An essential function of the immune system is the ability to defend against pathogenic infections. Immune cells can identify foreign antigens expressed on the surface of an infected cell, such as viral or bacterial proteins, and target these cells for destruction. Mutations and/or alterations in normal cellular proteins that arise in a cancerous cell also result in the display of unique antigens on the surface of these cells. When fully functional, the immune system has the capability to identify cancer cells as "non-self" and eliminate them from the body. It is self-evident, however, that clinically apparent tumors avoid effective antitumor immune responses; in fact, cancer patients often exhibit an immune-compromised phenotype that extends beyond an inability to recognize tumor antigens.
Tumor cells have developed a variety of cellular and molecular mechanisms to avoid antitumor immune responses,[2-8] including host alterations in T-cell receptor/CD3 complex expression and function, decreased major and minor histocompatibility complex expression by the tumor, and loss of tumor epitopes. Virtually all branches of the immune system can be affected. Tumor cells also secrete a variety of soluble factors that are capable of inhibiting immune cell function, such as interleukin (IL)-10, tumor necrosis factor (TNF), transforming growth factor-beta (TGF-beta), and vascular endothelial growth factor (VEGF). The effects of these factors appear to be twofold: to inhibit immune cell effector function and to impair the development of immune cells by acting on earlier stages of immunopoiesis.
VEGF and its receptors have profound effects on the early development and differentiation of both vascular endothelial and hematopoietic progenitors. It induces proliferation of mature endothelial cells and is an important component in the formation of tumor neovasculature. VEGF is abundantly expressed by a large percentage of solid tumors; this overexpression is closely associated with a poor prognosis.[11,12] Some of the earliest hematopoietic progenitors express receptors for VEGF; we have demonstrated that VEGF causes a defect in the functional maturation of dendritic cells from progenitors, resulting in defective antigen presentation. This developmental defect is associated with impaired activation of NF-kappaB.[14-17]
In addition to defects in the myeloid lineage, VEGF also plays a key role in mediating the development of lymphoid lineage cells. VEGF induces dramatic thymic atrophy resulting in decreased numbers of mature T cells in the periphery, and the loss of the effector cells may also significantly impair an antitumor response (unpublished data).
This article will attempt to provide the reader with an understanding of the major problems that can lead to a failure of antitumor immune induction, with special emphasis on our ongoing research into the important role VEGF plays in mediating this effect. We demonstrate that VEGF is not only important for tumor vascularization, but is also a key factor produced by solid tumors to inhibit recognition and destruction of tumor cells by the immune system.
A primary role of the immune system is to distinguish "self" from "non-self" proteins. Foreign antigens expressed by viruses or bacteria can be presented on the surface of an infected cell, and identify that cell as non-self for destruction by the immune system. Similarly, unique or altered versions of normal cellular proteins produced by tumor cells can be presented to cytotoxic T cells, resulting in a host response against the tumor. Chemical or physical carcinogens can induce tumor antigens or they may originate in spontaneous tumors. To date, a large number of tumor antigens have been identified.[18-23] These endogenous tumor antigens may be derived from fetal or embryonic genes, mutant oncogenes, or oncogenic viral genes such as human papillomavirus.
The display of tumor antigens on the cell surface is essential for the recognition and destruction of a tumor cell by the immune system. Tumor or foreign antigens must be degraded, along with normal cellular proteins, into small peptides by the proteosome. These peptides associate with class I MHC (MHC-I) in the lumen of the endoplasmic reticulum and are transported to the cell surface for presentation to CD8-positive cytotoxic T cells. In cases where a structural defect has occurred within the tumor cell, a genetic mutation is often responsible for disrupting the normal display of tumor antigens on the cell surface. These mutations may result in the inability of a cell to produce transporter molecules, such as TAP1, or other molecules essential for this process, such as MHC-I or beta-2-microglobin, and will lead to a failure of the cell to present all antigens. However, structural defects of this nature are only found in approximately 5% to 10% of human tumors, and the majority of human tumors are ineffective at directly inducing an immune response despite adequate display of tumor antigens on their cell surface.
What causes this lack of an antitumor immune response in the remaining 90% to 95% of human tumors? Induction of an effective immune response is a complex process that involves many cell types and cytokine mediators. Tumor-bearing hosts have acquired deficiencies in several of the host elements responsible for this induction. We have found that defects in both myeloid lineage and lymphoid lineage cells are major components of this problem, and the remainder of this article will focus on our studies in this area.
Professional antigen-presenting cells are responsible for the presentation of tumor antigens to both B and T lymphocytes, and can therefore induce both humoral and cell-mediated responses against a tumor (Figure 1). Several studies have described the defects in the function of antigen-presenting cells in tumor-bearing hosts.[24-26] Dendritic cells are the most potent antigen-presenting cells; for this reason, they are potential targets for tumor vaccines and immunotherapies. Because of the central role that dendritic cells play in induction of antitumor immunity, research in our laboratory has focused on the hypothesis that defects in dendritic cell function may potentially account for the immunoresistance of certain tumors.
Tumor-derived factors with the potential to interfere with the development or function of immune cells play an important role in the escape of tumors from normal immune surveillance. We have demonstrated that tumor cells secrete soluble factors that can inhibit the maturation of CD34-positive hematopoietic progenitor cells into functional dendritic cells when cultured in vitro.[14,27] CD34-positive hematopoietic progenitor cells were isolated from human cord blood and cultured in vitro in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, and TNF-alpha.
Tumor-cell supernatants, derived from colon and breast adenocarcinoma cell lines, were added to hematopoietic progenitor cells to determine the effect of tumor-derived soluble factors on dendritic cell maturation in vitro. Dendritic cell function was then measured by two distinct assays: (1) the ability of mature dendritic cells to stimulate proliferation of allogeneic T cells in mixed leukocyte reactions; and (2) the ability to take up fluorescein(Drug information on fluorescein) isothiocyanate (FITC)-dextran. Using both assays, we found that tumor-cell supernatants dramatically reduced dendritic cell function. Dendritic cells obtained after the culture of hematopoietic progenitor cells with tumor-cell supernatants were not only functionally impaired, but also morphologically distinct from mature dendritic cells.
Overall, the number of mature dendritic cells present in the tumor-cell supernatant cultures were reduced two- to threefold. These cells expressed reduced levels of mature dendritic cell surface markers and exhibited several characteristics of immature myeloid cells. Tumor-cell supernatants did not inhibit proliferation of CD34-positive progenitors, nor did they affect the total number of CD34-positive or CD34-positive/CD38-negative progenitor cells, indicating that tumor-cell supernatant-induced defects did not result from the loss of multipotent progenitor cells. Furthermore, inhibition of dendritic cell function was observed only when tumor-cell supernatants were added within the first 4 days of in vitro culture, indicating an effect on early dendritic cell development.
Size fractionation experiments demonstrated that dendritic cell-inhibitory action was restricted to the 30 to 50 kD size fraction of tumor-cell supernatants. Neutralizing antibodies to proteins within this size range, and known to be produced by tumor cells, were added to mixed leukocyte reactions in an attempt to identify the dendritic cell-inhibitory factor. Neutralizing antibodies to VEGF, but not antibodies against TGF-beta, IL-10, or c-kit, blocked the ability of dendritic cells to stimulate proliferation of allogeneic T cells (Figure 2). Furthermore, there was a tight correlation between VEGF concentrations and the inhibitory activity of tumor-cell supernatants in 12 tumor cell lines observed. These data indicate that inhibition of dendritic cell function by tumor-cell supernatants is substantially mediated by VEGF.