TNF-a and TGF-b Counter-Regulate PD-L1
Expression on Monocytes in Systemic
Jing-Ni Ou1, Alice E. Wiedeman2& Anne M. Stevens1,3
1Seattle Children’s Research Institute,2Department of Immunology,3Department of Pediatrics, University of Washington, Seattle,
Monocytes in patients with systemic lupus erythematosus (SLE) are hyperstimulatory for T lymphocytes.
We previously found that the normal program for expression of a negative costimulatory molecule
programmed death ligand-1 (PD-L1) is defective in SLE patients with active disease. Here, we investigated
the mechanism forPD-L1dysregulation on lupusmonocytes.Wefound that PD-L1expressiononcultured
SLE monocytes correlated with TNF-a expression. Exogenous TNF-a restored PD-L1 expression on lupus
on healthy monocytes. Therefore, PD-L1 expression in monocytes is regulated by opposing actions of
TNF-a and TGF-b. As PD-L1 functions to fine tune lymphocyte activation, dysregulation of cytokines
resulting in reduced expression could lead to loss of peripheral T cell tolerance.
elucidated, but most likely depends on differential levels of positive and negative costimulatory molecules
expressed on antigen presenting cells (APCs)4,5. PD-L1 (B7-H1 or CD274) functions as a critical regulatory
protein to maintain T cell self-tolerance6, and could play a major role in determining monocyte activity in SLE.
PD-L1, expressed on hematopoietic and parenchymal cells, binds to programmed death-1 (PD-1) to inhibit T
cell receptor-mediated proliferation and induce T cell anergy6. Engagement of the PD-1 pathway is essential in
suppressing autoimmunity, as originally demonstrated in mice lacking PD-1 expression that developed a disease
similar to SLE7. Blockade of PD-1 has been shown to affect disease activity in a lupus mouse model8,9. PD-L1
deficiency does not by itself lead to SLE, but exacerbates systemic autoimmunity in lupus-prone mice6,10. The
mechanistic link between PD-1 or PD-L1 expression and the pathogenesis of human SLE is not well understood.
Polymorphisms in the PD-1 gene are associated with SLE susceptibility in some populations of adults and
children11–15. However, PD-L1 gene polymorphisms were not associated with SLE16,17.
encompasses two abnormal processes that occur in SLE: the APC response to apoptotic cells and lymphocyte
response to autologous antigens. The failure of APCs to up-regulate PD-L1 may occur in vivo and contribute to
the breakdown of self-tolerance in SLE patients.
Cytokines abnormally expressed in SLE have been implicated in the regulation of PD-L118,20–26. Specifically,
TNF-a has been associated with increased PD-L1 expression on synovial and peripheral macrophages derived
from patients with rheumatoid arthritis24, whereas TGF-b inhibits PD-L1 expression on renal tubular cells21.
In this report we investigated the role of cytokines in regulating expression of PD-L1 in SLE. During active
disease, the overexpression of TGF-b correlated with decreased levels of PD-L1 surface protein on lupus mono-
cytes. Deficient PD-L1 expression could be restored in vitro by TNF-a, a factor required to induce PD-L1
that PD-L1 expression was not inhibited by SLE lymphocytes. TNF-a induced expression of PD-L1 mRNA in
onocytes and dendritic cells from SLE patients display aberrant phenotypes, namely abnormal cytokine
T cells1–3. The mechanism which leads to dysregulated lymphocyte activation in SLE has not been
11 October 2011
6 February 2012
2 March 2012
requests for materials
should be addressed to
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
lupus cells, while TGF-b suppressed induction of the mRNA in
healthy control cells, suggesting opposing transcriptional regulation
by these two cytokines. These findings demonstrate that abnormal
cytokine production may lead to poor PD-L1 expression on mono-
cytes, contributing to the hyperstimulatory phenotype found in SLE.
Aberrant expression of TNF-a and TGF-b correlates with PD-L1
levels on SLE monocytes. To investigate the mechanism for dys-
regulation of PD-L1 expression in SLE, PBMC from patients and
healthy controls were cultured for 24 hours without stimulation.
PD-L1 surface protein on monocytes was assayed by flow cyto-
metry (Supplemental Figure 1). We observed no PD-L1 expression
at the initiation of culture on either monocytes or myeloid DC from
increased at 24 hours, and remained high until day five (Sup-
plemental Figure 2). Deficient PD-L1 expression on cultured SLE
monocytes during active disease was not related to specific me-
dications, and is unlikely to be a gene defect, as most patients were
able to restore PD-L1 protein during remission19. Hence we
postulated that dysregulated cytokine production in SLE may lead
to decreased expression of PD-L1 on SLE APCs. Supernatants from
control and SLE PBMC cultures were assayed for expression of
cytokines known to associate with the severity of SLE, including
IFN-c, IFN-a, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-a, and TGF-b.
The most significant differences between SLE patients and healthy
controls were found in the expression of TNF-a and TGF-b.
Interestingly, TNF-a, reported to be increased in SLE patient
serum25, was expressed at 2.7-fold higher levels by PBMC from
healthy controls compared to SLE patients in our experiments
(mean, 129.4 pg ml21vs. 44.2 pg ml21, Figure 1A). Though
production of TNF-a was restored in some SLE patients during
remission, the mean was still significantly reduced compared to
controls (81.7 pg ml21). In contrast, TGF-b was induced to higher
levelsinbothactiveSLE(2393 pgml21)andremission(3914 pgml21)
compared to controls (1684 pg ml21, Figure 1B). Among other
cytokines tested, IFN-c, IL-4, IL-10, IFN-a and IL-2 were
undetectable in supernatants from most subjects, an expected result
considering that T lymphocytes were unstimulated. Moreover,
expression of neither IL-6 nor IL-8 was significantly different in
controls compared to SLE patients (data not shown).
We then tested for correlations between TNF-a, TGF-b and PD-L1
expression. The expression of PD-L1 significantly correlated with
TNF-a during disease remission (Figure 1C), suggesting that express-
ion of TNF-a may be required to restore PD-L1 expression on lupus
monocytes. In contrast, high expression of TGF-b was significantly
correlated with low PD-L1 levels during active disease (Figure 1D). In
PBMC from healthy controls, there was no correlation between
Figure 1 | PD-L1 levels correlated with TNF-a in remission and with TGF-b in patients with active disease. (A) and (B) Levels of TNF-a and TGF-b
detected in supernatants from PBMC cultured overnight without stimulation. Horizontal lines represent mean values. Cytokine levels between healthy
controls and SLE patient groups were compared using the Wilcoxon-Mann-Whitney test. Significance was assigned where p,0.05; N.S., not significant.
(C) PD-L1 protein levels on monocytes in the same culture was assayed by flow cytometry; TNF-a positively correlated with PD-L1 expression on
Correlations were analyzed by the Spearman’s rank correlation test.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
cytokines and PD-L1 expression (data not shown), as most cells
expressed PD-L1. Thus, correlation analyses suggested that TGF-b
suppresses PD-L1 expression during active SLE, while TNF-a induces
PD-L1 expression during remission and in healthy controls.
PD-L1 surface protein is induced by TNF-a and down-regulated
by TGF-b. We have previously shown that cells from patients in
remission restored PD-L1 expression, suggesting a reversible me-
chanism for regulation of PD-L1 during the disease course of
SLE19. Therefore, we tested the hypothesis that expression of PD-
L1 on SLE cells can be restored by TNF-a. To determine whether
TNF-a is normally required for induction of PD-L1, we examined
the change of PD-L1 expression on healthy cells treated with a TNF-
a neutralizing antibody. Blocking TNF-a resulted in significantly
decreased expression of PD-L1 surface protein on monocytes from
healthy controls (Figure 2A). Likewise, treatment with TGF-b
significantly suppressed PD-L1 surface protein on healthy mono-
cytes. Addition of neither recombinant TNF-a nor TGF-b block-
ing antibody affected PD-L1 expression on healthy cells.
We found that TNF-a significantly induced PD-L1 protein ex-
pression on SLE remission monocytes (Figure 2B). However, induc-
tion of PD-L1 by TNF-a was not significant in patients with active
disease. Of note, TNF-a treatment resulted in little induction of PD-
L1 on myeloid DCs (data not shown), indicating that expression of
PD-L1 is differentially regulated on monocytes and myeloid DCs.
Blocking TGF-b with a mAb was not sufficient to restore PD-L1 on
L1 expression on SLE monocytes may require TNF-a and other
positive regulatory factors in addition to blocking inhibitory TGF-
b. TNF-a is normally required for PD-L1 induction, whereas TGF-b
may oppose the induction in active SLE. As SLE monocytes express
low levels of PD-L1, inhibition of TNF-a by mAb or addition of
recombinant TGF-b was not able to reduce the protein expression
further (Figure 2B).
PD-L1 expression is induced by monocyte-derived factors.
Cytokines that dysregulate expression of PD-L1 can be derived
from either aberrant SLE myeloid or lymphoid cells2,27. To identify
other cell populations affecting PD-L1 expression on monocytes, we
compared the expression of PD-L1 on isolated CD141cells cultured
0 (Supplemental Figure 2). PD-L1 was induced on isolated healthy
CD141monocytes cultured overnight without stimulation, indi-
cating that monocyte-intrinsic factors are sufficient for induction
of surface PD-L1 (Figure 3A and B). However, the depletion of
CD142cells decreased expression of PD-L1 on myeloid DCs and
monocytes from healthy donors that had higher PD-L1 expression
(Figure 3B), suggesting that induction of PD-L1 could be enhanced
by lymphocytes. Both regulatory T cells and NKT cells have been
previously reported to induce PD-L1 expression, and may play roles
in defective PD-L1 induction in SLE28,29. There was no change in
the level of PD-L1 expression on either isolated SLE monocytes or
myeloid DCs as compared to total PBMCs (Figure 3C and D),
demonstrating that PD-L1 expression is not suppressed by SLE
lymphocytes. Rather, SLE APCs lack intrinsic factors required for
We next tested the direct effect of cytokines on isolated CD141
monocytes after treatment of total PBMCs with TNF-a that we
showed in Figure 2 was similarly demonstrated in isolated SLE
monocytes, where expression of PD-L1 was significantly induced
in four out of five patients (Figure 3E). Blocking TGF-b did not
changed expression of PD-L1 protein on isolated SLE monocytes
(Figure 3F), whereas blocking TNF-a significantly decreased PD-
L1 expression on isolated healthy monocytes (Figure 3G).
Moreover, TGF-b treatment of isolated monocytes resulted in sig-
nificant repression of PD-L1 in healthy donors (Figure 3H). These
findings support the hypothesis that intrinsic defects in SLE mono-
cyte cytokine expression result in poor PD-L1 induction.
Expression of PD-L1 correlated with TNF-a in monocyte culture
supernatants. In order to determine whether or not PD-L1 is
induced by monocyte-intrinsic factors, we assayed levels of TNF-a
and TGF-b in purified monocyte culture supernatants. Increased
TNF-a was detected in supernatants from healthy monocytes
(mean, 157 pg/ml) compared to those from patients with active
disease (Figure 4A, p 5 0.027). TNF-a expression was higher in
SLE remission (109 pg/ml) compared to active disease (37 pg/ml),
but the difference was not statistically significant (p 5 0.067).
Expression of PD-L1 on isolated monocytes from remission pa-
tients also significantly correlated with TNF-a (Figure 4B). These
Figure 2 | PD-L1 surface protein on SLE APCs is differentially regulated by TNF-a and TGF-b. (A) Control PBMC were treated for 24 hours.
ofPD-L1onmonocytes inSLEPBMCtreatedfor24 hours orculturedinmediaalone.InductionofPD-L1proteinbycytokinetreatmentswastestedfor
significance by the Wilcoxon signed-rank test. Results were derived from multiple independent experiments.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
data provide evidence that monocytes produce TNF-a required to
induce PD-L1. Conversely, substantial expression of TGF-b was
detected in monocyte supernatants (Figure 4C). Isolated mono-
cytes from SLE patients produce higher level of TGF-b compared
to healthy controls (active disease 5 3527 pg/ml; remission 5
2189 pg/ml; healthy controls 5 1446 pg/ml), but the difference was
insignificant. No correlation was found between PD-L1 protein level
andTGF-b(Figure 4D).Thesedata suggestthatcelltypesother than
monocytes can also produce TGF-b to counter-regulate level of PD-
L1 on monocytes.
Opposing effects of TNF-a and TGF-b on PD-L1 mRNA
expression. PD-L1 expression has been reported to be regulated at
both transcriptional and translational levels in different model
systems30–32. PD-L1 mRNA expression was assayed in cultured
PBMC from SLE patients and healthy donors. We found that PD-
remission), although the differences were not significant. Parallel
flow cytometry assays demonstrated that some patients with active
disease and high PD-L1 mRNA expression had little to no PD-L1
protein on the surface of monocytes, myeloid DCs, or lymphocytes.
Moreover, there was no correlation between PD-L1 mRNA and pro-
tein expression in any subject group (data not shown), indicating that
restoration of PD-L1 surface protein was not entirely dependent upon
mRNA expression, but may require additional translational signals, as
demonstrated in other biological models30,31.
The signaling pathways mediated by TNF-a and TGF-b are well
documented33,34. However, it is unclear how these distinct pathways
converge to counter-regulate PD-L1 expression. We next demon-
strated that PD-L1 mRNA is differentially regulated by TGF-b and
TNF-a. PD-L1 mRNA was significantly induced in SLE cells treated
with TNF-a (Figure 5B), whereas blocking TNF-a prevented induc-
tion of PD-L1 mRNA in three of six healthy donors (Figure 5C).
Similarly, TGF-b significantly inhibited expression of PD-L1 mRNA
in control PBMC (Figure 5D). Overall, these data support a model
in which PD-L1 gene expression is tightly regulated by the action
of two opposing cytokines, TNF-a and TGF-b.
Although functional defects in monocytes and DCs are well known
in SLE, the underlying molecular mechanisms are not fully under-
stood. The present study addresses the mechanism for regulating
Figure 3 | ExpressionofPD-L1onisolatedCD141cellsisnotentirelydependentonlymphocytes. (A)and(C)Representativehistogramsdemonstrate
PD-L1 induction on total PBMC or isolated CD141cells from a control subject and an active SLE patient. Protein expression was assayed by flow
cytometry after culturing cells for 24 hours without stimulation. (B) and (D) PD-L1 expression on monocytes gated from total PBMC compared to
isolated monocytes and myeloid DCs in eight healthy controls and eight SLE patients. (E) and (F) Fold induction of PD-L1 MFI on isolated CD141SLE
monocytes treated withTNF-a or withanti-TGF-bmAb. (G)and(H) Foldinduction ofPD-L1 MFI onisolated CD141monocytes from healthy donors
cells, and tested for significance by the Wilcoxon signed-rank test.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
versus anti-inflammatory activity of APCs. PD-L1 expression is
regulated by a balance of cytokines with known importance in
SLE21,35. In other inflammatory diseases including rheumatoid arth-
ritis, infection, and malignancies, PD-L1 expression has been
reported to be up-regulated by inflammatory cytokines36,37. In con-
trast, we previously demonstrated deficient PD-L1 surface express-
ion on monocytes and myeloid DCs with active SLE19. We have
shown here that the defect on SLE monocytes can be attributed
partially to overexpression of TGF-b, which inhibits PD-L1 express-
tion of PD-L1 mRNA and surface protein expression.
In the current study, some patients with active SLE produced less
TNF-a, a trait reported to correlate with SLE-associated HLA-DR
alleles38–40. Whether children with SLE expressing less TNF-a in
correlation with decreased PD-L1 expression on monocytes carry
the polymorphisms leading to low TNF-a expression is not known.
Expression of PD-L1 may also be influenced by the availability of
in the TNF receptor signaling pathway. TNF receptor expression is
reportedly normal in SLE, though decreased expression of TNF-a
signaling proteins in SLE patients has been reported41,42, and could
SLE monocytes from some patients, although most patient mono-
cytes respond well to TNF-a. Anti-TNF-a therapy is effective in
treatingrheumatoid arthritis, auto-inflammatory diseases, and some
SLE patients. However, TNF-a blockers have been shown to trigger
lupus in some patients, and induce lupus-related autoantibody pro-
duction. The onset of autoimmunity triggered by TNF-a blockers
may be in part through activation of plasmacytoid dendritic cells,
which lead to enhanced production of IFN-a43. Our data suggests
contribute to disease. These studies underline the complex role of
TNF-a in autoimmunity, with different effects at distinct cellular
The role of TGF-b in the pathogenesis of SLE remains unclear.
Previous studies have shown decreased TGF-b production by SLE
lymphocytes44, in contrast to our increase in TGF-b production by
therapies in lupus-prone mice have yielded mixed results45, perhaps
because TGF-b contributes to the development of both proinflam-
matory Th17 cells and regulatory T cells (Treg)46. TGF-b is essential
for differentiation of murine Treg, but its role in human Treg is less
well defined. PD-L1 has been shown to induce Treg in mice and
humans28,47, suggesting defective induction of PD-L1 may lead to
reduced number of functional Treg observed in SLE patients48.
Figure 4 | MonocytesproduceTNF-atoinduceexpressionofPD-L1. (A)TNF-awasassayedinsupernatantsfromisolatedCD141monocytescultured
overnight without stimulation. Horizontal lines represent mean values. (B) PD-L1 protein levels on monocytes in the same culture was assayed by flow
cytometry; TNF-a positively correlated with PD-L1 expression on monocytes in SLE patients in remission. (C) TGF-b protein levels in monocyte
supernatants. (D) Correlation betweenTGF-bandPD-L1 protein levelson monocytes. Cytokine levelsbetweenhealthy controls and SLEpatient groups
were compared using the Wilcoxon-Mann-Whitney test. Correlations were determined by the Spearman’s rank correlation test.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
in SLE49, though whether or not this leads to increased surface pro-
tein expression and higher sensitivity to TGF-b signaling is not
with a previous finding that TGF-b suppresses PD-L1 expression on
of CD81cytotoxic T cells21. Our data suggest that overexpression of
TGF-b during active SLE can suppress PD-L1 by inhibiting protein
and mRNA expression. It has been shown that TGF-b can inhibit
production of TNF-a in mouse macrophages50, but further work is
required to determine whether TGF-b suppresses PD-L1 expression
directly, or through inhibition of TNF-a. TGF-b and TNF-a could
directly counter-regulate gene expression, as occurs in mucosal epi-
thelial cells, where TGF-b inhibits recruitment of the transcription
factor NFkB to the IL-6 promoter51.
Our results differed from previous findings, which have shown
increased levels of TNF-a and diminished expression of TGF-b in
SLE patients53. In our experimental system, levels of cytokines were
measured in culture supernatants of unstimulated cells, whereas
supernatants stimulated by adding apoptotic cells53. Our model sys-
tem may measure in part response to apoptotic cells; more apoptotic
cells were found in SLE PBMC cultures compared to healthy cells.
expression, and preliminary experiments showed no effect of addi-
tional apoptotic cells on PD-L1 expression on healthy monocytes.
Thus, SLE monocytes may be resistant to apoptotic cell signals.
Subject selection could also influence reported cytokine profiles,
affected cytokine or PD-L1 expression in pediatric SLE patients in
normally contribute to expression of PD-L1 on APCs. We noted
decreased PD-L1 expression on cultured isolated myeloid DCs and
monocytes in some control subjects and patients. Interestingly, we
found that PD-L1 expression on SLE monocytes and myeloid DCs
could be partially restored by co-culturing with allogenic CD41T
cells from healthy donors (preliminary data). SLE T cells present
multiple signaling aberrations27, and are reduced in number of func-
tional Treg48, which could be required for induction of PD-L1 on
APCs28. It remains to be determined whether correcting SLE T cell
defects can alter expression of PD-L1 on APCs.
Although most SLE patients with active disease expressed PD-L1
mRNA, surface protein expression was generally low or undetect-
able. The lack of correlation between mRNA and protein suggests
Figure 5 | PD-L1mRNAisinducedbyTNF-aandsuppressedbyTGF-b. TotalPBMCwereculturedfor24 hours.PD-L1mRNAexpressionwasassayed
by RT-qPCR for each subject in triplicate. (A) Relative PD-L1 mRNA expression in controls and lupus patients was represented by the scatter plot.
compared using the Wilcoxon-Mann-Whitney test. P.0.05 between all groups. (B) PD-L1 mRNA in SLE PBMC treated with recombinant TNF-a. (C)
and (D) PD-L1 mRNA levels in healthy donor PBMC treated with anti-TNF-a or TGF-b. Induction of PD-L1 mRNA by cytokines was tested for
significance by the Wilcoxon signed-rank test.
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
that additional translational mechanisms may be required for effec-
L1 could be cleaved from the cell surface. However, soluble PD-L1
was undetectable in culture supernatants and in SLE plasma when
assayed with a sensitivity of 60 pg/ml. Post-transcriptional and
translational regulation of PD-L1 has been shown in different bio-
logical models. In human trophoblast cells, epidermal growth factor
IFN-c induces PD-L1 protein expression by inhibiting miRNA in
cholangiocytes30. We are currently investigating whether additional
translational regulatory mechanisms are involved in determining
expression of PD-L1 surface protein on APCs.
We cannot rule out a role for additional cytokines in PD-L1 regu-
lation in SLE. Type I and II IFNs are critical mediators of SLE1, and
known inducers of PD-L1 expression20,22. However, neither IFN-c
nor IFN-a were detected in unstimulated PBMC cultures in our
experiments or by others54, and thus are unlikely to be involved in
the regulation of PD-L1 in our model system. IL-10 signaling
through STAT3 has been shown to up-regulate PD-L1 protein in
dendritic cells55,56, but in our experiments IL-10 was only detected
at minimal levels in some unstimulated PBMC cultures (data not
shown). IL-6 also signals through STAT3 to induce PD-L1 express-
ion56. While IL-6 was detected in SLE culture supernatants (data not
shown), the direct role of IL-6 in the regulation of PD-L1 in SLE has
yet to be explored. Deficient PD-L1 expression on SLE APCs may
result from the cytokine environment in vivo. Therefore, we assayed
levels of 16 plasma cytokines in 66 SLE patients in remission, 48
patients with active disease, and 65 healthy donors. We found no
correlation between plasma cytokine levels and PD-L1 expression.
These data do not exclude the possibility that cytokines may have an
effect on phenotypes of APCs in lymphoid tissues, which are inac-
cessible for human studies.
In the current study, we identified TNF-a and TGF-b as counter-
regulators of PD-L1 expression. Monocytes are programmed to
respond in autologous culture to generate intrinsic factors required
of proper induction of PD-L1 may contribute to the hyperstimula-
tory phenotype of SLE APCs, resulting in reduced peripheral T cell
tolerance. Better characterization of mediators regulating PD-L1
may lead to promising new therapeutic targets aimed at restoring
PD-L1 expression in SLE patients.
Human subjects and blood samples. The research protocol was approved by the
recruited in the Seattle Children’s Hospital Rheumatology Clinic; age-matched
healthy pediatric volunteers were recruited through an ongoing project to study
pediatric autoimmune diseases. Informed assent and consent were obtained. All
lupus patients fulfilled the current American College of Rheumatology (ACR)
classification criteria for SLE diagnosis57. Subjects were excluded for infections,
malignancy, or other autoimmune diseases that may affect PD-L1 expression.
Peripheral venous blood was collected into Cell Preparation Tubes (CPT, BD
Biosciences, San Jose, CA). PBMC were isolated and frozen in 7% DMSO (Sigma-
ml21in culture medium consisting of RPMI 1640 supplemented with L-glutamine
(CellGro, Manassas, VA), and 10% heat-inactivated human AB serum (Valley
Biomedical, Winchester, VA), 1% penicillin/streptomycin (CellGro) and 0.1% b-
mercaptoethanol. All assays were performed using frozen PBMC samples, as we
previously determined that freezing does not influence PD-L1 expression. Cells were
plated in round-bottom 96-well plates, and cultured for 24 hours without
stimulation. Between 60–85% of total PBMC were recovered after overnight
culturing. PBMC were surface-stained using fluorochrome-conjugated mAb,
including: anti-CD3 (UCHT1), anti-PD-L1 (MIH1) (eBioscience, San Diego, CA),
anti-CD11c (B-ly6), and anti-CD14 (MjP9) (BD Biosciences), with isotype-matched,
fluorochrome-labeled antibodies as controls. All samples were blocked using 0.5%
human AB serum and anti-FcR antibody (Miltenyi, Bergisch GladbachGermany)
prior and during staining. Flow cytometry was performed using an LSR II cytometer
(BD Biosciences), and the data analyzed using FlowJo software (Tree Star, Ashland,
OR). Examination ofsurface markers on normalPBMC determinedthat the PD-L11
cells segregated into monocyte (CD14highCD11c1) and immature myeloid DC
(CD14lowCD11c1) populations (Supplemental Figure 1)19,59. A similar expression
compared to polystyrene-treated plates. For cytokine experiments, PBMC were
cultured for 24 hours in the culture medium, or with 10 ng ml-1of recombinant
cytokines TNF-a (eBioscience) or TGF-b (R&D systems). In some experiments, cells
were treated with 10 mg ml21of mAb neutralizing TNF-a or TGF-b (R&D systems).
CD141monocytes and myeloid DCs were sorted by negative selection using the
EasySep human monocyte enrichment kit (StemCell Technologies, Inc., Vancouver,
BC, Canada), with purity .90%. Endotoxin levels in PBMC and isolated monocytes
were determined to be ,1 EU ml21by Limulus amoebocyte lysate clot assay
(Associates of Cape Cod, East Falmouth, MA). Expression of PD-L1 on monocytes
and myeloid DC was determined by subtracting the background PD-L1 mean
fluorescence intensity (MFI) of the CD32CD142CD11c2subset, which was
consistently negative for PD-L1 expression, similar to the isotype controls.
Cytokine analysis. All cytokine assays were performed on culture supernatants
(24 hours PBMC culture) by the Cytokine Analysis Laboratory at the Fred
system with a latex bead-based multianalyte system (Luminex, Austin, TX). TGF-b
was assayed by ELISA (R&D Systems).
Real-time reverse transcriptase-qPCR (RT-qPCR). Total RNA was extracted from
PBMC using the RNeasy Plus Micro Kit (Qiagen, Germantown, MD). Real-time RT-
qPCR analysis was performed using a Bio-Rad iCycler, in 25-ml reaction mixtures
for amplifying PD-L1 mRNA (Gene Bank accession number: NM_014143) were
sense: 59-GGCATTTGCTGAACGCAT-39; antisense: 59-CAATTAGTGCAGCCA-
GGT-39. For GAPDH, the primers were sense: 59-TGCACCACCAACTGCTTA-39;
antisense: 59-GGATGCAGGGATGATGTTC-39. Standard curves were determined
for each primer set by serial dilution. Expression of each cDNA was calculated from
the cycle threshold (CT). The relative PD-L1 mRNA expression was determined by
sample, where PD-L1 is predominantly expressed.
Statistical analyses. STATA statistical software was used to perform all analyses
(Stata Corporation, College Station, TX).
1. Blanco, P., Palucka, A. K., Gill, M., Pascual, V. & Banchereau, J. Induction of
dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus.
Science 294, 1540–1543 (2001).
2. Katsiari, C. G., Liossis, S. N. & Sfikakis, P. P. The Pathophysiologic Role of
Monocytes and Macrophages in Systemic Lupus Erythematosus: A Reappraisal.
Semin Arthritis Rheum 39, 491–503 (2009).
3. Zhu, J. et al. T cell hyperactivity in lupus as a consequence of hyperstimulatory
antigen-presenting cells. J Clin Invest 115, 1869–1878 (2005).
4. Ding, D., Mehta, H., McCune, W. J. & Kaplan, M. J. Aberrant phenotype and
function of myeloid dendritic cells in systemic lupus erythematosus. J Immunol
177, 5878–5889 (2006).
5. Monrad, S. & Kaplan, M. J. Dendritic cells and the immunopathogenesis of
systemic lupus erythematosus. Immunol Res 37, 135–145 (2007).
6. Keir, M. E., Butte, M. J., Freeman, G. J. & Sharpe, A. H. PD-1 and its ligands in
tolerance and immunity. Annu Rev Immunol 26, 677–704 (2008).
like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM
motif-carrying immunoreceptor. Immunity 11, 141–151 (1999).
8. Kasagi, S. et al. Anti-programmed cell death 1 antibody reduces CD41PD-11 T
cells and relieves the lupus-like nephritis of NZB/W F1 mice. J Immunol 184,
9. Wong, M., La Cava, A., Singh, R. P. & Hahn, B. H. Blockade of programmed
activity of suppressive CD81 T cells that protect from lupus-like disease. J
Immunol 185, 6563–6571 (2010).
10. Lucas, J. A. et al. Programmed death ligand 1 regulates a critical checkpoint for
autoimmune myocarditis and pneumonitis in MRL mice. J Immunol 181, 2513–
11.Bertsias, G.K. etal.Genetic, immunologic,and immunohistochemical analysis of
lupus erythematosus. Arthritis Rheum 60, 207–218 (2009).
12. Thorburn, C. M. et al. Association of PDCD1 genetic variation with risk and
clinical manifestations of systemic lupus erythematosus in a multiethnic cohort.
Genes Immun 8, 279–287 (2007).
13. Velazquez-Cruz,R. et al. Association of PDCD1 polymorphisms with childhood-
onset systemic lupus erythematosus. Eur J Hum Genet 15, 336–341 (2007).
cell death 1 polymorphisms and systemic lupus erythematosus: a meta-analysis.
Lupus 18, 9–15 (2009).
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295
15. Suarez-Gestal, M., Ferreiros-Vidal, I., Ortiz, J. A., Gomez-Reino, J. J. & Gonzalez,
A. Analysis of the functional relevance of a putative regulatory SNP of PDCD1,
PD1.3, associated with systemic lupus erythematosus. Genes Immun 9, 309–315
16. Abelson, A. K. et al. No evidence of association between genetic variants of the
PDCD1 ligands and SLE. Genes Immun 8, 69–74 (2007).
17. Wang, S. C. et al. Ligands for programmed cell death 1 gene in patients with
systemic lupus erythematosus. J Rheumatol 34, 721–725 (2007).
18. Brown, J. A. et al. Blockade of programmed death-1 ligands on dendritic cells en-
19. Mozaffarian, N., Wiedeman, A. E. & Stevens, A. M. Active systemic lupus
erythematosus is associated with failure of antigen-presenting cells to express
programmed death ligand-1. Rheumatology (Oxford) 47, 1335–1341 (2008).
20. Chen, Y. et al. Expression of B7-H1 in inflammatory renal tubular epithelial cells.
Nephron Exp Nephrol 102, e81–92 (2006).
21. Starke, A., Wuthrich, R. P. & Waeckerle-Men, Y. TGF-beta treatment modulates
PD-L1 and CD40 expression in proximal renal tubular epithelial cells and en-
22. Waeckerle-Men, Y., Starke, A. & Wuthrich, R. P. PD-L1 partially protects renal
tubular epithelial cells from the attack of CD81 cytotoxic T cells. Nephrol Dial
Transplant 22, 1527–1536 (2007).
23. Schreiner, B. et al. Interferon-beta enhances monocyte and dendritic cell
expression of B7-H1 (PD-L1), a strong inhibitor of autologous T-cell activation:
relevance for the immune modulatory effect in multiple sclerosis. J
Neuroimmunol 155, 172–182 (2004).
24. Wan, B. et al. Aberrant regulation of synovial T cell activation by soluble
costimulatory molecules in rheumatoid arthritis. J Immunol 177, 8844–8850
25. O’Shea, J. J., Ma, A. & Lipsky, P. Cytokines and autoimmunity. Nat Rev Immunol
2, 37–45 (2002).
26.Kinter,A. L.etal.Thecommongamma-chain cytokinesIL-2, IL-7,IL-15, andIL-
21 induce the expression of programmed death-1 and its ligands. J Immunol 181,
27. Crispin, J. C., Kyttaris, V. C., Terhorst, C. & Tsokos, G. C. T cells as therapeutic
targets in SLE. Nat Rev Rheumatol 6, 317–325 (2010).
28.Amarnath, S. etal.RegulatoryTcells and human myeloiddendritic cells promote
tolerance via programmed death ligand-1. PLoS Biol 8, e1000302 (2010).
29. Hegde, S. et al. Human NKT cells direct the differentiation of myeloid APCs that
regulate T cell responses via expression of programmed cell death ligands. J
Autoimmun 37, 28–38 (2011).
30. Gong, A. Y. et al. MicroRNA-513 regulates B7-H1 translation and is involved in
IFN-gamma-induced B7-H1expression incholangiocytes. JImmunol182,1325–
31. Holets, L.M., Carletti, M. Z., Kshirsagar, S. K., Christenson, L. K. & Petroff, M. G.
Differentiation-induced post-transcriptional control of B7-H1 in human
trophoblast cells. Placenta 30, 48–55 (2009).
32. Marzec, M. et al. Oncogenic kinase NPM/ALK induces through STAT3
expression of immunosuppressive protein CD274 (PD-L1, B7-H1). Proc Natl
Acad Sci U S A 105, 20852–20857 (2008).
33. Parameswaran, N. & Patial, S. Tumor necrosis factor-alpha signaling in
macrophages. Crit Rev Eukaryot Gene Expr 20, 87–103 (2010).
34.Massague, J.&Gomis, R.R.Thelogic ofTGFbetasignaling.FEBSLett580,2811–
35. Ronnblom, L. & Elkon, K. B. Cytokines as therapeutic targets in SLE. Nat Rev
Rheumatol 6, 339–347 (2010).
36. Brown, K. E., Freeman, G. J., Wherry, E. J. & Sharpe, A. H. Role of PD-1 in
regulating acute infections. Curr Opin Immunol 22, 397–401 (2010).
37. Dong, H. & Chen, X. Immunoregulatory role of B7-H1 in chronicity of
inflammatory responses. Cell Mol Immunol 3, 179–187 (2006).
38. Atsumi, T. [Tumor necrosis factor alpha in systemic lupus erythematosus:
evaluation by restriction fragment length polymorphism and production by
peripheral blood mononuclear cells]. Hokkaido Igaku Zasshi 67, 408–419 (1992).
39. Jacob, C. O. et al. Heritable major histocompatibility complex class II-associated
differences in production of tumor necrosis factor alpha: relevance to genetic
predisposition to systemic lupus erythematosus. Proc Natl Acad Sci U S A 87,
40. McHugh, N. J., Owen, P., Cox, B., Dunphy, J. & Welsh, K. MHC class II, tumour
necrosis factor alpha, and lymphotoxin alpha gene haplotype associations with
serological subsets of systemic lupus erythematosus. Ann Rheum Dis 65,488–494
41. Zhu, L. et al. Decreased expressions of the TNF-alpha signaling adapters in
peripheral blood mononuclear cells (PBMCs) are correlated with disease activity
in patients with systemic lupus erythematosus. Clin Rheumatol 26, 1481–1489
42. Zhu, L. J. et al. Altered expression of TNF-alpha signaling pathway proteins in
systemic lupus erythematosus. J Rheumatol 37, 1658–1666 (2010).
43. Palucka, A. K., Blanck, J. P., Bennett, L., Pascual, V. & Banchereau, J. Cross-
regulation ofTNFandIFN-alphainautoimmunediseases. ProcNatlAcadSciUS
A 102, 3372–3377 (2005).
44. Ohtsuka, K., Gray, J. D., Stimmler, M. M., Toro, B. & Horwitz, D. A. Decreased
production of TGF-beta by lymphocytes from patients with systemic lupus
erythematosus. J Immunol 160, 2539–2545 (1998).
45. Saxena, V. et al. Dual roles of immunoregulatory cytokine TGF-beta in the
pathogenesis of autoimmunity-mediated organ damage. J Immunol 180, 1903–
46. Ghoreschi, K., Laurence, A., Yang, X. P., Hirahara, K. & O’Shea, J. J. T helper 17
47. Francisco, L. M. et al. PD-L1 regulates the development, maintenance, and
function of induced regulatory T cells. J Exp Med 206, 3015–3029 (2009).
48. La Cava, A. T-regulatory cells in systemic lupus erythematosus. Lupus 17, 421–
49. Hrycek, A., Kusmierz, D., Dybala, T. & Swiatkowska, L. Expression of messenger
RNA for transforming growth factor-beta1 and for transforming growth factor-
beta receptors in peripheral blood of systemic lupus erythematosus patients
treated with low doses of quinagolide. Autoimmunity 40, 23–30 (2007).
50. Khera, T. K., Dick, A. D. & Nicholson, L. B. Fragile X-related protein FXR1
controls post-transcriptional suppression of lipopolysaccharide-induced tumour
negative bacteria-induced NF-kappa B recruitment to the interleukin-6 gene
Biol Chem 278, 23851–23860 (2003).
systemic lupus erythematosus. Br J Rheumatol 35, 1067–1074 (1996).
53. Sule, S., Rosen, A., Petri, M., Akhter, E. & Andrade, F. Abnormal production of
pro- and anti-inflammatory cytokines by lupus monocytes in response to
apoptotic cells. PLoS One 6, e17495 (2011).
IFN-alpha and chemokines by autoantibodies in the cerebrospinal fluid of
patients with neuropsychiatric lupus. J Immunol 182, 1192–1201 (2009).
55. Saito, M. et al. Defective IL-10 signaling in hyper-IgE syndrome results in
Exp Med 208, 235–249 (2011).
Eur J Immunol 41, 413–424 (2011).
for the classification of systemic lupus erythematosus. Arthritis Rheum 40, 1725
58. Bombardier, C., Gladman, D. D., Urowitz, M. B., Caron, D. & Chang, C. H.
Derivation of the SLEDAI. A disease activity index for lupus patients. The
Committee on Prognosis Studies in SLE. Arthritis Rheum 35, 630–640 (1992).
59. O’Doherty, U. et al. Human blood contains two subsets of dendritic cells, one
immunologically mature and the other immature. Immunology 82, 487–493
This project was supported by grants from the Lupus Research Institute, and Lupus
Foundation of America to A.M.S., and the Thrasher Research Foundation (award number:
NR-0109) to J-N.O. Support was derived in part by Clinical and Translational Science
Award (CTSA) (Grant Number: I ULI RR025014-02) from the National Center for
Research Resources (NCRR), a component of the National Institutes of Health (NIH). Its
contents are solely the responsibility of the authors and do not necessarily represent the
official view of NCRR or NIH.
We thank Dr. Veronika Groh-Spies, Dr. Keith Elkon and Dr. Jeffrey Ledbetter for helpful
analyses, Matthew Crabtree and Michelle Stanley for technical assistance, Elizabeth
Ocheltree and Dr. Rebecca Howsmon for proofreading of the manuscript and Gretchen
Henstorf for collection of clinical samples.
JNO and AMS performed study design, contributed to data analyses and wrote the
manuscript. JNO performed experiments; AEW participated in study design and reviewed
Supplementary information accompanies this paper at http://www.nature.com/
Competing financial interests: The authors declare no competing financial interests.
License: This work is licensed under a Creative Commons
Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this
license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/
How to cite this article: Ou, J., Wiedeman, A.E. & Stevens, A.M. TNF-a and TGF-b
Counter-Regulate PD-L1 Expression on Monocytes in Systemic Lupus Erythematosus. Sci.
Rep. 2, 295; DOI:10.1038/srep00295 (2012).
SCIENTIFIC REPORTS | 2 : 295 | DOI: 10.1038/srep00295