CpG motifs as proinflammatory factors render autochthonous tumors permissive for infiltration and destruction

In a transgenic mouse model expressing SV40 T Ag (Tag) as a de novo tumor Ag, immune surveillance fails and islet cell carcinomas grow progressively. To develop an anticancer strategy that would be effective in eradicating solid, autochthonously growing tumors, we evaluated the effectiveness of immunostimulatory oligodeoxynucleotides (ODN) with cytosine-guanine-rich (CpG) motifs (CpG-ODN). In a classical vaccination protocol, Tag was administered with CpG-ODN as adjuvant. The antitumor vaccination, however, was only effective in a prophylactic setting, despite the successful activation of a Tag-specific CTL response in vivo. Histological examination demonstrated that even primed immune cells failed to infiltrate tumors once a malignant environment was established. To ensure that effector cells were not limiting, highly activated tumor Ag-specific T cells were transferred into tumor-bearing mice. However, this treatment also failed to result in tumor infiltration and rejection. Therefore, we further tested the efficacy of CpG-ODN as a proinflammatory agent in combination with the transfer of preactivated Tag-specific CD4(+) and CD8(+) T cells. Indeed, this combination therapy proved to be highly effective, because CpG-ODN rendered insulinomas permissive for massive infiltration and destruction. The opening of tumor tissue correlated with uptake of CpG-ODN by tissue-resident macrophages and a strong up-regulation of adhesion molecules such as ICAM and VCAM on blood vessel endothelia. These data demonstrate that systemic application of proinflammatory reagents drastically enhances extravasation of effector cells into tumor tissue, an observation that is of general importance for immunotherapy of solid tumors in a clinical setting.     

Cutting edge: CD8+ effector T cells reject tumors by direct antigen recognition but indirect action on host cells

CD8(+) effector T cells recognize malignant cells by monitoring their surface for the presence of tumor-derived peptides bound to MHC class I molecules. In addition, tumor-derived Ags can be cross-presented to CD8(+) effector T cells by APCs. IFN-gamma production by CD8(+) T cells is often critical for tumor rejection. However, it remained unclear whether 1) CD8(+) T cells secrete IFN-gamma in response to Ag recognition on tumor cells or APCs and 2) whether IFN-gamma mediates its antitumor effect by acting on host or tumor cells. We show in this study that CD8(+) effector T cells can reject tumors in bone marrow-chimeric mice incapable of cross-presenting Ag by bone marrow-derived APCs and that tumor rejection required host cells to express IFN-gammaR. Together, CD8(+) effector T cells recognize Ag directly on tumor cells, and this recognition is sufficient to reject tumors by IFN-gamma acting on host cells.     

Stimulation of the glucocorticoid-induced TNF receptor family-related receptor on CD8 T cells induces protective and high-avidity T cell responses to tumor-specific antigens

Treatment of tumor-bearing mice with a stimulatory Ab to glucocorticoid-induced TNFR family-related receptor (GITR) has previously been shown to elicit protective T cell responses against poorly immunogenic tumors. However, the role of GITR stimulation on CD8 T cells and the nature of tumor rejection Ags have yet to be determined. In this study, we show that a stimulatory mAb to GITR (clone DTA-1) acts directly on CD8 T cells, but not on CD4(+)CD25(+) regulatory T (T(reg)) cells, in B16 tumor-bearing mice to induce concomitant immunity against secondary B16 tumors, as well as protective memory following surgical excision of the primary tumor. Melanoma growth itself induced GITR expression on tumor-specific CD8 T cells, providing a mechanism whereby these cells may respond to stimulatory anti-GITR. Unexpectedly, in contrast to T(reg) cell depletion therapy with anti-CD4, GITR stimulation induced very weak CD8 T cell responses to melanocyte differentiation Ags expressed by the tumor, and did not induce autoimmune vitiligo. Accordingly, GITR-stimulated hosts that were primed with B16 melanoma rejected B16, but not the unrelated JBRH melanoma, indicating that tumor rejection Ags are tumor-specific rather than shared. In support of this, we show that GITR stimulation induces CD8 T cell responses to a tumor-specific Ag, and that these responses are of higher functional avidity compared with those induced by T(reg) cell depletion. We conclude that stimulation of GITR on effector CD8 T cells results in high-avidity T cell responses to tumor-specific Ags, thereby inducing potent antitumor immunity in the absence of autoimmunity.     

TRAIL/DR5 plays a critical role in NK cell-mediated negative regulation of dendritic cell cross-priming of T cells

Dendritic cells (DCs) are critical in initiating immune responses by cross-priming of tumor Ags to T cells. Previous results showed that NK cells inhibited DC-mediated cross-presentation of tumor Ags both in vivo and in vitro. In this study, enhanced Ag presentation was observed in draining lymph nodes in TRAIL(-/-) and DR5(-/-) mice compared with that of wild-type mice. NK cells inhibit DC cross-priming of tumor Ags in vitro, but not direct presentation of endogenous Ags. NK cells lacking TRAIL, but not perforin, were not able to inhibit DC cross-priming of tumor Ags. DCs that lack expression of TRAIL receptor DR5 were less susceptible to NK cell-mediated inhibition of cross-priming, and cross-linking of DR5 receptor led to reduced generation of MHC class I-Ag peptide complexes, followed by attenuated cross-priming of CD8(+) T cells. In addition, key molecules involved in the TRAIL/DR5 pathway during DC/NK cell interactions were determined. In summary, these data indicate a novel alternative pathway for DC/NK cell interactions in antitumor immunity and may reflect homeostasis of both DCs and NK cells for regulation of CD8(+) T cell function in physiological conditions.     

Antigen localization controls T cell-mediated tumor immunity

Effective antitumor immunotherapy requires the identification of suitable target Ags. Interestingly, many of the tumor Ags used in clinical trials are present in preparations of secreted tumor vesicles (exosomes). In this study, we compared T cell responses elicited by murine MCA101 fibrosarcoma tumors expressing a model Ag at different localizations within the tumor cell in association with secreted vesicles (exosomes), as a nonsecreted cell-associated protein, or as secreted soluble protein. Remarkably, we demonstrated that only the tumor-secreting vesicle-bound Ag elicited a strong Ag-specific CD8(+) T cell response, CD4(+) T cell help, Ag-specific Abs, and a decrease in the percentage of immunosuppressive regulatory T cells in the tumor. Moreover, in a therapeutic tumor model of cryoablation, only in tumors secreting vesicle-bound Ag could Ag-specific CD8(+) T cells still be detected up to 16 d after therapy. We concluded that the localization of an Ag within the tumor codetermines whether a robust immunostimulatory response is elicited. In vivo, vesicle-bound Ag clearly skews toward a more immunogenic phenotype, whereas soluble or cell-associated Ag expression cannot prevent or even delay outgrowth and results in tumor tolerance. This may explain why particular immunotherapies based on these vesicle-bound tumor Ags are potentially successful. Therefore, we conclude that this study may have significant implications in the discovery of new tumor Ags suitable for immunotherapy and that their location should be taken into account to ensure a strong antitumor immune response.     

Tumor immunotherapy by epicutaneous immunization requires langerhans cells

A role for Langerhans cells (LC) in the induction of immune responses in the skin has yet to be conclusively demonstrated. We used skin immunization with OVA protein to induce immune responses against OVA-expressing melanoma cells. Mice injected with OVA-specific CD8(+) T cells and immunized with OVA onto barrier-disrupted skin had increased numbers of CD8(+) T cells in the blood that produced IFN-gamma and killed target cells. These mice generated accelerated cytotoxic responses after secondary immunization with OVA. Prophylactic or therapeutic immunization with OVA onto barrier-disrupted skin inhibited the growth of B16.OVA tumors. LC played a critical role in the immunization process because depletion of LC at the time of skin immunization dramatically reduced the tumor-protective effect. The topically applied Ag was presented by skin-derived LC in draining lymph nodes to CD8(+) T cells. Thus, targeting of tumor Ags to LC in vivo is an effective strategy for tumor immunotherapy.     

Role of TRAIL and IFN-gamma in CD4+ T cell-dependent tumor rejection in the anterior chamber of the eye

Although the anterior chamber of the eye expresses immune privilege, some ocular tumors succumb to immune rejection. Previous studies demonstrated that adenovirus-induced tumors, adenovirus type 5 early region 1 (Ad5E1), underwent immune rejection following transplantation into the anterior chamber of syngeneic mice. Intraocular tumor rejection required CD4(+) T cells, but did not require the following: 1) CD8(+) T cells, 2) B cells, 3) TNF, 4) perforin, 5) Fas ligand, or 6) NK cells. This study demonstrates that CD4(+) T cell-dependent tumor rejection does not occur in IFN-gamma-deficient mice. Ad5E1 tumor cells expressed DR5 receptor for TRAIL and were susceptible to TRAIL-induced apoptosis. Although IFN-gamma did not directly induce apoptosis of the tumor cells, it rendered them 3-fold more susceptible to TRAIL-induced apoptosis. Both CD4(+) T cells and corneal endothelial cells expressed TRAIL and induced apoptosis of Ad5E1 tumor cells. The results suggest that Ad5E1 tumor rejection occurs via TRAIL-induced apoptosis as follows: 1) tumor cells express TRAIL-R2 and are susceptible to TRAIL-induced apoptosis, 2) IFN-gamma enhances TRAIL expression on CD4(+) T cells and ocular cells, 3) IFN-gamma enhances tumor cell susceptibility to TRAIL-induced apoptosis, 4) apoptotic tumor cells are found in the eyes of rejector mice, but not in the eyes of IFN-gamma knockout mice that fail to reject intraocular tumors, 5) CD4(+) T cells and corneal endothelial cells express TRAIL and induce apoptosis of tumor cells, and 6) apoptosis induced by either CD4(+) T cells or corneal cells can be blocked with anti-TRAIL Ab.     

Cellular and molecular requirements for rejection of B16 melanoma in the setting of regulatory T cell depletion and homeostatic proliferation

We have recently demonstrated that adoptive transfer of regulatory T cell-depleted polyclonal T cells into lymphopenic mice leads to rejection of B16 melanoma, which generated an opportunity to study host requirements for tumor rejection when it effectively occurred. CD8(+) T cell priming and tumor rejection required tumor Ag cross-presentation, as evidenced by tumor outgrowth in Kb(-/-) bone marrow chimeric or B71/2(-/-) mice. CD4(+) T cells were additionally required for optimal tumor control, although not through classical CD4 "help," as the frequency of primed CD8(+) T cells was similar in the absence of CD4(+) T cells, and tumor rejection did not depend upon CD40-CD40L interactions or on IL-2 production by CD4(+) T cells. Rather, CD4(+) T cells appeared to act at the effector phase of tumor rejection and responded to B16-derived Ags in vitro. At the effector phase, IFN-γ production by transferred T cells, but not host cells, was necessary. IFN-γ acted either on host or tumor cells and was associated with reduced tumor vascularity. Finally, tumor rejection occurred after transfer of TNF-α, perforin, or FasL-deficient T cells. However, perforin/FasL double-knockout T cells failed to reject, arguing that the killing of B16 melanoma cells could occur either via the cytotoxic granule or Fas pathways. Collectively, these results support a model in which host tumor Ag cross-presentation primes adoptively transferred T cells, which remain functional in the setting of homeostatic proliferation and regulatory T cell depletion, and which promote tumor rejection via IFN-γ and lysis via cytotoxic granules and/or FasL.     

Activation of dendritic cells that cross-present tumor-derived antigen licenses CD8+ CTL to cause tumor eradication

The fate of naive CD8(+) T cells is determined by the environment in which they encounter MHC class I presented peptide Ags. The manner in which tumor Ags are presented is a longstanding matter of debate. Ag presentation might be mediated by tumor cells in tumor draining lymph nodes or via cross-presentation by professional APC. Either pathway is insufficient to elicit protective antitumor immunity. We now demonstrate using a syngeneic mouse tumor model, expressing an Ag derived from the early region 1A of human adenovirus type 5, that the inadequate nature of the antitumor CTL response is not due to direct Ag presentation by the tumor cells, but results from presentation of tumor-derived Ag by nonactivated CD11c(+) APC. Although this event results in division of naive CTL in tumor draining lymph nodes, it does not establish a productive immune response. Treatment of tumor-bearing mice with dendritic cell-stimulating agonistic anti-CD40 mAb resulted in systemic efflux of CTL with robust effector function capable to eradicate established tumors. For efficacy of anti-CD40 treatment, CD40 ligation of host APC is required because adoptive transfer of CD40-proficient tumor-specific TCR transgenic CTL into CD40-deficient tumor-bearing mice did not lead to productive antitumor immunity after CD40 triggering in vivo. CpG and detoxified LPS (MPL) acted similarly as agonistic anti-CD40 mAb with respect to CD8(+) CTL efflux and tumor eradication. Together these results indicate that dendritic cells, depending on their activation state, orchestrate the outcome of CTL-mediated immunity against tumors, leading either to an ineffective immune response or potent antitumor immunity.     

Th1 and Th2 cell clones to a poorly immunogenic tumor antigen initiate CD8+ T cell-dependent tumor eradication in vivo

Although CD8(+) T cells play a central role as immune effectors, CD4(+) T cells act to control the activation and persistence of the CD8(+) T cell response in autoimmune disease, antiviral immunity, and experimental systems with immunogenic model tumor Ag. However, little information is available on the effects of CD4(+) T cells on the function of endogenous CD8(+) T lymphocytes recognizing authentic tumor rejection Ag with limited immunogenicity. We report here that the prophylactic or postchallenge administration of T helper Th1-type and Th2-type CD4(+) clones specific for an unmutated rejection Ag (murine P815AB, resembling tumor-specific shared Ag in humans) leads to the induction of P815AB-specific reactivity in vivo and concomitant tumor destruction, with quantitative rather than qualitative differences characterizing the antitumor activity of Th1 vs Th2 cells. Because the transferred CD4(+) cells lacked direct antitumor activity in vitro and required the de novo generation of P815AB-specific CD8(+) T cells in vivo, these findings suggest that CD4(+) lymphocytes can enhance the ability of host APC to initiate an endogenous CD8(+) T cell response to authentic, poorly immunogenic tumor rejection Ag.     

IL-2-activated CD8+CD44high cells express both adaptive and innate immune system receptors and demonstrate specificity for syngeneic tumor cells

CD8(+) T cells depend on the alphabeta TCR for Ag recognition and function. However, Ag-activated CD8(+) T cells can also express receptors of the innate immune system. In this study, we examined the expression of NK receptors on a population of CD8(+) T cells expressing high levels of CD44 (CD8(+)CD44(high) cells) from normal mice. These cells are distinct from conventional memory CD8(+) T cells and they proliferate and become activated in response to IL 2 via a CD48/CD2-dependent mechanism. Before activation, they express low or undetectable levels of NK receptors but upon activation with IL-2 they expressed significant levels of activating NK receptors including 2B4 and NKG2D. Interestingly, the IL-2-activated cells demonstrate a preference in the killing of syngeneic tumor cells. This killing of syngeneic tumor cells was greatly enhanced by the expression of the NKG2D ligand Rae-1 on the target cell. In contrast to conventional CD8(+) T cells, IL-2-activated CD8(+)CD44(high) cells express DAP12, an adaptor molecule that is normally expressed in activated NK cells. These observations indicate that activated CD8(+)CD44(high) cells express receptors of both the adaptive and innate immune system and may play a unique role in the surveillance of host cells that have been altered by infection or transformation.     

The surprising kinetics of the T cell response to live antigenic cells

Cooperation between CD4(+) and CD8(+) T cells is required for the proper development of primary effector and memory CD8(+) T cells following immunization with noninflammatory immunogens. In this study, we characterized murine CD4(+) and CD8(+) T cell responses to male-specific minor histocompatibility (HY) Ags following injection of live male cells into females of the same strain. Male cells are rejected 10-12 days after transfer, coinciding with the expansion and effector function of CD8(+) CTLs to two H-2D(b)-restricted epitopes. Although anti-HY CD4(+) T cell responses are readily detectable day 5 posttransfer, CD8(+) responses are undetectable until day 10. The early CD4(+) response is not dependent on direct presentation of Ag by donor male cells, but depends on presentation of the male cells by recipient APC. The CD4(+) T cell response is required for the priming of CD8(+) T cell effector responses and rejection of HY-incompatible cells. Unexpectedly, HY-specific CD4(+) T cells are also capable of efficiently lysing target cells in vivo. The delay in the CD8(+) T cell response can be largely abrogated by depleting T cells from the male inoculum, and donor male CD8(+) T cells in particular suppress host anti-HY CD8(+) responses. These data demonstrate dramatic differences in host T cell responses to noninflammatory Ags compared with responses to pathogens. We explain the delayed CD8(+) response by proposing that there is a balance between cross-presentation of Ag by helper cell-licensed dendritic cells, on the one hand, and veto suppression by live male lymphocytes on the other.     

Activation of CD8+ regulatory T cells by human placental trophoblasts

The immunological basis by which a mother tolerates her semi-allogeneic fetus remains poorly understood. Several mechanisms are likely to contribute to this phenomenon including active immune regulation by regulatory T cells. In this article, we report that human placental trophoblasts activate a clonal population of CD8(+) T cells with regulatory function. These cells are not MHC class I restricted, but require costimulation through a member of the carcinoembryonic Ag family present on early gestation trophoblasts. These regulatory T cells express the mucosal markers CD101 and CD103 and display selective usage of the TCR gene Vbeta9. CD8(+) T cells isolated from the peripheral blood of pregnant mothers (16-28 wk) also demonstrate expansions in the same Vbeta family (Vbeta9), signaling a possible role for these cells in preventing fetal rejection in vivo. We have previously characterized a subset of CD8(+) regulatory T cells activated by the combination of the nonclassical class I molecule CD1d and a costimulatory molecule of the carcinoembryonic Ag family present on the intestinal epithelium. These data support the concept that distinct regulatory T cell populations exist at different sites and may be regulated locally by unique restriction elements, costimulatory signals, and Ags.     

Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity

Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.     

In vivo manipulation of dendritic cells overcomes tolerance to unmodified tumor-associated self antigens and induces potent antitumor immunity

Most tumor-associated Ags are self proteins that fail to elicit a T cell response as a consequence of immune tolerance. Dendritic cells (DCs) generated ex vivo have been used to break tolerance against such self Ags; however, in vitro manipulation of DCs is cumbersome and difficult to control, resulting in vaccines of variable potency. To address this problem we developed a method for loading and activating DCs, in situ, by first directing sufficient numbers of DCs to peripheral tissues using Flt3 ligand and then delivering a tumor-associated Ag and oligonucleotide containing unmethylated CG motifs to these tissues. In this study, we show in three different tumor models that this method can overcome tolerance and induce effective antitumor immunity. Vaccination resulted in the generation of CD8(+) T and NK cell effectors that mediated durable tumor responses without attacking normal tissues. These findings demonstrate that unmodified tumor-associated self Ags can be targeted to DCs in vivo to induce potent systemic antitumor immunity.     

Passive and active mechanisms trap activated CD8+ T cells in the liver

The liver is a site where activated CD8(+) T cells are trapped and destroyed at the end of an immune response. The intrahepatic accumulation of activated murine TCR transgenic CD8(+) T cells was significantly reduced when either ICAM-1 or VCAM-1 was blocked by specific Ab. These two adhesion mechanisms account for essentially all the trapping of activated CD8(+) T cells in the mouse liver. Although the ICAM-1-mediated trapping depends on the capacity of the vasculature and/or the parenchymal cells to present Ag, the accumulation of cells through VCAM-1 does not require Ag recognition. Thus, Ags expressed by the non-bone marrow-derived cells in the liver actively cause CD8(+) T cell accumulation through TCR-activated ICAM-1 adhesion, but the liver can also passively sequester activated CD8(+) T cells that do not recognize intrahepatic Ag, through VCAM-1 adhesion.     

Cutting edge: CD4+ T cell control of CD8+ T cell reactivity to a model tumor antigen

Neoantigens resulting from the inherent genomic instability of tumor cells generally do not trigger immune recognition. Similarly, transfection of tumors with model Ags often fails to elicit CD8+ T cell responses or alter a tumor's growth rate or lethality. We report here that the adoptive transfer of activated Th1-type CD4+ T cells specific for a model tumor Ag results in the de novo generation of CD8+ T cells with specificity to that Ag and concomitant tumor destruction. The anti-tumor effects of the CD4+ T cells required the presence of both MHC class I and class II on host cells, as evidenced by experiments in knockout mice, suggesting that CD4+ T cells enhanced the ability of host APC to activate endogenous CD8+ T cells. These results indicate that the apparent inability of tumor cells expressing highly immunogenic epitopes to activate tumor-specific CD8+ T cells can be altered by activated CD4+ T cells.     

IL-17-dependent, IFN-gamma-independent tumor rejection is mediated by cytotoxic T lymphocytes and occurs at extraocular sites, but is excluded from the eye

Although intraocular tumors reside in an immune-privileged site where immune responses are suppressed, some tumors are rejected. An example of this is the rejection of intraocular adenovirus-induced (adenovirus type 5 early region 1 [Ad5E1]) tumors in C57BL/6 mice. We previously identified an Ad5E1 tumor clone in which the rejection is IFN-γ dependent and culminates in the destruction of both the tumor and the eye. Although Ad5E1 tumors are not rejected when transplanted into the eyes of IFN-γ KO mice, they are rejected after s.c. transplantation. Thus, outside of the eye Ad5E1 tumors elicit a form of tumor immunity that is IFN-γ independent. In this article, we demonstrate that IFN-γ-independent s.c. rejection requires both CD4(+) and CD8(+) T cells. Furthermore, s.c. tumor rejection requires IL-17, which is produced by IFN-γ-deficient CD4(+) T cells in response to tumor Ags (TAs). Splenocytes from CD4-depleted IFN-γ KO mice produce significantly less IL-17 compared with splenocytes from isotype-treated IFN-γ KO animals in response to TAs. Furthermore, depletion of IL-17 decreases CTL activity against Ad5E1 tumor cells. In this model we propose that, in the absence of IFN-γ, CD4(+) T cells produce IL-17 in response to TAs, which increases CTL activity that mediates tumor rejection; however, this does not occur in the eye. IL-6 production within the eye is severely reduced, which is consistent with the failure to induce Th17 cells within the intraocular tumors. In contrast, the s.c. environment is replete with IL-6 and supports the induction of Th17 cells. Therefore, IFN-γ-independent tumor rejection is excluded from the eye and may represent a newly recognized form of ocular immune privilege.     

CD8+ T cells circumvent immune privilege in the eye and mediate intraocular tumor rejection by a TNF-alpha-dependent mechanism

Although intraocular tumors reside in an immune-privileged environment, T cells can circumvent immune privilege and mediate tumor rejection without inducing damage to normal ocular tissue. In this study, we used a well-characterized tumor, Ad5E1 (adenovirus type 5 early region 1), to analyze the role of CD8+ T cells in the pristine rejection of intraocular tumors. It has been previously documented that Ad5E1 tumor rejection can occur in the absence of CD8+ T cells. However, here we find that CD8+ T cells infiltrated intraocular Ad5E1 tumors in C57BL/6 mice. Surprisingly, CD8+ T cells from tumor-rejector mice could mediate intraocular tumor rejection following adoptive transfer to SCID mice. In determining the mechanisms behind CD8+ T cell-mediated tumor rejection, we discovered that antitumor CTL activity was neither observed nor necessary for rejection of the intraocular tumors. CD8+ T cells from rejector mice did not produce IFN-gamma in response to Ad5E1 tumor Ags or use FasL to mediate intraocular tumor rejection. Also, CD8+ T cells did not use perforin or TRAIL, as CD8+ T cells from perforin knockout (KO) and TRAIL KO mice conferred protection to SCID recipient mice following adoptive transfer. We discovered that CD8+ T cells used TNF-alpha to mediate tumor rejection, because Ad5E1 tumor cells were highly sensitive to TNF-alpha-induced apoptosis and CD8+ T cells from TNF-alpha KO mice did not protect SCID mice from progressive Ad5E1 tumor growth. The results indicate that CD8+ T cells circumvent immune privilege and mediate intraocular tumor rejection by a TNF-alpha-dependent manner while leaving the eye intact and vision preserved.