This information is current as
of June 16, 2017.
Characterization of Human Lung
Tumor-Associated Fibroblasts and Their
Ability to Modulate the Activation of
Tumor-Associated T Cells
Michael R. Nazareth, Lori Broderick, Michelle R.
Simpson-Abelson, Raymond J. Kelleher, Jr., Sandra J.
Yokota and Richard B. Bankert
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2007 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2007; 178:5552-5562; ;
doi: 10.4049/jimmunol.178.9.5552
http://www.jimmunol.org/content/178/9/5552
The Journal of Immunology
Characterization of Human Lung Tumor-Associated
Fibroblasts and Their Ability to Modulate the Activation
of Tumor-Associated T Cells1
Michael R. Nazareth, Lori Broderick, Michelle R. Simpson-Abelson, Raymond J. Kelleher, Jr.,
Sandra J. Yokota, and Richard B. Bankert2
L
ung tumors are the leading cause of cancer deaths in the
United States (1). Human non-small cell lung cancers
(NSCLC),3 including squamous cell carcinomas, large
cell carcinomas, and adenocarcinomas, are the most prevalent type
of lung tumor, and account for 80 –90% of all lung cancer cases.
Unfortunately, while lung cancer research progresses at a rapid
pace, we are still short of understanding the causes and progression
of this devastating disease. Typically, human tumors develop
slowly over a prolonged period of time and they often progress and
spread despite the presence, within the tumor microenvironment,
of large numbers of CD4⫹ and CD8⫹ T cells with activated or
memory cell phenotypes. The T cells in the microenvironment of
human non-small cell lung tumors are hyporesponsive to stimula-
Department of Microbiology and Immunology, Witebsky Center for Microbial Pathogenesis and Immunology, University at Buffalo, State University of New York, Buffalo, NY 14214
Received for publication July 27, 2006. Accepted for publication February 9, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by U.S. Public Health Service Grants R01CA10897, The John R. Oishei Foundation, and The Mary Kay Ash Foundation
(to R.B.B.).
2
Address correspondence and reprint requests to Dr. Richard B. Bankert, Department
of Microbiology and Immunology, University at Buffalo, State University of New
York, 138 Farber Hall, 3435 Main Street, Buffalo, NY 14214. E-mail address:
[email protected]
3
Abbreviations used in this paper: NSCLC, non-small cell lung cancer; TAT, tumorassociated T cell; TAF, tumor-associated fibroblast; ECM, extracellular matrix; IP-10,
IFN-␥-inducible protein 10; MIG, monokine induced by IFN-␥; FAP, fibroblast activation protein; ␣-SMA, ␣-smooth muscle actin.
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
www.jimmunol.org
tion via the TCR under conditions that fully activate T cells derived from the peripheral blood of normal donors or from cancer
patients (2). A combination of factors both intrinsic and extrinsic
have been identified that may contribute to the T cell hyporesponsiveness and to their inability to kill tumor cells (3). The hyporesponsiveness of memory T cells in the human NSCLC microenvironment was found to be partly due to TGF-␤1 that is produced
by and is present on cells within the tumor microenvironment (4).
The arrest of T cell function was shown to be reversible both in
vitro and in vivo by IL-12 (2).
Considerable effort has been given to identifying cells in the tumor
microenvironment that are responsible for the nonresponsiveness of
the tumor-associated T cells (TAT). This effort has been focused
largely upon the tumor cells themselves, and has established that
both soluble and membrane-associated factors have the capacity to
suppress the antitumor response of T cells (5–7). Several different
nonmalignant cell types within the tumor microenvironment have
also been identified to have potent immunosuppressive effects
upon TAT. These include subsets of both myeloid and plasmacytoid dendritic cells (8 –10) and CD4⫹CD25⫹ regulatory T cells
(11, 12). Another nonmalignant tumor-associated cell with potential immunomodulatory effects is the fibroblast. These cells are an
extremely heterogeneous and multifunctional cell population that
often constitute a majority of cells within the tumor microenvironment (13). Therefore, it is puzzling why this population of cells
has received comparatively little attention as a potential modulator
of TAT function (14).
Through their production of cytokines, chemokines, and other biologically active factors, it is known that fibroblasts play a significant
role in attracting and retaining inflammatory leukocytes within
sites of chronic inflammation (15). Fibroblasts in inflammatory
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The tumor microenvironment of human non-small cell lung cancer (NSCLC) is composed largely of stromal cells, including
fibroblasts, yet these cells have been the focus of few studies. In this study, we established stromal cell cultures from primary
NSCLC through isolation of adherent cells. Characterization of these cells by flow cytometry demonstrated a population which
expressed a human fibroblast-specific 112-kDa surface molecule, Thy1, ␣-smooth muscle actin, and fibroblast activation protein,
but failed to express CD45 and CD11b, a phenotype consistent with that of an activated myofibroblast. A subset of the tumorassociated fibroblasts (TAF) was found to express B7H1 (PD-L1) and B7DC (PD-L2) constitutively, and this expression was
up-regulated by IFN-␥. Production of cytokines and chemokines, including IFN-␥, monokine induced by IFN-␥, IFN-␥-inducible
protein-10, RANTES, and TGF-␤1 was also demonstrated in these cells. Together, these characteristics provide multiple opportunities for the TAF to influence cellular interactions within the tumor microenvironment. To evaluate the ability of TAF to
modulate tumor-associated T cell (TAT) activation, we conducted coculture experiments between autologous TAF and TAT. In
five of eight tumors, TAF elicited a contact-dependent enhancement of TAT activation, even in the presence of a TGF-␤1-mediated
suppressive effect. In the three other tumors, TAF had a net suppressive effect upon TAT activation, and, in one of these cases,
blockade of B7H1 or B7DC was able to completely abrogate the TAF-mediated suppression. We conclude that TAF in human
NSCLC are functionally and phenotypically heterogeneous and provide multiple complex regulatory signals that have the potential to enhance or suppress TAT function in the tumor microenvironment. The Journal of Immunology, 2007, 178: 5552–5562.
The Journal of Immunology
5553
FIGURE 1. The human non-small cell lung tumor
microenvironment. The images are histological sections of non-small cell lung tumors illustrating the
constituents of the tumor microenvironment. A and B
are stained with H&E to illustrate general tissue components and architecture. C and D are stained with
trichrome, which stains collagen and connective tissue in an aquamarine color. TAF are in close apposition to tumor cells, lymphocytes, and the other cellular and noncellular constituents of the human lung
tumor microenvironment. The images shown are representative of more than 20 human non-small cell
lung tumors tested.
these cells to modulate the function of the TAT. We report here
that the TAF produce both soluble factors (e.g., TGF-␤1, IFN-␥,
IFN-␥-inducible protein 10 (IP-10), and monokine induced by
IFN-␥ (MIG)) and membrane-associated molecules (e.g., B7H1
and B7DC) that have been shown to have immunomodulatory effects upon lymphocytes. Cocultures of the TAF with autologous
TAT establish that the heterogeneous stromal fibroblasts have the
capacity to modulate (i.e., both enhance and suppress) the ability
of the TAT to respond to activation via the TCR.
Materials and Methods
Patient samples
Primary human non-small cell lung tumor tissue from patients undergoing
surgery for the removal of lung tumors was obtained from local surgical
sites, including the Kenmore Mercy Hospital Pathology Laboratory, Millard Fillmore Gates Department of Surgery, and the Tissue Procurement
Facility of Roswell Park Cancer Institute. Tissue was transported in sterile
containers in DMEM/F12 medium supplemented with 10% FCS and penicillin/streptomycin antibiotics (henceforth referred to as “standard culture
medium”) to facilitate tissue survival until cell isolation could be performed. In some cases, patient peripheral blood was also obtained along
with the tumor tissue. Postsurgical histological and pathological diagnoses
were obtained for the tumor specimens anonymously. All tumor and patient
blood specimens were obtained with institutional review board-approved
protocols.
TAF and TAT isolation
A single-cell suspension was created from the tumor tissue by mechanical
disruption of the tissue using a Teflon policeman to force cells through a
size 50 mesh. Cell suspensions were layered over Ficoll-Paque Plus (Amersham Biosciences) and the resulting interface was removed and centrifuged at 1500 ⫻ g, and washed once in PBS. The interface, an enriched
population of CD45⫹ mononuclear cells containing the TAT, was resuspended in a freezing medium consisting of human serum albumin:PBS:
DMSO (8:1:1) and was frozen at ⫺80°C in a “Mister Frosty” slow freezing
device before being transferred to liquid nitrogen storage tanks 1 day later.
The pellet remaining after the Ficoll-Paque Plus separation, along with the
tumor tissue that did not pass through the gross mesh, was put into culture
in 6-well tissue culture treated plates to grow fibroblasts. Within 1–2 wk,
confluent fibroblasts were removed by treatment with trypsin/EDTA for
5–10 min and seeded into tissue culture flasks. All fibroblast cultures were
maintained in standard culture medium and were characterized and used in
coculture experiments after four or five passages.
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microenvironments have also been shown to be capable of modulating leukocyte function, and, in this respect, have been implicated as a regulator of the switch from acute to chronic inflammation (16). In addition to the secretion of various inflammatory
cytokines, fibroblasts have also been shown to modulate inflammatory leukocytes through the production of chemical mediators,
such as PGE2, and through cell surface molecules such as CD40
(17, 18). Fibroblasts derived from sites of chronic inflammation,
such as the inflamed synovial joints in rheumatoid arthritis patients, possess an imprinted phenotype that is stable in culture even
after a number of passages (18, 19). The rheumatoid fibroblasts
exhibit important differences in their expression of cell signaling
molecules, cell adhesion molecules, and cell proliferation. For example, rheumatoid arthritis synovial fibroblasts display enhanced
NF-␬B activation status, up-regulated production of matrix metalloproteinases, and increased cell proliferation (20). These phenotypic differences also result in distinct functional differences between fibroblasts from rheumatoid and nonrheumatoid joints in
terms of the ability of inflammatory fibroblasts to influence lymphocyte homing, survival, and differentiation in the rheumatoid
synovial joints (20).
In addition to their role in shaping the inflammatory microenvironment of the synovial joints in rheumatoid arthritis, fibroblasts
have also been shown to regulate the Th1-Th2 balance in a murine
model of granulomatous inflammation by producing different cytokines, chemokines, and extracellular matrix (ECM) components.
Other studies with transgenic mice have demonstrated that the spatial and temporal production of various cytokines, such as IL-1 and
TGF-␤, can regulate the constituents and persistence of inflammatory
infiltrates resulting in tissue damage and fibrosis. In addition to their
direct and indirect roles in mediating inflammation, fibroblasts also
play a role in normal lymphocyte developmental regulation in the
thymic microenvironment along with thymic epithelial cells (21).
Specifically, the maturation of CD4⫺CD8⫺ T cell precursors to the
CD4⫹CD8⫹ stage is dependent upon the presence of fibroblasts
and MHC class II⫹ thymic epithelial cells (22).
In view of the many well-established effects of fibroblasts on
lymphocytes in nonmalignant tissues, we have focused here upon
the characterization of tumor-associated fibroblasts (TAF) derived
from human non-small cell lung tumors, and upon the capacity of
5554
TAF MODULATE TAT
Table I. Summary of the histological diagnoses for each of the human
non-small cell lung tumors from which TAF cultures were derived
Histological Type of Tumor
Human Lung Tumor Patient Numbers
Adenocarcinoma
Squamous cell carcinoma
Large cell carcinoma
Bronchioalveolar carcinoma
Unknown type of NSCLC
Carcinoid tumor
Metastatic non-small cell
lung carcinoma
Small cell lung carcinoma
1, 2, 4, 6, 7, 12, 17, 18, 19, 26
3, 5, 8, 13, 16, 21, 22, 24, 25
15, 20
11
9, 14
10
23
27
Patient peripheral blood was also separated by centrifugation over a
layer of Ficoll-Paque Plus. The interface was washed once in PBS and
frozen in the same manner as the tumor-associated mononuclear cells.
Phenotyping the primary fibroblast cultures
Assessment of TAF cytokine production
Production of IFN-␥, MIG, IP-10, RANTES, IFN-␣, TGF-␤1, and IFN-␤
by TAF was assessed by intracellular flow cytometry and ELISAs on the
TAF culture supernatants. Intracellular flow cytometry was performed as
described above and the following mouse anti-human Abs were used at a
concentration of 1 ␮g/assay: IFN-␥, MIG, IP-10, RANTES (PE conjugated; BD Biosciences), and IFN-␣ (PE conjugated; Chromaprobe). Appropriate isotype controls for each of the Abs were also purchased from BD
Biosciences. Results were analyzed in our laboratory by WinList software
(Verity) and reflect the percentage of positive cells compared with the
isotype controls. A human TGF-␤1 Quantikine ELISA kit was acquired
from R&D Systems (to assess TAF production of TGF-␤1, and ELISAs for
the presence of human IFN-␥, MIG, and IP-10 in the TAF culture supernatant fluids were also performed, using Abs from Endogen and 96-well
plates from Nunc, on the TAF culture supernatant fluids.
TAF expression of coregulatory molecules
Fibroblasts were isolated from culture flasks as described above and
stained with PE-conjugated primary Abs (eBioscience) that bind to the
following B7 family coregulatory molecules: B7H1 (PD-L1), B7DC (PDL2), B7H2 (B7h, B7RP1), B7H3, B7H4 (B7S1, B7x), PD-1, and inducible
costimulator. All of the Abs were mouse anti-human, except for the B7H3
Ab, which was rat anti-mouse, though this particular Ab is also crossreactive with human B7H3. Flow cytometry and analysis were performed
as previously described.
Immunohistochemistry for TGF-␤1 in tumor tissue sections
Immunohistochemistry was performed on fixed tissue sections of original
lung tumors to assess the presence of TGF-␤1. Fresh pieces of tumor were
processed by the University at Buffalo Histology Service Laboratory as
FIGURE 2. Flow cytometry for fibroblast surface markers to define their phenotype and coregulatory molecule expression. A, Primary cultures of TAF
were stained extracellularly with the human fibroblast-specific SM1214P Ab, HLA-A, -B, -C (class I), CD45, CD11b/Mac-1, or Thy1/CD90. B, They were
also stained for FAP, ␣-SMA, B7H1 (PD-L1), or B7DC (PD-L2) both before and after 24 h treatment with IFN-␥. In the representative histograms, the
unshaded region represents the isotype control or autofluorescence and the shaded region represents the human-specific Ab tested. The percentage of
positive cells is shown for each Ab tested.
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Fibroblasts after four or five passages were isolated from culture flasks by
treatment with trypsin/EDTA for 5–10 min. Fresh medium was then added
to quench the trypsin and the cells were pelleted by centrifugation. Mouse
IgG (for directly conjugated Abs) or goat IgG (for unconjugated Abs) was
used to block nonspecific binding before the addition of the following
primary mouse anti-human Abs: SM1214P (unconjugated) (Acris Antibodies), CD45 (FITC or allophycocyanin conjugated; BD Biosciences),
HLA-A, -B, -C (class I; allophycocyanin conjugated; BD Biosciences),
Thy1/CD90 (allophycocyanin conjugated; BD Biosciences), and fibroblast
activation protein (FAP; unconjugated) (Calbiochem; EMD Biosciences).
After incubation on ice, cells were washed in PBS and then pelleted by
centrifugation. Goat anti-mouse Alexa 488 was then added to samples that
contained unconjugated primary Abs, and the cells were incubated on ice,
washed once in PBS, and then pelleted by centrifugation. A Fix and Perm
Cell Permeabilization kit (Caltag Laboratories; Invitrogen Life Technologies) was used to perform intracellular staining for ␣-smooth muscle actin
(␣-SMA; PE conjugated; R&D Systems), and mouse IgG was used as a
block during the permeabilization steps. After completion of cell staining,
all samples were fixed in 2% ultrapure formaldehyde. Flow cytometry was
performed using a FACSCalibur flow cytometer (BD Biosciences; University at Buffalo, State University of New York) and analyzed in our laboratory by WinList software (Verity). Appropriate isotype control Abs were
acquired from BD Biosciences and the results reflect the percentage of
positive cells compared with the isotype controls.
The Journal of Immunology
FIGURE 3. TAF constitutively produce IFN-␤, IFN-␥, and IP-10. The
culture supernatant fluids of a number of TAF cultures was collected and
assessed by ELISA for the presence of IFN-␤, IFN-␥, IP-10, and MIG.
TAF constitutively produced and secreted IFN-␤, IFN-␥, and IP-10, although MIG (ⴱ) was below the level of detection in the collected supernatant fluids.
Coculture experiments of autologous TAF and TAT
Coculture experiments were performed between eight autologous sets of
TAF and TAT. More autologous pairs were not available due to the inability to culture TAF from every tumor, the limited amount of TAT due
to a lack of sufficient tumor tissue, and the use of TAT for other experiments. Five of the eight TAT used in these experiments were analyzed by
FACS for their expression of CD3, CD4, CD8, and CD20. The percentage
of CD3⫹ T cells in these populations ranged from 15 to 33%, and included
both CD4⫹ (6 –21%) and CD8⫹ (3–15%) T cells. In four of the cell preparations, the percentage of CD20⫹ B cells was 1.5% or below. A more
complete phenotypic analysis of two of the TAT populations revealed that
the T cells had a phenotype of memory cells, i.e., CD3⫹, CD45RO⫹,
CXCR3⫹, and CD62L⫺. Previous studies from our laboratory have more
extensively characterized the phenotype of T cells isolated from six different NSCLC (including three of the major histologic types) and shown
them to express a similar phenotype that is associated with effector memory
T cells, i.e., CD3⫹, CD45RO⫹, CXCR3⫹, CD11a⫹, CD44⫹, IL-12R⫹,
CD28⫹, and CD62L⫺ with a subset expressing CD161 (23, 24). Less than
1% of the cells derived from the tumor tissues were CD56⫹ and monocytes
ranged from 0.4 to 8.1% of the cells in the TAT population. TAF were split
into 12-well tissue culture-treated plates and grown to confluence in
DMEM/F12 plus 10% FCS medium. The population of enriched CD45⫹
mononuclear cells containing the TAT was thawed and added to the wells,
along with 20 ␮l of DynaBeads coated with anti-CD3 and anti-CD28 (T
Cell Expander kit; Dynal Biotech; Invitrogen Life Technologies). The total
volume in each well was 2 ml and fresh medium was added back at each
time point to maintain the same volume throughout the experiment. In
some experiments, additional factors were added to the coculture conditions, including anti-TGF-␤1 function blocking Ab (5 ␮g/ml; R&D Systems), recombinant human TGF-␤1 (10 ng/ml; R&D Systems), anti-B7H1
(5 ␮g/ml; eBioscience), or anti-B7DC (5 ␮g/ml; eBioscience). Aliquots of
each well were taken at days 3, 5, and 7, and the various factors that were
being tested were also added, in addition to medium, on days 3 and 5.
Coculture experiments were also performed in the same manner with
Transwell plates (Corning; Costar). To maintain cell separation, a polyester
membrane with a 0.4-␮m pore size was used. TAF were grown to confluence in the lower chamber and TAT and DynaBeads were added to either
the upper or lower chamber. At the start of the coculture experiments, 1 ml
of medium was added to the upper chamber and 1 ml to the lower chamber.
Aliquots were taken from both the upper and lower chambers at the various
time points. All other conditions, including other factors added to the conditions, were the same as for the regular 12-well plates, as described previously. Culture samples were assayed for IFN-␥ production by ELISA as
previously reported (25).
Results
Establishment of primary TAF cultures
The human non-small cell lung tumor microenvironment is a complex tissue involving dynamic interactions between a number of
cellular and noncellular components (Fig. 1, A and B). Tumor cells
are characterized by their large irregular shape with heterochromatic nuclei. Stromal cells including lymphocytes, plasma cells,
blood vessels and fibroblasts are labeled in the sections (Fig. 1).
The fibroblast is one of the major cellular constituents of the tumor
microenvironment in tumors of the breast, colon, stomach, and
pancreas (13). To demonstrate the predominance of fibroblasts in
our lung tumor specimens, a trichrome stain, for collagen and fibroblasts, was applied to NSCLC tissue sections. The dominant
presence of fibroblasts and ECM and their juxtaposition to the
tumor cells in all of the NSCLC studied was clearly established in
the trichrome images (Fig. 1, C and D).
To characterize and study the role of TAF in the lung tumor
microenvironment, we established primary cell cultures directly
from 27 different lung tumor biopsy tissues. The adherent cells,
which possessed a fibroblast-like morphology (i.e., adherent,
flat, elongated cells with cytoplasmic extensions and flat, ovalshaped nuclei) were successfully cultivated from NSCLC tissue
including adeno, squamous, and large cell carcinomas. Table I
shows the histological diagnosis for each of the 27 tumors from
FIGURE 4. TAF constitutively produce IFN-␥, IP-10, MIG, and RANTES intracellularly. Cells from several TAF cultures were assayed for the presence
of intracellular cytokines by flow cytometry. TAF constitutively produced IFN-␥, IP-10, RANTES, and low, but detectable, levels of MIG. The results
presented are representative of three different cultures, that yielded average values of 14.3 ⫾ 2.84% for IFN-␥, 67.62 ⫾ 11.62% for IP-10, 8.05 ⫾ 3.63%
for MIG, and 40.66 ⫾ 1.46% for RANTES.
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previously described and numerous serial sections of paraffin-embedded
tissue blocks were generated on glass slides for use with immunohistochemistry protocols. A DakoCytomation HRP kit (DakoCytomation) and
an unconjugated mouse anti-human TGF-␤1 mAb (BioSource International; Invitrogen Life Technologies) were used to stain different tumors, and
an appropriate isotype control was also run on a serial section. The slides
were assessed by light microscopy and the expression of TGF-␤1 was
monitored in the stroma, tumor cells, fibroblasts, and lymphocytes in the
section. Images were taken with a Sony color video camera CCD SSC-S20
mounted on an Olympus BX40 light microscope using Snappy Video
Snapshot 4.0 software (Play).
5555
5556
TAF MODULATE TAT
Table II. Constitutive presence of TGF-␤1 detected in TAF culture
supernatant fluids by ELISA
Primary TAF Culture Supernatant
Fluid, Patient No.
TGF-␤1 (pg/ml/24 h) as
Detected by ELISA
5
6
7
8
9
10
27
292.3
473.9
182.4
197.3
168.7
101.1
144.9
TAF expression of the B7 family coregulatory molecules B7H1
(PD-L1) and B7DC (PD-L2)
The B7 family of coregulatory molecules contains many different
members, some of which transmit stimulatory signals to T cells
and others which transmit inhibitory signals (7, 36). The expression of B7H1 has been noted previously on a number of different
cell types, including tumor cells, dendritic cells, and certain lymphocyte subsets (37– 44), but it has not been previously reported to
be expressed on fibroblasts. Using flow cytometry, we establish
here that a subset of lung TAF constitutively express the B7 family
coregulatory molecules B7H1 (PD-L1) and B7DC (PD-L2) (Fig.
2B). As has been previously demonstrated in other cell types, the
expression of these molecules is up-regulated on the TAF by treatment with IFN-␥ (Fig. 2B). The expression of B7H1 ranged from
63 to 96% of the fibroblasts and for B7DC the range was 13– 82%.
TAF did not express B7H2 (B7h, B7RP1), B7H3, or B7H4 (B7S1,
Characterization of TAF based upon their expression of other
cell surface markers
Fibroblasts found in normal tissues are known to be heterogeneous
with respect to their function and phenotype. One of the major
distinguishing cell surface phenotypic markers of a subset of human and murine fibroblasts is Thy1 or CD90 (29, 30). Thy1⫹ and
Thy1⫺ subpopulations can be isolated from fibroblasts derived
from the orbit, spleen, female reproductive tract, and the lung (30 –
34). Thy1⫹ and Thy1⫺ fibroblasts differ in their ability to produce
cytokines and express MHC class II peptides, though they do not
differ in terms of growth rate. All 15 of the primary TAF cultures
Table III. TGF-␤1 detected in situ by immunohistochemistry in original
NSCLC tissue sections
TGF-␤1 Expression as Detected by IHCa
Histological Diagnoses
of Tumor
Fibroblasts
ECM
Tumor Cells
Lymphocytes
Adenocarcinoma
Adenocarcinoma
Squamous
Unknown type
Unknown type
Squamous
Unknown type
Adenocarcinoma
⫹
⫹
⫹
⫺
⫺
⫺
⫹
⫹
⫹
⫹
⫹
⫹
⫹
⫺
⫹
⫹
⫺
⫹
⫺
⫺
⫹
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
⫺
a
There was no positive staining with a mouse IgG1 isotype control Ab.
FIGURE 5. Immunohistochemistry demonstrates the in situ presence of
TGF-␤1 in non-small cell lung tumor-derived fibroblasts and ECM, but not
tumor cells or lymphocytes. A, A ⫻100 image of one representative tumor,
showing fibroblasts/ECM, tumor cells, and lymphocytes. B, Dense staining, indicating the presence of TGF-␤1, is seen in this ⫻400 image of
fibroblasts/ECM. C, In this ⫻400 image of the tumor cells, there is no
positive staining for TGF-␤1. D, As is the case with tumor cells, no staining for TGF-␤1 is visible in this ⫻400 image of the tumor-associated
lymphocytes. Although not shown, a mouse IgG1 isotype control was also
run. There was no nonspecific staining with the isotype control Ab. tu,
tumor cells; ly, lymphocytes; fb, fibroblasts.
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which we derived primary TAF cultures. There were 26 nonsmall cell tumors, including squamous cell carcinomas, adenocarcinomas, and large cell carcinomas, in addition to the 1 small
cell carcinoma. To confirm that these cells were indeed fibroblasts, and not contaminated with tumor cells or tumor-associated leukocytes, the adherent cells were assayed by flow cytometry and immunofluorescence microscopy using the
SM1214P Ab, which is specific for a 112-kDa molecule found
on the surface of human fibroblasts (26 –28). As shown in Fig.
2A, the adherent cells stained positively with the SM1214P Ab
by flow cytometry in each of the 15 primary cultures tested.
Immunofluorescence microscopy with the SM1214P Ab demonstrated diffuse punctate staining on the entire surface of the
adherent cells (data not shown). Furthermore, all of the 20 primary NSC tumor lines tested, as well as normal human PBMC,
did not stain positively using the SM1214P Ab (data not
shown). The primary fibroblasts in our cultures were positive
for HLA class I, thereby confirming that they were human cells
(Fig. 2A), and were negative for CD45 and CD11b/Mac-1 (Fig.
2A), thereby establishing that they were not contaminated with
human lymphocytes and monocytes/macrophages. These studies confirm the identity of the cultures as TAF for further characterization of cell surface marker expression.
tested were shown to stain positively for Thy1 by flow cytometry
(92–100%) (Fig. 2A), suggesting they have the potential to play an
active role in the tumor microenvironment.
We also assessed the expression of FAP, a serine protease that
can potentiate tumor growth, and ␣-SMA, a characteristic marker
associated with differentiated myofibroblasts that have been reported to be present in other tumor microenvironments (35). Twenty-three to 39% of the cells in the TAF cultures were positive for
FAP, while the majority of the fibroblasts (65%) were positive for
␣-SMA (Fig. 2B). These results suggest that the fibroblasts from
the human lung tumor microenvironment are less heterogeneous
than fibroblasts derived from normal tissues and express proteins
found in other tumor microenvironments. Having established and
characterized primary cultures of human TAF that were not contaminated with other cell types, we set out to determine whether
the lung TAF express B7 family coregulatory molecules and produce biologically active molecules which have the potential to alter lymphocyte function.
The Journal of Immunology
5557
B7x) either constitutively or inducibly following treatment with
IFN-␥ (data not shown). The lung TAF also did not express B7
family receptors, such as PD-1 or inducible costimulator (data not
shown). The expression of B7H1 and B7DC on the TAF represents
one potential mechanism by which fibroblasts in the tumor microenvironment could be regulating lymphocyte function. This possibility is addressed later.
TAF production and secretion of cytokines and chemokines
The expression of Thy1, shown to be associated with cytokine
expression and cell differentiation, and B7 regulatory molecules
suggests that the TAF could play an active role in the tumor microenvironment. Both intracellular expression using FACS and
ELISA analysis of culture supernatants were used to evaluate cytokine production by TAF. Fibroblasts are known to produce type
I IFN. Consistent with this finding, the TAF produced low levels
of IFN-␤ (Fig. 3). Surprisingly, the lung TAF produced IFN-␥
(Fig. 3), a type II IFN previously believed to be produced primarily
if not exclusively by lymphocytes. Secretion of IP-10 by TAF was
also observed (Fig. 3). The lung TAF were further assayed for
chemokines production by FACS analysis. A significant percentage of TAF stained positively for IFN-␥ (12.5%), RANTES
(40.2%) and IP-10 (60.2%), and fewer cells were positive for MIG
(6.2%) (Fig. 4). The presence of these chemokines in the TAF,
particularly IP-10 and MIG, are considered significant, as they are
responsible for attracting and retaining activated T cells into the
tumor microenvironment (45).
In addition to the production of the proinflammatory cytokines
and chemokines, the TAF were evaluated for production or immunosuppressive cytokines, specifically TGF-␤1. This cytokine,
shown to have potent immunosuppressive effects upon T cell function in the tumor microenvironment (46, 47), was produced by
TAF, as shown in Table II. Although TGF-␤ was produced by all
of the 10 different TAF tested so far, this cytokine was produced
by only two of four NSCLC tumor cell lines that have been assayed and the amount produced by the tumor cell line was 2- to
5-fold less than that produced by TAF (data not shown).
Because the in vitro cultivation of the TAF and tumor cell lines
may have influenced the cells ability to produce TGF-␤, the fibroblasts and tumor cells were monitored for the expression of this
cytokine before being put into culture. The in situ production of
TGF-␤1 by fibroblasts, tumor cells, and lymphocytes was assayed
by immunohistochemistry of fresh lung tumor biopsy tissues.
TGF-␤1 was present in the fibroblasts or the ECM in seven of
eight tumors tested (Table III). Images for a representative tumor
on which immunohistochemistry for TGF-␤1 was performed are
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FIGURE 6. TAF enhance activated TAT IFN-␥ production in a contact-dependent manner. A, TAF enhance
TAT IFN-␥ production following TCR activation. This
enhancing effect was noted in five of the eight tumors
tested. B, The TAF-mediated enhancement of TAT
IFN-␥ production following TCR activation is dependent upon cell contact. When the autologous coculture
of TAF and TAT was performed in Transwell plates
with cell impermeable 0.4-␮m polyester membrane
pores, no enhancing effect was observed indicating that
the TAF-mediated enhancement requires cell contact
between the TAF and the TAT. The results from two
representative tumors are depicted in B.
5558
TAF MODULATE TAT
shown in Fig. 5. A ⫻100 image is shown in Fig. 5A, which
illustrates the microenvironment of this tumor, and higher magnification images (⫻400) from this section containing fibroblasts/ECM, tumor cells, and lymphocytes are shown in Fig. 5,
B–D, respectively. In this particular tumor, positive staining for
TGF-␤1 was present in the fibroblasts and the ECM (Fig. 5B), but
not in the tumor cells (Fig. 5C) or lymphocytes (Fig. 5D). Tumor
cells stained positive for TGF-␤1 in only two of the eight tumors
tested, and no TGF-␤1 staining of lymphocytes was observed in
any of the tumors tested (Table III). No staining was observed in
any of the tumors using an isotype control Ab (data not shown).
These results, that fibroblasts in the tumor microenvironment
rather than the tumor cells, are the major cell type producing
TGF-␤ in addition to their close association of fibroblasts with
lymphocytes, strongly support the potential of fibroblasts to modulate lymphocyte function within the microenvironment of human
non-small cell lung tumors.
To establish whether fibroblasts had the capacity to modulate lymphocyte function, coculture experiments were initiated. CD45⫹
cells were enriched from single-cell suspensions derived from
fresh human non-small cell lung tumor biopsy tissues. These cell
suspensions, which contained CD3⫹CD4⫹ and CD3⫹CD8⫹ T
cells with an effector memory phenotype (24), were frozen and
stored in liquid nitrogen until primary TAF cultures were established from the same tumor biopsy tissues. At this point, the enriched CD45⫹ population of cells was thawed, and the response of
the TAT to activation via the TCR was assessed either with or
without cocultivation of the TAT with autologous TAF. Activation
of the TAT within the CD45⫹ cell suspension was initiated by the
persistent cross-linking of CD3 and CD28 on the TAT by antiCD3/anti-CD28 Abs immobilized on synthetic beads. The response to this prolonged activation signal was monitored by quantifying the level of IFN-␥ in the culture supernatant fluids
(picograms per milliliter) at days 3, 5, and 7 following activation.
In five of the eight coculture pairs tested, TAF enhanced the response of TAT to activation via the TCR. Fig. 6A depicts all five
of the patient tumors in which TAF enhanced autologous TAT
activation. The increase in the level of IFN-␥ was observed at all
three time intervals (i.e., days 3, 5, and 7). Each panel in Fig. 6A
represents a single experiment with a pair of TAT and TAF derived from a different tumor. Although the amount of IFN-␥ produced in response to TCR activation varied in each coculture, the
increase in IFN-␥ when TAF were present was observed in all five
pairs of cells and this pattern was repeated at least twice with each
of the cell pairs. Differences in the amount of IFN-␥ produced was
likely due in part to both the percentage of T cells (which ranged
from 15 to 20%) and the viability of the cells that varied from 26
to 33% at the end of each experiment. Less than 1% of the CD45⫹
enriched cells were NK cells and 0.4 – 8% were monocytes. All of
the fibroblast lines expressed B7H1 and B7DC and produced
TGF-␤1.
Consistent with the assumption that the response being monitored is that of the T cells to TCR stimulation, no IFN-␥ was
detected when TAT, either alone or in coculture with TAF, were
cultured without the anti-CD3/anti-CD28 stimulus. We conclude
that TAF from the majority of lung tumors tested have the capacity
to enhance the response of T cells derived from the same tumor.
No correlation was observed with the ability of the TAF to augment the TAT response and the histological type of the NSCLC.
To obtain further insight with respect to how the fibroblasts were
able to modulate the lymphocyte response to activation, a protocol
FIGURE 7. In some tumors, TAF suppress the activation of TAT. A, In
three of the eight tumors tested, TAF suppressed the activation of TAT
when cocultured together. B, In one of the three tumors for which TAF
mediated a net suppressive response on TAT activation, blockade of B7H1
and/or B7DC was able to completely reverse the suppression.
was designed to determine whether the enhancing effect was due to
a diffusible factor or required a direct cell-to-cell interaction.
TAF enhancement of TAT IFN-␥ production in response to TCR
stimulation requires direct cell contact
To determine whether the enhancing response was cell contact
dependent, the coculture experiments with TAF and TAT were
repeated in Transwell plates with the two cell types separated by a
cell-impermeable, 0.4-␮m polyester membrane. It was determined
that the enhancing effect of TAF on TAT response to TCR stimulation was only observed when the two cell types were in direct
cell contact, as the enhancing effect was totally abrogated when
they were separated by the Transwell membrane (Fig. 6B). In fact,
suppression was sometimes noted when the two cell types were
separated by the Transwell membrane. This observed suppression
suggests that a soluble factor may mediate a down-regulation of
the T cell activation potential that is not recognized when the TAF
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Coculture of TAF with TAT has an enhancing effect upon the
response of the T cells to activation via the TCR
The Journal of Immunology
5559
of TAT and TAF. This scenario further illustrates the fact that the
interaction of TAF with TAT is complex and that there are coexisting positive and negative factors that are present enabling fibroblast modulation of T cell activation and function.
Discussion
FIGURE 8. The role of endogenous and exogenous TGF-␤1 in TAF
regulation of TAT. The addition of recombinant human TGF-␤1
suppresses TAT IFN-␥ production, and the addition of an anti-TGF-␤1blocking Ab enhances TAT IFN-␥ production. The results for one representative tumor are shown.
Coculture of TAF with TAT can also have an inhibitory effect
upon the response of TAT to activation via the TCR
As indicated above, TAF derived from five of eight tumors tested
had a net enhancing effect upon the response of autologous TAT to
activation. However, TAF derived from three of the eight tumors
had a suppressive effect upon T cell activation when cocultured
with the TAT derived from the same tumor. The three panels in
Fig. 7A represent data derived from a single experiment with a pair
of TAT and TAF derived from three different tumors. Each of
these experiments were repeated at least two times and the same
pattern (i.e., inhibition of IFN-␥) was again observed in each of the
three coculture experiments. We were unable to establish a correlation of the inhibitory activity of the fibroblasts and their expression of coregulatory molecules and the production of TGF-␤1. The
percentage of T cells in the CD45⫹-enriched population varied
from 14 to 33% and the viability of the cells ranged from 14 to
46%. Again, no correlation was observed with the ability of the
TAF to suppress the TAT response and the histological type of the
NSCLC.
In one of the three tumors for which suppression of the TAT
response by TAF was noted, we observed that this inhibition was
reversed by anti-B7H1- and anti-B7DC-specific function-blocking
Abs (Fig. 7B). This indicates that one of the mechanisms by which
TAF can suppress TAT activation involves interactions among
members of the B7 family of coregulatory ligands and their cognate receptor(s).
Inhibitory effects of TGF-␤ on TAT response to TCR stimulation
when cocultured with autologous TAF
Because we had established that TAF constitutively produce significant quantities of the inhibitory cytokine TGF-␤1, we set up
experiments to assess whether this cytokine could be dampening
the contact-dependent enhancing effect of TAF in coculture with
the autologous TAT. Although it was observed that coculture of
TAT with autologous TAF resulted in an increased IFN-␥ response
by TAT (in the presence of TCR stimulation) when the cells were
in direct contact, the response could be partially suppressed by the
addition of exogenous TGF-␤1 (Fig. 8). Blocking endogenous
TGF-␤1 using a function blocking Ab was able to further enhance
the response (Fig. 8). This suggests that TAF-produced TGF-␤1 is
capable of exerting an inhibitory effect on TAT activation, despite
the fact that a net enhancement is observed in five autologous pairs
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and TAT are in direct contact due to the dominant-enhancing effect
that is dependent on cell-to-cell contact. This possibility was addressed and our findings are presented in the last section of the
results.
We establish here, for the first time, that fibroblasts derived from
the microenvironment of human lung tumors have the capacity to
modulate the function of T cells derived from the same tumor
microenvironment. The effect of the TAF on the response of TAT
to a strong and persistent stimulation via the TCR is complex and
includes both enhancing and inhibiting activities. In coculture experiments of autologous TAF and TAT, a net enhancing effect on
the activation of TAT was observed with five of the eight pairs of
cells tested. The augmentation of the response was manifested by
an increase in the amount of IFN-␥ that was produced in response
to the cross-linking of CD3 and CD28. This enhanced response
persisted for at least 7 days and was dependent on the direct interaction of the TAF with the TAT. When the TAF and TAT cells
were separated in coculture experiments in Transwell plates, augmentation of TAT activation was eliminated.
Superimposed on the contact-dependent net augmentation of the
TAT response, we observed an inhibitory effect of the TAF that
was mediated by TGF-␤1. This was determined from the observation that IFN-␥ levels were increased when the activated TAT
were cocultured with TAF in the presence of function blocking
anti-TGF-␤1 Abs. This increase due to the blockade of endogenous TGF-␤1 in the coculture system was observed whether the
TAT and TAF were incubated together or separated by a Transwell
cell impermeable membrane. Furthermore, the addition of exogenous TGF-␤1 was also able to decrease the IFN-␥ levels observed
in the TAF/TAT cocultures. Previous studies established that T
cells isolated from the microenvironment of human lung tumors
are hyporesponsive to a brief stimulation of the TCR (2). This T
cell hyporesponsiveness was determined to be due to endogenous
TGF-␤1 present within the tumor microenvironment (4). We conclude that TAF have the capacity to modulate the response of the
TAT to activation and that this fibroblast effect includes both providing positive signals that require close association between TAF
and TAT and negative signals mediated, at least in part, by soluble
factors including TGF-␤1, to the TAT.
A consistent net inhibitory effect was observed in coculture experiments with three of the autologous TAF and TAT pairs. Two
coregulatory molecules, B7H1 (PD-L1) and B7DC (PD-L2), were
observed on all of the TAF tested, and their expression was enhanced by treatment with IFN-␥. Because both of these members
of the B7 family have been shown to suppress T cell activity (7),
the possibility that they could be involved in the TAF-mediated
suppression of TAT was addressed in two of the autologous TAF/
TAT pairs for which the net inhibitory effect was observed. In one
of these pairs, this inhibitory effect was completely reversed when
the TAF and TAT were cocultured with either anti-B7H1 or antiB7DC function blocking Abs. Although this reversal of the TAF
inhibition was observed in only one of the two TAF/TAT pairs
tested, it demonstrates yet another potential mechanism by which
fibroblasts can alter the response of T cells within the tumor microenvironment and highlights the complexity of the influence that
stromal fibroblasts have on T cells within the tumor microenvironment. Previous studies have demonstrated the abundant presence of B7H1 on human carcinomas of the lung, ovary, breast,
head/neck, and colon, in addition to melanomas (39, 42, 48, 49).
These studies have also established the ability of coregulatory molecules to promote the apoptotic cell death of activated tumor Agspecific T cells in vitro (42, 48). We also observed the expression of
5560
TAF production and secretion of the chemokines IP-10 and
MIG is potentially highly significant for two reasons. Both of these
glutamate-leucine-arginine-negative CXC chemokines have the
capacity to attract and retain activated T cells (58, 59), and they
both have been shown to have significant angiostatic activity that
contributes to the inhibition of human NSCLC tumorigenesis and
spontaneous metastases (60, 61). Collectively, these findings illustrate the importance and potential capacity of fibroblasts in the
tumor microenvironment to attract, retain, and modulate the activation of tumor-associated lymphocytes and to directly and indirectly alter the local progression and metastasis of human tumors.
In view of the multiple biologically active molecules produced by
fibroblasts and their recognized heterogeneity, our observation of
both enhancing and suppressing effects of TAF on autologous TAT
is not surprising. Although we are not aware of any other reports
on the cocultivation of human TAF and TAT, there are several
reports on the cocultivation of TAF with tumor cells in vitro and
in vivo. These studies have revealed that fibroblasts can both inhibit and enhance tumor growth. The cocultivation of fibroblasts
with mammary carcinoma cells exhibits a more orderly histological differentiation (62, 63) and basal cell carcinoma cells grown in
association with stromal cells demonstrated a loss of their malignant properties (64). In contrast, other studies revealed that TAF
have a growth-stimulating effect upon tumors in vitro (65– 67) and
in vivo (68 –70). These studies support the concept that TAF contribute directly to the promotion of tumor growth but also have the
capacity to inhibit tumor growth.
The accumulating evidence that fibroblasts are able to modulate
the behavior of malignant cells has provoked interest in developing
novel cancer therapeutic strategies that target TAF (71). Clinical
trials that 1) target tumor signals responsible for the recruitment or
development of TAF, 2) target TAF signals that initiate and promote tumor growth and facilitate invasion and metastasis, and 3)
eliminate the TAF to abolish all of the fibroblast activities, have
already been instituted or are at the preclinical development stage
(54)). In view of the results presented here that some TAF are able
to enhance the function of TAT and produce and secrete chemokines that are known to inhibit the progression of human tumors,
caution should be exercised in the development and testing of
these new therapeutic approaches. This would be particularly true
for those therapies that are designed to completely eliminate TAF.
Although much remains unknown about the actual mechanisms
by which fibroblasts in the tumor microenvironment, and other
sites of chronic inflammation as a whole, regulate lymphocytic
responses, efforts will be made to further elucidate the complex
possibilities. We show here a variety of ways in which TAF can
affect the function of TAT and we expect that additional mechanisms that govern the TAF/TAT interactions will be discovered.
Based upon differences in function and the varying expression levels of cytokines, chemokines, and coregulatory molecules in TAF,
we expect that subsets of fibroblasts exist within the tumor microenvironment with different functional capacities enabling them to
enhance or suppress T cell activation. Efforts are currently underway with the production of mAbs to fibroblasts that will help to
define these subsets by surface markers that would be unique to
each subset. We do not believe that fibroblast heterogeneity and
their interaction with lymphocytes are properties unique to the tumor microenvironment, and, as such, we expect that fibroblasts
from other tissues, particularly at sites of chronic inflammation,
will have similar characteristics in terms of their phenotype and
functional ability to regulate lymphocytes. This could partially be
attributable to the conditioning of fibroblasts by inflammatory signals within a microenvironment or to the recruitment of specific
subsets of fibroblasts to sites of inflammation, wound repair, or
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both B7H1 and B7DC, in addition to other B7 family coregulatory
molecules such as B7H4 and B7H2, on tumor cell lines derived
from our primary human non-small cell lung tumors (data not
shown). The finding of B7DC on both fibroblasts and tumor cell
lines is significant, because this coregulatory molecule has been
thought to be restricted to monocytes, macrophages, dendritic
cells, and other cells of the myeloid lineage (50 –53). The failure of
anti-B7H1 and anti-B7DC function blocking Abs to reverse the inhibition in one of the TAF/TAT pairs tested may reflect differences in
the expression of PD-1 or other putative receptors for these ligands
on the surface of the TAT. The amount of tumor biopsy tissue
obtained and the number of TAT derived differs for each of the
lung tumors and these variables limit the number of conditions that
can be tested in each experiment. We intend to explore the role of
PD-1 in future experiments using ovarian tumors and tumor ascites
where we are able to obtain greater numbers of TAT from most
patients.
Our findings that fibroblasts have pleiotropic (i.e., both enhancing and inhibitory) effects on the T cells in the tumor microenvironment are not unexpected given the heterogeneity of the TAF
with respect to membrane-associated and soluble factors produced
by these cells. The phenotype of all of the fibroblasts that we
derived from numerous human non-small cell lung tumors was
very similar, as they expressed molecules previously associated
with fibroblasts derived from chronic inflammatory tissues and
other tumors. This phenotype includes a combination of FAP and
␣-SMA (54). Fibroblasts with this phenotype are termed myofibroblasts and these cells are also distinguished by their positivity
for Thy1/CD90 (55). Myofibroblasts from sites of chronic, persistent inflammation have been shown to produce a wide variety of
potentially immunomodulatory factors, including tenacin C,
thrombospondin-1, many different peptide growth factors, PGs,
and high levels of cytokines and chemokines, including IL-1, IL-6,
IL-8, IL-10, TNF-␣, and MCP-1 (16, 56). Relatively few studies so
far have examined whether fibroblasts are a relevant source of
cytokines or chemokines in tumors (14). We demonstrate here that
the TAF are producing at least three significant chemokines, IP-10,
MIG, and RANTES, in addition to cytokines including TGF-␤1,
IFN-␤, and IFN-␥. Finding IFN-␥ production by TAF was unexpected, because there is only one previous report indicating the
production of type II IFN by human fibroblasts (57), and this cytokine has been thought to be produced primarily, if not exclusively, by lymphocytes. The possibility that cultivating the human
fibroblasts in FCS activates the fibroblasts and contributes to their
production of cytokines and chemokines was addressed. Three different TAF primary cultures grown in medium with human serum
produced similar levels of cytokines and chemokines including
TGF-␤, IFN-␥, and IP-10.
Our finding that TAF produce IFN-␥ raises the question of
whether IFN-␥ detected in the TAF and TAT coculture experiments is produced by the TAT or both the TAT and the TAF. Two
things support the notion that the IFN-␥ in the coculture experiments is produced by the TAT. First, control wells containing TAF
alone run as part of the coculture experiments did not test positive
for IFN-␥ production, and second, in coculture experiments with
TAF and TAT, no IFN-␥ was produced unless TCR stimulation
(by anti-CD3/anti-CD28-coated DynaBeads) was provided. Although these findings suggest that the IFN-␥ that we measure in
the cocultures is derived from TCR-activated T cells it remains
possible that other cell types in the CD45⫹-enriched tumor-associated population or TAF in response to soluble factors produced
by the activated lymphocytes are contributing to the production
of IFN-␥.
TAF MODULATE TAT
The Journal of Immunology
cancer. Future studies are required to better understand and define
fibroblast-mediated 1) contact-dependent cell surface receptor/ligand interactions, 2) chemical mediators (i.e., PGs, NO, etc.), and
3) soluble biological mediators (i.e., chemokines/cytokines) that
modulate lymphocyte function.
Acknowledgments
We thank Drs. Hiroshi Takita, Todd Demmy, and Harry Slocum and the
Tissue Procurement Facility at Roswell Park Cancer Institute for providing
us with patient tumor samples, and Liza Pope, Jenni Loyall, and Robert
Parsons for technical assistance.
Disclosures
The authors have no financial conflict of interest.
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