Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

13.3 Identification of a Specific Receptor for CpG motifs, Toll-like Receptor (TLR)-9
Furthermore, the bases flanking CpGs in vertebrate genomes are not random: the
most common base preceding a CpG is a C and the most common base following a
CpG is a G [34]. It is therefore noteworthy that these types of CpG motifs (CCG and
CGG) do not support strong immune stimulation, but can actually neutralize other
immune stimulatory CpG motifs [35]. Thus, vertebrate DNA inhibits the immune
stimulatory effects of bDNA [36]. In addition to these differences in CpG content,
CpG dinucleotides are not methylated in bDNA, but are routinely methylated at the
5 position of about 70 % of the cytosines in vertebrate DNAs [33], which increases
their immune inhibitory effects [36].
The immune stimulatory effects of bDNA are due to the unmethylated CpG motifs,
since methylation with CpG methylase completely abolished its activity [32]. Extracts
of Babesia bovis, like those of many other microbes, are immune stimulatory. These
effects result from the DNA, since they are abolished by treatment of the extracts
with nuclease and can be reproduced with purified B. bovis DNA [37]. Immune-stimulatory
DNA is not unique to microbes; Drosophila extracts are also immune stimulatory,
due to the unmethylated CpG motifs in insect DNA [38]. A survey of different
genomic DNAs has shown immune stimulation by nematode and mollusk
DNAs and confirmed that hypomethylation of CpG is required for immune stimulation
[39].
13.3
Identification of a Specific Receptor for CpG motifs, Toll-like Receptor (TLR)-9
ªPattern recognition receptorsº (PRRs) give the innate immune system a general
ability to detect certain molecular structures that are conserved among pathogens,
but are not present in self tissues [21, 40]. Examples of PRR ligands include endotoxins,
high manose proteins, double-stranded viral RNAs and, most recently, CpG motifs
[21, 40, 41]Ç The immune system uses specific PRRs to detect the presence of particular
types of pathogens, coupling this detection to the activation of appropriate defense
pathways. A major group of PRRs is the TLR family, named for their homology
to the fruit fly Toll protein, that coordinates insect innate immune responses to infection
[42, 43]Ç This family of proteins appears to have at least 10 members in the human,
that couple the detection of microbial or viral products to cell activation via the
adaptor molecule MyD88 and TRAF6, that are linked to the IkB kinase complex and
to the MAPKs.
Some PRRs are expressed on the cell surface, where they may easily detect pathogens.
For example, some peptidoglycans are detected by TLR-2, most LPS are detected
via TLR-4 and flagellin is detected via TLR-5 [42]. In contrast, it appears that
CpG ODN must be taken up by cells and bind to an intracellular receptor [32, 44±47].
Drugs that interfere with the endosomal acidification/processing of ODN, such as
chloroquine, specifically block the immune stimulatory effects of CpG DNA, but not
those of LPS [45, 48, 49]. These data suggest that an endosomal step is required for
the CpG-induced signal transduction pathways [45, 49]. Recently, mice deficient in a
member of the TLR family, TLR-9, were shown to be unresponsive to a phosphor-
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