Spontaneous Formation of Nucleic Acid-based Nanoparticles Is Responsible for High Interferon-α Induction by CpG-A in Plasmacytoid Dendritic Cells

Abstract

Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-α and IFN-β) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-α (IFN-α) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5′ and 3′ end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-α (CpG-B) gained the potency of CpG-A to induce IFN-α. Higher ODN uptake in PDC was not responsible for the higher IFN-α-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-α in PDC. Plasmacytoid dendritic cells (PDC) represent a highly specialized immune cell subset that produces large quantities of the anti-viral cytokines type I interferons (IFN-α and IFN-β) upon viral infection. PDC employ a member of the family of toll-like receptors, TLR9, to detect CpG motifs (unmethylated CG dinucleotides in certain base context) present in viral DNA. A certain group of CpG motif-containing oligodeoxynucleotides (CpG ODN), CpG-A, was the first synthetic stimulus available that induced large amounts of interferon-α (IFN-α) in PDC. However, the mechanism responsible for this activity remained elusive. CpG-A is characterized by a central palindrome and poly(G) at the 5′ and 3′ end. Here we demonstrate that CpG-A self-assembles to higher order tertiary structures via G-tetrad formation of their poly(G) motifs. Spontaneous G-tetrad formation of CpG-A required the palindrome sequence allowing structure formation in a physiological environment. Once formed, G-tetrad-linked structures were stable even under denaturing conditions. Atomic force microscopy revealed that the tertiary structures formed by CpG-A represent nucleic acid-based nanoparticles in the size range of viruses. Similarly sized preformed polystyrene nanoparticles loaded with a CpG ODN that is otherwise weak at inducing IFN-α (CpG-B) gained the potency of CpG-A to induce IFN-α. Higher ODN uptake in PDC was not responsible for the higher IFN-α-inducing activity of CpG-A or of CpG-B-coated nanoparticles as compared with CpG-B. Based on these results we propose a model in which the spatial configuration of CpG motifs as particle is responsible for the virus-like potency of CpG-A to induce IFN-α in PDC. The induction of natural type I interferons (IFNs) 1The abbreviations used are: IFN, interferon; PDC, plasmacytoid dendritic cell(s); TLR, toll-like receptor; ODN, oligodeoxynucleotide(s); PBS, phosphate-buffered saline without calcium and magnesium; AFM, atomic force microscopy; TBE, Tris-borate-EDTA. (anti-viral cytokines, IFN-α and IFN-β) in vivo may pave the way for improved therapies for viral infection such as chronic hepatitis or for malignancies. A particular immune cell subset, the so-called plasmacytoid dendritic cell (PDC), has been identified as the major type I interferon-producing cell upon viral infection (1Cella M. Jarrossay D. Facchetti F. Alebardi O. Nakajima H. Lanzavecchia A. Colonna M. Nat. Med. 1999; 5: 919-923Crossref PubMed Scopus (1454) Google Scholar, 2Siegal F.P. Kadowaki N. Shodell M. Fitzgerald-Bocarsly P.A. Shah K. Ho S. Antonenko S. Liu Y.J. Science. 1999; 284: 1835-1837Crossref PubMed Scopus (1929) Google Scholar). Viruses for a long time have been the only stimuli to induce large amounts of IFN-α in PDC. Recently it became clear that certain members of the family of toll-like receptors (TLRs) are involved in the regulation of IFN-α synthesis in PDC. In general, TLRs (TLR1–TLR10) are specialized for the detection of conserved pathogen-associated molecules such as endotoxin (component of Gram-negative bacteria, detected by TLR4) leading to activation of the corresponding immune cell subset (3Akira S. Takeda K. Kaisho T. Nat. Immunol. 2001; 2: 675-680Crossref PubMed Scopus (4021) Google Scholar). The only two TLRs expressed in PDC are TLR7 and TLR9 (4Hornung V. Rothenfusser S. Britsch S. Krug A. Jahrsdorfer B. Giese T. Endres S. Hartmann G. J. Immunol. 2002; 168: 4531-4537Crossref PubMed Scopus (1654) Google Scholar, 5Krug A. Towarowski A. Britsch S. Rothenfusser S. Hornung V. Bals R. Giese T. Engelmann H. Endres S. Krieg A.M. Hartmann G. Eur. J. Immunol. 2001; 31: 3026-3037Crossref PubMed Scopus (673) Google Scholar, 6Kadowaki N. Ho S. Antonenko S. Malefyt R.W. Kastelein R.A. Bazan F. Liu Y.J. J. Exp. Med. 2001; 194: 863-869Crossref PubMed Scopus (1699) Google Scholar). Consequently, PDC can be stimulated by the corresponding ligands for TLR7 and TLR9. Although DNA containing so-called CpG motifs (unmethylated CG dinucleotides with certain flanking bases, CpG-DNA) has been identified as a ligand for TLR9 early on (7Hartmann G. Weiner G.J. Krieg A.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 9305-9310Crossref PubMed Scopus (565) Google Scholar), CpG motif containing synthetic oligodeoxynucleotides (CpG ODN) only induce large amounts of IFN-α in PDC when they fulfill certain sequence requirements (palindrome, two poly(G) ends, phosphorothioate-modified linkages at the 5′ and 3′ ends) in addition to the CpG motif; this group of CpG ODN is termed CpG-A (8Krug A. Rothenfusser S. Hornung V. Jahrsdo ̈rfer B. Blackwell S. Ballas Z.K. Endres S. Krieg A.M. Hartmann G. Eur. J. Immunol. 2001; 31: 2154-2163Crossref PubMed Scopus (778) Google Scholar, 9Krieg A.M. Annu. Rev. Immunol. 2002; 20: 709-760Crossref PubMed Scopus (2263) Google Scholar). The prototype sequence of CpG-A is ODN 2216 (8Krug A. Rothenfusser S. Hornung V. Jahrsdo ̈rfer B. Blackwell S. Ballas Z.K. Endres S. Krieg A.M. Hartmann G. Eur. J. Immunol. 2001; 31: 2154-2163Crossref PubMed Scopus (778) Google Scholar). ODN 2006 (10Hartmann G. Weeratna R.D. Ballas Z.K. Payette P. Blackwell S. Suparto I. Rasmussen W.L. Waldschmidt M. Sajuthi D. Purcell R.H. Davis H.L. Krieg A.M. J. Immunol. 2000; 164: 1617-1624Crossref PubMed Scopus (546) Google Scholar, 11Hartmann G. Krieg A.M. J. Immunol. 2000; 164: 944-953Crossref PubMed Scopus (561) Google Scholar), the sequence currently being tested in a number of clinical trials (ProMune, ODN 7909), belongs to the group of CpG-B. CpG-B are weak at inducing IFN-α but are more active than CpG-A to stimulate costimulatory molecules and proinflammatory cytokines and to promote survival and differentiation of PDC (5Krug A. Towarowski A. Britsch S. Rothenfusser S. Hornung V. Bals R. Giese T. Engelmann H. Endres S. Krieg A.M. Hartmann G. Eur. J. Immunol. 2001; 31: 3026-3037Crossref PubMed Scopus (673) Google Scholar). The most recent group of CpG ODN is CpG-C, which induces intermediate levels of IFN-α in PDC and directly activates B cells (12Poeck H. Wagner M. Battiany J. Rothenfusser S. Wellisch D. Hornung V. Jahrsdorfer B. Giese T. Endres S. Hartmann G. Blood. 2004; 103: 3058-3064Crossref PubMed Scopus (247) Google Scholar, 13Hartmann G. Battiany J. Poeck H. Wagner M. Kerkmann M. Lubenow N. Rothenfusser S. Endres S. Eur. J. Immunol. 2003; 33: 1633-1641Crossref PubMed Scopus (263) Google Scholar, 14Marshall J.D. Fearon K. Abbate C. Subramanian S. Yee P. Gregorio J. Coffman R.L. Van Nest G. J. Leukocyte Biol. 2003; 73: 781-792Crossref PubMed Scopus (234) Google Scholar, 15Vollmer J. Weeratna R. Payette P. Jurk M. Schetter C. Laucht M. Wader T. Tluk S. Liu M. Davis H.L. Krieg A.M. Eur. J. Immunol. 2004; 34: 251-262Crossref PubMed Scopus (512) Google Scholar). Regulation of type I IFN production in PDC by the different classes of CpG ODN is not well understood. In studies with TLR9-deficient mice it has been demonstrated that both CpG-A and CpG-B depend on TLR9 (16Hemmi H. Kaisho T. Takeuchi O. Sato S. Sanjo H. Hoshino K. Horiuchi T. Tomizawa H. Takeda K. Akira S. Nat. Immunol. 2002; 3: 196-200Crossref PubMed Scopus (2114) Google Scholar). However, CpG-B shows much higher activity in TLR9-transfected cell lines than CpG-A. 2S. Akira, personal communication. Because TLR9 is required for both CpG-A and CpG-B, it is intriguing that ligation of the same receptor, TLR9, can elicit distinct responses in PDC. ODNs with poly(G) motifs (at least four guanosines) are known to self-associate via Hoogsteen base pairing into quadruplex structures, so-called G-tetrads (17Williamson J.R. Raghuraman M.K. Cech T.R. Cell. 1989; 59: 871-880Abstract Full Text PDF PubMed Scopus (1067) Google Scholar, 18Sen D. Gilbert W. Nature. 1988; 334: 364-366Crossref PubMed Scopus (1538) Google Scholar, 19Keniry M.A. Biopolymers. 2000; 56: 123-146Crossref PubMed Scopus (317) Google Scholar). The formation and the stability of quadruplex ODNs depend on the sequence as well as on the concentrations of monovalent cations such as sodium and potassium in the ODN diluent (20Hardin C.C. Henderson E. Watson T. Prosser J.K. Biochemistry. 1991; 30: 4460-4472Crossref PubMed Scopus (244) Google Scholar, 21Marathias V.M. Bolton P.H. Biochemistry. 1999; 38: 4355-4364Crossref PubMed Scopus (112) Google Scholar). It has been suggested that the formation of G-tetrad-stabilized structures is involved in immunoglobulin switch regions and in chromosomal telomeres, both of which contain guanosine-rich sequences (22Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Crossref PubMed Scopus (130) Google Scholar, 23Balagurumoorthy P. Brahmachari S.K. J. Biol. Chem. 1994; 269: 21858-21869Abstract Full Text PDF PubMed Google Scholar). Here we demonstrate that the CpG-A ODN 2216 spontaneously and under physiological conditions forms nanoparticles via G-tetrads. Based on our results we propose a concept in which the palindrome sequence of CpG-A via duplex formation of two CpG-A molecules facilitates the generation of G-tetrads and in which the spatial configuration of CpG motifs as a nanoparticle is critical for the high IFN-α inducing activity characteristic for CpG-A. Media and Reagents—RPMI cell culture medium (PAA Laboratories, Linz, Austria) supplemented with 8% human serum (BioWhittaker, Walkersville, MD), 1.5 mm l-glutamine, 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma, Munich, Germany) was used throughout the studies. All compounds were purchased endotoxin-free. ODN were provided by Coley Pharmaceutical Group (Wellesley, MA) or purchased from Metabion (Martinsried, Germany). Sequences of the ODN used in this study are provided in Table I. No endotoxin could be detected in ODN preparations the detection ODN were used at a of μg/ml as (8Krug A. Rothenfusser S. Hornung V. Jahrsdo ̈rfer B. Blackwell S. Ballas Z.K. Endres S. Krieg A.M. Hartmann G. Eur. J. Immunol. 2001; 31: 2154-2163Crossref PubMed Scopus (778) Google Scholar). was from 5′ and were purchased from saline without calcium and was from were from Germany). was purchased from of ODN used 3′ of the 5′ to in a were at the 5′ with the 5′ to the provided by the Higher order structure formation of ODN in was by TBE, The was at a of denaturing TBE, The was at a of denaturing was with the of structure formation in different and ODN were purchased from Metabion (Martinsried, Germany). detection of ODN was by the and the and from to the provided by the formation under the were (22Sen D. Gilbert W. Methods Enzymol. 1992; 211: 191-199Crossref PubMed Scopus (130) Google mm mm mm mm mm mm mm mm mm were under conditions with mm and were for at 100 by at V. Atomic in without and (PAA were on mm for for with and under as Kerkmann M. Hartmann G. Endres S. S. 2004; PubMed Scopus Google Scholar). All were a Germany) in at conditions with a and a of The was was of and from to tested to be for human hepatitis B and hepatitis were provided by the of the of cells were from by as S. Hornung V. M. Britsch S. Towarowski A. Krug A. A. Lubenow N. D. Endres S. Hartmann G. Blood. 2004; 103: PubMed Scopus Google Scholar). PDC were by cell the dendritic cell from Germany). PDC were with to and a and The of PDC and was was as by PDC or cells were in in the of different stimuli as CpG ODN were used at a of were different of time for and of nanoparticles were by under conditions as by H. M. E. J. Sci. PubMed Scopus Google Scholar). The was to induce was by the addition of the The size of the was by a and amounts to of CpG ODN to polystyrene nanoparticles was as by H. M. E. J. Sci. PubMed Scopus Google Scholar). ODN were purchased from Metabion (Martinsried, Germany). the of ODN uptake PDC were with ODN or ODN to polystyrene nanoparticles of ODN, at of ODN was by at 1.5 cells were and was by with two at and cells were from the by were of IFN-α from range and the human from were are as of was by the were for and were of IFN-α in Plasmacytoid by CpG-A G-tetrad is characterized by to induce large amounts of IFN-α in PDC. However, the for this of CpG-A is not well understood. In addition to CpG motifs that are present in both CpG-A and CpG-B, the sequence of CpG-A a palindrome in the and poly(G) motifs at the 5′ and the 3′ end. that sequence motifs of CpG-A were required for of the CG dinucleotides to IFN-α induction in PDC The of only two of the palindrome motif the activity of CpG-A of the poly(G) motifs at both by to of activity compared with CpG-A motifs the sequence of CpG-A may to the IFN-α-inducing activity by different immune or G-tetrad G-tetrad formation can be by in the poly(G) motifs M. A. S. PubMed Scopus Google Scholar). of base in of the poly(G) motifs of CpG-A the IFN-α inducing activity of CpG-A that G-tetrad formation is required for the activity of CpG-A. of CpG-A Spontaneous of of at least four poly(G) motifs via Hoogsteen base Because CpG-A two poly(G) G-tetrad formation at least two CpG-A the of G-tetrad formation by CpG-A, ODN were on a a high of CpG-A a to the the formation of large and tertiary structures from the of CpG-A to for by to the formation of tertiary structures of CpG-A this the of the CpG-A on the No tertiary structures were for CpG-B or when the poly(G) motifs of CpG-A were by and from the structures were when the palindrome of CpG-A was by the of two from the structures were with a ODN containing two poly(G) motifs at both but palindrome 1.5 Henderson E. Biochemistry. 1994; 33: PubMed Scopus Google results that CpG-A but not CpG-B has a high to spontaneously large tertiary structures and that this structure formation on the of poly(G) motifs and on the palindrome and can be to the molecules by to high conditions the of DNA on the is not to the size of the base allowing a of the ODN Although large of CpG-A were to be under denaturing conditions by tertiary structures formed by CpG-A were by and A of CpG-A is in the by However, the first of higher in higher and that the first stable tertiary structure is a quadruplex (4Hornung V. Rothenfusser S. Britsch S. Krug A. Jahrsdorfer B. Giese T. Endres S. Hartmann G. J. Immunol. 2002; 168: 4531-4537Crossref PubMed Scopus (1654) Google than a duplex of CpG-A. under denaturing large tertiary structures to the were It has been that the conditions for G-tetrad formation is a of sodium and in a to D. Gilbert W. Nature. PubMed Scopus Google Scholar). compared formation of tertiary structures by CpG-A under different concentrations CpG-A was first to and on and at or for in the of or C. formation of tertiary structures by CpG-A was at in conditions tested However, tertiary structure formation was when CpG-A was at results that of CpG-A at physiological conditions under both high sodium potassium concentrations for and sodium high potassium concentrations for the of Spontaneous CpG-A by Atomic of a large of CpG-A in the high and the of the suggested that CpG-A may used high atomic force microscopy for of structures formed by CpG-A. of the size and the of the of formed under different conditions. CpG-A or CpG-B were in without and and on with mm was in the in that CpG-A forms of and The size of structures was the of structures 100 The of structures was and CpG-B higher order structures were not with CpG-B on the of that nanoparticle formation of CpG-A under physiological conditions may be critical for the of CpG-A to induce large amounts of IFN-α in PDC. Consequently, preformed nanoparticles with CpG-B to higher order structures by may activity as CpG-A. this CpG-A, CpG-B, and CpG-B were to the of polystyrene The of CpG-B to polystyrene nanoparticles in a more than in IFN-α induction in PDC compared with CpG-B CpG-B, IFN-α induction by CpG-B was even higher than with CpG-A. The of CpG-A to induce IFN-α was by to polystyrene No induction of IFN-α was with the corresponding of CpG-B with and without polystyrene nanoparticles that IFN-α induction in PDC is and that polystyrene nanoparticles have IFN-α inducing activity In to IFN-α both CpG-A and CpG-B were to induce in PDC. The of in PDC with polystyrene nanoparticles was much as compared with the in IFN-α that the induction of was on the of CpG motifs on of and CpG-A in Plasmacytoid of amounts of induced by CpG-A and CpG-B and the of induction for CpG-B when loaded on polystyrene nanoparticles it that uptake of the ODN in the of nanoparticles is responsible for the in IFN-α induction in PDC. this we of ODN in PDC with and without polystyrene of ODN was as the of at the of of ODN at that the uptake of CpG-B with and without polystyrene nanoparticles and of CpG-A were in the same range 1.5 A and suggested that uptake of ODN is not the mechanism by which nanoparticle formation or CpG-B to the of ODN to induce IFN-α in PDC. Although type I IFN induction by has been known for CpG-A was the first synthetic stimulus to large amounts of IFN-α in human PDC (8Krug A. Rothenfusser S. Hornung V. Jahrsdo ̈rfer B. Blackwell S. Ballas Z.K. Endres S. Krieg A.M. Hartmann G. Eur. J. Immunol. 2001; 31: 2154-2163Crossref PubMed Scopus (778) Google Scholar), CpG ODN were of PDC and B cell differentiation but not of IFN-α The mechanism the IFN-α inducing potency of CpG-A has remained Here we that CpG-A spontaneously and under physiological conditions forms stable nanoparticles of a size to to 100 has been used for of DNA I. K. PubMed Scopus Google Scholar). In our study we used to the formation of higher order structures with to the size and the of the structures formed by CpG-A. that CpG-A but not CpG-B forms both and structures but structures have been by group the G-tetrad formation of a poly(G) containing ODN sequence containing poly(G) but in Henderson E. Biochemistry. 1994; 33: PubMed Scopus Google Scholar, J. Henderson E. PubMed Scopus Google Scholar). physiological conditions used in our 1.5 was weak at higher order structures when compared with CpG-A. In the quadruplex formation has been demonstrated to depend on the of sodium and potassium in the ODN diluent (20Hardin C.C. Henderson E. Watson T. Prosser J.K. Biochemistry. 1991; 30: 4460-4472Crossref PubMed Scopus (244) Google Scholar, 21Marathias V.M. Bolton P.H. Biochemistry. 1999; 38: 4355-4364Crossref PubMed Scopus (112) Google Scholar). In as study by and S. E. PubMed Scopus Google Scholar), quadruplex structure formation of poly(G) containing ODN was at under sodium high potassium high sodium potassium or physiological sodium concentrations without quadruplex formation in that study was only at that a palindrome formation of nucleic nanoparticles of poly(G) containing sequences under physiological conditions is Spontaneous formation of higher order structures by CpG-A was at and for concentrations for both the or high to our results the palindrome to G-tetrad formation of the poly(G) ends, allowing structures to be formed even under conditions that otherwise not G-tetrad Based on our and we propose a model in which two CpG-A a duplex via base pairing of their palindrome four poly(G) of the two are via G-tetrads in Because the formation of G-tetrads that four poly(G) sequences are to at the same duplex formation via the of such by the of the of the of the by may G-tetrad formation is by a palindrome the as in our are two for of duplex is via G-tetrad in a of the or two to the quadruplex by base pairing leading to a is base pairing of the is by Hoogsteen base pairing of the G-tetrad G. B. Science. 1994; PubMed Scopus Google Scholar). model is by the of both and structures formed by CpG-A in the from the of CpG-A under denaturing conditions. In denaturing higher order structures of CpG-A as a a duplex of two CpG-A molecules was not with four required for a the stable on a denaturing was a only but of higher order denaturing that such G-tetrads. In with our it has been that G-tetrad formed are to Henderson E. Biochemistry. 1994; 33: PubMed Scopus Google Scholar). It has been that CpG-A a or a M. D. I. J. Leukocyte Biol. 2002; Google Scholar, D. R.A. B. J. Immunol. 2002; 168: PubMed Scopus Google Scholar). However, in our study the concept that structure formation than a different is responsible for the distinct of CpG-A as compared with CpG-B. The to induce IFN-α in PDC was to the of higher order formation by CpG-A. of the palindrome sequence by the of two or of poly(G) with or of G-tetrad in poly(G) motif both structure formation and IFN-α nanoparticles loaded with CpG-B palindrome or were as CpG-A at inducing IFN-α in PDC. IFN-α inducing activity of both CpG-A and CpG-B loaded nanoparticles was highly the corresponding were A for higher IFN-α inducing activity of CpG-A may be uptake of CpG-A as compared with CpG-B. It has been that the higher activity of poly(G) containing CpG is by uptake of the M. D. I. J. Leukocyte Biol. 2002; Google Scholar, S. I. K. 2002; PubMed Scopus Google Scholar). of poly(G) to receptors was to in cells K. E. T. S. T. T. T. J. 1994; PubMed Scopus Google Scholar, M.K. J.K. C. J. Immunol. 2000; PubMed Scopus Google Scholar). group that A to cell of DNA but was not for the activity of CpG ODN 2001; 103: PubMed Scopus Google Scholar). In study it was that CpG a high to the of PDC K. J.D. Abbate C. Subramanian S. Yee P. Gregorio J. G. G. S. Van Nest G. Coffman R.L. Eur. J. Immunol. 2003; 33: PubMed Scopus Google Scholar). However, the of CpG ODN has not been in this In our study with PDC, levels of were for CpG-A, CpG-B, and CpG-B loaded on polystyrene A was not expressed on PDC. the different potency of CpG-A and CpG-B to induce IFN-α in PDC is not by a in ODN is by our that the large in IFN-α induction CpG-A and CpG-B induce the same of not could be responsible for higher IFN-α inducing activity of when in a be that the spatial configuration of CpG motifs in to and of TLR9 in The concept of TLR9 was by group on their that ODN gained they contain poly(G) motif in addition to a CpG motif and that this of activity was with formation of the ODN C.C. J. E. M. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). However, palindrome was present in their ODN and the activity production in of these ODN in their study not even the potency of a CpG-B The CpG-B ODN of the most CpG-B ODN known to in our study was by active than CpG-A, the ODN by in the of IFN-α induction by CpG-A and CpG-B may to the of IFN-α induction with both of CpG In a study we have demonstrated that IFN-α induction by CpG-A but not CpG-B was on the type I IFN M. Rothenfusser S. Hornung V. Towarowski A. Wagner M. A. Giese T. Endres S. Hartmann G. J. Immunol. 2003; PubMed Scopus Google Scholar). Because of of nucleic CpG-A, CpG motifs be present in the of PDC for a of time required to of the type I IFN In our results that nucleic nanoparticle formation is for the distinct activity of CpG-A to induce large amounts of IFN-α in PDC. The on the of CpG-A as in this is for the of CpG-A as a synthetic stimulus for IFN-α induction in as well as for clinical in of CpG-A for both in studies and for clinical not only have to the and sequence of the but to and the size of the Lubenow and from the of and at the of for