IMMUNOLOGY
ORIGINAL ARTICLE
Extended sequence preferences for oligodeoxyribonucleotide activity
Petar Lenert, Adam J. Goeken and Robert F. Ashman Division of Rheumatology, Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, IA, USA
doi:10.1111/j.1365-2567.2006.02320.x Received 19 May 2005; revised 22 November 2005; accepted 24 November 2005. Correspondence: Dr Petar Lenert, C312GH, Department of Internal Medicine, Carver College of Medicine, The University of Iowa, Iowa City, IA 52242, USA. Email:
[email protected] Senior author: Robert Ashman, email:
[email protected]
Summary Synthetic type B phosphorothioate oligodeoxyribonucleotides (ODN) activate mouse B cells via Toll-like receptor 9 (TLR9). Starting with closely related 15-mer prototype ODN, the sequence requirements for stimulatory (ST-) and inhibitory (IN-) activity were contrasted, by measuring apoptosis protection, G1 entry and interleukin-6 secretion. ST-ODN and IN-ODN differ in that (1) ST-ODN require a 50 T, (2) the central CG is obligatory, (3) CG must be flanked 30 specifically by TT at the position where IN-ODN have GG, and (4) IN-ODN tolerate truncation of the 30 end better than ST-ODN. Features shared by ST-ODN and IN-ODN include (1) requiring CC adjacent to the 50 end, and (2) avoiding CC immediately 50 to the CG. This pattern is used to create a model of how ST-ODN binding might function to aggregate TLR9 so as to initiate the signal, and how the 50 ends of ST-ODN and IN-ODN compete for binding. Further justification for considering TLR9 to be the ODN receptor was provided by a demonstration that in HEK293 cells transfected with TLR9, the potency of a panel of ODN for activating NF-jB roughly parallels that seen in the biological assays in mouse B cells. Keywords: apoptosis; B cells; cell cycle entry; CpG-DNA; interleukin-6
Introduction Our innate immune receptors have evolved to recognize a variety of molecules made by pathogens but not by us. The importance of these ‘danger signals’ for accelerating and directing the adaptive immune response is increasingly being recognized.1 Among these receptors is Toll-like receptor 9 (TLR9), which mediates the response to bacterial DNA.2,3 Short, single-stranded oligonucleotides are able to duplicate the response to bacterial DNA.2 A target motif was identified by a computer search for short sequences that are present less frequently in mammalian DNA than in bacterial DNA, where frequencies approximate the chance distribution. CG pairs were shown to be about one-quarter as frequent in mammalian DNA. If the C were unmethylated, and the adjacent bases were two purines on the 50 side and two pyrimidines on the 30 side, the motif frequency in mammalian DNA was decreased to about one-sixteenth that of bacterial DNA.4 An alternative
formulation expressed the motif as ‘not C, unmethylated C, G, not G’.5 Because of the ease of degradation of the native oligodeoxyribonucleotides (ODN) with phosphodiester (O-) backbones by nucleases, the nuclease-resistant sulphur-substituted phosphorothioate (S-) series has been commonly used in experiments and in vaccine protocols. In mice, on a molar basis, the stimulatory S-ODN are approximately 100–200 times more potent than O-ODN with identical sequences.3 Quantitative species-related sequence preferences exist such that GACGTT appears in a variety of S-ODN that stimulate mouse cells best, and GTCGTT appears in those that stimulate human cells best.6 ODN-responsive cell types (B cells and dendritic cells in both species, and macrophages in mice) all appear to share this preference. However, species-related preferences are inapparent with O-ODN.7 Previous attempts to define the optimal stimulatory sequence have focused mainly on the central five or six bases, noting major potency loss associated with methylating the central C, reversing the CG to GC, and reversing
Abbreviations: DC, dendritic cell; IN, inhibitory; O-, phosphodiester; ODN, oligodeoxyribonucleotides; S-, phosphorothioate; ST, stimulatory; TLR, toll-like receptor. 474
2006 Blackwell Publishing Ltd, Immunology, 117, 474–481
Sequence requirements for CpG-ODN activity the adjacent base substitutions to give CCG or CGG.4 Our surprising discovery that the sequence requirements for inhibitory (IN-) ODN are restricted to only six bases8 led us to re-examine the sequence requirements for stimulatory activity, which proved to be much more extensive than previously thought. These new insights generated a model of the putative TLR9-ODN binding site.
coated with anti-IL-6 antibodies MP5-20-F3 (Bio Legend, San Diego, CA) overnight, then washed three times. After washing MP5-32C11-biotin and extravidin-peroxidase were used to detect bound IL-6. The assay sensitivity was 16 pg/ml using recombinant IL-6 (BioLegend) as standard. Examples of dose–response curves for these three assays appear in Figure 1 in ref. 9.
Materials and methods
NF-jB activation
Source and characterization of resting B cells B6 D2 F1 female mice at 8–12 weeks of age from Jackson Laboratories, Bar Harbor, ME, were housed in a specific pathogen-free facility with HEPA-filtered air, autoclaved bedding and water. Spleen cell suspensions underwent red blood cell lysis, then negative selection with antiCD43-coated magnetic beads (Midi-Macs by Miltenyi Biotec, Auburn, CA). The yield of 25 · 106 total B cells per spleen consisted of > 97% CD19+ cells, of which 1% were apoptotic, 1% in G1 and 98% in G0 by acridine orange flow cytometry.9 A human embryonic kidney cell line (HEK293) stably transfected with TLR9 and a nuclear factor-jB (NF-jB) -promoter luciferase construct10 were the kind gifts of Dr Grayson Lipford (Coley Pharmaceuticals, Wellesley, MA). HEK293 cells expressing the NF-jB-promoter-luciferase construct but lacking TLR9 were purchased from Panomics.
Oligonucleotides (ODN) Synthetic phosphorothioate (S-) ODN were purchased from Integrated DNA Technologies, Coralville, IA. The prototype ST-ODN was 20845 and appeared for reference in all experiments: TCCTGACGTTGAAGT. Titrations were usually performed with five concentrations spread 0.5 log apart in the 10–1000 nM range.
Flow cytometry By a modification of the method of Traganos and colleagues11 apoptosis and phases of the cell cycle were determined after 18 hr of culture by acridine orange flow cytometry, which measures the RNA and DNA content of each cell. For each run, freshly isolated B cells were used to set the G0/G1 and G0/apoptosis boundaries. Cells in G1, G2, S and M phases were included in ‘% cell cycle entry’; the denominator was total cells counted.
Interleukin-6 enzyme-linked immunosorbent assay (IL-6 ELISA) Supernatants collected at 18 hr were incubated for 2 hr at room temperature in 10% fetal calf serum/phosphatebuffered saline on Greiner Bio-One plates which were 2006 Blackwell Publishing Ltd, Immunology, 117, 474–481
Parental HEK293 cells and HEK293 cells stably expressing TLR9 and an NF-jB-promoter-luciferase construct were incubated for 24 hr with a panel of ODN with different stimulatory potencies for B cells or with 50 ng/ml of tumour necrosis factor-a as a positive control. After three washes in Ca2+/Mg2+-free medium, the cells were resuspended in lysis buffer containing 1% Triton-X-100 and centrifuged to remove debris. Twenty microlitres of supernatant was then mixed with 100 ll luciferin using the Promega assay system and light was quantified by luminometry.
Data analysis The geometric mean of the concentrations giving 50% of a maximal response to 2084 in each of three experiments was taken as the potency of an ODN. The maximal IL-6 response was taken as that given by 100 nM 2084, whereas the other curves gave distinct plateaus. The potency of the prototype ODN 2084 was set at 100% activity. The % activity was given by the equation: (potency of 2084/the potency of a test ODN) · 100%.
Results Apoptosis protection, entry into G1 and IL-6 secretion yielded the same rank order of ODN activity. Apoptosis protection required the least ODN 2084 for 50% maximal activity (14 nM), IL-6 secretion required the most (119 nM) and G1 entry an intermediate amount (34 nM), a rank order that was also seen with all the other ST-ODN (Fig. 1).
Truncation studies Base positions were numbered according to the scheme at the top of Table 1 (Fig. 1). Loss of only the T at )7 (4168) decreased activity by 98%. At the 30 end, loss of the first base (+ 8) only reduced activity by 25% (4170), but activity was progressively lost with further 30 truncation (4209, 4167). Interestingly, the length, not the base sequence, was critical at the 30 end, because ODN 4215 with random bases from + 5 to + 8 had normal activity. When an ODN lacked the T at ) 7, the loss of one or two bases at the 30 end made no further difference (4149, 475
P. Lenert et al. nM 10
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100% Truncation 2% 3% 75% 36% 11% 3%
