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tumors. The best results have been reported for patients with metastatic skin melanoma. In spite of this progress, many significant obstacles remain for cancer immunotherapies: (1) the frequency of patients responding to these new treatments is extremely low; (2) the cost of the treatments is enormous—they are also timeconsuming, and technically very demanding; and (3) there is no direct correlation between the response to therapy and the markers of immunity [3]. This last problem is particularly troubling. Many patients receiving therapy generate a vigorous tumorspecific T-cell response directed against a tumor antigen expressed on the tumor, but the T-cell response fails to control tumor growth. In addition, a few patients with partial or complete tumor responses fail to display any detectable specific antitumor immunity. For these reasons, immunologists are currently focusing on determining why the current cancer immunotherapies are not more effective.

There are a number of reasons why few if any of the current immunotherapies have been attempted on uveal melanoma patients: (1) the number of patients is relatively low; (2) there is no large source of tumor tissue available for immunological studies; (3) the immune response has not been characterized extensively; (4) patients with metastatic disease are highly resistant to chemotherapy, giving oncologists the impression these patients will respond poorly to immunotherapy; and (5) the immune-privileged environment of the eye, as compared with other anatomical sites, will decrease the effectiveness of immunotherapy. Although this last issue may be a potent critical barrier for immunotherapeutic treatment of uveal melanomas, the difficulties associated with eliminating tumors from an immuneprivileged site may reveal important insights into why cancer immunotherapies in general are not more effective. We propose that the reason cancer immunotherapies fail is also the reason that the eye succeeds at maintaining immune privilege. If we can learn how to terminate immune privilege in the eye, we will know how to make cancer immunotherapies succeed.

This chapter outlines a strategy for the development of a tumor cell vaccine for uveal melanomas that utilizes a unique characteristic of these tumors. In order to fully appreciate this strategy, a preliminary review of the research and background that form the basis for this approach is in order.

II.EFFECTOR T CELLS IN ADAPTIVE IMMUNITY

The principle behind adaptive antitumor immunity is deceptively simple. Tumors express specific antigens recognized by antigen-specific immune effector cells that eliminate only the malignant tumor and leave the normal tissue intact. It is now well established that tumors express specific antigens found on malignant cells but absent from normal cells. There are variations of this scenario, such as the following: tumor antigens may be expressed on normal cells at low levels, but at much higher levels on tumor cells. However, the important issue is that the tumor cells display a target antigen that allows the immune system to recognize and differentiate between normal and malignant cells. Since tumors express these antigens but still grow progressively, it is obvious that these antigens are unable to elicit protective immunity. Immunologists distinguish between ‘‘antigenic’’ and ‘‘immunogenic.’’ An antigen is anything that can be recognized by the immune system, but not all antigens are capable of eliciting an immune response. Only immunogenic antigens

induce immunity. Essentially all of the antigens identified to date on spontaneous human tumors are not immunogenic. Therefore the goal of adaptive antitumor immunotherapy is to manipulate the immune response so that effective immunity is directed against the tumor antigens, resulting in elimination of the tumor. How to manipulate the tumor-specific immune response to produce protective immunity is a complicated issue, in which some but not all of the important mechanisms have been identified.

There are two components to the immune response, the innate phase and the adaptive phase. This chapter focuses on the role of adaptive immunity in immunotherapy. There are many excellent reviews on the role of innate immunity in tumor immunology [4,5]. Innate immunity (1) is immediate, (2) is nonspecific, and

(3) has no memory. The main cellular components are natural killer (NK) cells, natural killer T (NKT) cells, macrophages, dendritic cells, and neutrophils. Although these cells are nonspecific, they display a limited level of specificity. They respond to certain groups of pathogens or ‘‘danger’’ signals through the expression of Toll receptors and pathogen-associated molecular patterns (PAMPS).

By contrast, adaptive immunity (1) is delayed, (2) is antigen-specific, and (3) has long-term memory. The main effector cells are T cells and B cells. In the past, B cells were not believed to participate effectively in antitumor immunity. However, this idea is beginning to change [6]. Tumor immunologists have focused almost exclusively on antigen-specific T cells in the past decade due to their antigen specificity and memory. Specificity is required to prevent destruction of normal tissue and memory is required to prevent recurrences of primary and metastatic tumors. Although T cells are clearly important in antitumor immunotherapy, it is becoming more obvious that innate immunity also plays a critical role in the development of a sustained antitumor T-cell response. The problems associated with current tumor immunotherapies may stem from the failure to include strategies to activate innate immunity.

Experiments from laboratories that study viral immunity indicate that protective immunity requires both innate and adaptive responses. Innate immunity alone is unable to control viral infections, and activation of adaptive immunity does not occur without an initial innate response. In addition, experimental animal models that study activation of antigen-specific T cells require that animals be immunized with antigen in conjunction with a potent adjuvant. If the animal is exposed to the antigen without the adjuvant, no T-cell response occurs. A major function of the adjuvant is to stimulate a local inflammatory response; it is therefore similar to the early innate immune response induced by most pathogens. It may not be surprising that recent studies suggest tumors go to great lengths to prevent local inflammation and activation of innate immunity [7].

The following section describes antigen-specific T-cell subpopulations, the tumor antigens expressed on human tumors, the antigen processing pathways used to express tumor antigens, and how specific T cells are normally activated. This information is important to understand how the current forms of cancer immunotherapy attempt to manipulate the immune response to generate protective antitumor immunity.

III.CD8zþ CYTOTOXIC T CELLS

There are two major T-cell subpopulations, CD8þ T cells and CD4þ T cells, that recognize distinct antigens and display distinct effector functions. CD8þ cytotoxic T cells (CTLs) display T-cell receptors (TcRs) that recognize small peptide antigens displayed by class I molecules. CD8 on T cells binds a nonpolymorphic region of class I that stabilizes the TcR-antigen complex (Fig. 1A). Two signals are required to successfully activate CD8þ T cells: (1) signal one is provided by TcR recognition of antigen in the context of class I and (2) signal two is provided by CD80 (B7.1) costimulatory molecules that are also expressed on antigen presenting cells (APCs). Successful activation of CD8þ T cells occurs only when both signal one and signal two are received. CTL activation proceeds through a series of distinct stages, starting with the clonal expansion of precursor T cells, differentiation into cells that acquire cytolytic capacity, and ending with fully mature CTLs. The mechanism of CTLmediated tumor cell lysis is rapid and involves several consecutive steps. It starts with the formation of the TcR antigen–class I complex, which is reinforced by ligation of adhesion molecules and colocalization of additional TcR. Signal transduction pathways trigger migration of intracellular vesicles that contain cytolytic factors to the site where the T cells bind the tumor cell. Cytolytic factors are then released from the T cell into the tumor cell membrane, causing cell death. CTLs are resistant to their own cytolytic factors and therefore are able to survive and proceed rapidly to bind and kill another tumor target cell. By this recycling mechanism, a small number of CTLs can eliminate a much larger tumor burden.

IV. CD4þ HELPER T CELLS

Helper T cells are restricted by MHC class II molecules, express CD4, and secrete a vast array of cytokines. These cytokines drive the differentiation and proliferation of other T helper cells to become CD4þ Th1 and CD4þ Th2 cells. The distinction between Th1 and Th2 cells is based on the cytokine secretion profile [8]. Th1 cells stimulate growth and proliferation of other T cells (cell-mediated immunity). Th2 cells activate B cells to differentiate into antibody-secreting plasma cells (humoral immunity). Tumor immunologists have focused on the Th1 subpopulation due to its important role in activating cytotoxic T cells.

CD4þ T cells display receptors (TcRs) that recognize peptide antigens that are

presented by MHC class II (Fig. 1B). Class II expression is highly regulated and

¨ þ restricted mainly to professional APCs responsible for activating naive CD4 T

cells. As was the case with CD8þ T cells, two signals are required to activate CD4þ T cells successfully: (1) signal one is provided by TcR recognition of antigen in the context of class II and (2) signal two is provided by CD80 costimulatory molecules that are also expressed on APCs. Primed CD4þ T cells display three effector functions in antitumor immunity: (1) secretion of ‘‘helper’’ lymphokines (such as IL- 2 and IFN-g), required to induce proliferation and differentiation of CD8þ T cells (Fig. 2); (2) indirect activation of CD8þ T cells via induction of costimulatory signals in APC (Fig. 3A and B); and (3) indirect killing of tumor cells via activation of macrophages to secrete cytokines (TNF-a and nitric oxide) that lyse tumor cells (Fig. 4). The second function is achieved through expression of CD40 ligand on activated CD4þ T cells. This ligand triggers CD40 receptors found on APCs,

Figure 2 Effector functions of CD4þ T cells—secretion of lymphokines. T-helper cells that are activated by antigen and costimulatory signals secrete lymphokines, such as IL-2 and IFN- g, that induce proliferation and activation of CD8þ cytotoxic T cells.

resulting in upregulation of other costimulatory receptors on the APC, such as CD137 (BB-1), that preferentially activates CD8þ CTL.

V.GEOGRAPHIC SPECIFICITY OF CD4þ T-HELPER CELLS

One of the criticisms against CD4þ T cells as effector T cells in antitumor immunotherapy is the lack of specificity. Although CD4þ T cells are triggered by specific antigens expressed on APCs, they activate APCs, to release TNF-a and nitric oxide, nonspecific cytokines that lack any specificity for malignant tumor cells. Once APCs release these cytokines into the surrounding environment, tumor cells and normal cells are equally vulnerable and can be destroyed. However, even though the effector cytokines are nonspecific, there is a ‘‘geographic’’ specificity to this response that may allow this type of immunity to work even within the eye (Fig. 5). In order for primed CD4þ T cells to be activated in peripheral tissues, they must recognize their specific antigen presented by class II. For uveal melanomas that rarely express class II, this occurs only when APCs infiltrate the tumor site and reprocess tumor antigens. Therefore CD4þ T cells and APCs are activated within the tumor site only when they are both within close proximity. This limits the release of nonspecific cytokines to a small geographic location within the tumor, diminishing the extent of nonspecific destruction of surrounding normal tissues.

Figure 3 Effector functions of CD4þ T cells—activation of APC. T-helper cells activate APC to express costimulatory signals that stimulate CD8þ T cells (A). CD40 ligand on T- helper cells triggers CD40 receptors on APC, resulting in activation of APC and upregulation of other costimulatory signals, such as CD137 ligand. CD8þ T cells preferentially express CD137 receptors that are triggered by activated APC, resulting in activation of CD8þ cytotoxic T cells (B).

VI. COSTIMULATORY MOLECULES

As mentioned above, naı¨ve CD4þ T cells and CD8þ T cells need to recognize antigen plus a second costimulatory signal to induce proliferation and differentiation

Figure 4 Effector functions of CD4þ T cells—activation of cytolytic APC. T-helper cells indirectly lyse tumor cells via activation of APC to secrete cytokines, such as TNF-a and nitric oxide, that nonspecifically lyse tumor cells.

into armed effector T cells. The best-characterized costimulatory molecules are the closely related B7 molecules B7.1 (CD80) and B7.2 (CD86). CD80 is a cell-surface protein that triggers CD28 receptors on T cells [9]. An important issue for antitumor immunity is that recognition of antigen in the absence of costimulation not only fails to stimulate naı¨ve T cells but also induces anergy—a form of unresponsiveness that makes T cells resistant to future activation [10]. Anergized T cells fail to respond even when triggered by professional APCs that present signals one and two. This is an important mechanism that could allow tumors to prevent activation of specific T cells.

T cells activated in the lymph node by APCs expressing both signals are ‘‘primed’’ and migrate into the periphery where they mediate their effector function when triggered by their specific antigen. Once T cells are successfully primed to an antigen, a second costimulatory signal is no longer required [11]. In recent years the number of costimulatory signals identified has increased dramatically. Now there are a number of positive and negative costimulatory signals that function during different stages of T-cell activation (reviewed in Ref. 12). This new information will clearly be important in future studies that refine our methods of activating tumorspecific T cells.