Intraocular tumor antigen drains specifically to submandibular lymph nodes, resulting in an abortive cytotoxic T cell reaction

Ocular immune privilege is considered essential in the protection against sight-threatening immune responses, as illustrated by the ability of the ocular environment to permit the growth of tumors that are rejected when implanted at other sites. Although several studies indicate that soluble Ag can drain directly into the spleen when injected into the anterior chamber, the primary site of intraocular tumor Ag presentation to tumor-specific CTLs has not been studied. To gain a better understanding of the mechanism involved in ocular immune privilege, we examined to which lymphoid organs anterior chamber tumor Ags primarily drain. Our data show that intraocular tumor Ag drains exclusively to the submandibular lymph nodes, resulting in activation of tumor-specific CTLs, whereas no Ag drainage was found in spleen. However, these tumor-specific CTLs do not distribute systemically and, as a consequence, intraocular tumor growth is unhampered. A similar lack of CTL efficacy has been observed in mice bearing s.c. tumors, which is converted to a systemic tumoricidal CTL response by administration of agonistic anti-CD40 mAb. In contrast, systemic anti-CD40 treatment of eye tumor-bearing mice did not result in mobilizing tumor-specific CTLs or tumor eradication. Together, these results show that intraocular tumor Ag drains to regional lymph nodes for activation of tumor-specific CTLs. However, the induced tumor-specific immunity is insufficient for tumor clearance, even combined with otherwise highly effective immune intervention protocols.     

Longevity of antigen presentation and activation status of APC are decisive factors in the balance between CTL immunity versus tolerance

Encounter of Ag by naive T cells can lead to T cell priming as well as tolerance. The balance between immunity and tolerance is controlled by the conditions of Ag encounter and the activation status of the APC. We have investigated the rules that govern this balance in case an environment that normally induces tolerance is reverted into a milieu that promotes T cell priming, using a minimal CTL epitope derived from human adenovirus type 5 E1A. Vaccination of mice s.c. with E1A peptide in IFA readily induces CTL tolerance, resulting in the inability to control E1A-expressing tumors. The present study shows that efficient CTL priming is achieved when this peptide vaccine is combined with systemic administration of APC-activating compounds like agonistic anti-CD40 mAb or polyriboinosinate-polyribocytidylate. Surprisingly, this CTL response is not long-lasting and therefore fails to protect against tumor outgrowth. Disappearance of CTL reactivity was strongly associated with systemic persistence of the peptide for >200 days. In contrast, peptide administered in PBS does not persist and generates long term CTL immunity capable of rejecting Ad5E1A-positive tumors, when combined with CD40 triggering. Thus, presentation of CTL epitopes in an appropriate costimulatory setting by activated APC, although being essential and sufficient for CTL priming, eventually results in tolerance when the Ag persists systemically for prolonged times. These observations are important for the development of immune intervention schemes in autoimmunity and cancer.     

Rejection of intraocular tumors by CD4(+) T cells without induction of phthisis.

Immune privilege of the eye protects against sight-threatening inflammatory events, but can also permit outgrowth of otherwise nonlethal immunogenic tumors. Nonetheless, ocular tumor growth can be controlled by cellular immune responses. However, this will normally result in phthisis of the eye, in case tumor rejection is mediated by a delayed-type hypersensitivity response orchestrated by CD4(+) T cells. We now show that intraocular tumors can be eradicated by CD4(+) Th cells without inducing collateral damage of neighboring ocular tissue. Injection of tumor cells transformed by the early region 1 of human adenovirus type 5 in the anterior chamber of the eye leads to intraocular tumor formation. Tumor growth is transient in immunocompetent mice, but lethal in immunodeficient nude mice, indicating that T cell-dependent immunity is responsible for tumor clearance. Tumor rejection has all the characteristics of a CD8(+) T cell-mediated immune response, as the tumor did not express MHC class II and only tumor tissue was the subject of destruction. However, analysis of the molecular and cellular mechanisms involved in tumor clearance revealed that perforin, TNF-alpha, Fas ligand, MHC class I, and CD8(+) T cells did not play a crucial role in tumor eradication. Instead, effective tumor rejection was entirely dependent on CD4(+) Th cells, as CD4-depleted as well as MHC class II-deficient mice were unable to reject their intraocular tumor. Taken together, these observations demonstrate that CD4(+) T cells are able to eradicate MHC class II-negative tumors in an immune-privileged site without affecting surrounding tissues or the induction of phthisis.     

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.     

Intracamerally induced concomitant immunity: mice harboring progressively growing intraocular tumors are immune to spontaneous metastases and secondary tumor challenge

Intracameral inoculation of allogeneic P815 mastocytoma cells (DBA/2) into BALB/c mice resulted in progressively growing intraocular tumors. Intraocular tumor cells disseminated rapidly to the spleen and cervical lymph nodes, yet extraocular nests of tumor cells never developed into fulminant tumors. Further experiments showed that tumor cells were continuously seeded from the primary intraocular tumor and were rapidly cleared from extraocular sites. Hosts harboring intraocular P815 mastocytomas rejected tumorigenic doses of P815 cells inoculated subcutaneously or even into the contralateral anterior chamber. This systemic tumor immunity was found to be radiosensitive and T cell dependent. Spleen cells from animals with progressively growing intraocular tumors protected recipient mice challenged with intracamerally inoculated tumor cells and thus suggests that a cell-mediated mechanism is the underlying basis for this form of tumor immunity. The data indicate that mice harboring progressively growing intraocular tumors develop a potent state of "concomitant immunity," that prevents the development of metastases, yet is ineffective in controlling the primary tumor.     

Adoptive T cell immunotherapy of human uveal melanoma targeting gp100

HLA-A*0201-restricted CTL against human gp100 were isolated from HLA-A*0201/K(b) (A2/K(b))-transgenic mice immunized with recombinant canarypox virus (ALVAC-gp100). These CTL strongly responded to the gp100(154-162) epitope, in the context of both the chimeric A2/K(b) and the wild-type HLA-A*0201- molecule, and efficiently lysed human HLA-A*0201(+), gp100(+) melanoma cells in vitro. The capacity of the CTL to eradicate these tumors in vivo was analyzed in A2/K(b)-transgenic transgenic mice that had received a tumorigenic dose of human uveal melanoma cells in the anterior chamber of the eye. This immune-privileged site offered the unique opportunity to graft xenogeneic tumors into immunocompetent A2/K(b)-transgenic mice, a host in which they otherwise would not grow. Importantly, systemic (i.v.) administration of the A2/K(b)-transgenic gp100(154-162)-specific CTL resulted in rapid elimination of the intraocular uveal melanomas, indicating that anti-tumor CTL are capable of homing to the eye and exerting their tumoricidal effector function. Flow cytometry analysis of ocular cell suspensions with HLA-A*0201-gp100(154-162) tetrameric complexes confirmed the homing of adoptively transferred CTL. Therefore, the immune-privileged state of the eye permitted the outgrowth of xenogeneic uveal melanoma cells, but did not protect these tumors against adoptive immunotherapy with highly potent anti-tumor CTL. These data constitute the first direct indication that immunotherapy of human uveal melanoma may be feasible.     

Systemic immunological tolerance to ocular antigens is mediated by TRAIL-expressing CD8+ T cells

Systemic immunological tolerance to Ag encountered in the eye restricts the formation of potentially damaging immune responses that would otherwise be initiated at other anatomical locations. We previously demonstrated that tolerance to Ag administered via the anterior chamber (AC) of the eye required Fas ligand-mediated apoptotic death of inflammatory cells that enter the eye in response to the antigenic challenge. Moreover, the systemic tolerance induced after AC injection of Ag was mediated by CD8(+) regulatory T cells. This study examined the mechanism by which these CD8(+) regulatory T cells mediate tolerance after AC injection of Ag. AC injection of Ag did not prime CD4(+) T cells and led to increased TRAIL expression by splenic CD8(+) T cells. Unlike wild-type mice, Trail(-/-) or Dr5(-/-) mice did not develop tolerance to Ag injected into the eye, even though responding lymphocytes underwent apoptosis in the AC of the eyes of these mice. CD8(+) T cells from Trail(-/-) mice that were first injected via the AC with Ag were unable to transfer tolerance to naive recipient wild-type mice, but CD8(+) T cells from AC-injected wild-type or Dr5(-/-) mice could transfer tolerance. Importantly, the transferred wild-type (Trail(+/+)) CD8(+) T cells were also able to decrease the number of infiltrating inflammatory cells into the eye; however, Trail(-/-) CD8(+) T cells were unable to limit the inflammatory cell ingress. Together, our data suggest that "helpless" CD8(+) regulatory T cells generated after AC injection of Ag enforce systemic tolerance in a TRAIL-dependent manner to inhibit inflammation in the eye.     

The tumoricidal activity of memory CD8+ T cells is hampered by persistent systemic antigen, but full functional capacity is regained in an antigen-free environment

Naive T cells can be tolerized in the periphery by diverse mechanisms. However, the extent to which memory T cells are susceptible to tolerance induction is less well defined. Vaccination of mice with a minimal CTL epitope derived from human adenovirus type 5 E1A in IFA s.c. readily tolerizes naive as well as recently activated CD8(+) T cells due to the overwhelming systemic and persistent presence of the peptide. We have now studied the effect of this peptide on established memory cells, which were induced at least 50 days before by virus vaccination. Memory cells did not undergo peripheral deletion and kept their ability to produce IFN-gamma as well as their cytolytic activity in response to Ag directly ex vivo. However, memory CTL responses in virus vaccinated mice injected with peptide ceased to control tumor outgrowth. Interestingly, functional capacities were regained when T cells were transferred to an Ag-free environment in vivo as determined by their ability to reject an otherwise lethal tumor challenge. Together, these findings indicate that memory CTL responses can be functionally incapacitated, but are not, in contrast to naive or recently activated T cells, irreversibly tolerized by persistent systemic Ag, as memory T cells quickly regain effector function upon disappearance of the Ag.     

Cytokine requirements for induction of systemic and mucosal CTL after nasal immunization.

Cholera toxin (CT) is frequently used as an experimental adjuvant intranasally for the induction of systemic and mucosal immunity. However, CT is highly reactogenic and not approved for use in humans. To define the cytokine requirements for the nasal activation of the systemic and mucosal immune system, and to design new adjuvants with efficacy similar to CT, we defined the cytokines that were able to replace CT as a nasal adjuvant for the induction of CTL. BALB/c mice were nasally immunized with an HIV immunogen that contains an MHC class I-restricted CTL epitope +/- cytokines and tested for HIV-specific immune responses. We found that combinations of IL-1alpha plus IL-18, IL-1alpha plus IL-12, and IL-1alpha plus IL-12 plus GM-CSF each induced optimal splenocyte anti-HIV CTL responses in immunized mice (range 60-71% peptide-specific (51)Cr release). Peak H-2D(d)-peptide tetramer-binding T cell responses induced by cytokine combinations were up to 5.5% of CD8(+) PBMC. Nasal immunization with HIV immunogen and IL-1alpha, IL-12, and GM-CSF also induced Ag-specific IFN-gamma-secreting cells in the draining cervical lymph node and the lung. The use of IL-1alpha, IL-12, and GM-CSF as nasal adjuvants was associated with an increased expression of MHC class II and B7.1 on nonlymphocytes within the nasal-associated lymphoid tissue/nasal mucosa. Thus, IL-1alpha, IL-12, IL-18, and GM-CSF are critical cytokines for the induction of systemic and mucosal CTL after nasal immunization. Moreover, these cytokines may serve as effective adjuvants for nasal vaccine delivery.     

Emergence of a dominant cytotoxic T lymphocyte antitumor effector from tumor-infiltrating cells in the anterior chamber of the eye

Summary Previous studies in mice revealed that resolving intraocular tumors (UV5C25 fibrosarcoma) were infiltrated with mononuclear cells and invoked potent systemic delayed-type hypersensitivity responses without nonspecific tissue destruction. The present study characterized the tumor-infiltrating lymphocyte (TIL) population and established its role as the mediator of specific intraocular tumor rejection. This was accomplished by (a) isolating TIL from resolving intraocular tumors; (b) identifying characteristic surface markers on TIL; and (c) demonstrating in vitro and in vivo antitumor functions. Fluorescence-activated cell sorter analysis of TIL showed 33.4% Thy1⁺, 19.8% CD8⁺, 11.1% CD4⁺, 17.2% MAC–1⁺, 10.4% F4/80⁺, and 7.7% B220⁺. Functional studies indicated that TIL were directly cytolytic for UV5C25 tumor cells. Additionally a tumor-necrosis-factor(TNF)-sensitive cell line (WEHI 164.1) was lysed on cocultivation with TIL, whereas UV5C25 tumor cells were insensitive to lysis by TNF. Precursor CTL analysis demonstrated a high frequency (1/251) of tumor-specific precursors and a low frequency of alloresponsive cells in the TIL population. In vivo analysis by a Winn-type assay demonstrated that only TIL could effect tumor resolution in immunosuppressed hosts. These results demonstrate that although CD4⁺ T cells and macrophages were present and TNF activity was detected in the TIL population, there was no evidence for nonspecific tissue destruction within the eye. Therefore, this pattern of intraocular tumor rejection is mediated by a lymphocyte population expressesing cell-surface phenotypes and functional characteristics of conventional cytotoxic T lymphocytes. Moreover, the results suggest that a regulatory mechanism within the eye allows for the emergence of one dominant antitumor effector (CTL) while controlling a more destructive mechanism (delayed-type hypersensitivity).Supported in part by USPHS grants EYO5631 and CA30276, an unrestricted grant from Research to Prevent Blindness Inc., and a grant from the Texas Department of the Ladies Auxiliary, Veterans of Foreign Wars. Dr. Niederkorn is a Research to Prevent Blindness Olga Keith Wiess Scholar     

Fas-mediated T cell deletion potentiates tumor antigen-specific tolerance in a mouse model of prostate cancer

A pivotal obstacle to cancer immunotherapy is peripheral T cell tolerance to tumor-associated antigens (TAAs). Tolerance induction among mature T cells in the periphery operates through a variety of mechanisms, including anergy and apoptosis. Although Fas-FasL-mediated apoptosis is a well-defined tolerance inducing mechanism, direct evidence of its interference with TAA-specific immunity in vivo is still lacking. In this report, we used the TRAMP mouse, which expresses SV40 large T antigen (Tag) preferentially in the prostate and develops prostate tumors, as a model system to address the role of Fas-mediated apoptosis in regulating peripheral T cell tolerance. Using RT-PCR and tetramer staining to quantify TAA-specific TCR-expressing cytolytic T lymphocytes (CTLs), we have shown the presence of TAA-specific CTLs at higher levels in TRAMP mice than in syngeneic C57Bl/6 mice. Tag-specific immunization led to the expansion of Tag-specific CTLs in C57Bl/6 mice, and to their elimination in TRAMP mice. Interestingly, in TRAMP mice with deficient Fas (Hybrid TRAMP-lpr/lpr), Tag-specific CTL elimination in response to Tag immunization did not take place. The results of cytolytic-function assays were consistent with induction and elimination patterns of TAA-specific CTLs and those of RT-PCR and tetramer staining. In conclusion, our data show that Fas-mediated TAA-specific CTL apoptosis contributes to T cell tolerance and suggest that such tolerance could be potentiated following TAA-specific immunization.     

Studies on the recovery from tolerance to tumor antigens

C3H/He mice were injected i.v. with heavily X-irradiated syngeneic X5563 tumor cells three times at 4-day intervals. This regimen resulted in the abrogation of the potential to generate X5563 tumor-specific T cell-mediated immunity as induced by i.d. inoculation of viable X5563 tumor cells followed by surgical resection of the tumor, representing the tolerance induction. Although such a tumor-specific tolerant state was long-lasting, the recovery of anti-X5563 effector T cell responses was observed when the above ordinary immunization procedure was performed 6 months after the tolerance induction. The present study investigated whether the recovery from the tolerance can be accelerated by applying a helper-effector T-T cell interaction model in which enhanced anti-X5563 immunity is obtained by priming mice with BCG and by immunizing X5563 tumor cells modified with BCG cross-reactive MDP hapten (designated as L4-MDP) in the presence of anti-L4-MDP helper T cells preinduced with BCG. The results demonstrated that BCG-primed mice which received the tolerance regimen failed to generate anti-X5563 immunity when the ordinary immunization was performed 2 or 3 months after the tolerance induction. In contrast, the immunization of BCG-primed and X5563-tolerant mice with L4-MDP-coupled X5563 tumor cells at comparable timing to that of the ordinary immunization were capable of generating potent X5563-specific in vivo protective T cell-mediated immunity. As control groups, BCG-primed or unprimed tolerant mice did not develop anti-X5563 immunity when immunized with L4-MDP-uncoupled or L4-MDP-coupled tumor cells, respectively. These results indicate that immunization of BCG-primed, tumor-tolerant mice with L4-MDP-modified tumor cells results in accelerated recovery from the tumor tolerance.     

Oral Tolerance to Cancer Can Be Abrogated by T Regulatory Cell Inhibition

Oral administration of tumour cells induces an immune hypo-responsiveness known as oral tolerance. We have previously shown that oral tolerance to a cancer is tumour antigen specific, non-cross-reactive and confers a tumour growth advantage. We investigated the utilisation of regulatory T cell (Treg) depletion on oral tolerance to a cancer and its ability to control tumour growth. Balb/C mice were gavage fed homogenised tumour tissue – JBS fibrosarcoma (to induce oral tolerance to a cancer), or PBS as control. Growth of subcutaneous JBS tumours were measured; splenic tissue excised and flow cytometry used to quantify and compare systemic Tregs and T effector (Teff) cell populations. Prior to and/or following tumour feeding, mice were intraperitoneally administered anti-CD25, to inactivate systemic Tregs, or given isotype antibody as a control. Mice which were orally tolerised prior to subcutaneous tumour induction, displayed significantly higher systemic Treg levels (14% vs 6%) and faster tumour growth rates than controls (p<0.05). Complete regression of tumours were only seen after Treg inactivation and occurred in all groups - this was not inhibited by tumour feeding. The cure rates for Treg inactivation were 60% during tolerisation, 75% during tumour growth and 100% during inactivation for both tolerisation and tumour growth. Depletion of Tregs gave rise to an increased number of Teff cells. Treg depletion post-tolerisation and post-tumour induction led to the complete regression of all tumours on tumour bearing mice. Oral administration of tumour tissue, confers a tumour growth advantage and is accompanied by an increase in systemic Treg levels. The administration of anti-CD25 Ab decreased Treg numbers and caused an increase in Teffs. Most notably Treg cell inhibition overcame established oral tolerance with consequent tumor regression, especially relevant to foregut cancers where oral tolerance is likely to be induced by the shedding of tumour tissue into the gut.     

Critical components of a DNA fusion vaccine able to induce protective cytotoxic T cells against a single epitope of a tumor antigen

DNA vaccines can activate immunity against tumor Ags expressed as MHC class I-associated peptides. However, priming of CD8(+) CTL against weak tumor Ags may require adjuvant molecules. We have used a pathogen-derived sequence from tetanus toxin (fragment C (FrC)) fused to tumor Ag sequences to promote Ab and CD4(+) T cell responses. For induction of CD8(+) T cell responses, the FrC sequence has been engineered to remove potentially competitive MHC class I-binding epitopes and to improve presentation of tumor epitopes. The colon carcinoma CT26 expresses an endogenous retroviral gene product, gp70, containing a known H2-L(d)-restricted epitope (AH1). A DNA vaccine encoding gp70 alone was a poor inducer of CTL, and performance was not significantly improved by fusion of full-length FrC. However, use of a minimized domain of FrC, with the AH1 sequence fused to the 3' position, led to rapid induction of high levels of CTL. IFN-gamma-producing epitope-specific CTL were detectable ex vivo and these killed CT26 targets in vitro. The single epitope vaccine was more effective than GM-CSF-transfected CT26 tumor cells in inducing an AH1-specific CTL response and equally effective in providing protection against tumor challenge. Levels of AH1-specific CTL in vivo were increased following injection of tumor cells, and CTL expanded in vitro were able to kill CT26 cells in tumor bearers. Pre-existing immunity to tetanus toxoid had no effect on the induction of AH1-specific CTL. These data demonstrate the power of epitope-specific CTL against tumor cells and illustrate a strategy for priming immunity via a dual component DNA vaccine.     

Destructive and nondestructive patterns of immune rejection of syngeneic intraocular tumors

Two immunogenic, syngeneic murine tumors were used to analyze the immunopathological processes associated with the immune rejection of primary intraocular tumors. Intracameral inoculation of P91 mastocytoma, an immunogenic variant of P815 mastocytoma, into DBA/2 mice resulted in progressive tumor growth for several weeks before immune rejection eradicated the intraocular neoplasm. The histopathologic characteristics of the tumor rejection included: a) destruction of the vascular endothelium of the microvasculature feeding the tumor; b) ischemic bulk necrosis; c) extensive innocent bystander damage to normal ocular structures; and d) absence of direct inflammatory cell-to-tumor cell contact. Thus, the immunopathological features resembled a delayed-type hypersensitivity (DTH) lesion. A second intraocular tumor model was similarly studied. UV5C25 fibrosarcoma grew slowly in the eyes of syngeneic BALB/c hosts. In sharp contrast to P91 tumors, a mononuclear cellular infiltrate was prominent within the tumor. After 5 wk, the intraocular tumors were completely rejected without detectable damage to normal ocular structures. The rejection of UV5C25 tumors did not produce scar tissue, damage to vascular endothelium, bulk necrosis, or atrophy of the globe. Although tumor-specific cytotoxic T lymphocytes (CTL) and DTH responses were readily detected, there was no histological evidence for DTH-mediated tumor rejection. Moreover, in situ immunoperoxidase staining indicated that the majority of the infiltrating lymphocytes were CTL, based on their characteristic phenotype: Thy-1+, Lyt-2+. Furthermore, the growth of UV5C25 fibrosarcoma in athymic, natural killer (NK) cell competent BALB/c nude mice demonstrated progressive tumor growth without infiltrating host cells. Collectively, the results indicate that immunogenic intraocular tumors can undergo strikingly different patterns of immune rejection with profoundly different pathological consequences. In one case (P91), tumor rejection occurs by a process that strongly resembles DTH and produces extensive nonspecific damage to normal tissues, resulting in irrevocable loss of vision. In contrast, the second intraocular tumor undergoes an immune rejection that is characterized by precision and a notable absence of damage to normal ocular tissues. The weight of evidence presented here strongly supports the hypothesis that the latter form of tumor rejection is mediated by CTL. Thus, the immunologic pathway invoked for tumor rejection in the eye has a profound effect on the fate of this delicate organ and the preservation of vision.     

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.     

The role of tumor-specific Lyt-1+2- T cells in eradicating tumor cells in vivo. I. Lyt-1+2- T cells do not necessarily require recruitment of host's cytotoxic T cell precursors for implementation of in vivo immunity

The present study determines the Ly phenotype of T cells mediating tumor cell rejection in vivo and investigates some of cellular mechanisms involved in the in vivo protective immunity. C3H/HeN mice were immunized to syngeneic X5563 plasmacytoma by intradermal (i.d.) inoculation of viable X5563 tumor cells, followed by the surgical resection of the tumor. Spleen cells from these immune mice were fractionated by treatment with anti-Lyt antibodies plus complement, and each Lyt subpopulation was tested for the reconstituting potential of in vivo protective immunity in syngeneic T cell-depleted mice (B cell mice). When C3H/HeN B cell mice were adoptively transferred with Lyt-1-2+ T cells from the above tumor-immunized mice, these B cell mice exhibited an appreciable cytotoxic T lymphocyte (CTL) response to the X5563 tumor, whereas they failed to resist the i.d. challenge of X5563 tumor cells. In contrast, the adoptive transfer of Lyt-1+2- anti-X5563 immune T cells into B cell mice produced complete protection against the subsequent tumor cell challenge. Although no CTL or antibody response against X5563 tumors was detected in the above tumor-resistant B cell mice, these mice were able to retain Lyt-1+2- T cell-mediated delayed-type hypersensitivity (DTH) responses to the X5563 tumor. These results indicate that Lyt-1+2- T cells depleted of the Lyt-2+ T cell subpopulation containing CTL or CTL precursors are effective in in vivo protective immunity, and that these Lyt-1+2- T cells implement their in vivo anti-tumor activity without inducing CTL or antibody responses. The mechanism(s) by which Lyt-1+2- T cells function in vivo for the implementation of tumor-specific immunity is discussed in the context of DTH responses to the tumor-associated antigens and its related Lyt-1+2- T cell-mediated lymphokine production.     

CD40 activation in vivo overcomes peptide-induced peripheral cytotoxic T-lymphocyte tolerance and augments anti-tumor vaccine efficacy.

The outcome of antigen recognition by naive CD8+ cytotoxic T lymphocytes (CTLs) in the periphery is orchestrated by CD4+ T-helper cells, and can either lead to priming or tolerization. The presence of T-helper cells favors the induction of CTL immunity, whereas the absence of T-helper cells can result in CTL tolerance. The action of T helper cells in CTL priming is mediated by CD40-CD40 ligand interactions. We demonstrate here that triggering of CD40 in vivo can considerably enhance the efficacy of peptide-based anti-tumor vaccines. The combination of a tolerogenic peptide vaccine containing a minimal essential CTL epitope with an activating antibody against CD40 converts tolerization into strong CTL priming. Moreover, CD40 ligation can provide an already protective tumor-specific peptide vaccine with the capacity to induce therapeutic CTL immunity in tumor-bearing mice. These findings indicate that the CD40-CD40 ligand pair can act as a 'switch', determining whether naive peripheral CTLs are primed or tolerized, and support the clinical use of CD40-stimulating agents as components of anti-cancer vaccines.     

Three different vaccines based on the 140-amino acid MUC1 peptide with seven tandemly repeated tumor-specific epitopes elicit distinct immune effector mechanisms in wild-type versus MUC1-transgenic mice with different potential for tumor rejection

Low-frequency CTL and low-titer IgM responses against tumor-associated Ag MUC1 are present in cancer patients but do not prevent cancer growth. Boosting MUC1-specific immunity with vaccines, especially effector mechanisms responsible for tumor rejection, is an important goal. We studied immunogenicity, tumor rejection potential, and safety of three vaccines: 1) MUC1 peptide admixed with murine GM-CSF as an adjuvant; 2) MUC1 peptide admixed with adjuvant SB-AS2; and 3) MUC1 peptide-pulsed dendritic cells (DC). We examined the qualitative and quantitative differences in humoral and T cell-mediated MUC1-specific immunity elicited in human MUC1-transgenic (Tg) mice compared with wild-type (WT) mice. Adjuvant-based vaccines induced MUC1-specific Abs but failed to stimulate MUC1-specific T cells. MUC1 peptide with GM-CSF induced IgG1 and IgG2b in WT mice but only IgM in MUC1-Tg mice. MUC1 peptide with SB-AS2 induced high-titer IgG1, IgG2b, and IgG3 Abs in both WT and MUC1-Tg mice. Induction of IgG responses was T cell independent and did not have any effect on tumor growth. MUC1 peptide-loaded DC induced only T cell immunity. If injected together with soluble peptide, the DC vaccine also triggered Ab production. Importantly, the DC vaccine elicited tumor rejection responses in both WT and MUC1-Tg mice. These responses correlated with the induction of MUC1-specific CD4+ and CD8+ T cells in WT mice, but only CD8(+) T cells in MUC1-Tg mice. Even though MUC1-specific CD4+ T cell tolerance was not broken, the capacity of MUC1-Tg mice to reject tumor was not compromised.     

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.