- Email: [email protected]
Targeting split vaccines to the endosome improves vaccination
Targeting split vaccines to the endosome improves vaccination Hermann Wagner, Antje Heit, Frank Schmitz and Stefan Bauer Compared to ‘live’ vaccines, the immunogenicity of ‘split’ vaccines based on recombinant antigen (Ag) is poor, presumably because exogeneous recombinant Ag fails to efficiently access the major histocompatibility complex (MHC) class I processing pathway needed for ‘cross-presentation’. Here we discuss recent evidence that targeting ligands of the Toll-like receptor 9 together with proteinaceous Ag to the endosome of dendritic cells conveys immunogenicity to Ag similar in magnitude to ‘live’ vaccines that produce Ag. Enforced endocytosis of Ag together with the adjuvant effect of Toll-like receptor 9 ligands might be key for the efficient cross-presentation of exogeneous Ag as well as for effective cross-priming of MHC class I restricted CD8 T effector cells. Addresses Institute of Medical Microbiology, Immunology and Hygiene, Trogerstrasse. 9, 81675 Munich, Germany e-mail: [email protected]
Current Opinion in Biotechnology 2004, 15:538–542 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Carlos A Guzman and Giora Z Feuerstein Available online 13th October 2004 0958-1669/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.09.006
Abbreviations Ag antigen APC antigen-presenting cell CpG-ODN CpG oligodeoxynucleotide CTL cytotoxic T lymphocyte DC dendritic cell ER endoplasmatic reticulum MHC major histocompatibility complex Ova ovalbumin rec recombinant TLR Toll-like receptor
Introduction Vaccines probably represent one of the greatest successes of modern medicine. This refers primarily to classical vaccines towards, for example, bacterial toxins, measles or polyomyelitis against which neutralizing antibodies are the key to attenuate pathophysiology. By contrast, efficient vaccines are still lacking against tuberculosis, parasitic diseases such as malaria and certain viral diseases such as that caused by human immunodeficiency virus [1,2]. In the latter spectrum of diseases, T-cell-mediated Current Opinion in Biotechnology 2004, 15:538–542
immunoprotection and protective antibodies come into play [3]. Whereas attenuated ‘live’ vaccines often recapitulate aspects of natural infection, such as the transient replication of antigens (Ag) in infected cells, ‘dead’ vaccines based on recombinant (rec) Ag cannot do so. However, many pathogens have developed particular strategies to subvert recognition by immune cells [4]; a problem conceptionally bypassed by using dead vaccines. Although the immunoglobulin receptors of both B cells and secreted antibodies directly recognize complex folded proteins or carbohydrates, T cells only recognize small peptides when presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs). Classical experiments have shown that T cells presenting CD8 markers are specific for fragments of proteins (peptides) that are synthesized and processed by the cell itself. These are subsequently presented by its MHC class I molecules [5]. By contrast, exogenous Ag first needs to be internalized and processed in the endolysosomal compartment of APCs to be presented to T cells by MHC class II molecules (endocytic pathway). Provided these rules are strict, the ‘peptide’ repertoire of exogenous Ag as provided by dead vaccines would not be accessible to MHC class I restricted CD8 T cells. Recent studies in dendritic cells (DCs), however, have shown that in addition to MHC class II, internalized exogenous Ag can also access the MHC class I presentation pathway, a process referred to as ‘cross-presentation’ [6,7]. Immunological memory not only provides long-term protection, but is necessary for robust vaccination. Longterm protection depends on maintenance of memory B and T cells [8]. How, for example, naı¨ve T cells encountering Ag make the decision to develop into either shortlived effector cells (required to control acute infection) or long-lived (central or effector) memory cells (typifying recall responses) is the subject of intensive investigations [9]; for example, recent observations imply that CD8aa expression characterizes CD8 memory T cells [10]. One of the practical implications of such data for vaccine design is that the ‘burst size’ (magnitude) of a primary T-cell response does not per se determine the magnitude of CD8 memory T-cell responses. Several lines of evidence define DCs as the principle, professional APCs both for T cell cross-priming and crosstolerization. While under steady-state conditions receptor-mediated uptake of exogenous Ag favors crosspresentation, which can lead to subsequent deletion www.sciencedirect.com
Targeting split vaccines to the endosome improves vaccination Wagner et al. 539
of Ag-reactive T cells [11], co-administration of a stimulus causing DC maturation triggers ‘cross-priming’ and thus favors induction of primary and memory effector T cells [6,12,13]. Considering the information given above, several checkpoints need to be overcome to design a robust vaccination protocol based upon rec Ag. These include Ag delivery to DCs resulting in improved cross-presentation. In addition, an adjuvant that efficiently activates cross-presenting DCs into professional APCs needs to be chosen that is able to generate long-lasting and protective memory T cells. This review focuses on recent developments that address these checkpoints of vaccination.
MHC class I restricted Ag presentation (cross-presentation) Exogenous Ag internalized by DCs via various pathways can access the MHC class I presentation pathway, yet their respective efficacy differs. Micropinocytosis of solvent Ag allows, for example, receptor-independent crosspresentation of soluble Ag by DCs [14], yet its efficacy is poor. By contrast, receptor-mediated endocytosis is a specific uptake process that is able to efficiently translocate macromolecules, viruses and small particles into the endosomal compartment of DCs. Large particles, however, such as microbial aggressors or apoptotic cells are taken up by actin-dependent phagocytosis, a process often initiated by germline-encoded phagocytotic receptors [15]. Both receptor-mediated endocytosis and phagocytosis shuttle their cargo into a multifaceted developing endosomal compartment. During biogenesis of the phago-endosome, the endoplasmatic reticulum (ER) comes into play; for example, part of the phago-endosomal membrane is donated by the ER in a dynamic process referred to as ‘ER-mediated phagocytosis’ [16]. During ER recruitment to, and fusion with, the developing phagosome/endosome the ER transiently ‘opens’ to the ‘outer space’ potentially translocating ER constituents such as calreticulin, calnexin and newly formed yet still empty MHC class I molecules to the developing endosome. Pioneering work in macrophages and DCs revealed that the efficacy of cross-presentation is strongly increased by linking Ag to latex beads and thus enforcing Ag internalization by phagocytosis [17]. Phagocytosis of bacteria also results in efficient cross-presentation [18], as does phagocytosis of apoptotic cells [19], tumor (melanoma) cells [20] or exosomes secreted by tumor cells [21]. In addition, targeting Ag to Fc receptors (FcRs) to trigger FcR-mediated uptake [22] allows cross-presentation at Ag concentrations three to five log below that needed for fluid phase uptake [23]. Whether receptor-mediated Ag internalization just improves Ag uptake or whether robust cross-presentation requires targeting of Ag to specific endocytotic compartments is not yet known. www.sciencedirect.com
In this context two recent publications have shed some light on the molecular mechanisms why receptormediated targeting of exogenous Ag to the endosome might improve cross-presentation [24,25]. Accordingly, ER fusion with the nascent phago-endosome not only provides ER-derived membrane for building of the phagosome membrane, but in addition confers to the endosome cross-presentation properties including access to (immuno)proteasomes and TAP- (transporter associated with antigen processing) dependent transport of peptides to be loaded onto MHC class I molecules. As a consequence, the phago-endosome not only acquires but displays competence for Ag cross-presentation [24,25].
Adjuvants — properties of ligands of Toll-like receptor family members In the mouse, the Toll-like receptor (TLR) family includes ten proteins (TLR1–9 and TLR11). The TLR family members share common structural features including an ectodomain (containing multiple leucinerich repeats and one cysteine-rich domain), a transmembrane region and a highly conserved cytoplasmic Tollinterleukin 1 receptor (TIR) domain. Ligand binding occurs within the ectodomain, whereas the TIR domain interacts with family members of MyD88 adaptor protein to initiate activation of innate immune cells [26]. Based on amino acid sequence similarities, the TLR family members may be divided into subfamilies. Members of the TLR9 subfamily (TLR7, 8 and 9) recognize pathogen-derived single-stranded RNA [27,28] and DNA motifs [29,30], whereas TLR3 recognizes doublestranded RNA. The bacterial protein flagellin acts as ligand for TLR5 and lipopolysaccharide is the prototypic ligand for TLR4 [31]. Members of the TLR2 subfamily (TLR1, 2, 6) heterodimerize, thus creating specificities for di- or tri-acetylated lipoproteins primarily from Grampositive bacteria [32]. Interestingly, both TLR3 and the TLR9 subfamily members are expressed intracellularly at the endosomal compartment [13], whereas TLR2, 4 and 5 are cell surface bound. It follows that cell surface bound TLRs rapidly sense bacterial infections by recognizing extracellular cell wall components, whereas bacterial or viral DNA or RNA motifs (ligands of the TLR3 or TLR9 subfamily members) are ‘liberated’ in the endosome upon receptor-mediated internalization of the respective pathogen. Alternatively, the respective ligands are generated intracellularly upon cell infection.
The endosome as ‘cross-road’ for Ag and TLR ligands Unmethylated CpG dinucleotides within immunostimulatory DNA sequences are ligands for TLR9 [29,30]. CpG oligodeoxynucleotides (CpG-ODN) mixed with proteinaceous Ag have been shown to act as powerful adjuvants for Th-1 biased immunoresponses and cytotoxic T lymphocyte (CTL) induction, both in murine infection and tumor model systems [33,34]. As CpG-ODN directly Current Opinion in Biotechnology 2004, 15:538–542
540 Pharmaceutical biotechnology
converts cross-presenting immature DCs into professional APCs [35], CpG-ODN ‘aided’ CTL induction basically is T-helper independent. Raz and colleagues were the first to describe a novel CpG-ODN-based vaccine composed of Ag (ovalbumin [Ova]) chemically linked to CpG-ODN [36]. These Ag–CpG conjugates turned out to be more potent in priming CTL activity compared to a mixture of Ag and CpG-ODN. In fact, the power of induction of Th-1-based immunity by Ag–CpG conjugates revealed prophylactic and therapeutic effects in a murine asthma model system. We, and others, have set out to unravel why CpG-DNA conjugated to Ag increases the immunogenicity of Ag 50- to 100-fold when compared to Ag mixed with CpG-ODN [37,38]. Overall, these studies have led to the following conclusions. First, by conjugating CpG-ODN to Ag (Ag–CpG complex) the conjugated CpG-ODN acts as ligand for DNA receptor mediated internalization of the Ag–CpG complex by
DCs. In other words, a currently ill-defined DNA sequence non-specific endocytosis receptor (i.e. uptake can be competed for by ‘third party’ ODN [37]) is utilized by DCs to efficiently translocate the Ag–CpG complex into endosomes. Second, upon endosome formation TLR9 is expressed at the endosome [39,40]. Third, recent evidence implies that during endosome biogenesis ER constituents, including newly formed TLR9 and empty MHC class I molecules, gain access to the endosomal compartment [24,25,40]. Fourth, the MHC class I processing machinery assembles around or gains access to the endosomal compartment [24,25]. Altogether, these dynamic processes donate competence to the endosome for cross-presentation and TLR9-driven DC activation. Upon acidification (maturation) of endolysomes CpG-ODN presumably becomes liberated from the Ag–CpG complex and thus is able to activate the endosomal membrane-bound TLR9 (Figure 1). As a
Figure 1
1
2
3
4
5
Endocytosis
Ag-CpG
Cytokines (IL-12) Peptide-loaded class I MHC
DNA Receptor
61?)
Cross-presentation
Endosomal maturation (acidification)
I cla ss
MHC class I
MyD88 recruitment
M
Delivery of
HC
TL
R9
(T A P? )
(Sec
Upregulation of co-stimulatory molecules
NFκB
p38, AP-1
Gene activation Current Opinion in Biotechnology
Targeting the endosome with Ag–CpG complexes. Schematic illustrating the close relationship between the biogenesis of the DC phago-endosome and the acquisition of competence for cross-presentation. TLR9 signalling drives the maturation of cross-presenting DCs into professional APCs with competence for cross-priming. Phase 1: CpG–Ova complexes bind to a DNA-binding receptor (not TLR9) that mediates internalization of the CpG–Ova complex into the endosome. Phase 2: during biogenesis of the endosome, endoplasmatic reticulum (ER) tubular structures (yellow) contact the cell membrane; the early endosome thus displays a hybrid of ER-derived (yellow) and cell-membrane-derived (blue) membrane. During this process the ER transiently opens, translocating ER constituents such as TLR9 and MHC class I molecules to the endosome. In addition, components of the MHC class I processing machinery including transporter associated with antigen processing (TAP) and the proteasome (green) are recruited to the cytosolic part of the endosome. Sec 61 represents a pore-forming protein and translocates proteins up to 40 kDa. Phase 3: Ag becomes processed, CD8 epitopes are loaded on MHC class I and loaded MHC class I molecules (red triangles) translocate to the membrane. Upon maturation (red colour [acidification]) of the endosome CpG-ODN liberated from the protein activates TLR9, which causes MyD88 recruitment and in turn (Phase 4) triggers the Toll/IL-1 receptor signal pathway. Phase 5: As a consequence, DCs mature into professional APCs by upregulating co-stimulatory molecules and cytokine production. Current Opinion in Biotechnology 2004, 15:538–542
www.sciencedirect.com
Targeting split vaccines to the endosome improves vaccination Wagner et al. 541
consequence, signaling is initiated by recruitment of the MyD88 family members [39,40] to the cytosolic site of the endosome membrane. According to this scenario, the endosome might be regarded as a subcellular site able to develop competence for both cross-presentation and cross-priming, the latter by utilizing the adjuvants effect of TLR9-mediated DC activation. In past work we have compared, for example, the efficacy of live vaccines (Listeria monocytogenes [Lm] transduced to produce Ova or vaccinia virus encoding an Ova cassette) with that of the Ova–CpG complex (as an example of a ‘dead’ vaccine). We quantified cross-priming by enumeration of SIINFEKL- (peptide-) specific T cells via tetramer technology. To our surprise we noted that in mice the primary and secondary burst size (clonal expansion versus contraction) of SIINFEKL-specific CD8 T cells triggered by Ova–CpG conjugate (‘dead vaccine’) was comparable to that caused by Lm–Ova (‘live vaccine’). Furthermore, mice primed with either ‘live’ or ‘dead’ vaccine turned out to be resistant to otherwise lethal doses of Lm–Ova, but not to wild-type Lm (A Heit et al., unpublished).
Conclusions What can we learn from this information in attempts to approve the immunogenicity of ‘dead’ vaccines based on recombinant proteinaceous Ag? First, the potential ‘peptide’ repertoire of exogenous Ag might be best unveiled by enforced internalization via receptormediated endocytosis of Ag by DCs. Second, the use of receptor-mediated endocytosis highlights the endosome as an organelle displaying competence for crosspresentation. Last, by utilizing the adjuvant activities of TLRs expressed at the endosome, cross-presenting immature DCs become activated into professional APCs. These are then able to initiate powerful primary and secondary CD8 T-cell responses. It follows that by targeting exogenous Ag together with an appropriate TLR ligand to the endosome provides robust immunogenicity to otherwise poorly immunogenic rec Ag.
Acknowledgements
5.
Cresswell P, Bangia N, Dick T, Diedrich G: The nature of the MHC class I peptide loading complex. Immunol Rev 1999, 172:21-28.
6.
Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S: Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 2002, 20:621-667.
7.
Bevan MJ: 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 1976, 143:1283-1288.
8.
Schluns KS, Lefrancois L: Cytokine control of memory T-cell development and survival. Nat Rev Immunol 2003, 3:269-279.
9.
Kaech SM, Tan JT, Wherry EJ, Konieczny BT, Surh CD, Ahmed R: Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat Immunol 2003, 4:1191-1198.
10. Madakamutil LT, Christen U, Lena CJ, Wang-Zhu Y, Attinger A, Sundarrajan M, Ellmeier W, von Herrath MG, Jensen P, Littman DR, Cheroutre H: CD8aa-mediated survival and differentiation of CD8 memory T cell precursors. Science 2004, 304:590-593. 11. Steinman RM, Hawiger D, Nussenzweig MC: Tolerogenic dendritic cells. Annu Rev Immunol 2003, 21:685-711. 12. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K: Immunobiology of dendritic cells. Annu Rev Immunol 2000, 18:767-811. 13. Wagner H: The immunobiology of the TLR9 subfamily. Trends Immunol 2004, 25:381-386. 14. Norbury CC, Chambers BJ, Prescott AR, Ljunggren HG, Watts C: Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells. Eur J Immunol 1997, 27:280-288. 15. Underhill DM, Ozinsky A: Phagocytosis of microbes: complexity in action. Annu Rev Immunol 2002, 20:825-852. 16. Gagnon E, Duclos S, Rondeau C, Chevet E, Cameron PH, Steele-Mortimer O, Paiement J, Bergeron JJ, Desjardins M: Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 2002, 110:119-131. This manuscript points out that the ER membrane fuses with the cell membrane during biogenetics of phago-endosomes. 17. Kovacsovics-Bankowski M, Clark K, Benacerraf B, Rock KL: Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. Proc Natl Acad Sci USA 1993, 90:4942-4946. 18. Rescigno M, Citterio S, Thery C, Rittig M, Medaglini D, Pozzi G, Amigorena S, Ricciardi-Castagnoli P: Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells. Proc Natl Acad Sci USA 1998, 95:5229-5234.
The work cited was supported by the Deutsche Forschungsgemeinschaft, ForImmun and the Coley Pharmaceutical Group.
19. Albert ML, Sauter B, Bhardwaj N: Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 1998, 392:86-89.
References and recommended reading
20. Berard F, Blanco P, Davoust J, Neidhart-Berard EM, Nouri-Shirazi M, Taquet N, Rimoldi D, Cerottini JC, Banchereau J, Palucka AK: Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med 2000, 192:1535-1544.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
21. Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, Flament C, Pouzieux S, Faure F, Tursz T et al.: Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 2001, 7:297-303.
1.
Rappuoli R, Miller HI, Falkow S: Medicine. The intangible value of vaccination. Science 2002, 297:937-939.
2.
Levine MM, Sztein MB: Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol 2004, 5:460-464.
3.
Wong P, Pamer EG: CD8 T cell responses to infectious pathogens. Annu Rev Immunol 2003, 21:29-70.
22. Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, Rescigno M, Saito T, Verbeek S, Bonnerot C, Ricciardi-Castagnoli P, Amigorena S: Fcg receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J Exp Med 1999, 189:371-380.
4.
Zinkernagel RM: On natural and artificial vaccinations. Annu Rev Immunol 2003, 21:515-546.
23. Machy P, Serre K, Leserman L: Class I-restricted presentation of exogenous antigen acquired by Fcg receptor-mediated
www.sciencedirect.com
Current Opinion in Biotechnology 2004, 15:538–542
542 Pharmaceutical biotechnology
endocytosis is regulated in dendritic cells. Eur J Immunol 2000, 30:848-857. 24. Guermonprez P, Saveanu L, Kleijmeer M, Davoust J, Van Endert P, Amigorena S: ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. Nature 2003, 425:397-402. [24] and [25] were the first articles to describe endosomes as organelles with competence for cross-presentation. 25. Houde M, Bertholet S, Gagnon E, Brunet S, Goyette G, Laplante A, Princiotta MF, Thibault P, Sacks D, Desjardins M: Phagosomes are competent organelles for antigen cross-presentation. Nature 2003, 425:402-406. See annotation for [24]. 26. Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev Immunol 2003, 21:335-376. 27. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S: Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004, 303:1526-1529. This article and [28] define single-stranded RNA motifs as ligand for TLR7 (mouse) and TLR8 (human). 28. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C: Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004, 303:1529-1531. See annotation for [27].
macrophage phagosomes and discriminates between pathogens. Nature 1999, 401:811-815. 33. Lipford GB, Bauer M, Blank C, Reiter R, Wagner H, Heeg K: CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants. Eur J Immunol 1997, 27:2340-2344. 34. Wagner H: Bacterial CpG DNA activates immune cells to signal infectious danger. Adv Immunol 1999, 73:329-368. 35. Sparwasser T, Koch ES, Vabulas RM, Heeg K, Lipford GB, Ellwart JW, Wagner H: Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol 1998, 28:2045-2054. 36. Cho HJ, Takabayashi K, Cheng PM, Nguyen MD, Corr M, Tuck S, Raz E: Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activity by a T-helper cell-independent mechanism. Nat Biotechnol 2000, 18:509-514. 37. Maurer T, Heit A, Hochrein H, Ampenberger F, O’Keeffe M, Bauer S, Lipford GB, Vabulas RM, Wagner H: CpG-DNA aided cross-presentation of soluble antigens by dendritic cells. Eur J Immunol 2002, 32:2356-2364.
29. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S: A Toll-like receptor recognizes bacterial DNA. Nature 2000, 408:740-745.
38. Heit A, Maurer T, Hochrein H, Bauer S, Huster KM, Busch DH, Wagner H: Cutting edge: Toll-like receptor 9 expression is not required for CpG DNA-aided cross-presentation of DNA-conjugated antigens but essential for cross-priming of CD8 T cells. J Immunol 2003, 170:2802-2805. This manuscript describes that TLR9 is not involved in endocytosis of CpG-DNA.
30. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, Wagner H, Lipford GB: Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA 2001, 98:9237-9242.
39. Ahmad-Nejad P, Hacker H, Rutz M, Bauer S, Vabulas RM, Wagner H: Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol 2002, 32:1958-1968. The first description of endosomally expressed TLR9.
31. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C et al.: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998, 282:2085-2088. 32. Underhill DM, Ozinsky A, Hajjar AM, Stevens A, Wilson CB, Bassetti M, Aderem A: The Toll-like receptor 2 is recruited to
Current Opinion in Biotechnology 2004, 15:538–542
40. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, Lien E, Nilsen NJ, Espevik T, Golenbock DT: TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 2004, 5:190-198. The first work to establish that TLR9 is expressed at the ER in nonactivated DCs.
www.sciencedirect.com
Current Opinion in Biotechnology 2004, 15:538–542 This review comes from a themed issue on Pharmaceutical biotechnology Edited by Carlos A Guzman and Giora Z Feuerstein Available online 13th October 2004 0958-1669/$ – see front matter # 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2004.09.006
Abbreviations Ag antigen APC antigen-presenting cell CpG-ODN CpG oligodeoxynucleotide CTL cytotoxic T lymphocyte DC dendritic cell ER endoplasmatic reticulum MHC major histocompatibility complex Ova ovalbumin rec recombinant TLR Toll-like receptor
Introduction Vaccines probably represent one of the greatest successes of modern medicine. This refers primarily to classical vaccines towards, for example, bacterial toxins, measles or polyomyelitis against which neutralizing antibodies are the key to attenuate pathophysiology. By contrast, efficient vaccines are still lacking against tuberculosis, parasitic diseases such as malaria and certain viral diseases such as that caused by human immunodeficiency virus [1,2]. In the latter spectrum of diseases, T-cell-mediated Current Opinion in Biotechnology 2004, 15:538–542
immunoprotection and protective antibodies come into play [3]. Whereas attenuated ‘live’ vaccines often recapitulate aspects of natural infection, such as the transient replication of antigens (Ag) in infected cells, ‘dead’ vaccines based on recombinant (rec) Ag cannot do so. However, many pathogens have developed particular strategies to subvert recognition by immune cells [4]; a problem conceptionally bypassed by using dead vaccines. Although the immunoglobulin receptors of both B cells and secreted antibodies directly recognize complex folded proteins or carbohydrates, T cells only recognize small peptides when presented by major histocompatibility complex (MHC) molecules on the surface of antigen-presenting cells (APCs). Classical experiments have shown that T cells presenting CD8 markers are specific for fragments of proteins (peptides) that are synthesized and processed by the cell itself. These are subsequently presented by its MHC class I molecules [5]. By contrast, exogenous Ag first needs to be internalized and processed in the endolysosomal compartment of APCs to be presented to T cells by MHC class II molecules (endocytic pathway). Provided these rules are strict, the ‘peptide’ repertoire of exogenous Ag as provided by dead vaccines would not be accessible to MHC class I restricted CD8 T cells. Recent studies in dendritic cells (DCs), however, have shown that in addition to MHC class II, internalized exogenous Ag can also access the MHC class I presentation pathway, a process referred to as ‘cross-presentation’ [6,7]. Immunological memory not only provides long-term protection, but is necessary for robust vaccination. Longterm protection depends on maintenance of memory B and T cells [8]. How, for example, naı¨ve T cells encountering Ag make the decision to develop into either shortlived effector cells (required to control acute infection) or long-lived (central or effector) memory cells (typifying recall responses) is the subject of intensive investigations [9]; for example, recent observations imply that CD8aa expression characterizes CD8 memory T cells [10]. One of the practical implications of such data for vaccine design is that the ‘burst size’ (magnitude) of a primary T-cell response does not per se determine the magnitude of CD8 memory T-cell responses. Several lines of evidence define DCs as the principle, professional APCs both for T cell cross-priming and crosstolerization. While under steady-state conditions receptor-mediated uptake of exogenous Ag favors crosspresentation, which can lead to subsequent deletion www.sciencedirect.com
Targeting split vaccines to the endosome improves vaccination Wagner et al. 539
of Ag-reactive T cells [11], co-administration of a stimulus causing DC maturation triggers ‘cross-priming’ and thus favors induction of primary and memory effector T cells [6,12,13]. Considering the information given above, several checkpoints need to be overcome to design a robust vaccination protocol based upon rec Ag. These include Ag delivery to DCs resulting in improved cross-presentation. In addition, an adjuvant that efficiently activates cross-presenting DCs into professional APCs needs to be chosen that is able to generate long-lasting and protective memory T cells. This review focuses on recent developments that address these checkpoints of vaccination.
MHC class I restricted Ag presentation (cross-presentation) Exogenous Ag internalized by DCs via various pathways can access the MHC class I presentation pathway, yet their respective efficacy differs. Micropinocytosis of solvent Ag allows, for example, receptor-independent crosspresentation of soluble Ag by DCs [14], yet its efficacy is poor. By contrast, receptor-mediated endocytosis is a specific uptake process that is able to efficiently translocate macromolecules, viruses and small particles into the endosomal compartment of DCs. Large particles, however, such as microbial aggressors or apoptotic cells are taken up by actin-dependent phagocytosis, a process often initiated by germline-encoded phagocytotic receptors [15]. Both receptor-mediated endocytosis and phagocytosis shuttle their cargo into a multifaceted developing endosomal compartment. During biogenesis of the phago-endosome, the endoplasmatic reticulum (ER) comes into play; for example, part of the phago-endosomal membrane is donated by the ER in a dynamic process referred to as ‘ER-mediated phagocytosis’ [16]. During ER recruitment to, and fusion with, the developing phagosome/endosome the ER transiently ‘opens’ to the ‘outer space’ potentially translocating ER constituents such as calreticulin, calnexin and newly formed yet still empty MHC class I molecules to the developing endosome. Pioneering work in macrophages and DCs revealed that the efficacy of cross-presentation is strongly increased by linking Ag to latex beads and thus enforcing Ag internalization by phagocytosis [17]. Phagocytosis of bacteria also results in efficient cross-presentation [18], as does phagocytosis of apoptotic cells [19], tumor (melanoma) cells [20] or exosomes secreted by tumor cells [21]. In addition, targeting Ag to Fc receptors (FcRs) to trigger FcR-mediated uptake [22] allows cross-presentation at Ag concentrations three to five log below that needed for fluid phase uptake [23]. Whether receptor-mediated Ag internalization just improves Ag uptake or whether robust cross-presentation requires targeting of Ag to specific endocytotic compartments is not yet known. www.sciencedirect.com
In this context two recent publications have shed some light on the molecular mechanisms why receptormediated targeting of exogenous Ag to the endosome might improve cross-presentation [24,25]. Accordingly, ER fusion with the nascent phago-endosome not only provides ER-derived membrane for building of the phagosome membrane, but in addition confers to the endosome cross-presentation properties including access to (immuno)proteasomes and TAP- (transporter associated with antigen processing) dependent transport of peptides to be loaded onto MHC class I molecules. As a consequence, the phago-endosome not only acquires but displays competence for Ag cross-presentation [24,25].
Adjuvants — properties of ligands of Toll-like receptor family members In the mouse, the Toll-like receptor (TLR) family includes ten proteins (TLR1–9 and TLR11). The TLR family members share common structural features including an ectodomain (containing multiple leucinerich repeats and one cysteine-rich domain), a transmembrane region and a highly conserved cytoplasmic Tollinterleukin 1 receptor (TIR) domain. Ligand binding occurs within the ectodomain, whereas the TIR domain interacts with family members of MyD88 adaptor protein to initiate activation of innate immune cells [26]. Based on amino acid sequence similarities, the TLR family members may be divided into subfamilies. Members of the TLR9 subfamily (TLR7, 8 and 9) recognize pathogen-derived single-stranded RNA [27,28] and DNA motifs [29,30], whereas TLR3 recognizes doublestranded RNA. The bacterial protein flagellin acts as ligand for TLR5 and lipopolysaccharide is the prototypic ligand for TLR4 [31]. Members of the TLR2 subfamily (TLR1, 2, 6) heterodimerize, thus creating specificities for di- or tri-acetylated lipoproteins primarily from Grampositive bacteria [32]. Interestingly, both TLR3 and the TLR9 subfamily members are expressed intracellularly at the endosomal compartment [13], whereas TLR2, 4 and 5 are cell surface bound. It follows that cell surface bound TLRs rapidly sense bacterial infections by recognizing extracellular cell wall components, whereas bacterial or viral DNA or RNA motifs (ligands of the TLR3 or TLR9 subfamily members) are ‘liberated’ in the endosome upon receptor-mediated internalization of the respective pathogen. Alternatively, the respective ligands are generated intracellularly upon cell infection.
The endosome as ‘cross-road’ for Ag and TLR ligands Unmethylated CpG dinucleotides within immunostimulatory DNA sequences are ligands for TLR9 [29,30]. CpG oligodeoxynucleotides (CpG-ODN) mixed with proteinaceous Ag have been shown to act as powerful adjuvants for Th-1 biased immunoresponses and cytotoxic T lymphocyte (CTL) induction, both in murine infection and tumor model systems [33,34]. As CpG-ODN directly Current Opinion in Biotechnology 2004, 15:538–542
540 Pharmaceutical biotechnology
converts cross-presenting immature DCs into professional APCs [35], CpG-ODN ‘aided’ CTL induction basically is T-helper independent. Raz and colleagues were the first to describe a novel CpG-ODN-based vaccine composed of Ag (ovalbumin [Ova]) chemically linked to CpG-ODN [36]. These Ag–CpG conjugates turned out to be more potent in priming CTL activity compared to a mixture of Ag and CpG-ODN. In fact, the power of induction of Th-1-based immunity by Ag–CpG conjugates revealed prophylactic and therapeutic effects in a murine asthma model system. We, and others, have set out to unravel why CpG-DNA conjugated to Ag increases the immunogenicity of Ag 50- to 100-fold when compared to Ag mixed with CpG-ODN [37,38]. Overall, these studies have led to the following conclusions. First, by conjugating CpG-ODN to Ag (Ag–CpG complex) the conjugated CpG-ODN acts as ligand for DNA receptor mediated internalization of the Ag–CpG complex by
DCs. In other words, a currently ill-defined DNA sequence non-specific endocytosis receptor (i.e. uptake can be competed for by ‘third party’ ODN [37]) is utilized by DCs to efficiently translocate the Ag–CpG complex into endosomes. Second, upon endosome formation TLR9 is expressed at the endosome [39,40]. Third, recent evidence implies that during endosome biogenesis ER constituents, including newly formed TLR9 and empty MHC class I molecules, gain access to the endosomal compartment [24,25,40]. Fourth, the MHC class I processing machinery assembles around or gains access to the endosomal compartment [24,25]. Altogether, these dynamic processes donate competence to the endosome for cross-presentation and TLR9-driven DC activation. Upon acidification (maturation) of endolysomes CpG-ODN presumably becomes liberated from the Ag–CpG complex and thus is able to activate the endosomal membrane-bound TLR9 (Figure 1). As a
Figure 1
1
2
3
4
5
Endocytosis
Ag-CpG
Cytokines (IL-12) Peptide-loaded class I MHC
DNA Receptor
61?)
Cross-presentation
Endosomal maturation (acidification)
I cla ss
MHC class I
MyD88 recruitment
M
Delivery of
HC
TL
R9
(T A P? )
(Sec
Upregulation of co-stimulatory molecules
NFκB
p38, AP-1
Gene activation Current Opinion in Biotechnology
Targeting the endosome with Ag–CpG complexes. Schematic illustrating the close relationship between the biogenesis of the DC phago-endosome and the acquisition of competence for cross-presentation. TLR9 signalling drives the maturation of cross-presenting DCs into professional APCs with competence for cross-priming. Phase 1: CpG–Ova complexes bind to a DNA-binding receptor (not TLR9) that mediates internalization of the CpG–Ova complex into the endosome. Phase 2: during biogenesis of the endosome, endoplasmatic reticulum (ER) tubular structures (yellow) contact the cell membrane; the early endosome thus displays a hybrid of ER-derived (yellow) and cell-membrane-derived (blue) membrane. During this process the ER transiently opens, translocating ER constituents such as TLR9 and MHC class I molecules to the endosome. In addition, components of the MHC class I processing machinery including transporter associated with antigen processing (TAP) and the proteasome (green) are recruited to the cytosolic part of the endosome. Sec 61 represents a pore-forming protein and translocates proteins up to 40 kDa. Phase 3: Ag becomes processed, CD8 epitopes are loaded on MHC class I and loaded MHC class I molecules (red triangles) translocate to the membrane. Upon maturation (red colour [acidification]) of the endosome CpG-ODN liberated from the protein activates TLR9, which causes MyD88 recruitment and in turn (Phase 4) triggers the Toll/IL-1 receptor signal pathway. Phase 5: As a consequence, DCs mature into professional APCs by upregulating co-stimulatory molecules and cytokine production. Current Opinion in Biotechnology 2004, 15:538–542
www.sciencedirect.com
Targeting split vaccines to the endosome improves vaccination Wagner et al. 541
consequence, signaling is initiated by recruitment of the MyD88 family members [39,40] to the cytosolic site of the endosome membrane. According to this scenario, the endosome might be regarded as a subcellular site able to develop competence for both cross-presentation and cross-priming, the latter by utilizing the adjuvants effect of TLR9-mediated DC activation. In past work we have compared, for example, the efficacy of live vaccines (Listeria monocytogenes [Lm] transduced to produce Ova or vaccinia virus encoding an Ova cassette) with that of the Ova–CpG complex (as an example of a ‘dead’ vaccine). We quantified cross-priming by enumeration of SIINFEKL- (peptide-) specific T cells via tetramer technology. To our surprise we noted that in mice the primary and secondary burst size (clonal expansion versus contraction) of SIINFEKL-specific CD8 T cells triggered by Ova–CpG conjugate (‘dead vaccine’) was comparable to that caused by Lm–Ova (‘live vaccine’). Furthermore, mice primed with either ‘live’ or ‘dead’ vaccine turned out to be resistant to otherwise lethal doses of Lm–Ova, but not to wild-type Lm (A Heit et al., unpublished).
Conclusions What can we learn from this information in attempts to approve the immunogenicity of ‘dead’ vaccines based on recombinant proteinaceous Ag? First, the potential ‘peptide’ repertoire of exogenous Ag might be best unveiled by enforced internalization via receptormediated endocytosis of Ag by DCs. Second, the use of receptor-mediated endocytosis highlights the endosome as an organelle displaying competence for crosspresentation. Last, by utilizing the adjuvant activities of TLRs expressed at the endosome, cross-presenting immature DCs become activated into professional APCs. These are then able to initiate powerful primary and secondary CD8 T-cell responses. It follows that by targeting exogenous Ag together with an appropriate TLR ligand to the endosome provides robust immunogenicity to otherwise poorly immunogenic rec Ag.
Acknowledgements
5.
Cresswell P, Bangia N, Dick T, Diedrich G: The nature of the MHC class I peptide loading complex. Immunol Rev 1999, 172:21-28.
6.
Guermonprez P, Valladeau J, Zitvogel L, Thery C, Amigorena S: Antigen presentation and T cell stimulation by dendritic cells. Annu Rev Immunol 2002, 20:621-667.
7.
Bevan MJ: 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 1976, 143:1283-1288.
8.
Schluns KS, Lefrancois L: Cytokine control of memory T-cell development and survival. Nat Rev Immunol 2003, 3:269-279.
9.
Kaech SM, Tan JT, Wherry EJ, Konieczny BT, Surh CD, Ahmed R: Selective expression of the interleukin 7 receptor identifies effector CD8 T cells that give rise to long-lived memory cells. Nat Immunol 2003, 4:1191-1198.
10. Madakamutil LT, Christen U, Lena CJ, Wang-Zhu Y, Attinger A, Sundarrajan M, Ellmeier W, von Herrath MG, Jensen P, Littman DR, Cheroutre H: CD8aa-mediated survival and differentiation of CD8 memory T cell precursors. Science 2004, 304:590-593. 11. Steinman RM, Hawiger D, Nussenzweig MC: Tolerogenic dendritic cells. Annu Rev Immunol 2003, 21:685-711. 12. Banchereau J, Briere F, Caux C, Davoust J, Lebecque S, Liu YJ, Pulendran B, Palucka K: Immunobiology of dendritic cells. Annu Rev Immunol 2000, 18:767-811. 13. Wagner H: The immunobiology of the TLR9 subfamily. Trends Immunol 2004, 25:381-386. 14. Norbury CC, Chambers BJ, Prescott AR, Ljunggren HG, Watts C: Constitutive macropinocytosis allows TAP-dependent major histocompatibility complex class I presentation of exogenous soluble antigen by bone marrow-derived dendritic cells. Eur J Immunol 1997, 27:280-288. 15. Underhill DM, Ozinsky A: Phagocytosis of microbes: complexity in action. Annu Rev Immunol 2002, 20:825-852. 16. Gagnon E, Duclos S, Rondeau C, Chevet E, Cameron PH, Steele-Mortimer O, Paiement J, Bergeron JJ, Desjardins M: Endoplasmic reticulum-mediated phagocytosis is a mechanism of entry into macrophages. Cell 2002, 110:119-131. This manuscript points out that the ER membrane fuses with the cell membrane during biogenetics of phago-endosomes. 17. Kovacsovics-Bankowski M, Clark K, Benacerraf B, Rock KL: Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. Proc Natl Acad Sci USA 1993, 90:4942-4946. 18. Rescigno M, Citterio S, Thery C, Rittig M, Medaglini D, Pozzi G, Amigorena S, Ricciardi-Castagnoli P: Bacteria-induced neo-biosynthesis, stabilization, and surface expression of functional class I molecules in mouse dendritic cells. Proc Natl Acad Sci USA 1998, 95:5229-5234.
The work cited was supported by the Deutsche Forschungsgemeinschaft, ForImmun and the Coley Pharmaceutical Group.
19. Albert ML, Sauter B, Bhardwaj N: Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 1998, 392:86-89.
References and recommended reading
20. Berard F, Blanco P, Davoust J, Neidhart-Berard EM, Nouri-Shirazi M, Taquet N, Rimoldi D, Cerottini JC, Banchereau J, Palucka AK: Cross-priming of naive CD8 T cells against melanoma antigens using dendritic cells loaded with killed allogeneic melanoma cells. J Exp Med 2000, 192:1535-1544.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest
21. Wolfers J, Lozier A, Raposo G, Regnault A, Thery C, Masurier C, Flament C, Pouzieux S, Faure F, Tursz T et al.: Tumor-derived exosomes are a source of shared tumor rejection antigens for CTL cross-priming. Nat Med 2001, 7:297-303.
1.
Rappuoli R, Miller HI, Falkow S: Medicine. The intangible value of vaccination. Science 2002, 297:937-939.
2.
Levine MM, Sztein MB: Vaccine development strategies for improving immunization: the role of modern immunology. Nat Immunol 2004, 5:460-464.
3.
Wong P, Pamer EG: CD8 T cell responses to infectious pathogens. Annu Rev Immunol 2003, 21:29-70.
22. Regnault A, Lankar D, Lacabanne V, Rodriguez A, Thery C, Rescigno M, Saito T, Verbeek S, Bonnerot C, Ricciardi-Castagnoli P, Amigorena S: Fcg receptor-mediated induction of dendritic cell maturation and major histocompatibility complex class I-restricted antigen presentation after immune complex internalization. J Exp Med 1999, 189:371-380.
4.
Zinkernagel RM: On natural and artificial vaccinations. Annu Rev Immunol 2003, 21:515-546.
23. Machy P, Serre K, Leserman L: Class I-restricted presentation of exogenous antigen acquired by Fcg receptor-mediated
www.sciencedirect.com
Current Opinion in Biotechnology 2004, 15:538–542
542 Pharmaceutical biotechnology
endocytosis is regulated in dendritic cells. Eur J Immunol 2000, 30:848-857. 24. Guermonprez P, Saveanu L, Kleijmeer M, Davoust J, Van Endert P, Amigorena S: ER-phagosome fusion defines an MHC class I cross-presentation compartment in dendritic cells. Nature 2003, 425:397-402. [24] and [25] were the first articles to describe endosomes as organelles with competence for cross-presentation. 25. Houde M, Bertholet S, Gagnon E, Brunet S, Goyette G, Laplante A, Princiotta MF, Thibault P, Sacks D, Desjardins M: Phagosomes are competent organelles for antigen cross-presentation. Nature 2003, 425:402-406. See annotation for [24]. 26. Takeda K, Kaisho T, Akira S: Toll-like receptors. Annu Rev Immunol 2003, 21:335-376. 27. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, Lipford G, Wagner H, Bauer S: Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 2004, 303:1526-1529. This article and [28] define single-stranded RNA motifs as ligand for TLR7 (mouse) and TLR8 (human). 28. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C: Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004, 303:1529-1531. See annotation for [27].
macrophage phagosomes and discriminates between pathogens. Nature 1999, 401:811-815. 33. Lipford GB, Bauer M, Blank C, Reiter R, Wagner H, Heeg K: CpG-containing synthetic oligonucleotides promote B and cytotoxic T cell responses to protein antigen: a new class of vaccine adjuvants. Eur J Immunol 1997, 27:2340-2344. 34. Wagner H: Bacterial CpG DNA activates immune cells to signal infectious danger. Adv Immunol 1999, 73:329-368. 35. Sparwasser T, Koch ES, Vabulas RM, Heeg K, Lipford GB, Ellwart JW, Wagner H: Bacterial DNA and immunostimulatory CpG oligonucleotides trigger maturation and activation of murine dendritic cells. Eur J Immunol 1998, 28:2045-2054. 36. Cho HJ, Takabayashi K, Cheng PM, Nguyen MD, Corr M, Tuck S, Raz E: Immunostimulatory DNA-based vaccines induce cytotoxic lymphocyte activity by a T-helper cell-independent mechanism. Nat Biotechnol 2000, 18:509-514. 37. Maurer T, Heit A, Hochrein H, Ampenberger F, O’Keeffe M, Bauer S, Lipford GB, Vabulas RM, Wagner H: CpG-DNA aided cross-presentation of soluble antigens by dendritic cells. Eur J Immunol 2002, 32:2356-2364.
29. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S: A Toll-like receptor recognizes bacterial DNA. Nature 2000, 408:740-745.
38. Heit A, Maurer T, Hochrein H, Bauer S, Huster KM, Busch DH, Wagner H: Cutting edge: Toll-like receptor 9 expression is not required for CpG DNA-aided cross-presentation of DNA-conjugated antigens but essential for cross-priming of CD8 T cells. J Immunol 2003, 170:2802-2805. This manuscript describes that TLR9 is not involved in endocytosis of CpG-DNA.
30. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, Wagner H, Lipford GB: Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA 2001, 98:9237-9242.
39. Ahmad-Nejad P, Hacker H, Rutz M, Bauer S, Vabulas RM, Wagner H: Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol 2002, 32:1958-1968. The first description of endosomally expressed TLR9.
31. Poltorak A, He X, Smirnova I, Liu MY, Van Huffel C, Du X, Birdwell D, Alejos E, Silva M, Galanos C et al.: Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 1998, 282:2085-2088. 32. Underhill DM, Ozinsky A, Hajjar AM, Stevens A, Wilson CB, Bassetti M, Aderem A: The Toll-like receptor 2 is recruited to
Current Opinion in Biotechnology 2004, 15:538–542
40. Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, Lien E, Nilsen NJ, Espevik T, Golenbock DT: TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 2004, 5:190-198. The first work to establish that TLR9 is expressed at the ER in nonactivated DCs.
www.sciencedirect.com