Targeting lymphotoxin beta receptor with tumor-specific T lymphocytes for tumor regression.

Purpose: One of the impediments of immunotherapy against cancer is the suppression of tumor-specific CTLs in the tumor microenvironment, partly due to the selective inhibition of the perforin pathway and the emergence of Fas-resistant tumors. Therefore, we sought to identify perforin- and Fas-independent cytotoxic pathways and explored the potential of targeting LTβR with tumor-specific CTLs to induce tumor rejection in vivo. Experimental Design: Fas-resistant tumors were examined for their susceptibility to perforin-deficient (pfp) CTLs via CTL adoptive transfer in mouse models of experimental lung metastasis. The specificity of LTβR, a cell surface death receptor, in causing tumor rejection by CTLs was analyzed by LTβR-specific neutralizing monoclonal antibody in vitro. The specificity and efficacy of LTβR in the suppression of established tumors was further investigated by silencing LTβR in tumor cells in vivo. Results: pfp CTLs exhibited significant cytotoxicity against Fas-resistant tumors in vivo. The perforin- and Fas-independent cytotoxicity was directly mediated, at least in part, by the adoptively transferred CTLs. It was observed that LTβR was expressed on the tumor cell surface, and LTα, LTβ, and LIGHT, all of which are ligands for LTβR, were either constitutively expressed or activated in the tumor-specific CTLs and primary CD8+ T cells. Blocking LTβR with LTβR-specific neutralizing monoclonal antibody decreased CTL cytotoxicity in vitro. Silencing LTβR using LTβR-specific short hairpin RNA reduced the ability of pfp CTLs to induce tumor rejection in vivo. Conclusion: LTβR directly mediates CTL-directed tumor rejection in vivo. Targeting LTβR with tumor-specific CTLs is a potential therapeutic approach.

CTLs lyse target tumor cells through two primary cellular effector mechanisms (1). The first cytolytic pathway depends on the polarized secretion of perforin and granzymes. The second effector mechanism involves the interaction of FasL on the activated CTL with its receptor Fas on the target cell surface (2 -6). Despite the fact that the perforin pathway is the dominant antitumor effector mechanism (4,5,7,8), recent studies have begun to shed light on the importance of other cytotoxic effector mechanisms of tumor-specific CTLs for the suppression of tumor growth (9,10). For example, Caldwell et al. (6) observed that the perforin pathway of tumor-specific CTLs mediated strong antitumor effects in a minimal disease setting, but that both the perforin and FasL-dependent effector mechanisms were essential for optimal tumor regression under conditions of extensive tumor burden. Seki et al. (3) have also observed that although perforin-mediated killing was of paramount importance for CTL-mediated lysis in vitro, some in vivo effector mechanisms were clearly independent of perforin as illustrated in a Renca pulmonary metastasis model. Dobrzanski et al. (11) reported that although tumor cytolysis was predominantly perforin-dependent in vitro, the therapeutic effects of CTL-based immunotherapy were dependent, in part, on effector cell -derived LTa in a B16 lung metastasis model. Furthermore, it has been shown that both D122 Lewis lung carcinoma and melanoma were rejected by tumor-specific CTLs through a cytolytic mechanism that was independent of both perforin and Fas pathways in vivo (12,13). These studies suggest that other cytotoxic effector pathways, in addition to perforin and Fas/FasL, play significant roles in the inhibition of tumor growth.
LThR is a member of the tumor necrosis factor receptor (TNFR) superfamily, and was initially identified as a critical mediator controlling the development and organization of the secondary lymphoid tissues (14). However, it is increasingly appreciated that the LThR signaling pathway is involved in numerous other biological processes, including the initiation of extrinsic apoptotic cell death in tumor cells (15 -19). Engagement of LThR with agonistic anti-LThR monoclonal antibody (mAb), or recombinant ligand proteins (LTa1LTh2 or LIGHT) induced the cell death of several types of tumor cells (15 -19). Moreover, Lukashev et al. recently examined LThR protein in clinical human tumor tissues and observed that 87% to 96% of colorectal, lung, larynx/pharynx, stomach, and melanoma tumors were LThR-positive (19). These authors further showed that anti-LThR agonistic mAb effectively inhibited human colorectal tumor growth in xenograft mouse models (19). In this study, we revealed that LThR directly mediates a CTLdirected perforin-and Fas-independent cytotoxic effector mechanism in vivo. We further showed that targeting LThR with tumor-specific CTLs via adoptive transfer is a potential therapeutic approach in the induction of tumor rejection in vivo.

Materials and Methods
Mice. Female BALB/c (H-2 d ) mice were obtained from Charles River Laboratories. Female perforin-deficient (pfp) mice on a BALB/c background were kindly provided by M. Smyth (Peter MacCallum Cancer Institute, East Melbourne, Australia) via R. Wiltrout (LEI, CCR, National Cancer Institute. Frederick, MD). All mice were housed, maintained, and studied in accordance with the approved guidelines of the NIH and Medical College of Georgia for animal use and handling.
Tumor cells. The CMS4 sarcoma line was kindly provided by A. Deleo (University of Pittsburgh, Pittsburgh, PA). The CMS4-met subline was produced from the parental CMS4 population by one in vivo passage in the lungs of normal BALB/c mice, as described (20,21). The mammary carcinoma cell line, 4T1, was obtained from the American Type Culture Collection.
Measurement of Fas-mediated cell death. Cell death was measured by propidium iodide (PI) staining as described previously (22). Tumor cells were treated with recombinant IFN-g (100 units/mL; R&D Systems) or TNFa (100 units/mL; R&D Systems), or both, overnight. The cytokine-treated cells were incubated with recombinant human FasL (100 ng/mL, PeproTech) for f22 h. Cells were then analyzed by PI staining and flow cytometry. The percentage of cell death was calculated by the formula: (% PI + cells with sFasL) -(% PI + cells without sFasL).
Cytotoxicity assay. CTL activity was assessed by 51 Cr release assays as previously described (23). To analyze the role of LThR, lytic assays were done in the absence or presence of anti-LThR mAb (clone AC.H6; PharMingen). 51 Cr-labeled CMS4-met.vFLIP cells (1 Â 10 4 cells) were preincubated with anti-LThR mAb (10 Ag/mL) in 100 AL culture medium in 96-well, U-bottomed plates at 4jC for 30 min. pfp CTLs (1 Â 10 5 cells in 100 AL medium) were then added, and cultured for f18 h. The percentage of specific 51 Cr release was calculated according to the following formula: % lysis = (experimental cpm -spontaneous cpm) / total cpm Â 100%.
CTL adoptive transfer. Treatment of tumor-bearing mice by CTL adoptive transfer was conducted as previously described (23). To inactivate endogenous host immune cells, mice were irradiated in a Gammacell 40 Exactor Radiator (Nordion International) for a total dose of 5 Gy. Four hours later, tumor cells were injected into mice as described above.
Reverse transcription-PCR analysis. Total RNA was isolated from tumor cells using RNA STAT-60 reagent (Tel-Test) according to the manufacturer's instruction, and used for first strand cDNA synthesis using the ThermoScript reverse transcription-PCR (RT-PCR) system (Invitrogen). The cDNA was then used as templates for PCR amplification. The sequences of the primers used are listed in Table 1.
Stable transfection of tumor cells. CMS4-met and 4T1 cells were transfected with the mammalian expression plasmid pEGFPN1 (Invitrogen) containing the vFLIP gene. Transfections were done using LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's instructions. The transfected cells were propagated and maintained in culture medium containing geniticin (0.75 mg/mL; Invitrogen).
Stable short hairpin RNA expression plasmid construction. To produce stable short hairpin RNA (shRNA) expression constructs, two complimentary pairs of oligonucleotides encoding a 21-bp mLThRspecific double-stranded siRNA (5 ¶-GGCACAGAAGCCGAGGTCACA-3 ¶) and a 20-bp scramble double-stranded siRNA (5 ¶-ATAGCGACTAAACA-CATCAA-3 ¶) were synthesized and cloned to psiRNA-h7SKgzGFP expression vector (Invivogen), respectively, according to the manufacturer's instructions. The resulting expression constructs express a 49 and a 48 bp RNA that forms a double-stranded RNA with a hairpin structure, termed shRNA. The shRNA expression vectors containing scramble shRNA and mLThR-specific shRNA were transfected into CMS4-met.vFLIP cells using LipofectAMINE 2000 according to the manufacturer's instructions. Stable transfectants were selected by using Zeocin. The transfectants were further selected by cell sorting based on the GFP intensity in a cell sorter (Dako MoFlo).

Results
Tumor-specific CTLs execute antitumor cytotoxicity through perforin-and Fas-independent effector mechanisms. CMS4-met tumor cells were stably transfected with a mammalian expression vector pEGFPN1 expressing Fas-mediated apoptosis   1). pfp CTLs also completely inhibited CMS4-met and CMS4-met.vector tumor growth because of their Fas sensitivity ( Fig. 1), as previously reported (23). Interestingly, pfp CTLs also significantly, but incompletely, inhibited CMS4-met.vFLIP tumor growth (Fig. 1). Because CMS4-met.vFLIP cells are completely resistant to Fas-mediated apoptosis (Fig. 2B), pfp CTLs should not reject CMS4-met.vFLIP cells if perforin and Fas-mediated cytotoxicity are the only two cytotoxic effector mechanisms. The inhibition of CMS4-met.vFLIP cells by pfp CTLs suggests the existence of a perforin-and Fas-independent cytotoxic pathway that functions in vivo.
To exclude the possibility that pfp CTL-mediated suppression of CMS4-met.vFLIP tumors in vivo was due to loss of vFLIP expression in tumor cells in vivo, we injected CMS4-met.vFLIP tumor cells into mice and recovered tumor cells from mouse lungs 17 days after tumor implantation. The recovered cells were analyzed for GFP expression (GFP exists as a fusion protein with vFLIP and is thereby a surrogate indicator of the presence of the expression vector in the cells) and sensitivity to Fas-mediated apoptosis. CMS4-met.vFLIP cells recovered from three separate mice all maintained GFP expression ( Fig. 2A) and were still resistant to Fas-mediated apoptosis (Fig. 2B). Therefore, we concluded that vFLIP is stably expressed in the transfected tumor cells in vivo, and that the cells retained resistance to Fas-mediated apoptosis.
The pfp CTLs were generated from perforin-knockout mice and are thus completely negative for wt perforin. To ensure that the pfp CTLs have no wt CTL contamination, we analyzed perforin expression by RT-PCR analysis in wt and pfp CTLs. The pfp mouse contains a DNA fragment insertion in its perforin coding sequence. We designed a pair of PCR primers to cover the coding region containing the insertion. RT-PCR analysis indicated, as expected, that the pfp CTLs contained no wt perforin (Fig. 2C). Therefore, we concluded that no wt CTL contamination contributed to the lysis of Fas-resistant CMS4met.vFLIP tumor cells by pfp CTLs.
Tumor-specific pfp CTLs are directly responsible for rejection of Fas-resistant tumors. In this experimental lung metastasis model, the experiment was completed in 17 days. Therefore, it is unlikely that endogenous T lymphocytes were responsible for the tumor rejection response. However, to preclude the likelihood that tumor rejection was mediated by host immune cells, recipient mice were sublethally irradiated prior to the implantation of CMS4-met.vFLIP cells and the adoptive transfer of pfp CTLs. The efficacy of pfp CTLs in the rejection of Fasresistant tumors was then examined in these irradiated mice.
The irradiated mice died more quickly than the nonirradiated mice after CMS4-met.vFLIP implantation (Fig. 2D), suggesting that the host immune cells, probably the innate immune cells, play a role in tumor suppression. Adoptive transfer of pfp CTLs effectively inhibited CMS4-met.vFLIP tumor growth in the lung, and the degree of inhibition was even greater in irradiated mice than in nonirradiated control mice (Fig. 2E), suggesting  that (a) the tumor-specific pfp CTLs possess perforin-and Fasindependent cytotoxic effector mechanisms, and that (b) radiation might eliminate immunosuppressive cells (24,25) in the host and thus enhance pfp CTL-mediated cytotoxicity against Fas-resistant CMS4-met.vFLIP cells.
IFN-g and TNFa do not induce direct tumor cell death in vitro. Tumor-specific T cells secrete abundant amounts of IFN-g and TNFa after interaction with antigen-bearing tumors. To determine whether IFN-g or TNFa play a direct role in the death of Fas-resistant CMS4-met.vFLIP cells, we examined the sensitivity of these Fas-resistant tumor cells to IFN-g or TNFa in vitro. RT-PCR analysis indicated that stimulation of pfp CTLs by tumor cells rapidly induced the expression of both IFN-g and TNFa (Fig. 3A). Immunostaining of tumor cells with mAb that are specific for IFN-gR and TNFaR revealed that both IFN-gR and TNFaR are expressed on the tumor cell surfaces (Fig. 3A). Furthermore, exposure of CMS4-met.vFLIP cells to TNFa or IFN-g up-regulated Fas, a gene known to be activated by IFN-g and TNFa, indicating that TNFaR and IFN-gR are functionally responsive (Fig. 3B). However, treatment with TNFa or IFN-g or with both TNFa and IFN-g did not induce any detectable cell death in CMS4-met.vFLIP cells in vitro ( Fig. 3C and D), suggesting that these soluble cytokines might not be the direct cause of pfp CTL-mediated cytotoxicity against CMS4-met.vFLIP cells.
Tumor-specific pfp CTLs suppress Fas-resistant mammary carcinoma tumor growth. To determine whether the inhibition of Fas-resistant tumor growth by pfp CTLs is tumor typespecific, we sought to extend our findings to another type of tumor. Because these tumor-specific CTLs are H-2L d -restricted and recognizes an epitope mapped to the MuLV gp70 protein (20), we screened various tumor cell lines and identified a mammary carcinoma cell line, 4T1, that expresses both gp70 (antigen) and H-2L d (Fig. 4A). Next, we transfected 4T1 cells with the empty vector (4T1.vector) or the vector containing vFLIP (4T1.vFLIP) and established stable sublines. Interestingly, although 4T1.vector cells were poorly Fas-sensitive in vitro (<10% cell death; data not shown) and the vFLIP-transfected cells were completely Fas-resistant, somewhat different results were observed in vivo, presumably due to the longer-term interactions in vivo. The stable transfectants were injected into syngeneic mice, followed by adoptive transfer of pfp CTLs. Examination of tumor growth in the lung indicated that the pfp CTLs exhibited significant cytotoxicity against 4T1.vector cells (Fig. 4B). However, the Fas-resistant 4T1.vFLIP cells also exhibited significant susceptibility to pfp CTLs, but not to the same level as seen with the vector control (Fig. 4B). Thus, these observations indicate that pfp CTLs could elicit cytotoxicity against Fas-resistant mammary tumors through a perforin-and Fas-independent effector mechanism.
Expression of LTbR in tumor cells and activation of LTbR ligands in T cells. The above results strongly suggest that these tumor-specific CTLs mediate an additional cell contactdependent cytotoxic pathway. In the literature, it has been shown that, like Fas, LThR is a death receptor that mediates apoptosis in different types of tumor cells (14 -19). Therefore, we hypothesized that LThR might be responsible for the perforin-and Fas-independent effector mechanism elicited by these tumor-specific CTLs. To test this hypothesis, we first analyzed LThR expression in CMS4-met and 4T1 tumor cells. Staining the cell surface of LThR with LThR-specific mAbs indicated that LThR is expressed in both tumors (Fig. 5A). Next, we analyzed the expression of LTa, LTh, and LIGHT, all of which are ligands for LThR, during CTL activation. We also analyzed the expression of key molecules in the perforin and Fas pathways during CTL activation. RT-PCR analysis revealed  -d). B, apoptosis assay of CMS4-met.vector and CMS4-met.vFLIP cells recovered from the three mice described in (A). Cells were preincubated with IFN-g and TNFa, followed by culture with sFasL (100 ng/mL). Cell death was measured by PI staining and analyzed by flow cytometry. Columns, mean of three separate experiments; bars, SD. C, RT-PCR analysis of perforin expression in wt and pfp CTLs. The PCR primers were designed to cover the disrupted (insertion) region of the perforin cDNA. The amplified wt perforin and mutant perforin are indicated. D, naI« ve BALB/c mice were sublethally irradiated (5 Gy) to inactivate host immune cells. CMS4-met.vFLIP cells (2.5 Â 10 5 cells/mouse) were injected i.v. into nonirradiated (control) and irradiated mice. Survival was recorded. E, CMS4-met.vFLIP cells (2.5 Â 10 5 cells/mouse) were injected i.v. into nonirradiated (control) and irradiated mice. Three days later, pfp CTLs (1 Â10 6 /mouse) or saline (HBSS) were also injected i.v. into tumor-bearing mice. Mice were euthanized 14 d after adoptive CTL transfer. The number of lung tumor nodules was enumerated. Dots, counts from independent mice. that three key molecules in the perforin pathway: perforin, granzyme A, and granzyme B, were all up-regulated in activated CTLs, as did FasL, the ligand for the Fas receptor (Fig. 5B). LTh was constitutively expressed, whereas LTa and LIGHT were upregulated in the activated CTLs (Fig. 5B). Therefore, it is clear that the key molecules involved in these three pathways were coordinately activated during CTL activation. Because the CTLs are established T cell lines, we purified primary CD8 + T cells from naBve mice and stimulated them with anti-CD3 and CD28 mAb. RT-PCR analysis of LTa, LTh, and LIGHT expression indicated that the activation kinetics of these ligands were very similar to that of the established CTLs (Fig. 5C). Therefore, we concluded that LTa, LTh, and LIGHT activation is associated with T cell activation and is a general phenomenon.
Tumor-specific CTLs execute antitumor cytotoxicity through LTbR. Our above findings indicate that LThR is expressed on the tumor cell surface and all ligands for LThR are either constitutively expressed or activated during T cell activation, suggesting that the LThR pathway might be an effector pathway that mediates the destruction of Fas-resistant tumor cells by pfp CTLs. To test this hypothesis, we first sought to block the function of LThR on the tumor cell surface using a LThRspecific neutralizing mAb (26). The Fas-resistant tumor cells were incubated with the neutralizing antibody and then coincubated with pfp CTLs in an in vitro CTL assay. Measurement of cell death revealed that blocking LThR on the tumor cell surface significantly decreased tumor cell sensitivity to CTL-mediated cytotoxicity (P = 0.007; Fig. 5D).
The above mAb neutralization experiments indicate that LThR directly mediates CTL killing. To further show that function, we used a second approach. We constructed a stable shRNA expression vector that constitutively expresses a shRNA specific for mouse LThR. Expression of LThR-specific shRNA significantly (P = 0.002) decreased LThR expression on the tumor cell surface (Fig. 6A) and silencing LThR did not alter tumor cell ability to colonize and grow in the lungs (Fig. 6B). It is important to point out that a lower dose of tumor cells (1 Â 10 5 cells/mouse) was used in this experiment to allow quantitative comparison of tumor nodule number between mice that received the various tumor sublines without CTL adoptive transfer.
To determine whether LThR mediates CTL killing, we injected mice with tumor sublines that constitutively express either a scramble shRNA (CMS4-met.vFLIP.psiRNA.scramble) or LThRspecific shRNA (CMS4-met.vFLIP.psiRNA.LThR) sequence. The pfp CTLs were then adoptively transferred to the tumor-bearing mice. It is important to point out here that a higher concentration of pfp CTL (3 Â 10 6 /mouse) was used (Fig. 6C) as compared with Figs. 1 and 2 (2.5 Â 10 6 and 1 Â 10 6 cells/ mouse, respectively) in an effort to reduce the variability in the observed antitumor response and to better unmask the potential contribution of an alternative CTL-mediated tumor rejection mechanism. At this higher pfp CTL concentration, we observed stronger antitumor effects against the Fas-resistant tumors (Fig. 6C). More importantly, the CMS4-met.vFLIP.psiRNA.LThR tumor cells became significantly less susceptible to pfp CTLs as compared with CMS4-met.vFLIP.psiRNA.scramble tumor cells (P = 0.001; Fig. 6C). Therefore, we concluded that the pfp CTLs lyse the Fas-resistant CMS4-met.vFLIP tumor cells through a LThR-mediated effector mechanism.

Discussion
The TNFR superfamily consists of at least 28 receptors and 18 ligands (27). Many of the members in this superfamily, including TNFa, LTa, LIGHT, and TRAIL, have been well  documented to be involved in tumor cell apoptosis (28 -32). The LThR was initially identified to be critical for the organization of lymphoid tissues, lymph nodes, and Peyer patches during embryogenesis and development, and maintenance of secondary lymphoid architectures in adults (33 -36). However, it has been well established that the LThR signaling pathway is involved in the initiation of apoptotic death in tumor cells (15 -19). Here, we showed that tumor-specific CTLs execute antitumor cytotoxicity through LThR and thereby revealed that LThR, in addition to the perforin and Fas pathways, mediates another cell contact -dependent antitumor effector mechanism.
Recent in vivo studies have indicated that T reg cells might play a role in selectively inhibiting the perforin pathway in vivo.
Chen et al. (37) reported that T reg cells effectively inhibited tumor-specific CTL-mediated tumor rejection whereas exhibited no inhibitory effects on CTL proliferation in vivo. Mempel et al. (38) further showed that the failure of the activated tumor-specific CTL to kill tumor cells in vivo was correlated with the impaired release of lytic granules. Therefore, selective inhibition of the perforin pathway by immunosuppressive cells in vivo might underlie the limited antitumor efficacy of the perforin pathway in vivo against certain tumors. If immunosuppressive cells do selectively suppress the perforin pathway, then other cytotoxic pathways, including Fas and the LThR pathways, could become more critically important in the suppression of tumor development or growth in vivo. This may explain the phenomenon of differential antitumor efficacy of  the perforin pathway in vitro and in vivo and the significant role of perforin-independent cytotoxicity (3).
Immunohistochemical analysis of clinical tumor tissues has shown that 87% to 96% of colorectal, lung, larynx/pharynx, stomach, and melanoma tumors were LThR-positive, and f50% of breast tumors showed certain degrees of LThR staining by anti-LThR mAb (19). Therefore, it seems that LThR is expressed in a broad range of solid tumors of diverse tissue origins and histologies. More importantly, the high frequency of LThR expression is well-correlated with the sensitivity of tumor cells to LThR-mediated apoptosis. Engagement of LThR with recombinant ligand protein complex LTa1LTh2 or LIGHT, or with agonistic anti-LThR mAb effectively induced apoptotic death of tumor cells in vitro (15,17,18,39) and suppressed tumor growth in vivo (19,40). Therefore, LThR is a common cell surface death receptor of tumor cells. Interestingly, the finding that LThR functions not only in the homeostasis of immune cells but also in tumor cell apoptosis resembles what is known for Fas, another member of the TNFR superfamily. Like LThR, Fas was originally identified as a critical factor for homeostasis and self-tolerance of immune cells (41). It has since then been revealed that Fas is widely expressed in various tumors and functions as an essential mediator of extrinsic apoptosis (42). Engagement of Fas with recombinant FasL or agonistic anti-Fas mAb induced apoptosis of tumor cells (43 -45). Therefore, although their signaling pathways are distinct, LThR and Fas are two common death receptors that mediate apoptosis and CTL-induced cytotoxicity in tumor cells.
The cell surface -bound heterotrimeric LTa1LTh2 complex and the membrane-anchored homotrimeric LIGHT complex are the two ligands that initiate signaling through the LThR (14). Both ligands are expressed on activated T lymphocytes (28,33,46). What we observed here is a coordinate activation of ligands for Fas and LThR. The activation kinetics of FasL mimics LTa and LIGHT during tumor-specific CTL activation. Moreover, this coordinate ligand activation kinetics was also observed in primary CD8 + T cells, suggesting that coordinate FasL, LTa, and LIGHT activation is a general phenomenon of T cell activation. Because FasL activation is a characteristic of CTL activation, including antitumor immune response (47), the synchronized activation of FasL, LTa, and LIGHT suggest that the LThR-mediated effector mechanism might be part of an adaptive immune response. Thus, in addition to perforin-and Fas-mediated cytotoxicity, the LThR-mediated signaling pathway represents another cell contact -dependent cytotoxic mechanism of activated T lymphocytes.
Although both engage LThR, experimental data obtained from LTa, LTh, and LThR-deficient mice indicate that LTa1LTh2 and LIGHT are not redundant in the development of lymphoid tissues (48 -51). It is clear that both LTa1LTh2 and LIGHT protein complexes are capable of inducing tumor cell apoptosis, but it is not clear whether the functions of LTa1LTh2 and LIGHT of activated CTL in promoting tumor cell death are distinct under physiologic conditions. Mauri et al. (52) showed that the activation signals required for activation of LIGHT and LTa1LTh2 by T lymphocytes are different. They observed that LIGHT activation requires stimulation with both phorbol 12-myristate 13-acetate and calcium ionophore, whereas LTa1LTh2 expression requires only phorbol 12-myristate 13-acetate and thus suggest that these two ligands are important for different T cell functions (52). We observed here that LIGHT and LTa are coordinately activated in tumorspecific T cells by stimulation with tumors, suggesting that these two ligands might both be involved in T cell -directed LThRmediated antitumor cytotoxicity in vivo.
Although our data showed that TNFa and IFN-g does not directly induce the apoptosis of CMS4 sarcoma tumor cells in vitro, these data do not imply that these cytokines do not function in vivo in the induction of tumor cell death. In fact, other TNFR family members have been shown to possess potent antitumor activity (53,54). However, the relative contribution of LThR and other TNFR family members in the suppression of tumor development and whether these perforinand Fas-independent pathways cooperate to inhibit tumor growth require further study.