Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

Figure 5 Geographic specificity of T-helper cells. APCs that infiltrate into tumors are activated only when they come into contact with CD4þ T-helper cells specific for antigens expressed on the APCs. This limits the release of nonspecific cytokines from APCs and CD4þ T-helper cells to a small geographical area within the tumor, resulting in limited destruction of normal tissue.

VII. TUMOR ANTIGENS

There are four general categories of tumor antigens (reviewed in Ref. 13): (1) tissuespecific differentiation antigens expressed on melanomas and normal melanocytes,

(2) tumor-selective antigens that are expressed in fetal tissues but not found on adult tissues (except the testis), (3) antigens derived from mutated proteins found only in tumors and not normal tissues, and (4) viral antigens found in virus-associated cancers. Uveal melanomas express tissue-specific tumor antigens that are found on skin melanomas, such as Mart-1, and tumor selective antigens, such as MAGE-1 [14]. Unfortunately, there has not been an extensive analysis of tumor antigens expressed on either primary uveal melanomas or liver metastases. Furthermore, it is unknown whether uveal melanomas express unique tumor antigens that are not found on skin melanomas. Considering the extensive differences in the clinical pattern of disease progression between skin and uveal melanomas and the functional differences between normal skin and choroidal melanocytes, it seems likely that unique tumor antigens will be expressed on uveal melanomas. Much more work is needed in this area if immunotherapies are to be used for treating patients with uveal melanomas.

Tumor immunologists have focused almost exclusively on the activation of CTLs for several reasons. First, tumor-specific CD8þ T cells can be recovered from cancer patients and activated in vitro. Second, almost all of the tumor antigens identified to date are recognized by CTLs. Third, the highest degree of specificity for

tumor target cells is displayed by CTLs. Fourth, numerous animal models demonstrate that CTLs are highly effective in eliminating progressively growing tumors. Finally, it was predicted that excessive amounts of costimulation plus tumor antigens would bypass the need for CD4þ T helper cells. In spite of these observations, immunotherapies utilizing CD8þ T cells are not as effective as originally predicted. Even though tumor-specific CD8þ CTLs are activated, in many cases they are unable to prevent continued progressive tumor growth. Although there are many potential reasons for the failure of CD8þ T-cell therapy, the lack of a sustained T-cell response is currently a primary focus of many immunologists. Therefore many researchers have begun to focus their attention on the activation of tumor-specific CD4þ T-helper cells.

VIII. ANTIGEN PROCESSING AND PRESENTATION PATHWAYS— MAJOR HISTOCOMPATIBILITY COMPLEX (MHC)

As a defense against foreign pathogens, mammals have evolved a sophisticated system that enables them to distinguish self from non-self. This was first demonstrated by the rejection of foreign tissue grafts in mice [15–17]. The genetic loci involved in graft rejection were subsequently mapped to a region on chromosome 17 [17], which became known as the major histocompatibility complex (MHC). The human MHC, also known as the human leukocyte antigen (HLA) system, is located on the short arm of chromosome 6 [18,19].

The MHC complex is divided into three different classes of genes: I, II, and III. There are a variety of class I genes, but three loci are typically identified in humans: HLA-A, HLA-B, and HLA-C. Class II genes are located in the HLA-D region and consist of at least three loci: HLA-DR, HLA-DQ, and HLA-DP [20,21]. The class III genes encode components of the complement system and a diverse collection of at least 20 other genes [22–24].

In order to detect and eliminate pathogens from different anatomical sites, the immune system has developed a mechanism to recognize foreign antigens derived from two different cellular sources. CD8þ T cells recognize antigens derived from intracellular/endogenous proteins presented by class I. By contrast, CD4þ T cells recognize antigens derived from extracellular/exogenous proteins presented by class II. The class I and class II pathways of antigen processing have evolved in order to present small peptide fragments of degraded proteins derived from these two different sources.

It is believed that there is a strong selective pressure to develop an immune system which can protect the host during the childbearing years from bacterial and viral infections threatening the propagation of the species. This selective advantage is absent for pathogens that threaten only older adults that are past the childbearing stage. Since cancer is mainly a disease of old age, it is frequently hypothesized that the immune system is not specifically designed to protect the host from malignant transformation. For this reason, it is important to understand the class I and class II pathways of antigen processing and how these pathways are used or not used to process and present tumor antigens. This is critical for understanding how these pathways can be manipulated in cancer immunotherapies.

is stably associated with class II is called CLIP [32–34]. Synthetic peptides corresponding to the CLIP region of Ii chain bind with high avidity to class II and compete with antigenic peptides for class II binding [35,36]. Following dissociation of the Ii chain from the class II a/b chains, the class II binding site becomes open for loading with peptides [37].

After migration through the Golgi system, the class II–Ii complex is transported into specialized endosomal compartments (MIIC). This MIIC is similar to late endosomes [38]. In these compartments, Ii chain undergoes proteolysis and dissociation from class II. If Ii chain degradation is blocked and remains bound to class II, then class II–Ii complexes are retained within the cell and fail to present antigen [39–41]. A potent additional cofactor necessary to promote class II–Ii dissociation, through the removal of CLIP, is DM [42]. Once CLIP is removed from the class II–peptide binding site, class II encounters antigen that binds the peptidebinding groove. Loaded class II–peptide complexes are now ready to travel to the cell surface where they are expressed and can be recognized by specific CD4þ T cells.

XI. CROSS-PRIMING VERSUS DIRECT RECOGNITION

Understanding how tumor antigens enter the class I and class II pathways is critical for the successful activation of tumorspecific CD4þ T cells and CD8þ T cells. For

Figure 8 Direct recognition of tumor antigens. CD8þ cytotoxic T cells recognize directly endogenous tumor antigens presented by class I on the surface of tumor cells.

Figure 10 Cross-priming of tumor antigens. Cross-priming occurs when APC infiltrate the tumor site and process and present tumor antigens on both class I and class II. APCs that use this route of antigen presentation activate CD4þ T cells and CD8þ T cells.

they process and present the antigens to responding naı¨ve T cells. This results in proliferation, differentiation, and clonal expansion of antigen-specific effector T cells that migrate systemically and eliminate infected cells. This same mechanism should also successfully eliminate spontaneous human tumors expressing tumor antigens. Although animal models demonstrate that cross-priming of tumor antigens can occur, there is hardly any evidence that host APCs in cancer patients successfully present tumor antigens. Several mechanisms used by tumors to escape immune detection and destruction have been identified (reviewed in Ref. 44). One important escape mechanism is blocking the activation of CD4þ T-helper cells.

Extensive studies using animal models indicate that, although either CD8þ or CD4þ T cells alone can independently mediate tumor rejection, the most effective and long-lasting protective immunity occurs when both CD4þ and CD8þ T cells are activated. For this reason, the current lack of tumor antigens presented by class II and recognized by CD4þ T cells is an obvious important deficiency. It has been proposed that tumor cells block APC activation of CD4þ T cells by releasing either

(1) suppressive factors that block APC antigen processing or (2) factors that block APC maturation. For example, vascular endothelial growth factor (VEGF) secreted by tumor cells has been shown to inhibit tumor antigen presentation by APCs [45]. Together with other reports, this evidence suggests that tumor cells create a local microenvironment that is hostile to infiltrating APCs and activation of tumorspecific CD4þ T cells.

Some approaches of experimental immunotherapy aim to modify host APCs in an attempt to increase antigen presentation to CD4þ T helper cells. The most widely studied host APCs are dendritic cells (DC). Mature DCs induce specific T-cell immunity and resistance to tumors in several different animal models. However,

since the inflammatory and microbial stimuli required for DC maturation are frequently absent in tumors, it is likely that tumor-infiltrating DCs fail to mature. Immature DCs downregulate adaptive T-cell responses by inducing regulatory T cells [46,47]. In an effort to avoid this, some cancer immunotherapy strategies use mature DCs that are pulsed with tumor antigens or transduced with genes encoding tumor antigens in vitro prior to injection into tumor-bearing patients. This strategy successfully generated APCs that were highly effective at inducing antitumor immunity in animal models [48–51]. Whether this strategy will be successful in patients is still unclear.

XIII. SUCCESSFUL ACTIVATION OF CTL REQUIRES T-HELPER CELLS

Primed tumor-specific CTIs can be found among peripheral blood lymphocytes and lymphocytes that infiltrate the tumor site [52–54]. In vitro, these primed T cells can be activated to become cytolytic cells by addition of exogenous lymphokines; however, there is little evidence that mature cytolytic T cells develop in situ. Because CD8þ T cells are capable of responding to lymphokines in vitro and it has been very difficult to detect tumor-specific CD4þ T cells in vivo, it seems that stimulation of specific cytotoxic T cells to differentiate into functional killer cells requires the presence of helper lymphokines. Many immunotherapy approaches have attempted to bypass the requirement for T helper cells and directly stimulate cytotoxic T cells. Tumor cells were genetically modified to express a variety of different cytokine genes: IL-2, IL-4, IL-7, TNF-a, IFN-g, and GM-CSF [55]. This approach was successful in activating antitumor lymphocytes with cytolytic activity. However, this antitumor immunity was time-limited and ineffective against progressive, growing tumors. Expression of cell membrane–bound costimulatory molecules was also attempted by expressing CD80 on tumor cells. This strategy induced significant antitumor immune responses and generated protection against a challenge from wild-type tumors at a distant site [56]. However, further analysis suggested that this approach was not successful for weakly immunogenic tumor antigens [57].

Increasing evidence from animal studies and clinical trials indicates that CD4þ T cells are indispensable in inducing host immunity against tumors [58–61]. Compared to the high number of reports on the role of specific cytotoxic T cells in antitumor immunity, much less attention has been given to activation of CD4þ T cells. There are at least two reasons for this: (1) most tumor cells express MHC class I but not MHC class II molecules and (2) few MHC class II–restricted tumor antigens have been identified. With increasing evidence supporting the hypothesis that CD4þ T cells play a central role in a successful antitumor immunity, it is necessary to develop strategies for the identification of MHC class II-restricted tumor antigens and activation of tumor-specific CD4þ T cells. For a number of reasons described below, human uveal melanomas are an ideal tumor to study the activation of tumor specific CD4þ T cells and may provide important information on the activation of this critical subpopulation of T cells.

vivo studies with tumor-bearing mice that demonstrate the significant activity of these vaccines as therapeutic agents for the treatment of established primary tumors and progressive metastatic disease. The experiments were performed in three animal models: sarcoma, melanoma, and mammary carcinoma.

The first-generation tumor cell vaccine was produced over a decade ago and

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protected naive animals from a subsequent challenge of wild-type tumor cells. This initial approach was based on the hypothesis that tumor cells expressing syngeneic MHC class II molecules would directly present tumor antigens to CD4þ T cells and thereby facilitate antitumor immunity. This hypothesis was tested by transfecting mouse SaI sarcomas (H-2a) with syngeneic MHC class II genes (Aak þ Abk genes; SaI/Ak tumor cells) and using the tumor cells to vaccinate syngeneic A/J mice. Mice immunized with several clones of this vaccine were completely protected against the wild-type tumor [65]. In vivo antibody depletion studies confirmed that CD4þ T cells were required for protection.

At the time of these initial studies 10 years ago, the role of costimulation was not understood. In several studies, it was demonstrated that antigen presentation by MHC class II molecules required the cytoplasmic domain of the class II heterodimer

[66].Since, in the above described experiments, the hypothesis was that class II-

transfected tumor cells functioned as APCs, SaI tumor cells transfected with MHC class II genes truncated for their cytoplasmic domains (SaI/Ak/tr) were generated. These transfectants were not effective vaccines and were tumorigeneic themselves

[67].However, the vaccine effect was restored if the SaI/Ak/tr cells were further

transfected with the gene encoding the CD80 (B7.1) costimulatory molecule (SaI/ Aktr/B7.1) [68]. These experiments, along with two other studies [69,70], provided the first evidence that tumor cell expression of costimulatory molecules enhanced antitumor immunity. They also suggested that one of the functions of MHC class II molecules in antigen presentation is induction of CD80 in APCs.

The second-generation vaccines were effective in treating mice with established primary sarcoma and metastatic mammary carcinoma. The vaccine’s efficacy was increased by using transfected tumor cells that constitutively expressed MHC class II plus CD80. Vaccines were generated and tested in a primary tumor model (SaI sarcoma) and in metastatic models (BALB/c-derived 4T1 mammary carcinoma and C57BL/6-derived B16melF10 melanoma). Mice were inoculated with the wild-type tumor cells and the tumors were allowed to grow progressively until they were well vascularized (approximately 3 weeks after initial inoculation). Weekly therapeutic injections of irradiated tumor cells were given for 3 weeks and tumor progression was monitored. The most dramatic antitumor effect was observed following vaccination with class II/CD80 transfectants in the SaI, 4T1, and B16melF10 tumor systems [71–72].

The third-generation vaccines were more effective in treating mice with

established metastatic disease. The enterotoxin B of Staphylococcus aureus (SEB) is a potent activator of CD4þ T cells [73]. The third-generation vaccines therefore consisted of SEB-transfected tumor cells combined with class II/B7 transfectants. This strategy was tested in the BALB/c-derived 4T1 mammary carcinoma model. Mice treated with this combination vaccine had significantly fewer lung metastases.

Together, these results support the hypothesis that MHC class II and CD80-

transfected tumor cell vaccines induce antitumor immunity because they directly present endogenously synthesized tumor antigens to CD4þ T cells [74]. If this MHC