Chronic immune activation in HIV-1 infection contributes to reduced interferon alpha production via enhanced CD40:CD40 ligand interaction.

Abstract

Although a signature of increased interferon (IFN-)alpha production is observed in HIV-1 infection, the response of circulating plasmacytoid dendritic cells (PDC) to Toll-like receptor ligand stimulation is substantially impaired. This functional PDC deficit, which we specifically observed in HIV-1 infected individuals with less than 500 CD4+ T cells/µl, is not well understood. We provide evidence that the peripheral IFN-alpha production in HIV-1 infection is actively suppressed by the enhanced interaction of CD40 ligand (CD40L), a member of the tumor necrosis factor family, and its receptor CD40, which are both upregulated upon immune activation. Plasma levels of soluble CD40L were significantly higher in untreated HIV-1 infected individuals (n = 52) than in subjects on long-term antiretroviral therapy (n = 62, p<0.03) and in uninfected control donors (n = 16, p<0.001). Concomitantly, cell-associated CD40L and the expression of the receptor CD40 on the PDC were significantly upregulated in HIV-1 infection (p<0.05). Soluble and cell-associated CD40L inhibited the PDC-derived IFN-alpha production by CpG oligodeoxynucleotides dose-dependently. This suppressive effect was observed at much lower, physiological CD40L concentrations in peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals compared to controls (p<0.05). The CpG-induced IFN-alpha production in PBMC of HIV-1 infected donors was directly correlated with PDC and CD4+ T cell counts, and inversely correlated with the viral loads (p<0.001). In HIV-1 infected donors with less than 500 CD4+ T cells/µl, the CpG-induced IFN-alpha production was significantly correlated with the percentage of CD40-expressing PDC and the level of CD40 expression on these cells (p<0.05), whereas CD40L plasma levels played a minor role. In addition, low-dose CD40L contributed to the enhanced production of interleukin 6 and 8 in PBMC of HIV-1 infected donors compared to controls. Our data support the conclusion that the chronic immune activation in HIV-1 infection impairs peripheral PDC innate immune responses at least in part via enhanced CD40:CD40L interactions.

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Chronic Immune Activation in HIV-1 Infection
Contributes to Reduced Interferon Alpha Production via
Enhanced CD40:CD40 Ligand Interaction
Norbert Donhauser1, Kathrin Pritschet1, Martin Helm2, Thomas Harrer3, Philipp Schuster1, Moritz Ries1,
Georg Bischof1, Jo ¨rg Vollmer4, Sigrun Smola5, Barbara Schmidt1*, for the German Competence Network
HIV/AIDS
1Institute of Clinical and Molecular Virology, German National Reference Centre for Retroviruses, Friedrich-Alexander-Universita ¨t Erlangen-Nu ¨rnberg, Erlangen, Germany,
2Praxis Dr. G. Abelein/Dr. M. Helm, Nu ¨rnberg, Germany, 3Department for Internal Medicine III, University Hospital Erlangen, Friedrich-Alexander-Universita ¨t Erlangen-
Nu ¨rnberg, Erlangen, Germany, 4Pfizer Oligonucleotide Therapeutics Unit, Coley Pharmaceutical GmbH, Du ¨sseldorf, Germany, 5Institute of Virology, Saarland University,
Homburg/Saar, Germany
Abstract
Although a signature of increased interferon (IFN-)alpha production is observed in HIV-1 infection, the response of
circulating plasmacytoid dendritic cells (PDC) to Toll-like receptor ligand stimulation is substantially impaired. This
functional PDC deficit, which we specifically observed in HIV-1 infected individuals with less than 500 CD4+ T cells/ml, is not
well understood. We provide evidence that the peripheral IFN-alpha production in HIV-1 infection is actively suppressed by
the enhanced interaction of CD40 ligand (CD40L), a member of the tumor necrosis factor family, and its receptor CD40,
which are both upregulated upon immune activation. Plasma levels of soluble CD40L were significantly higher in untreated
HIV-1 infected individuals (n=52) than in subjects on long-term antiretroviral therapy (n=62, p,0.03) and in uninfected
control donors (n=16, p,0.001). Concomitantly, cell-associated CD40L and the expression of the receptor CD40 on the PDC
were significantly upregulated in HIV-1 infection (p,0.05). Soluble and cell-associated CD40L inhibited the PDC-derived IFN-
alpha production by CpG oligodeoxynucleotides dose-dependently. This suppressive effect was observed at much lower,
physiological CD40L concentrations in peripheral blood mononuclear cells (PBMC) of HIV-1 infected individuals compared
to controls (p,0.05). The CpG-induced IFN-alpha production in PBMC of HIV-1 infected donors was directly correlated with
PDC and CD4+ T cell counts, and inversely correlated with the viral loads (p,0.001). In HIV-1 infected donors with less than
500 CD4+ T cells/ml, the CpG-induced IFN-alpha production was significantly correlated with the percentage of CD40-
expressing PDC and the level of CD40 expression on these cells (p,0.05), whereas CD40L plasma levels played a minor role.
In addition, low-dose CD40L contributed to the enhanced production of interleukin 6 and 8 in PBMC of HIV-1 infected
donors compared to controls. Our data support the conclusion that the chronic immune activation in HIV-1 infection
impairs peripheral PDC innate immune responses at least in part via enhanced CD40:CD40L interactions.
Citation: Donhauser N, Pritschet K, Helm M, Harrer T, Schuster P, et al. (2012) Chronic Immune Activation in HIV-1 Infection Contributes to Reduced Interferon
Alpha Production via Enhanced CD40:CD40 Ligand Interaction. PLoS ONE 7(3): e33925. doi:10.1371/journal.pone.0033925
Editor: Roberto F. Speck, University Hospital Zurich, Switzerland
Received August 30, 2011; Accepted February 20, 2012; Published March 21, 2012
Copyright: ? 2012 Donhauser et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the ELAN Fonds (VI-08-06.18.1), the German Research Foundation (SCHM1702/2-2, SCHM1702/3-1), the graduate college
1071 (‘‘Viruses of the immune system’’), the ‘‘Interdisziplina ¨res Zentrum fu ¨r Klinische Forschung, Erlangen’’ (TP50), the German Competence Network HIV/AIDS,
and the ‘‘Akademie der Wissenschaften und Literatur zu Mainz’’. The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: Jo ¨rg Vollmer has a potential conflict of interest as he is an employee of Pfizer and provided the CpG oligodeoxynucleotides used in the
study. This does not alter the authors’ adherence to all the PLoS ONE policies on sharing data and materials.
* E-mail: baschmid@viro.med.uni-erlangen.de
Introduction
Evidence is accumulating from human and simian studies that
chronic immune activation with enhanced T-cell turnover and
apoptosis plays a crucial role in lentiviral pathogenesis [1]. An
important trigger of immune activation are the type I interferons
(IFN), mainly produced by plasmacytoid dendritic cells (PDC)
[2,3]. PDC express Toll-like receptors (TLR) 7 and 9 for the
recognition of single-stranded RNA and CpG-like DNA, respec-
tively. Upon stimulation, proinflammatory cytokines are secreted
that initiate early immune responses. High-titered HIV-1 and in
particular HIV-1 infected cells induce major IFN-alpha produc-
tion [4–6]. The antiviral activity, however, is counteracted by the
apoptosis of uninfected CD4+ bystander cells via enhanced
expression of the tumor necrosis factor (TNF)-related apoptosis-
inducing ligand and its death receptor 5 [7].
The signature of increased expression of IFN-stimulated genes
in peripheral cells and lymphatic tissue [8,9] faces an impaired
IFN-alpha production upon TLR stimulation in circulating
mononuclear cells and PDC of HIV-1 infected individuals [10–
14]. This reduced responsiveness to stimulation was recently
associated with prior activation of PDC via type I IFNs or virions
in vivo [15]. Another cause may be a cellular factor induced upon
immune activation. In this respect, we focused on the costimula-
tory molecule CD40 and its ligand, CD40L (CD154), because we
and others have shown that CD40L reduced the IFN-alpha
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production at high concentrations [5,16]. CD40 was identified on
B cells, monocytes, dendritic cells, endothelial and epithelial cells
[17]. CD40L, a member of the TNF family, is primarily expressed
on activated CD4+ T cells and on a small proportion of CD8+ T
cells and platelets [17]. The interaction of CD40 and CD40L is
important for activating antigen presenting cells, T cells and
macrophages, antibody isotype switching, and germinal center
formation. CD40L may be expressed as a heteromultimeric
complex [18]. After cleavage of the transmembrane protein by
naturally occurring metalloproteinases, the soluble form (sCD40L)
still binds to CD40 and delivers biological signals like a cytokine
[19].
In HIV-1 infection, CD40 is upregulated on myeloid and
plasmacytoid dendritic cells in the peripheral blood [20] and
lymphoid tissue [21,22]. Increased sCD40L levels are detected in
the serum of HIV-1 infected subjects and cerebrospinal fluid of
patients with AIDS dementia [23,24]. Controversial data are
reported for cell-associated CD40L (cCD40L). Enhanced expres-
sion at baseline was observed on CD4+ cells of HIV-1 infected
patients, reversed by antiretroviral therapy [25], and on platelets
[26]. In contrast, others reported comparable cCD40L levels on
resting CD4+ T cells in HIV-1 infected children and uninfected
controls [27]. Upon stimulation, CD40L was not sufficiently
upregulated on CD4+ cells of HIV-1 infected subjects, which may
result in deficient activation of dendritic cells and an ineffective
cellular immune response [28–30].
In our study, we hypothesized that the HIV-1 induced immune
stimulation is directly linked with the decreased PDC IFN-alpha
production upon stimulation. We focused on the role of CD40L
and provide evidence that physiological concentrations of this
molecule reduce the TLR-induced IFN-alpha production in HIV-
1 infection.
Materials and Methods
Ethics statement
All protocols for this study were approved by the ethical
committee of the Medical Faculty, Friedrich-Alexander-Universi-
ta ¨t Erlangen-Nu ¨rnberg (Ref. no. 3375). Ethical guidelines accord-
ing to the declaration of Helsinki were adhered to, and written
informed consent was obtained from the study participants.
Patient characteristics
The HIV-1 infected subjects (n=44) were monitored at two
specialized outpatient centers in Nu ¨rnberg and Erlangen,
Germany. None of them had received antiretroviral therapy.
Controls (n=16) were age-matched healthy volunteers at the
Institute of Virology, Erlangen, Germany (patients vs. controls,
median 41.3 vs. 41.1 years, range 25–68 vs. 25–67 years, p=0.73,
n.s.). Data of some participants were reported recently [31].
Details on baseline cell counts of HIV-1 infected patients and
controls are given in Fig. 1a. Since the study developed over time,
Figure 1. Levels of soluble and cell-associated CD40 ligand in HIV-1 infected patients (pat.) and controls (ctrl.). (a) Baseline counts of
natural killer (NK) cells, monocytes, CD4+ and CD8+ T cells, B cells, plasmacytoid (PDC) and myeloid dendritic cells (MDC) in HIV-1 infected individuals
and uninfected controls, given as percentages (%) of peripheral blood mononuclear cells (PBMC). (b) Plasma levels of interleukin (IL-)3 and soluble
CD40 ligand (sCD40L), the latter also analyzed in patients on antiretroviral therapy (HAART). (c) Expression of cell-associated CD40L (cCD40L) and (d)
CD40 at the cell surface, evaluated by flow cytometry as percentage of positive cells and delta mean fluorescence intensity after subtraction of
isotype controls. Data are presented as median, interquartile ranges (boxes), and 10% and 90% values (whiskers). *p,0.05, **p,0.01, ***p,0.001
(Mann-Whitney test).
doi:10.1371/journal.pone.0033925.g001
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not all values were obtained from the beginning and could not be
repeated later on, because antiretroviral therapy was initiated.
However, all values that were obtained are reported.
Isolation and stimulation of cells
EDTA-anticoagulated blood was processed within a few hours
(h). PBMC were isolated using Ficoll (Biochrom AG, Berlin,
Germany) density gradient centrifugation, and plated at 106cells/
500 ml in 24-well flat bottom plates. Viability of cells was checked
by trypan blue staining. PBMC were cultivated in RPMI 1640
media (Gibco, Eggenstein, Germany) plus 10% heat-inactivated
fetal calf serum (FCS) (Lonza, Basel, Suisse), 50 mg/ml glutamine,
200 U/ml penicillin, and 90 U/ml streptomycin. IL-3 (R&D
Systems, Wiesbaden-Nordenstadt, Germany) was added at
indicated concentrations. Cells were stimulated directly after
isolation. Supernatants were harvested and stored at 220uC. For
the induction of CD40L, cells were stimulated with 1 mg/ml
ionomycin und 10 ng/ml PMA (both Sigma-Aldrich) [32]. CD4+
and CD8+ T cells as well as PDC were purified from PBMC by
positive selection (Miltenyi Biotec, Bergisch-Gladbach, Germany).
Determination of cytokines
Cell culture supernatants were analyzed using an IFN-alpha 2a/
2b ELISA module set (Bender MedSystems, Vienna, Austria).
Samples were measured within the linear range of the assay. IL-3
and sCD40L concentrations were determined using the Quanti-
kine Immunoassay human IL-3 (R&D Systems) and the human
sCD40L module set (Bender Medsystems), respectively. Other
cytokines were measured using the FlowCytomix technology
(Bender MedSystems).
Generation of the viral stock
The X4-tropic primary virus isolate HIV-1SF33was grown on
PHA-stimulated CD4+ T cells. At peak viral replication,
supernatants were transferred to PHA-stimulated PBMC. Super-
natants were filtered through 0.22 mm filter devices (Millipore,
Schwalbach, Germany), aliquoted and stored at 280uC. The
TCID50was quantified in PHA- and IL-2-stimulated PBMC [33].
Cell lines
CD40L-expressing BHK and control cells [34] were grown in
Dulbecco’s modified Eagles medium (DMEM) (Gibco), supple-
mented with 10% FCS and antibiotics (see above). For the
production of sCD40L-containing or control supernatants, BHK
cells were grown to subconfluency, washed twice with DPBS, and
fed with serum-free DMEM. After 24 h, supernatants were
harvested and concentrated 100-fold using the Centricon Plus-
70-10 Centrifugal Filter devices (Millipore). Aliquots were stored at
280uC. To determine the effects of cCD40L, BHK cells were
plated at different concentrations into 24-well flat bottom plates.
The next day, the plates were incubated on ice for a few minutes.
Cells were harvested, washed and fixed in 1.5 ml cryotubes
(Eppendorf, Hamburg, Germany) using 4% PFA in DPBS, washed
again six times in DPBS, and then added to the PBMC.
Biologic reagents
Cell-culture grade sCD40L and the respective CD40L antibody
were obtained from R&D Systems. Endotoxin-free CpG-A (ODN
6016,59-T*C-G-A-C-G-T-C-G-T-G-G*G*G*G-39,
phosphorothioate and – phosphodiester bonds) and CpG-P
(ODN 21798, 59-T*C-G*T*C-G*A*C-G*A*T*C-G*G*C*G*C-
G*C*G*C*C*G-39) were provided by the Pfizer Oligonucleotide
Therapeutics Unit – Coley Pharmaceutical GmbH (Du ¨sseldorf,
*indicates
Germany) and used at 0.75 mM and 0.25 mM, respectively. The
synthetic TLR7 agonist S-27609, kindly provided by Richard
Miller, 3M Pharmaceuticals (St. Paul, UK), was used at 5 mM.
Western Blotting
The SDS-denatured sCD40L preparations were separated on a
10% polyacrylamide gel, transferred to a PVDF membrane,
incubated with anti-CD40L over night, and then a secondary
horseradish peroxidase-conjugated antibody (Dako, Hamburg,
Germany; 1:1,000). The protein size was assessed using the Page
Ruler Prestained Protein Ladder (Fermentas, St. Leon-Rot,
Germany). Luminescence was developed by adding ECL solution
containing luminol (Sigma-Aldrich, Taufkirchen, Germany),
documented using the Fujifilm LAS-1000 plus gel documentation
system.
FACS analysis
Isolated PBMC were washed with DPBS supplemented with 1%
FCS and 0.5 mM EDTA (Sigma-Aldrich). FcR-blocking reagent
(Miltenyi Biotec) was added at 4uC for 10 minutes (min) to reduce
unspecific staining. PDC and myeloid dendritic cells were stained
using a cocktail of PE-conjugated lineage antibodies (CD3, CD14,
CD16, and CD20; Immunotools, Friesoythe, Germany), PE-Cy5-
conjugated anti-CD11c, and PE-Cy7-conjugated anti-CD4 (both
BD Bioscience, Heidelberg, Germany). B cells were identified
using PE-Cy5 conjugated anti-CD19 and APC-conjugated anti-
CD20 (Immunotools). CD4+ and CD8+ T-lymphocytes, mono-
cytes, and natural killer (NK) cells were stained using a cocktail of
PE-conjugated anti-CD16 (BD Bioscience), PE-Cy5-conjugated
anti-CD14 (Immunotools), Pacific Blue-conjugated anti-CD3, PE-
Cy7-conjugated anti-CD4 (BD Bioscience) and APC-conjugated
anti-CD8 (Immunotools). CD40 and CD40L (CD154) were
stained using FITC-conjugated antibodies (eBioscience, Frankfurt,
Germany). CD4+ T cell memory subsets were identified using
CD3-PB and CD4-PE-Cy7 (both BD Bioscience) in addition to
CD45RO-APC and CD197(CCR7)-PE (both eBioscience). Re-
spective mouse IgG antibodies served as isotype controls. After
staining at 4uC for 20 min, cells were washed and fixed in 4%
paraformaldehyde (Sigma-Aldrich, Taufkirchen). Depending on
the expected cell frequency, a total of 100,000–400,000 events
were collected using a four-color FACS Calibur with CellQuest
3.3 software (BD Biosciences). CD4+ T cell memory subsets were
dissected using an LSR II. The data were analyzed using FCS
Express V3.
Statistics
The Mann-Whitney U-test was used for comparisons between
patients and controls. Independently repeated experiments were
compared using the paired and unpaired Student’s t-test. All
statistical calculations assumed a two-sided significance at p values
, 0.05.
Results
Patient characteristics
We studied 52 untreated HIV-1 infected subjects (48 male, 4
female). All males except two were infected through sex with men.
The participants were classified into CDC categories A1 (n=16),
A2 (n=20), A3 (n=2), B1 (n=4), B2 (n=6), B3 (n=1), C2 (n=1),
and C3 (n=2). AIDS-defining illnesses were pneumocystis
pneumonia (n=1), chronic diarrhea and wasting (n=1), candida
oesophagitis (n=1), and HIV-1 associated encephalopathy (n=2).
Eight patients suffered from chronic bronchitis and/or allergic
coryza, four from lues, two from chronic hepatitis, and one each
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from proctitis, anal fistula, sigma diverticulitis, skin mycosis,
recurrent herpes genitalis, condylomata accuminata, nephro-
pathia, and pancreatitis. Two patients were diabetic; one
hemophiliac, one addicted to illegal drugs, and three addicted to
alcohol. The median CD4+ T cell count was 397/ml (interquartile
range, IQR, 273–627; range, 19–1780), the median viral load 4.4
log10 (IQR, 3.9–5.1 log10; range, 1.3–5.8 log10). Baseline
percentages of the different cell types are included in Fig. 1a.
TLR-ligand induced IFN-alpha production is decreased in
HIV-1 infection
The function of PDC was investigated using the oligodeox-
ynucleotides (ODN) CpG-A (6016) and CpG-P (21798) [35] as
TLR9 stimuli (Fig. 2). PBMC of HIV-1 infected patients secreted
significantly less IFN-alpha than controls upon stimulation with
both CpG ODN (p,0.001). When the patients were divided into
three groups according to their CD4+ T cell counts, it became
obvious that only patients with less than 500 CD4+ T cells/ml
contributed to this difference (p,0.01). When the CpG-induced
IFN-alpha production was corrected for the percentage of PDC,
PBMC of HIV-1 infected patients with CD4+ T cell counts below
500/ml still produced significantly less IFN-alpha compared to
controls (p,0.05), whereas for HIV-1 infected patients with more
than 500 CD4+ T cells/ml, the normalized IFN-alpha production
was not significantly different from control donors. Thus, the
functional response of circulating PDC to TLR9 stimulation was
Figure 2. Deficits in interferon (IFN) alpha production in HIV-1 infected patients (pat.) versus control donors (ctrl.). Peripheral blood
mononuclear cells (PBMC) were exposed to (a) the CpG-A oligonucleotide 6016 (0.75 mM) and (b) the CpG-P oligodeoxynucleotide 21798 (0.25 mM)
for 20 hours (h). The IFN-alpha production is given before and after normalization for the percentage of plasmacytoid dendritic cells (PDC). The data
of HIV-1 infected patients were also analyzed for samples with CD4+ T cell counts below 250/ml (,250), between 250–500/ml (250–500), and above
500/ml (.500). Data are presented as median, interquartile ranges (boxes), and 10% and 90% values (whiskers). *p,0.05, **p,0.01, ***p,0.001
(Mann-Whitney test). Part of the data were presented in another study [31]. Similar data were obtained using S-27609 (data not shown).
doi:10.1371/journal.pone.0033925.g002
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substantially impaired in HIV-1 infected subjects with less than
500 CD4+ T cells/ml. The response of PBMC to stimulation with
the TLR7 agonist S-27609 was also significantly lower in HIV-1
infected patients compared to controls, before and after correction
for the percentage of PDC (both p,0.05; data not shown). In
conclusion, both TLR7 and TLR9 responses are impaired in
HIV-1 infection.
sCD40L and cCD40L are significantly elevated in HIV-1
infection
In the search for a cellular factor which is upregulated upon
immune activation and suppresses IFN-alpha production, we
investigated two molecules: IL-3, secreted by activated CD8+ T
cells, which plays an important role in PDC survival and
maturation [36]; and CD40L, mainly produced by activated
CD4+ T cells, which inhibits the PDC-derived IFN-alpha
production at high concentrations [5,16]. IL-3 plasma levels were
similar in HIV-1 infected subjects (n=15) and controls (n=10)
(median, 6.0 vs. 7.4 pg/ml; IQR 3.4–15.5 pg/ml vs. 1.0–31.0 pg/
ml, respectively) (p=0.89, n.s.) (Fig. 1b). The sCD40L plasma
levels, however, were significantly elevated in HIV-1 infected
patients (n=52) compared to controls (n=16) (median, 7.9 vs.
3.7 ng/ml; IQR 5.3–10.3 ng/ml vs. 2.0–5.7 ng/ml, respectively)
(p,0.001) (Fig. 1b). These data compare to 2-fold increased
Figure 3. Suppression of interferon (IFN) alpha production by CD40 ligand. (a) Preincubation of peripheral blood mononuclear cells (PBMC)
of controls without (w/o) or with (w/t) a cell-culture grade commercial soluble CD40 ligand (sCD40L; 3 mg/ml) for 48 hours (h), followed by
stimulation with CpG-P 21798. Data represent 11 and 5 separate experiments for IL-3 levels of 0 and 5–5,000 pg/ml, respectively. The arrow indicates
physiological IL-3 levels. (b) Neutralization of sCD40L with a cell-culture grade anti-CD40L antibody with subsequent CpG-P stimulation in two
donors. Similar data were obtained using CpG-A (data not shown). (c) Direct effect of sCD40L (3 mg/ml) on the CpG-P induced IFN-alpha production
by plasmacytoid dendritic cells (PDC) of control donors. (d) Expression of cell-associated CD40L (cCD40L) on baby hamster kidney (BHK) cells. (e)
Shedding of sCD40L into the cell culture supernatants after 24 h and 48 h of culture. (f) Western Blot of concentrated supernatants of CD40L-
expressing BHK cells and a commercial sCD40L preparation (R&D Systems) using reducing conditions. Data represent three separate experiments. No
bands were detected in the supernatants of BHK wild type cells (data not shown). (g) Inhibition of CpG-P induced IFN-alpha production using sCD40L
from R&D Systems and BHK cells. Statistics were calculated between PBMC of HIV-1 infected patients (pat.) (n=5) and controls (ctrl.) (n=10) exposed
to sCD40L (R&D Systems). (h) cCD40L expression on CD4+ and CD8+ T cells of control donors after stimulation with mock (grey filled curve) or PMA/
ionomycin (black dotted curve). (i) Inhibition of CpG-P induced IFN-alpha production by increasing counts of untreated (unstim.) or PMA/ionomycin-
stimulated (stim.) CD4+ T cells, which were left unfixed or fixed used 4% paraformaldehyde, and coincubated with 16106PBMC of five control
donors. Similar results were obtained with CpG-A (data not shown). (j) Inhibition of CpG-P induced IFN-alpha production by paraformaldehyde-fixed
cCD40L-expressing and wild type (WT) BHK cells using PBMC of eight control donors. Data are presented as mean and standard error. *p,0.05,
**p,0.01, ***p,0.001 (Student’s t-test).
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