Elsevier

Oral Oncology

Volume 42, Issue 3, March 2006, Pages 268-274
Oral Oncology

Predominant expression of B7-H1 and its immunoregulatory roles in oral squamous cell carcinoma

https://doi.org/10.1016/j.oraloncology.2005.07.013Get rights and content

Summary

We examined cell surface expression of five B7 costimulatory molecules (B7-H1, B7-DC, B7h, CD80 and CD86) in human oral squamous cell carcinoma (SCC) lines. Most human SCC cell lines expressed various levels of B7-H1 and B7-DC. Their expression was further upregulated by interferon (IFN)-γ stimulation. Immunohistochemical staining revealed substantial and predominant expression of B7-H1 on human primary oral SCC. A murine SCC line, NR-S1, neither expressed B7-H1 nor B7-DC, but induced B7-H1 by IFN-γ stimulation in culture and the inoculation in vivo. Although NR-S1 tumors grew progressively in immunocompetent syngeneic mice, the administration of blocking anti-B7-Hl or anti-PD-1 mAb significantly inhibited the tumor growth, suggesting the negative regulation of host immune responses by the PD-1:B7-H1 pathway. Our results demonstrate that B7-H1 is predominantly induced on oral SCC within the B7 family molecules. A successful inhibition of tumor growth by blockade of the PD-1:B7-H1 pathway may implicate a new approach for immunotherapy of oral SCC.

Introduction

Now five B7 family molecules and their receptors have been identified.1 B7h/B7-RP1,2 B7-H1/PD-L1,3 and B7-DC/PD-L24 are new members of the B7 family molecules. B7h can interact with ICOS expressed on activated T cells, that induces positive costimulation. In contrast, both B7-H1 and B7-DC can interact with PD-1 expressed on activated T cells, that induces negative costimulation. B7h is constitutively expressed on B cells and induced on activated macrophages and dendritic cells (DCs).2 B7-H1 is expressed broadly in most lymphocytes, whereas expression of B7-DC is limited to mature DCs and activated macrophages.5 All three new molecules share about 20–30% homologous to well-known B7 molecules, CD80 and CD86.1 Unlike the limited expression of CD80 and CD86 on hematopoietic immune cells, more abundant expression of these new B7 molecules was observed in tissue cells. B7-H1 was induced by inflammatory stimuli on non-lymphoid tissue cells, such as epithelial cells and muscle cells.6, 7, 8 B7h was also induced on fibroblasts and endothelial cells.9 These molecules are also expressed on tumor cells. Especially, B7-H1 was expressed on various tumor cells3, 10 and was induced or upregulated following treatment with IFN-γ.11 It has been shown that tumor-associated B7-H1 increased apoptosis of tumor-reactive T cells and reduced their immunogenicity.11 The blockade of B7-H1 by neutralizing mAb enhanced anti-tumor T cell-responses by inhibiting immunosuppression in B7-H1-expressing APCs and by preventing tumor-mediated apoptosis through tumor-associated B7-H1.12, 13, 14, 15

In this report, we performed a parallel assessment for five B7 molecules on human squamous cell carcinoma (SCC) cells and examined the functional role of B7-H1 using a murine oral SCC line.

Section snippets

SCC cell lines and culture

The human oral SCC cell lines NA and HSC2, HSC3 and HSC4 were kindly provided by Drs. Y. Kimura (Showa University) and F. Momose (Tokyo Medical and Dental University), respectively. The cell lines Ca922, ZA and HOC313 were established from tumor specimens of oral SCC in our department as previously described.16 KE3 and KE4 were human esophageal SCC generously provided by Dr. K. Itoh (Kurume University). Cells were maintained in D-MEM or RPMI-1640 (Sigma) supplemented with 10% FBS and

Monoclonal antibodies and flow cytometry

The mAbs against human B7-H1 (MIH1),18 B7-DC (MIH14),18 CD86 (IT2.2, mouse IgG2b),19 and CD80 (L307, mouse IgGl)20 were obtained as described previously. A mAb against human B7h (MIH12, mouse IgG1) was generated by immunizing BALB/c mice with human B7h-L cell transfectants and fusing immune splenocytes with P3U1 myeloma cells. The mAbs against mouse B7-H1 (MIH5, rat IgG2a),21 B7-DC (TY25, rat IgG2a, kindly provided by Dr. K. Okumura, Juntendo University),5 B7h (HK5.3, rat IgG2a),22 PD-1 (J43,

Mice

Female 5–8 week-old C3H/HeN (C3H) mice were obtained from Japan Charles River Breeding Laboratories (Kanagawa, Japan), and were maintained under specific pathogen-free conditions. All mice procedures were reviewed and approved by the Animal Care and Use Committee at the Tokyo Medical and Dental University.

Immunohistochemistry

Human tumor specimens were obtained from patients with tongue SCC undergoing surgery in our university hospital, with the approval of the human research review committee and with patients’ informed consent. Mouse NR-S1 tumor specimens were resected from the mice 30 days after NR-S1 inoculation. Frozen tissues were embedded in Tissue-Tek (Sakura, Tokyo, Japan), frozen, and stored at −80 °C until use. Cryostat sections were fixed in absolute acetone and stained with indicated mAbs. For blocking,

Tumor inoculation and evaluation of tumor growth

NR-S1 cells at 5 × 105, 1 × 106, and 5 × 106 cells/mouse were injected subcutaneously into the shaved left back of syngeneic C3H mice, and tumor volumes were evaluated.17 In the experiments to examine the contributions of PD-1 and B7-H1, 100 μg/mouse of anti-PD-1 (J43) mAb, 200 μg/mouse of anti-B7-H1 (MIH5) mAb, or control rat or hamster Ig, was injected i.p. every other day for 30 days after tumor inoculation.

Preferential expression of B7-H1 on human SCC cell lines

To investigate the expression of new B7 family molecules on human tumor cells, we examined expression of a panel of B7 molecules on human SCC lines. Most SCC lines substantially express B7-H1 and B7-DC at various levels when cultured with medium alone (Fig. 1A and Table 1). B7-H1 expression was enhanced efficiently by treatment with IFN-γ. Two other cytokines, TNF-α and IL-1-α had minimal effects on B7-H1 expression (Fig. 1B and Table 1). B7-DC was induced by the stimulation with IFN-γ,

De novo induced B7-H1 on tumors regulates antitumor immunity

NR-S1 is a transplantable and immunogenic oral SCC tumor line. Cultured NR-S1 cells did not express B7-H1, B7-DC, B7h, or CD86, but substantially expressed CD80 and MHC class I (Fig. 3A). To examine the potential for the induction of B7-H1, NR-S1 cells were stimulated with IFN-γ in vitro. A substantial induction of B7-H1 was observed (Fig. 3B). Furthermore, the tumor mass 30 days after tumor inoculation showed the abundant expression of B7-H1 within the tissues (Fig. 3C). These results suggest

Discussion

We compared the protein expression levels of five B7 family molecules (B7-H1, B7-DC, B7h, CD80 and CD86) in human oral SCC. B7-H1 was expressed abundantly on human SCC cell lines, and its expression was efficiently enhanced by IFN-γ. The dominant expression of B7-H1 was further confirmed in specimens from oral SCC tumors. Most SCC cell lines also expressed B7-DC like B7-H1, this was enhanced by IFN-γ stimulation; however, tumor cells expressing B7-DC were not found in biopsy specimens. The

Acknowledgements

We thank Drs. Y. Kimura, F. Momose, K. Itoh, K. Ando, L. Chen, D.M. Pardoll, K. Maruyama, K. Okumura, and T. Honjo for cell lines, plasmids, a vector, and mAbs. This study was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology in Japan.

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  • Cited by (0)

    1

    Present address: Dermatoimmunology Laboratory, Johns Hopkins Medical Institutions, 600N, Wolfe Street, Baltimore, MD 21287, USA.

    2

    Present address: Department of Oral Medicine, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Road, Pathumwan, Bangkok 10330, Thailand.

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