Ординатура / Офтальмология / Английские материалы / Ocular Oncology_Albert, Polans_2003

chemical staining has confirmed that the TILs express the CD8 molecule, the most common surface marker for CTLs. TILs isolated from the intraocular tumors kill the tumor cells in an antigen-specific manner in vitro. Adoptive transfer of the TILs has demonstrated that they are able to recapitulate the regression of intraocular tumors in third party hosts. That is, TILs expanded in vitro and coinjected with tumor cells into the eyes of immunoincompetent hosts produced intraocular tumor resolution that mimicked the original phenotype, both clinically and histopathologically.

Results from two other studies lend support to the hypothesis that T cell– dependent immune mechanisms can mediate intraocular tumor rejection without jeopardizing the anatomical and functional integrity of the eye [44–46]. Using an adenovirus-induced tumor model in syngeneic C57BL/6 mice, Schurmans and coworkers demonstrated that intravenously injected tumor-specific CTLs entered the intraocular tumors and produced tumor resolution in a manner reminiscent of the aforementioned UV5C25 tumor rejection [45]. That is, tumor resolution occurred in a nonnecrotizing manner that culminated in tumor rejection and minimal damage to normal ocular tissues. An almost identical pattern of intraocular tumor resolution was observed in an SV40 large-T-antigen transgenic retinal pigmented epithelial (RPE) tumor model in FVB/N mice [44]. Tumor resolution was accompanied by infiltration of CD8þ TILs that displayed tumor-specific cytolytic activity in vitro and produced intraocular tumor rejection following adoptive transfer to third-party hosts. However, further examination of the aforementioned adenovirus-induced intraocular tumor model in syngeneic C57BL/6 mice indicated that, in contrast to the rejection induced by adoptively transferred immune lymphocytes, spontaneous immune rejection proceeded unabatedly in hosts who were depleted of CD8þ T cells. By contrast, intraocular tumors grew progressively in either C57BL/6 mice depleted of CD4þ T cells or in MHC class II-deficient C57BL/6 mice [46]. Intraocular tumor rejection did not require TNF- a, Fas ligand, perforin, transporters associated with antigen processing (TAP), or B cells, as mice with disruptions of the respective genes encoding these molecules were able to reject their intraocular tumors in a manner that was indistinguishable from that seen in wild-type C57BL/6 mice. These results indicate that syngeneic intraocular tumors can undergo immune rejection by a CD4dependent mechanism that (1) does not involve CD8þ T cells; (2) does not bear the hallmarks of a DTH response in situ; and (3) does not culminate in damage to innocent bystander cells. Thus, immunogenic intraocular tumors can undergo immune rejection in the eye by two distinct patterns. In one pattern, the mechanism involves ischemic necrosis by a DTH-like process that carries a heavy burden of innocent bystander damage. A second pattern is characterized by piecemeal necrosis of the tumor cells and culminates in the preservation of the normal ocular architecture. This nonnecrotizing pattern of rejection can be mediated by classical CD8þ CTL or by an unconventional CD4-dependent process that does not require either perforin, MHC class I antigen expression or the participation of CD8þ T cells. Understanding the immunoregulatory processes that determine which pathway is invoked could have enormous clinical applications in devising immunotherapeutic strategies for managing intraocular tumors without jeopardizing vision.

III.THE ROLE OF THE INNATE IMMUNE SYSTEM IN UVEAL MELANOMA

In addition to T cell-mediated tumor immunity, natural resistance mechanisms may also be important in the immune response to neoplasms [47]. Natural resistance mechanisms involve macrophages, NK cells, and granulocytes. NK cells seem particularly suited for the surveillance of neoplasms, as these cells do not require priming or clonal expansion in order to exert antitumor effects. Like T cells, NK cells produce cell-mediated cytotoxicity and secrete a diverse array of cytokines and chemokines. However, unlike those of B and T cells, the development and function of NK cells do not require gene rearrangement [47]. Results from animal studies over the past two decades have provided compelling evidence that NK cells play an important role in limiting the growth and metastasis of various rodent tumors, especially skin melanomas. The importance of NK cells in the surveillance of human tumors is less clear. Human melanoma cell lines are susceptible to NK cell-mediated cytolysis in vitro and in vivo [48]. NK cells have been detected within skin melanoma lesions [49], and a positive correlation between NK cell activity and disease-free survival time in cutaneous melanoma patients has been observed [50].

The significance of NK cells in uveal melanomas is just emerging. Preliminary findings suggest that the innate immune system, especially the NK-cell arm, may play an important role in controlling metastases arising from uveal melanomas. Human uveal melanoma cells display varying degrees of susceptibility to NK cellmediated lysis in vitro [51,52]. Moreover, there is a close correlation between the expression of the MHC class I antigen on uveal melanoma cells and a reduced susceptibility to NK cell-mediated cytolysis [51,52]. These findings are in keeping with the ‘‘missing self’’ hypothesis, which proposes that MHC class I molecules on a potential target cell transmit an inhibitory signal to NK cells and thereby prevent cytolysis [53]. However, cells failing to express MHC class I molecules do not send an ‘‘off’’ signal to NK cells; as a result, they are killed. Unlike skin melanomas, intraocular melanomas reside in an environment containing a myriad of factors that might influence MHC class I expression. The AH in particular is richly endowed with TGF-b [54,55], a cytokine noted for its capacity to downregulate MHC class I expression [56,57]. Uveal melanoma cells incubated in TGF-b display significantly reduced levels of MHC class I molecules and a proportional increased susceptibility to NK cell-mediated lysis in vitro [51]. Analogous effects have been reported with uveal melanomas that constitutively express low levels of MHC class I molecules. Stimulation of class I antigen expression by incubation with IFN-g results in a sharp increase in MHC class I expression and a comparable diminution in NK cellmediated lysis [51]. Thus it appears that human uveal melanomas are indeed susceptible to NK cell-mediated lysis in vitro. The capacity of TGF-b, an intraocular cytokine, to downregulate class I expression and thereby increase the vulnerability of melanoma cells to NK-mediated cytotoxicity suggests that melanomas within the eye might be particularly susceptible to NK cell-mediated surveillance. This begs the question as to whether cells of the immune system, especially NK cells, can enter the eye and penetrate intraocular tumors.

There is considerable evidence that lymphocytes enter tumor-containing eyes. Lymphocytes have been found infiltrating 7–20% of the uveal melanomas examined [58–60]. However, whether the lymphocyte population infiltrating uveal melanomas

displays antigen-specificity is uncertain. Nitta and coworkers demonstrated that TILs present in 7 of 8 human uveal melanomas expressed the same Va T cell receptor (TCR) gene, which is suggestive of a melanoma-specific T-cell response [60]. By contrast, others have reported that TIL populations from uveal melanomas express a diversity of TCR Vb genes [61]. Thus it is not clear whether the lymphocytes that infiltrate uveal melanomas are oligoclonal in nature or represent a random array of lymphocytes. The former proposition is supported by in vitro studies demonstrating that TILs from uveal melanoma patients can mediate uveal melanoma–specific cytolysis [39,40]. Much less is known about the presence and functional capacities of NK cells that infiltrate uveal melanomas. In two studies, less than 10% of the TILs in uveal melanomas expressed surface markers indicative of NK cells [62,63]. However, Ksander and coworkers reported that 41% of the TIL population from a choroidal melanoma expressed the CD16 NK-cell marker and lysed NK-sensitive K562 target cells in vitro [40].

Once NK cells enter the eye, they must negotiate the multiple layers of immune privilege. Until only recently, it was not known whether ocular immune privilege affected NK cell-mediated immune mechanisms. An important contributor to the immune privilege in the eye is TGF-b, a cytokine present within both the anterior and posterior compartments of the eye. TGF-b is a pleiotropic molecule, and among its many functions is its capacity to inhibit T- and B-cell proliferation and NK cellmediated cytotoxicity [17]. Macrophage migration inhibitory factor (MIF) is another potent inhibitor of NK cell-mediated cytotoxicity, which is produced by many cells within the eye, including those of the uveal tract [64]. Importantly, both of these molecules are present in ocular tissues at concentrations known to inhibit NK activity in vitro [15–17]. The in vivo significance of TGF-b and MIF in influencing the fate of NK cell-sensitive uveal melanoma cells has been confirmed in a nude mouse model of intraocular melanoma [14]. Although nude mice are deficient in T cell-mediated and T cell-dependent immune responses, they exhibit above normal NK cell-mediated immunity. NK-sensitive human uveal melanomas are promptly rejected following subcutaneous transplantation in nude mice. The rejection of the subcutaneous tumors by nude mice is mediated by NK cells, as the same tumors grow progressively in nude mice whose NK-cell populations have been depleted by systemic treatment with antibodies specific for murine NK cells [14]. By contrast, NK-sensitive tumors, which are rejected at subcutaneous sites, grow progressively in the eyes of nude mice, even at doses 50-fold lower than those that are rejected following subcutaneous transplantation [14]. Thus, NK cells can enter at least some intraocular melanomas. However, the intraocular milieu stifles the NK cell-mediated cytolytic machinery and allows the tumors to grow progressively.

Although the intraocular milieu provides relief from NK cell-mediated attack, once metastatic uveal melanoma cells leave the eye, they must evade NK cells within the bloodstream and the parenchyma of the liver—two sites of intense NK activity. Studies in mice have shown that in vivo depletion of NK cells results in a 200-fold increase in liver metastases in hosts challenged intravenously with B16 skin melanoma cells [65]. If NK cell-mediated immune surveillance plays a significant role in controlling uveal melanoma metastases, one might predict that successful metastases must be equipped with properties that inhibit NK-cell activity outside of the immunologically privileged confines of the eye. Two possibilities come to mind. First, the expression of MHC class I molecules is known to protect tumor cells from

NK cell-mediated lysis and might be an effective strategy for metastases to escape destruction in the bloodstream and liver. A second strategy is for the melanoma cells to create their own immune-privileged nidus by secreting NK-inhibitory factors such as TGF-b and MIF. Two studies on human melanoma patients offer support for the hypothesis that MHC class I antigen expression favors the development of uveal melanoma metastases. Verbik and coworkers demonstrated that only 4% of the tumor cells from a primary uveal melanoma expressed MHC class I molecules. By contrast, expression of MHC class I was nine times higher in melanoma cells isolated from four different liver metastases in the same patient [66]. Further support for this hypothesis comes from studies by Blom and coworkers, who examined 30 primary uveal melanomas and found a significant correlation between the expression of MHC class I molecules on primary uveal melanomas and poor prognosis [67].

Some cell lines from uveal melanomas and their metastases escape NK cellmediated lysis by producing MIF [68]. In a recent study, it was reported that the uveal melanoma cell lines that produced the most MIF were those that were isolated from metastases [68]. The capacity of uveal melanoma cells to express MHC class I antigens or to secrete MIF spontaneously suggests that metastases might arise from subpopulations of cells within primary uveal melanomas that are preadapted to escape NK cell-mediated assault once they leave the sanctuary of the eye.

One can only speculate as to whether activation of NK cells will be an effective adjunct therapy for uveal melanoma patients. The profound inhibitory effect of TGF-b and MIF within the eye casts doubt on the feasibility of activating NK cells as a means of eradicating primary uveal melanomas. Likewise, NK cells might be faced with an insurmountable task if liver metastases express high MHC class I antigens and secrete MIF (and perhaps other inhibitor molecules such as TGF-b). Efforts to utilize NK cells to treat liver metastases should consider strategies for dismantling these barriers, thereby allowing NK cells to perform their role in immune surveillance.

IV. CONCLUSIONS

The immune privilege of the eye gives uveal melanomas sanctuary from some forms of immunological attack. However, once uveal melanomas depart from the eye and begin their metastatic journey, they are confronted with a full array of immunological defense mechanisms. Metastatic uveal melanoma cells must evade the watchful eye of NK cells in the bloodstream and in the liver—two sites of the most intense NK activity in the body [69–71]. Uveal melanomas express melanomaspecific and melanoma-associated antigens that are potential targets of CTLs and antibody [35–37]. However, uveal melanoma metastases employ strategies that serve to thwart both antibody-mediated and CTL-mediated immune surveillance. Goslings and coworkers [26] reported that all 10 human uveal melanoma cell lines tested expressed at least one of the complement regulatory proteins and, as a result, resisted complement-mediated lysis. Uveal melanoma cells are also able to evade immune surveillance mediated by CD8þ CTLs. CTL-mediated lysis of target cells can occur only if the relevant antigens are displayed in the groove of MHC class I molecules. Expression of melanoma-specific antigens is reduced or absent in many uveal melanomas. Moreover, uveal melanoma cells express little or no MHC class I

molecules; as a result, they are invisible to CTL-mediated surveillance. The absence of MHC class I molecules, however, renders uveal melanoma cells potentially vulnerable to NK cell-mediated attack. Some uveal melanoma metastases appear to overcome this vulnerability by secreting TGF-b [72] and MIF [68], two cytokines that strongly inhibit NK cell-mediated cytolysis [15–17].

Although there has been a rebirth of interest in tumor immunotherapy, it might be worthwhile to ponder the other side of the coin and reconsider an iconoclastic hypothesis proposed by Richmond Prehn over 30 years ago [73]. Prehn raised the disconcerting proposition that, under certain circumstances, the immune system enhanced rather than hindered tumor progression. Two observations regarding the behavior of uveal melanomas are consistent with the Prehn ‘‘immune stimulation’’ hypothesis of tumor development. The first observation is that the presence of TILs carries a poor prognosis in uveal melanoma patients [58]. If confirmed, this conclusion is counterintuitive and contradicts the immune surveillance theory. What benefit can TILs convey to uveal melanoma cells? Two possibilities come to mind. The first is that TILs produce cytokines, such as IFN-g, that upregulate MHC class I molecules and thereby protect uveal melanoma cells from NK cell-mediated attack once the tumor cells metastasize from the eye. The second explanation is that TILs produce IL-2, which serves as a growth factor for the uveal melanoma cells. Importantly, human uveal melanoma cells and murine skin melanoma cells express IL-2 receptors [74] (Niederkorn et al., unpublished findings). Studies in mice have shown that IL-2 stimulates the proliferation of murine skin melanoma cells and increases their resistance to NK cell-mediated lysis in vitro [74,75]. Moreover, murine skin melanoma cells incubated in IL-2 and injected intravenously produced three times as many liver metastases as untreated melanoma cells [75]. It remains to be determined whether uveal melanoma cells behave in a similar manner. If they do, it would explain the paradoxical association between TILs and a poor prognosis in uveal melanoma patients.

A second paradoxical observation is the correlation between the presence of tumor-infiltrating macrophages and increased malignancy in uveal melanoma patients [76]. It has been recognized for over three decades that activated macrophages are capable of killing a wide variety of tumor cells in vitro, and maneuvers that activate macrophages in vivo often produce favorable results in controlling the growth and metastasis of murine tumors. However, in the case of intraocular tumors, the macrophages that infiltrate uveal melanomas may unwittingly contribute to the generation of an aberrant immune response that hinders the expression of conventional tumor immunity, including the generation of tumoricidal macrophages. One of the unique features of the intraocular milieu is the abundance of immunosuppressive cytokines, especially TGF-b. This cytokine is expressed throughout the eye and is also produced by uveal melanoma cells [13,68]. Several studies have demonstrated that macrophages exposed to antigens in the presence of TGF-b present antigens to T cells in a manner that leads the development of ACAID [77–80]. Moreover, TGF-b acts in an autocrine fashion to induce macrophages to secrete additional TGF-b [81] and IL-10 [78]. In addition to facilitating the induction of ACAID, TGF-b and IL-10 are potent anti-inflammatory molecules that inhibit Th1 immune responses to tumors. Thus, uveal melanomas have the capacity to coerce tumor-infiltrating macrophages to promote systemic downregulation of Th1

immune responses (i.e., ACAID) and thereby to paralyze the immune surveillance apparatus.

It is not surprising that in spite of three decades of intense investigation, the immunology of uveal melanoma remains a conundrum. Unraveling which immunological modalities are effective against uveal melanomas and—equally important—identifying those immune elements that betray the immune surveillance machinery is daunting but remain important goals.

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