Feb 27, 1995 - of collagenase per ml (Sigma)/1 mM Hepes buffer (GIBCO) in HBSS (GIBCO) for 2-4 h with continual stirring. Lympho- cytes were isolated from ...
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 6547-6551, July 1995
Immunology
T lymphocytes that infiltrate tumors and atherosclerotic plaques produce heparin-binding epidermal growth factor-like growth factor and basic fibroblast growth factor: A potential pathologic role GEORGE E. PEOPLES*, SRULLY BLOTNICKt, KAZUE TAKAHASHIt, MICHAEL R. FREEMAN§, MICHAEL KLAGSBRUNI, AND TIMOTHY J. EBERLEIN*II *Department of Surgery, Division of Surgical Oncology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115; tDepartments of Cell and Developmental Biology, Harvard Medical School, and Surgical Research, Children's Hospital, Boston, MA 02115; tDepartment of Pediatrics, Division of Hematology/Oncology, Children's Hospital, Harvard Medical School, Boston, MA 02115; §Department of Surgery, Urology Research Laboratory, Harvard Medical School, Boston, MA 02115; and IDepartment of Surgery, Children's Hospital, Harvard Medical School, Boston, MA 02115
Communicated by Joseph Murray, Harvard Medical School, Boston, MA, February 27, 1995
ABSTRACT Despite significant infiltration into tumors and atherosclerotic plaques, the role of T lymphocytes in these pathological conditions is still unclear. We have demonstrated that tumor-infiltrating lymphocytes (TILs) and plaqueinfiltrating lymphocytes (PILs) produce heparin-binding epidermal growth factor-like growth factor (HB-EGF) and basic fibroblast growth factor (bFGF) in vitro under nonspecific conditions and in vivo in tumors by immunohistochemical staining. HB-EGF and bFGF derived from TILs and PILs directly stimulated tumor cells and vascular smooth muscle cells (SMCs) in vitro, respectively, while bFGF displayed angiogenic properties. Therefore, T cells may play a critical role in the SMC hyperplasia of atherosclerosis and support tumor progression by direct stimulation and angiogenesis.
fibroblasts, SMCs, and endothelial cells (ECs) (16). bFGF is a powerful mediator of angiogenesis and has been found to play a role in wound healing (17) and tumor vascularization (18). In this study, we investigate the possibility that T lymphocytes may participate in the progression of atherosclerosis and cancer by production and release of HB-EGF and bFGF at the sites of disease.
MATERIALS AND METHODS Materials. Recombinant human HB-EGF corresponding to amino acids 73-147 of the HB-EGF precursor (6) was obtained from J. Abraham (Scios-Nova, Mountain View, CA). Recombinant human bFGF was obtained from Takeda, Osaka. EP-170 cells (19) which are interleukin 3 dependent 32D myeloid cells transfected with the EGF receptor were obtained from J. Pierce (National Institutes of Health, Bethesda). F32 cells (20), which are interleukin 3 dependent BaF3 lymphoid cells transfected with the FGF receptorflg, were obtained from P. Leder (Harvard Medical School, Boston). 197 is an antiHB-EGF polyclonal antibody raised in goats directed against the recombinant 76-amino acid human HB-EGF (containing
T lymphocytes are known to play a key, yet incompletely defined, role in such significant pathological conditions as atherosclerosis (1) and cancer (2). Atherosclerosis is responsible for -50% of the deaths in the United States each year (1). The pathogenesis of this disease has been extensively studied and can be traced to the initial event of macrophages and lymphocytes infiltrating the arterial wall presumably secondary to endothelial injury (3, 4). Macrophages have been shown to produce, among many other cytokines and growth factors, platelet-derived growth factor (5) and heparin-binding epidermal growth factor-like growth factor (HB-EGF) (6), both of which are potent smooth muscle cell (SMC) mitogens. These growth factors are currently believed to be involved in the progression of atherosclerosis (1). The role of lymphocytes in this disease has not been demonstrated. Likewise, T lymphocytes are known to infiltrate tumors (7), but only a small minority of tumor-infiltrating lymphocytes (TILs) have been shown to specifically recognize tumor and, therefore, play a protective role (8-10). Thus far, it has been difficult to correlate the degree of lymphocytic infiltrate with clinical outcome (11). Clearly, in most cancers, despite the lymphocytic infiltrate, the tumors progress. We have recently shown that T lymphocytes isolated from the peripheral blood of healthy donors have the ability to produce HB-EGF and basic fibroblast growth factor (bFGF) (12). HB-EGF is a well-characterized 22-kDa glycoprotein that binds the EGF receptor with high affinity and is mitogenic for fibroblasts, SMCs, and epithelial cells (6, 13). HB-EGF has been shown to play a role in wound healing (14) and the SMC hyperplasia related to pulmonary hypertension (15). bFGF is an 18-kDa ubiquitous protein with strong mitogenic effects on
an additional N-terminal methionine and made in Escherichia
coli) and was kindly provided by D. Damm and J. Abraham (Scios-Nova). 2911 is an anti-HB-EGF polyclonal antibody raised in rabbits against human HB-EGF (Scios-Nova). 77R is an anti-bFGF polyclonal antibody raised in sheep and directed against the recombinant 154-amino acid human bFGF produced in E. coli. The murine anti-human bFGF monoclonal antibody (mAb) used for immunohistochemistry was obtained from Takeda. TILs and Plaque-Infiltrating Lymphocytes (PILs). Five solid tumor (three ovarian and two breast) and three atherosclerotic arterial specimens were minced manually to fragments of 97% CD3+. TIL and PIL CM was collected after 5 days and analyzed for heparin-binding growth factors by HAC. The FPLC fractions were tested for mitogenic activity by thymidine incorporation assays to measure DNA synthesis in 3T3 fibroblasts; EP-170, an EGF receptor-transfected myeloid cell line; and F32, a FGF1 receptor-transfected lymphoid cell line. 0, TIL-derived fractions; 0, PIL-derived fractions. (B) Immunoneutralization of mitogenic activities. Five microliters of the peak fraction from peak 1 and peak 2 was added to 3T3 with increasing concentrations of 197 and 77R antibodies. Recombinant HB-EGF and bFGF were used as controls. Experiments were performed for both TILs and PILs with similar results. Data shown are from TILs.
Immunology: Peoples et aL
Proc. Natl. Acad. Sci. USA 92 (1995)
Perry Laboratories). Primary antibodies were applied simultaneously. Also, frozen sections were labeled with a murine anti-bFGF mAb, GAM/AP, and red substrate followed by rabbit anti-CD3 antibody (Dako), GAR/AP, and blue substrate. Simultaneous negative controls were prepared by omission of the first antibody.
Immunoneutralization of Growth Factor Activities. bFGF was neutralized by using various concentrations of anti-bFGF antibody 77R incubated for 1 h at room temperature with 5 ,ul of the relevant FPLC fractions before addition of the mixture to 3T3 cells. For neutralization of HB-EGF, various concentrations of anti-HB-EGF antibody 197 and 5 ,ul of samples were added directly to 3T3 cells. DNA synthesis was measured by [3H]thymidine incorporation. Recombinant bFGF and HBEGF were used as controls. Immunohistochemistry. Cultured PILs and TILs were labeled on poly(L-lysine)-coated slides rehydrated in Trisbuffered saline with either rabbit anti-HB-EGF antibody 2911 (Scios-Nova) or murine anti-bFGF mAb (Takeda) followed by a goat anti-rabbit mAb (GAR) or a goat anti-mouse mAb (GAM) conjugated with alkaline phosphatase (AP), respectively, and color developed with red substrate (Kirkegaard & Perry Laboratories). Fresh frozen surgical specimens from ovarian and breast cancers were analyzed for T-cell infiltrates by histological staining with hematoxylin and immunohistochemical staining with special attention to blocking endogenous AP activity using citric acid (0.2 M for 15 min and then washed). Sections were labeled with 15 ,ul of a murine antiCD3 mAb (Dako) at a final dilution of 1:50 at room temperature for 1-2 h, GAM/AP (1:1000 dilution for 30 min), and red substrate for at least 10 min to demonstrate the T-cell infiltrate. Frozen sections of tumors were also double-labeled with rabbit anti-HB-EGF antibody (15 gl of 1:100 dilution), GAR/AP (1:1000 dilution), and red substrate followed by murine anti-CD3 mAb (15 ,ll of 1:50 dilution), GAM/AP, and blue substrate with a double AP labeling kit (Kirkegaard &
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RESULTS TILs and PILs Produce HB-EGF and bFGF. Initially, TILs and PILs were directly isolated from fresh surgical tumor and atherosclerotic arterial specimens, respectively. TILs and PILs were maintained for 2-3 weeks in individual cultures after anti-CD3 antibody selection and were demonstrated to be >97% CD3+ prior to use. The CM was collected from the TILs and PILs after 5 days of routine culture without any T-cell receptor-mediated stimulus and was analyzed for heparinbinding growth factors by HAC. Two peaks of mitogenic activity for 3T3 fibroblasts were found eluting at 1.0-1.2 M NaCl and 1.6-1.9 M NaCl, suggestive of HB-EGF and bFGF, respectively (Fig. 1A), in all cultures. To further characterize the 1.0 M peak (peak 1), the fractions were found to stimulate myeloid cells transfected with the EGF receptor EP 170 (Fig. 1A). Likewise, the 1.6 M fractions (peak 2) were mitogenic for F32 lymphoid cells transfected with the FGF1 receptor (Fig. 1A). To confirm the identity of these peaks, antibodies specific to HB-EGF and bFGF significantly neutralized peak 1 and peak 2, respectively (Fig. 1B). Therefore, TILs and PILs appear to produce and release HB-EGF and bFGF-like activities. The release of bFGF is atypical (16, 18), but T cells may
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FIG. 2. Mitogenic activity of TIL- and PIL-derived HB-EGF and bFGF. (A) Mitogenic activity on tumor cells. CM was collected from 2 x 106 TILs after 5 days of routine culture and analyzed by HAC. Fractions of peak 1 and peak 2 containing maximum activity were added to multiple ovarian and breast cancer cell lines in increasing amounts and mitogenic activity was measured by thymidine incorporation assays. Shown are data from two breast cancer cell lines (ATCC 1897 and 1902) and the fresh ovarian cancer cell line from which the TILs were isolated. (B) Mitogenic activity on 3T3, SMCs, and ECs. Increasing amounts of peak 1 and peak 2 fractions from TILs and PILs were added to 3T3 fibroblasts, bovine aortic SMCs, and bovine adrenal capillary ECs in thymidine incorporation assays as described (6, 12, 13). Target cell specificities of TILs and PILs were identical, and the PIL result is shown.
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Proc. Natl. Acad. Sci. USA 92 (1995)
Immunology: Peoples et al.
released in tumors, they could support the growth of the cancer by directly stimulating the tumor and by mediating vascularization of the neoplasm through angiogenesis. PIL-derived HB-EGF and bFGF were mitogenic for vascular SMCs in a dose-response fashion (Fig. 2B), suggesting that, likewise, if these factors were released into the arterial wall of atherosclerotic plaques, they could mediate the SMC hyperplasia characteristic of this disease. Immunohistochemical Staining of PIL and TIL Production of HB-EGF and bFGF. To test directly whether T cells produce HB-EGF and bFGF in vivo, we utilized immunohistochemical staining with growth factor-specific antibodies. We first stained cultured PILs with anti-HB-EGF (Fig. 3A) and antibFGF (Fig. 3B) antibodies to confirm the usefulness of these reagents. Similar staining patterns were seen in cultured TILs, but to investigate the in vivo production of these growth factors we obtained frozen sections of fresh tumors from three ovarian and two breast cancer patients. The sections were histologically stained and labeled with a pan T-cell anti-CD3 mAb to
be uniquely equipped to export bFGF, or T cells may produce a unique factor closely related to bFGF, or both (12). Mitogenic Activity of TIL- and PIL-Derived HB-EGF and bFGF. The effects of the TIL-derived HB-EGF and bFGF were tested directly on tumor cell lines established from breast and ovarian carcinomas. These types of cancer are derived from epithelial cell origin and have been shown by others (21) to be responsive to EGF. Fig. 24 demonstrates the effect of TIL-derived HB-EGF on two breast cancer cell lines and the freshly cultured ovarian tumor from which the TILs were derived. The maximum amount of HB-EGF used in this experiment was the equivalent of a T cell/tumor cell ratio of 1:1, and this dose significantly stimulated these tumors. TILderived bFGF was also mitogenic for these cancer cells (Fig. 2A). In addition, TIL-derived bFGF was mitogenic for ECs (Fig. 2B), suggesting the ability of bFGF to mediate angiogenesis, consistent with the findings of others (16, 18). These mitogenic activities could also be neutralized as in Fig. 1B (data not shown). Therefore, if these growth factors were
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