We describe a novel sequencing approach that combines non-gel-based signature sequencing with in vitro cloning of millions of templates on separate 5 microm diameter microbeads. After constructing a microbead library of DNA templates by in vitro cloning, we assembled a planar array of a million template-containing microbeads in a flow cell at a density greater than 3x10(6) microbeads/cm2. Sequences of the free ends of the cloned templates on each microbead were then simultaneously analyzed using a fluorescence-based signature sequencing method that does not require DNA fragment separation. Signature sequences of 16-20 bases were obtained by repeated cycles of enzymatic cleavage with a type IIs restriction endonuclease, adaptor ligation, and sequence interrogation by encoded hybridization probes. The approach was validated by sequencing over 269,000 signatures from two cDNA libraries constructed from a fully sequenced strain of Saccharomyces cerevisiae, and by measuring gene expression levels in the human cell line THP-1. The approach provides an unprecedented depth of analysis permitting application of powerful statistical techniques for discovery of functional relationships among genes, whether known or unknown beforehand, or whether expressed at high or very low levels.

Recent reports have identified two major classes of CpG motif-containing oligodeoxynucleotide immunostimulatory sequences (ISS): uniformly modified phosphorothioate (PS) oligodeoxyribonucleotides (ODNs), which initiate B cell functions but poorly activate dendritic cells (DCs) to make interferon (IFN)-alpha, and chimeric PS/phosphodiester (PO) ODNs containing runs of six contiguous guanosines, which induce very high levels of plasmacytoid DC (PDC)-derived IFN-alpha but poorly stimulate B cells. We have generated the first reported ISS, C274, which exhibits very potent effects on all human immune cells known to recognize ISS. C274 is a potent inducer of IFN-gamma/IFN-alpha from peripheral blood mononuclear cells and exhibits accelerated kinetics of activity compared with standard ISS. This ODN also effectively stimulates B cells to proliferate, secrete cytokines, and express costimulatory antigens. In addition, C274 specifically activates PDCs to undergo maturation and secrete cytokines, including very high levels of IFN-alpha. Sequence variation studies based on C274 were used to identify the general motif requirements for this novel and distinct class of ISS. In contrast, chimeric PO/PS CpG-containing ODNs with polyguanosine sequences exert a differential pattern of ISS activity compared with C274, perhaps in part as a result of their greatly different structural nature. This pattern is composed of high IFN-alpha/IFN-gamma induction and low DC maturation in the absence of B cell stimulation. In conclusion, we have generated a novel class of ISS that transcends the limitations ascribed to classes described previously in that it provides excellent stimulation of B cells and simultaneously activates PDCs to differentiate and secrete large amounts of type I IFN.

In parallel with the discovery of the immunostimulatory activities of CpG-containing oligodeoxynucleotides, several groups have reported specific DNA sequences that could inhibit activation by CpG-containing oligodeoxynucleotides in mouse models. We show that these inhibitory sequences, termed IRS, inhibit TLR-9-mediated activation in human as well as mouse cells. This inhibitory activity includes proliferation and IL-6 production by B cells, and IFN-α and IL-12 production by plasmacytoid dendritic cells. Our studies of multiple cell types in both mice and humans show the optimal IRS to contain a GGGG motif within the sequence, and the activity to require a phosphorothioate backbone. Although the GGGG motif readily itself leads to formation of a tetrameric oligodeoxynucleotide structure, inhibitory activity resides exclusively in the single-stranded form. When coinjected with a CpG oligodeoxynucleotide in vivo, IRS were shown to inhibit inflammation through a reduction in serum cytokine responses. IRS do not need to be injected at the same site to inhibit, demonstrating that rapid, systemic inhibition of TLR-9 can be readily achieved. IRS can also inhibit a complex pathological response to ISS, as shown by protection from death after massive systemic inflammation induced by a CpG-containing oligodeoxynucleotides.

IntroductionImmunostimulatory sequences (ISS) are short oligonucleotides (ODNs) that mimic the innate immune response to microbial DNA. 1 ISS contain one or more cytosine-phosphate-guanine (CpG) dinucleotide-containing motifs with unmethylated cytosine residues and are recognized by Toll-like receptor-9 (TLR-9), one of a family of receptors prominent in innate responses to microbial pathogens. 2,3 TLR-9 expression is not widely distributed, and ISS-responsive cells in human peripheral blood mononuclear cells (PBMCs) are limited to B cells and plasmacytoid dendritic cells (PDCs). ISS activate B cells to proliferate, secrete interleukin (IL)-6, and differentiate to plasma cells. 4 PDCs respond to ISS by secreting type I interferons, tumor necrosis factor ␣ (TNF-␣), and, upon signaling through CD40, IL-12. 2,5 In both cell types, ISS are potent enhancers of antigen-presenting cell (APC) function and induce key costimulatory molecules, such as CD40, CD80, and CD86. 6 In mixed cell populations, such as human PBMCs, the direct response to ISS initiates a cascade of secondary responses, including activation of macrophages and natural killer (NK) cells and the induction of interferon ␥ (IFN-␥) and a wide range of other cytokines and chemokines characteristic of inflammatory responses. [6][7][8][9][10] At least 3 classes of ISS can be distinguished on the basis of both structure and function. Uniformly modified phosphorothioate (PS) ISS, called CpG-B, strongly activate B cells but are weak stimulators of IFN-␣ from human PBMCs. Phosphodiester (PO)-linked sequences flanked by PS-linked poly-G ends, called CpG-A ISS, in contrast, are potent inducers of IFN-␣ and IFN-␥ but are weak activators of B cells. 11 Recently, a third class of ISS has been defined, called CpG-C. CpG-C ISS retain distinctive properties of both CpG-A and -B ISS with respect to IFN-␣ production and B-cell activation. 6 Preliminary data have shown that ISS affect PDC survival, maturation, and cytokine production, but the differential properties of these ISS on PDCs are otherwise undefined. 5,6,12 The efficient activation of APCs and induction of IL-12, IFN-␣, and IFN-␥ explains the potent ability of ISS to act as a strong T-helper cell 1 (Th)1-polarizing adjuvant. 13 Besides inducing Th1 responses, administration of ISS mixed or covalently linked to antigens can inhibit Th2 responses 14-17 and stimulate CD8 T-cell responses. 7 These activities have stimulated much interest in the clinical use of ISS as a vaccine adjuvant and in the treatment of allergy, asthma, cancer, and infectious diseases, and clinical trials are currently being conducted in all of these disease areas.IL-10 is a potent anti-inflammatory cytokine that can act as a feedback regulator of the inflammatory response to many microbial stimuli. 18 IL-10 can be produced by a number of different cell types and can inhibit both Th1 and Th2 responses by affecting APC function and dendritic cell (DC) maturation. 18-20 IL-10 inhibits activation and induces death of PDCs in vitro and can reduce PDC...

CpG-C are a novel class of CpG motif-containing immunostimulatory sequences (ISS) that includes both a 5'-TCG element and a CpG-containing palindrome. CpG-C drive all known ISS activities and, in particular, are potent enhancers of IFN-alpha from plasmacytoid dendritic cells (PDCs). In our examination of CpG-C sequence requirements, we determined that optimal IFN-alpha-inducing activity could be achieved with longer palindromes. Longer palindromes also correlated with maintenance of the double-stranded (ds) form despite concentration and pH changes, indicating a preference for ds oligodeoxynucleotides (ODNs) by the ISS-induced signaling mechanism for IFN-alpha synthesis. This correlation did not hold for all arms of the ISS-induced immune response, since we did not observe increased B cell activity with the longer palindrome CpG-C ODNs. We further demonstrated that CpG-C retained activity in an in vitro primate system and induced the expression of several cytokines and IFN-alpha-inducible genes when CpG-C were administered in vivo to mice and primates. In conclusion, we have shown CpG-C to exert several types of immune functions across multiple species, and this novel class is thus an attractive candidate for ISS-based therapeutic strategies.