HI, I want help with my methodology to make some add and change in two parts that I already writ it ( sample and data collection and protocol of the lab work ) in this project. ( I already writ my impact and introduction) I will send one of the article to let you understand the criteria of how I want the the protocol look like.My supervisor dose not want the protocol like toking about the producer, he want to writ about the principle. also, in the sample collection and data, I just used normal healthy human sample, I did not use any sample from B CELL LYMPHOMA patient, that’s why I want to make change on this part.
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Article
Leukocyte Ig-Like receptor B1 restrains
dendritic cell function through increased
expression of the NF-kB regulator
ABIN1/TNIP1
Rahul C. Khanolkar,*,1 Michail Kalogeropoulos,* Alistair Lawrie,* Ali Roghanian,†,‡,§
Mark A. Vickers,* and Neil T. Young*
*Division of Applied Medicine, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdom; †David H. Koch
Institute for Integrative Cancer Research and ‡Department of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts, USA; and §Antibody and Vaccine Group, Cancer Sciences Unit, Faculty of Medicine, University of Southampton,
Southampton General Hospital, Southampton, United Kingdom
RECEIVED SEPTEMBER 15, 2015; REVISED APRIL 4, 2016; ACCEPTED APRIL 6, 2016. DOI: 10.1189/jlb.1A0915-420RRR
ABSTRACT
Introduction
Inhibitory receptors of the human leukocyte
immunoglobulin-like receptor family are constitutively
expressed on all myeloid cell types and regulate their
functional activity. We demonstrate that ligation of the
human leukocyte antigen class I-specific receptor
LILRB1, during the differentiation of monocytes to dendritic cells in vitro, results in increased expression of the
nuclear factor kB inhibitor protein ABIN1 (also known as
TNIP1). Similarly increased expression of ABIN1/TNIP1
was observed in the “immunosuppressive” monocyte
populations of patients with non–Hodgkin lymphoma ex
vivo. Reducing expression of ABIN1/TNIP1 using small
interfering ribonucleic acid allows dendritic cells and
immunosuppressive monocytes to respond to stimulation by allowing nuclear factor kB translocation to the
nucleus (P , 0.001), increasing cell surface expression
of antigen presentation and costimulatory molecules
(P , 0.01), increasing phagocytic capacity (P , 0.001),
secreting proinflammatory cytokines (P , 0.01), and an
increasing ability to stimulate T cell responses (P ,
0.05). Our study, therefore, identifies an important
functional role for ABIN1/TNIP1 in mediating the effects
of LILRB1 ligation-induced inhibitory effects on immune
responses. Our findings suggest that inhibiting the
LILRB1-ABIN1/TNIP1 pathway in antigen-presenting
cells could be a therapeutic approach to stimulate
antitumor immune responses. Conversely, stimulation
of the pathway may also ameliorate autoimmune diseases in which TNIP1 is a susceptibility gene.
J. Leukoc. Biol. 100: 737–746; 2016.
DCs have a central role in the initiation, regulation, and
maintenance of immune responses. Recognition of “danger”
signals by a variety of pattern-recognition receptors expressed by
DCs initiates a program of cellular maturation, creating potent
antigen-presenting cells, which are capable of stimulating
antigen-speci?c, na??ve T lymphocytes and establishing adaptive,
antigen-speci?c immune responses. Conversely, immature DCs
are thought to be involved in the prevention of inappropriate
immune responses against “self” antigens by secreting immunosuppressive cytokines and interacting with regulatory T cell
populations [1]. The regulation of DC maturation is controlled
by several levels of molecular control to ensure that DCs respond
appropriately and that immune homeostasis is maintained [2].
Most myeloid lineage cells, including DCs, constitutively
express transmembrane cell surface receptors of the LILR gene
family. The LILR genes comprise part of the leukocyte receptor
cluster on human chromosome band 19q13.4 and include
isoforms with either inhibitory or activating functions, dependent
on their possession of ITIM motifs within their cytoplasmic tail
[3]. ITIM motifs recruit phosphatase enzymes, such as src
homology region 2 domain-containing phosphatase 1 (SHP1),
which diminish the intracellular-signaling phosphorylation
events initiated by the activating stimuli.
We have previously demonstrated that the HLA class I–speci?c
inhibitory receptor LILRB1 is involved in maintaining human
monocyte–derived DCs in a quiescent state, regulating the
capacity of DCs to increase levels of cell-surface antigen
presentation and costimulatory molecules, controlling the
secretion of cytokines, conferring resistance to FAS-mediated
apoptosis and in?uencing T cell reactivity by interacting with a
population of CD4+ CD25+ CD1272 regulatory T cells [4]. Other
reports have described similar functions for additional members
Abbreviations: ABIN1 = A20-binding inhibitor of NF-kB, DC = dendritic cell,
LAT = linker for activation of T cells, LILR = leukocyte Ig-like receptors,
MDSC = myeloid-derived suppressor cell, NHL = non–Hodgkin lymphoma,
siRNA = small interfering ribonucleic acid, SNP = single-nucleotide polymorphisms, TNIP1 = tumor necrosis factor a-induced protein 3 interacting protein 1
The online version of this paper, found at www.jleukbio.org, includes
supplemental information.
1. Correspondence: Institute of Medical Sciences, University of Aberdeen,
Aberdeen AB25 2ZD, United Kingdom. E-mail: [email protected]
0741-5400/16/0100-737 © Society for Leukocyte Biology
Volume 100, October 2016
Journal of Leukocyte Biology 737
of the LILR family [5–8], suggesting an important role for this
family of receptors in establishing activation thresholds and
regulating myeloid lineage induction of immune responses. The
murine homolog of inhibitory LILR, Pirb, has also been shown to
control the survival and function of MDSCs [9], a label applied to
a diverse range of immature myeloid-lineage cells with regulatory
properties [10], often associated with reduced immune responsiveness to tumor transformation. Mice transgenic for both
LILRB1 and its ligand HLA-G have an expanded population of
MDSCs [11], which can prolong skin allograft survival.
In an effort to delineate the molecular mechanisms underlying
the quiescent nature of monocyte-derived DCs by the ligation of
LILRB1, we have performed genome-wide mRNA expression
analysis on human monocyte derived DCs cultured with LILRB1speci?c mAb or isotype control. mRNA expression studies on
human monocyte–derived DCs cultured with LILRB1-speci?c
mAbs (henceforth, referred to as LILRB1 DC) revealed the upregulation of mRNA that encodes NF-kB–inhibiting proteins—
ABIN1 (also known as TNIP1) and ABIN3 (also known as TNIP3)
(unpublished data). Here, we describe our analysis of the
ABIN1/TNIP1 protein (A20-binding inhibitor of NF-kB/TNFa–induced protein 3 interacting protein 1) [12] after ligation of
LILRB1 during DC differentiation and show that altering the
expression level of ABIN1/TNIP1 by siRNA-mediated knockdown of gene expression has a signi?cant in?uence on the
monocyte-derived DC phenotype and function in vitro. We also
examine the expression and function of ABIN1/TNIP1 in
“immunosuppressive” monocytes [13] from ex vivo analysis of
patients with NHL. Our results suggest that LILRB1 is a
potential target to manipulate ABIN1/TNIP1 protein levels
and modulate DC responsiveness in lymphomas and immunemediated diseases.
MATERIALS AND METHODS
Isolation of monocytes from peripheral blood and
nodal biopsies
Healthy donors were identi?ed at the Institute of Medical Sciences
(Aberdeen, United Kingdom). Patients with suspected follicular lymphoma,
mantle cell lymphoma, or nodular lymphocyte-predominant Hodgkin
lymphoma undergoing a lymph node biopsy were identi?ed through the
Haematology Outpatient Clinic, Aberdeen Royal In?rmary (Aberdeen,
United Kingdom), and samples were obtained after full informed consent was
given (North of Scotland Ethics Committee, Integrated Research Application
System, project 9412). Peripheral blood obtained from the healthy controls, as
well as lymph node biopsies obtained from patients, were subjected to
negative selection using Depletion MyOne SA Dynabeads (Thermo Fisher
Scienti?c, Waltham, MA, USA) to obtain a single-cell suspension of
monocytes.
Differentiation of monocytes into DCs
Monocytes isolated from peripheral blood of healthy donors were differentiated into DCs for a period of 7 d using a 50 ng/ml concentration of
recombinant human IL-4 and GM-CSF (PeproTech, Inc, Rocky Hill, NJ, USA).
Half of the medium was replaced every 2 d with fresh cytokines, and on day 6,
1 ng/ml of bacterial LPS (Sigma-Aldrich, St. Louis, MO, USA) was added for
24 h where required. Ligation of the LILRB1 receptor was mediated by the
addition of puri?ed anti-LILRB1 (clone 292305, R&D Systems, Minneapolis,
MN, USA; or clone HPF1, eBioscience, San Diego, CA, USA) at a ?nal
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Journal of Leukocyte Biology
Volume 100, October 2016
concentration of 5 mg/ml in the presence of 1 mg/ml Protein G (SigmaAldrich). Mouse IgG1k (MOPC21; Sigma-Aldrich) was used as an isotype
control at the same concentration as previously described [4].
siRNA-mediated knockdown of ABIN1/TNIP1 expression
The cells of interest were seeded into 6-well plates at 2 3 105 cells/well in 2 ml
of antibiotic-free RPMI 1640 medium supplemented with 10% heatinactivated human AB serum for 24 h to ensure con?uence. For each
transfection, 60 pM ABIN1 siRNA (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA) was used along with 100 ml siRNA transfection medium (Santa
Cruz Biotechnology) and 100 ml siRNA transfection reagent (Santa Cruz
Biotechnology). The cells were then incubated at 37°C, 5% CO2 for an
additional 5 h; following which, the medium in each well containing the
siRNA-treated cells was changed to RPMI 1640 supplemented with 10% heatinactivated human AB serum and 50 U/ml of penicillin-streptomycin and was
incubated for an additional 24 h at 37°C, 5% CO2.
Flow cytometry
The following antibodies were used for ?ow cytometry: anti-CD3-PE (clone
SK7), anti-CD4-AF700 (clone RPA-T4), anti-CD8-PE-Cy5 (clone RPA-T8), antiCD80-FITC (clone L307), anti-CD86-APC (clone 2331 FUN-1), anti-HLA-DRPE-Cy5 (clone TU36), anti-HLA-ABC-PE (clone G46-2.6), anti-pERK1/2-PE
(clone 25/MEK1), anti-pLAT-AF488 (clone 158-1169), anti-IFN-g-AF700
(clone B27), anti-IL-12p70–PE (clone 20C2) (all BD Biosciences, Franklin
Lakes, NJ, USA); anti-IL-10–PE-Cy7 (clone JES3-9D7; BioLegend, San Diego,
CA, USA); anti-IFN-a-PE (clone 1-D1K; Mabtech, Cincinnati, OH, USA). AntiTNIP1 (ABIN1) antibody (clone 5C4, Abcam, Cambridge, MA, USA) was used
in conjunction with goat anti-mouse IgG (heavy and light chain)-PE
(Beckman Coulter, Brea, CA, USA). Monocytes, DCs, or PBMCs were washed
and incubated at room temperature for 30 min with the above antibodies. The
cells were then ?xed with 4% paraformaldehyde, washed and analyzed on a
BD LSR II ?ow cytometer (BD Biosciences) using FlowJo software (Tree Star,
Ashland, OR, USA). DCs and monocytes were identi?ed using forward and
side light scatter characteristics. A minimum of 10,000 events were acquired
on all samples.
To assess the macropinocytic capacity of different DC populations, the cells
were incubated with FITC-conjugated dextran molecules at a concentration of
50 mg/ml for 24 h before ?xation and ?ow cytometric analysis.
To quantify cytokine production, DCs, monocytes or PBMCs were treated
with 1 mg/ml monensin (BD Biosciences), ?xed with 4% paraformaldehyde,
and permeabilized with methanol before staining for ?ow cytometry. PBMCs
were stained with T cell-speci?c markers to analyze cytokine production from
T cell that were stimulated with different DC populations.
To assess T cell proliferation when stimulated by different DC populations,
CFSE (Thermo Fisher Scienti?c)-stained PBMCs were incubated with DCs at a
ratio of 10:1 at 37°C for 5 d; following which, the cells were stained with T cellspeci?c markers, ?xed, washed, and analyzed on a BD LSR II ?ow cytometer
using FlowJo software. A minimum of 100,000 events were acquired on all
samples.
Fluorescence and light microscopy
To assess the expression levels of NF-kB p65 in the nucleus of different DC
populations, DCs were seeded into poly-L-lysine–treated wells of a 48 well plate
and left to adhere at 37°C for 2 h. The cells were then ?xed with 4%
formaldehyde, permeabilized with Triton X-100, and blocked with 1% BSA in
PBS containing 0.05% Tween 20, all at room temperature. The cells were
stained with an anti–NF-kB p65 antibody (Abcam) for 1 h at room
temperature; after which, they were washed and incubated with a goat antimouse heavy and light chain secondary antibody conjugated to FITC (Abcam)
for 2 h at room temperature. The nucleus of the cells was stained with Hoechst
stain (ImmunoChemistry Technologies, Bloomington, MN, USA) for 5 min.
The cells were then washed with PBS and analyzed on a Zeiss Axio Observer
Z1 inverted microscope (Carl Zeiss Microscopy, Thornwood, NY, USA).
Nuclear expression of NF-kB p65 was analyzed using the ImageJ software (U.S.
www.jleukbio.org
Khanolkar et al. LILRB1 control of dendritic cell function
National Institutes of Health, Bethesda, MD, USA) that quanti?ed the
?uorescence emitted by ?uorochrome-conjugated antibodies speci?c to the
NF-kB subunit p65. A maximum of 50 cells were analyzed per treatment per
donor, set up in triplicates.
To assess the phagocytic population of different DC populations, cells were
incubated with 6 mm polystyrene beads at a ratio of 1:3 for 3 h. The cells were
washed with PBS, ?xed with 4% formaldehyde, and analyzed on an EVOS 3l
transmitted light microscope (Thermo Fisher Scienti?c).
Statistical analysis
Differences in ABIN1 protein expression, NF-kB/p65 translocation,
phagocytic/macropinocytic capacity, cytokine production, expression of
antigen presentation, TLRs, and costimulatory molecules in DC populations,
and phosphorylation of intracellular molecules, and proliferation of T cells
was analyzed using 1-way ANOVA test, followed by the Bonferroni multiple
comparison test. Differences in ABIN1 expression, expression of antigen
presentation and costimulatory molecules, and cytokine production between
monocytes obtained from healthy donors and those obtained from patients
with NHL were analyzed using unpaired t tests. Differences in cytokine
production and expression of antigen presentation and costimulatory
molecules in control monocytes and monocytes obtained from patients with
NHL after treatment with ABIN1 siRNA were analyzed using paired t tests.
RESULTS
Increased expression levels of ABIN1/TNIP1 protein
in monocyte-derived DCs after LILRB1 ligation
mRNA expression studies and intracellular ?ow cytometry in
monocyte-derived DCs, in which LILRB1 had been ligated
throughout in vitro differentiation, demonstrated enhanced
expression of ABIN1/TNIP1 on an mRNA level (data not shown)
and protein level (Fig. 1A and C) when compared with DCs
cultured with the isotype control antibody. ABIN1/TNIP1
protein levels were signi?cantly increased after exposure to the
TLR4 ligand bacterial lipopolysaccharide (P , 0.001) (Fig. 1A
and C). Signi?cant differences (P , 0.05) were also noted after
transfection of LILRB1 DCs with siRNA speci?c for ABIN1/
TNIP1, where a reduction in protein expression was observed
Figure 1. LILRB1 ligation during in vitro DC differentiation results in higher levels of ABIN1/TNIP1 protein levels. (A and C) Higher ABIN1/
TNIP1 protein levels are evident in LILRB1 DCs and can be signi?cantly increased (P , 0.01) after LPS exposure. (B and C) ABIN1/TNIP1 levels
can be signi?cantly reduced in LILRB1 DCs using siRNA. (D) LILRB1 DCs display signi?cantly elevated levels of CD14 when compared with
control DCs. In all experiments (n = 4 individual donors), bars depict means 6 SEM. CI, con?dence interval; Geo-MFI, geometric mean
?uorescence intensity; Gep-MFI, granulin-epithelin precursor mean ?uorescence intensity; neg, negative. *P , 0.05 (95% CI), **P , 0.01
(99% CI), ***P , 0.001 (99.9% CI).
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Volume 100, October 2016
Journal of Leukocyte Biology 739
(Fig. 1B and C). LILRB1 DCs expressed signi?cantly elevated
levels of CD14 when compared with the isotype control DCs
(Fig. 1D).
ABIN1/TNIP1 in?uences NF-kB nuclear
translocation and macropinocytic/phagocytic
functions of LILRB1 DCs
In accordance with ABIN1/TNIP1’s known cellular function, NFkB translocated to the nucleus less ef?ciently in LILRB1 DCs
than it did in isotype control DCs (P , 0.001), but translocation
signi?cantly (P , 0.001) increased after siRNA-mediated reduction in ABIN1/TNIP1 expression levels (Fig. 2A).
DCs are characterized by their antigen sampling abilities and
their capacity to secrete cytokines rapidly after recognition of
pathogens or damage-associated molecular patterns. After incubation with FITC-labeled dextran as a surrogate for a ?uid
phase antigen, LILRB1 DCs dramatically increased their uptake
capacity after the reduction in ABIN1/TNIP1 levels, as measured
by ?ow cytometry (P , 0.001; Fig. 2B, upper panel), whereas
phagocytic uptake of 6 mM polystyrene beads, measured by
light microscopy, also signi?cantly increased (P , 0.05, Fig.
2B, lower panel).
ABIN1/TNIP1 in?uences expression levels of
antigen-presentation molecules and cytokine
secretion in LILRB1 DCs
Flow cytometric analysis of cell surface levels of important ligands
in DC antigen presentation and T cell costimulation demonstrated signi?cant increases in amounts of HLA- ABC (P , 0.001)
and HLA-DR (P , 0.05) when ABIN1/TNIP1 levels were
reduced by siRNA-mediated knockdown. This ?nding was
particularly strong for HLA-ABC because LILRB1 DCs did not
increase expression after exposure to LPS, unlike isotype control
DCs, but HLA-ABC levels almost quadrupled after reduction in
ABIN1/TNIP1 levels, even in immature LILRB1 DCs (Fig. 3A,
lower panel). Although CD80 and CD86 levels also increased,
this relationship did not reach statistical signi?cance (Fig. 3A,
upper panel).
Likewise, siRNA-mediated knockdown of ABIN1/TNIP1 expression signi?cantly increased LILRB1 DC production of IL12p70 (P , 0.001) after stimulation with the TLR4 ligand LPS
(Fig. 3B, upper panel). Signi?cant increases were also observed
in LILRB1 DC’s capacity to produce IL-10 (P , 0.05) and IFN-a
(P , 0.05) (Fig. 3B, upper and lower panels) following ABIN1/
TNIP1 knockdown.
ABIN1/TNIP1 in?uences the capacity of
LILRB1-ligated DCs to activate allogeneic
T lymphocytes
The prime function of DCs is to stimulate speci?c T lymphocytes
to initiate an adaptive, antigen-speci?c immune response. We
investigated the role that ABIN1/TNIP1 has in regulating the
function of LILRB1 DCs by coculturing the isotype control or
LILRB1 DCs with allogeneic CD4+ and CD8+ T lymphocytes after
siRNA-mediated reduction in ABIN1/TNIP1 expression.
T lymphocyte responses were assessed by ?ow cytometric analysis
of phosphorylation of key T cell signaling molecules ERK1/2 and
740
Journal of Leukocyte Biology
Volume 100, October 2016
Figure 2. Reducing ABIN1/TNIP1 expression signi?cantly increases
NF-kB nuclear translocation and the macropinocytic and phagocytic
capacity of LILRB1 DC. Fluorescence microscopy was used to determine
nuclear expression levels of NF-kB/p65, as described in the Materials and
Methods section. (A) Nuclear expression of NF-kB/p65 was signi?cantly
lower in LILRB1 DCs than it was in isotype control DCs (P , 0.001)
and was signi?cantly restored on siRNA knockdown of ABIN1/TNIP1
(P , 0.001) in semimature LILRB1 DCs. Following ABIN1/TNIP1
knockdown, LILRB1 DCs increased their capacity for macropinocytic/
phagocytic uptake of ?uorescently labeled dextran (B, upper panel)
(P , 0.001), as determined by ?ow cytometry and 6 mm polystyrene beads
(B, lower panel) (P , 0.05), as determined by light microscopy. In all
experiments (n = 6 individual donors), bars depict means 6 SEM. CI,
con?dence interval; Geo-MFI, geometric mean ?uorescence intensity; neg,
negative; ns, not signi?cant. *P , 0.05 (95% CI), **P , 0.01 (99% CI),
***P , 0.001 (99.9% CI). Representative examples of images used to
obtain data depicted in (A) are provided in Supplemental Fig. 1.
www.jleukbio.org
Khanolkar et al. LILRB1 control of dendritic cell function
Figure 3. Reducing ABIN1/TNIP1 expression in LILRB1 DCs increases expression of cell-surface antigen presentation and costimulatory
molecules, with increased expression of proin?ammatory cytokines. CD80 (ns) and CD86 (ns) (A, upper panel) and HLA-ABC (P , 0.001)
and HLA-DR (P , 0.05) (A, lower panel) were increased in LILRB1 DCs following siRNA-mediated knockdown of ABIN1/TNIP1 expression.
Production of proin?ammatory cytokines IL-12 p70 (B, upper panel) (P , 0.001) and IFN-a (B, lower panel) (P , 0.05) were also increased
in LILRB1 DCs following treatment with ABIN1 siRNA. In all experiments (n = 6 individual donors), bars depict means 6 SEM. Geo-MFI,
geometric mean ?uorescence intensity; neg, negative; ns, not signi?cant. *P , 0.05 (95% CI), ** P , 0.01 (99% CI), *** P , 0.001 (99.9%
CI). Representative examples of the combined data are provided in Supplemental Fig. 2.
signi?cant changes were observed with respect to T cells capacity
to produce IFN-g when treated with the different DC populations
(Fig. 4C).
LAT using phosphospeci?c antibodies. T cell proliferation was
also measured by the loss of CFSE ?uorescence from proliferating T cells after 5 d of culture.
We observed no signi?cant differences in level of phosphorylation of ERK1/2 (pERK1/2) in both responding CD4 (Fig. 4A,
left panel) and CD8 T lymphocytes (Fig. 4B, left panel) when
stimulated by control or wild-type LILRB1-ligated DCs. However,
a signi?cant (P , 0.05) increase in pERK1/2 was seen in both
T cell subsets (Fig. 4A and B, left panel) when stimulated by
LILRB1 DCs carrying the ABIN1/TNIP1 expression knockdown.
A similar result was observed for the phosphorylation of LAT
residue 171 after ABIN1/TNIP1 expression knockdown in
LILRB1 DCs (Fig. 4A and B, middle panel). Analysis of
phosphorylation of LAT residue 226 did not show any speci?c
differences (data not shown).
LILRB1 ligated DCs were poor stimulators of allogeneic T cell
proliferation in mixed lymphocyte cultures in comparison to
isotype control DCs, and this was reversed by a reduction in
ABIN1/TNIP1 levels. LILRB1 DCs after ABIN1/TNIP1 expression knockdown stimulated a signi?cant increase in the proliferation of CD4+ T cells (Fig. 4A, right panel). No statistically
signi?cant changes were observed with respect to CD8+ T cell
proliferation (Fig. 4B, right panel). Furthermore, no statistically
Several studies have reported the presence of an immunosuppressive monocyte population in patients with NHL. Patients with
NHL presenting with increased percentages of these suppressive
monocytes usually display a more progressive disease. We
examined expression levels of ABIN1/TNIP1 in monocytes
obtained from patients with NHL (follicular, mantle cell, or
nodular lymphocyte predominant Hodgkin lymphoma) (n = 12).
Intracellular levels of ABIN1/TNIP1 were signi?cantly higher
(P , 0.01) in monocytes obtained from patient samples than
they were from peripheral population of monocytes of healthy
donors and increased after stimulation with TLR4 agonists
(Fig. 5A, left panel). ABIN1/TNIP1 levels were signi?cantly
reduced in monocytes obtained from patients with NHL after
siRNA treatment (Fig. 5A, right panel). Patient-derived monocytes expressed lower cell surface levels of CD80 (P , 0.01),
CD86 (not signi?cant, [ns]), and HLA-DR (P , 0.001) than did
those of healthy controls (Fig. 5B). Similarly, monocytes from
www.jleukbio.org
Volume 100, October 2016
ABIN1/TNIP1 is expressed at high levels in monocytes
isolated from lymph nodes involved by NHL
Journal of Leukocyte Biology 741
Figure 4. Reducing ABIN1/TNIP1 expression restores the ability of LILRB1 DCs to stimulate allogeneic CD4+ and CD8+ T lymphocytes. No
statistically signi?cant difference were observed in the phosphorylation of ERK1/2 in CD4+ (A) and CD8+ (B) T cells when control or LILRB1 DCs
were used as stimulators. Following ABIN1/TNIP1 knockdown in LILRB1 DCs, immature LILRB1 DCs stimulated signi?cantly elevated
phosphorylation of ERK1/2 (P , 0.05) in allogeneic T cells (A and B, left panel), whereas the semimature LILRB1 DC population stimulated
signi?cantly elevated phosphorylation of LAT171 (P , 0.05) in allogeneic T cells (A and B, middle panel). Knockdown of ABIN1/TNIP1 resulted
in a signi?cantly increased capacity of both immature (P , 0.01) and semimature (P , 0.05) LILRB1 DCs to stimulate CD4+ T cell proliferation
(A, right panel). Results for CD8+ T cell proliferation were not signi?cant (B, right panel). (C) Reducing ABIN1/TNIP1 levels in LILRB1 DCs had
no statistically signi?cant effect in stimulating production of IFN-g in CD3+ T cells. In all experiments, statistical comparisons were made between
unstimulated T cells and T cells stimulated with different monocyte-derived DC populations. In all experiments (n = 6 individual donors), bars
depict means 6 SEM. Geo-MFI, geometric mean ?uorescence intensity; CI, con?dence interval; neg, negative; ns, not signi?cant. *P , 0.05
(95% CI), **P , 0.01 (99% CI). Representative examples of the combined data are provided in Supplemental Fig. 3.
patients produced signi?cantly less IL-12p70 (P , 0.01) and IFN-a
(P , 0.05) (Fig. 5C). Signi?cant differences were not observed in
IL-10 production of monocytes between those of patients with
NHL and those from healthy controls (Fig. 5C).
ABIN1/TNIP1 regulates the phenotype and function of
monocytes from patients with NHL
Monocytes obtained from healthy controls after LPS stimulation
signi?cantly increased their expression of CD80 (P , 0.01),
CD86 (P , 0.05), HLA-DR (P , 0.05), IL-12p70 (P , 0.05), and
IFN-a (P , 0.05) (Fig. 6A and B). Monocytes obtained from
patients with NHL after LPS stimulation did not signi?cantly
increase expression of CD80, CD86, HLA-DR, IL-12p70, or IFN-a
possibly because of elevated levels of ABIN1/TNIP1. siRNA742
Journal of Leukocyte Biology
Volume 100, October 2016
mediated reduction in ABIN1/TNIP1 expression levels and LPS
stimulation produced statistically signi?cant increases in expression levels of CD80 (P , 0.05), CD86 (P , 0.01), and HLADR (P , 0.05) in the monocytes isolated from patients with
NHL (Fig. 6A). Additionally, signi?cant increases (P , 0.05) in
production of IL-12p70 and IFN-a were observed in monocytes
of patients with NHL, after stimulation with LPS, and reduction
in ABIN1/TNIP1 levels. No signi?cant changes were observed
for IL-10 (Fig. 6B).
DISCUSSION
The plasticity of DCs, with respect to its evolving phenotype or
function as an initiator of immune responses or an inducer of
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Khanolkar et al. LILRB1 control of dendritic cell function
Figure 5. Monocytes from patients with lymphoma express higher levels of intracellular ABIN1/TNIP1 ex vivo, poorly express cell-surface antigen
presentation and costimulatory molecules, and produce limited amounts of proin?ammatory cytokines. (A, left panel) Monocytes obtained from
patients with NHL expressed ABIN1/TNIP1 at signi?cantly (P , 0.01) higher levels than did monocytes obtained from healthy controls. (A, right
panel) ABIN1/TNIP1 levels in patient monocytes could be signi?cantly reduced following treatment with ABIN1 siRNA. Patient monocytes had
lower cell-surface levels of CD80 (P , 0.01), CD86 (ns), and HLA-DR (P , 0.001) (B) and produced signi?cantly less IFN-a (P , 0.05) and IL12p70 (P , 0.01) (C). Results for IL-10 were not signi?cant. (B and C) Black bars indicate samples were stimulated with 1 ng/ml LPS, whereas
white bars are unstimulated samples. In all experiments (n = 12 individual donors), bars depict means 6 SEM. Geo-MFI, geometric mean
?uorescence intensity; CI, con?dence interval; HC, healthy control; neg, negative; ns, not signi?cant. *P , 0.05, **P , 0.01 at 95% CI,
***P , 0.001 at 99.9% CI. Representative examples of the combined data are provided in Supplemental Fig. 4.
tolerance, has made it an attractive cellular target for immunotherapy in cancer and autoimmunity. A key component of DC
activation and maturation, after pattern recognition, is activation
of the transcription factor NF-kB, which is released from a
complex, regulated control system in the cytoplasm and relocates
to the nucleus, where it initiates the expression of hundreds of
genes encoding proin?ammatory proteins. Inhibition of canonical NF-kB signaling has been shown to reduce the functionality
of both CD34+ myeloid DCs and monocyte-derived DCs by
regulating the survival, differentiation, and maturation of both
DC populations [2, 14, 15].
We have demonstrated higher expression levels of the NF-kB
regulator ABIN1/TNIP1 in DCs in which the HLA class I–speci?c
inhibitory receptor LILRB1 has been ligated during in vitro
differentiation from PBMCs. We have con?rmed previous
?ndings that LILRB1 DCs retain surface expression of CD14 after
their in vitro differentiation from monocytes [4]. In this study, we
showed that modulating the expression levels of the ABIN1/
TNIP1 protein reversed the functional effects of LILRB1 ligation,
allowing the monocyte-derived DCs to partially up-regulate cell
surface expression of antigen-presenting and costimulatory
molecules, to produce cytokines, and to stimulate T cell
activation in response to challenge with LPS. The reduction of
ABIN1/TNIP1 in LILRB1-ligated, monocyte-derived DCs leave
the resultant DC population with a phenotype that bares
resemblance to the “semimature” DC population that is
extensively reviewed in the following study [16]. Our results
suggest ABIN1/TNIP1 is a potential regulator of monocytederived DC activation, and the increased ABIN1/TNIP1 expression after self-HLA recognition by LILRB1 is a mechanism
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Volume 100, October 2016
Journal of Leukocyte Biology 743
Figure 6. Reducing expression of ABIN1/TNIP1 in monocytes from patients with lymphoma restores cell-surface expression of CD80 and HLA-DR
and signi?cantly increases expression of IL-12p70 and IFN-a. (A) Lowering ABIN1/TNIP1 expression resulted in signi?cantly increased expression
of CD80 (P , 0.05), CD86 (P , 0.01), and HLA-DR (P , 0.05) in the monocytes of patients with lymphoma after stimulation with LPS. (B) After
ABIN1/TNIP1 knockdown and LPS stimulation, the monocytes of patients with NHL produced signi?cantly greater amounts of IL-12p70 (P , 0.05)
and IFN-a (P , 0.05), whereas results for IL-10 were not signi?cant. Black bars indicate samples were stimulated with 1 ng/ml LPS, whereas white
bars are unstimulated samples. In all experiments (n = 12 individual donors), bars depict means 6 SEM. Geo-MFI, geometric mean ?uorescence
intensity; CI, con?dence interval; HC, healthy control; neg, negative; ns, not signi?cant. *P , 0.05, **P , 0.01, ***P , 0.001 at 95% CI.
Representative examples of the combined data are provided in Supplemental Fig. 4.
establishing a threshold for NF-kB translocation and DC
maturation in response to triggering of pattern recognition
receptors. The increased production of IFN-a we detected is
consistent with a report of ABIN1/TNIP1 involvement in
regulating cellular antiviral responses [17].
ABIN1/TNIP1 functions in cooperation with the ubiquitinsensing regulatory protein TNFAIP3 (TNF-a–induced protein 3),
also known as A20, to regulate the ubiquitination of the NF-kB
inhibitory protein Ik B kinase-g [18], thus controlling its
proteasomal degradation. ABIN1/TNIP1 also