A. 99 , 3165–3170 10.1073/pnas.052702799 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Van den Berg B., Clemons W. M., Jr., Collinson I., Modis Y., Hartmann E., Harrison S. C., Rapoport T. A. (2004). X-ray structure of a protein-conducting channel . Nature 427 , 36–44 10.1038/nature02218 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• van Montfoort N., Camps M. G., Khan S., Filippov D. V., Weterings J. J., Griffith J. M., Geuze H. J., van Hall T., Verbeek J. S., Melief C. J., Ossendorp F. (2009). Antigen storage compartments in mature dendritic cells facilitate prolonged cytotoxic T lymphocyte cross-priming capacity . Proc. Natl. Acad. Sci. U.S.A. 106 , 6730–6735 10.1073/pnas.0900969106 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wakim L. M., Bevan M. J. (2011). Cross-dressed dendritic cells drive memory CD8+ T-cell activation after viral infection . Nature 471 , 629–632 10.1038/nature09863 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Weck M. M., Appel S., Werth D., Sinzger C., Bringmann A., Grunebach F., Brossart P. (2008). hDectin-1 is involved in uptake and cross-presentation of cellular antigens . Blood 111 , 4264–4272 10.1182/blood-2006-10-051375 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Werneburg N. W., Guicciardi M. E., Bronk S. F., Gores G. J. (2002). Tumor necrosis factor-alpha-associated lysosomal permeabilization is cathepsin B dependent . Am. J. Physiol. Gastrointest. Liver Physiol. 283 , G947–G956 [ PubMed ] [ Google Scholar ]
• Wiertz E. J., Tortorella D., Bogyo M., Yu J., Mothes W., Jones T. R., Rapoport T. A., Ploegh H. L. (1996). Sec61-mediated transfer of a membrane protein from the endoplasmic reticulum to the proteasome for destruction . Nature 384 , 432–438 10.1038/384432a0 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Wolkers M. C., Brouwenstijn N., Bakker A. H., Toebes M., Schumacher T. N. (2004). Antigen bias in T cell cross-priming . Science 304 , 1314–1317 10.1126/science.1096268 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ye Y., Meyer H. H., Rapoport T. A. (2003). Function of the p97-Ufd1-Npl4 complex in retrotranslocation from the ER to the cytosol: dual recognition of nonubiquitinated polypeptide segments and polyubiquitin chains . J. Cell Biol. 162 , 71–84 10.1083/jcb.200302169 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Ye Y., Shibata Y., Yun C., Ron D., Rapoport T. A. (2004). A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol . Nature 429 , 841–847 10.1038/nature02656 [ PubMed ] [ CrossRef ] [ Google Scholar ]
• Zehner M., Chasan A. I., Schuette V., Embgenbroich M., Quast T., Kolanus W., Burgdorf S. (2011). Mannose receptor polyubiquitination regulates endosomal recruitment of p97 and cytosolic antigen translocation for cross-presentation . Proc. Natl. Acad. Sci. U.S.A. 108 , 9933–9938 10.1073/pnas.1102397108 [ PMC free article ] [ PubMed ] [ CrossRef ] [ Google Scholar ]

ORIGINAL RESEARCH article

The purinergic receptor p2x7 as a modulator of viral vector-mediated antigen cross-presentation.

• 1 Institute of Virology, Universitätsklinikum Düsseldorf, Düsselorf, Germany
• 2 Institute of Molecular Medicine II, Universitätsklinikum Düsseldorf, Düsseldorf, Germany
• 3 Department of Medical Sciences, University of Ferrara, Ferrara, Italy
• 4 Institute of Infection Immunity, University of Utrecht, Utrecht, Netherlands
• 5 Biological and Medical Research Center (BMFZ), Medical Faculty, Heinrich-Heine-University, Düsseldorf, Germany

Introduction: Modified Vaccinia Virus Ankara (MVA) is a safe vaccine vector inducing long- lasting and potent immune responses. MVA-mediated CD8 + T cell responses are optimally induced, if both, direct- and cross-presentation of viral or recombinant antigens by dendritic cells are contributing.

Methods: To improve the adaptive immune responses, we investigated the role of the purinergic receptor P2X7 (P2RX7) in MVA-infected feeder cells as a modulator of cross-presentation by non-infected dendritic cells. The infected feeder cells serve as source of antigen and provide signals that help to attract dendritic cells for antigen take up and to license these cells for cross-presentation.

Results: We demonstrate that presence of an active P2RX7 in major histocompatibility complex (MHC) class I (MHCI) mismatched feeder cells significantly enhanced MVA-mediated antigen cross-presentation. This was partly regulated by P2RX7-specific processes, such as the increased availability of extracellular particles as well as the altered cellular energy metabolism by mitochondria in the feeder cells. Furthermore, functional P2RX7 in feeder cells resulted in a delayed but also prolonged antigen expression after infection.

Discussion: We conclude that a combination of the above mentioned P2RX7-depending processes leads to significantly increased T cell activation via cross- presentation of MVA-derived antigens. To this day, P2RX7 has been mostly investigated in regards to neuroinflammatory diseases and cancer progression. However, we report for the first time the crucial role of P2RX7 for antigen- specific T cell immunity in a viral infection model.

Introduction

The P2X7 receptor (P2RX7) belongs to the ionotropic purinergic P2X subfamily and is mostly expressed in immune, endothelial and epithelial cells ( 1 , 2 ). High concentrations of adenosine triphosphate (ATP) are known to activate the ion channel as a danger-associated molecular pattern (DAMP), leading to the intracellular increase of Na + and Ca 2+ and the efflux of K + . P2RX7 has been shown to be crucial for the regulation of various signaling pathways, such as the inflammasome pathway or those that lead to the release of cytokines, cell death or mitochondrial activation ( 3 – 6 ). Next to its contribution to various pathological diseases, P2RX7 activation is also associated with the release of extracellular vesicles (EVs) or particles (EPs) ( 7 – 9 ). They contain various immunostimulatory molecules known to be pivotal for the activation of antigen presentation processes ( 10 , 11 ). Recently, functional P2RX7 expression has been linked to increased viral loads of human herpes virus 6A ( 12 ). Furthermore, data from our lab ( 13 , 14 ) indicate increased expression of the P2X7 receptor during infection with Modified Vaccinia Virus Ankara (MVA).

MVA is a highly attenuated double-stranded DNA virus belonging to the family of the Poxviridiae and the genus Orthopoxvirus ( 15 – 17 ). For the generation of MVA, the parental strain Chorioallantois Vaccinia Virus (CVA) was passaged over 570 times in chicken embryo fibroblasts, leading to six large deletions in the MVA genome and the inability to replicate in most mammalian cells ( 17 – 20 ). Since MVA fails to generate infectious particles in humans, it has been developed as a suitable vector for vaccine design ( 21 – 23 ). It is able to express a large amount of recombinant DNA, and it induces strong humoral and cellular immune responses upon vaccination ( 22 , 24 ).

Interestingly, robust and long-lived cytotoxic T cell (CTL) immunity is dependent on cross-presentation during MVA infection ( 25 , 26 ). Upon infection with MVA, cells undergo apoptosis, containing and releasing antigens to be phagocytosed by professional antigen-presenting cells (APCs) ( 27 , 28 ). Upon internalization of the antigen, two distinct pathways can lead to the loading of MHC class I molecules (MHCI): the vacuolar and the cytosolic antigen-processing pathway ( 29 ). A peptide-MHCI-complex can either be generated by TAP interacting with internalized phagosomes containing the peptide to be processed or by processing already internalized peptides via the endoplasmic reticulum ( 30 ). In the vacuolar pathway, the processing and loading, both will occur in the vacuoles themselves ( 31 ). The preformed MHCI-peptide complex is then released and exported to the cell surface where CTL can be activated and release inflammatory cytokines, such as IFNү and TNFα ( 32 ). Since cross-presentation is essential for optimal CD8 + T cell priming for various pathogens as well as vector delivery systems, its molecular regulation has encouraged intense investigations. More evidence suggests that the stimulus for successful cross-presentation does not originate from the non-infected antigen-presenting cell, but rather from the bystanding initially infected cell, which we term feeder cell. We have recently shown that STING in feeder cells is involved in regulating CD8 + T cell responses via type I interferon production acting on the cross-presenting APC ( 33 ).

In this study, we aim to analyze the role of other innate triggers in feeder cells for MVA-induced antigen cross-presentation. The innate immune system serves as the first line of defense once a pathogen is encountered and P2RX7 as a member of the innate system has been shown to be potently activated by extracellular ATP, which is released by different stimuli. ATP is essential during vaccinia virus infection and therefore for the regulation of immune responses ( 34 , 35 ). P2RX7 has been described to be involved in antigen presentation ( 36 ). It alters the secretome in cells bearing the active P2RX7, such as the production of extracellular vesicles that might contain antigens or the production and release of varying inflammatory cytokines and chemokines ( 10 , 37 , 38 ). Additionally, P2RX7 activity has been associated with the expression of Nfatc1 , belonging to the group of primary response genes modulated by immune signals ( 39 – 41 ). Therefore, we investigated the involvement of the P2X7 receptor, as a member of the innate immune system, in infected feeder cells during cross-presentation of MVA-derived antigens.

The regulation of cross-presentation has been intensively studied for years, however, detailed knowledge about the molecular mechanisms that underlie the relevant pathways as well as about the innate triggers to initiate the process in the cross-presenting APC is lacking. In the present study, we aimed to investigate the potential role of P2RX7 as an innate stimulus in infected feeder cells for the initiation and modulation of cross-presentation in the non-infected bystander APC. We show that the ATP-sensitive P2RX7 from the BALB/c strain in feeder cells is essential for enhancing CD8 + T cell responses via cross-presentation in vitro . We show that various P2RX7-dependent pathways that we analyzed and which are crucial for the initiation of immune responses, such as the release of inflammatory cytokines, mRNA and the presence of apoptotic stimuli, were modulated in the presence of functional P2RX7 in infected feeder cells. Our findings suggest that the improved cross-presentation capacity of antigen-presenting cells co-cultured with infected feeder cells bearing active P2RX7 might be due to the activation of several pathways in feeder cells that may act together to orchestrate the immune response. To our knowledge, this is the first report instigating the function of the P2RX7 for regulation of MVA-mediated T cell immunity.

The plasma membrane P2X7 receptor is not functional in Cloudman (CM) cells and reconstitution with active P2RX7 from BALB/c mice enhances the release of extracellular particles after MVA infection

In line with the literature ( 42 ) and our sequencing data ( Supplementary Figure 1A ), the fluorometric analysis of intracellular Ca 2+ influx failed to demonstrate activation of the plasma membrane-located P2RX7 in Cloudman (CM) mock-or MVA-PK1L-Ova infected cells upon Bz-ATP specific stimulus ( Figure 1A ). Although recent New Generation Sequencing analysis ( 14 ) has shown that the expression of the P2X7 receptor was upregulated after MVA infection, qRT-PCR analysis of infected CM cells failed to demonstrate an upregulation of expression but indicated a stable constitutive expression after infection ( Supplementary Figure 1B ). For the matter of simplification, we distinguish between active and inactive P2RX7, referring to the BALB/c P2RX7 or DBA/C57BL/6 P2RX7 (less sensitive to ATP stimuli ( 42 )), respectively. To exclude that the lack of intracellular Ca 2+ increase was due to faulty loading of the fluorescent indicator FURA-2-AM, we additionally stimulated the cells with ionomycin, which is a receptor-independent trigger for a maximal increase of intracellular Ca 2+ . Even higher Bz-ATP stimuli could not increase intracellular Ca 2+ concentrations ( Supplementary Figure 1C ). Interestingly, when stimulating the CM cells with ATP, known to activate additional purinergic receptors besides P2RX7, we observed a gain in intracellular Ca 2+ amounts, suggesting the activity of other receptors of this or the P2Y receptor family ( 43 ) ( Supplementary Figure 1D ). Next, we were interested in studying the role of active P2RX7 and transfected our CM cells with the fully functional P2RX7 expressed by BALB/c mice ( 42 ). We were able to demonstrate its activity after transfection, by an increase in the concentration of intracellular Ca 2+ upon Bz-ATP stimulus ( Figure 1B ). This response was reversed when the reconstituted cells were treated with the P2RX7-specific competitive inhibitor A740003 at 20µM. Transfection with the empty vector control, similar to CM WT cells, did not alter the amount of intracellular Ca 2+ upon Bz-ATP treatment. Toxicity of A740003 was excluded ( Supplementary Figure 1E ). Furthermore, the receptor activity represented by an increased Ca 2+ influx in the P2RX7-transfected cells appeared to be significantly higher at earlier time points during MVA infection after 4 h.p.i. ( Supplementary Figure 1F ), suggesting that the infection itself modulates the activity of the receptor. However, calcium levels did not change after 20h MVA infection when compared to uninfected cells indicating that the receptor activity is only transiently increased after infection ( Figure 1C ). Interestingly, we did not observe P2RX7-specific pore function in P2RX7 transfected cells ( Supplementary Figure 1G ) ( 44 ). The release of extracellular particles, which is reported to be partly P2RX7 dependent ( 7 ), was enhanced when P2RX7 transfected cells were infected with MVA, supporting the regulation of P2RX7-specific functions during MVA infection ( Figures 1D, E ). Overall, we confirmed the reconstitution and functionality of P2RX7 in CM cells after transfection.

Figure 1 The P2X7 receptor is inactive in Cloudman (CM) cells (DBA background) and transfection with functional P2RX7 from BALB/c mice restores P2RX7-specific functions. (A) Fluorometric analysis of intracellular Ca 2+ (iCa 2+ ) influx as a known marker of P2RX7 activity to demonstrate the function of the plasma membrane-located P2RX7 in CM cells infected with MVA or mock. Activity of P2RX7 in CM wildtype (WT) cells infected either with MVA-PK1l-Ova (20hpi/MOI1) expressing ovalbumin under the control of the vaccinia virus early promoter PK1L or mock-infected was investigated by measurement of intracellular Ca 2+ concentrations of FURA-2-AM loaded cells upon stimulus with 200µM Bz-ATP. (B) CM cells were transfected with empty vector (CM pcDNA3) or P2RX7 containing plasmid DNA. Activity was assessed at least one week post transfection by fluorometric assay. CM P2RX7 cells were additionally pre-treated for 5min with 20µM A740003 (CM P2RX7 A740003), a P2RX7 specific inhibitor. (C) Intracellular calcium concentrations were further tested in CM P2RX7-transfected cells 20h post-MVA infection (MOI1) (CM P2RX7 MVA) or mock-infected cells (CM P2RX7). (D) Quantification of extracellular particles per total number of cells was started after addition of 200µM Bz-ATP for a time frame of approximately 30sec (three subsequent frames) from (E) MVA- (MOI1) or mock-infected CM WT or transfected cells (CM P2RX7 or CM pcDAN3) cells. Cells were stained with quinacrine nucleic acid stain and PKH26 membrane stain and then stimulated with 200µM Bz-ATP to visualize the release of particles using confocal image analysis. Data shown, represent one from at least n=3 (A–C) or n=2 independent experiments (D, E) . Statistical significance (P) ***P ≤ 0.001.

Active P2RX7 in feeder cells promotes MVA antigen cross-presentation

Recent studies have shown that innate triggers derived from infected feeder cells are relevant for the activation of T cells by antigen-presenting cells ( 33 ). We were interested in investigating whether the presence of a functional P2X7 receptor in feeder cells may have an impact on the antigen uptake and presentation capacity of bone marrow-derived dendritic cells (BMDCs) for activation of CD8 + T cells. We demonstrate that using MVA-OVA-infected feeder cells bearing the active P2X7 receptor led to a significantly higher CD8 + T cell activation as determined by IFNү production in B8R- specific T cells or by TNFα production in either B8R- or OVA-specific T cell lines when co-cultured with uninfected dendritic cells as cross-presenting APC ( Figure 2A ). These data are in line with the P2RX7-dependent release of these cytokines in mice ( 45 ). The frequency of cross-presenting BMDCs with SIINFEKL/H2-K b complexes on the cell surface, as well as the amount of these peptide/MHCI complexes per cell, was significantly increased ( Figure 2B left, middle). Interestingly, the presence of the active P2RX7 in CM cells led to the increase of MHCII surface expression in co-cultured BMDCs ( Figure 2B right). The expression of other maturation markers, such as CD40 or CD86 on co-cultured BMDCs was not affected (data not shown). Furthermore, pre-treatment of CM P2RX7 cells with A740003 before co-culture with BMDCs led to a significantly reduced CD8 + T cell activation ( Supplementary Figure 2A ) and SIINFEKL/H2-K b expression ( Supplementary Figure 2B ) which was comparable to CM WT or CM pcDNA3 cells. To corroborate the specific function of P2RX7, we used HEK293 as feeder cells expressing active human P2RX7 ( Figure 2D ). Indeed, the co-incubation of BMDCs with MVA-infected HEK293 hP2RX7 feeder cells resulted in increased SIINFEKL/H2-K b expression ( Figure 2C ). In sum, we demonstrated that the presence of functional P2RX7 in feeder cells aids in improving antigen cross-presentation upon MVA infection.

Figure 2 Antigen cross-presentation is enhanced in the presence of active P2RX7 in CM feeder cells. GM-CSF bone marrow derived-dendritic cells (BMDCs) were co-cultured with CM WT (CM), control plasmid (CM pcDNA3) P2RX7 transfected (CM P2RX7) feeder cells infected with MVA-PK1L-Ova expressing ovalbumin under control of the viral early promoter PK1L (MVA) at MOI1 or mock-infected (Ø). At 20hpi, B8R- or Ova-specific CD8 + T cells were added to the co-culture for 4h. Presence of intracellular activation markers was determined by flow cytometric analysis (FACS) of (A) IFNү (left) or TNFα (right) production. (B) Frequency (left) and mean fluorescent intensity (MFI) (middle) of SIINFEKL/H2-K b surface expression (FACS). MHCII (%) expression in BMDCs (right). (C) SIINFEKL/Kb expression was also assessed in BMDC that had been co-cultured with HEK293 cells (WT or hP2RX7 transfected) infected with either mock (Ø) or MVA-PK1L-Ova (MVA) at MOI1 for 20h. (D) Fluorometric analysis of intracellular calcium concentrations in HEK293 WT (HEK293) and hP2RX7 transfected (HEK293 hP2RX7) cells with or without MVA-PK1L-OVA (MOI1, 20h) infection and 200µM Bz-ATP stimulus. Data are pooled from at least n=3 independent experiments (n=3-5) and shown as means ± SD. P values indicate statistical significance (P) with *P ≤ 0.05 **P ≤ 0.01; ***P ≤ 0.001.

Functional P2RX7 does not alter antigen availability or replication capacity of MVA but impacts the gene expression of viral antigens

We first hypothesized that the increased SIINFEKL/H2-K b surface expression on antigen-presenting cells and the improved CD8 + T cell activation that we found when BMDCs were co-cultured with P2RX7 feeder cells, might be due to an increased amount of viral antigens. We analyzed the expression of early antigens, such as B8R , a native MVA antigen, or Ova , expressed under the control of the early MVA promoter PK1L, or the late viral antigen A19L in CM P2RX7 or CM pcDNA3 transfected cells and compared it to CM WT cells. Expression of these antigens was initially lower in CM P2RX7 cells compared to CM WT or the empty vector control CM pcDNA3. However, at 24hpi B8R , Ova and A19L mRNA fold change in CM P2RX7 cells was significantly higher when compared to CM WT or CM pcDNA3 cells ( Figure 3A ). We concluded that the expression kinetics of viral genes in CM P2RX7 was delayed, although mRNA expression at later time points in these cells was significantly higher. This was further confirmed when analyzing the replication capacity of MVA in the different CM feeder cell lines. We also observed a higher residual viral titer at 0hpi in CM P2RX7 ( Figure 3B ), suggesting a possible role of the P2X7 receptor for viral entry, as previously stated ( 12 ). Since MVA has lost its ability to replicate in most mammalian cells ( 19 ), we wanted to exclude the possibility that the presence of this receptor was affecting viral replication behavior and, as a consequence, promote antigen cross-presentation. However, at 24hpi viral particle amounts in CM P2RX7 cells were comparable to CM WT or CM pcDNA3 cells ( Figure 3B right). Similarly, the synthesis of OVA protein in CM P2RX7 cells was initially reduced at 8hpi but comparable at 20hpi ( Figure 3C ), indicating that expression of viral genes and synthesis of corresponding proteins might be delayed in CM P2RX7 transfected cells.

Figure 3 Expression of active P2RX7 in feeder cells alters several signaling pathways that may be correlated with enhanced antigen availability and cross-presentation by BMDCs. (A) CM WT (CM) or pcDNA3 or P2RX7 transfected cells (CM pcDNA3 or CM P2RX7) were infected with MVA-PK1L-Ova at MOI1 for 0h to 24h to assess the expression of viral antigens B8R and A19L or recombinant antigen Ova, respectively. mRNA fold change is shown as expression of the respective gene at each time point compared to the 0h time point of CM WT cells. (B) Viral growth kinetics of MVA-p7.5-GFP expressing green fluorescent protein (GFP) under control of the viral early/late promoter p7.5. (Left) Wildtype or transfected CM cells were infected for 0h to 24hpi and viral titers were determined by back titration on DF1 cells at the indicated hpi as tissue culture infectious dose (TCID 50 ). (Right) Final viral loads after 24hpi were measured by subtracting viral output at 24hpi with viral input at 0hpi. (C) Western Blot analysis of WT or transfected CM cells upon MVA-PK1L-Ova infection at 8hpi or 20hpi (left). Quantification of OVA (middle) or cleaved CASPASE-8 (right) protein amounts in cellular extracts. (D) Phosphatidylserine residues on the cell surface of mock- (Ø) or MVA-infected (MOI1) WT or transfected CM cells at either 6hpi or 20hpi. Quantification of either APC-negative (APC-) (live/dye non-permissive) (left) or APC-positive (APC+) (dead/dye permissive) (right) within phosphatidylserine positive cells, depicting either early or late apoptotic cells, respectively. Experiments are shown as means with SEM (A) or SD of n=3 independent biological replicates with statistical significance (P) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

The above data exclude that altered protein amounts in feeder cells as the source of antigenic uptake by antigen-presenting cells contributed to the improved antigen presentation by BMDCs co-cultured with infected CM P2RX7 cells. It has been postulated that the antigen has to be released but needs to be still cell-associated for the antigen-presenting cells to phagocytose and process it for cross-presentation to CD8 + T cells ( 46 – 48 ). We confirmed the activation of the extrinsic apoptosis pathway by cleavage of Caspase 8 ( Figure 3C ). The exposure of phosphatidylserine residues on the cell surface is important for the activation of phagocytosis by APCs and has been linked to P2RX7 activation ( 49 – 51 ). We demonstrate that the presence of the functional P2X7 receptor led to the increased surface expression of phosphatidylserine residues in cells having a permeable cell membrane ( Figure 3D right) when mock-infected or infected with MVA for 6h. In contrast, MVA-infected CM P2RX7 with intact cell membrane, hence alive cells, displayed reduced phosphatidylserine levels as compared to CM WT or CM pcDNA3 cells after 20h MVA infection. Overall, we speculate that expression of the functional P2X7 receptor in CM feeder cells does not modulate the antigen-presentation capacity by APCs by increasing the total amount of antigenic protein in feeder cells, but rather allows for increased viral mRNA levels in infected cells at later time points as well as by altering apoptotic pathways. This depicts the importance of further analyses on the RNA level and the possible altered localization of P2RX7 affecting other cellular pathways.

Extracellular particles as well as supernatant released from feeder cells with active P2RX7 promote MVA antigen cross-presentation

Extracellular particles are known to contain crucial regulatory molecules for cell-to-cell communication ( 11 ). Similarly, supernatant from P2RX7 transfected cells differs from control cells ( 37 ). Here we hypothesized that both the extracellular particle fraction (EP-fraction), as well as the supernatant fraction (sup-fraction) from CM P2RX7 cells, are responsible for the enhanced antigen cross-presentation we observed. We first isolated the EP-fraction and the supernatant fraction as depicted in Figure 4A and determined both, mRNA and protein content. OVA protein amounts were comparable in wildtype and transfected CM cells after overnight infection in both, the EP- and the sup-fraction ( Figure 4B ). Interestingly, mRNA levels of the MVA early antigen B8R , but not Ova or A19L expression, were significantly higher in the EP-fraction at 0 hours post-infection leading to the hypothesis that the lower mRNA expression we observed in the cell extracts ( Figure 3A ) might be due to the release of RNA in extracellular particles ( Figure 4D ). This finding highlights the importance of focusing on the role of RNA in the regulation of MVA antigen presentation. We also showed that CM P2RX7 transfected cells allowed the secretion of significantly higher amounts of inflammatory cytokines pre- as well as post-infection ( Figure 4C ), demonstrating that the presence of functional P2RX7 modulates other signaling pathways as well. A detailed overview of the secreted cytokines can be seen in Supplementary Figure 3A . To further investigate the function of these culture sub-fractions from P2RX7 transfected cells, we delivered EP- and sup-fractions to the co-culture of uninfected CM cells and BMDCs to monitor subsequent CD8 + T cell activation and expression of SIINFEKL/H2-K b complexes on dendritic cells. The EP-fraction from CM P2RX7 was not able to induce stronger CD8 + T cell IFNү or TNFα production as compared to the EP-fraction derived from pcDNA3 transfected CM cells. However, the sup-fraction led to increased IFNү and TNFα production when added to the co-culture of CM and BMDCs ( Figure 4F ). Interestingly, both fractions from CM P2RX7 cells were able to induce a significantly higher SIINFEKL/H2-K b expression as compared to the empty vector control-derived fractions ( Figure 4G ). To establish whether these fractions could initiate similar CD8 + T cell activation as the infection of CM P2RX7 cells, these were added to MVA-infected CM WT cells ( Supplementary Figures 3B–E ). Even though both fractions seem to contribute to the increased expression of SIINFEKL/H2-K b on BMDCs, the addition of EPs or supernatants from infected P2RX7 CM cells could not further increase CD8 + T cell activation or SIINFEKL/H2-K b expression on BMDCs significantly. In addition, we filtered supernatants from infected feeder cells to remove any cell components larger than 0.2µM, such as apoptotic bodies (but leaving exosomes and microvesicles in the fraction) and added this filtered supernatant (fil sup) to either uninfected CM or MVA-infected cells ( Figure 4H left, middle). We found a significant contribution of the filtered supernatant from CM P2RX7 cells for the activation of dendritic cells and subsequent presentation of the SIINFEKL peptide on its MHCI. The total amount of cross-presenting BMDCs (frequency) with SIINFEKL/H2-Kb complexes at the cell surface as well as the amount of SIINFEKL/H2-Kb complexes per BMDC (MFI) was significantly increased ( Figure 4H left, middle). Notably, MHC I expression (total H2-K b ) of the antigen-presenting cells was not altered upon the addition of this filtered supernatant ( Figure 4H right). To sum this up, we demonstrate that the cellular fraction of P2RX7 transfected cells as well as other subcellular fractions are critically involved in enhancing antigen cross-presentation.

Figure 4 Subcellular fractions of P2RX7 transfected CM feeder cells enhance MVA-mediated antigen cross-presentation upon co-cultivation with cross-presenting BMDCs. (A) Experimental setup to extract extracellular particle (EP) fraction and supernatant fraction (sup) from infected cells. Figure created in Biorender with permission. (B) Both fractions were analyzed for OVA protein content at 20 hours post MVA-PK1L-Ova infection (MVA) at MOI1.5 (EP) or MOI1 (sup). (C) Expression and release of inflammatory chemokines was assessed in the supernatant fraction of CM, CM pcDNA3 or CM P2RX7 cells that were either mock- or MVA-infected (MOI1 for 20h) by Legendplex assay. (D) mRNA expression. Quantification of Ova (upper) or B8R mRNA (lower) at 0hpi and 20hpi in EP-fractions of MVA-PK1L-OVA infected cells (MOI1.5). (E, F) EP- or sup-fractions from MVA-PK1L-Ova infected CM pcDNA3 or CM P2RX7 cells that were added to the co-culture of uninfected CM WT feeder cells with uninfected BMDCs which were then co-cultured with B8R- or Ova-specific CD8 + T cells for 4h to assess IFNү or TNFα production upon antigen-specific activation. (G) SIINFEKL/H2-K b surface expression of BMDCs was determined after adding either the EP- or the sup-fraction from control (pcDNA3) or P2RX7 transfected (P2RX7) cells to uninfected BMDCs co-cultured with uninfected CM WT (CM) cells. (H) SIINFEKL/H2-K b surface expression of BMDCs by frequency (left) or mean fluorescence intensity (middle) after the addition of filtered supernatants from MVA-infected CM pcDNA3 or CM P2RX7 cells to either mock-infected (Ø) or MVA-PK1L-OVA (MVA) at MOI1 infected CM cells. (Right) MHCI expression (total H2-K b ) of BMDCs was assessed as a control. Plots show the mean of n=3 independent experiments with SD and statistical significance (P) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001. ns, not significant.

The presence of active P2RX7 modulates mitochondrial function

As a plasma membrane receptor, P2RX7 is associated with the activation of the canonical inflammasome pathway ( 52 ). Recent studies, however, show that P2RX7 can also localize to and function intracellularly on mitochondrial structures ( 53 ). We confirmed the presence of intracellular P2X7 receptors in our feeder cells. These were significantly increased in P2RX7 transfected CM cells ( Figure 5A ). Mitochondria and energy metabolism are impacted during viral infection and since the presence of P2RX7 on mitochondrial surfaces of HEK293 hP2RX7 and N13 microglial cells has been shown recently by others ( 53 , 54 ), we determined the mitochondrial activity in our feeder cells. As expected, maximal respiration and spare capacity were significantly increased in CM cells bearing the active P2X7 receptor as compared to CM WT or empty vector-transfected cells. Basal respiration was only increased in CM P2RX7 cells when compared to CM pcDNA3 cells. Also after infection, the maximal respiration and spare capacity were significantly upregulated in CM P2RX7 cells ( Figure 5B ). In addition, the extracellular acidification rate (ECAR), an indicator for glycolysis processes ( 55 ), was significantly higher in both, mock-infected and MVA-infected CM P2RX7 cells, as compared to CM WT or CM pcDNA3 cells ( Figure 5C ). Interestingly, intracellular ATP concentrations in the uninfected CM P2RX7 cells were comparable to CM WT cells but slightly lower than in CM pcDNA3 cells. After infection, however, intracellular ATP was significantly increased in CM P2RX7 cells only ( Figure 5D ), while extracellular ATP inversely correlated after infection showing a significant decrease for CM P2RX7 cells only ( Figure 5E ). In summary, we report that presence of P2RX7 significantly alters the energy metabolism of the cells by increasing the availability of mitochondrial ATP after MVA infection.

Figure 5 Presence of functional P2RX7 affects mitochondrial functions. (A) Intracellular expression of P2RX7. Mean fluorescent intensities (MFI) in mock- (Ø) or MVA-infected (MOI1, 20hpi) (MVA) CM WT (CM), empty vector control (CM pcDNA3) or P2RX7 (CM P2RX7) transfected cells. (B) Mitochondrial activity was assessed by measurement of the oxygen consumption rate (OCR) after treatment of CM WT, CM pcDNA3 or CM P2RX7 transfected cells with modulators of the electron transport chain such as Oligomycin, FCCP, and Rotenone/Antimycin A) (upper graphs) with (right) or without MVA infection (left) according to the Mitostress test kit (Seahorse). (lower graph) Mock-infected or MVA-infected (MOI5 for 6h) CM WT, CM pcDNA3, or CM P2RX7 cells were used to determine basal and maximal respiration as well as spare capacity upon addition of above mentioned modulators. (C) Comparable conditions were used to assess the extracellular acidification rate (ECAR) as an indicator for glycolysis processes in the indicated mock or MVA-infected (MOI5 for 6h) cells. (D) Quantification of intracellular or (E) extracellular ATP in CM WT, CM pcDNA3, or CM P2RX7 transfected cells either mock or MVA-infected (MOI5 for 6h). Data shown are of at least n=3 independent experiments, depicted as means with SD or SEM (D, E) and statistical significance (P) *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. ns, not significant.

Studies have demonstrated the significant role of the P2X7 receptor in response to viral infections, exhibiting both protective and pathological functions ( 56 ). However, the function of P2RX7 during MVA infection and its relevance for antigen cross-presentation has not been investigated yet. Our findings suggest that the presence of an active P2X7 receptor in MVA-infected feeder cells can lead to a strongly increased antigen-presentation capacity by cross-presenting dendritic cells. This highlights a new function of purinergic receptor signaling in feeder cells serving as antigenic source for cross-presentation by dendritic cells.

Before delving into the specific role of the P2X7 receptor, we ensured its lack of function in the CM feeder cells ( Figure 1A , Supplementary Figure 1C ). As expected and as described in the literature, there was no discernible increase in intracellular Ca 2+ in these cells, despite the stimulus with high concentrations of Bz-ATP, an agonist of P2X7 receptors ( 42 , 43 ). Interestingly, when CM cells were treated with ATP, we could observe a desensitizing peak, which was ATP-dependent and strongly indicated the activation of other P2 receptors ( 43 ). However, when we reconstituted CM cells with the wild type, fully ATP-sensitive P2RX7 derived from BALB/c mice, we observed an increase in intracellular Ca 2+ , which was reversible upon treatment with A740003, a P2RX7-specific inhibitor ( 57 ). It also appeared that P2RX7 may play an important role during the initial phase of MVA infection, as infection for 4h resulted in a slight increase in intracellular Ca 2+ values ( Supplementary Figure 1G ), while infection for 20h left intracellular Ca 2+ levels unaffected ( Figure 1C ).

Based on our studies, MVA infection regulates P2RX7-specific functions, since the release of extracellular particless, which has been reported to be at least partly P2RX7-dependent ( 7 , 8 ), was enhanced after MVA infection in the presence of an active P2RX7 ( Figures 1D, E ). We anticipate, however, that wildtype or empty vector-transfected CM cells with inactive P2RX7 would release apoptotic bodies instead of EPs, as MVA has been shown to trigger the initiation of apoptosis pathways in infected cells ( 11 , 27 ). Further CM cell-specific studies are underway to understand the molecular pathways by which P2RX7 signaling modulates EP release mechanisms.

The presence of active P2RX7 in feeder cells during MVA infection led to a dramatic increase in CD8 + T cell activation and enhanced expression of SIINFEKL/H2-K b complexes on murine antigen-presenting cells via cross-presentation ( Figures 2A, B ). This finding was corroborated in human cells by using a human feeder cell line, namely HEK293 cells, expressing a functional human P2RX7, while pre-treatment of CM P2RX7 with the P2RX7-specific inhibitor A740003 abrogated it ( Supplementary Figures 2A, B ). These results provide substantial evidence for the involvement of P2RX7 in feeder cells during MVA infection for antigen cross-presentation by dendritic cells. Since the presence of active P2RX7 in CM cells led to increased MHC II expression in dendritic cells ( Figure 2B right) ( 58 , 59 ), we hypothesize that P2RX7 alters the microenvironment e.g. by secretion of cytokines which stimulate non-infected bystander dendritic cells and has been previously suggested to be relevant for antigen presentation ( 60 ). Expression of P2RX7 has been associated with improved antigen presentation, especially due to the release of P2RX7-dependent extracellular particles containing inflammatory molecules and antigens ( 61 , 62 ). Our data indicates a substantial P2RX7-dependent modulation of the production of MVA-derived antigen in feeder cells and its release or presentation to DCs enhancing MVA-mediated antigen cross-presentation.

Recent studies imply that feeder cells play a crucial role for antigen cross-presentation ( 33 ). In order to better understand this process, we decided to investigate the expression of viral antigens in feeder cells. Interestingly, viral particles were attached to the cell surface, but not internalized in CM P2RX7 cells as demonstrated by the increased viral particle load at 0hpi ( Figure 3B left). In line with this finding, mRNA levels of all antigens tested (viral B8R and A19L as well as recombinant Ova ) were initially lower in CM P2RX7 compared to CM WT cells, but were significantly increased at 20 hpi ( Figure 3A ) indicating that viral antigen expression kinetics is delayed in P2RX7 cells. This altered kinetics was corroborated by western blot analysis of OVA antigen synthesis in infected CM P2RX7 cells compared to the controls ( Figure 3C left and middle). This implies that P2RX7 might play a role for viral entry, as previously described for other viruses such as HHV-6A and HBV/HDV ( 12 , 63 ). Importantly, the MVA replication capacity in feeder cells was not altered in the absence or presence of P2RX7 resulting in comparable viral titers/multiplication rates ( Figure 3B right) ( 19 ), thereby excluding that increased amounts of antigen due to increased viral replication in CM P2RX7 cells altered the cross-presentation capacities of APCs.

APCs require cell-associated antigens to be phagocytosed and processed for antigen cross-presentation ( 28 , 47 ). It has been shown that expression of P2RX7 activates Caspase-8-mediated apoptosis and leads to the exposure of phosphatidylserine at the cell surface ( 64 , 65 ). In fact, our studies demonstrate high expression of active Caspase-8 ( Figure 3C left/right) and an increase of late apoptosis in CM P2RX7 cells at early time (8hpi) after mock or MVA infection ( Figure 3D right) in the presence of P2RX7 thereby enhancing the decoration of the cell with the early apoptotic marker phosphatidylserine. Interestingly, at later time (20hpi), only cells without functional P2RX7 showed expression of phosphatidylserine on the cell surface (early apoptotic) ( Figure 3D ). Phosphatidylserine at the cell surface is required for vaccinia virus including MVA to enter cells ( 66 ). Vaccinia virus entry is facilitated by ‘apoptotic mimicry’, hence by flagging phosphatidylserine on mature virions and identifying it as apoptotic debris for uptake ( 67 ). Additionally, vaccinia virus is able to transfer phosphatidylserine molecules from the lipid bilayer of cell membranes to increase its infectivity ( 68 ), likely because the exposure of phosphatidylserine on the cell surface may act as an ‘eat-me’ signal for phagocytes ( 69 ) which potentially explains the increased expression of the BMDC maturation marker CD40 when co-cultured with uninfected CM P2RX7 cells. EPs, which emerge from the cell membrane, incorporate membrane-specific molecules, including phosphatidylserine ( 11 , 70 ). Rausch and colleagues have proposed that vesicles bearing phosphatidylserine can trigger CD8 + T cell activation ( 70 ). Interestingly, in an experimental setting where either EP- or sup-fractions serve as the only source of antigen for cross-presentation ( Figures 4E, F ), we observed an increased CD8 + T cell activation which was accompanied by enhanced antigen-specific peptide/MHC class I surface expression in cross-presenting DC ( Figure 4G ). The above findings suggest that expression of a functional P2X7 receptor in feeder cells not only modulates antigen cross-presentation in APCs at the protein level, as previously suggested ( 71 ), but also influences viral gene expression and viral entry in feeder cells as well as phagocytosis by BMDCs.

P2RX7-expressing cells feature an altered secretome e.g. released extracellular particles might contain antigens, cytokines and regulatory RNAs that are important for antigen-presentation ( 8 , 37 , 39 , 61 ). Although the amount of OVA protein in isolated extracellular particles or in the supernatant of MVA-infected cells was comparable in the absence or presence of P2RX7 ( Figure 4B ), the composition and expression level of (pro)inflammatory cytokines and chemokines in the supernatant of P2RX7 expressing cells was significantly altered ( Figure 4C , Supplementary Figure 3A ) in the presence of P2RX7. These results support the importance of P2RX7 during the initial phase of MVA infection as well as at the later stage, when infected feeder cells are co-cultured with antigen-presenting cells and continuously supply the microenvironment with stimulatory molecules and enhance cross-presentation ( 7 , 72 ). The EP fraction and, significantly stronger the supernatant fraction of CM P2RX7 cells increased CD8 + T cell activation and SIINFEKL/H2-K b expression by cross-presenting BMDCs when added to mock-infected feeder cells ( Figures 4F, G ). This effect was less pronounced when these fractions were added to MVA-infected feeder cells ( Supplementary Figure 3B–E ). The feeder cells produce apoptotic bodies upon MVA infection, which may contain antigens for phagocytosis. Since these were not eliminated by our isolation method ( 46 ), we filtered the supernatant (0.2µM) to exclude apoptotic bodies in this fraction. We confirmed that the secretome of CM P2RX7 cells significantly contributed to improved antigen cross-presentation by BMDCs ( Figure 4H left/middle). This effect could be attributed to both, the secreted pro-inflammatory cytokines as well as small vesicles such as exosomes or micro vesicles released due to the presence of P2RX7 ( 8 , 73 ). Additional soluble as well as cell-associated factors from infected feeder cells may be needed to fully license DCs for enhanced cross-presentation, as the total MHCI expression of BMDCs remained unchanged upon the addition of filtered supernatant fractions ( Figure 4D right).

The P2RX7 protein is known to be expressed on the plasma membrane as well as on intracellular membrane structures, suggesting that it may have multiple functions depending on the compartment within the cell. Sarti and colleagues have previously described the enhancement of mitochondrial metabolism by P2RX7 ( 53 ). We confirmed the intracellular presence of P2RX7 in our feeder cells ( Figure 5A ) as well as an increase in mitochondrial activity in the presence of P2RX7 in our MVA-infection model ( Figures 5B, C ). P2RX7 expression correlated with maximal respiration rate and spare capacity in MVA- and mock-infected cells, demonstrating enhanced ability of the cells to respond to stress ( 74 ). Furthermore, the extracellular acidification rate (ECAR) was significantly enhanced in the presence of active P2RX7, delineating the altered glycolysis pathway in these cells ( Figure 5C ), in line with previously reported glycolytic activity attributed to P2RX7 ( 75 ). These results suggest that P2RX7 is able to change the bioenergetics state of cells ( 76 ), with or without MVA infection. ATP is required for efficient vaccinia virus production ( 77 ). Importantly, we observed higher ATP levels within P2RX7 competent feeder cells which were significantly increased after MVA infection ( Figure 5D ). In contrast, basal secretion of ATP by these cells (extracellular ATP level) was less or comparable when infected with MVA, while cells with inactive P2RX7 released significantly higher amounts of ATP into the supernatant after MVA infection ( Figure 5E ). As shown before, cells infected with MVA undergo apoptosis. Since ATP is released during cell death processes ( 27 , 78 ), we suggest in line with others that ATP regulation is P2RX7-dependent in BALB/c P2RX7-bearing feeder cells, but it is apoptosis-dependent in feeder cells lacking the fully functional P2RX7 ( 79 ). Further studies are required to analyze if the available ATP can act in an autocrine manner and reactivate P2X7 receptors of the feeder cell, as previously reported ( 80 ). It has been described that infection of viral pathogens may drive metabolic reprogramming to allow for adaptation of the cell to biosynthetic and energetic needs required for viral replication ( 81 , 82 ). We demonstrate that cells expressing a functional P2RX7 seem to handle MVA infections better due to prolonged active cell metabolism and increased energy levels resulting in increased overall cell fitness and delayed apoptotic cell death. The lack of ethidium bromide pore opening ( Supplementary Figure 1G ), another potential characteristic of P2RX7 plasma membrane expression ( 44 ), indicates a rather protective role of P2RX7 in feeder cells. In this respect, active Caspase-8 found in cells with functional P2RX7 seems to be activated in the absence of cell death, leading to the release of inflammatory cytokines and the restriction of pathogen growth ( 83 ). A signaling pathway associated with these processes and affected by P2RX7-dependent modulation may involve NF-κB ( 84 ).

In this study, we have uncovered significant factors influenced by the ATP-sensitive P2X7 receptor in MVA-infected feeder cells that promote antigen cross-presentation by dendritic cells. These factors include (i) the secretion of pro-inflammatory cytokines, (ii) delayed viral entry during MVA infection of CM P2RX7 cells associated with delayed viral gene expression and subsequent viral antigen synthesis. We further identified (iii) the release of small vesicles (<0.2µM) such as exosomes and microvesicles as well as viral mRNA containing EPs, as a potential source of viral antigens challenging the fact that only cell-associated antigens may be involved in the activation of CD8 + T cells by antigen cross-presentation. Additionally, we found that (iv) the presence of late apoptotic markers in feeder cells as well as (v) improved mitochondrial functions in feeder cells contribute to a favorable microenvironment for enhanced cross-presentation. Based on our results, we suggest that various signaling pathways triggered by active P2RX7 in infected CM feeder cells interplay and significantly contribute to the increased antigen cross-presentation capability of BMDCs leading to enhanced SIINFEKL//H2-K b expression in BMDCs and subsequent CD8 + T cell activation.

Limitations of study

Since the ECAR measured in this assay is only a quantitative measurement of the total amount of acid (H + ) produced during both the tricarboxylic acid cycle and glycolysis, further assays may help to determine more specific alterations in the glycolytic metabolism ( 55 ). This study did not include an in-depth analysis of different extracellular particle fractions such as apoptotic bodies, microvesicles and exosomes. Further fractionation and subsequent characterization will allow to determine the exact content of these particles. Further work should address the role of DBA P2RX7 in CM cells. Even though the plasma membrane receptor is not functional in cells with a DBA background due to the P451L mutation ( 42 ), further studies may try to characterize other functions of this receptor during MVA infection in vitro and in vivo . Up to now, animal models such as appropriate P2RX7 knockout mice are lacking to investigate the role of the receptor in vivo . Since ATP may play a role during the co-culture of immune cells ( 85 ), future studies should assess its role in feeder cells, APCs and during co-culture. In addition, understanding how the altered mitochondrial metabolism affects EP release and composition as well as cross-presentation on the molecular level would be important for future studies. Due to the limitations of our in vitro murine model for the cross-presentation of MVA antigens, future research should include human cells.

Materials and methods

The identifiers of all reagents and resources used are listed in Supplementary Table 1 ( Supplementary Material ).

For isolation of bone marrow female 12-to 16-week adult C57BL/6N mice were purchased from Janvier and were allowed to acclimate for a minimum of one week in the in-house animal facility. For weekly T cell stimulation, the spleen of adult C57BL/6N mice was used. Animals were maintained at the Zentrale Einrichtung für Tierversuchsanstalt (ZETT) at the University of Düsseldorf under specific pathogen-free conditions. Experimental procedures have been approved by the regional authorities (North Rhine-Westphalia State Environment Agency - LUA NRW, Germany) and the animal use committee at the University of Düsseldorf (Reg. No O119/11).

Recombinant MVA were generated by homologous recombination as previously described ( 33 , 86 ). All stock preparations of MVA used in this study were diluted to a concentration of 1x10 9 viral particles/mL and maintained at -80°C. Viral aliquots were thawed in a water bath, sonicated for one minute, briefly vortexed and spun down for usage. Freeze/thawed aliquots were not used more than three times.

Infection of cells

Unless differently stated, cells were harvested, pelleted in a falcon and infected with MVA (MOI1, unless otherwise specified) for one to two hours at 37°C, 5% CO 2 with intermittent shaking every 15min. Cells were washed twice before incubation with other cells for cross-presentation experiments. For the remaining experiments cells were seeded and incubated immediately. Since harvesting at different time points was required for expression kinetics analyses and titration experiments, cells were seeded and allowed to adhere before infection. Infection was then performed directly on the plate with intermittent shaking and washing after one hour of incubation at 37°C and 5% CO 2 . For infection in 96-well plates (Mitochondrial function assay and intracellular ATP determination assay) virus was added to each well of the plate, shaken every 15min for 1h and subsequently incubated for the remaining time frame at 37°C and 5% CO 2.

Fluorometric analysis of intracellular Ca 2+ levels or EtBr-guided pore opening

1x10 6 cells were either mock- or MVA (MVA-PK1L-OVA) infected at an MOI of 1 for either 4h or 20h. For Ca 2+ measurements infected cells were placed in a falcon for loading with 4µM FURA-2 AM (Sigma-Aldrich) in saline solution (12.5mM NaCl, 0.5mM KCl, 0.1mM MgSO 4 , 2mM HEPES, 0.55mM D-glucose, 0.5mM NaHCO 3 (all Sigma-Aldrich)) supplemented with 0.5mM CaCl 2 (pH 7.4, Merck) and 250µM sulfinpyrazone (Sigma-Aldrich) at 37°C for 20min. Cells were then washed, resuspended in saline solution and stimulated with the indicated concentrations of Bz-ATP (Sigma-Aldrich) and 1µM ionomycin (Invitrogen) for the recording of intracellular Ca 2+ release. For detection of pore opening at the cell membrane, cells were loaded with 2µL ethidium bromide (EtBr (Sigma-Aldrich)) and stimulated with 200µM Bz-ATP and 100µM digitonin (Sigma-Aldrich). Measurements were done in a thermostat quartz cuvette using a Perkin-Elmer KS50 rotating and heating system at a wavelength of 340/380nm (excitation) and 505nm (emission) for intracellular Ca 2+ release and at a wavelength of 360nm (excitation) and 580nm (emission) for EtBr- pore opening assay.

Generation of stably transfected cell lines

Cloudman S91 cells (ATCC CCL-53.1) were seeded at a density of 1.5x10 5 cells per well in a 6-well plate and transfected with either 3µG pcDNA3 control or P2RX7 encoding plasmid DNA using Lipofectamine reagent (Invitrogen) according to the manufacturer’s instructions. Briefly, plasmid DNA was dissolved in medium, incubated with 1µL PlusReagent for 5min and then 3µL Lipofectamine was added and further incubated for 30min at room temperature. The Lipofectamine-DNA mixture was then added dropwise to the cells and cells were selected for geneticin (0.2mg/mL) resistance two days post-transfection. Transfection efficacy was confirmed by Western Blot analysis of P2RX7 synthesis and by fluorometric analysis measuring the P2RX7-dependent intracellular Ca 2+ increase.

Live cell confocal imaging

Cells were grown on a round cover dish placed in a 6-well plate at a density of 5x10 5 cells per well. Cells were stained with 2µM PKH-26 (Sigma-Aldrich) and 2µM Quinacrine (Sigma-Aldrich) for 10min at 37°C and 5% CO 2 in saline saccharose solution (30mM saccharose, 0.1mM K 2 HPO 4 , 0.1mM MgSO 4 , 0.5mM D-glucose, 0.2mM HEPES (all Sigma-Aldrich)) supplemented with CaCl 2 (pH7.4). Cells were placed in a holder device for round cover glasses and stimulated with 200µM Bz-ATP. Images were acquired in 6-second intervals for approximately 15min. Images were taken at 60x magnification of the Olympus Fluoview FV3000 (Olympus). Data visualization was achieved using OMERO software (Open microscopy imaging).

Generation of bone marrow-derived dendritic cells

Bone marrow was obtained from 12-to 16-week-old C57BL/6N and 5x10 6 bone marrow cells were seeded with 10% GM-CSF (obtained from supernatant of B16 cells expressing GM-CSF, originally kindly provided by Georg Häcker, Freiburg) in RPMI-medium (Gibco) containing 10% heat-inactivated FBS and 50µM 2-mercaptoethanol (M2 Medium) in 10cm Petri-dishes. On day three fresh M2 Medium and GM-CSF was added to the primary culture and on day six 10mL medium was replaced with fresh M2 Medium containing GM-CSF. BMDCs were used on day seven for all experiments.

T cell restimulation

CD8 + T cell lines were generated as described recently ( 33 ). For weekly T cell stimulation, both EL4 cells (ATCC TIB-39) and naïve splenocytes from C57BL/6N were irradiated with 100Gy or 30Gy, respectively. EL4 cells were loaded with 1µg/mL B8R-peptide (TSYKFESV; immunodominant peptide derived from the B8 protein from vaccinia virus) or Ova-peptide (SIINFEKL; derived from ovalbumin) and then co-incubated with splenocytes, CD8 + specific T cells and M2 Medium containing 5% TCGF (T-cell growth factor). Both peptides are H2-K b -restricted.

Cross-presentation assay

Cloudman S91 murine melanoma (CM) cells (MHC I haplotype H2-d) were used as feeder cells for antigen cross-presentation assays. A total of 2x10 6 cells were either mock- or MVA-PK1L-OVA (MOI1) infected for 20h, washed and subsequently incubated with psoralen (1µg/mL) (Sigma-Aldrich) for 15min at 37°C and 5% CO 2 and treated with UV-A light (PUVA) for further 15min. Cells were harvested, transferred to a falcon and washed with medium. CM feeder cells were co-incubated with uninfected BMDCs, which were previously generated from bone marrow of C57BL/6N mice (MHC I haplotype H2-K b ) at a ratio of 1:1 in a 6 cm dish for 18h. The next day, the co-culture of CM and BMDCs was harvested, washed in M2 Medium and resuspended in M2 medium in a final volume of 1mL. One part of the co-culture suspension (200µL) was immediately stained for the surface expression of peptide/MHCI complexes (SIINFEKL peptide within H2-K b ) on BMDCs, while 100µL of the CM-BMDCs co-culture (containing 2x10 5 BMDCs as antigen-presenting cells) was further incubated with 2x10 5 B8R- or Ova- specific CD8 + T cells in the presence of 1µg/mL Brefeldin A (Sigma Aldrich) for 4h at 37°C and 5% CO 2 . Further analysis of CD8 + T-cell activation is described below.

Intracellular cytokine staining (ICS)

To determine the antigen presentation capacity of dendritic cells in the cross-presentation setting, peptide-specific T cell lines were used as a read out system. After 4h incubation (see above cross-presentation assay), cells were washed with PBS and dead cells were excluded by staining with Fixable viability dye eFluor 506 (Invitrogen) (1:600) for 20min on ice. Cells were washed with FACS buffer (PBS supplemented with 1% BSA and 0.02% sodium azide) and then stained using anti-mouse CD8α eFluor 450 (eBioscience) (1:300) for 20min on ice. Subsequently, cells were permeabilized with BD Cytofix (BD Biosciences) for 15min on ice and then stained with Anti-mouse IFNy APC (Invitrogen) (1:400) and Anti-mouse TNFα PE-Cyanine7 (Invitrogen) (1:300) in 1:10 diluted BD Perm/Wash for 30min on ice. Cells were washed twice and resuspended in 1% PFA for subsequent analysis using the FACS Canto II device (BD Biosciences).

SIINFEKL/H2-K b surface staining/MHC II maturation staining

Antigen processing and presentation capacity was also assessed by measuring the MHCI/peptide complex formation as SIINFEKL/H2-K b expression on the surface of the dendritic cells. For this co-cultured BMDCs (see above cross-presentation assay) were washed with PBS and dead cells were stained with Fixable viability dye eFluor 660 (Invitrogen) (1:2000) for 20min on ice. Fc-receptors were blocked using anti-mouse CD16/CD32 (eBioscience) (1:200). After Fc-blocking, surface staining was performed for 30min on ice using anti-mouse CD11c PE (Invitrogen), anti-mouse H2-Kb FITC (Biolegend) and anti-mouse SIINFEKL/H2-K b PE-Cyanine 7 (eBioscience) (all 1:300 in FACS buffer). Cells were washed twice and resuspended in 1% PFA for subsequent analysis using FACS Canto II. Alternatively, cells were stained with Fixable viability dye eFluor 506 (Invitrogen) (1:600) for 20min on ice, followed by the Fc-blocking step and surface staining with CD11c APC-Cyanine 7 (BD Pharmingen) and MHCII PE (all 1:300 in FACS buffer).

Viral or cellular gene expression

For kinetic analysis of viral gene expression (0h to 24hpi), 2x10 6 cells were infected with MVA-PK1L-Ova (MOI1) for 1h at 4°C, resulting in the virus attachment to the cell surface. After washing cells were harvested at the indicated time points, spun down and the pellet was resuspended for total RNA isolation as described in the manufacturer´s protocol (RNeasy Mini Kit (Qiagen)). Briefly, cells were lysed using RLT buffer containing 10µL 2-ß-mercaptoethanol and mixed with one volume of 70% ethanol for subsequent isolation using the RNeasy Mini spin column. cDNA was then transcribed using the Revert Aid H minus first strand cDNA synthesis (Thermo Fisher Scientific) according to the manufacturer’s instructions and used as a template for subsequent quantitative PCR reaction with PowerUp SYBR Green Master Mix (Applied Biosciences). Expression of viral B8R , Ova , A19L and cellular P2rx7 genes was normalized to expression of 18S-rRNA housekeeping gene and ΔΔCT was calculated by further comparison of ΔCT values with the 0h time point of CM wildtype cells. Primer sequences are listed in Supplementary Table 1 .

Viral replication

In order to determine the replication capacity of MVA, 1x10 6 CM cells (WT, pcDNA3- or P2RX7- transfected) were infected with MVA-p7.5-GFP at MOI5 for 0h (1h at RT), washed and further incubated until harvested at 4h, 8h or 24hpi. Collected samples were vortexed and subjected to three rounds of freeze-thaw-sonication cycles to release viral particles. Viral suspensions were then used to prepare serial dilutions that were plated on 96-well plates containing MVA-permissive DF-1 cells (ATCC CRL-12203) (80% confluent). Fluorescent signal and cytopathic effect was monitored for seven days post-infection to determine the 50% endpoint titer of viral particles per milliliter by using the Spearman-Karber method to calculate the tissue culture infectious dose 50 (TCID 50 ).

Western Blot analysis

For extraction of proteins, 2x10 6 cells were infected (see above “infection of cells”) and harvested at the indicated time points. Collected cells were spun down by centrifugation, washed with PBS and resuspended in RIPA buffer (Thermo Fisher Scientific) containing HALT Protease & Phosphatase Inhibitor cocktail (Thermo Fisher Scientific) (1:100). After three rounds of freeze-thaw-sonication cycles, supernatants were harvested after a single centrifugation step at full speed for five minutes at 4°C. Protein content was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). SDS-PAGE and blotting on nitrocellulose membranes was performed as described elsewhere ( 87 ). Membranes were incubated with Anti-Ovalbumin (Rockland) (1:20 000); Anti-Cleaved caspase-8 (Cell signaling) (1:1000); Anti-P2RX7 (Sigma-Aldrich) (1:200) and Anti-ß- Actin (Sigma-Aldrich) (1:50 000). Relative quantification of specific proteins was done by calculating ratio of the protein of interest with the ß-Actin loading control using the ImageJ analysis tool (US National Institutes of Health, Bethesda, USA).

Phosphatidylserine exposure analysis

Surface staining of phosphatidylserine residues on MVA-infected cells was done according to Apotracker-Green protocol (Biolegend) at either 6hpi or 20hpi. Briefly, 2x10 5 cells were washed with FACS buffer and incubated in 400nM Apotracker-Green staining solution for 20min at room temperature. Cells were subsequently stained with fixable viability dye eFluor 660 (1:2000) for 20min on ice, washed and immediately analyzed by FACS. Cells were either gated for APC-negative (non-permissive for viability dye) and FITC-positive (Apotracker Green-positive) populations, designating early apoptotic cells or gated for APC- and FITC-double-positive populations, indicating late apoptotic cells.

Isolation of extracellular particles and supernatant fractions

For extracellular particle isolation the protocol was adapted according to Pegoraro and colleagues ( 8 ). Four T75 flasks (approximately 8x10 6 cells/flask) were seeded with CM cells one day before infection to obtain 90% confluency. Before infection, one flask per cell line was counted in order to calculate the respective MOI. Cells were allowed to rest for 30min at room temperature and after washing 3mL medium was added in each flask. MVA-PK1L-Ova (MOI 1.5) was added and flasks were shaken every 15min for one hour (at 4°C for RNA isolation). After one hour cells were washed and harvested (0h value) or further incubated for a total of 20h at 37°C and 5% CO 2 . For harvesting, medium was discarded, cells were washed with PBS and 3mL saline solution supplemented with 0.05mM CaCl 2 was added. Cells were stimulated with 200µM Bz-ATP for 30min at 37°C. Thereafter, the supernatant was aspirated and centrifuged at 300g for five minutes at 4°C to remove cell debris. The cleared supernatant was harvested, aliquoted in Eppendorf tubes and centrifuged at 20.000 g for one hour at 4°C. The supernatant was discarded and the remaining extracellular particle fraction (EP-fraction) was either used for quantitative RNA analyses (resuspended in RLT buffer with 2-ß-mercaptoethanol), western blot analyses (resuspended in RIPA buffer with HALT Protease & Phosphatase Inhibitor cocktail) or for cross-presentation assays (resuspended in PBS). For cross-presentation assays, EP fractions were additionally PUVA treated prior to the last centrifugation step, as described above.

For isolation of supernatants, 2x10 6 cells per condition tested were used. Cells were either MVA-PK1L-Ova (MOI1) or mock-infected for the indicated time (8h or 20h for western blot analysis; 20h for Legendplex and cross-presentation assays), harvested and supernatants (sup-fraction) were collected after centrifugation at 300g for five minutes. For indicated experiments, supernatant fractions were further passed through a 0.2µM size pore filter (fil sup-fraction) to be used for cross-presentation experiments. All supernatant fractions (sup- or fil sup-fractions) used for cross-presentation assays were additionally PUVA treated as described above.

Cytokine and chemokine analysis in supernatants

The release of cytokines/chemokines was analyzed using the Legendplex MU anti-virus response panel (Biolegend). Briefly, 2x10 6 cells were either MVA-PK1L-Ova (MOI1) or mock-infected for 20h. After harvesting the cell suspensions, supernatants were collected after centrifugation at 300g for 5min and processed according to the manufacturer’s instructions. Data was analyzed using the Biolegend LEGENDplex Data Analysis Software (Biolegend).

Cross-presentation assays using EP- or supernatant-fractions

Infection of CM feeder cells was performed as described above for cross-presentation assays. On day two, CM WT feeder cells (either mock- or MVA-PK1L-OVA infected, MOI1) were co-incubated with BMDCs and, additionally, pulsed with either EP-, sup- or fil sup-fractions. These fractions were isolated from either infected CM pcDNA3 (transfected cells with inactive P2RX7) or infected CM P2RX7 cells (transfected cells with active P2RX7) after 20hpi as described above. On day 3, cross-presentation assays were continued as described above.

Intracellular quantification of P2RX7

To assess the expression of P2RX7, 2x10 5 CM cells (WT, pcDNA3- or P2RX7-transfected) were mock- or MVA-PK1L-OVA (MOI1) infected. After 20hpi, cells were stained with fixable viability dye eFluor 660 (1:2000) for 20min on ice, permeabilized with BD Cytofix for 15min on ice and then stained with anti-P2RX7 (1:200) for one hour on ice to quantify the intracellular presence of P2RX7. Cells were further incubated with anti-mouse-IgG-PE (Jackson laboratories) (1:200) secondary antibody for 30min on ice, washed and immediately used for FACS analysis by FACS Canto II.

Mitochondrial metabolism analysis

The day prior to infection, 2x10 4 cells were seeded in a Seahorse XF96 Cell culture Microplate (Agilent Technologies). Cells were allowed to adhere for 1h at room temperature and were further incubated at 37°C at 5% CO 2 overnight. The next day cells were infected with MVA-PK1L-Ova (MOI5, 6h). Mitochondrial function was assessed using the Seahorse XF Cell Mito Stress test (Agilent technologies) according to the manufacturer’s instructions. Compounds have been used at the concentration of 15µM for Oligomycin, 5µM for FCCP and 5µM for Rot/AA.

Intracellular ATP measurements

The day prior to infection, 5x10 4 cells were seeded in a 96 flat well chimney base plate and incubated overnight at 37°C and 5% CO 2 . Cells were infected with MVA-PK1L-Ova for 6h (MOI5) before intracellular ATP concentrations were determined using the Luminescent ATP detection assay kit (Abcam) as described in the supplier´s protocol. Briefly, cells were lysed and ATP was stabilized by a detergent during a shaking step. After the addition of the substrate solution, prompted luminescence was measured and compared to ATP standard samples using a Spark plate reader (Tecan).

Extracellular ATP measurement

1x10 6 CM cells (WT, pcDNA3- or P2RX7-transfected either MVA-PK1L-Ova or mock-infected, MOI5) were seeded in 1mL in a 6-well plate. The supernatant was harvested after 6hpi. For each condition, 50µL supernatant was incubated with 50µL of FirezymeB Diluent buffer (Firezyme). Samples as well as an ATP standard (Sigma- Aldrich) were compared in a standard curve at serial dilutions run by the Luminometer Victor 3 1420 Multiwell counter (Perkin Elmer) with automated addition of 100µL Enliten Luciferase/Luciferin reagent (Promega) to detect emitted luminescence.

Quantification and statistical analysis

Details on statistical analyses are integrated in figure legends. When indicated, data was normalized to untreated or WT control cells. Unpaired two-tailed student’s t-test was used to calculate statistical significances using Prism 8 (GraphPad Software). Extracellular particles ( Figure 1C ) were quantified by counting three adjacent frames of each replicate upon Bz-ATP stimulus and normalized to cell numbers (determined by quinacrine staining) per frame. Graphical data represent mean values with error bars indicating SD or SEM with P-values of ≤ 0.05 (*), ≤ 0.01 (**), ≤ 0.001 (***) and ≤ 0.0001 (****) indicating significant differences between groups.

Data availability statement

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding author.

Ethics statement

Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by North Rhine-Westphalia State Environment Agency - LUA NRW, Germany) and the animal use committee at the University of Düsseldorf (Reg. No O119/11). The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

YL: Writing – review & editing, Data curation, Formal analysis, Investigation, Validation, Writing – original draft. SM: Investigation, Writing – review & editing. GA: Investigation, Writing – review & editing. JW: Investigation, Methodology, Writing – review & editing. IK: Investigation, Methodology, Writing – review & editing. EDM: Investigation, Methodology, Writing – review & editing. AP: Investigation, Methodology, Writing – review & editing. RL: Methodology, Writing – review & editing. KK: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. PP: Data curation, Formal Analysis, Investigation, Methodology, Validation, Writing – review & editing. RT: Investigation, Methodology, Writing – review & editing. FDV: Methodology, Writing – review & editing. EA: Methodology, Supervision, Validation, Writing – review & editing. ID: Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review & editing.

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Grants GK1949/2 and DR632/2-1 project No 452147069 to ID and by the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No. 812915 to ID. EA and FDV were supported by grants from the Italian association for cancer research (AIRC grant numbers: IG 22837, IG 13025 and IG 18581).

Acknowledgments

We thank the Laboratory of Clinical Pathology, especially Luigia Ruo (Department of Medical Sciences, University of Ferrara) for support in fluorometric assays and Sha Tao and Cornelia Barnowski (Institute of Virology, Universitätsklinikum Düsseldorf) for answering scientific and technical questions. We acknowledge Professor Massimo Bonora (Department of Medical Sciences, University of Ferrara) for support with Live Confocal Imaging. Computational infrastructure and support were provided by the Centre for Information and Media Technology at Heinrich-Heine-University Düsseldorf.

Conflict of interest

FDV is a member of the Scientific Advisory Board of Biosceptre Ltd, a biotech Company involved in the development of anti-P2X7 antibodies, and a Consultant with Breye Therapeutics.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2024.1360140/full#supplementary-material

1. Gu BJ, Zhang WY, Bendall LJ, Chessell IP, Buell GN, Wiley JS. Expression of P2X(7) purinoceptors on human lymphocytes and monocytes: evidence for nonfunctional P2X(7) receptors. Am J Physiol Cell Physiol . (2000) 279:C1189–97. doi: 10.1152/ajpcell.2000.279.4.C1189

PubMed Abstract | CrossRef Full Text | Google Scholar

2. Burnstock G, Knight GE. Cellular Distribution and Functions of P2 Receptor Subtypes in Different Systems. In: International Review of Cytology , vol. 240. London, UK: Academic Press (2004). p. 31–304.

Google Scholar

3. Kaczmarek-Hájek K, Lörinczi E, Hausmann R, Nicke A. Molecular and functional properties of P2X receptors–recent progress and persisting challenges. Purinergic signalling . (2012) 8:375–417. doi: 10.1007/s11302-012-9314-7

4. Kopp R, Krautloher A, Ramirez-Fernandez A, Nicke A. P2X7 interactions and signaling - making head or tail of it. Front Mol Neurosci . (2019) 12:183. doi: 10.3389/fnmol.2019.00183

5. Bianco F, Perrotta C, Novellino L, Francolini M, Riganti L, Menna E, et al. Acid sphingomyelinase activity triggers microparticle release from glial cells. EMBO J . (2009) 28:1043–54. doi: 10.1038/emboj.2009.45

6. Nuttle LC, Dubyak GR. Differential activation of cation channels and non-selective pores by macrophage P2z purinergic receptors expressed in Xenopus oocytes. J Biol Chem . (1994) 269:13988–96. doi: 10.1016/S0021-9258(17)36744-3

7. Pizzirani C, Ferrari D, Chiozzi P, Adinolfi E, Sandona D, Savaglio E, et al. Stimulation of P2 receptors causes release of IL-1beta-loaded microvesicles from human dendritic cells. Blood . (2007) 109:3856–64. doi: 10.1182/blood-2005-06-031377

8. Pegoraro A, De Marchi E, Ferracin M, Orioli E, Zanoni M, Bassi C, et al. P2X7 promotes metastatic spreading and triggers release of miRNA-containing exosomes and microvesicles from melanoma cells. Cell Death Dis . (2021) 12:1088. doi: 10.1038/s41419-021-04378-0

9. Guo J, Yang P, Li YF, Tang JF, He ZX, Yu SG, et al. MicroRNA: Crucial modulator in purinergic signalling involved diseases. Purinergic signalling . (2023) 19:329–41. doi: 10.1007/s11302-022-09840-y

10. Lindenbergh MFS, Stoorvogel W. Antigen presentation by extracellular vesicles from professional antigen-presenting cells. Annu Rev Immunol . (2018) 36:435–59. doi: 10.1146/annurev-immunol-041015-055700

11. Battistelli M, Falcieri E. Apoptotic bodies: particular extracellular vesicles involved in intercellular communication. Biol (Basel) . (2020) 9. doi: 10.3390/biology9010021

CrossRef Full Text | Google Scholar

12. Pegoraro A, Bortolotti D, Marci R, Caselli E, Falzoni S, De Marchi E, et al. The P2X7 receptor 489C>T gain of function polymorphism favors HHV-6A infection and associates with female idiopathic infertility. Front Pharmacol . (2020) 11:96. doi: 10.3389/fphar.2020.00096

13. Barnowski C. The Role of Innate Immune Triggers for the Induction of Poxviral Vectored Cytotoxic T Cell Responses . Düsseldorf: Heinrich-Heine-Universität (2022).

14. Longo Y. Impact of inflammasome activation on cross-presentation of viral antigens after MVA infection . Düsseldorf: Heinrich-Heine-Universität (2020).

15. Geshelin P, Berns KI. Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J Mol Biol . (1974) 88:785–96. doi: 10.1016/0022-2836(74)90399-4

16. Baroudy BM, Venkatesan S, Moss B. Structure and replication of vaccinia virus telomeres. Cold Spring Harbor Symp quantitative Biol . (1983) 47 Pt 2:723–9. doi: 10.1101/SQB.1983.047.01.083

17. Gilbert SC. Clinical development of Modified Vaccinia virus Ankara vaccines. Vaccine . (2013) 31:4241–6. doi: 10.1016/j.vaccine.2013.03.020

18. Mayr A, Stickl H, Müller HK, Danner K, Singer H. [The smallpox vaccination strain MVA: marker, genetic structure, experience gained with the parenteral vaccination and behavior in organisms with a debilitated defence mechanism (author's transl)]. Zentralblatt fur Bakteriologie Parasitenkunde Infektionskrankheiten und Hygiene Erste Abteilung Originale Reihe B: Hygiene Betriebshygiene praventive Medizin . (1978) 167:375–90.

PubMed Abstract | Google Scholar

19. Blanchard TJ, Alcami A, Andrea P, Smith GL. Modified vaccinia virus Ankara undergoes limited replication in human cells and lacks several immunomodulatory proteins: implications for use as a human vaccine. J Gen Virology . (1998) 79:1159–67. doi: 10.1099/0022-1317-79-5-1159

20. Drexler I, Heller K, Wahren B, Erfle V, Sutter G. Highly attenuated modified vaccinia virus Ankara replicates in baby hamster kidney cells, a potential host for virus propagation, but not in various human transformed and primary cells. J Gen virology . (1998) 79:347–52. doi: 10.1099/0022-1317-79-2-347

21. Sutter G, Moss B. Nonreplicating vaccinia vector efficiently expresses recombinant genes. Proc Natl Acad Sci United States America . (1992) 89:10847–51. doi: 10.1073/pnas.89.22.10847

22. de Vries RD, Altenburg AF, Nieuwkoop NJ, de Bruin E, van Trierum SE, Pronk MR, et al. Induction of cross-clade antibody and T-cell responses by a modified vaccinia virus ankara–based influenza A(H5N1) vaccine in a randomized phase 1/2a clinical trial. J Infect Diseases . (2018) 218:614–23. doi: 10.1093/infdis/jiy214

23. Kaynarcalidan O, Moreno Mascaraque S, Drexler I. Vaccinia virus: from crude smallpox vaccines to elaborate viral vector vaccine design. Biomedicines . (2021) 9. doi: 10.3390/biomedicines9121780

24. Samreen B, Tao S, Tischer K, Adler H, Drexler I. ORF6 and ORF61 expressing MVA vaccines impair early but not late latency in murine gammaherpesvirus MHV-68 infection. Front Immunol . (2019) 10:2984. doi: 10.3389/fimmu.2019.02984

25. Gasteiger G, Kastenmuller W, Ljapoci R, Sutter G, Drexler I. Cross-priming of cytotoxic T cells dictates antigen requisites for modified vaccinia virus Ankara vector vaccines. J Virol . (2007) 81:11925–36. doi: 10.1128/JVI.00903-07

26. Wong YC, Croft S, Smith SA, Lin LCW, Cukalac T, La Gruta NL, et al. Modified vaccinia virus ankara can induce optimal CD8(+) T cell responses to directly primed antigens depending on vaccine design. J Virol . (2019) 93. doi: 10.1128/JVI.01154-19

27. Chahroudi A, Garber DA, Reeves P, Liu L, Kalman D, Feinberg MB. Differences and similarities in viral life cycle progression and host cell physiology after infection of human dendritic cells with modified vaccinia virus Ankara and vaccinia virus. J Virol . (2006) 80:8469–81. doi: 10.1128/JVI.02749-05

28. Gros M, Amigorena S. Regulation of antigen export to the cytosol during cross-presentation. Front Immunol . (2019) 10:41. doi: 10.3389/fimmu.2019.00041

29. Joffre OP, Segura E, Savina A, Amigorena S. Cross-presentation by dendritic cells. Nat Rev Immunol . (2012) 12:557–69. doi: 10.1038/nri3254

30. Rock KL, Shen L. Cross-presentation: underlying mechanisms and role in immune surveillance. Immunol Rev . (2005) 207:166–83. doi: 10.1111/j.0105-2896.2005.00301.x

31. Ma W, Stroobant V, Heirman C, Sun Z, Thielemans K, Mulder A, et al. The vacuolar pathway of long peptide cross-presentation can be TAP dependent. J Immunol (Baltimore Md 1950) . (2019) 202:451–9. doi: 10.4049/jimmunol.1800353

32. Embgenbroich M, Burgdorf S. Current concepts of antigen cross-presentation. Front Immunol . (2018) 9. doi: 10.3389/fimmu.2018.01643

33. Barnowski C, Ciupka G, Tao R, Jin L, Busch DH, Tao S, et al. Efficient induction of cytotoxic T cells by viral vector vaccination requires STING-dependent DC functions. Front Immunol . (2020) 11:1458. doi: 10.3389/fimmu.2020.01458

34. Broyles SS, Moss B. DNA-dependent ATPase activity associated with vaccinia virus early transcription factor. J Biol Chem . (1988) 263:10761–5. doi: 10.1016/S0021-9258(18)38036-0

35. Feng L-l, Cai Y-q, Zhu M-c, Xing L-j, Wang X. The yin and yang functions of extracellular ATP and adenosine in tumor immunity. Cancer Cell Int . (2020) 20:110. doi: 10.1186/s12935-020-01195-x

36. Mutini C, Falzoni S, Ferrari D, Chiozzi P, Morelli A, Baricordi OR, et al. Mouse dendritic cells express the P2X7 purinergic receptor: characterization and possible participation in antigen presentation. J Immunol (Baltimore Md 1950) . (1999) 163:1958–65. doi: 10.4049/jimmunol.163.4.1958

37. de Torre-Minguela C, Barbera-Cremades M, Gomez AI, Martin-Sanchez F, Pelegrin P. Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process. Sci Rep . (2016) 6:22586. doi: 10.1038/srep22586

38. Barrera-Avalos C, Briceno P, Valdes D, Imarai M, Leiva-Salcedo E, Rojo LE, et al. P2X7 receptor is essential for cross-dressing of bone marrow-derived dendritic cells. iScience . (2021) 24:103520. doi: 10.1016/j.isci.2021.103520

39. Adinolfi E, Callegari MG, Cirillo M, Pinton P, Giorgi C, Cavagna D, et al. Expression of the P2X7 receptor increases the Ca2+ content of the endoplasmic reticulum, activates NFATc1, and protects from apoptosis. J Biol Chem . (2009) 284:10120–8. doi: 10.1074/jbc.M805805200

40. Bhattacharyya S, Deb J, Patra AK, Thuy Pham DA, Chen W, Vaeth M, et al. NFATc1 affects mouse splenic B cell function by controlling the calcineurin–NFAT signaling network. J Exp Med . (2011) 208:823–39. doi: 10.1084/jem.20100945

41. Rudolf R, Busch R, Patra A, Muhammad K, Avots A, Andrau J-C, et al. Architecture and expression of the nfatc1 gene in lymphocytes. Front Immunol . (2014) 5. doi: 10.3389/fimmu.2014.00021

42. Adriouch S, Dox C, Welge V, Seman M, Koch-Nolte F, Haag F. Cutting edge: a natural P451L mutation in the cytoplasmic domain impairs the function of the mouse P2X7 receptor. J Immunol (Baltimore Md 1950) . (2002) 169:4108–12. doi: 10.4049/jimmunol.169.8.4108

43. North RA. Molecular physiology of P2X receptors. Physiol Rev . (2002) 82:1013–67. doi: 10.1152/physrev.00015.2002

44. Di Virgilio F. The P2Z purinoceptor: an intriguing role in immunity, inflammation and cell death. Immunol Today . (1995) 16:524–8. doi: 10.1016/0167-5699(95)80045-X

45. De Marchi E, Pegoraro A, Turiello R, Di Virgilio F, Morello S, Adinolfi E. A2A receptor contributes to tumor progression in P2X7 null mice. Front Cell Dev Biol . (2022) 10:876510. doi: 10.3389/fcell.2022.876510

46. Arina A, Tirapu I, Alfaro C, Rodrıíguez-Calvillo M, Mazzolini G, Inogés S, et al. Clinical implications of antigen transfer mechanisms from Malignant to dendritic cells: Exploiting cross-priming. Exp Hematology . (2002) 30:1355–64. doi: 10.1016/S0301-472X(02)00956-6

47. Norbury CC, Basta S, Donohue KB, Tscharke DC, Princiotta MF, Berglund P, et al. CD8+ T cell cross-priming via transfer of proteasome substrates. Sci (New York NY) . (2004) 304:1318–21. doi: 10.1126/science.1096378

48. Schulz O, Diebold SS, Chen M, Naslund TI, Nolte MA, Alexopoulou L, et al. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature . (2005) 433:887–92. doi: 10.1038/nature03326

49. MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA, Surprenant A. Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity . (2001) 15:825–35. doi: 10.1016/S1074-7613(01)00229-1

50. Borisenko GG, Matsura T, Liu SX, Tyurin VA, Jianfei J, Serinkan FB, et al. Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells–existence of a threshold. Arch Biochem Biophys . (2003) 413:41–52. doi: 10.1016/S0003-9861(03)00083-3

51. Adinolfi E, Callegari MG, Ferrari D, Bolognesi C, Minelli M, Wieckowski MR, et al. Basal activation of the P2X7 ATP receptor elevates mitochondrial calcium and potential, increases cellular ATP levels, and promotes serum-independent growth. Mol Biol Cell . (2005) 16:3260–72. doi: 10.1091/mbc.e04-11-1025

52. Di Virgilio F, Dal Ben D, Sarti AC, Giuliani AL, Falzoni S. The P2X7 receptor in infection and inflammation. Immunity . (2017) 47:15–31. doi: 10.1016/j.immuni.2017.06.020

53. Sarti AC, Vultaggio-Poma V, Falzoni S, Missiroli S, Giuliani AL, Boldrini P, et al. Mitochondrial P2X7 receptor localization modulates energy metabolism enhancing physical performance. Function . (2021) 2. doi: 10.1093/function/zqab005

54. Brokatzky D, Häcker G. Mitochondria: intracellular sentinels of infections. Med Microbiol Immunol . (2022) 211:161–72. doi: 10.1007/s00430-022-00742-9

55. Ferrick DA, Neilson A, Beeson C. Advances in measuring cellular bioenergetics using extracellular flux. Drug Discovery Today . (2008) 13:268–74. doi: 10.1016/j.drudis.2007.12.008

56. Soare AY, Freeman TL, Min AK, Malik HS, Osota EO, Swartz TH. P2RX7 at the host-pathogen interface of infectious diseases. Microbiol Mol Biol Rev MMBR . (2021) 85. doi: 10.1128/MMBR.00055-20

57. Adinolfi E, Raffaghello L, Giuliani AL, Cavazzini L, Capece M, Chiozzi P, et al. Expression of P2X7 receptor increases in vivo tumor growth. Cancer Res . (2012) 72:2957–69. doi: 10.1158/0008-5472.CAN-11-1947

58. Baroja-Mazo A, Barberà-Cremades M, Pelegrín P. P2X7 receptor activation impairs exogenous MHC class I oligopeptides presentation in antigen presenting cells. PloS One . (2013) 8:e70577. doi: 10.1371/journal.pone.0070577

59. Toki Y, Takenouchi T, Harada H, Tanuma S, Kitani H, Kojima S, et al. Extracellular ATP induces P2X7 receptor activation in mouse Kupffer cells, leading to release of IL-1β, HMGB1, and PGE2, decreased MHC class I expression and necrotic cell death. Biochem Biophys Res Commun . (2015) 458:771–6. doi: 10.1016/j.bbrc.2015.02.011

60. Larsson M, Fonteneau JF, Bhardwaj N. Dendritic cells resurrect antigens from dead cells. Trends Immunol . (2001) 22:141–8. doi: 10.1016/S1471-4906(01)01860-9

61. Arima Y, Liu W, Takahashi Y, Nishikawa M, Takakura Y. Effects of localization of antigen proteins in antigen-loaded exosomes on efficiency of antigen presentation. Mol Pharm . (2019) 16:2309–14. doi: 10.1021/acs.molpharmaceut.8b01093

62. Lombardi M, Gabrielli M, Adinolfi E, Verderio C. Role of ATP in extracellular vesicle biogenesis and dynamics. Front Pharmacol . (2021) 12:654023. doi: 10.3389/fphar.2021.654023

63. Taylor JM, Han Z. Purinergic receptor functionality is necessary for infection of human hepatocytes by hepatitis delta virus and hepatitis B virus. PloS One . (2010) 5:e15784. doi: 10.1371/journal.pone.0015784

64. Mackenzie AB, Young MT, Adinolfi E, Surprenant A. Pseudoapoptosis induced by brief activation of ATP-gated P2X7 receptors. J Biol Chem . (2005) 280:33968–76. doi: 10.1074/jbc.M502705200

65. Aguirre A, Shoji KF, Sáez JC, Henríquez M, Quest AF. FasL-triggered death of Jurkat cells requires caspase 8-induced, ATP-dependent cross-talk between Fas and the purinergic receptor P2X(7). J Cell Physiol . (2013) 228:485–93. doi: 10.1002/jcp.24159

66. Luteijn RD, Praest P, Thiele F, Sadasivam SM, Singethan K, Drijfhout JW, et al. A broad-spectrum antiviral peptide blocks infection of viruses by binding to phosphatidylserine in the viral envelope. Cells . (2020) 9. doi: 10.3390/cells9091989

67. Mercer J, Helenius A. Vaccinia virus uses macropinocytosis and apoptotic mimicry to enter host cells. Sci (New York NY) . (2008) 320:531–5. doi: 10.1126/science.1155164

68. Ichihashi Y, Oie M. The activation of vaccinia virus infectivity by the transfer of phosphatidylserine from the plasma membrane. Virology . (1983) 130:306–17. doi: 10.1016/0042-6822(83)90085-5

69. Segawa K, Nagata S. An apoptotic ‘Eat me’ Signal: phosphatidylserine exposure. Trends Cell Biol . (2015) 25:639–50. doi: 10.1016/j.tcb.2015.08.003

70. Rausch L, Flaskamp L, Ashokkumar A, Trefzer A, Ried C, Buchholz VR, et al. Phosphatidylserine-positive extracellular vesicles boost effector CD8+ T cell responses during viral infection. Proc Natl Acad Sci . (2023) 120:e2210047120. doi: 10.1073/pnas.2210047120

71. Blander JM. Regulation of the cell biology of antigen cross-presentation. Annu Rev Immunol . (2018) 36:717–53. doi: 10.1146/annurev-immunol-041015-055523

72. Nair-Gupta P, Baccarini A, Tung N, Seyffer F, Florey O, Huang Y, et al. TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation. Cell . (2014) 158:506–21. doi: 10.1016/j.cell.2014.04.054

73. Qu Y, Franchi L, Nunez G, Dubyak GR. Nonclassical IL-1 beta secretion stimulated by P2X7 receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages. J Immunol (Baltimore Md 1950) . (2007) 179:1913–25. doi: 10.4049/jimmunol.179.3.1913

74. Gu X, Ma Y, Liu Y, Wan Q. Measurement of mitochondrial respiration in adherent cells by Seahorse XF96 Cell Mito Stress Test. STAR Protoc . (2021) 2:100245. doi: 10.1016/j.xpro.2020.100245

75. Amoroso F, Falzoni S, Adinolfi E, Ferrari D, Di Virgilio F. The P2X7 receptor is a key modulator of aerobic glycolysis. Cell Death Dis . (2012) 3:e370. doi: 10.1038/cddis.2012.105

76. Borges da Silva H, Beura LK, Wang H, Hanse EA, Gore R, Scott MC, et al. The purinergic receptor P2RX7 directs metabolic fitness of long-lived memory CD8+ T cells. Nature . (2018) 559:264–8. doi: 10.1038/s41586-018-0282-0

77. Chang C-W, Li H-C, Hsu C-F, Chang C-Y, Lo S-Y. Increased ATP generation in the host cell is required for efficient vaccinia virus production. J Biomed Science . (2009) 16:80. doi: 10.1186/1423-0127-16-80

78. Elliott MR, Chekeni FB, Trampont PC, Lazarowski ER, Kadl A, Walk SF, et al. Nucleotides released by apoptotic cells act as a find-me signal to promote phagocytic clearance. Nature . (2009) 461:282–6. doi: 10.1038/nature08296

79. Amores-Iniesta J, Barberà-Cremades M, Martínez CM, Pons JA, Revilla-Nuin B, Martínez-Alarcón L, et al. Extracellular ATP activates the NLRP3 inflammasome and is an early danger signal of skin allograft rejection. Cell Rep . (2017) 21:3414–26. doi: 10.1016/j.celrep.2017.11.079

80. Yip L, Woehrle T, Corriden R, Hirsh M, Chen Y, Inoue Y, et al. Autocrine regulation of T-cell activation by ATP release and P2X7 receptors. FASEB J . (2009) 23:1685–93. doi: 10.1096/fj.08-126458

81. Munger J, Bajad SU, Coller HA, Shenk T, Rabinowitz JD. Dynamics of the cellular metabolome during human cytomegalovirus infection. PloS pathogens . (2006) 2:e132. doi: 10.1371/journal.ppat.0020132

82. Chambers JW, Maguire TG, Alwine JC. Glutamine metabolism is essential for human cytomegalovirus infection. J Virol . (2010) 84:1867–73. doi: 10.1128/JVI.02123-09

83. Brokatzky D, Dörflinger B, Haimovici A, Weber A, Kirschnek S, Vier J, et al. A non-death function of the mitochondrial apoptosis apparatus in immunity. EMBO J . (2019) 38. doi: 10.15252/embj.2018100907

84. Yu Y, Feng S, Wei S, Zhong Y, Yi G, Chen H, et al. Extracellular ATP activates P2X7R-NF-κB (p65) pathway to promote the maturation of bone marrow-derived dendritic cells of mice. Cytokine . (2019) 119:175–81. doi: 10.1016/j.cyto.2019.03.019

85. De Marchi E, Orioli E, Pegoraro A, Sangaletti S, Portararo P, Curti A, et al. The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment. Oncogene . (2019) 38:3636–50. doi: 10.1038/s41388-019-0684-y

86. Staib C, Drexler I, Sutter G. Construction and isolation of recombinant MVA. Methods Mol Biol (Clifton NJ) . (2004) 269:77–100. doi: 10.1385/1-59259-789-0:077

87. Thiele F, Tao S, Zhang Y, Muschaweckh A, Zollmann T, Protzer U, et al. Modified vaccinia virus Ankara-infected dendritic cells present CD4+ T-cell epitopes by endogenous major histocompatibility complex class II presentation pathways. J Virol . (2015) 89:2698–709. doi: 10.1128/JVI.03244-14

Keywords: Modified Vaccinia Virus Ankara, cross-presentation, P2RX7, extracellular vesicles, cytokines

Citation: Longo Y, Mascaraque SM, Andreacchio G, Werner J, Katahira I, De Marchi E, Pegoraro A, Lebbink RJ, Köhrer K, Petzsch P, Tao R, Di Virgilio F, Adinolfi E and Drexler I (2024) The purinergic receptor P2X7 as a modulator of viral vector-mediated antigen cross-presentation. Front. Immunol. 15:1360140. doi: 10.3389/fimmu.2024.1360140

Received: 22 December 2023; Accepted: 05 April 2024; Published: 22 April 2024.

Reviewed by:

Copyright © 2024 Longo, Mascaraque, Andreacchio, Werner, Katahira, De Marchi, Pegoraro, Lebbink, Köhrer, Petzsch, Tao, Di Virgilio, Adinolfi and Drexler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Ingo Drexler, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

Cross-presentation by dendritic cells

Affiliation.

• 1 INSERM U932, 26 Rue d'Ulm, 75005 Paris, France.
• PMID: 22790179
• DOI: 10.1038/nri3254

The presentation of exogenous antigens on MHC class I molecules, known as cross-presentation, is essential for the initiation of CD8(+) T cell responses. In vivo, cross-presentation is mainly carried out by specific dendritic cell (DC) subsets through an adaptation of their endocytic and phagocytic pathways. Here, we summarize recent advances in our understanding of the intracellular mechanisms of cross-presentation and discuss its role in immunity and tolerance in the context of specialization between DC subsets. Finally, we review current strategies to use cross-presentation for immunotherapy.

Publication types

• Research Support, Non-U.S. Gov't
• CD8-Positive T-Lymphocytes / cytology
• CD8-Positive T-Lymphocytes / immunology*
• Cross-Priming / immunology*
• Dendritic Cells / cytology
• Dendritic Cells / immunology*
• Histocompatibility Antigens Class I / immunology
• Immune Tolerance / immunology
• Histocompatibility Antigens Class I

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Positive 11 Month Topline Efficacy Data Showing Significant Clinical Improvement from enVVeno Medical's VenoValve(R) Pivotal Trial to be Presented Today at the 46th Annual Charing Cross Symposium

Published: Apr 24, 2024

• 9.29 Points for Patients at the Two-Year Milestone
• 8.08 Points for Patients at the One-Year Milestone
• 8.71 Points for Patients at the Six-Month Milestone
• 72% of the Study Patients Showing Clinical Meaningful Benefit from the VenoValve at a Weighted Average of 11 Months Post Surgery
• 94% of VenoValve Study Patients Showing Clinical Improvement at a Weighted Average of Eleven Months Post Surgery (rVCSS Improvement ≥ 1 point)
• Company on Track to File Application Seeking VenoValve FDA Approval in Q4 2024

IRVINE, CA / ACCESSWIRE / April 24, 2024 / enVVeno Medical Corporation (NASDAQ:NVNO) ("enVVeno" or the "Company"), a company setting new standards of care for the treatment of venous disease, today announced the presentation of positive topline efficacy data showing significant clinical improvement from the SAVVE U.S. pivotal trial for the VenoValve at the 2024 Charing Cross International Symposium in London, UK.

The data being presented shows that for patients experiencing a Clinical Meaningful Benefit (Revised Venous Clinical Severity Score (rVCSS) improvement ≥ 3 points), the overall average rVCSS improvement was 8.46 points, including 9.29 points for patients at the two-year milestone, 8.08 points for patients at the one-year milestone, and 8.71 points for patients at the six-month milestone. All rVCSS evaluations were based on the patient's most recent clinical visit, compared to baseline, for a weighted average of eleven months following VenoValve implantation for the Clinical Meaningful Benefit patient cohort.

Overall, 94% of the study patients receiving the VenoValve have shown clinical improvement as measured by rVCSS at a weighted-average patient follow-up of 11.04 months for the clinical improvement cohort, and 72% of the study patients have improved the three or more rVCSS points needed to demonstrate the VenoValve's Clinical Meaningful Benefit, at a weighted-average patient follow-up of 11.64 months for the Clinical Meaningful Benefit cohort. Total patient follow-up was 762 months and 582 months, respectively, for the two patient cohorts.

"To see patients with a more than 9-point average rVCSS improvement at 24 months post VenoValve surgery is extremely encouraging and exceeds our expectations," said Robert Berman, enVVeno Medical's CEO. "While we would have been satisfied with merely maintaining the clinical improvement levels demonstrated at six-months as patients approach the one-year and two-year post-surgery milestones, instead we are seeing even higher levels of clinical improvement. It is so exciting to be achieving what was previously thought to be impossible, and to be continually raising the bar for the potential of the VenoValve. We are hopeful and determined to bring relief with the VenoValve to the millions of patients suffering from severe deep venous CVI, who have no effective treatment options."

The rVCSS is an objective grading system used by vascular specialists throughout the world to report clinical outcomes and responses to treatments for venous diseases such as Chronic Venous Insufficiency (CVI). The score consists of 10 categories graded from 0 to 3 and includes patient reported outcomes and physician assessments.

In assessing the benefit and risk of a novel technology such as the VenoValve, which addresses an unmet medical need, the FDA considers a variety of factors including whether a medical device provides a clinical meaningful benefit compared to existing technologies. Patients who were enrolled in the SAVVE trial all showed little or no improvement after at least three months of conventional treatment with existing technologies (compression therapy, leg elevation, and wound care for venous ulcer patients). For severe CVI patients, an improvement in the rVCSS of 3 or more points is considered by the FDA to be evidence of clinical meaningful benefit.

Severe CVI is a debilitating disease that is most often caused by blood clots (deep vein thromboses or DVTs) in the deep veins of the leg. When valves inside of the veins of the leg fail, blood flows in the wrong direction and pools in the lower leg, causing pressure within the veins of the leg to increase (venous hypertension). Symptoms of severe CVI include leg swelling, pain, edema, and in the most severe cases, recurrent open sores known as venous ulcers. The disease can severely impact everyday functions such as sleeping, bathing, and walking, and is known to result in high rates of depression and anxiety. There are currently no effective treatments for severe CVI of the deep vein system caused by valvular incompetence and the Company estimates that there are approximately 2.5 million new patients each year in the U.S. that could be candidates for the VenoValve.

The FDA has asked the Company to collect a minimum of one year of data on all SAVVE patients prior to filing its PMA application seeking FDA approval, which the Company expects to have completed collecting in September of 2024. As of December 31, 2023, the Company had cash and investments of $46.4 million on hand, which the Company expects to be sufficient capital to fund operations through an FDA decision on the VenoValve and the end of 2025.

The S urgical A nti-reflux V enous V alve E ndoprosthesis (SAVVE) U.S. pivotal study for the VenoValve is a prospective, non-blinded, single arm, multi-center study of seventy-five (75) CVI patients enrolled at 21 U.S. sites. The presentation, entitled Efficacy Results of the SAVVE Trial: Long-term Results for Use of a Bioprosthetic Valve for Patients with Chronic Deep Venous Reflux, will be made by primary investigator Dr. David Dexter, Sentara Hospital, Norfolk, Virginia and Associate Professor of Surgery, Eastern Virginia Medical School. A copy of the VenoValve CX Symposium slides will be made available after the presentation on Company's website.

About enVVeno Medical Corporation enVVeno Medical (NASDAQ:NVNO) is an Irvine, California-based, late clinical-stage medical device Company focused on the advancement of innovative bioprosthetic (tissue-based) solutions to improve the standard of care for the treatment of venous disease. The Company's lead product, the VenoValve®, is a first-in-class surgical replacement venous valve being developed for the treatment of deep venous Chronic Venous Insufficiency (CVI). The Company is also developing a non-surgical, transcatheter based replacement venous valve for the treatment of deep venous CVI called enVVe®. CVI occurs when valves inside of the veins of the leg become damaged, resulting in the backwards flow of blood (reflux), blood pooling in the lower leg, increased pressure in the veins of the leg (venous hypertension) and in severe cases, venous ulcers that are difficult to heal and become chronic. Both the VenoValve and enVVe are designed to act as one-way valves, to help assist in propelling blood up the leg, and back to the heart and lungs. The VenoValve is currently being evaluated in the SAVVE U.S. pivotal trial and the company is currently performing the final testing necessary to seek approval for the enVVe pivotal trial.

Cautionary Note on Forward-Looking Statements This press release and any statements of stockholders, directors, employees, representatives and partners of enVVeno Medical Corporation (the "Company") related thereto contain, or may contain, among other things, certain "forward-looking statements" within the meaning of the Private Securities Litigation Reform Act of 1995. Such forward-looking statements involve significant risks and uncertainties. Such statements may include, without limitation, statements identified by words such as "projects," "may," "will," "could," "would," "should," "believes," "expects," "anticipates," "estimates," "intends," "plans," "potential" or similar expressions. These statements are based upon the current beliefs and expectations of the Company's management and are subject to significant risks and uncertainties, including those detailed in the Company's filings with the Securities and Exchange Commission. Actual results and timing may differ significantly from those set forth or implied in the forward-looking statements. Forward-looking statements involve certain risks and uncertainties that are subject to change based on various factors (many of which are beyond the Company's control). The Company undertakes no obligation to publicly update any forward-looking statements, whether as a result of new information, future presentations or otherwise, except as required by applicable law.

INVESTOR CONTACT: Jenene Thomas, JTC Team, LLC [email protected] (833) 475-8247

SOURCE: enVVeno Medical Corporation

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Did you mean..., diploma of arts and social sciences, art/science collaboration wins waterhouse natural science art prize, southern cross university researchers take our backyard to the world.

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Three projects from Southern Cross University’s VC Flood Recovery Project Scheme will feature in the River Cities Network’s online presentation, 'Splashing around in our backyard', on Wednesday 24 April 2024.

The team, comprising researchers from Southern Cross University and the Richmond Riverkeeper Association, will showcase three of the University’s VC flood recovery projects to the River Cities Network’s global audience.

The projects include:

Talking about the Richmond River - community values for river health in a post-flood environment ( Professor Amanda Reichelt-Brushett )

Create an online data repository to understand how to manage the Richmond River catchment and restore its health ( Associate Professor Adele Wessell )

Create and coordinate an ongoing citizen science program to assess riverine ecosystem health across the catchment ( Mr Brendan Cox )

Established in 2022, the VC Flood Recovery Project Scheme explores the ways we live with a dynamic and highly modified river in our landscape.

"It’s great to see Southern Cross University’s VC Flood Recovery Projects being featured in the River Cities Network presentation series", said Senior Deputy Vice-Chancellor, Professor Mary Spongberg.

"This not only recognises the significance and quality of our researchers’ work, but also takes that research to a worldwide audience. This in turn opens the door to knowledge-sharing with other experts in this important field, and to potential collaborative projects in the future."

The presentation will also highlight the work of Living Lab Northern Rivers , a joint initiative of Southern Cross University, University of Technology Sydney and the NSW Government. Located in the Lismore CBD, Living Lab Northern Rivers provides a space for research and community to meet and create solutions for the region’s future.

Professor Reichelt-Brushett is also conducting research into the Richmond River through the Global Estuaries Monitoring Programme (GEM) , which is investigating pharmaceutical residues in estuaries around the world.

You can join the Splashing around in our backyard presentation through this link .

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Sound solution for struggling coral reefs: new documentary charts Our Changing Planet

Solving our global waste problem: SCU Buzz

Masters degree opens up life-changing opportunities for naturopath, Carla

The Conversation: No, beetroot isn’t vegetable Viagra. But here’s what else it can do

Southern Cross health expert gives marathon runners the edge

The Conversation: ‘It doesn’t matter where you come from’: regional youth orchestras help fight music education inequality

Southern Cross unveils bold campus vision

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The Conversation: Considering taking a weight-loss drug like Ozempic? Here are some potential risks and benefits

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Structural Behaviour of Balloon Type Cross Laminated Timber: Student presentation on April 26

The  GCS Centre for Structural Safety and Resilience (CSSR)  is proud to announce its Monthly Student Presentation event. Please find below the details of this month's Research Students' Presentation Event:

Date and Time: Friday, April 26 from 4 to 5 p.m. (Montreal EST time)

Title:  Structural Behaviour of Balloon Type Cross Laminated Timber

Gayan Kandethanthri, Master’s Student, Department of Building, Civil, and Environmental Engineering, Gina Cody School of Engineering and Computer Science, Concordia University 

Supervisor : Dr. Farah Hafeez

Zoom link details:

https://concordia-ca.zoom.us/j/81701785123 Meeting ID: 817 0178 5123

© Concordia University

Comscore's 2024 State of Digital Commerce Report

Comscore has released its annual, research-based overview of key insights and trends from 2023 on digital e-commerce, mobile shopping, social and how brands are engaging consumers throughout the consumer journey.

Download the report to see the latest analysis on emerging trends and ways that brands are leveraging a cross-platform strategy to drive total awareness and engagement.

Key takeaways include:

• Record $1.3 Trillion spend in 2023
• Desktop growth outpaced mobile growth
• Q4 2023 achieved the highest quarter since Comscore reporting began

Download Report

To get in touch with our team about this report and how we can support your e-commerce growth, click on this link .

The 2024 State of Digital Commerce report was unveiled at ARF Shopper 2024 in Chicago on April 18.

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Highlights from miart 2024, milan’s modern and contemporary art fair.

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Artworks by Italian artists Michelangelo Pistoletto ("Stracci della Pace") and Lucio Fontana on show ... [+] at Miart in Milan, Italy.

The 28th annual edition of the international art fair Miart last week kicked off a month of art, architecture, design and fashion in Italy’s most fashionable city. With 180 galleries from 28 countries showing more than 1,000 artworks, Milan’s international fair has a well-deserved reputation for carefully selected galleries. The fair has made a stellar effort to stand out among the hundreds of annual art fairs that include mammoth fairs like Frieze and Art Basel by focusing on Italian galleries and by taking the unusual decision to have the emerging galleries, rather than the established blue chip galleries, right at the front of the fair.

An artwork by Italian artist Michele Gabriele, during Miart 2024 in Milan, Italy.

The galleries at Miart are vetted by Nicola Ricciardi, the fair director and a panel of judges who choose around half of the applicants. While the focus is on Italy, this year they’ve also accepted a good range of international galleries including Helena Anrather (New York), Galerie Buchholz (Cologne, Berlin), Emanuela Campoli (Paris, Milan), Fabienne Levy (Lausanne, Geneva), Fortes D'Aloia & Gabriel (São Paulo, Rio de Janeiro), Greengrassi (London) and Galerie Neu (Berlin).

Fondazione Prada in Milan, Italy.

For foreign visitors, Miart offers the chance to see the great Italian art galleries that make up half of the fair, plus a wide range of art events around the city. A sensational retrospective show at the stunning Rem Koolhaas designed Fondazione Prada features 49 mostly sculptural works by Pino Pascali, who created so much before dying at age 32 in a motorcycle accident in 1968, the year he represented Italy at the Venice Biennale . And, at Pirelli HangarBicocca , a vast former tire factory, is a monumental Anselm Kiefer installation and two temporary shows: Ground Break by Nari Ward and Call and gather by Chiara Camoni.

Huawei s Pura 70 Ultra Beats iPhone With Pioneering New Feature

Sh gun episode 10 review a powerful finale but not what i was expecting, trump ally warned ex-president about keeping classified docs—over a year before indictment, fbi filing says.

David Hockney, Carribbean Tea Time, 1987 at the Lelongi gallery, Miart 2024

Among the big ticket items at Miart this year were works by Italian artists like Giorgio de Chirico, Michelangelo Pistoletto, Lucio Fontana, Alighiero Boetti and Carribbean Tea Time , a gorgeous painted screen by David Hockney that sold for US$480,000 within the first two hours of the fair opening.

Francesco Arena, Swing, for Galleria Raffaella Cortese (Milan) Miart 2024

One of the most memorable booths was the Milanese Galleria Raffaella Cortese with a single artwork, a bronze swing that visitors were invited to try out. Created by Francesco Arena, the swing was available in an edition of three, all of which were sold the first day for $US 35,000 each.

Alexis Soul-Gray in the Emergent section at Miart, 2024

In the Emergent section, a solo presentation by Alexis Soul-Gray was a real standout at Los Angeles gallery Bel Ami’s booth. The UK-based artist’s works combine painting, drawing and collage, using imagery from Italian Renaissance painting and advertisements idealizing family life from popular British magazines.

Galerie Buchholz, (Cologne - Berlin - New York) won Best booth at Miart

The Herno Prize, a 10,000-euro prize, for the art fair’s best booth went to Galerie Buchholz, (Cologne - Berlin - New York), with a well thought out presentation of works by Paul Thek and Isa Genzken, Lukas Duwenhögger and Lutz Bacher.

Robert Smithson: Spiral Jetty, Utah,1970, Vintage Gelatine Silver Print, from Repetto Gallery, ... [+] Lugano at Miart

At a painting-heavy fair, at Lugano-based gallery Repetto’s stand, it was good to be reminded of Robert Smithson’s monumental 1970 installation Spiral Jetty , brilliantly captured on film by the late, great photojournalist, Gianfranco Gorgoni.

Installation view of Vivian Suter, “Tintin Nina Disco,” 2024, Kaufmann Repetto gallery at Miart

Tintin Nina disco , an installation of Argentinian artist Vivian Suter’s richly colored, large scale works at the Kaufmann Repetto (Milan – New York) booth was the culmination of decades of exploration into the treasures of the Guatemalan rainforest.

“La Nascita” (“The birth”), an installation by French artist JR, in Piazza Duca d’Aosta, in front of ... [+] the Central Station, Milan, Italy.

If you arrive by train in Milan this month, you’ll be greeted by a spectacular installation by French street artist JR French artist. La Nascita just outside Milan Central Station consists of a series of huge printed images of rock formations plastered onto aluminum slats and is on view until 1 May 2024.

Kutnia fabric covered pillars at Interni Cross Vision

A free public design exhibition at the University of Milan is another must visit this month. Interni Cross Vision , hosted by Interni magazine, is on until 28 April in and above the glorious historic courtyard of the University. Over 40 architecture and interior design exhibitions and installations showcase the best of Italian and international design including 70 designers from Brazil and from Turkey, Kutnia ’s Weaving Inside Out, a dramatic draping of the imposing columns with luxurious swathes of striped and Ikat patterned fabrics.

Practicalities

British Airways and Ita Airways have numerous daily flights to Milan Linate airport, a smaller and delightfully easy to navigate airport, only 20 minutes from the city center.

Where to Stay

Hotel Principe di Savoia, Milan

For a luxury stay in Milan, an excellent option in the Porta Nuova district, is the super stylish Principe di Savoia , part of the Dorchester Collection. For smaller budgets, the four star NH Milano City Life is right beside the art fair and the metro or in the city center, Sonder Missori is a chic choice.

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• Published: 31 March 2021

TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming

• Gaëtan Barbet   ORCID: orcid.org/0000-0002-0152-3650 1 , 2   na1   nAff15 ,
• Priyanka Nair-Gupta 3 , 4   na1   nAff16 ,
• Michael Schotsaert 5 , 6 ,
• Stephen T. Yeung 7   nAff17 ,
• Julien Moretti   ORCID: orcid.org/0000-0002-3415-9058 1 , 2 ,
• Fabian Seyffer 8 ,
• Giorgi Metreveli 5 , 6 ,
• Thomas Gardner 9   nAff18 ,
• Angela Choi 5 , 6   nAff19 ,
• Domenico Tortorella 6 ,
• Robert Tampé   ORCID: orcid.org/0000-0002-0403-2160 10 ,
• Kamal M. Khanna   ORCID: orcid.org/0000-0002-9328-3817 8 , 11 ,
• Adolfo García-Sastre   ORCID: orcid.org/0000-0002-6551-1827 4 , 5 , 6 , 7 &
• J. Magarian Blander   ORCID: orcid.org/0000-0001-9207-1700 1 , 2 , 12 , 13 , 14  

Nature Immunology volume  22 ,  pages 497–509 ( 2021 ) Cite this article

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• Infectious diseases
• MHC class I
• Viral infection

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8 + T cells. Priming CD8 + T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8 + T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor–regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER–Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment–dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8 + T cell priming.

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Data availability.

Source data and uncropped immunoblot images are provided with this paper. All other data supporting the findings of the paper are available from the corresponding author upon reasonable request.

Blum, J. S., Wearsch, P. A. & Cresswell, P. Pathways of antigen processing. Annu. Rev. Immunol. 31 , 443–473 (2013).

Article   CAS   PubMed   PubMed Central   Google Scholar  

Neefjes, J., Jongsma, M. L., Paul, P. & Bakke, O. Towards a systems understanding of MHC class I and MHC class II antigen presentation. Nat. Rev. Immunol. 11 , 823–836 (2011).

Article   CAS   Google Scholar  

Blees, A. et al. Structure of the human MHC-I peptide-loading complex. Nature 551 , 525–528 (2017).

Article   CAS   PubMed   Google Scholar  

Mayerhofer, P. U. & Tampe, R. Antigen translocation machineries in adaptive immunity and viral immune evasion. J. Mol. Biol. 427 , 1102–1118 (2015).

Praest, P., Liaci, A. M., Forster, F. & Wiertz, E. New insights into the structure of the MHC class I peptide-loading complex and mechanisms of TAP inhibition by viral immune evasion proteins. Mol. Immunol. 113 , 103–114 (2019).

Blander, J. M. Regulation of the cell biology of antigen cross-presentation. Annu. Rev. Immunol. 36 , 717–753 (2018).

Ackerman, A. L., Kyritsis, C., Tampe, R. & Cresswell, P. Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens. Proc. Natl Acad. Sci. USA 100 , 12889–12894 (2003).

Delamarre, L., Holcombe, H. & Mellman, I. Presentation of exogenous antigens on major histocompatibility complex (MHC) class I and MHC class II molecules is differentially regulated during dendritic cell maturation. J. Exp. Med. 198 , 111–122 (2003).

Nair-Gupta, P. et al. TLR signals induce phagosomal MHC-I delivery from the endosomal recycling compartment to allow cross-presentation. Cell 158 , 506–521 (2014).

Montealegre, S., Abramova, A., Manceau, V., de Kanter, A.F. & van Endert, P. The role of MHC class I recycling and Arf6 in cross-presentation by murine dendritic cells. Life Sci. Alliance 2 , e201900464 (2019).

Article   PubMed   PubMed Central   Google Scholar  

Oliveira, C. C. & van Hall, T. Alternative antigen processing for MHC class I: multiple roads lead to Rome. Front. Immunol. 6 , 298 (2015).

Rock, K. L. & Shen, L. Cross-presentation: underlying mechanisms and role in immune surveillance. Immunol. Rev. 207 , 166–183 (2005).

Sengupta, D., Graham, M., Liu, X. & Cresswell, P. Proteasomal degradation within endocytic organelles mediates antigen cross-presentation. EMBO J. 38 , e99266 (2019).

Cruz, F. M., Colbert, J. D., Merino, E., Kriegsman, B. A. & Rock, K. L. The biology and underlying mechanisms of cross-presentation of exogenous antigens on MHC-I molecules. Annu. Rev. Immunol. 35 , 149–176 (2017).

Heath, W. R. & Carbone, F. R. Cross-presentation in viral immunity and self-tolerance. Nat. Rev. Immunol. 1 , 126–134 (2001).

Joffre, O. P., Segura, E., Savina, A. & Amigorena, S. Cross-presentation by dendritic cells. Nat. Rev. Immunol. 12 , 557–569 (2012).

Gutiérrez-Martínez, E. et al. Cross-presentation of cell-associated antigens by MHC class I in dendritic cell subsets. Front. Immunol. 6 , 363 (2015).

Helft, J. et al. Cross-presenting CD103 + dendritic cells are protected from influenza virus infection. J. Clin. Investig. 122 , 4037–4047 (2012).

Silvin, A. et al. Constitutive resistance to viral infection in human CD141 + dendritic cells. Sci. Immunol. 2 , eaai8071 (2017).

Sigal, L. J., Crotty, S., Andino, R. & Rock, K. L. Cytotoxic T-cell immunity to virus-infected non-haematopoietic cells requires presentation of exogenous antigen. Nature 398 , 77–80 (1999).

Whitney, P. G. et al. Effective priming of herpes simplex virus-specific CD8 + T cells in vivo does not require infected dendritic cells. J. Virol. 92 , e01508–e01517 (2018).

Donaldson, J. G. & Williams, D. B. Intracellular assembly and trafficking of MHC class I molecules. Traffic 10 , 1745–1752 (2009).

Nebenfuhr, A., Ritzenthaler, C. & Robinson, D. G. Brefeldin A: deciphering an enigmatic inhibitor of secretion. Plant Physiol. 130 , 1102–1108 (2002).

Cebrian, I. et al. Sec22b regulates phagosomal maturation and antigen crosspresentation by dendritic cells. Cell 147 , 1355–1368 (2011).

Hewitt, E. W. The MHC class I antigen presentation pathway: strategies for viral immune evasion. Immunology 110 , 163–169 (2003).

Noriega, V., Redmann, V., Gardner, T. & Tortorella, D. Diverse immune evasion strategies by human cytomegalovirus. Immunol. Res. 54 , 140–151 (2012).

Shen, L., Sigal, L. J., Boes, M. & Rock, K. L. Important role of cathepsin S in generating peptides for TAP-independent MHC class I crosspresentation in vivo. Immunity 21 , 155–165 (2004).

Tiwari, N. et al. A transporter associated with antigen-processing independent vacuolar pathway for the MHC class I-mediated presentation of endogenous transmembrane proteins. J. Immunol. 178 , 7932–7942 (2007).

Bertholet, S. et al. Leishmania antigens are presented to CD8 + T cells by a transporter associated with antigen processing-independent pathway in vitro and in vivo. J. Immunol. 177 , 3525–3533 (2006).

Merzougui, N., Kratzer, R., Saveanu, L. & van Endert, P. A proteasome-dependent, TAP-independent pathway for cross-presentation of phagocytosed antigen. EMBO Rep. 12 , 1257–1264 (2011).

Lawand, M., Abramova, A., Manceau, V., Springer, S. & van Endert, P. TAP-dependent and -independent peptide import into dendritic cell phagosomes. J. Immunol. 197 , 3454–3463 (2016).

Blander, J. M. & Medzhitov, R. Toll-dependent selection of microbial antigens for presentation by dendritic cells. Nature 440 , 808–812 (2006).

Huang, A. Y., Bruce, A. T., Pardoll, D. M. & Levitsky, H. I. In vivo cross-priming of MHC class I-restricted antigens requires the TAP transporter. Immunity 4 , 349–355 (1996).

Lankat-Buttgereit, B. & Tampe, R. The transporter associated with antigen processing: function and implications in human diseases. Physiol. Rev. 82 , 187–204 (2002).

Halenius, A. & Hengel, H. Human cytomegalovirus and autoimmune disease. Biomed. Res. Int. 2014 , 472978 (2014).

Fu, Y., Yan, G., Shi, L. & Faustman, D. Antigen processing and autoimmunity. Evaluation of mRNA abundance and function of HLA-linked genes. Ann. N. Y. Acad. Sci. 842 , 138–155 (1998).

Aldrich, C. J. et al. Positive selection of self- and alloreactive CD8 + T cells in Tap-1 mutant mice. Proc. Natl Acad. Sci. USA 91 , 6525–6528 (1994).

Van Kaer, L., Ashton-Rickardt, P. G., Ploegh, H. L. & Tonegawa, S. TAP1 mutant mice are deficient in antigen presentation, surface class I molecules, and CD4 – 8 + T cells. Cell 71 , 1205–1214 (1992).

Behar, S. M., Dascher, C. C., Grusby, M. J., Wang, C. R. & Brenner, M. B. Susceptibility of mice deficient in CD1D or TAP1 to infection with Mycobacterium tuberculosis . J. Exp. Med. 189 , 1973–1980 (1999).

Alloatti, A. et al. Critical role for Sec22b-dependent antigen cross-presentation in antitumor immunity. J. Exp. Med. 214 , 2231–2241 (2017).

Wu, S. J. et al. A critical analysis of the role of SNARE protein SEC22B in antigen cross-presentation. Cell Rep. 19 , 2645–2656 (2017).

Sigal, L. J. & Rock, K. L. Bone marrow–derived antigen-presenting cells are required for the generation of cytotoxic T lymphocyte responses to viruses and use transporter associated with antigen presentation (Tap)-dependent and -independent pathways of antigen presentation. J. Exp. Med. 192 , 1143–1150 (2000).

Geng, J., Zaitouna, A. J. & Raghavan, M. Selected HLA-B allotypes are resistant to inhibition or deficiency of the transporter associated with antigen processing (TAP). PLoS Pathog. 14 , e1007171 (2018).

Cerundolo, V. & de la Salle, H. Description of HLA class I- and CD8-deficient patients: insights into the function of cytotoxic T lymphocytes and NK cells in host defense. Semin. Immunol. 18 , 330–336 (2006).

de la Salle, H. et al. HLA class I deficiencies due to mutations in subunit 1 of the peptide transporter TAP1. J. Clin. Investig. 103 , R9–R13 (1999).

Article   PubMed   Google Scholar  

Garrido, G. et al. Tumor-targeted silencing of the peptide transporter TAP induces potent antitumor immunity. Nat. Commun. 10 , 3773 (2019).

Marijt, K. A., Van Der Burg, S. H. & van Hall, T. TEIPP peptides: exploration of unTAPped cancer antigens. Oncoimmunology 8 , 1599639 (2019).

Marijt, K. A. & van Hall, T. To TAP or not to TAP: alternative peptides for immunotherapy of cancer. Curr. Opin. Immunol. 64 , 15–19 (2020).

Uger, R. A. & Barber, B. H. Presentation of an influenza nucleoprotein epitope incorporated into the H-2Db signal sequence requires the transporter-associated with antigen presentation. J. Immunol. 158 , 685–692 (1997).

Zhong, W., Reche, P. A., Lai, C. C., Reinhold, B. & Reinherz, E. L. Genome-wide characterization of a viral cytotoxic T lymphocyte epitope repertoire. J. Biol. Chem. 278 , 45135–45144 (2003).

Perez, O. A. et al. CD169 + macrophages orchestrate innate immune responses by regulating bacterial localization in the spleen. Sci. Immunol. 2 , eaah5520 (2017).

Suchin, E. J. et al. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question. J. Immunol. 166 , 973–981 (2001).

Drutman, S. B. & Trombetta, E. S. Dendritic cells continue to capture and present antigens after maturation in vivo. J. Immunol. 185 , 2140–2146 (2010).

Moltedo, B., Li, W., Yount, J. S. & Moran, T. M. Unique type I interferon responses determine the functional fate of migratory lung dendritic cells during influenza virus infection. PLoS Pathog. 7 , e1002345 (2011).

Rodriguez-Madoz, J. R. et al. Inhibition of the type I interferon response in human dendritic cells by dengue virus infection requires a catalytically active NS2B3 complex. J. Virol. 84 , 9760–9774 (2010).

Noriega, V. M. et al. Human cytomegalovirus modulates monocyte-mediated innate immune responses during short-term experimental latency in vitro. J. Virol. 88 , 9391–9405 (2014).

Parham, P., Barnstable, C. J. & Bodmer, W. F. Use of a monoclonal antibody (W6/32) in structural studies of HLA-A,B,C, antigens. J. Immunol. 123 , 342–349 (1979).

Bolte, S. & Cordelieres, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224 , 213–232 (2006).

Husebye, H. et al. The Rab11a GTPase controls Toll-like receptor 4-induced activation of interferon regulatory factor-3 on phagosomes. Immunity 33 , 583–596 (2010).

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Acknowledgements

We thank V. Gillespie for expert pathology on mouse lung tissues. We thank S. Trombetta (Boehringer Ingelheim); K. Rock (University of Massachusetts); P. Cresswell (Yale University); and W. Li, T. M. Moran, D. B. Rubiov and A. Fernandez-Sesma (Icahn School of Medicine at Mount Sinai) for reagents and technical advice. We are grateful to current Blander laboratory members and to H. Gupta, M. A. Blander and S. J. Blander for discussions and support. The ISMMS-Microscopy Shared Resource Facility was supported by grants no. NIH 5R24 CA095823-04, no. S10 RR0 9145-01 and no. NSF DBI-9724504. This work was supported by NIH grants no. AI073899 and no. AI123284 to J.M.B.; an NIH/NIAID Center for Research on Influenza Pathogenesis contract as part of the CEIRS Network no. HHSN266200700010C to A.G.-S., grant nos. AI101820 and AI112318 to D.T., and grant no. AI143861 to K.M.K.; German Research Foundation grants no. SFB 807–Membrane Transport and Communication and no. TA157/7; and by the European Research Council (ERC Advanced Grant no. 789121) to R.T. Support to J.M.B. was also provided by NIH grants no. DK111862 and no. AI127658, the Burroughs Wellcome Fund, and the Leukemia and Lymphoma Society. G.B. has been supported by a fellowship and is currently supported by a career development award from the Crohn’s and Colitis Foundation. T.G. was supported by an American Heart Association pre-doctoral fellowship and NIH grant no. F32CA224438.

Author information

Gaëtan Barbet

Present address: The Child Health Institute of New Jersey, and Department of Pediatrics, Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA

Priyanka Nair-Gupta

Present address: Janssen Research and Development LLC, Spring House, PA, USA

Stephen T. Yeung

Present address: Division of Infectious Disease, Department of Medicine, Weill Cornell Medicine, New York, NY, USA

Thomas Gardner

Present address: ArsenalBio, San Francisco, CA, USA

Angela Choi

Present address: Moderna Inc., Cambridge, MA, USA

These authors contributed equally: Gaëtan Barbet, Priyanka Nair-Gupta.

Authors and Affiliations

The Jill Roberts Institute for Research in Inflammatory Bowel Disease, Weill Cornell Medicine, Cornell University, New York, NY, USA

Gaëtan Barbet, Julien Moretti & J. Magarian Blander

Division of Gastroenterology and Hepatology, Department of Medicine, Weill Cornell Medicine, New York, NY, USA

Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Priyanka Nair-Gupta & Adolfo García-Sastre

Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Michael Schotsaert, Giorgi Metreveli, Angela Choi & Adolfo García-Sastre

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Michael Schotsaert, Giorgi Metreveli, Angela Choi, Domenico Tortorella & Adolfo García-Sastre

The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

Stephen T. Yeung & Adolfo García-Sastre

Perlmutter Cancer Center, New York University Langone Health, New York, NY, USA

Fabian Seyffer & Kamal M. Khanna

Molecular Pharmacology and Chemistry Program, Sloan Kettering Institute, New York, NY, USA

Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt am Main, Germany

Robert Tampé

Department of Microbiology, New York University School of Medicine, New York, NY, USA

Kamal M. Khanna

Department of Microbiology and Immunology, Weill Cornell Medicine, New York, NY, USA

J. Magarian Blander

Sandra and Edward Meyer Cancer Center, Weill Cornell Medicine, New York, NY, USA

Immunology and Microbial Pathogenesis Program, Weill Cornell Graduate School of Medical Sciences, Weill Cornell Medicine, New York, NY, USA

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Contributions

G.B., P.N.-G. and J.M.B. designed experiments, directed the study and wrote the manuscript. P.N.-G. and G.B. performed most in vitro and in vivo experiments, respectively, and curated data. M.S., G.M., A.C. and A.G.-S. provided influenza A virus animal model expertise and management and conducted animal weight loss and lung viral titer measurements. F.S. and R.T. prepared and provided recombinant soluble TAP inhibitor US6. J.M. and G.B. performed immunoblots for knockdown validation. T.G. and D.T. provided reagents and methodology for HCMV infections. S.T.Y. and K.M.K. sectioned, stained and imaged lung tissues by confocal microscopy. J.M.B. supervised and conceived of the study.

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Correspondence to J. Magarian Blander .

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Additional information

Peer review information Nature Immunology thanks Malini Raghavan, Scheherazade Sadegh-Nasseri and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended data fig. 1 steady state residual expression of mhc-i at the plasma membrane of tap –/– dc..

a , Confocal micrographs of WT or Tap −⁄− resting DCs stained for cholera toxin B subunit (CTB), H2-K b and ERGIC-53. b , FACS analyses of WT or Tap −⁄− DCs for surface H2-K b . Scale bars represent 10 µm. Data represent at least three independent experiments.

Extended Data Fig. 2 HCMV protein expression is detected only in DC treated with live and not UV-inactivated virus.

Confocal micrographs of human DCs stained for immediate early (IE) viral protein along with CTB, 48h post infection with either HCMV TB40/E or its UV-irradiated counterpart at a MOI=10. Scale bars represent 10 µm. Data represent at least three independent experiments.

Extended Data Fig. 3 Impaired TAP function blocks classic MHC-I presentation but not cross-presentation.

a , Classic MHC-I presentation by WT DCs of SIINFEKL derived from influenza PR/8-OTI virus to OVA-specific CD8 + T cells. DCs were infected with influenza PR/8-OT-I virus for 5h and incubated in the presence of active or inactive US6. b , c , WT and Tap −⁄− DC cross-presentation of SIINFEKL from E. coli -OVA at several time points ( b ), or at 4.5h post-phagocytosis of either E. coli -OVA or 5h post-infection with influenza PR/8-OTI virus ( c ). Data represent at least three independent experiments except for ( b ) depicting two independent experiments. The mean ± SEM are presented and each symbol represents biological replicate. ** P  < 0.01; N.S.= non statistically significant ( P >0.05) using an unpaired two-tailed t -test.

Source data

Extended data fig. 4 classic mhc-i presentation of viral antigen by wt dc is not affected by sec22b or rab11a inhibition..

a , Immunoblots for the expression of β-actin, Sec22b and Rab11a on whole cell extracts prepared from DCs seven days post-differentiation of bone marrow progenitors that had been transduced with lentiviruses encoding for control shRNA or shRNA targeting either Rab11a or Sec22b . Immunoblots were cropped to show indicated proteins. b , WT DC progenitors were transduced with recombinant lentiviruses expressing scrambled, Sec22b or Rab11a specific shRNA. Classic MHC-I presentation of SIINFEKL at 5h after infection with recombinant SIINFEKL-expressing Influenza-OTI (PR/8-OTI) (left panels, no secondary treatment) or cross-presentation of SIINFEKL from heat-inactivated Influenza-OTI given to DCs at 3h following infection by Influenza-OTI virus. Cross-presentation was assessed 2h later. Data represent at least three independent experiments. The mean ± SEM are presented and each symbol represents biological replicate. N.S.= non statistically significant ( P >0.05) using an unpaired two-tailed t -test.

Extended Data Fig. 5 Lung CD8 T cells during lethal challenge with influenza A virus.

a , Percent of influenza A specific CD8 + T cells in the lungs of WT → WT and Tap ⎯ / ⎯ → WT mice at days 3 and 5 post lethal challenge with 75 p.f.u. influenza A PR8 virus. Data show two different H-2D d tetramers loaded with either a polymerase acidic protein epitope (SSLENFRAYV) or a nucleoprotein epitope (ASNENMETM). b , Representative FACS plot and % CD8 + CD44 + cells in the lung of naïve chimeric mice. c , Flow cytometry plots showing CD8 + T cells in the lungs or spleens of WT mice on days 1 and 2 following injection with anti-CD8α antibody (clone 2.43) intranasally (i.n., 200µg), intraperitoneally (i.p., 100µg), or via both routes (i.p.+i.n.). Percent of CD3 + CD8β + T cells remaining are indicated in the blue gates. The mean ± SEM are presented and each symbol represents a mouse. N.S.= non statistically significant ( P >0.05) using an unpaired two-tailed t -test.

Extended Data Fig. 6 CD8 T cells mediate protection of Tap ⎯ / ⎯ chimeric mice against lethal challenge with influenza A virus.

a , Confocal micrographs at 20X magnification of lung sections from indicated anti-CD8α-treated mice stained for CD8α, CD11c, EpCAM and influenza A virus. Legend to the right shows the color code for each antibody. Scale bar represents 50 µm. b , Lung PR8 viral titers at days 3 and 5 in WT → WT and Tap ⎯ / ⎯ → WT chimeric mice. Each symbol is one mouse. c , Pathology scores of infected mice at different time points post lethal PR8 challenge in WT → WT and Tap ⎯ / ⎯ → WT mice. Slides were scored by a blinded pathologist for perivascular, bronchiolar or alveolar inflammation, epithelial degeneration or necrosis, and intraluminal debris or hemorrhage. Each symbol is one mouse. d , Hematoxylin-and-eosin staining of lung sections from WT → WT and Tap ⎯ / ⎯ → WT mice on days 3 or 5 post PR8 challenge. Scale bar represents 100 μm. The mean ± SEM are presented and each symbol represents a mouse. Data represent 2 experiments (for a total n of 140 mice).

Supplementary information

Supplementary information.

Supplementary gating strategy.

Reporting Summary

Supplementary video 1.

A schematic illustrating the cell biology of noncanonical cross-presentation based on the data presented in this study. The model phagocytic cargo shown here is a bacterium that engages TLR signaling. Based on previous work, two pathways of vesicular traffic contribute to the cross-presenting phagosome: TLR-regulated traffic from the ERC to the phagosome mediated by TLR–MyD88–IKK2-dependent phosphorylation of SNAP23 (not shown, refer to ref. 9 ) which delivers MHC-I molecules, and TLR-independent traffic from the ERGIC to phagosomes requiring the ER-SNARE Sec22b, which delivers the components of the peptide-loading complex, including TAP and Sec61 (refs. 9 , 24 ). Accumulation of MHC-I molecules in the ERGIC upon TAP dysfunction enables their Sec22b-dependent recruitment to phagosomes to prime CD8 + T cells through noncanonical cross-presentation.

Supplementary Video 2

3D reconstruction of confocal stacks showing CD11c + cells with infected epithelial cell inclusions (white merge) in the lungs of WT  →  WT at day 3 post influenza A/PR8 challenge. Staining for CD11c is in red, influenza in green, CD8 in cyan and EpCAM in magenta.

Supplementary Video 3

3D reconstruction of confocal stacks showing CD11c + cells with infected epithelial cell inclusions (white merge) in the lungs of Tap ⎯ / ⎯  → WT at day 3 post influenza A/PR8 challenge. Staining for CD11c is in red, influenza in green, CD8 in cyan and EpCAM in magenta.

Source Data Fig. 1

Numerical data used for plots.

Source Data Fig. 2

Source data fig. 3, source data fig. 4.

Unprocessed immunoblots.

Source Data Fig. 5

Source data fig. 6, source data fig. 7, source data fig. 8, source data extended data fig. 3, source data extended data fig. 4, source data extended data fig. 5, source data extended data fig. 6, rights and permissions.

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Barbet, G., Nair-Gupta, P., Schotsaert, M. et al. TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming. Nat Immunol 22 , 497–509 (2021). https://doi.org/10.1038/s41590-021-00903-7

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Received : 27 April 2020

Accepted : 22 February 2021

Published : 31 March 2021

Issue Date : April 2021

DOI : https://doi.org/10.1038/s41590-021-00903-7

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