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Pivotal antitumor role of the immune checkpoint molecule B7-H1 in pancreatic cancer


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Immune checkpoint molecule B7-H1 plays a decisive immune regulatory role in different pathologies including cancer, and manipulation of B7-H1 expression became an attractive approach in cancer immunotherapy. Pancreatic cancer (PDAC) is characterized by pronounced immunosuppressive environment and B7-H1 expression correlates with PDAC prognosis. However, the first attempts to diminish B7-H1 expression in patients were not so successful. This points the complicity of PDAC immunosuppressive network and requires further examinations. We investigated the effect of B7-H1 deficiency in PDAC. Our results clearly show that partial or complete B7-H1 inhibition in vivo let to reduced tumor volume and improved survival of PDAC-bearing mice. This oncological benefit is due to the abrogation of immunosuppression provided by MDSC, macrophages, DC and Treg, which resulted in simultaneous restoration of anti-tumor immune response, namely improved accumulation and functionality of effector-memory CD4 and CD8 T cells. Our results underline the potential of B7-H1 molecule to control immunosuppressive network in PDAC and provide new issues for further clinical investigations.
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Pivotal antitumor role of the immune checkpoint molecule B7-H1 in pancreatic cancer
Alexandr V. Bazhin
, Katharina von Ahn
, Jasmin Fritz
, Henriette Bunge
, Caroline Maier
, Orkhan Isayev
, Florian Ne
Jens T. Siveke
, and Svetlana Karakhanova
Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg, Germany;
Department of Cytology, Embryology
and Histology, Azerbaijan Medical University, Baku, Azerbaijan;
Division of Solid Tumor Translational Oncology, German Cancer Research Center
(DKFZ) and German Cancer Consortium (DKTK), partner site University Hospital Essen, Heidelberg, Germany;
Bridge Institute of Experimental Tumor
Therapy, West German Cancer Center, University Hospital Essen, University Duisburg-Essen, Essen, Germany
Immune checkpoint molecule B7-H1 plays a decisive immune regulatory role in dierent pathologies
including cancer, and manipulation of B7-H1 expression became an attractive approach in cancer
immunotherapy. Pancreatic cancer (PDAC) is characterized by pronounced immunosuppressive environ-
ment and B7-H1 expression correlates with PDAC prognosis. However, the rst attempts to diminish B7-
H1 expression in patients were not so successful. This points the complicity of PDAC immunosuppressive
network and requires further examinations. We investigated the eect of B7-H1 deciency in PDAC. Our
results clearly show that partial or complete B7-H1 inhibition in vivo let to reduced tumor volume and
improved survival of PDAC-bearing mice. This oncological benet is due to the abrogation of immuno-
suppression provided by MDSC, macrophages, DC and Treg, which resulted in simultaneous restoration of
anti-tumor immune response, namely improved accumulation and functionality of eector-memory CD4
and CD8 T cells. Our results underline the potential of B7-H1 molecule to control immunosuppressive
network in PDAC and provide new issues for further clinical investigations.
Received 1 March 2021
Revised 10 February 2022
Accepted 10 February 2022
B7-H1 (PD-L1); B7-H1
knockout; immune
checkpoint molecules;
immunosuppression; cancer
immunotherapy; pancreatic
Immunotherapy has become nowadays a paradigm-shifting
approach for patients with advanced malignancies. Particularly,
the so-called immune checkpoint molecules (ICPM) attract an
enormous attention of scientists and clinicians due to the fact
that immunotherapy using antibody or inhibitors targeting these
molecules could demonstrate spectacular clinical responses.
These exciting results led to the approval of antibody against
cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), pro-
grammed cell death protein 1 (PD-1), and programmed cell
death-ligand 1 (PD-L1, B7-H1) for treatment of patients with
melanoma, non-small cell lung cancer, and renal cell
Unfortunately, only restricted cohorts of patients
benefit from these treatments probably due to an intricate net-
work of strong immunosuppression.
Pancreatic ductal adeno-
carcinoma (PDAC) is one of such malignancies, which have not
responded to therapies with ICPM inhibitors in pilot clinical
The regulatory cell-surface proteins of the B7-H family play
an important role in the modulation of immune responses.
B7-homologue 1 (B7-H1, CD274, PD-L1), a ligand for PD-1
receptor, was described by Dong.
B7-H1 expression has been
found on the surface of macrophages, dendritic cells (DC), and
activated T cells, B cells, endothelial, and epithelial cells.
Moreover, B7-H1 is ubiquitously present in tumor cells in
different types of cancer,
and its expression correlates with
a poor prognosis for the patient and contributes to tumor
immune evasion.
This makes B7-H1 and its regulation an
important target for ongoing investigations in the field of
cancer immunotherapy, with ICPM inhibitors.
B7-H1-deficient mice (B7-H1KO) were generated by Dong
and colleagues.
In our recent research, we deeply investigated
this mouse strain and showed that B7-H1 deficiency in vivo
modulates several immunological parameters, including the
amount and composition of Gr1
myeloid population,
the composition and activation state of the DC compartment,
the frequency and status of natural killer (NK) and NKT cells,
B cells, naïve/memory state of CD8 T cells, production of IL-2
and IL-10 cytokines, and increased PD-1 expression in the
immune cells.
All these data underline the importance of B7-
H1 as a decisive immune regulatory molecule.
PDAC is one of the deadliest cancers in the world, with
5-year survival rates of only ~1% and median survival of 4–
6 months.
The reasons for such a poor prognosis are multi-
ple, including rapid tumor dissemination, latent nonspecific
symptoms associated with a delayed diagnosis
and a highly
immunosuppressive milieu.
Immunotherapy might be con-
sidered as an attractive approach to combat PDAC.
one phase I trial using an antibody against B7-H1 did not
reveal any objective response by the patients,
pointing the
complicity of immunosuppressive network induced by PDAC.
Despite B7-H1 supposed to be one of the crucial actors in this
immunosuppressive structure, the immunological outcome of
B7-H1 deficiency in PDAC is not fully characterized yet.
CONTACT Svetlana Karakhanova Department of General, Visceral and Transplantation Surgery, University of Heidelberg, Heidelberg,
Supplemental data for this article can be accessed on the publisher’s website.
2022, VOL. 11, NO. 1, e2043037 (14 pages)
© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (, which permits
unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
In this work, we used both in vivo and ex vivo approaches to
inhibit B7-H1 and demonstrated that such inhibition indeed
reduces tumor volume and improves survival of PDAC-
bearing mice. This oncological benefit was due to the abroga-
tion of general immunosuppression and simultaneous restora-
tion of an antitumor immune response.
Materials and methods
Antibodies used are listed in the Suppl. Table 1. FoxP3 staining
Buffer Set was purchased from eBioscience. Collagenases III
and IV and Trypan Blue 0.5 % were obtained from Biochrom
AG. DNAse I was purchased from Roche. Dulbecco’s PBS
Solution (10x), HBSS Buffer, and RPMI 1640 were obtained
from PAA Laboratories GmbH. RBC Lysis Buffer (10x) was
purchased from BioLegend and Hyaluronidase by Linaris
GmbH. CD11b MicroBeads as well as Myeloid-Derived
Suppressor Cell Isolation mouse kit were purchased by
Miltenyi Biotec GmbH. Milliplex® MAP Kit, Mouse
Cytokine/Chemokine Magnetic Bead Panel, was purchased
from EMD Millipore Corporation Merck KGaA.
FluoSpheres®Carboxylate, Yellow-green (505/515) conjugated,
was obtained from Life Technologies. CFSE (5-(and6)-
Carboxyl-fluorescein diacetate succinimidyl eyter, CFDA SE)
and CD274 (B7-H1) Functional Grade Purified antibody were
purchased from eBioscience.
Two mouse strains in the age range 8–12 weeks were used. The
C57BL/6 wild-type mice were purchased by Charles River,
Sulzbach. The B7-H1KO mice were originally created by
Dong and colleagues by homolog recombination in embryonic
stem cells in a C57/Bl6 background
and kindly provided by
Dr. Linda Diehl and Dr. Percy Knolle. The mice were kept in
the animal facility of University Heidelberg (IBF, Heidelberg)
and in the Department of Neuropathology (University
Hospital, LMU Munich) under specific pathogen-free (SPF)
conditions. Homozygous B7-H1KO mice were checked for
the KO genotype stability in regular intervals. Experiments
with animals were carried out after approval by the authorities
(Regierungspraesidium Karlsruhe and Regierungspraesidium
Orthotopic mouse model
The murine PDAC model has been induced as previously
The i.p. treatment with anti-B7-H1 antibody or
IgG control (Functional Grade purified B7-H1 and IgG
control Ab, eBioscience; 150 µg/mouse in 150 µl) was
performed at day (d) 5, 8, 11, 15, 19 after cancer cell
transplantation. All in vivo primary results were obtained
from repetitive independent experiments, and “n” reflects
total amount of mice per group. Each in vivo experiment
data is shown in Suppl. Figure S1.
Preparation of tumor, spleen, and blood samples
Four weeks after the cell implantation, the mice were sacrificed
by cervical dislocation. Spleens and tumors were dissected, and
the size was determined using a slide vernier caliper. The
abdomen was examined for the presence of metastasis in the
liver, intestines, and peritoneum, as well as any adhesions or
abnormalities. The size of the metastasis was graded from + to
+++. Blood was allowed to clot and centrifuged at 600 g for
25 min. The clear serum supernatant was centrifuged at 9300 g
for 10 min and frozen at −20°C.
Preparation of a single-cell suspension
Isolated tumors were cut into small pieces and incubated in
5 ml of the collagenases digestion solution at 37°C. Tissue was
pressed through a 100 µm cell strainer and flushed with 10 ml
of PBS. The samples were centrifuged at 4°C, 400 g, 5 min, and
the supernatant was discarded. After adding 1 ml of erythro-
lysis buffer, the samples were resuspended, incubated for
2 min, and 10 ml of PBS was added to stop erythrolysis. After
another centrifugation step, the cells were resuspended in
10 ml of PBS and flowed twice through a 40 µm cell strainer.
The cell concentration was adjusted to the 2 × 10
cells/50 µl.
Immunophenotyping with FACS
The splenocytes and tumor cells were stained with different
antibody combinations in several panels. In tumor-bearing
mice, CD45 antibody was used to differentiate between tumor
cells (CD45
) and tumor-infiltrating leukocytes (CD45
). CD4/
CD8 panel: CD4/CD8 T cells as well as their subsets, naïve
T cells (CD62L
), effector T cells (CD62L
), cen-
tral memory T cells (CD62L
), and effector memory
T cells (CD62L
) were analyzed. DC panel: conventional
DC (CD11c
, cDC) and plasmacytoid DC
, pDC) were characterized, and the expres-
sion of MHC-II (I-A[b]), B7-H1, and CD80 and CD86 mole-
cules was investigated. For the analysis of suppressive cell
populations, the Treg panel and the MDSC (Myeloid-derived
suppressor cells) panel were used. Treg panel: the T regulatory
cells were gated as Foxp3
within the CD4 T-cell popula-
tion. Total MDSC were gated as CD11b
. The granulocytic
MDSC are characterized as Ly6C
and the monocytic
MDSC as Ly6C
. To test functional status of MDSC,
the expression of Arginase-1 (Arg-1) and inducible Nitric Oxide
Synthase (iNOS) was examined.
To assess phagocytosis activity of macrophages (Gr1
cells), Gr1
cells were isolated in
two MACS-isolation steps, using Gr1-negative- followed by
CD11b-positive selection. Isolated cells were incubated for
1 h at 37°C with the FluoSpheres®Carboxylate yellow-green
beads (Life Technology), at a concentration of 7.7x10
cells and examined by flow cytometry. The amount of
phagocytized beads was assessed by analyzing the fluorescence
intensity of the cells in the FITC channel. For the detailed
gating strategy, see Suppl. Figure S2A.
e2043037-2 A. V. BAZHIN ET AL.
Extracellular staining
The cells were preincubated for 10 min with anti-CD16/CD32
mixture, and appropriate antibodies for cell surface molecules
were added to 50 µl of the cell suspension and incubated for
15 min at 4°C in the dark. The samples were washed twice,
resuspended in stain buffer, and analyzed on the BD Canto II
Flow cytometer.
Intracellular staining
First, extracellular staining with non-conjugated antibodies was
performed and the cells were fixed with 1 ml of the Fixation/
Permeabilization Buffer. The samples were then incubated for at
least 3 hours at 4°C in the dark. Afterwards, two washing steps
with Permeabilization Buffer were performed at 4°C, 400 g,
5 min and the intracellular and the conjugated extracellular
antibodies were pipetted to the samples. After two washing
steps with Permeabilization Buffer, the cells were resuspended
in stain buffer and analyzed on the BD FACS Canto II.
MACS (Magnetic Activated Cell Sorting)
MACS separation was performed as described elsewhere,
according to manufacturer instructions. For MDSC isolation,
cells were magnetically labeled by Anti-Ly6G-
Biotin antibody and Anti-Biotin MicroBeads. The first flow-
through was pre-enriched in Gr1
cells. The positive
cells from the column were removed from the outside magnetic
field using the MACS buffer and a plunger. To increase the
purity of Gr1
cells, the cells were passed through
using a new column. The first flow-through pre-enriched in
cells was further labeled with Anti-Gr1-Biotin
antibodies and Streptavidin MicroBeads to isolate
cells by positive selection. For macrophage iso-
lation, Gr1
cells were magnetically labeled by Anti-Gr1-Biotin
antibody and Anti-Biotin MicroBeads and removed from the
suspension by positive selection. The Gr1
cells in the first
flow-through from the column were subsequently labeled by
Anti-CD11b- MicroBeads. To increase the purity of
cells, they were run through a new second
The CFSE-labeled splenocytes were seeded at a concentration
of 2x10
/200 µl medium and with the cells of interests in 96-
well round bottom plates. One hour later, CD3 antibody (1 µg/
ml) and CD28 antibody (2 µg/ml) were added to activate the
cells. After 72 hours at 37°C, the supernatant was collected and
stored at −20°C for the Luminex assay. The cells were har-
vested, stained with appropriate antibodies, and analyzed by
flow cytometry.
LUMINEX (Bioplex) assay
LUMINEX assay was performed as described elsewhere,
according to the manufacturer instructions, and measured by
the Luminex® 100/200 System.
Statistical analysis
All statistical analyses were performed using GraphPad Prism
Version 5.01 or 7.01. Distributions of continuous variables
were described by means, SE, median, 25% and 75% percen-
tiles, and were presented as indicated in the Figure Legends.
D’Agostino and Pearson omnibus normality tests were con-
ducted to estimate the distribution of data. The null hypothesis
(mean values were equal) versus the alternative hypothesis
(mean values were not equal) was tested by unpaired, two-
tailed T-test for normal distributed variants or by the Mann–
Whitney test for nonparametric distributed data. Survival ana-
lysis was done with Kaplan–Meier curves and statistically ana-
lyzed with the Log-rank test. All statistical tests were two-tailed.
The significance level was α = 5%.
In this work, we used two approaches for B7-H1 inhibition
based on the Panc02 orthotopic model of PDAC. This model is
characterized by strong immunosuppression,
which makes
it similar to human PDAC.
For the first approach, the murine PDAC cells of the Panc02
cell line were orthotopically injected in the B7-H1KO mice or
in the wild-type (WT) animals (control). For the second one,
only WT mice were used for Panc02 cell injection. In this case,
one group of the animals was treated with an antibody against
B7-H1 and another group became an isotype antibody
Notably, both cultured PDAC Panc02 cells and the tumors
generated from the orthotopic injection of Panc02 cells demon-
strated the expression of B7-H1 molecule (Suppl. Figure S2B).
Absence of B7-H1 reduces tumor volume and appearance
of peritoneal carcinosis and improves survival of
PDAC-bearing mice
First, B7-H1KO and WT BL6 animals (co) were transplanted
orthotopically with Panc02 cells, and tumor volume, metas-
tases, and survival of the tumor-bearing mice were analyzed
(Figure 1). B7-H1KO mice showed a decrease in the PDAC
tumor volume and a reduction of peritoneal carcinosis
(Figure 1A and C). No difference in liver and colon metastases
was observed (Suppl. Figure S3). Importantly, B7-H1KO
tumor-bearing mice demonstrated a clear survival benefit com-
pared to the WT animals (Figure 1B). For the second approach,
we operated WT mice and treated them with a monoclonal
antibody against B7-H1 (abB7-H1) or with an isotype control
IgG (co). The abB7-H1 treatment reduced tumor volume and
improved survival of PDAC-bearing mice (Figure 1D and E).
We detected also a decrease in a number of liver metastasis, but
the difference was not significant (Suppl. Figure S3). Thus,
inhibition of B7-H1 leads to an improvement of oncological
parameters of PDAC-bearing hosts.
We supposed that the oncological improvement in PDAC-
bearing mice observed was due to a restoration of the antitu-
mor immune response. To prove or disprove this hypothesis,
a profound investigation of antitumor immunity in the PDAC-
bearing mice has been carried out.
Inhibition of B7-H1 leads to a partial abrogation of
As we showed previously, tumors of the Panc02 PDAC model
are strongly immunosuppressive due to the tumor accumula-
tion of high amount of Treg and MDSC,
as well as due to
a building of an immunosuppressive cytokine milieu.
we investigated pro- and anti-inflammatory cytokines in the
serum of tumor-bearing mice studied in the previous section.
In the sera of B7-H1KO PDAC-bearing mice, we detected less
VEGF as well as a trend to diminished production of KC
(CXCL1) and IL10 (Suppl. Figure S4). Amount of IL1b,
TGFβ, IL4, IL6, and IL13 were not changed significantly
(Suppl. Figure S4). Interestingly, in the serum of PDAC-
bearing mice treated with the B7-H1 antibody, we could in
addition detect a significant decrease in the TGFβ concentra-
tion (Suppl. FigureS4). These results reflect a lower level of
immunosuppression on the cytokine level in the tumor-
bearing hosts without B7-H1.
At the next step, we profoundly investigated immunosuppres-
sion on the cellular level in the tumor-bearing mice with or
without B7-H1 expression. We found less Treg in tumors of B7-
H1KO mice and in WT PDAC-bearing animals treated with
abB7-H1 (Figure 2A and Suppl. Figure S2A). It should be noted
that in spleen, we could not see any difference in the Treg amount
between the mouse strains analyzed (data not shown). Also an
analysis of Tumor Infiltrating Leukocytes (TILs) did not reveal
any change in the amount of MDSC (Suppl. Figure S5). However,
splenocytes from the tumor-bearing mice demonstrated
a decrease in the amount of MDSC in B7-H1KO mice as well
as in WT PDAC-bearing animals treated with antibody against
B7-H1 (Figure 2B and Suppl. Figure S2A). It is important to note
that granulocytic MDSC (gMDSC) was more often detected in
TILs of WT tumor-bearing mice compared to B7-H1KO animals,
whereas monocytic MDSC (mMDSC) was prominently found in
TILs of B7-H1KO mice (Figure 2C and Suppl. Figure S2A).
Besides, based on the downregulation of their inducible nitric
oxide synthase (iNOS) and Arginase (Arg) expression, B7-H1KO
MDSC have to possess a low immunosuppressive capacity
(Figure 2D and Suppl. Figure S2A). Indeed, a proliferation assay
using T-lymphocytes as target cells revealed that MDSC from WT
mice are stronger immunosuppressive then MDSC from B7-
H1KO mice (Figure 3A and Suppl. Figure S2A).
Thus, the general PDAC immunosuppression depends on
the status of B7-H1 expression manifesting in modulation of
Treg and MDSC as well as their immunosuppressive function.
Since MDSC express high amount of B7-H1 (Figure 3B), it
was intriguing to investigate whether this immunosuppressive
molecule could reinforce the immunosuppressive effect of these
cells. We performed once more a proliferative assay, as men-
tioned above, using MDSC isolated from WT mice and incu-
bated them with T lymphocytes with or without an antibody
against B7-H1. We registered no effect of the antibody on pro-
liferation (data not shown). However, the addition of anti-B7-
H1 antibody in vitro led to an increase in the interferon-γ (IFNγ)
concentration in the co-culture supernatants (Figure 3C), indi-
cating that B7-H1 seems to be partially involved in the immu-
nosuppression by MDSC in these experimental settings. Thus,
B7-H1 could be engaged in the interplay of MDSC and T cells.
Inhibition of B7-H1 leads to a restoration of the antitumor
immune response in PDAC-bearing mice
We supposed that low level of immunosuppression in B7-
H1KO tumor-bearing mice can lead to the restoration of
the antitumor immune response, which can be responsible
for a better oncological outcome of tumor-bearing animals.
Therefore, we investigated in detail lymphocyte populations
generally involved in the antitumor response. For this pur-
pose, T cells and their subgroups were analyzed both in
spleen and tumor of WT and B7-H1KO tumor-bearing
mice. The analysis revealed a high amount of CD4
phocytes in spleens of B7-H1KO tumor-bearing animals
Figure 1. Absence of B7-H1 reduces tumor volume and appearance of peritoneal carcinosis and improves survival of PDAC-bearing mice. (A) Tumor volume, (B) survival,
and (C) signs of peritoneal carcinosis of WT (co) and B7-H1KO (B7-H1) tumor-bearing mice; (D) tumor volume and (E) survival of WT tumor-bearing mice treated with an
antibody against B7-H1 (abB7-H1) or with an isotype control (co). Data are presented (A and D) as a scatter plot (mean with SD), n = 11–18, analyzed with the unpaired
T-test; (C) as stacked bars analyzed with the fisher’s exact test; (B and E) with a Kaplan-Meier curves and analyzed with the log-rank (mantel-cox) test. **p < 0.01,
***p < 0.001, and ****p < 0.0001.
e2043037-4 A. V. BAZHIN ET AL.
and in tumors of WT mice (Figure 4A,C and Suppl. Figure
S2A). Importantly, a stronger accumulation of effector-
memory CD4
lymphocytes was detected in the B7-H1KO
mice compared to WT animals (Figure 4B and Suppl.
Figure S2A). Regarding CD8
lymphocytes, their high
amount was detected both in tumor and spleen of B7-
H1KO animals compared to WT mice (Figure 4D,F and
Suppl. Figure S2A). Besides, a pronounced accumulation of
effector-memory CD8
lymphocytes was found in tumor
and in spleen of the B7-H1KO mice (Figure 4E,G and
Suppl. Figure S2A). Higher tumoral accumulation of
lymphocytes and their effector-memory subpopula-
tion were seen in the WT tumor-bearing mice treated with
an antibody against B7-H1, while control treated animals
showed lower accumulation of these cells (Suppl. Figure
S6). It should be noted that no difference in the B-cell
and NK-cell distribution either in tumor or spleen was
detected in the tumor-bearing animals investigated (data
not shown).
IFNγ production by T cells serves as a marker of their
activation. Therefore, we measured intracellular IFNγ produc-
tion by TILs and splenocytes obtained from PDAC-bearing
WT and B7-H1KO mice. We found higher level of activation
of different subpopulation of immune cells in TILs (Figure 5A)
and splenocytes (Suppl. Figure S7) isolated from B7-H1KO
animals compared to WT ones. Another important antitumor
characteristic of immune cells represents the cytotoxicity of
TILs against tumor cells. We hypothesized that the TILs from
B7-H1KO mice would possess better cytotoxicity then TILs
obtained from WT animals. For this purpose, CD45
were isolated from tumors of both mice strains and co-
cultivated with the Panc02 cells. The experiments proved our
hypothesis (Figure 5B). The amount of IFNγ-producing CD4
and CD8 cells was indeed much higher in the TILs from B7-
H1KO tumor-bearing mice as well as the percent of killed
tumor cells in the respective co-cultures. This means that
abrogation of B7-H1 even only on the non-tumor cells
improves antitumor cytotoxicity of TILs.
Figure 2. Inhibition of B7-H1 leads to a decrease in amount of Treg and MDSC in tumor-bearing mice. (A) Amount of Treg is diminished in the tumor of B7-H1KO mice
(B7-H1) and of WT mice treated with antibody against B7-H1 (abB7-H1): data are presented as a scatter plot (mean with SD), n = 7–15, analyzed with the unpaired T-test.
(B) Amount of MDSC is diminished in the spleen of B7-H1KO mice (B7-H1) and of WT mice treated with antibody against B7-H1 (abB7-H1): data are presented as a scatter
plot (mean with SD), n = 8–15, analyzed with the unpaired T-test. (C) Distribution of MDSC subpopulations in tumor of mice, data are presented as a scatter plot (mean
with SD), n = 6–17, analyzed with the unpaired T-test. (D) Expression of iNOS and Arg is lower in the MDSC of B7-H1KO mice (B7-H1): data are presented as a colon bar
graph (mean with SD), n = 4–12, analyzed with the unpaired T-test. MFI – fluorescence intensity. *p < 0.05, **p < 0.01.
Thus, the antitumor immune response in the absence
of B7-H1 seems to be restored due to higher immune cell
activation and improved cytotoxicity, as well as accumu-
lation of effector-memory lymphocyte in the tumor-
bearing mice.
Inhibition of B7-H1 improves functionality of DC
It is well-known that B7-H1 molecule is important for the
impeccable function of DC. Therefore, we investigated this
immune cell compartment more precisely. While the plasma-
cytoid (p) DC amount was reduced in the spleen of B7-H1KO
tumor-bearing mice compared to the WT ones, these DC
showed an improvement in their maturation state
(Figure 6A). However, the CD80 co-expression on the surface
of pDC was diminished in the absence of B7-H1 (Figure 6A).
In regard of CD80 expression on conventional (c) DC, we
detected a decrease in the CD80 expression intensity
(Figure 6B). Splenocytes of the B7-H1KO tumor-bearing
mice showed also an increase in the amount of CD86
cDC (Figure 6B). However, their expression intensity was
reduced (Figure 6B). In addition, in the spleen of mice treated
with abB7-H1, a reduction of the cDC amount was registered
(Figure 6B). Finally, we saw no phenotypical differences in the
tumor DC of both models (data not shown). Activation and
proliferation of CD4
lymphocytes after co-incubation with
DC serves as a readout for the functionality of DC. Therefore,
we isolated DC from tumor and CD4
cells from spleen from
control WT tumor-bearing mice and co-incubated them with
or without an antibody against B7-H1. It should be noted that
the antibody alone did not affect the expression of CD69 and
CD25 markers on CD4
lymphocytes and their proliferation
(data not shown). However, blocking of B7-H1 with the anti-
body in co-cultures led to better activation of lymphocytes
(expression of CD69 and CD25) and influenced their prolifera-
tion (Suppl. Fig8).
Thus, the inhibition of B7-H1 expression improves the
function of PDAC tumor DC.
Inhibition of B7-H1 reduces amount of splenic and tumor
M2 macrophages in tumor-bearing mice and induces the
macrophage phagocytic activity
Macrophages play a dual role in tumor-bearing hosts: M1
macrophages are generally involved in antitumor immune
response, while M2 favorite tumor progression.
In our
PDAC model, splenic and tumor macrophages also
expressed B7-H1 on their surface (Figure 7A). Therefore,
Figure 3. MDSC from B7-H1KO tumor-bearing mice are less immunosuppressive as compared to their WT counterparts. (A) MDSC from tumors of B7-H1KO tumor-
bearing mice (B7-H1 MDSC) have lower suppressive capacity on the T-cell proliferation then MDSC from tumor of WT tumor-bearing mice (co MDSC). (B) Flow cytometry
analysis of the B7-H1 expression on MDSC in tumor and spleen. (C) Analysis of IFNγ production by T cells co-cultured with MDSC with or without abB7-H1. A summary of
results of two independent experiments, each with 3–5 mouse probes presented as a colon bar graph (mean with SD), analyzed with the unpaired T-test. **p < 0.01 and
****p < 0.0001.
e2043037-6 A. V. BAZHIN ET AL.
we questioned whether the B7-H1 inhibition would influ-
ence this group of immune cells. We found that the amount
of macrophages (CD11b
cells) is decreased in tumor
and spleen from B7-H1KO mice compared to control WT
animals (Figure 7B). Importantly, the amount of M2
) macrophages was also reduced both in spleen
and tumor of the B7-H1KO mice (Figure 7B). Since phago-
cytic activity is an important feature of macrophages, we
analyzed it in the tumor macrophages obtained from both
tumor-bearing mice strains. Indeed, the macrophages from
tumor of B7-H1KO mice possessed better phagocytic activity
then CD11b
TILs from tumor-bearing WT hosts.
These results suggest that B7-H1 expression is important
for macrophages and that the M2 immunosuppressive
macrophages are low presented in the absence of this check-
point molecule.
In this study, we investigated for the first time in detail the
effect of total and partial B7-H1 deficiency in PDAC on the
regulation of immune response. Recently, we provided
a detailed expression pattern of PD-L1 and its receptor PD-1
on specific healthy mouse immune cells.
Taking in account
Figure 4. Accumulation of effector-memory lymphocytes in the tumor-bearing B7-H1KO mice. CD4
or CD8
lymphocytes in tumor (A and D) and spleen (C and F) and
their effector-memory subpopulation (B, E and G). Data are presented as a scatter plot (mean with SD), n = 7–19, analyzed with the unpaired T-test. *p < 0.05,
**p < 0.01, ***p < 0.001, and ****p < 0.0001.
the ubiquitous PD-L1 expression demonstrated in this project
and in different studies on immune cells like MDSC,
Treg cells,
and of PD-L1 receptor
PD-1 on non-myeloid immune cells (CD8, CD4 and Treg),
we expected that the absence of B7-H1 on immune cell could
lead to a decrease in general immunosuppression and conse-
quently to reduced tumor growth.
Indeed, we show here for the first time that the reduction of
even only the host non-tumor B7-H1 in PDAC leads to the
restoration of immune response, tumor reduction, and survival
benefit. These results are in line with the recent observations in
colon and sarcoma tumor models.
They also highlight the
intriguing discussion about the crucial role of the host immune
cells in general and particularly B7-H1
-positive immune cells in
the regulation of antitumor response.
In our study, the favor-
able immune effects of B7-H1 antibody were surprisingly not
more, but sometimes even less pronounced then effects observed
in B7-H1KO tumor-bearing mice. This implies that aside from
various parameters determining the efficacy of immune check-
point inhibition in PDAC like tumor immunogenicity, initial
T-cell priming, immunosuppressive network, and T-cell anergy
and exhaustion, some limitations concerning explicit usage of
antibody could exist. Antibody delivery to the target tumor and
nontumor cells requires not only favorable pharmacokinetics
but also sufficient micro-vessel permeability, efficient penetra-
tion, and retention in the targeted tissue, which is controlled by
multiple characteristics of antibody and by clinical settings.
Moreover, the initial absence of B7-H1 molecule in B7-H1KO
might contribute to the development of more favorable, in con-
text of antitumor response, tumor microenvironment (TME).
Previous reports from Winograd et al. and Ma et al. demon-
strated that single anti-PD-1 blockade failed to show anti-
cancer activity in murine pancreatic cancer model.
In the
first study, other models of pancreatic cancer were used,
namely the KPC mouse spontaneous PDA and the subcuta-
neous PDA tumor model, which might have a certain differ-
ences in organization of TME compared with our orthotopic
Moreover, in contrast to our approach, anti-PD-1
antibodies were used in both studies to block PD-1/B7-H1
axis. Taking in account that (i) PDAC tumor cells express B7-
H1, (ii) B7-H1 expression could be shown on the majority of
immune cells, whereby PD-1 expression was more restricted,
and (iii) various immune cells expressing B7-H1 could be
modulated by B7-H1 blockage in their phenotypes and func-
tions even in healthy mice,
the direct reduction of B7-H1
expression might be more encouraging. Besides, B7-H1 was
described to have an appreciable affinity for the CD80 costi-
mulatory molecule,
and recent evidence suggests also that
a reverse signaling may exist downstream of B7-H1 in both
tumor and immune cells.
These new B7-H1 characteristics
are not thoroughly investigated yet, but could make as well an
input into the outcome of the direct B7-H1 inhibition.
Figure 5. Immune cells from tumors of B7-H1KO PDAC-bearing mice produce high amount of IFNγ. (A) Ratio of intracellular IFNγ production in different subpopulation
of immune cells (WT (co), B7-H1KO (B7-H1)) is presented as a colon bar graph (mean with SD), n = 4–8, analyzed with the unpaired T-test – left panel, and as
a representative FACS picture – right panel. (B) Cytotoxicity of TILs (WT(co), B7-H1KO (B7-H1)) is presented as a percent of cell death in anc02 cells in a colon bar graph
(mean with SD) summarizing two independent experiments, each with 3–5 mouse probes, and analyzed with the unpaired T-test. **p < 0.01 and ***p < 0.001.
e2043037-8 A. V. BAZHIN ET AL.
Our Panc02 PDAC model has several benefits like presence
of an intact immune system and of strongly immunosuppressive
TME as well as scarcity in effector CD8+ T cells, making it
comparable with human PDAC and suitable for immunother-
apeutic preclinical studies. Other sides, this model (i) does not
develop a strong desmoplastic reaction and therefore differs in
TME and (ii) demonstrates, due to its carcinogen-derived
tumorigenesis, a high mutational burden.
This may limit the
use of Panc02 model in immunotherapeutic approaches, since
these characteristics can modulate the efficiency of immunother-
apy. For example s.c. Panc02 tumor-bearing mice usually
respond better to immune checkpoint blockade.
In contrast,
in the work of Luheshi et al., Panc02 pancreatic tumor cells were
surgically implanted in matrigel to produce more robust TME,
similar to human PDAC. This study demonstrated that modula-
tion of the TME by a CD40 agonist antibody correlates with
improved antitumor reactions to, otherwise, poor responsive,
PD-L1 blockade, underlining relevance of TME variations in
therapeutic studies.
Since human PDAC has generally lower mutation burden
unless it has mismatch repair (MMR) deficiency (d-MMR), the
promising results obtained in PDAC model might be
particularly relevant for this cohort of patients. Supportively,
d-MMR, including MMR protein loss and/or microsatellite
instability, was shown to be predictive of response to immu-
notherapy in pancreatic cancer.
Recent clinical trials reported no clinical benefit of single
agent B7-H1 blockade by antibody Durvalumab in pancreatic
However, patients in clinical studies are
much more heterogenic then the experimental mice, which
are of the same line, age, tumor inoculation time, early injec-
tion time of antibody, etc. They often, like in this trial, repre-
sent a population of patients with mPDAC who had poor
prognoses and rapidly progressing disease. Most of the studies
enroll an unselected group of patients to treat, and the number
of respondents might appear insufficient to establish the asso-
ciation between clinical outcomes and B7-H1 expression.
line, in recent years in PDAC studies, multiple evaluated tar-
geted therapies and cancer vaccines failed to show efficacy in
late-stage clinical trials.
Despite preclinical tumor models
partially reflect the human disease, the complex tumor envir-
onment of PDAC, which is genetically heterogenic, hypoxic,
fibrotic, immunosuppressive, and not highly immunogenic,
still differs in humans.
Figure 6. B7-H1 has an influence on the composition of splenic DC, their maturation state, and expression of co-stimulatory molecules. Amount of pDC (A) and cDC (B),
their maturation state (A and B), and expression of CD80 (A and B) as well as of CD86 (B) molecules on mature DC. WT (co), B7-H1KO (B7-H1), WT treated with antibody
against B7-H1 (abB7-H1). (A, B (CD86)) are presented as a scatter plot (mean with SD), n = 8–9; amount of cDC (B) presented as a scatter plot (mean with SD), n = 14–15,
and MFI of CD80 and CD86 expression (B) are presented as a column bar graph (mean with SD), n = 9; all data are analyzed with the unpaired T-test. *p < 0.05,
***p < 0.001, and ****p < 0.01.
Our study clearly demonstrates that B7-H1 inhibition
approach has a therapeutic potential, despite disappointing
outcome in the first clinical trials. We believe that various
parameters and clinical settings should be further examined
and optimized in order to achieve in patients similar effects of
B7-H1 inhibition on immunosuppressive network and
immune response as were observed in our PDAC model.
Future studies should as well evaluate the possibilities to iden-
tify patients most likely to benefit from B7-H1 inhibition and
to develop the best combined therapies with B7-H1 blockade
and other agents. Hereby, the simultaneous targeting of other
molecular or cellular components of PDAC immunosuppres-
sive network as well as individualized treatment for certain
patient populations could be promising approaches.
One of the crucial immunosuppressive axes is provided by
MDSC. We demonstrated previously that a decrease in MDSC
frequencies and in the systemic VEGF level leads to
a restoration of anti-cancer immune responses and an
improved survival of PDAC-bearing female mice and hypothe-
sized that MDSC are strongly involved in the PDAC-associated
Also pancreatic cancer patients with
up-regulated B7-H1 display lymphocyte exhaustion and are
more enriched in MDSC.
In this study, we observed in the
sera less VEGF in B7-H1KO. In spleens, we found a decrease in
the amount of MDSC in B7-H1KO tumor-bearing mice as well
as in WT PDAC-bearing animals treated with antibody against
B7-H1. This is also aligned with our foregoing observation that
B7-H1KO healthy mice have a reduced Gr1
ment within myeloid cells
and with strong indications that
signaling through B7-H1 can make an input in cancer immune
escape functionally related to MDSC.
Not only the expansion of MDSC but also their iNOS /Arg
production and suppressive activity were partially B7-H1-
dependent in our study. In accordance, the role of B7-H1 in
MDSC function was highlighted in several other models. In vitro
studies with human gMDSC showed that targeting B7-H1 par-
tially impaired MDSC-mediated T-cell suppression.
bone marrow-derived B7-H1
MDSC are responsible for
immune suppression through a mechanism involving Arg-1
and IDO expression, and B7-H1 and MHC class II expression
Figure 7. Absence of B7-H1 abolishes the accumulation of M2 macrophages. (A) Flow cytometry analysis of the B7-H1 expression on macrophages in tumor and spleen.
Representative FACS pictures are shown. (B) Macrophage and M2 macrophage accumulation in spleen and tumor of mice, WT (co), B7-H1KO (B7-H1). Data are presented
as a scatter plot (mean with SD), n = 4–8, analyzed with the unpaired T-test. (C) Phagocytic activities of macrophages were evaluated as an uptake of fluorescent-labeled
beads analyzed by flow cytometry, WT (co), B7-H1KO (B7-H1). Data are presented as a column bar graph summarized from two independent experiments, each with 3–5
mouse probes, and analyzed with the unpaired T-test. *p < 0.05, **p < 0.01, and ****p < 0.0001.
e2043037-10 A. V. BAZHIN ET AL.
on in vitro produced bone marrow-derived MDSC and MDSC
from patients with melanoma and colorectal cancer correlated
with T-cell dysfunction.
In hepatocellular carcinoma (HCC)
patients, higher percentages of B7-H1
MDSC was found, which
were inversely correlated with disease-free survival and were
reduced by HCC treatment.
Moreover, the majority of liver
MDSC, which are associated with liver metastases and suppress
antitumor immunity, express high level of B7-H1 and demon-
strate B7-H1-dependent mode of suppression.
Also in multiple
myeloma, MDSC were characterized by high expression of B7-
H1, and PD-1/B7-H1 blockade made an input in the inhibition
of MDSC-mediated immune suppression and multiple myeloma
Interestingly, age as a known factor encouraging
immunosuppression promoted accumulation of B7-H1
in aged mice in lung cancer and enhanced tumor growth. These
pro-tumor effects could be diminished by inhibition of B7-H1
with specific antibody.
MDSC compartment is divided into two
subpopulations, and gMDSC was strongly presented in the WT
tumor-bearing mice while mMDSC was predominantly found in
the B7-H1KO mice. In contrast, in healthy B7-H1KO mice, the
ratio between Gr1
and Gr1
subpopulations was shifted toward Gr1
Which role the ratio between gMDSC and mMDSC could play in
general and particularly in anti-cancer immunity is still the
matter of debate. However, several observations pointed to the
specific characteristics of gMDSC subset. For example, in pros-
tate cancer, gMDSC is supposed to exhibit a high pSTAT3 levels
and a high degree of immunosuppressive activity and high levels
of B7-H1.
Increased levels of gMDSC were also found in
individuals with initial phase of HIV infection, which correlated
negatively with CD4 T-cell levels. Moreover, this B7-H1 expres-
sion was utilized by gMDSC to inhibit proliferation and IFNγ
secretion of CD8 T cells.
Other closely related myeloid cells – macrophages – were as
well reduced in their numbers in tumor and spleen from
tumor-bearing B7-H1KO mice, but possessed better phagocy-
tic activity. Tumor-associated macrophages can restrict
immune engagement and therefore became an attractive target
in PDAC treatment.
In PDAC, the high number of macro-
phages was associated with poor prognosis.
Phagocytosis and
intracellular killing activity of macrophages in tuberculosis can
be as well as significantly increased with PD-1/PD-L1
Development and progression of oral squamous
cell carcinoma are characterized by the augmented presence of
M2 macrophages with upregulated B7-H1 expression and cor-
related with increased capacity to induce T-cell apoptosis.
Particularly important in our experiments is the concomitant
reduction of tumor-promoting M2 macrophages in spleen and
tumor of B7-H1KO mice, which are an important part of tumor
immune suppressive environment
and are associated with
decreased survival and poor prognosis in pancreatic cancer.
Also in gastric adenocarcinoma cells, M2-like macrophage infil-
tration is highly associated with B7-H1 expression.
In this
work, we did not investigate other potentially important part
of tumor environment, namely cancer-associated fibroblasts
(CAFs), which represent a heterogeneous population of cells.
Recent studies, however, suggested that in some cancers, CAFs
express B7-H1, which can serve as a prognostic marker
described a CAF subset that is associated with a poor response
to anti–B7-H1 therapy.
Thus, further investigation of interplay
between tumor cells, CAFs, and immune cells could explore our
knowledge regarding immune checkpoint blockade therapy.
DC is one of the major cell compartments that express B7-H1
and can further up-regulate it in response to various
but less is known about the effect of B7-H1 on
DC per se. We demonstrated that while in the tumor-bearing
mice the pDC subpopulation was reduced in the spleen of B7-
H1KO but showed an improvement in their maturation state, in
healthy B7-H1KO, the percentage of pDC was increased.
addition, in the spleen of PDAC mice treated with abB7-H1,
a reduction of the cDC amount was registered. This means that
B7-H1 molecule is involved in the regulation of DC subdivisions,
but the outcome is different in case of tumor presence or healthy
conditions. The CD80 co-expression on the surface of pDC and
cDC was diminished in the absence of B7-H1 in tumor-bearing
hosts. In line, the same effect we previously observed in healthy
Thus, this intrinsic regulation occurs independently
from tumor environment. Splenocytes of the B7-H1KO tumor-
bearing mice showed in this study an increase in the amount of
mature cDC but reduction in the intensity of CD86
expression. Interestingly, the amount of CD86
pDC and
CD86 expression intensity was higher in B7-H1KO healthy
mice, whereas cDC did not demonstrate any variations.
the modulation of maturation stage upon B7-H1 deficiency
seems to be as well partially tumor dependent.
In accordance with the observations of other research-
ers and our own previous findings discussed, we demon-
strated here that B7-H1 expression on DC correlates with
their stimulation capacity toward T cells, since the inhibi-
tion of B7-H1 expression improves the activation ability of
PDAC tumor DC. In our recent work, we showed that DC
represented only a small population in PDAC TILs, did
not possess tolerogenic phenotype and still could improve
the proliferation capacity of the co-cultured splenocytes.
This let us assume that DC are not a major player in the
context of immunosuppression in PDAC but rather a soft
However, it is known that DC has reduced func-
tions in several types of cancer, including PDAC, and
many clinical approaches aim to improve their stimulatory
Another important pivot point of cellular immunosuppres-
sion is Treg cells, which are highly enriched in the PDAC.
Various observations demonstrated that B7-H1 expression
could encourage accumulation of Treg and that manipulating
of the PD-1/B7-H1 interaction can enhance the in vitro and ex
vivo expansion and function of Tregs.
In context of pancreatic tumor, we accordingly found less
Treg in tumors of B7-H1KO mice and in WT PDAC-bearing
animals treated with abB7-H1, while in healthy B7-H1KO
mice, we have rather seen a percentile increase in Treg within
the CD4 compartment.
These results propose that the
absence of B7-H1 can modulate Treg incidental to tumor
Taken together, our findings demonstrate that B7-H1 defi-
ciency affects the most prominent suppression actors in
PDAC, by influencing their amount, constellation, and func-
tions. Consequently, we observed restoration of immune
response manifested in the increased accumulation of
effector-memory CD4 and CD8 cells and improved function-
ality of CD4 and CD8 compartments. Our results highlight
the great potential of B7-H1 molecule to control sophisticated
immunosuppressive network in PDAC. They also provide
new impulses for further clinical investigations to improve
the immunotherapy with checkpoint B7-H1 inhibition and its
clinical outcome.
The authors thank Ms. Tina Maxelon, Ms. Inna Schwarting, Mr. Markus
Herbst, and Ms. Michaela Svihla for their excellent technical assistance.
This study was supported by Funding Program “Foundations and Prizes”
of the Medical Faculty Heidelberg to S.K. The J.T.S. is supported by the
German Cancer Consortium (DKTK), the Deutsche
Forschungsgemeinschaft (DFG) through grant SI1549/3–1 (Clinical
Research Unit KFO337) and SI1549/4–1 and the German Cancer Aid
(#70112505/PIPAC, #70113834/PREDICT-PACA).
Disclosure statement
No potential conflict of interest was reported by the author(s). J.T.S.
receives honoraria as consultant or for continuing medical education
presentations from AstraZeneca, Bayer, Immunocore, Roche, Servier.
His institution receives research funding from Bristol-Myers Squibb,
Celgene, Roche; He holds ownership and serves on the Board of
Directors of Pharma15, all outside the submitted work.
This work was supported by the Funding Program ”Foundations and
Prizes” of the Medical Faculty Heidelberg to S.K. and work in the lab of
J.T.S. is supported by the German Cancer Consortium (DKTK), the
Deutsche Forschungsgemeinschaft (DFG;SI1549/3–1 (Clinical Research
Unit KFO337), SI1549/4–1); the Deutsche Krebshilfe (German Cancer
Aid; #70112505, PIPAC, #70113834, PREDICT-PACA).
Jens T. Siveke
Author contributions:
SK and AB participated in the conceptualization and research design; All
authors participated in carrying out the research and analyzing the data;
AB and SK participated in writing the manuscript and critical correction
of the manuscript. AB and SK administered this work. All authors dis-
cussed the results and implications and gave constructive feedback on the
manuscript at all stages.
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e2043037-14 A. V. BAZHIN ET AL.
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Background Cancer-associated fibroblasts (CAFs) are some of the most abundant components of the tumour microenvironment. A recent study suggested that in some cancers, CAFs express programmed death ligand 1 (PD-L1), which can act as a prognostic marker. The aim of this study was to investigate the clinicopathological significance of CAF PD-L1 expression in patients with triple-negative breast cancer (TNBC) and to identify the most suitable primary antibody for immunostaining for CAF PD-L1. Methods Immunohistochemical staining (primary antibodies of 73–10, SP142, and E1L3N) and tissue microarrays were used to analyse the expression profiles of PD-L1 in CAF in 61 patients with TNBC who underwent surgery. Overall survival (OS) was compared based on CAF PD-L1 expression, and the risk factors for OS were analysed. The relationship between clinicopathological parameters and survival was also examined. Results Thirty-four (55.7%) patients were positive for CAF PD-L1 (73–10) expression. Compared with CAF PD-L1 negativity, there was a significant correlation between CAF PD-L1 positivity and better OS (p = 0.029). CAF PD-L1 expression, evaluated using SP-142 or E1L3N, did not correlate with OS. CAF PD-L1-positivity (73–10) correlated significantly with better prognosis in multivariate analyses (hazard ratio: 0.198; 95% confidence interval: 0.044–0.891; p = 0.035). Conclusions CAF PD-L1 expression is a novel marker for a better prognosis of patients with TNBC, and the 73–10 assay may be suitable for immunostaining CAF PD-L1.
Full-text available
The advent of immunotherapy has transformed the treatment landscape for several human malignancies. Antibodies against immune checkpoints, such as anti-PD-1/PD-L1 and anti-CTLA-4, demonstrate durable clinical benefits in several cancer types. However, checkpoint blockade has failed to elicit effective anti-tumor responses in pancreatic ductal adenocarcinoma (PDAC), which remains one of the most lethal malignancies with a dismal prognosis. As a result, there are significant efforts to identify novel immune-based combination regimens for PDAC, which are typically first tested in preclinical models. Here, we discuss the utility and limitations of syngeneic and genetically-engineered mouse models that are currently available for testing immunotherapy regimens. We also discuss patient-derived xenograft mouse models, human PDAC organoids, and ex vivo slice cultures of human PDAC tumors that can complement murine models for a more comprehensive approach to predict response and resistance to immunotherapy regimens.
Full-text available
Background Aims Advanced pancreatic ductal adenocarcinoma (PDAC) is resistant to therapy, including immune checkpoint inhibitors. We evaluated the effects of a neutralizing antibody against programmed cell death 1 (PDCD1, also called PD1) and an agonist of OX40 (provides a survival signal to activated T cells) in mice with pancreatic tumors. Methods We performed studies in C57BL/6 mice (controls), KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, and mice with orthotopic tumors grown from Panc02 cells, KrasG12D;P53flox/flox;PDX-1-Cre;Luciferase (KPC-Luc) cells, or mT4 cells. After tumors developed, mice were given injections of control antibody or anti-OX40 and/or anti-PD1 antibody. Some mice were then given injections of antibodies against CD8, CD4, or NK1.1 to deplete immune cells, and IL4 or IL7RA to block cytokine signaling. Bioluminescence imaging was used to monitor tumor growth. Tumor tissues collected and single-cell suspensions were analyzed by time of flight mass spectrometry analysis. Mice that were tumor-free 100 days after implantation of orthotopic tumors were rechallenged with PDAC cells (KPC-Luc or mT4) and survival was measured. Median levels of PD1 and OX40 mRNAs in PDACs were determined from the Cancer Genome Atlas and compared with patient survival times. Results In mice with orthotopic tumors, all those given control antibody or anti-PD1 died within 50 days, whereas 43% of mice given anti-OX40 survived for 225 days; almost 100% of mice given the combination of anti-PD1 and anti-OX40 survived for 225 days, and tumors were no longer detected. KPC mice given control antibody, anti-PD1, or anti-OX40 had median survival times of 50 days or less, whereas mice given the combination of anti-PD1 and anti-OX40 survived for a median 88 days. Mice with orthotopic tumors that were given the combination of anti-PD1 and anti-OX40 and survived 100 days were rechallenged with a second tumor; those re-challenged with mT4 cells survived an additional median 70 days and those re-challenged with KPC-Luc cells survived long term, tumor free. The combination of anti-PD1 and anti-OX40 did not slow tumor growth in mice with antibody-mediated depletion of CD4+ T cells. Mice with orthotopic tumors given the combination of anti-PD1 and anti-OX40 that survived after complete tumor rejection were re-challenged with KPC-Luc cells; those with depletion of CD4+ T cells before the re-challenge had uncontrolled tumor growth. Furthermore, KPC orthotopic tumors from mice given the combination contained an increased number of CD4+ T cells that expressed CD127, compared with mice given control antibody. The combination of agents reduced the proportion of T-regulatory and exhausted T cells and decreased T-cell expression of GATA3; tumor size was negatively associated with numbers of infiltrating CD4+ T cells, CD4+CD127+ T cells, and CD8+CD127+ T cells and positively associated with numbers of CD4+PD1+ T cells, CD4+CD25+ T cells, and CD8+PD1+ T cells. PDACs with high levels of OX40 and low levels of PD1 were associated with longer survival times of patients. Conclusions Pancreatic tumors appear to evade the immune response by inducing development of immune-suppressive T cells. In mice, the combination of anti-PD1 inhibitory and anti-OX40 agonist antibodies reduces the proportion of T-regulatory and exhausted T cells in pancreatic tumors and increases numbers of memory CD4+ and CD8+ T cells, eradicating all detectable tumor. This information might be used in development of immune-based combination therapies for PDAC.
Full-text available
The so-called immune checkpoints are pathways that regulate the timing and intensity of the immune response to avoid an excessive reaction and to protect the host from autoimmunity. Immune checkpoint inhibitors (ICIs) are designed to target the negative regulatory pathways of T cells, and they have been shown to restore anti-tumor immune functions and achieve considerable clinical results. Indeed, several clinical trials have reported durable clinical response in different tumor types, such as melanoma, renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLC). Nonetheless, after the initial enthusiasm, it is now evident that the majority of patients do not benefit from ICIs, due to innate or acquired tumor resistance. It is therefore mandatory to find ways to identify those patients who will respond and to find ways to induce response in those who at present do not benefit from ICIs. In this regard, the expression of programmed death ligand 1 (PD-L1) on neoplastic cells was the first, and most obvious, biomarker exploited to predict the activity of anti-programmed death 1 (PD-1) and/or anti-PD-L1 antibodies. As expected, a correlation was confirmed between the levels of PD-L1 and the efficacy of anti-PD-1 therapy in melanoma, NSCLC and RCC. However, further results from clinical trials showed that some patients display a clinical response regardless of tumor cell PD-L1 expression levels, while others do not benefit from ICI treatment despite the expression of PD-L1 on neoplastic elements. These findings strongly support the notion that other factors may be relevant for the efficacy of ICI-based treatment regimens. Furthermore, although the current dogma indicates that the PD-1/PD-L1 axis exerts its regulatory effects via the signal transduced in PD-1-expressing T cells, recent evidence suggests that a reverse signaling may also exist downstream of PD-L1 in both tumor and immune cells. The reverse signaling of PD-L1, but also of other immune checkpoints, might contribute to the pro-tumoral/immune suppressive environment associated with tumor development and progression. Clarifying this aspect could facilitate the prediction of patients’ clinical outcomes, which are so far unpredictable and result in response, resistance or even hyper-progressive disease in some cases.
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Interferon-α (IFNα) has one of the longest histories of use amongst cytokines in clinical oncology and has been applied for the treatment of many types of cancers. Due to its immune-activating properties, IFNα is also an attractive candidate for combinatory anti-cancer therapies. Despite its extensive use in animal tumor models as well as in several clinical trials, the different mechanisms underlying patient responses and affecting desirable clinical benefits are still under investigation. Here we show that in addition to its immune-activating properties, IFNα induces the expression of a key negative regulator, immunosuppressive PD-L1 molecule, in the majority of the specific immune cell populations, particularly in the dendritic cells (DC). DC can modulate immune responses by a variety of mechanisms, including expression of T-cell regulatory molecules and cytokines. Our results showed that treatment of DC with IFNα-2b led to pronounced up-regulation of surface expression of PD-L1 molecules, increased IL-6 and decreased IL-12 production. Moreover, we present evidence that IFNα-treated DC exhibited a reduced capacity to stimulate interferon-γ production in T cells compared to control DC. This T-cell response after treatment of DC with IFNα was recovered by a pre-treatment with an anti-PD-L1 blocking antibody. Further analyses revealed that IFNα regulated PD-L1 expression through the STAT3 and p38 signaling pathways, since blocking of STAT3 and p38 activation with specific inhibitors prevented PD-L1 up-regulation. Our findings underline the important roles of p38 and STAT3 in the regulation of PD-L1 expression and prove that IFNα induces STAT3/p38-mediated expression of PD-L1 and thereby a reduced stimulatory ability of DC. The augmentation of PD-L1 expression in immune cells through IFNα treatment should be considered by use of IFNα in an anti-cancer therapy.
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Pancreatic ductal adenocarcinoma (PDAC), as the most frequent form of pancreatic malignancy, still is associated with a dismal prognosis. Due to its late detection, most patients are ineligible for surgery, and chemotherapeutic options are limited. Tumor heterogeneity and a characteristic structure with crosstalk between the cancer/malignant cells and an abundant tumor microenvironment (TME) make PDAC a very challenging puzzle to solve. Thus far, targeted therapies have failed to substantially improve the overall survival of PDAC patients. Immune checkpoint inhibition, as an emerging therapeutic option in cancer treatment, shows promising results in different solid tumor types and hematological malignancies. However, PDAC does not respond well to immune checkpoint inhibitors anti-programmed cell death protein 1 (PD-1) or anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) alone or in combination. PDAC with its immune-privileged nature, starting from the early pre-neoplastic state, appears to escape from the antitumor immune response unlike other neoplastic entities. Different mechanisms how cancer cells achieve immune-privileged status have been hypothesized. Among them are decreased antigenicity and impaired immunogenicity via both cancer cell-intrinsic mechanisms and an augmented immunosuppressive TME. Here, we seek to shed light on the recent advances in both bench and bedside investigation of immunotherapeutic options for PDAC. Furthermore, we aim to compile recent data about how PDAC adopts immune escape mechanisms, and how these mechanisms might be exploited therapeutically in combination with immune checkpoint inhibitors, such as PD-1 or CTLA-4 antibodies.
With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFβ and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15+ CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15+ CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15+ CAF signature correlated with poor response to anti–PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy. Significance This study describes the single-cell landscape of CAFs in pancreatic cancer during in vivo tumor evolution. A TGFβ-driven, LRRC15+ CAF lineage is associated with poor outcome in immunotherapy trial data comprising multiple solid-tumor entities and represents a target for combinatorial therapy. This article is highlighted in the In This Issue feature, p. 161
Objectives: Cancer-associated fibroblasts (CAFs) are a dominant cell type in tumor stroma and support the generation of pro-tumorigenic microenvironment. CAFs have frequent opportunities to interact with immune cells infiltrating the tumor stroma, but the process remains to be determined. In this study, we focused on immune checkpoint mechanism. We also examined the induction of programmed cell death-ligand 1 (PD-L1) on CAFs by immune cell, and the clinical significance of PD-L1-expressed CAFs in non-small cell lung cancer (NSCLC). Materials and methods: CAFs were isolated from human NSCLC tissues, and PD-L1 expression levels in CAFs were analyzed by real-time polymerase chain reaction and flow-cytometry. Following immunohistochemical analysis of PD-L1 in surgically resected pN0M0 NSCLC (n = 125, including 88 invasive adenocarcinomas and 37 squamous cell carcinomas), the correlation of PD-L1-positive CAFs with clinicopathological features was investigated. Results: PD-L1 mRNA and protein expression on CAFs was upregulated by exogenously supplemented interferon-gamma (IFN-γ) and downregulated through the depletion of IFN-γ. PD-L1 expression on CAFs was upregulated by co-culture with activated lymphocytes releasing IFN-γ. Immunohistochemistry revealed that PD-L1-positive CAFs were observed in 31 cases (24.8%). Postoperative relapse-free survival was significantly prolonged in patients with PD-L1-positive CAFs as compared with those with PD-L1-negative CAFs, with 5-year relapse-free probabilities of 84.5% and 66.3%, respectively (P = 0.031). Multivariate analysis revealed that PD-L1 expression on CAFs was an independent prognostic factor of longer relapse-free survival after surgery (hazard ratio: 3.225, P = 0.027). Conclusion: PD-L1 expression on CAFs is reversibly regulated by environmental stimuli including IFN-γ from activated lymphocytes. In the non-metastatic NSCLC, PD-L1 expression on CAFs suggests the induction of anti-tumor immune responses, contributing to better prognosis after surgery.
Importance New therapeutic options for patients with metastatic pancreatic ductal adenocarcinoma (mPDAC) are needed. This study evaluated dual checkpoint combination therapy in patients with mPDAC. Objective To evaluate the safety and efficacy of the anti–PD-L1 (programmed death-ligand 1) antibody using either durvalumab monotherapy or in combination with the anticytotoxic T-lymphocyte antigen 4 antibody using durvalumab plus tremelimumab therapy in patients with mPDAC. Design, Setting, and Participants Part A of this multicenter, 2-part, phase 2 randomized clinical trial was a lead-in safety, open-label study with planned expansion to part B pending an efficacy signal from part A. Between November 26, 2015, and March 23, 2017, 65 patients with mPDAC who had previously received only 1 first-line fluorouracil–based or gemcitabine-based treatment were enrolled at 21 sites in 6 countries. Efficacy analysis included the intent-to-treat population; safety analysis included patients who received at least 1 dose of study treatment and for whom any postdose data were available. Interventions Patients received durvalumab (1500 mg every 4 weeks) plus tremelimumab (75 mg every 4 weeks) combination therapy for 4 cycles followed by durvalumab therapy (1500 mg every 4 weeks) or durvalumab monotherapy (1500 mg every 4 weeks) for up to 12 months or until the onset of progressive disease or unacceptable toxic effects. Main Outcomes and Measures Safety and efficacy were measured by objective response rate, which was used to determine study expansion to part B. The threshold for expansion was an objective response rate of 10% for either treatment arm. Results Among 65 randomized patients, 34 (52%) were men and median age was 61 (95% CI, 37-81) years. Grade 3 or higher treatment-related adverse events occurred in 7 of 32 patients (22%) receiving combination therapy and in 2 of 32 patients (6%) receiving monotherapy; 1 patient randomized to the monotherapy arm did not receive treatment owing to worsened disease. Fatigue, diarrhea, and pruritus were the most common adverse events in both arms. Overall, 4 of 64 patients (6%) discontinued treatment owing to treatment-related adverse events. Objective response rate was 3.1% (95% CI, 0.08-16.22) for patients receiving combination therapy and 0% (95% CI, 0.00-10.58) for patients receiving monotherapy. Low patient numbers limited observation of the associations between treatment response and PD-L1 expression or microsatellite instability status. Conclusion and Relevance Treatment was well tolerated, and the efficacy of durvalumab plus tremelimumab therapy and durvalumab monotherapy reflected a population of patients with mPDAC who had poor prognoses and rapidly progressing disease. Patients were not enrolled in part B because the threshold for efficacy was not met in part A. Trial Registration identifier: NCT02558894