Although the exact physiological function of cFLIP has yet to be completely elucidated, a role in disease was rapidly suspected. Indeed, several viruses, some of which are oncogenic, and human malignant melanomas express high levels of FLIP 45. It was therefore postulated that virally infected cells and tumor cells may thereby acquire a certain degree of immune privilege by becoming resistant to FasL and perhaps other death ligands. The in vivo relevance of this hypothesis has since remained open and uncertain, however, as CTLs can lyse their targets through both Fas- and perforin-dependent pathways, and FLIP has only been shown to inhibit the former, at least in vitro 7.

In this issue, papers by Medema et al. 8 and Djerbi et al. 9 have clarified this issue. Both clearly demonstrate in different tumor models that in vivo expression of FLIP confers an advantage to tumors within an immunocompetent setting. In addition, they show that induction of tumor cell death by death receptor triggering is a more important mechanism of defense against tumors than had been suspected to date.

Djerbi et al. 9 generated mouse A20 lymphoma transfectants that stably overexpress viral FLIP from HHV8. These vFLIP-A20 cells, when compared with mock-transfected A20 cells, were shown to be resistant to Fas-mediated apoptosis by inhibition of caspase-8 activation but also showed decreased caspase-9 and -3 activation after triggering by FasL. Additionally, when grown in the continuous presence of death stimuli (agonistic anti-Fas antibody), vFLIP selectively allowed A20 cells to grow clonally. When tested in vivo using syngeneic (BALB/c) or semiallogeneic (BALB/c x C57BL/6) F1 recipients injected subcutaneously, both the frequency of tumor appearance and the time to tumor appearance were drastically higher for vFLIP-A20 cells as compared with mock-A20 cells. Depending on the vFLIP-A20 clone tested, in syngeneic mice vFLIP-A20 cells induced tumors in roughly 90% of mice versus 32% for mock-A20 cells. Similarly, in semiallogeneic mice, vFLIP-A20 cells induced tumors in roughly 65–80% of mice, depending on the clone used, versus 17% for mock-A20 cells. Further experiments revealed that this difference is most likely due to T cell immune escape conferred by vFLIP, as tumor establishment and growth of transfected and mock A20 cells was virtually identical in SCID mice.

The experimental approach used by Medema et al. 8 is quite different to that of Djerbi et al. 9 but provides us with exciting complementary information. These authors have assessed the effect of cellular FLIP in tumorigenesis and immune escape using stable cFLIP transfectants of two distinct cell lines: (a) a Fas-transfected Moloney murine leukemia virus–induced lymphoma (MBL2-Fas) and (b) a mouse embryo cell tumor line (AR6) generated by transfection of adenovirus type 5 E1A and mutant EJ-ras oncogenes. The latter express low but detectable levels of Fas, whereas the former express high levels of Fas. Independent cFLIP transfectant clones from both the MBL2-Fas and AR6 cell lines were shown to be resistant to Fas-mediated cell death in vitro as compared with mock-transfected counterparts. In agreement with previous results 7, these FLIP transfectants are, however, sensitive to CTL-induced apoptosis in vitro, the perforin pathway being able to induce target cell apoptosis despite caspase-8 (and -10) inhibition by FLIP.

When these cell lines are injected in vivo, similar to the results of Djerbi et al. 9, Medema et al. 8 show that transfectants expressing little or no FLIPs are rejected in the majority of mice, whereas injection of the same number of cells expressing high amounts of FLIP consistently resulted in tumor development. When injected into nude mice, both cell types result in tumors that grow equally fast no matter how much FLIP they express. Thus, FLIP offers significant protection from the in vivo immune response to these tumors, and protection is not limited to tumors with high Fas expression (MBL2-Fas). Additionally and interestingly, when the same experiments were performed in perforin-deficient mice with the AR6 cell line, AR6-FLIP tumors grew nearly as efficiently as in wild-type mice, suggesting that elimination of this tumor by the immune system depends almost exclusively on the Fas pathway. Last but not least, when the rare tumors that had developed in mice injected with MBL2-Fas cells expressing low amounts of FLIP were analyzed in vitro, they were shown to have become Fas resistant and high expressors of FLIP. Thus, tumor cells appear to be selected in vivo for elevated FLIP expression, most likely due to selective pressure by the immune system.

The novel and complementary experimental data presented in the papers by Medema et al. and Djerbi et al. bring about two important new pieces of information. The first is that Fas-mediated apoptosis is a more important mechanism of defense against tumors than had been suspected to date. The second is that FLIP expression by tumors is a significant and novel mechanism of immune escape from T cell immunity in vivo (Fig. 2). This may appear to be a surprise, as FLIP does not affect perforin-mediated lysis by CTLs in vitro. However, as suggested by Medema et al. 8, this discrepancy may be due to limitations of classical in vitro cytolysis assays that may not reflect the situation occurring in the tumor microenvironment as accurately as currently conceived. In line with this observation, recent evidence suggests that to trigger the release of perforin/granzyme B from cytoplasmic granules, stronger and more persistent TCR signals may be required than for the release of FasL (Fig. 2). Furthermore, whereas partial agonistic MHC peptides are capable of eliciting FasL but not perforin cytotoxicity, strong signals from fully agonistic MHC peptides trigger both pathways 10. Consequently, in vivo CTL recognition of tumor cells as "nonself" may be inefficient, leading to preferential activation of FasL and leaving the perforin pathway virtually unimplicated.

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Figure 2 Inhibition of death receptor signaling as a mechanism of tumor immune escape. CTLs use two major pathways (perforin/granzyme granule exocytosis and Fas) to induce target cell (tumor) death by apoptosis after TCR–MHC–peptide complex engagement. By downregulating Fas surface expression or by producing FLIP, tumor cells can escape immune destruction mediated by the Fas death receptor signaling pathway in vivo, despite the persistence of a functional perforin/granzyme granule exocytosis pathway.

Current attempts to improve cancer survival depend essentially on early diagnosis and the development of new treatment modalities, one of the most promising being immunotherapy. Given the new findings described herein, strategies to inhibit FLIP expression and/or FLIP-mediated inhibition of death receptor signaling may prove to be a useful complementary approach to the treatment of cancer.