Elsevier

Vaccine

Volume 24, Issue 23, 5 June 2006, Pages 4975-4986
Vaccine

4-1BB ligand enhances tumor-specific immunity of poxvirus vaccines

https://doi.org/10.1016/j.vaccine.2006.03.042Get rights and content

Abstract

Purpose

Recombinant poxvirus vaccines have been explored as tumor vaccines. The immunogenicity of these vaccines can be enhanced by co-expressing costimulatory molecules and tumor-associated antigens. While the B7–CD28 interaction has been most comprehensively investigated, other costimulatory molecules utilize different signaling pathways and might provide further cooperation in T cell priming and survival. 4-1BB (CD137) is a TNF family member and is critical for activation and long-term maintenance of primed T cells. This study was conducted to determine if a poxvirus expressing the ligand for 4-1BB (4-1BBL) could further improve the immune and therapeutic responses of a previously reported poxvirus vaccine expressing a triad of costimulatory molecules (B7.1, ICAM-1, and LFA-3).

Experimental design

A recombinant vaccinia virus expressing 4-1BBL was generated and characterized in an in vitro infection system. This vaccine was then used alone or in combination with a vaccinia virus expressing CEA, B7.1, ICAM-1, and LFA-3 in CEA-transgenic mice bearing established MC38 tumors. Tumor growth and immune responses against CEA and other tumor-associated antigens were determined. The level of anti-apoptotic proteins in responding T cells was determined by flow cytometry on tetramer selected T cells.

Results

The combination of 4-1BBL with B7.1-based poxvirus vaccination resulted in significantly enhanced therapeutic effects against CEA-expressing tumors in a CEA-transgenic mouse model. This was associated with an increased level of CEA-specific CD4+ and CD8+ T cell responses, induction of antigen spreading to p53 and gp70, increased accumulation of CEA-specific T cells in the tumor microenvironment, and increased expression of bcl-XL and bcl-2 in CD4+ and CD8+ T cells in vaccinated mice.

Conclusion

4-1BBL cooperates with B7 in enhancing anti-tumor and immunologic responses in a recombinant poxvirus vaccine model. The inclusion of costimulatory molecules targeting distinct T cell signaling pathways provides a mechanism for enhancing the therapeutic effectiveness of tumor vaccines.

Introduction

The magnitude of antigen specific T cell activation has been a focus of vaccine therapy for cancer. The interaction of the costimulatory molecule B7 with its ligand CD28 has been the most comprehensively investigated with several studies demonstrating potent anti-tumor immunity when B7.1 is co-expressed with tumor-associate antigens. This suggests that engagement of costimulatory molecules normally expressed on antigen-presenting cells (APCs) with cognate ligands on T cells could be an important factor in tumor eradication [1]. There have been few studies evaluating the effects of combining costimulatory molecules that target distinct signaling pathways within naïve T cells.

The TNF/TNFR family is a large group of related molecules that can act as costimulatory molecules for activation and maintenance of T cells [2]. In this family, 4-1BBL is a well-characterized costimulatory molecule expressed by APC, including B cells, macrophages, and DCs [3]. APC-expressed 4-1BBL binds to 4-1BB (CD137), a member of the TNFR superfamily expressed on monocytes, neutrophils, DC, NK cells and recently activated T cells. 4-1BB signaling has profound effects on T cells, including activation of both CD4+ and CD8+ T cells, increased levels of cytokine secretion [4], [5], enhanced clonal expansion [4], [6], [7] and increased survival [6], [7], [8]. Recent studies have noted strong anti-tumor effects mediated by 4-1BB stimulation using agonistic anti-4-1BB antibodies [9], [10], [11], [12] and adenoviruses expressing 4-1BBL [13], [14], [15]. Previous reports suggested that 4-1BB was superior to B7.1/CD28 signaling for stimulation of CD8+ T cell expansion [16] and that 4-1BB might activate T cells in the absence of B7.1/CD28 interactions [17], [18], [19] or APC stimulation [5]. Other reports, however, have demonstrated that combining B7.1/CD28 stimulation and 4-1BB signaling was required for optimal induction of CD25 and bcl-XL expression in CD8+ T cells [5], increased CTL activity [20] and protective anti-tumor immunity [21].

We have previously demonstrated that the addition of ICAM-1 and LFA-3 to replication-competent poxviruses expressing B7.1 improves antigen-specific therapeutic responses in murine models, especially when low levels of Signal 1 are provided [22]. In this study we describe the additional benefit of expressing 4-1BBL with a triad of costimulatory molecules, including B7.1, ICAM-1, and LFA-3 (designated TRICOM) in a carcinoembryonic antigen (CEA) transgenic mouse model. A recombinant vaccinia virus containing 4-1BBL transgene was generated (rV-4-1BBL) and resulted in enhanced T cell proliferation and survival in co-culture in vitro assays of virus-infected DC and naïve T cells. The rV-4-1BBL vaccine induced non-specific tumor growth arrest in an established murine tumor treatment model. When rV-4-1BBL was combined with a vaccinia virus expressing CEA and TRICOM a significant enhancement in responses were observed with a single immunization against both 4- and 7-day established CEA-bearing tumors in CEA-transgenic mice. This was associated with an increased level of CEA-specific CD4+ T cell proliferation, Th1 cytokine release, improved CD8+ T cell cytotoxicity and avidity, and induction of an antigen cascade against other tumor-associated antigens, i.e. p53 and gp70. In addition, the use of 4-1BBL with TRICOM resulted in increased expression of the anti-apoptotic proteins bcl-XL and bcl-2 in both CD4+ and CD8+ T cells and accumulation of CEA-specific T cells within the tumor microenvironment. These results have implications for the rational design of more potent vaccines in the treatment of established human tumors.

Section snippets

Animals and cell lines

Female C57BL/6 mice transgenic for human CEA were obtained from a breeding pair, provided by Dr. John Thompson (Institute of Immunobiology, University of Freiburg, Germany). The generation and characterization of the CEA-transgenic mouse has been previously described [23], [24]. Female C57BL/6 and BALB/c mice were obtained from the National Cancer Institute-Frederick Cancer Research Animal Facility (Frederick, MD). Mice were housed and maintained under pathogen-free conditions in microisolator

Characterization of rV-4-1BBL

We constructed a recombinant vaccinia virus expressing the full-length murine 4-1BBL gene. To confirm 4-1BBL protein expression, immature bone marrow-derived DCs were infected with rV-4-1BBL at MOI = 1 for 16 h. More than 30% of infected DCs expressed 4-1BBL on their surfaces by flow cytometry analysis (Fig. 1A). There was a dose-dependent increase in the percentage of 4-1BBL-positive cells with 60% of DCs expressing 4-1BBL at an MOI of 4 (data not shown). The expression of 4-1BBL was not noted on

Discussion

4-1BBL–4-1BB costimulation activates T cells through increased cytokine production [4], [5], enhanced clonal expansion [4], [6], [7] and induction of anti-apoptotic molecule expression [6], [7], [8]. Here, we extended these observations to a therapeutic poxvirus vaccine system in vitro utilizing DCs infected with rV-4-1BBL and in vivo utilizing T cells from tumor-bearing mice vaccinated with rV-4-1BBL. The results are important since poxviruses are currently in Phase III clinical trials and

Acknowledgments

This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and in part by NIH grant K08 CA79881 (H. Kaufman).

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