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* Department of Internal Medicine, Division of Clinical Pharmacology, University of Munich, Munich, Germany; and
Institute of Immunology, University of Heidelberg, Heidelberg, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IFN-
) upon viral infection (10, 11). Based on their distinct activity on PDC, two different
types of CpG ODN have been defined: CpG type A (prototype ODN 2216)
(7), which induces large amounts of IFN- in PDC; and
CpG type B (prototype ODN 2006) (7, 12), which is weak at
inducing IFN- but promotes survival, activation, and maturation of
PDC. Besides PDC, B cells are primary target cells for CpG ODN
(13, 14, 15). Direct activation of other human cell types is
controversial to date. CpG ODN activates monocytes in the context of
PBMC (16), but there are also reports that isolated
monocytes respond to CpG ODN (5, 17). Both direct and
indirect actions of CpG ODN have been proposed for human NK cells and T
cells (18, 19, 20). Although the CpG motifs differ between mice and humans (6, 13), in both species Toll-like receptor (TLR)9 seems to be involved in the recognition of CpG motifs (6, 9, 21). TLR9 belongs to the family of TLR, which established a combinatorial repertoire to discriminate among a wide spectrum of pathogen-associated microbial molecules (for a detailed review see Ref. 22). So far, 10 members of the TLR family (TLR1 to TLR10) have been reported (23, 24, 25, 26, 27). It has been demonstrated that the cytoplasmatic domains of TLR1, TLR2, and TLR6 form functional pairs to recognize a variety of microbial molecules (28). TLR cooperation not only extends the spectrum of ligands but also modulates the response toward a specific ligand. For example, TLR2-mediated response to phenol-soluble modulin is enhanced by TLR6 but inhibited by TLR1, indicating a functional interaction between these receptors (29). Although TLR9 seems to be essential for the recognition of CpG ODN in mice (21), other TLRs might be involved in modulating its activity.
In this study we used quantitative real-time PCR to examine the expression of TLR1 to TLR10 in subsets of human immunocompetent cells. In addition, we evaluated the sensitivity of these cell populations to CpG ODN and examined the modulation of TLR expression in CpG ODN-sensitive cells. The quantitative level of different TLRs in conjunction with sensitivity to CpG ODN allowed us to identify candidate TLRs potentially involved in specific recognition of CpG motifs or in its modulation. Furthermore, our study reveals PDC as a key sensor of CpG motifs in the human immune system, which regulates the activity of other cell types such as monocytes, NK cells, and T cells via PDC-derived cytokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following completely and partially phosphorothioate-modified ODN were provided by Coley Pharmaceutical Group (Wellesley, MA) and used at a final concentration of 6 µg/ml (small letters represent phosphorothioate linkage, capital letters represent phosphodiester linkage 3' of the base, and boldface represents CpG dinucleotides): ODN 2006, 5'-tcgtcgttttgtcgttttgtcgtt-3' (12); and ODN 2216, 5'-ggGGGACGATCGTCgggggG-3' (7). ODN were tested for endotoxin using the Limulus amebocyte lysate assay (lower detection limit, 0.1 EU/ml; BioWhittaker, Walkersville, MD).
Preparation, isolation, and culture of cells
Human PBMC were isolated from buffy coats provided by the blood bank of the University of Greifswald (Greifswald, Germany). Blood donors were 18- to 65-year-old healthy men and women who were tested to be negative for HIV, hepatitis B virus, and hepatitis C virus. Further exclusion criteria are manifest infections during the last 4 wk, fever, symptomatic allergies, abnormal blood cell counts, increased liver enzymes, or medication of any kind except vitamins and oral contraceptives. PBMC were prepared from buffy coats by Ficoll-Hypaque density gradient centrifugation (Biochrom, Berlin, Germany). PDC were positively isolated using an anti-blood DC Ag (BDCA)-4 Ab according to the manufacturer’s protocol (BDCA-4 cell isolation kit; Miltenyi Biotec, Bergisch Gladbach, Germany). Untouched primary B cells were prepared by an indirect magnetic labeling system using Abs against CD2, anti-IgE, CD4, CD11b, CD16, and CD36 to deplete T cells, NK cells, monocytes, granulocytes, platelets, and erythroid precursor cells from PBMC (B cell isolation kit; Miltenyi Biotec) (purity > 95% and no PDC detectable). Monocytes were isolated by depletion of non-monocytes (T cells, granulocytes, NK cells, B cells, DCs, and basophils) by using a mixture of Abs against CD3, CD7, CD19, CD45RA, CD56, and anti-IgE (monocyte isolation kit; Miltenyi Biotec) (purity > 97%). Untouched T cells were prepared from PBMC with a mixture of CD11b, CD16, CD19, CD36, and CD56 Abs (Pan T Cell Isolation kit; Miltenyi Biotec) (purity > 96%; depletion of non-T cells). Depletion of non-NK cells (T cells, B cells, and myeloid cells) was used to isolate untouched NK cells (CD3, CD14, CD19, CD36, and anti-IgE Abs; purity > 95%) (NK cell isolation kit; Miltenyi Biotec). When necessary, PDC were depleted before isolation of the other cell types by using the anti-BDCA-4 Ab (Miltenyi Biotec). CD8+ memory T cells were isolatedby positive sorting of CD8+CD45R0+ double-positive cells on a FACS sorter (FACSVantage SE-DIVA; BD Biosciences, Heidelberg, Germany) (purity > 99%). Cells were resuspended in IMDM supplemented with 8% human AB serum (BioWhittaker), 1.5 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all Sigma, Munich, Germany). All compounds purchased were endotoxin tested. Viability of cells was determined by trypan blue exclusion. In some experiments a transwell culture system was used for coculture of either purified NK cells or T cells with PBMC (0.2-µm Anapore membrane device; Nunc, Roskilde, Denmark). For the generation of PDC-derived supernatant, purified PDC (200,000 cells/ml) were stimulated with 3 µg/ml ODN 2006 or ODN 2216. Cell-free supernatant was harvested after 48 h and added to purified T cells and NK cells at a final concentration of 5%.
Generation of peptide-specific CD8+ T cell clones
Melan A26–35 A27L peptide-specific
CD8+ T cell clones were generated from PBMCs of
HLA-A*0201-positive healthy volunteers. PBMCs were stimulated in vitro
for 14 days with Melan A26–35 A27L peptide to
increase the frequency of Ag-specific cells. Melan
A26–35 A27L peptide-specific cells were labeled
after restimulation using the IFN-
secretion assay (Miltenyi
Biotec), subsequently sorted directly into 96-well plates using a
FACStarPlus flow cytometer (BD Biosciences) at a
frequency of 1 cell per well, and expanded as previously described
(30). Peptide-specific clones were simultaneously
stimulated at a ratio of 10:1 with TAP-deficient T2 cells (lymphoblast
cell line ATCC CRL-1992 (American Type Culture Collection,
Manassas, VA); T2 cells present only exogenous peptides) loaded with
their cognate peptide and CpG ODN (6 µg/ml) either in the presence or
absence of PBMC separated by a transwell cell culture device (0.2-µm
Anapore membrane; Nunc).
Flow cytometry
At the indicated time points, cells were harvested and surface Ag staining was performed as previously described (31). Anti-human CD3 (UCHT19), CD8 (RPA-T8), CD14 (3E2), CD19 (HIB19), CD69 (FN50), CD80 (L307.4), CD86 (IT2.2), CDw123 (7G3), and HLA DR (L243) were purchased from BD PharMingen (Heidelberg, Germany). Flow cytometric data were acquired on a FACSCalibur equipped with two lasers (BD Biosciences). Analysis was performed on viable cells. Data were analyzed using CellQuest software (BD Biosciences).
RT-PCR
Purified cell populations were cultured for 3 or 15 h,
respectively, in RPMI 1640 with 10% FCS. Cells were lysed and RNA was
extracted using the total RNA isolation kit (High Pure; RAS, Mannheim,
Germany). An aliquot of 8.2 µl RNA was reverse transcribed using
avian myeloblastosis virus-reverse transcriptase (RT) and
oligo(dT) as primer (First Strand cDNA Synthesis kit; Roche, Mannheim,
Germany). The obtained cDNA was diluted 1/25 with water and 10 µl
were used for amplification. Parameter-specific primer sets optimized
for the LightCycler (RAS) were developed by and purchased from
Search-LC (Heidelberg, Germany). The primer positions and amplification
efficiency are shown in Table I
. The PCR
was performed with the LightCycler FastStart DNA SYBR GreenI kit (RAS)
according to the protocol provided in the parameter-specific kits. To
control for specificity of the amplification products, a melting curve
analysis was performed. No amplification of unspecific products was
observed. The copy number was calculated from a standard curve,
obtained by plotting known input concentrations of four different
plasmids at log dilutions to the PCR cycle number (CP) at which the
detected fluorescence intensity reaches a fixed value. Using >300 data
points, the actual copy number per microliter of cDNA was calculated as
follows: X = e(-0.6553 x CP +
20.62). This approach dramatically reduced
variations due to dilution errors over several logarithmic dilution
steps. The amplification efficiency of the PCR was determined by
running log dilutions of standards. The slope of the standard curve was
converted to the amplification efficiency E by the following
algorithm: E = 10-1/slope. All
used primer sets had an efficiency >1.86 (Table I). The data of two
independent analyses for each sample and parameter were averaged. The
copy number of the different TLRs and of IL-8 was normalized by the
housekeeping gene cyclophilin-B and is presented as number of
transcripts per 103 copies of cyclophilin-B.
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1500 genomic copies. As shown in
Table I
, the copy number detected by the TLR primer sets is within the
range of 563-1791 copies (Table I). This range is close to the expected
value of 1500 genomic copies, considering a 16 million-fold
amplification demonstrating how precisely our assay performed. To
exclude contamination of cDNA with genomic DNA in randomly selected
samples we performed controls in which the RT was replaced by water
during the cDNA synthesis. Such "minus RT" controls did not contain
genomic DNA, as shown by a lack of PCR amplification. As a positive
control for the amplification of TLR we measured the number of TLR
transcripts in the cDNA prepared from 2 x
106 PBMC (Table I). As
TLRlow expressing control we used HEK293 cells
(Table I). Statistical analysis
Data are expressed as mean values ± SEM.
Statistical significance of differences was determined by the paired or
unpaired two-tailed Student t test. Differences were
considered statistically significant for p < 0.05.
Statistical analyses were performed using StatView 4.51 software
(Abacus Concepts, Calabasas, CA). In
Figs. 3–5, an asterisk indicates
values of p < 0.05 between medium control and
stimulation with CpG ODN.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We applied quantitative real-time PCR to study expression of
TLR transcripts in cellular subsets of human PBMC. A detailed
description of the technique is provided in Materials and
Methods. The characteristics of primer sets and controls are
provided in Table I. Human PDC, B cells, NK cells, T cells, and
monocytes were purified from freshly isolated PBMC, and TLR1 through
TLR10 were measured after 3 h of cell culture in the absence of
stimulation (Fig. 1).
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). Among these, TLR9 was most
prominent. B cells expressed a broad range of TLRs, which included the
TLR expressed in PDC, namely TLR1, TLR6, TLR7, TLR9, and TLR10 (Fig. 1). Compared with PDC, TLR1 and TLR6 were higher and TLR9 was lower in
B cells. In addition to PDC, B cells expressed considerable levels of
TLR2 and high levels of TLR10, while expression of TLR4 was relatively
low. Monocytes were characterized by an extremely high expression of
TLR2 (Fig. 1), with intermediate levels of TLR1 and TLR4, moderate
levels of TLR5, TLR6, and TLR8, and weak but detectable TLR9
expression. In NK cells expression levels of TLR1 were highest,
followed by moderate levels of TLR2, TLR3, TLR5, and TLR6 (Fig. 1). TLR
expression in T cells in general was low, with TLR5 being the only TLR
that was expressed at higher levels (Fig. 1). Low levels of TLR9 could
be detected in both NK cells and T cells.
Comparison of the expression levels of single TLRs in different cell
types (Fig. 1, vertical comparison) revealed the following
characteristic and statistically significant differences
(p < 0.05) between the cell subsets:
TLR1 was higher in B cells, monocytes, and NK cells as
compared with PDC and T cells, while between PDC and T cells, as well
as among monocytes, NK cells, and T cells, no significant difference
was found. TLR2 was higher in monocytes than in all other
subsets. Expression of TLR3 was higher in NK as compared
with other cell types. Regarding TLR4, monocytes expressed
higher levels than other cell types. In B cells, TLR4 expression seemed
to be more prominent than in PDC, NK cells, and T cells, but this
difference was not significant. Marked levels of TLR5 were
found in monocytes, NK cells, and T cells, all of which were
significantly higher than in PDC and B cells lacking TLR5.
TLR6 was highest in B cells as compared with all other cell
types. Moreover, monocytes and NK cells expressed higher TLR6 levels
than did T cells. TLR7 showed the opposite expression
pattern from TLR5; TLR7 was significantly higher in PDC and B cells as
compared with monocytes, NK cells, and T cells, which showed only
marginal levels of TLR7. Furthermore, TLR7 was significantly higher in
PDC than in B cells. Considerable levels of TLR8 were only
expressed in monocytes, which were higher than in PDC, B cells, and T
cells. The difference of TLR8 expression between monocytes and NK cells
did not reach statistical significance (p =
0.06). TLR9 showed a very similar expression pattern among
cell subsets as TLR7, but on a higher level. As for TLR7, PDC expressed
higher levels of TLR9 than did B cells; both PDC and B cells showed
higher TLR9 expression than did monocytes, NK cells, and T cells, which
only expressed marginal levels of TLR9 and among themselves showed no
significant differences. TLR10 was prominent on B cells as
compared with all other cell types (p <
0.001). Besides B cells, only PDC consistently expressed low levels of
TLR10, which were significantly higher than in monocytes, NK cells, and
T cells, which lacked TLR10.
In the absence of PDC, purified monocytes are not sensitive to CpG ODN-mediated activation
Sensitivity of different purified immune cell subsets to CpG ODN in conjunction with the expression pattern of TLRs may allow the identification of TLRs potentially involved in recognition of CpG ODN. In previous studies we and others demonstrated that both purified PDC and B cells are directly sensitive to CpG ODN-mediated activation (4, 5, 6, 7, 9, 13, 14, 15). We were interested in whether monocytes are directly or indirectly activated by CpG ODN. Two CpG ODN with distinct biological properties were used for these studies. ODN 2006 is the prototype of a CpG ODN which potently activates human B cells and PDC (5, 7, 12). The specific characteristic of ODN 2216 is to induce high amounts of type I IFN in PDC (7).
Monocytes were purified from freshly isolated PBMC by depletion of other cell types using magnetic beads (purity > 95%; see Materials and Methods). We found that this procedure led to an enrichment of PDC within the resulting monocyte population (increase of PDC from 0.2 to 0.4% in PBMC up to >1% in isolated monocytes). B cells were not detected within isolated monocytes. To avoid contamination with PDC, PDC were depleted by a magnetically labeled PDC-specific Ab (anti-BDCA-4 Ab) before the standard monocyte isolation protocol was applied to PBMC. The PDC-containing monocyte population (standard protocol) as well as the PDC-free monocyte population were incubated with CpG ODN.
After 48 h of culture, expression of CD80, CD86, and MHC
class II was assessed by flow cytometry (Fig. 2). In the monocyte preparation without
PDC, monocytes did not respond to stimulation by CpG ODN (Fig. 2, open
bars). In contrast, in the presence of PDC (Fig. 2, filled bars), CpG
ODN induced a marked increase of CD80 (ODN 2006 MFI, 64 vs 11;
n = 3), CD86 (ODN 2006 MFI, 106 vs 75;
p < 0.05; n = 3) and MHC class II (ODN
2006 MFI, 130 vs 74; n = 3) (Fig. 2, filled bars).
These results indicated that purified monocytes were not sensitive to
CpG ODN unless PDC were present.
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Previous studies demonstrated that, within PBMC, NK cells are
activated by CpG ODN (12, 18, 20). However, there has been
controversy over whether this activation is direct (20) or
indirect (12, 18). We separated NK cells from PBMC and
confirmed that, to the limits of detection, isolated NK cells did not
contain PDC or B cells. NK cells were incubated with ODN 2006 and ODN
2216 in a transwell system in the presence or absence of PBMC.
Expression of CD69 was examined after 24 h of culture. In the
absence of PBMC none of the CpG ODN up-regulated CD69 expression in NK
cells (Fig. 3A, open bars),
demonstrating that purified NK cells are not sensitive to CpG ODN. In
contrast, NK cells cocultivated with PBMC were strongly activated by
ODN 2216 (Fig. 3A, filled bars). ODN 2006 was less
effective. Because the transwell system inhibited cell to cell contact,
PBMC-derived soluble factors were responsible for indirect activation
of NK cells with CpG ODN. Next we tested whether PDC-derived cytokines
contribute to activation of NK cells. Supernatants were collected from
purified PDC, which were stimulated for 2 days with ODN 2006 or ODN
2216. In the presence of these supernatants containing PDC-derived
cytokines, isolated NK cells strongly up-regulated CD69 expression
within 2 days (Fig. 3B). Similar activation of NK cells was
found in the presence of rIFN- (Fig. 3B, right
bar), which is known to be produced by PDC in response to CpG ODN
(4, 5, 7).
For CpG ODN-mediated activation of T cells, direct (20, 32) and indirect (19, 33) mechanisms have been
proposed. To address this question we studied the effects of ODN 2006
and ODN 2216 on memory T cells and on Ag-specific activation of
peptide-specific CD8+ T cell clones.
CD45R0+CD8+ memory T cells
were isolated from PBMC by fluorescence-activated cell sorting and were
stimulated with ODN 2006, ODN 2216, the supernatants of CpG
ODN-activated PDC, or rIFN- (5000 IU/ml). After 48 h of cell
culture, expression of CD69 was assessed by flow cytometry. Although no
stimulation of purified T cells was observed in the presence of CpG
ODN, both the supernatants of CpG-activated PDC and rIFN- strongly
activated memory T cells, as indicated by up-regulation of CD69 (Fig. 4A).
To test the ability of CpG ODN to promote Ag-specific T cell responses,
CD8+ T cell clones with specificity for the Melan
A26–35 A27L peptide were generated and used as a
model system. Clones were restimulated in the presence or absence of
PBMC in a transwell cell culture. As expected, restimulation of T cell
clones with their cognate peptide (loaded on T2 cells) for 24 h
led to an increased expression of CD69 both with and without PBMC (Fig. 4B). In the absence of PBMC none of the CpG ODN up-regulated
CD69 on CD8+ T cells (Fig. 4B, open
bars). However, when PBMC were present in the transwell system CD69
expression on CD8+ T cells was markedly increased
in the presence of ODN 2216. The effect of ODN 2006 again was lower.
Soluble factors were responsible for indirect activation in the
presence of PBMC, as cell to cell contact was inhibited by the
transwell system. Together, these results indicated that purified NK
cells as well as CD8+ T cells are not sensitive
to CpG ODN but are activated by CpG ODN-induced PDC-derived cytokines.
ODN 2216 was more potent than ODN 2006 to indirectly stimulate NK
cells, memory CD8+ T cells, and CD8 T cell
clones.
Comparison of sensitivity to CpG ODN and the TLR expression pattern
To confirm that both PDC and B cells are directly activated by CpG
ODN in our system, we examined whether IL-8 as one representative
indicator of activation is up-regulated in response to CpG ODN. We
incubated purified PDC and B cells with or without CpG ODN. After
3 h and again after 15 h, cells were harvested and mRNA was
prepared. Real-time PCR analysis revealed that IL-8 mRNA was rapidly
up-regulated in B cells (Fig. 5A, left panel) and
PDC (Fig. 5B, left panel) within 3 h. These
results indicated that PDC and B cells are able to recognize CpG
motifs.
To identify TLRs possibly involved in recognition of CpG motifs, we
were interested in TLRs that were expressed in both PDC and B cells but
not in monocytes, NK cells, and T cells. According to Fig. 1, only TLR7
and TLR9 match these criteria. Because TLR1 and TLR6 are expressed in
both PDC and B cells, but are also expressed in other cell types, they
must not be sufficient for the recognition of CpG motifs but are
potential candidates involved in modulating the recognition of CpG
motifs.
Modulation of TLR9 and TLR7 expression in B cells and PDC in response to CpG ODN
It has been described that microbial stimuli affect the expression
of their cognate TLR (34, 35, 36, 37). We hypothesized that the
expression of a TLR involved in the recognition of CpG ODN would be
modulated upon stimulation with CpG ODN. We quantified TLR1, TLR6,
TLR7, and TLR9 mRNA after 3 h and again after 15 h of
stimulation of B cells and PDC with CpG ODN. Because ODN 2216 is weak
at stimulating B cells (our unpublished observation), B
cells were stimulated with ODN 2006, while PDC were stimulated with
both ODN 2006 and ODN 2216. In B cells both TLR9 and TLR7 were
down-regulated within 15 h after exposure to ODN 2006 (Fig. 5A, middle and right panels). In PDC,
almost a complete loss of TLR9 expression was seen after stimulation
with ODN 2006 as well as ODN 2216 (Fig. 5B, middle
panel, hatched bars). A similar decrease of TLR9 was found in the
presence of the growth factor IL-3, which, like CpG ODN, is known to
induce differentiation of PDC (Fig. 5B, middle
panel, open bars). IL-3 also decreased TLR7 (Fig. 5B,
right panel, open bars). However, unlike for TLR9,
both CpG ODN increased the expression of TLR7 (Fig. 5B,
hatched bars). The mRNA levels of TLR1 and TLR6 did not show
significant changes upon stimulation with CpG ODN (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In our study, the CpG sensitivity in conjunction with the TLR expression pattern of purified subsets of PBMC was consistent with the concept that TLR9 is primarily involved in recognition of CpG motifs in B cells and PDC. Interestingly, we found that CpG ODN rapidly down-regulated the expression of TLR9 in PDC. However, a similar decrease of TLR9 was observed during differentiation of PDC with IL-3 in the absence of CpG ODN. Consequently, the CpG-induced decrease in TLR9 cannot be separated from the reduction of TLR9 as a general aspect of PDC differentiation and thus provides no additional evidence that TLR9 is involved in recognition of CpG motifs. Similarly, it has been described that monocyte-derived immature DCs down-regulate TLR4 upon maturation with the corresponding cognate ligand LPS, but also in response to the unrelated maturation stimulus TNF (35).
It has been reported that TLR9 is essential for recognition of CpG ODN
in mice (21) and confers responsiveness to CpG ODN in
human cell lines (6). According to the model of
combinatorial recognition of microbial molecules by TLR
(28), other members of the TLR family might exist which
modulate the activity of TLR9. Besides TLR9, we found that marked
levels of TLR7 were expressed in CpG-sensitive cell types (PDC and B
cells) but not in the other cell types, which are not directly
sensitive to CpG ODN. Of note, in PDC TLR7 was up-regulated in response
to CpG ODN as opposed to decreased levels of TLR7 in the presence of
IL-3. In contrast to PDC, in B cells TLR7 was down-regulated in
response to CpG ODN. One might speculate that CpG-induced regulation of
TLR7 expression may be involved in positively or negatively modulating
the recognition of CpG motifs by TLR9. Furthermore, other TLRs such as
TLR1 and TLR6 may still be involved in modulating the recognition of
CpG motifs despite not being regulated upon CpG-mediated stimulation.
Cotransfection experiments are currently being performed to study a
cooperative role of TLR7 and TLR9 in the context of recognition of CpG
motifs. It has been reported that transfection of HEK293 cells with
TLR9 conferred responsiveness to CpG motif containing DNA
(6). We found that the relative lack of TLR1–10
expression in HEK293 cells (see Table I) provides a valuable
transfection model to examine cooperation between different
TLRs.
Our study represents the first analysis of TLR expression using real-time PCR, which is both a sensitive and a quantitative method to assess the number of transcripts of the target mRNA. We demonstrate that each cell type examined displays a characteristic profile of TLRs that was consistent between the different donors as demonstrated by statistical analysis. These results support the concept that different types of infections induce distinct types of immune responses based on activation of the subsets of immune cells that express the corresponding profile of TLRs. Because our studies were performed with cells from healthy blood donors, it will be interesting to study whether conditions such as a history of allergies or infectious disease affect the TLR profile.
In a previous study Muzio et al. (36) examined expression of TLR1 through TLR5 in human leukocyte subsets by Northern blot analysis. We confirm their results and, by using a more sensitive method, extend their study, demonstrating that TLR2 and TLR5 are not restricted to myelomonocytic cells. In our study, TLR2 was also present in B cells, NK cells, and T cells, and TLR5 was present in T cells and NK cells. TLR3 was also expressed in NK cells and is absent in PDC. Consequently, expression of TLR3 seems not to be a general feature of DCs.
Consistent with an earlier study (28), we found that TLR1 and TLR6 are expressed in monocytes. In addition, we detected high levels of TLR1 and TLR6 in B cells, suggesting a particular role of these two TLRs in B cells. TLR10, which is phylogenetically closely related to TLR1 and TLR6 (27), was present in B cells at similarly high levels.
In earlier studies we and others demonstrated by using semiquantitative RT-PCR that PDC express TLR9, while monocytes expressed TLR2 and TLR4 (6, 9). This study confirms and complements these results on a quantitative level. It has been suggested that isolated human monocytes are activated by CpG ODN and bacterial DNA (5, 17). In contrast, in the present study, evaluation of PDC within the monocyte population revealed that, in the absence of PDC, monocytes were nonresponsive to both CpG ODN tested, and that very low numbers of PDC were sufficient to activate monocytes via PDC-derived cytokines. Therefore, our early results of monocyte activation within human PBMC were likely due to secondary effects of CpG ODN mediated by PDC (16).
Of note, there seem to be significant differences between mice and humans regarding sensitivity of myeloid cells to CpG ODN. In mice macrophages and myeloid DC are directly activated upon recognition of CpG motifs (38, 39, 40, 41). In humans, besides monocytes as discussed above, monocyte-derived DC and peripheral blood myeloid DC are not activated by CpG ODN (6, 7, 8). However, the detection of baseline levels of TLR9 in human monocytes in this study suggests that monocytes might be able to modulate TLR9 expression along distinct differentiation pathways they enter and the cytokine milieu and the microbial molecules they encounter.
In conclusion, quantitative assessment of TLR expression in immunocompetent cells at different stages of differentiation and activation will open new avenues in the field of TLR research. As new members join the growing list of TLRs, the approach presented in this study will help to assign the large variety of microbial molecules to their cognate innate receptors.
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Acknowledgments |
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Footnotes |
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2 V.H. and S.R. contributed equally to this manuscript.
3 Address correspondence and reprint requests to Dr. Gunther Hartmann, Abteilung für Klinische Pharmakologie, Medizinische Klinik Innenstadt, Klinikum der Ludwig-Maximilians-Universität München, Ziemssenstrasse 1, 80336 Munich, Germany. E-mail address: ghartmann{at}lrz.uni-muenchen.de
4 Abbreviations used in this paper: ODN, oligodeoxynucleotide; TLR, Toll-like receptor; RT, reverse transcriptase; DC, dendritic cell; PDC, plasmacytoid DC; BDCA, blood DC Ag.
Received for publication October 24, 2001. Accepted for publication February 20, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M. M. Petzke, A. Brooks, M. A. Krupna, D. Mordue, and I. Schwartz Recognition of Borrelia burgdorferi, the Lyme Disease Spirochete, by TLR7 and TLR9 Induces a Type I IFN Response by Human Immune Cells J. Immunol., October 15, 2009; 183(8): 5279 - 5292. [Abstract] [Full Text] [PDF]
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E. Gaudreault and J. Gosselin Leukotriene B4 Potentiates CpG Signaling for Enhanced Cytokine Secretion by Human Leukocytes J. Immunol., August 15, 2009; 183(4): 2650 - 2658. [Abstract] [Full Text] [PDF]
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 M. V. Pahl, S. Gollapudi, L. Sepassi, P. Gollapudi, R. Elahimehr, and N. D. Vaziri Effect of end-stage renal disease on B-lymphocyte subpopulations, IL-7, BAFF and BAFF receptor expression Nephrol. Dial. Transplant., August 14, 2009; (2009) gfp397v1. [Abstract] [Full Text] [PDF]
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S. C. Lindner, U. Kohl, T. J. Maier, D. Steinhilber, and B. L. Sorg TLR2 ligands augment cPLA2{alpha} activity and lead to enhanced leukotriene release in human monocytes J. Leukoc. Biol., August 1, 2009; 86(2): 389 - 399. [Abstract] [Full Text] [PDF]
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 A. Goodchild, N. Nopper, A. Craddock, T. Law, A. King, G. Fanning, L. Rivory, and T. Passioura Primary Leukocyte Screens for Innate Immune Agonists J Biomol Screen, July 1, 2009; 14(6): 723 - 730. [Abstract] [PDF]
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A. F. de Vos, J. M. Pater, P. S. van den Pangaart, M. D. de Kruif, C. van 't Veer, and T. van der Poll In Vivo Lipopolysaccharide Exposure of Human Blood Leukocytes Induces Cross-Tolerance to Multiple TLR Ligands J. Immunol., July 1, 2009; 183(1): 533 - 542. [Abstract] [Full Text] [PDF]
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P. Zhang, C. J. Cox, K. M. Alvarez, and M. W. Cunningham Cutting Edge: Cardiac Myosin Activates Innate Immune Responses through TLRs J. Immunol., July 1, 2009; 183(1): 27 - 31. [Abstract] [Full Text] [PDF]
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 H. Liang, R. S. Russell, N. L. Yonkers, D. McDonald, B. Rodriguez, C. V. Harding, and D. D. Anthony Differential Effects of Hepatitis C Virus JFH1 on Human Myeloid and Plasmacytoid Dendritic Cells J. Virol., June 1, 2009; 83(11): 5693 - 5707. [Abstract] [Full Text] [PDF]
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A. Ablasser, H. Poeck, D. Anz, M. Berger, M. Schlee, S. Kim, C. Bourquin, N. Goutagny, Z. Jiang, K. A. Fitzgerald, et al. Selection of Molecular Structure and Delivery of RNA Oligonucleotides to Activate TLR7 versus TLR8 and to Induce High Amounts of IL-12p70 in Primary Human Monocytes J. Immunol., June 1, 2009; 182(11): 6824 - 6833. [Abstract] [Full Text] [PDF]
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D. M. Miller, T. B. Thornley, T. Pearson, A. J. Kruger, M. Yamazaki, L. D. Shultz, R. M. Welsh, M. A. Brehm, A. A. Rossini, and D. L. Greiner TLR Agonists Prevent the Establishment of Allogeneic Hematopoietic Chimerism in Mice Treated with Costimulation Blockade J. Immunol., May 1, 2009; 182(9): 5547 - 5559. [Abstract] [Full Text] [PDF]
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J. Jendholm, M. Morgelin, M. L. A. Perez Vidakovics, M. Carlsson, H. Leffler, L.-O. Cardell, and K. Riesbeck Superantigen- and TLR-Dependent Activation of Tonsillar B Cells after Receptor-Mediated Endocytosis J. Immunol., April 15, 2009; 182(8): 4713 - 4720. [Abstract] [Full Text] [PDF]
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H. Shin, Y. Zhang, M. Jagannathan, H. Hasturk, A. Kantarci, H. Liu, T. E. Van Dyke, L. M. Ganley-Leal, and B. S. Nikolajczyk B cells from periodontal disease patients express surface Toll-like receptor 4 J. Leukoc. Biol., April 1, 2009; 85(4): 648 - 655. [Abstract] [Full Text] [PDF]
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I. Douagi, C. Gujer, C. Sundling, W. C. Adams, A. Smed-Sorensen, R. A. Seder, G. B. Karlsson Hedestam, and K. Lore Human B Cell Responses to TLR Ligands Are Differentially Modulated by Myeloid and Plasmacytoid Dendritic Cells J. Immunol., February 15, 2009; 182(4): 1991 - 2001. [Abstract] [Full Text] [PDF]
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K. L. Good, D. T. Avery, and S. G. Tangye Resting Human Memory B Cells Are Intrinsically Programmed for Enhanced Survival and Responsiveness to Diverse Stimuli Compared to Naive B Cells J. Immunol., January 15, 2009; 182(2): 890 - 901. [Abstract] [Full Text] [PDF]
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C. K. Pfaller and K.-K. Conzelmann Measles Virus V Protein Is a Decoy Substrate for I{kappa}B Kinase {alpha} and Prevents Toll-Like Receptor 7/9-Mediated Interferon Induction J. Virol., December 15, 2008; 82(24): 12365 - 12373. [Abstract] [Full Text] [PDF]
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O. M. Grauer, J. W. Molling, E. Bennink, L. W. J. Toonen, R. P. M. Sutmuller, S. Nierkens, and G. J. Adema TLR Ligands in the Local Treatment of Established Intracerebral Murine Gliomas J. Immunol., November 15, 2008; 181(10): 6720 - 6729. [Abstract] [Full Text] [PDF]
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 B. Kapitein, M. O. Hoekstra, E. H. J. Nijhuis, D. J. Hijnen, H. G. M. Arets, J. L. L. Kimpen, and E. F. Knol Gene expression in CD4+ T-cells reflects heterogeneity in infant wheezing phenotypes Eur. Respir. J., November 1, 2008; 32(5): 1203 - 1212. [Abstract] [Full Text] [PDF]
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 P. A. Taylor, M. J. Ehrhardt, C. J. Lees, A. Panoskaltsis-Mortari, A. M. Krieg, A. H. Sharpe, W. J. Murphy, J. S. Serody, H. Hemmi, S. Akira, et al. TLR agonists regulate alloresponses and uncover a critical role for donor APCs in allogeneic bone marrow rejection Blood, October 15, 2008; 112(8): 3508 - 3516. [Abstract] [Full Text] [PDF]
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D. Klinman, H. Shirota, D. Tross, T. Sato, and S. Klaschik Synthetic oligonucleotides as modulators of inflammation J. Leukoc. Biol., October 1, 2008; 84(4): 958 - 964. [Abstract] [Full Text] [PDF]
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M. Parcina, C. Wendt, F. Goetz, R. Zawatzky, U. Zahringer, K. Heeg, and I. Bekeredjian-Ding Staphylococcus aureus-Induced Plasmacytoid Dendritic Cell Activation Is Based on an IgG-Mediated Memory Response J. Immunol., September 15, 2008; 181(6): 3823 - 3833. [Abstract] [Full Text] [PDF]
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D. Chiron, I. Bekeredjian-Ding, C. Pellat-Deceunynck, R. Bataille, and G. Jego Toll-like receptors: lessons to learn from normal and malignant human B cells Blood, September 15, 2008; 112(6): 2205 - 2213. [Abstract] [Full Text] [PDF]
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E. Marcenaro, B. Ferranti, M. Falco, L. Moretta, and A. Moretta Human NK cells directly recognize Mycobacterium bovis via TLR2 and acquire the ability to kill monocyte-derived DC Int. Immunol., September 1, 2008; 20(9): 1155 - 1167. [Abstract] [Full Text] [PDF]
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J. P. Wang, G. N. Bowen, C. Padden, A. Cerny, R. W. Finberg, P. E. Newburger, and E. A. Kurt-Jones Toll-like receptor-mediated activation of neutrophils by influenza A virus Blood, September 1, 2008; 112(5): 2028 - 2034. [Abstract] [Full Text] [PDF]
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W. Jiang, M. M. Lederman, R. J. Mohner, B. Rodriguez, T. M. Nedrich, C. V. Harding, and S. F. Sieg Impaired Naive and Memory B-Cell Responsiveness to TLR9 Stimulation in Human Immunodeficiency Virus Infection J. Virol., August 15, 2008; 82(16): 7837 - 7845. [Abstract] [Full Text] [PDF]
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 E. L. J. M. Smits, P. Ponsaerts, Z. N. Berneman, and V. F. I. Van Tendeloo The Use of TLR7 and TLR8 Ligands for the Enhancement of Cancer Immunotherapy Oncologist, August 1, 2008; 13(8): 859 - 875. [Abstract] [Full Text] [PDF]
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A. Malaspina, S. Moir, A. C. DiPoto, J. Ho, W. Wang, G. Roby, M. A. O'Shea, and A. S. Fauci CpG Oligonucleotides Enhance Proliferative and Effector Responses of B Cells in HIV-Infected Individuals J. Immunol., July 15, 2008; 181(2): 1199 - 1206. [Abstract] [Full Text] [PDF]
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 B. G. Molenkamp, B. J.R. Sluijter, P. A.M. van Leeuwen, S. J.A.M. Santegoets, S. Meijer, P. G.J.T.B. Wijnands, J. B.A.G. Haanen, A. J.M. van den Eertwegh, R. J. Scheper, and T. D. de Gruijl Local Administration of PF-3512676 CpG-B Instigates Tumor-Specific CD8+ T-Cell Reactivity in Melanoma Patients Clin. Cancer Res., July 15, 2008; 14(14): 4532 - 4542. [Abstract] [Full Text] [PDF]
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 S. Nierkens, M. H. den Brok, R. P.M. Sutmuller, O. M. Grauer, E. Bennink, M. E. Morgan, C. G. Figdor, T. J.M. Ruers, and G. J. Adema In vivo Colocalization of Antigen and CpG within Dendritic Cells Is Associated with the Efficacy of Cancer Immunotherapy Cancer Res., July 1, 2008; 68(13): 5390 - 5396. [Abstract] [Full Text] [PDF]
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L. N. Henning, A. K. Azad, K. V. L. Parsa, J. E. Crowther, S. Tridandapani, and L. S. Schlesinger Pulmonary Surfactant Protein A Regulates TLR Expression and Activity in Human Macrophages J. Immunol., June 15, 2008; 180(12): 7847 - 7858. [Abstract] [Full Text] [PDF]
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C. J. Blohmke, R. E. Victor, A. F. Hirschfeld, I. M. Elias, D. G. Hancock, C. R. Lane, A. G. F. Davidson, P. G. Wilcox, K. D. Smith, J. Overhage, et al. Innate Immunity Mediated by TLR5 as a Novel Antiinflammatory Target for Cystic Fibrosis Lung Disease J. Immunol., June 1, 2008; 180(11): 7764 - 7773. [Abstract] [Full Text] [PDF]
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D. B. Rosen, W. Cao, D. T. Avery, S. G. Tangye, Y.-J. Liu, J. P. Houchins, and L. L. Lanier Functional Consequences of Interactions between Human NKR-P1A and Its Ligand LLT1 Expressed on Activated Dendritic Cells and B Cells J. Immunol., May 15, 2008; 180(10): 6508 - 6517. [Abstract] [Full Text] [PDF]
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W. Shen, K. Stone, A. Jales, D. Leitenberg, and S. Ladisch Inhibition of TLR Activation and Up-Regulation of IL-1R-Associated Kinase-M Expression by Exogenous Gangliosides J. Immunol., April 1, 2008; 180(7): 4425 - 4432. [Abstract] [Full Text] [PDF]
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L. K. Juckem, K. W. Boehme, A. L. Feire, and T. Compton Differential Initiation of Innate Immune Responses Induced by Human Cytomegalovirus Entry into Fibroblast Cells J. Immunol., April 1, 2008; 180(7): 4965 - 4977. [Abstract] [Full Text] [PDF]
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A. Forsbach, J.-G. Nemorin, C. Montino, C. Muller, U. Samulowitz, A. P. Vicari, M. Jurk, G. K. Mutwiri, A. M. Krieg, G. B. Lipford, et al. Identification of RNA Sequence Motifs Stimulating Sequence-Specific TLR8-Dependent Immune Responses J. Immunol., March 15, 2008; 180(6): 3729 - 3738. [Abstract] [Full Text] [PDF]
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V. Athie-Morales, G. M. O'Connor, and C. M. Gardiner Activation of Human NK Cells by the Bacterial Pathogen-Associated Molecular Pattern Muramyl Dipeptide J. Immunol., March 15, 2008; 180(6): 4082 - 4089. [Abstract] [Full Text] [PDF]
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M. P. Gantier, S. Tong, M. A. Behlke, D. Xu, S. Phipps, P. S. Foster, and B. R. G. Williams TLR7 Is Involved in Sequence-Specific Sensing of Single-Stranded RNAs in Human Macrophages J. Immunol., February 15, 2008; 180(4): 2117 - 2124. [Abstract] [Full Text] [PDF]
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T. Ukai, H. Yumoto, F. C. Gibson III, and C. A. Genco Macrophage-Elicited Osteoclastogenesis in Response to Bacterial Stimulation Requires Toll-Like Receptor 2-Dependent Tumor Necrosis Factor-Alpha Production Infect. Immun., February 1, 2008; 76(2): 812 - 819. [Abstract] [Full Text] [PDF]
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V. Veckman and I. Julkunen Streptococcus pyogenes activates human plasmacytoid and myeloid dendritic cells J. Leukoc. Biol., February 1, 2008; 83(2): 296 - 304. [Abstract] [Full Text] [PDF]
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M. Fukata, K. Breglio, A. Chen, A. S. Vamadevan, T. Goo, D. Hsu, D. Conduah, R. Xu, and M. T. Abreu The Myeloid Differentiation Factor 88 (MyD88) Is Required for CD4+ T Cell Effector Function in a Murine Model of Inflammatory Bowel Disease J. Immunol., February 1, 2008; 180(3): 1886 - 1894. [Abstract] [Full Text] [PDF]
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F. Capolunghi, S. Cascioli, E. Giorda, M. M. Rosado, A. Plebani, C. Auriti, G. Seganti, R. Zuntini, S. Ferrari, M. Cagliuso, et al. CpG Drives Human Transitional B Cells to Terminal Differentiation and Production of Natural Antibodies J. Immunol., January 15, 2008; 180(2): 800 - 808. [Abstract] [Full Text] [PDF]
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T. Shimizu, Y. Kida, and K. Kuwano Mycoplasma pneumoniae-Derived Lipopeptides Induce Acute Inflammatory Responses in the Lungs of Mice Infect. Immun., January 1, 2008; 76(1): 270 - 277. [Abstract] [Full Text] [PDF]
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 E. F. M. Wouters, K. H. Groenewegen, M. A. Dentener, and J. H. J. Vernooy Systemic Inflammation in Chronic Obstructive Pulmonary Disease: The Role of Exacerbations Proceedings of the ATS, December 1, 2007; 4(8): 626 - 634. [Abstract] [Full Text] [PDF]
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A. Z. Dudek, C. Yunis, L. I. Harrison, S. Kumar, R. Hawkinson, S. Cooley, J. P. Vasilakos, K. S. Gorski, and J. S. Miller First in Human Phase I Trial of 852A, a Novel Systemic Toll-like Receptor 7 Agonist, to Activate Innate Immune Responses in Patients with Advanced Cancer Clin. Cancer Res., December 1, 2007; 13(23): 7119 - 7125. [Abstract] [Full Text] [PDF]
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F. Ishikawa, H. Niiro, T. Iino, S. Yoshida, N. Saito, S. Onohara, T. Miyamoto, H. Minagawa, S.-i. Fujii, L. D. Shultz, et al. The developmental program of human dendritic cells is operated independently of conventional myeloid and lymphoid pathways Blood, November 15, 2007; 110(10): 3591 - 3660. [Abstract] [Full Text] [PDF]
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J. P. Leonard, B. K. Link, C. Emmanouilides, S. A. Gregory, D. Weisdorf, J. Andrey, J. Hainsworth, J. A. Sparano, D. E. Tsai, S. Horning, et al. Phase I Trial of Toll-Like Receptor 9 Agonist PF-3512676 with and Following Rituximab in Patients with Recurrent Indolent and Aggressive Non Hodgkin's Lymphoma Clin. Cancer Res., October 15, 2007; 13(20): 6168 - 6174. [Abstract] [Full Text] [PDF]
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 Y.-C. Chen, E. Giovannucci, P. Kraft, R. Lazarus, and D. J. Hunter Association between Toll-Like Receptor Gene Cluster (TLR6, TLR1, and TLR10) and Prostate Cancer Cancer Epidemiol. Biomarkers Prev., October 1, 2007; 16(10): 1982 - 1989. [Abstract] [Full Text] [PDF]
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C.-L. Ku, H. von Bernuth, C. Picard, S.-Y. Zhang, H.-H. Chang, K. Yang, M. Chrabieh, A. C. Issekutz, C. K. Cunningham, J. Gallin, et al. Selective predisposition to bacterial infections in IRAK-4 deficient children: IRAK-4 dependent TLRs are otherwise redundant in protective immunity J. Exp. Med., October 1, 2007; 204(10): 2407 - 2422. [Abstract] [Full Text] [PDF]
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S. Thibault, M. R. Tardif, C. Barat, and M. J. Tremblay TLR2 Signaling Renders Quiescent Naive and Memory CD4+ T Cells More Susceptible to Productive Infection with X4 and R5 HIV-Type 1 J. Immunol., October 1, 2007; 179(7): 4357 - 4366. [Abstract] [Full Text] [PDF]
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H. J. Martin, J. M. Lee, D. Walls, and S. D. Hayward Manipulation of the Toll-Like Receptor 7 Signaling Pathway by Epstein-Barr Virus J. Virol., September 15, 2007; 81(18): 9748 - 9758. [Abstract] [Full Text] [PDF]
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G. Alter, T. J. Suscovich, N. Teigen, A. Meier, H. Streeck, C. Brander, and M. Altfeld Single-Stranded RNA Derived from HIV-1 Serves as a Potent Activator of NK Cells J. Immunol., June 15, 2007; 178(12): 7658 - 7666. [Abstract] [Full Text] [PDF]
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M. Severa, M. E. Remoli, E. Giacomini, V. Annibali, V. Gafa, R. Lande, M. Tomai, M. Salvetti, and E. M. Coccia Sensitization to TLR7 Agonist in IFN-beta-Preactivated Dendritic Cells J. Immunol., May 15, 2007; 178(10): 6208 - 6216. [Abstract] [Full Text] [PDF]
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B. G. Molenkamp, P. A.M. van Leeuwen, S. Meijer, B. J.R. Sluijter, P. G.J.T.B. Wijnands, A. Baars, A. J.M. van den Eertwegh, R. J. Scheper, and T. D. de Gruijl Intradermal CpG-B Activates Both Plasmacytoid and Myeloid Dendritic Cells in the Sentinel Lymph Node of Melanoma Patients Clin. Cancer Res., May 15, 2007; 13(10): 2961 - 2969. [Abstract] [Full Text] [PDF]
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U. Schleicher, J. Liese, I. Knippertz, C. Kurzmann, A. Hesse, A. Heit, J. A.A. Fischer, S. Weiss, U. Kalinke, S. Kunz, et al. NK cell activation in visceral leishmaniasis requires TLR9, myeloid DCs, and IL-12, but is independent of plasmacytoid DCs J. Exp. Med., April 16, 2007; 204(4): 893 - 906. [Abstract] [Full Text] [PDF]
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R. C. Gray, J. Kuchtey, and C. V. Harding CpG-B ODNs potently induce low levels of IFN-{alpha}{beta} and induce IFN-{alpha}{beta}-dependent MHC-I cross-presentation in DCs as effectively as CpG-A and CpG-C ODNs J. Leukoc. Biol., April 1, 2007; 81(4): 1075 - 1085. [Abstract] [Full Text] [PDF]
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B. Berghofer, G. Haley, T. Frommer, G. Bein, and H. Hackstein Natural and Synthetic TLR7 Ligands Inhibit CpG-A- and CpG-C-Oligodeoxynucleotide-Induced IFN-{alpha} Production J. Immunol., April 1, 2007; 178(7): 4072 - 4079. [Abstract] [Full Text] [PDF]
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C. Bourquin, L. Schmidt, V. Hornung, C. Wurzenberger, D. Anz, N. Sandholzer, S. Schreiber, A. Voelkl, G. Hartmann, and S. Endres Immunostimulatory RNA oligonucleotides trigger an antigen-specific cytotoxic T-cell and IgG2a response Blood, April 1, 2007; 109(7): 2953 - 2960. [Abstract] [Full Text] [PDF]
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A. R. M. Kraft, F. Krux, S. Schimmer, C. Ohlen, P. D. Greenberg, and U. Dittmer CpG oligodeoxynucleotides allow for effective adoptive T-cell therapy in chronic retroviral infection Blood, April 1, 2007; 109(7): 2982 - 2984. [Abstract] [Full Text] [PDF]
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J. P. Wang, D. R. Asher, M. Chan, E. A. Kurt-Jones, and R. W. Finberg Cutting Edge: Antibody-Mediated TLR7-Dependent Recognition of Viral RNA J. Immunol., March 15, 2007; 178(6): 3363 - 3367. [Abstract] [Full Text] [PDF]
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S. Hamm, A. Heit, M. Koffler, K. M. Huster, S. Akira, D. H. Busch, H. Wagner, and S. Bauer Immunostimulatory RNA is a potent inducer of antigen-specific cytotoxic and humoral immune response in vivo Int. Immunol., March 1, 2007; 19(3): 297 - 304. [Abstract] [Full Text] [PDF]
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J. Sawaki, H. Tsutsui, N. Hayashi, K. Yasuda, S. Akira, T. Tanizawa, and K. Nakanishi Type 1 cytokine/chemokine production by mouse NK cells following activation of their TLR/MyD88-mediated pathways Int. Immunol., March 1, 2007; 19(3): 311 - 320. [Abstract] [Full Text] [PDF]
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I. Bekeredjian-Ding, S. Inamura, T. Giese, H. Moll, S. Endres, A. Sing, U. Zahringer, and G. Hartmann Staphylococcus aureus Protein A Triggers T Cell-Independent B Cell Proliferation by Sensitizing B Cells for TLR2 Ligands J. Immunol., March 1, 2007; 178(5): 2803 - 2812. [Abstract] [Full Text] [PDF]
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A. K. Mayer, M. Muehmer, J. Mages, K. Gueinzius, C. Hess, K. Heeg, R. Bals, R. Lang, and A. H. Dalpke Differential Recognition of TLR-Dependent Microbial Ligands in Human Bronchial Epithelial Cells J. Immunol., March 1, 2007; 178(5): 3134 - 3142. [Abstract] [Full Text] [PDF]
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 P. Winkler, D. Ghadimi, J. Schrezenmeir, and J.-P. Kraehenbuhl Molecular and Cellular Basis of Microflora-Host Interactions J. Nutr., March 1, 2007; 137(3): 756S - 772S. [Abstract] [Full Text] [PDF]
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 A. Osterloh, U. Kalinke, S. Weiss, B. Fleischer, and M. Breloer Synergistic and Differential Modulation of Immune Responses by Hsp60 and Lipopolysaccharide J. Biol. Chem., February 16, 2007; 282(7): 4669 - 4680. [Abstract] [Full Text] [PDF]
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A. M.G. van der Aar, R. M. R. Sylva-Steenland, J. D. Bos, M. L. Kapsenberg, E. C. de Jong, and M. B. M. Teunissen Cutting Edge: Loss of TLR2, TLR4, and TLR5 on Langerhans Cells Abolishes Bacterial Recognition J. Immunol., February 15, 2007; 178(4): 1986 - 1990. [Abstract] [Full Text] [PDF]
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R. Zhou, H. Wei, and Z. Tian NK3-Like NK Cells Are Involved in Protective Effect of Polyinosinic-Polycytidylic Acid on Type 1 Diabetes in Nonobese Diabetic Mice J. Immunol., February 15, 2007; 178(4): 2141 - 2147. [Abstract] [Full Text] [PDF]
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J. Huggins, T. Pellegrin, R. E. Felgar, C. Wei, M. Brown, B. Zheng, E. C. B. Milner, S. H. Bernstein, I. Sanz, and M. S. Zand CpG DNA activation and plasma-cell differentiation of CD27- naive human B cells Blood, February 15, 2007; 109(4): 1611 - 1619. [Abstract] [Full Text] [PDF]
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D. Zipris, E. Lien, A. Nair, J. X. Xie, D. L. Greiner, J. P. Mordes, and A. A. Rossini TLR9-Signaling Pathways Are Involved in Kilham Rat Virus-Induced Autoimmune Diabetes in the Biobreeding Diabetes-Resistant Rat J. Immunol., January 15, 2007; 178(2): 693 - 701. [Abstract] [Full Text] [PDF]
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 M. Pashenkov, G. Goess, C. Wagner, M. Hormann, T. Jandl, A. Moser, C. M. Britten, J. Smolle, S. Koller, C. Mauch, et al. Phase II Trial of a Toll-Like Receptor 9-Activating Oligonucleotide in Patients With Metastatic Melanoma J. Clin. Oncol., December 20, 2006; 24(36): 5716 - 5724. [Abstract] [Full Text] [PDF]
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P. J. Popovic, R. DeMarco, M. T. Lotze, S. E. Winikoff, D. L. Bartlett, A. M. Krieg, Z. S. Guo, C. K. Brown, K. J. Tracey, and H. J. Zeh III High Mobility Group B1 Protein Suppresses the Human Plasmacytoid Dendritic Cell Response to TLR9 Agonists J. Immunol., December 15, 2006; 177(12): 8701 - 8707. [Abstract] [Full Text] [PDF]
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J. Tabiasco, E. Devevre, N. Rufer, B. Salaun, J.-C. Cerottini, D. Speiser, and P. Romero Human Effector CD8+ T Lymphocytes Express TLR3 as a Functional Coreceptor J. Immunol., December 15, 2006; 177(12): 8708 - 8713. [Abstract] [Full Text] [PDF]
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H. Bartz, N. M. Avalos, A. Baetz, K. Heeg, and A. H. Dalpke Involvement of suppressors of cytokine signaling in toll-like receptor-mediated block of dendritic cell differentiation Blood, December 15, 2006; 108(13): 4102 - 4108. [Abstract] [Full Text] [PDF]
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J. Wang, Y. Shao, T. A. Bennett, R. A. Shankar, P. D. Wightman, and L. G. Reddy The Functional Effects of Physical Interactions among Toll-like Receptors 7, 8, and 9 J. Biol. Chem., December 8, 2006; 281(49): 37427 - 37434. [Abstract] [Full Text] [PDF]
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K. A. Shirey, J.-Y. Jung, and J. M. Carlin Up-Regulation of Gamma Interferon Receptor Expression Due to Chlamydia-Toll-Like Receptor Interaction Does Not Enhance Signal Transducer and Activator of Transcription 1 Signaling Infect. Immun., December 1, 2006; 74(12): 6877 - 6884. [Abstract] [Full Text] [PDF]
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V. Hoene, M. Peiser, and R. Wanner Human monocyte-derived dendritic cells express TLR9 and react directly to the CpG-A oligonucleotide D19 J. Leukoc. Biol., December 1, 2006; 80(6): 1328 - 1336. [Abstract] [Full Text] [PDF]
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P. Paladino, D. T. Cummings, R. S. Noyce, and K. L. Mossman The IFN-Independent Response to Virus Particle Entry Provides a First Line of Antiviral Defense That Is Independent of TLRs and Retinoic Acid-Inducible Gene I J. Immunol., December 1, 2006; 177(11): 8008 - 8016. [Abstract] [Full Text] [PDF]
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K. K. B. Gorden, X. Qiu, J. J. L. Battiste, P. P. D. Wightman, J. P. Vasilakos, and S. S. Alkan Oligodeoxynucleotides Differentially Modulate Activation of TLR7 and TLR8 by Imidazoquinolines J. Immunol., December 1, 2006; 177(11): 8164 - 8170. [Abstract] [Full Text] [PDF]
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A. Dolganiuc, S. Chang, K. Kodys, P. Mandrekar, G. Bakis, M. Cormier, and G. Szabo Hepatitis C Virus (HCV) Core Protein-Induced, Monocyte-Mediated Mechanisms of Reduced IFN-{alpha} and Plasmacytoid Dendritic Cell Loss in Chronic HCV Infection J. Immunol., November 15, 2006; 177(10): 6758 - 6768. [Abstract] [Full Text] [PDF]
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P. S. Patole, R. D. Pawar, M. Lech, D. Zecher, H. Schmidt, S. Segerer, A. Ellwart, A. Henger, M. Kretzler, and H.-J. Anders Expression and regulation of Toll-like receptors in lupus-like immune complex glomerulonephritis of MRL-Fas(lpr) mice Nephrol. Dial. Transplant., November 1, 2006; 21(11): 3062 - 3073. [Abstract] [Full Text] [PDF]
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E. Hartmann, H. Graefe, A. Hopert, R. Pries, S. Rothenfusser, H. Poeck, B. Mack, S. Endres, G. Hartmann, and B. Wollenberg Analysis of Plasmacytoid and Myeloid Dendritic Cells in Nasal Epithelium Clin. Vaccine Immunol., November 1, 2006; 13(11): 1278 - 1286. [Abstract] [Full Text] [PDF]
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