758 Annika E. Langeneckert et al. DOI: 10.1002/eji.201847965 Eur. J. Immunol. 2019.

49: 758–769
Clinical

Allergy and inflammation

Research Article
CCL21-expression and accumulation of CCR7+ NK cells
in livers of patients with primary sclerosing cholangitis
Annika E. Langeneckert1 , Sebastian Lunemann1 , Glòria Martrus1 ,
Wilhelm Salzberger1 , Leonard U. Hess1 , Annerose E. Ziegler1 ,
Tobias Poch2 , Gevitha Ravichandran3 , Urte Matschl1 , Jens B. Bosse4 ,
Gisa Tiegs3 , Lutz Fischer5 , Martina Koch8 , Johannes Herkel2 ,
Karl J. Oldhafer6 , Christoph Schramm2,7 and Marcus Altfeld1

1
Research Department of Virus Immunology, Heinrich Pette Institute, Hamburg, Germany
2
I. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg,
Germany
3
Institute for Experimental Immunology and Hepatology, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
4
Research Department of Structural Cell Biology of Viruses, Heinrich Pette Institute,
Hamburg, Germany
5
Department of Hepatobiliary Surgery and Transplant Surgery, University Medical Center
Hamburg-Eppendorf, Hamburg, Germany
6
Department of General and Abdominal Surgery, Asklepios Hospital Barmbek, Semmelweis
University of Medicine Hamburg, Germany
7
Martin Zeitz Center for Rare Diseases, University Medical Center Hamburg-Eppendorf,
Hamburg, Germany
8
Department for General, Visceral and Transplant Surgery, University Hospital Mainz,
Germany

The pathogenesis of primary sclerosing cholangitis (PSC), an autoimmune liver disease,

remains unknown. The aim of this study was to characterize peripheral blood and intra-
hepatic NK cells from patients with PSC. Peripheral blood samples from patients with
PSC, other autoimmune liver diseases, and from healthy control individuals were used,
as well as liver tissues from PSC patients undergoing liver transplantation. Multipa-
rameter flow cytometry showed that peripheral blood NK cells from PSC patients were
significantly enriched for CCR7+ and CXCR3+ cells, and CCR7+ but not CXCR3+ cells were
also significantly increased within intrahepatic NK cells. PSC patients undergoing liver
transplantation furthermore had significantly higher plasma levels of the CCR7-ligand
CCL21, and the CXCR3-ligands CXCL10 and CXCL11, and significantly higher levels of
CCL21, but not CXCL10, were detected in liver tissues. CCR7+ and CXCR3+ NK cells from
PSC patients exhibited significantly higher functional capacity in peripheral blood, but
not liver tissues, consistent with chronic activation of these NK cells in the inflamed
liver. These data show that PSC is characterized by intrahepatic CCL21 expression and
accumulation of CCR7+ NK cells in the inflamed liver tissue.

Correspondence: Prof. Marcus Altfeld

e-mail: marcus.altfeld@leibniz-hpi.de


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Eur. J. Immunol. 2019. 49: 758–769
Keywords: Chemokines r Innate immunity r Liver inflammation r NK cells r Primary sclerosing
cholangitis
 Additional supporting information may be found online in the Supporting Information section
at the end of the article.
Introduction
Primary sclerosing cholangitis (PSC) is a chronic liver disease char-
acterized by inflammation and fibrosis of intrahepatic and extra-
hepatic bile ducts, ultimately leading to liver cirrhosis [1]. PSC
is associated with inflammatory bowel disease in approximately
80% of patients [1–4] and a high risk for cholangiocarcinoma [5].
To date, liver transplantation represents the only effective ther-
apy [1], although, recurrent PSC occurs within 4 years after trans-
plantation in over 20% of cases [6]. The pathogenesis of PSC is
not well understood, and environmental, genetic, and immuno-
logical factors have been implicated. Genome-wide association
studies have identified PSC risk-genes associated with immune
cell function [7, 8] and infiltrates of immune cells are observed
in the proximity of bile ducts of patients with PSC [9, 10]. A dys-
regulated cross-talk between activated cholangiocytes [11–14],
and infiltrating immune cells has been implicated in the patho-
genesis of PSC [10, 15–20]. As the infiltration of liver portal
areas by immune cells is one of the characteristic features of
PSC, chemokines might play an important role in this process
[21]. However, little is known about the role of NK cells in PSC
pathogenesis.
The main functions of NK cells are the defense against
pathogens and cancers [22], as well as regulation of inflam-
matory immune responses, thereby linking innate and adaptive
immune responses [23]. NK cells are highly enriched among
lymphocytes in human liver tissues and have been suggested to
participate in balancing immune tolerance and defense against
pathogens [24–27]. Expression of HLA-Bw4 and -C2, which
serve as ligands for the inhibitory killer-cell immunoglobulin-like
receptors (KIR) KIR3DL1 and KIR2DL1, respectively, was signif-
icantly reduced in PSC patients [28]. Furthermore, MIC-A and
MIC-B, which serve as ligands for activating NK-cell receptors,
were upregulated in PSC [29, 30], suggesting that NK cells
display a more activated status. In addition, higher frequencies
of intrahepatic NK (ihNK) cells have been described in PSC
livers with lower fibrotic status [31], and suggested to have
anti-fibrotic properties by inducing apoptosis in profibrogenic
myofibroblasts [32–34]. However, the precise role of NK cells and
their interactions with other immune cells in livers affected by PSC
still remain unknown. The aim of this study was to characterize
peripheral blood NK (pNK) cells and ihNK cells from PSC patients
and compare these to NK cells derived from patients with other
inflammatory liver diseases or nondiseased control individuals, to
identify changes in the NK-cell compartment associated with PSC
pathogenesis.

C 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Allergy and inflammation 759
Results
Increased frequencies of CXCR3+ and CCR7+ NK cells
in peripheral blood of patients with PSC
Earlier studies have indicated a role of chemokine receptors CCR7
and CXCR3 in the T-cell infiltration of inflamed liver tissues in
PSC and primary biliary cirrhosis (PBC) patients [35, 36]. How-
ever, only few studies have investigated the role of NK cells,
which are highly enriched within human liver-resident lympho-
cytes. We characterized NK cell populations derived from PBMCs
from patients with PSC, PBC, or AIH (autoimmune hepatitis),
and from healthy control individuals using multi-parameter flow
cytometry. Gating strategies to identify NK cell subpopulations
and expression of surface molecules on NK cells are shown in Sup-
porting Information Fig. 1. Overall frequencies of peripheral blood
natural killer cells (pNK) cells was similar between PSC patients
and healthy control individuals (Fig. 1A), while frequencies of
CD56bright CD16dim pNK cells were higher in PSC patients com-
pared to healthy control individuals (Fig. 1B, p = 0.02) and PBC
patients (p = 0.05). CD56dim CD16bright pNK cells were decreased
in PSC patients compared to healthy control individuals (Fig. 1B,
p = 0.01). Additionally tested markers expressed on the surface of
NK cells did not show any significant differences between patient
groups, with the exception of NKp44, which was less expressed
on pNK cells of PSC patients (Supporting Information Fig. 2,
p = 0.004) and NKp46, which was more highly expressed on ihNK
cells of PSC patients (Supporting Information Fig. 2, p = 0.01).
A t-distributed stochastic neighbor embedding (t-SNE) analysis of
high-dimensional data generated by multi-parameter flow cytom-
etry showed higher expression of the chemokine receptors CCR7
and CXCR3 on pNK cells of PSC patients compared to healthy
controls (Fig. 1C). Quantitative analysis of multi-parameter flow
cytometry data demonstrated a significantly higher proportion of
pNK cells expressing CCR7 and CXCR3 (Fig. 1D, p = 0.001 and
p = 0.003), as well as higher expression levels (MFI) of CCR7
and CXCR3 (Fig. 1D, p = 0.04 and p = 0.02), in PSC patients
compared to healthy control individuals. Also, a tendency toward
higher proportions of CXCR3+ pNK cells in PSC patients when
compared to PBC patients (p = 0.08) and to AIH patients (p
= 0.07) was observed. A more detailed boolean-gating analysis
showed a significantly higher frequency of pNK cells coexpressing
CCR7 and CXCR3 (Fig. 1E, p = 0.0003) and a significantly lower
frequency of CCR7− CXCR3− NK cells (Fig. 1E, p = 0.007) in indi-
viduals with PSC. Altogether, a phenotype of CCR7+ and CXCR3+
pNK cells was observed in peripheral blood from PSC patients.
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760 Annika E. Langeneckert et al. Eur. J. Immunol. 2019. 49: 758–769

Figure 1. Comparison of pNK cells from healthy control individuals, PSC, PBC, and AIH patients using flow cytometry. (A) NK cell frequencies
in peripheral blood from healthy control individuals, PSC, PBC, and AIH patients. (B) CD56bright and CD56dim distribution in pNK cells of healthy
control individuals, PSC, PBC, and AIH patients. (C) PMBCs from each group were gated for NK cells and concatenated. t-SNE plots show density and
the expression of CD56, CD16, CCR7, and CXCR3 on NK cells from healthy control individuals, PSC, PBC, and AIH patients. Color coding indicates
the density and marker expression. (D) Quantitative analysis of flow cytometry data of pNK cells expressing CCR7 and CXCR3 and the expression
levels on CCR7+ and CXCR3+ NK cells. (E) Boolean-gating showing all possible combinations of marker coexpression of CCR7 and CXCR3 on pNK
cells from healthy control individuals and PSC patients. For graphs (A–E): Data were pooled from five independent experiments with a maximum
of two donor samples for each group measured in one experiment; Mann–Whitney test was used for all statistical analyses and a p-value <0.05
was considered statistically significant; white circles: control (n = 10); black circles: PSC (n = 10); dark gray triangles: PBC (n = 10); light gray hexagon
AIH (n = 9); red lines indicate the median.


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Eur. J. Immunol. 2019. 49: 758–769
Liver-derived NK cells from PSC patients exhibited
higher expression of tissue-residency markers
To determine whether changes in peripheral blood NK cell sub-
sets were reflective of changes in ihNK cells, liver tissue from
PSC patients undergoing liver transplantation and nontumorous
liver tissue from patients with liver metastases were analyzed
using flow cytometry. Total ihNK cell frequencies and distribu-
tions of CD56bright CD16dim and CD56dim CD16bright NK cells did
not significantly differ between PSC patients and nondiseased con-
trols (Supporting Information Fig. 3A–C). Further comparisons to
liver samples derived from individuals with alcoholic liver disease
(ALD) and assessment of the influence of ulcerative colitis (UC)
as well as of treatment with ursodeoxycholic acid (UDCA) within
PSC patients did not show significant differences within NK cell
populations (Supporting Information Fig. 3A–C). Comparing NK
cells derived from peripheral blood and intrahepatic tissues using
t-SNE analysis, we observed that tissue-residency markers were
expressed to a higher extend on liver-derived NK cells (Fig. 2A),
in line with previous data [37–39]. Quantitative analysis revealed
a significantly higher expression of CD16 (Fig. 2B, p = 0.02), of
the tissue-residency markers CD49a and CD69 (Fig. 2B, CD49a:
p = 0.01; CD69: p = 0.03), and a trend toward higher CXCR6-
expression (Fig. 2B, p = 0.06) on ihNK cells derived from PSC
patients. Furthermore, ihNK cells also contained a significantly
higher frequency of CD49a+ cells in PSC patients compared to
control individuals (Fig. 2B, p = 0.01). Boolean-gating of tissue-
residency markers expressed on ihNK cells showed a significantly
decreased population of NK cells lacking tissue residency markers
(CD49− CD69− CXCR6− ) in PSC (Fig. 2C, p = 0.04). In summary,
ihNK cells showed a significantly higher expression of liver res-
idency markers CD49a, CD69, and CXCR6 when comparing NK
cells from PSC and from nondiseased control livers.
IhNK cells from PSC patients exhibited higher
expression of the chemokine receptor CCR7
IhNK cells from PSC patients were subsequently compared
to nondiseased control individuals to assess whether higher
chemokine-receptor expression observed on pNK cells from PSC
patients also occurred in liver tissues. Similar to peripheral blood,
ihNK cells from PSC patients were significantly enriched for CCR7+
NK cells (Fig. 3A and B, p = 0.009) although the MFI for CCR7 was
similar in both groups (Fig. 3B). Interestingly, while the frequency
of CXCR3+ ihNK cells did not differ between PSC patients and
nondiseased control individuals (Fig. 3B, p = 0.4), CXCR3 expres-
sion levels were significantly decreased in PSC (Fig. 3B, p = 0.01).
In addition, the frequencies of CCR7+ and CXCR3+ ihNK cells were
also significantly higher in PSC patients compared to patients with
ALD (Supporting Information Fig. 3D, p = 0.02 and p = 0.02).
No influence on CCR7 and CXCR3 expression was observed by the
presence of UC or treatment with UDCA within PSC patients (Sup-
porting Information Fig. 3D). Boolean-gating revealed an increase
of single CCR7+ ihNK cells (Fig. 3C, p = 0.002) and a decrease

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Allergy and inflammation 761
in single CXCR3+ ihNK cells in PSC patients (Fig. 3C, p = 0.03).
Of note, CCR7- as well as CXCR3-expression on ihNK cells did not
differ between CD56bright CD16dim and CD56dim CD16bright NK cell
subsets (Supporting Information Fig. 3E).
To assess possible functional differences between CCR7+ and
CXCR3+ NK cells in PSC patients, we quantified degranulation
(CD107a expression) and intracellular cytokine production of NK
cells following stimulation with K562 cells [40]. In peripheral
blood of PSC patients, CCR7+ and CXCR3+ NK cells showed a
significantly higher proportion of CD107a+ cells following stim-
ulation with K562 cells compared to CCR7− and CXCR3− NK
cells (Fig. 3D and E, p = 0.02, p = 0.007). In contrast, intra-
hepatic CCR7+ and CXCR3+ NK cells from PSC patients exhibited
a slightly lower proportion of CD107a+ cells compared to intra-
hepatic CCR7− and CXCR3− NK cells (Fig. 3D and E, p = 0.12,
p = 0.06). Furthermore, CXCR3− ihNK cells exhibited higher fre-
quencies of IFNγ+ cells compared to CXCR3+ ihNK cells (Fig.
3E, p = 0.06), while no differences were observed for CCR7+
NK cell subsets (Fig. 3D and E). No significant differences were
observed in the production of TNFα between NK cell subsets (Fig.
3D and E), or in the functional capacities of CCR7+ /CXCR3+
and CCR7− /CXCR3− pNK cell and ihNK cell subsets from other
autoimmune diseases (Supporting Information Fig. 4). Finally,
assessment of ADCC capability did not show any significant dif-
ferences between CCR7+ /CXCR3+ and CCR7− /CXCR3− NK cells
from PSC patients, or between NK cells derived from PSC patients
and the other study groups (p > 0.05 for all comparisons, data not
shown). Taken together, ihNK cells from PSC patients exhibited
higher proportions of CCR7+ NK cells, but lower levels of CXCR3-
expression compared to nondiseased controls, and CCR7+ and
CXCR3+ NK cells derived from peripheral blood of PSC patients
showed increased degranulation.
Increased plasma levels of CCL21, CXCL10, and
CXCL11, and increased intrahepatic levels of CCL21 in
PSC patients
To determine whether changes in chemokines levels were associ-
ated with observed changes in chemokine-receptor expression on
NK cells, we quantified levels of CCL21, CXCL10, and CXCL11 in
plasma samples of PSC patients, late stage PSC patients receiving
liver transplantation (lsPSC), PBC patients and AIH patients, as
well as healthy control individuals. Significant changes in plasma
chemokine levels were observed for CCL21 between lsPSC patients
and healthy control individuals, and also between lsPSC patients
and AIH patients (Fig. 4A, p = 0.03 and p = 0.02). CXCL10 and
CXCL11 plasma levels were furthermore significantly elevated in
patients with early-stage PSC (Fig. 4A, p = 0.02 and p = 0.001)
and lsPSC (Fig. 4A, p = 0.003 and p = 0.04) compared to healthy
control individuals. These data demonstrate that plasma levels of
ligands for the chemokine receptors CCR7 and CXCR3 are elevated
in patients at early and late stages of PSC disease. To investigate
whether local chemokine expression in liver tissues might account
for these observations, immunofluorescence staining of liver
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762 Annika E. Langeneckert et al. Eur. J. Immunol. 2019. 49: 758–769

Figure 2. Tissue-residency marker expression in pNK cells and ihNK cells. (A) t-SNE Plots from one representative PSC patient and one represen-
tative nondiseased control show density and expression of CD56, CD16, CD49a, CD69, and CXCR6 on pNK cells and ihNK cells. (B) Quantitative
analysis of flow cytometry data from surface markers CD16, CD49a, CD69, and CXCR6 on ihNK cells from PSC patients and nondiseased controls.
(C) Results of boolean-gating represented in pie charts showing all possible combinations of marker coexpression of CD49a, CD69, and CXCR6 on
ihNK cells from PSC patients and nondiseased controls. For graphs B and C: data were pooled from five independent experiments in which two
donor samples for each group were measured in one experiment; medians were used for boolean-gating analyses; Mann–Whitney test was used
for statistical analyses and a p-value <0.05 was considered statistically significant; white circles: control (n = 10); black circles: PSC (n = 10); red
lines indicate the median.


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Eur. J. Immunol. 2019. 49: 758–769 Allergy and inflammation 763

Figure 3. Chemokine receptor expression on ihNK cells from nondiseased controls and PSC patients. (A) t-SNE plots from one representative
individual showing density and expression of CCR7 and CXCR3 on ihNK cells. (B) Proportions of CCR7+ and CXCR3+ ihNK cells and MFI of CCR7+
and CXCR3+ NK cells from PSC patients and nondiseased controls. (C) Boolean-gating of CCR7 and CXCR3 on ihNK cells. For graphs (A–C): Data were
pooled from five independent experiments in which two donor samples per group were measured in one experiment; Mann–Whitney test was
used for statistical analyses and a p-value <0.05 was considered statistically significant; White circles: nondiseased control (n = 10); black circles:
PSC (n = 10); Red lines indicate the median. Plots representing CD107a, IFNγ, and TNFα expression in CCR7+ (red) and CCR7− (black) pNK cells and
ihNK cells (D) or in CXCR3+ (red) and CXCR3− (black) pNK cells and ihNK cells (E) from PSC patients upon coincubation with K562 cells. For graphs
(D and E): pNK cells (n = 8); ihNK cells (n = 5); data were pooled from two independent experiments with four donor samples per group measured
in one experiment for pNK cells in graph D; for ihNK cells in graph E, data were pooled from two independent experiments with maximum of
three samples per group measured in one experiment; cells incubated without target cells were used as controls; values were normalized to
unstimulated control; Wilcoxon signed rank test was used for statistical analyses and a p-value <0.05 was considered as statistically significant.


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764 Annika E. Langeneckert et al. Eur. J. Immunol. 2019. 49: 758–769

Figure 4. Chemokine concentration levels in plasma, CCL21, and CXCL10 expression in liver sections and migration toward CCL21. (A) Plasma
concentration level of CCL21, CXCL10, and CXCL11 from nondiseased controls, PSC patients, lsPSC patients, PBC patients, and AIH patients was
determined by a Luminex assay. White circles: nondiseased control (n = 8); black circles: PSC (n = 8); gray squares: lsPSC (n = 8); dark gray triangles:
PBC (n = 7); light gray hexagon: AIH (n = 7). Red lines indicate the median. Mann–Whitney test was used for statistical analyses and a p-value
<0.05 was considered as statistically significant. Data were pooled from one experiment with two technical replicates per donor measured in one
experiment. (B) Dual immunostaining of cryosections from one representative PSC patient and one representative nondiseased control individual
with anti-CCL21, donkey anti-goat NL557-conjugated antibody and DAPI. Plot shows quantitative analysis of CCL21 expression in % of total cells.
(C) Dual immunostaining of cryosections from one PSC patient and one nondiseased control individual with anti-CXCL10, donkey anti-goat NL557-
conjugated antibody, and DAPI. Plot shows quantitative analysis of CXCL10 expression in % of total cells. For B and C: scale bar is set to 30 μm;
a Nikon Ti-E with Yokogawa W1 Spinning Disk, an Andor 888 camera, an objective 100 × 1.49 NA and Nikon NIS software was used for analysis;
further analyses were assessed using Fiji software; data were pooled from three different experiments using three or more independent images
per donor; for the analysis of assays using replicate images linear-mixed effect regression models with a random intercept were used to take into
account the intraparticipant correlation of the repeated measurements; a p-value <0.05 was considered as statistically significant. (D) Quantitative
analysis of flow cytometry data from transmigration assays using isolated pNK cells from control individuals (n = 5, white circles) and PSC patients
(n = 5, black circles) migrating toward medium containing CCL21. RP10 medium without CCL21 served as control. Wilcoxon signed rank test was
used for statistical analyses. A p-value <0.05 was considered as statistically significant. Data were pooled from three independent experiments
with a maximum of two measured samples in one group and two technical replicates per donor measured in one experiment.


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Eur. J. Immunol. 2019. 49: 758–769
tissue cryosections from PSC patients and nondiseased control
individuals was performed using CCL21 and CXCL10 antibodies.
As shown in Fig. 4B, the CCR7-ligand CCL21 was significantly
higher expressed in liver sections from PSC patients compared
to nondiseased control livers (p = 0.006), while CXCL10 was
expressed to a slightly lower extend on liver sections from PSC
patients (Fig. 4C, p = 0.33). To begin to assess whether elevated
CCL21 expression in liver tissues might attract CCR7+ NK cells
to the liver, we performed a NK cell migration assay using trans-
well experiments. NK cells isolated from PBMCs from PSC patients
showed enhanced migration toward the chemokine CCL21 com-
pared to control medium without CCL21 (Fig. 4D, p = 0.06), while
there was no difference in the migration of NK cells from control
individuals (Fig. 4D, p = 0.19). Taken together, these data sug-
gest that local production of the CCR7-ligand CCL21 may result in
recruitment of CCR7+ NK cells into liver tissues of patients with
PSC.
Discussion
PSC is a chronic progressive inflammatory disease characterized
by the infiltration of liver portal areas with immune cells [15, 17,
20]. Previous studies have shown an increased expression of the
chemokine receptors CCR7 and CXCR3 on peripheral blood and
intrahepatic T cells during PSC and PBC [21, 35], as well as an
accumulation of the CCR7 ligand CCL21 within portal areas of PSC
livers [21] and the CXCR3 ligands CXCL9 and CXCL10 in portal
tracts of PBC livers [35]. NK cells express chemokine receptors
that can attract them to sites of tissue inflammation, but the role
of NK cells in the pathogenesis of PSC remains unknown. Here, we
describe higher proportions of CCR7+ NK cells in peripheral blood
and livers of PSC patients that were associated with elevated levels
of the CCR7-ligand CCL21, suggesting that CCL21-expression and
infiltration of CCR7+ NK cells might contribute to progressive liver
inflammation observed in PSC.
Several studies of chronic progressive liver inflammation
have assessed changes in lymphocyte populations in periph-
eral blood of PSC patients, and shown elevated T-cell num-
bers [21] and increased NK cell frequencies [17, 41]. Com-
paring pNK cells between healthy control individuals and indi-
viduals with PSC, PBC, and AIH, we observed a significantly
increased CD56bright CD16dim pNK cell population in PSC patients,
but no changes in overall NK cell numbers. This might be due
to increased inflammation, as previously suggested [42–45]. PSC
patients exhibited higher frequencies of CCR7+ and CXCR3+ NK
cells in the peripheral blood, and higher surface expression of
these two chemokine receptors. Our data are in line with previous
studies demonstrating an increased expression of these chemokine
receptors on CD4+ T cells of PSC patients [21, 35]. CCR7 is mainly
expressed on CD56bright CD16dim NK cells, enabling these cells to
respond to CCL21 [45], and has been shown to be required for the
trafficking to lymph nodes [45]. CXCR3 serves as a receptor for
several inflammatory chemokines (CXCL9, CXCL10, and CXCL11)
and has been described to be upregulated in inflammatory liver

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Allergy and inflammation 765
diseases [46]. While the cross-sectional design of our study did
not allow us to determine whether CCR7- and CXCR3-expression
on NK cells occurred before or as a consequence of liver inflam-
mation, these data suggest a role of these chemokine receptors in
the progressive pathogenesis of PSC.
Studies of peripheral blood immune cells provide limited infor-
mation into tissue-specific inflammatory processes. Only few stud-
ies have characterized ihNK cells in PSC. Berglin et al. observed
scattered NK cells preferentially located within the parenchyma
distant from fibrotic areas of PSC livers using immunohistochem-
istry, and no increased ihNK cell numbers compared to normal
controls [31]. In contrast, another study showed lower numbers
of ihNK cells in PSC [17]. In this study, we observed no significant
differences in ihNK cell numbers and subsets (CD56bright CD16dim ,
CD56dim CD16bright ) between PSC and nondiseased control individ-
uals using multi-parameter flow cytometry, but observed higher
frequencies of liver-resident NK cells in PSC livers. While the use
of nontumorous liver tissue from liver resections due to hepatic
metastases as nondiseased controls has caveats, as liver-resident
lymphocyte populations can be affected by the underlying diseases
and treatments, the accumulation of NK cells expressing markers
of liver residency appears to be a common feature of inflammatory
liver diseases [38, 47, 48]. We furthermore observed a significant
higher frequency of CCR7+ NK cells in livers, but not higher fre-
quencies of CXCR3+ NK cells. In contrast to peripheral blood,
CXCR3-expression on NK cells in PSC livers was reduced, possibly
suggesting a ligand-induced downregulation of this receptor. As
CXCR3 is essential for cellular trans-migration from hepatic sinu-
soids into the liver parenchyma [49], expression of CXCR3 might
be expendable for ihNK cells. Although the expression of CCR7 has
been implicated into the cellular egress from tissue into lymphatic
vessels [50], CCR7+ NK cell egress from livers of PSC patients
might be prevented due to expression of CCL21 within hepatic
portal fields. Taken together, these data suggest that expression
of liver-homing receptors and chemokine receptors contributes to
the accumulation of CCR7+ ihNK cells in PSC patients.
Chemokines play a critical role in the recruitment of lympho-
cytes to inflamed tissues. We detected increased plasma levels of
CCL21 (CCR7-ligand) in PSC patients compared to healthy con-
trol individuals and patients with other autoimmune liver dis-
eases, suggesting specific upregulation of this chemokine during
late stages of PSC disease. CCL21 is usually expressed on high
endothelial venules that have been observed in inflammatory liver
diseases like PSC, and a subset of CD11c+ cells in portal tracts of
PSC livers have been identified as a major source of CCL21 [21].
Previous studies showed that isolated blood lymphocytes and
intrahepatic lymphocytes (IHLs) from PSC patients responded to
CCL21 in micro chemotaxis chamber assays in vitro, but these
studies used total lymphocyte populations [21]. In this study, we
observed that isolated pNK cells from PSC patients but not from
healthy control individuals exhibit a trend toward higher migra-
tion toward CCL21. We furthermore observed that CCL21 was
significantly higher expressed in liver sections from PSC patients
compared to nondiseased control livers, in line with a previous
study in PSC [21], further supporting a role of this chemokine in
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766 Annika E. Langeneckert et al.
NK cell recruitment during PSC. Ligands for CXCR3 have also been
shown to be overexpressed in inflammatory liver diseases, which
can lead to an infiltration of CXCR3-expressing cells [35, 36]. We
indeed detected significantly elevated plasma levels of the CXCR3-
ligands CXCL10 and CXCL11 in PSC patients. However, we did
not detect elevated CXCL10-expression in PSC livers, but rather
lower expression than in nondiseased livers. We cannot exclude
that other CXCR3-ligands, such as CXCL11 or CXCL9, might have
been upregulated in liver tissues and triggered the downregula-
tion of CXCR3-expression observed on liver-resident NK cells in
PSC livers.
Very little is known regarding the functional capacity of NK
cells in inflammatory liver diseases. We analyzed the ability of
pNK cells and ihNK cells to degranulate following stimulation with
MHC-devoid K562 cells. Peripheral blood CCR7+ and CXCR3+
NK cells from PSC patients both exhibited higher proportions of
CD107a+ NK cells, suggesting that CCR7+ and CXCR3+ NK cells
might contribute to the inflammation observed during PSC. Fur-
thermore, comparing CCR7+ /CXCR3+ and CCR7− /CXCR3− pNK
cells and ihNK cells from patients with AIH and PBC for their
ability to degranulate and to produce cytokines did not show any
significant differences, indicating that the differences observed
in NK cell function were most pronounced in patients with PSC.
In contrast to these observation in peripheral blood, CCR7+ and
CXCR3+ ihNK cells exhibited less activity against K562 cells than
CCR7− and CXCR3− NK cells. These data are in line with a previ-
ous study reporting no cytotoxicity of NK cells against K562 cells
in PSC [17]. The inability of intrahepatic CCR7+ NK cells as well
as the overall ihNK cell population from PSC patients to strongly
react to additional target cell stimulation might be due to increased
baseline activation, as we observed a higher baseline of intracellu-
lar cytokine levels in CCR7+ ihNK cells compared to CCR7− ihNK
cells and also CCR7+ pNK cells. Additional studies will be required
to determine whether interventions targeting these chemokine-
receptor-expressing NK cells accumulating in inflamed livers might
help to prevent the progressive inflammation that is a character-
istic of PSC. Taken together, our data suggest liver recruitment of
highly functional CCR7+ NK cells associated with elevated CCL21-
expression within PSC livers, indicating that T and NK cells might
play in concert during inflammatory processes that lead to the
progressive destruction of the bile ducts. These data provide a
rational for the targeting of lymphocyte trafficking and homing
into inflamed liver tissues as a strategy to disrupt the detrimental
inflammatory cycle that is characteristic of PSC pathogenesis.
Materials and methods
Patient samples
A total of 75 study subjects were included in this study. Peripheral
blood samples were derived from individuals diagnosed with PSC
(n = 10), PBC (n = 10), and AIH (n = 9) recruited at the Uni-
versity Medical Center Hamburg-Eppendorf (UKE), and healthy

C 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Immunol. 2019. 49: 758–769
control donors enrolled into the Hamburger Gesundkohorte (n
= 10). Liver tissue samples were collected from patients under-
going liver transplantation in the Department of Hepatobiliary
and Transplant Surgery of the UKE (PSC n = 10; ALD n = 11;
PBC n = 2; AIH n = 2 and PBC/AIH overlap n = 1) or individ-
uals undergoing liver resection due to liver tumor metastases in
the Department of General and Visceral Surgery at the Asklepios
Clinic Hamburg-Barmbek (nondiseased n = 10). Characteristics
of study participants are summarized in Supporting Information
Table 1 and Supporting Information Table 2.
Ethics
All study subjects provided informed written consent according to
study protocols approved by the Ärztekammer Hamburg (PV4898,
PV4081, PV4780).
Isolation of lymphocytes from peripheral blood and
liver tissue
PMBCs were isolated from peripheral blood using standard den-
sity gradient centrifugation (Ficoll, Biochrom GmbH, Berlin,
Germany). Cells were cryopreserved in 90%fetal bovine serum
(FBS)/10%DMSO and stored in liquid nitrogen. IHLs were
isolated as previously described [51] and cryopreserved in
90%FBS/10%DMSO in liquid nitrogen.
Flow cytometry
Cryopreserved PBMC and liver samples were thawed, washed with
PBS and stained with surface antibodies and live dead marker
Zombie AquaTM (BioLegend, San Diego, CA, USA) for 20 min at
RT in the dark, fixed in 4%PFA/PBS and subsequently analyzed
using a LSR FortessaTM (BD Bioscience, San Jose, CA, USA). A
list of antibodies used for staining of immune cells is provided in
Supporting Information Table 3. For flow cytometry analysis, we
adhered to the “Guidelines for the use of flow cytometry and cell
sorting in immunological studies” [52].
Immunohistochemistry and fluorescence microscopy
Liver tissues from PSC patients and patients undergoing liver
resection were snap-frozen at –80°C. Tissues were mounted and
cut into 8 μm sections at –10°C using a cryostat (CM3050, Leica,
Wetzlar, Germany). Slides were air-dried and stored at –80°C.
Liver tissue was fixed with 4%PFA/PBS for 20 min at 4°C and per-
meabilized with 0.1%TritonX100/D for 5 min at RT. Slides were
blocked with 1%donkey serum/PBS for 1 h at RT. Anti-CCL21
and anti-CXCL10 were applied as first antibodies overnight at
4°C. Control slides were incubated without the aforementioned
antibodies (Supporting Information Fig. 5). Secondary antibodies
www.eji-journal.eu
Eur. J. Immunol. 2019. 49: 758–769
were applied for 60 min at RT. Slides were then stained with
DAPI (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at
RT and mounted with Fluorescence Mounting Medium (Agilent,
Santa Clara, CA, USA). Samples were visualized using fluores-
cence microscopy (Nikon Ti-E with Yokogawa W1 Spinning Disk,
Andor 888 camera, objective 100 × 1.49 NA, Nikon NIS software).
Chemokine quantification in plasma samples
Plasma samples from PSC, PBC, and AIH patients as well as healthy
control donors and PSC patients undergoing liver transplantation
(late stage PSC) were stored at –80°C. Plasma concentrations of
CCL21, CXCL10, and CXCL11 were quantified using a Bio-Plex Pro
Human Cytokine Kit, a Bio-Plex200 analyzer, and Bioplex Analysis
Software (Biorad Laboratories, Herkules, CA, USA).
NK cell degranulation and ADCC assay
Cryopreserved PBMCs (n = 8) and IHLs (n = 5) from PSC patients
were thawed and rested overnight in R10 supplemented with
5 ng/mL IL-15. Cells were cocultured with K562 cells (DSMZ)
and Raji cells (in the presence of Rituximab, Invivogen, Toulouse,
France) at an effector to target (E:T) ratio of 5:1 in the presence
of anti-CD107a, as previously described [40]. After 1 h incuba-
tion at 37°C, Brefeldin A (Sigma-Aldrich, Taufkirchen, Germany)
and Monensin (BD Bioscience, San Jose, CA, USA) were added
followed by four additional hours of incubation. Cells were sub-
sequently stained with surface antibodies and live-dead marker
Zombie AquaTM (Biolegend, San Diego, CA, USA) for 20 min at
RT. For intracellular cytokine staining a Cytofix/Cytoperm kit (BD
Bioscience, San Jose, CA, USA), anti-IFNγ and anti-TNFα (Biole-
gend, San Diego, CA, USA) was used. Samples were then analyzed
by flow cytometry.
NK cell migration assay
PBMCs from control individuals and PSC patients were thawed
and cultured with IL15 (5 ng/mL) in RPMI medium with 10%
FCS for 2 h at 37°C. NK cells were isolated and migration toward
the chemokine CCL21 (10 ng/mL) was assessed using a transwell
system with a 5.0 μm pore polycarbonate membrane (Fisher Sci-
entific GmbH, Schwerte, Germany). NK cells with a concentration
of 6,7 cells/mL in RP10 were incubated for 4 h at 37°C and the
amount of migrated cells was determined using CountBrightTM
Absolute Counting Beads (Fisher Scientific GmbH, Schwerte,
Germany).
Statistical analyses
Flow cytometry data were analyzed using FlowJoTM Version
10.4.2. (Ashland, OR, USA). Additional visualization of flow

C 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Allergy and inflammation 767
cytometry data was done using viSNE (Cytobank, Inc., Santa Clara,
CA, USA). Plots were generated and statistical comparisons per-
formed in Prism7 (GraphPad Sofware, La Jolla, CA, USA). For
comparisons between two groups Mann–Whitney test was used.
For comparisons between two paired groups Wilcoxon signed rank
test was used. For all tests, p < 0.05 was considered statistically
significant. Significant and close to significant p values are shown
in all figures. Medians are shown as red lines in all Figures, in
Supporting Information Figures median is shown in black. Due
to limited sample sizes from different patient cohorts no techni-
cal or biological replicates were measured except for data in Fig.
4A and D. The analysis of fluorescence microscopy images was
carried out using Fiji software. For the analysis of assays using
replicate images, linear-mixed effect regression models with a ran-
dom intercept were used to take into account the intraparticipant
correlation of the repeated measurements.
Acknowledgments: The authors thank all the donors from the
LTX cohort at the University Clinical Center Hamburg-Eppendorf
(UKE), the AKB cohort at the Asklepios Hospital Barmbek (AKB)
and the healthy donor cohort at the Heinrich Pette Institute (HPI)
in Hamburg for their participation in our study. Furthermore, the
authors would like to thank all nurses and surgeons of the Depart-
ment of Hepatobiliary and Transplant Surgery (UKE) and of the
Department of General and Abdominal Surgery (AKB) for their
involvement. M.A., J.H., C.S., G.T., and S.L. received funding from
the SFB841 of the Deutsche Forschungsgesellschaft. C.S. is funded
by the Helmut and Hannelore Greve Foundation. A.L., M.A., C.S.,
J.H., G.T., T.P., and G.R. are supported by the CRU306 of the
Deutsche Forschungsgesellschaft. L.H. and A.Z. received financial
support from the graduate school of the SFB841. The funders had
no influence on study design, data collection, and analysis, the
decision to publish or contents of the manuscript. A.L., S.L., M.A.
designed the study. A.L., G.M., L.H., G.R., U.M., W.S., T.P., S.L.
performed experiments. A.L. analyzed the data. A.L. and M.A.
wrote the manuscript. M.K., K.O., L.F. provided patient samples.
All authors critically reviewed the manuscript.
Conflict of interest: J.B.B. is a consultant of Crescenta Bio-
sciences, NJ, USA and holds options in this company. All
other authors declare no commercial or financial conflict of
interest.
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46 Shields P. L., Morland C. M., Salmon M., Qin S., Hubscher S. G. and The peer review history for this article is available at
Adams D. H., Chemokine and chemokine receptor interactions pro- https://publons.com/publon/10.1002/eji.201847965
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Received: 11/10/2018
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Revised: 22/1/2019
47 Zhu, J., Feng, A., Sun, J., Jiang, Z., Zhang, G., Wang, K., Hu, S. et al., Accepted: 19/2/2019
Increased CD4(+) CD69(+) CD25(-) T cells in patients with hepatocellular Accepted article online: 20/2/2019


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