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

272 13 Applications of CpG Motifs from Bacterial DNA in Cancer Immunotherapy
othioate CpG ODN despite normal activation by LPS [50]. It is therefore intriguing
that TLR-9appears to be localized within the endosome [51]. Human 293 (embryonic
kidney) cells do not express TLR-9, and normally are not activated by a CpG ODN.
However, these cells become CpG responsive when they are transfected to express
TLR-9[51]. Among human cell types, TLR-9expression is highest in B cells and plasmacytoid
DC, which are also the only cell types that have been reproducibly shown
to be directly activated by phosphorothioate CpG ODN [51, 52]. Perhaps the most direct
evidence for the role of TLR-9in the direct recognition of a CpG motif is the fact
that 293 cells transfected to express the mouse TLR-9 protein become optimally responsive
to the preferred mouse CpG motif, GACGTT, while 293 cells transfected to
express the human TLR-9protein become optimally responsive to the preferred human
CpG motif, GTCGTT. Thus, the TLR-9protein determines the species specificity
of CpG motifs and presumably either binds to the CpG motif directly or in close
association with some unidentified cofactor.
CpG DNA activates the MAPK pathways and NFkB within minutes of exposure in
B cells, DCs and mouse macrophage-like cell lines [45, 53±55]Ç Although these signaling
pathways are also used in responses to LPS, differences in their biologic effects
demonstrate that there must be some additional specific signal(s) provided by LPS
and/or CpG [56±59]Ç Independent signaling activities are also suggested by the observations
that CpG DNA and LPS synergize for inducing macrophage NO production
[60] and TNF-a expression in vitro [61], and for inducing the systemic inflammatory
response syndrome (SIRS; ªcytokine stormº) in vivo [62].
13.4
Backbone-dependent Immune Effects of CpG Motifs and Delineation of CpG-A versus
CpG-B Classes of ODN
The immune effects of a CpG ODN are determined not only by the bases flanking
the CpG dinucleotide, but also by the backbone of the ODN. ODN synthesized with
the normal phosphodiester DNA backbone (PO) are rapidly degraded inside lymphocytes
[63]. The susceptibility of phosphodiester ODN to degradation not only reduces
their ability to drive B cell proliferation, but can even result in an artifact in studies
using a [ 3 H]thymidine incorporation assay to measure B cell proliferation. Degradation
of the phosphodiester DNA releases free thymidine which competes for the
[ 3 H]thymidine, causing an apparent decrease in cell proliferation [64]. This non-specific
effect is most marked if thymidine nucleotides are present at the 3¢ end of an
ODN, but can also be observed with high-molecular-weight DNA such as bacterial
genomic DNA. At least one exonuclease is specifically expressed in B lymphocytes,
indicating that results obtained using phosphodiester DNA in one cell type cannot
necessarily be extrapolated to other cell types [65]. Moreover, nuclease activities appear
to be higher in human than in mouse cells, with the result that phosphodiester
DNA can appear non-stimulatory unless it is added repeatedly [66, 67].
Replacement of one of the non-bridging oxygen atoms around the phosphodiester
linkage with a sulfur atom, which is called a phosphorothioate (PS) linkage, provides