Integrase-Defective Lentiviral Vector Is an Efficient Vaccine Platform for Cancer Immunotherapy.

Integrase-defective lentiviral vectors (IDLVs) have been used as a safe and efficient delivery system in several immunization protocols in murine and non-human primate preclinical models as well as in recent clinical trials. In this work, we validated in preclinical murine models our vaccine platform based on IDLVs as delivery system for cancer immunotherapy. To evaluate the anti-tumor activity of our vaccine strategy we generated IDLV delivering ovalbumin (OVA) as a non-self-model antigen and TRP2 as a self-tumor associated antigen (TAA) of melanoma. Results demonstrated the ability of IDLVs to eradicate and/or controlling tumor growth after a single immunization in preventive and therapeutic approaches, using lymphoma and melanoma expressing OVA. Importantly, LV-TRP2 but not IDLV-TRP2 was able to break tolerance efficiently and prevent tumor growth of B16F10 melanoma cells. In order to improve the IDLV efficacy, the human homologue of murine TRP2 was used, showing the ability to break tolerance and control the tumor growth. These results validate the use of IDLV for cancer therapy.

Skin immunization for effective treatment of multifocal melanoma refractory to PD1 blockade and Braf inhibitors.

BACKGROUND: Despite the remarkable benefits associated with the interventional treatment of melanomas (and other solid cancers) with immune checkpoint and Braf inhibitors (Brafi), most patients ultimately progress on therapy. The presence of multifocal/disseminated disease in patients increases their mortality risk. Hence, the development of novel strategies to effectively treat patients with melanomas that are resistant to anti-PD1 mAb (αPD1) and/or Brafi, particularly those with multifocal/disseminated disease remains a major unmet clinical need. METHODS: Mice developing induced/spontaneous BrafV600E/Pten-/- melanomas were treated by cutaneous immunization with a DNA vaccine encoding the melanoma-associated antigen TRP2, with Brafi or αPD1 alone, or with a combination of these treatments. Tumor progression, tumor-infiltration by CD4+ and CD8+ T cells, and the development of TRP2-specific CD8+ T cells were then monitored over time. RESULTS: Vaccination led to durable antitumor immunity against PD1/Brafi-resistant melanomas in both single lesion and multifocal disease models, and it sensitized PD1-resistant melanomas to salvage therapy with αPD1. The therapeutic efficacy of the vaccine was associated with host skin-resident cells, the induction of a systemic, broadly reactive IFNγ+CD8+ T cell repertoire, increased frequencies of CD8+ TIL and reduced levels of PD1hi/intCD8+ T cells. Extended survival was associated with improved TIL functionality, exemplified by the presence of enhanced levels of IFNγ+CD8+ TIL and IL2+CD4+ TIL. CONCLUSIONS: These data support the use of a novel genetic vaccine for the effective treatment of localized or multifocal melanoma refractory to conventional αPD1-based and/or Brafi-based (immune)therapy.

Functional tumor specific CD8 + T cells in spleen express a high level of PD-1.

The inhibitory effects of programmed cell death 1 (PD-1) receptor on tumor specific T cells were mainly investigated at tumor site. While PD-1 expression can be rapidly unregulated upon T cell activation at lymphoid tissues, little is known about where PD-1 signal exerts its inhibitory function in tumor-bearing host. To address this issue, we assessed the effects of PD-1 on vaccine induced activation of splenic CD8 + T cells in mice. The vaccine consisted of mice CD8 + T cell epitope peptide and poly IC. After vaccination, spleen or tumor tissues were dissociated, IFN-γ synthesis and PD-1 expression by CD8 + T cells were detected by flow cytometry. We found that CD8 + T cells could be successfully activated in spleen after immunization, characterized by the capability of producing IFN-γ when encountering relevant peptide. These activated splenic CD8 + T cells also expressed a high level of PD-1. Although PD-L1 expression in spleen parenchyma was also increased after vaccination, PD-1 blockade did not affect the activation of splenic CD8 + T cells, but enhanced the anti-tumor effects of peptide vaccine. This synergetic effect of peptide vaccine plus PD-1 blockade was coupled with increased aggregation of IFN-γ + CD8 + tumor infiltrated lymphocytes (TILs), rather than CD4 + TILs. The results indicated that for tumor-bearing host, PD-1 pathway exerted its inhibitory function at tumor site and PD-1 expression on the splenic CD8 + T cells correlated positively with IFN-γ synthesis.

Sulfated archaeal glycolipid archaeosomes as a safe and effective vaccine adjuvant for induction of cell-mediated immunity.

Archaeosomes are liposomal vesicles composed of ether glycerolipids unique to the domain of Archaea. Unlike conventional ester-linked liposomes, archaeosomes exhibit high stability and possess strong adjuvant and immunostimulatory properties making them an attractive vaccine delivery vehicle. Traditionally comprised of total polar lipids (TPL) or semi-synthetic phospho-glycerolipids of ether-linked isoprenoid phytanyl cores with varied glycol- and amino-head groups, archaeosomes can induce robust and long-lasting humoral and cell-mediated immune responses against antigenic cargo and provide protection in murine models of infectious disease and cancer. However, traditional TPL archaeosome formulations are relatively complex comprising several lipid species. Semi-synthetic archaeosomes tested previously contain a combination of several phospho-glycolipids (negative and neutral charged) to produce a stable, uniform-sized liposome formulation. Moreover, they involve many synthetic steps to arrive at the final desired glycolipid composition. Herein, we present a novel adjuvant formulation comprising a sulfated saccharide group covalently linked to the free sn-1 hydroxyl backbone of an archaeal core lipid (sulfated S-lactosylarchaeol, SLA). SLA individually or mixed with uncharged glyolipid (lactosylarchaeol, LA) constituted efficacious carrier vesicles for entrapped antigens (ovalbumin or melanoma associated tyrosinase-related protein 2 [TRP-2]) and induction of strong cell-mediated responses in mice and protection against subsequent B16 melanoma tumor challenge. Thus, semi-synthetic sulfated glycolipid archaeosomes represent a new class of adjuvants that will potentially ease manufacturing and scale-up, while retaining immunostimulatory activity.

Synthetic TRP2 long-peptide and α-galactosylceramide formulated into cationic liposomes elicit CD8+ T-cell responses and prevent tumour progression.

The lipid antigen α-galactosylceramide (α-GalCer) is a potent activator of invariant natural killer T-cells (iNKT cells) and can stimulate cytotoxic and anti-tumour immune responses. However optimal responses appear to be induced by α-GalCer when cell-based vaccines are delivered intravenously. Here we investigated if co-delivery of protein and peptide antigens along with α-GalCer in a liposomal formulation could stimulate therapeutic anti-tumour immune responses. Cationic liposomes were inherently immune-stimulatory and induced cytotoxic immune responses when delivered both by intravenous and subcutaneous injection. However, only vaccine delivered intravenously stimulated therapeutic anti-tumour immune responses to a peptide antigen. Surface modification with polyethylene glycol (PEG) did not improve immune responses to either intravenously or subcutaneously delivered vaccines. Immune responses to short and long peptide sequences (CD8 and CD4 epitopes) of the self-antigen tyrosinase-related protein 2 (TRP2) as a vaccine antigen, co-delivered with α-GalCer in either cationic liposomes or PBS were further examined. Enhanced production of IFN-γ, increased cytotoxic T-cell responses and tumour survival were observed when a long TRP2-peptide was delivered with α-GalCer in cationic liposomes.

Prostaglandin D synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ.

The antitumor activity of prostaglandin (PG) D2 has been demonstrated against some types of cancer, including gastric cancer. However, exogenous PGD2 is not useful from a clinical point of view because it is rapidly metabolized in vivo. The aim of this study was to clarify the antitumor efficacy of an alternative, PGD synthase (PGDS), on gastric cancer cells. The effects of PGD2 and PGDS on the proliferation of gastric cancer cells were examined in vivo and in vitro. The expression levels of PGD2 receptors and peroxisome proliferator-activated receptor γ (PPARγ) were evaluated by RT-PCR. The effects of a PPARγ antagonist or siPPARγ on the proliferation of cancer cells and the c-myc and cyclin D1 expression were examined in the presence or absence of PGD2 or PGDS. PPARγ was expressed in gastric cancer cell lines, but PGD2 receptors were not. PGD2 and PGDS significantly decreased the proliferation of gastric cancer cells that highly expressed PPARγ. PGDS increased the PGD2 production of gastric cancer cells. A PPARγ antagonist and siPPARγ transfection significantly suppressed the growth-inhibitory effects of PGD2 and PGDS. Expression of c-myc and cyclin D1 was significantly decreased by PGD2 ; this inhibitory effect was suppressed by PPARγ antagonist. Both PGD2 and PGDS significantly decreased subcutaneous tumor growth in vivo. Tumor volume after PGDS treatment was significantly less than PGD2 treatment. These findings suggest that PGDS and PGD2 decrease the proliferation of gastric cancer cells through PPARγ signaling. PGDS is a potentially promising therapeutic agent for gastric cancers that express PPARγ.

Macrophage migration inhibitory factor deficiency augments doxorubicin-induced cardiomyopathy.

BACKGROUND: Recent evidence has depicted a role of macrophage migration inhibitory factor (MIF) in cardiac homeostasis under pathological conditions. This study was designed to evaluate the role of MIF in doxorubicin-induced cardiomyopathy and the underlying mechanism involved with a focus on autophagy. METHODS AND RESULTS: Wild-type (WT) and MIF knockout (MIF(-/-)) mice were given saline or doxorubicin (20 mg/kg cumulative, i.p.). A cohort of WT and MIF(-/-) mice was given rapamycin (6 mg/kg, i.p.) with or without bafilomycin A1 (BafA1, 3 µmol/kg per day, i.p.) for 1 week prior to doxorubicin challenge. To consolidate a role for MIF in the maintenance of cardiac homeostasis following doxorubicin challenge, recombinant mouse MIF (rmMIF) was given to MIF(-/-) mice challenged with or without doxorubicin. Echocardiographic, cardiomyocyte function, and intracellular Ca(2+) handling were evaluated. Autophagy and apoptosis were examined. Mitochondrial morphology and function were examined using transmission electron microscopy, JC-1 staining, MitoSOX Red fluorescence, and mitochondrial respiration complex assay. DHE staining was used to evaluate reactive oxygen species (ROS) generation. MIF knockout exacerbated doxorubicin-induced mortality and cardiomyopathy (compromised fractional shortening, cardiomyocyte and mitochondrial function, apoptosis, and ROS generation). These detrimental effects of doxorubicin were accompanied by defective autophagolysosome formation, the effect of which was exacerbated by MIF knockout. Rapamycin pretreatment rescued doxorubicin-induced cardiomyopathy in WT and MIF(-/-) mice. Blocking autophagolysosome formation using BafA1 negated the cardioprotective effect of rapamycin and rmMIF. CONCLUSIONS: Our data suggest that MIF serves as an indispensable cardioprotective factor against doxorubicin-induced cardiomyopathy with an underlying mechanism through facilitating autophagolysosome formation.

Window of opportunity for neuroprotection with an antioxidant, allene oxide synthase, after hypoxia-ischemia in adult male rats.

AIMS: Oxidative stress is an early event in the cascade leading in neuronal damage after hypoxic-ischemic (HI) brain injury. In the present study, we examined the dose response and window of opportunity for neuroprotection after HI injury with Allene Oxide Synthase (AOS), an anti-oxidative enzyme of the member of cytochrome P450 family. METHODS: Adult male rats received intra-cerebro-ventricular infusions of either saline (vehicle) or AOS (1 µg or 10 µg or 100 µg per rat, intracerebroventricular n = 16 all groups) either 45 min or 3 h after unilateral HI brain injury. Brains were collected 5 days later. The extent of brain damage, neuronal survival, apoptosis, and glial reactions were assessed in the striatum, hippocampus, and cortex. RESULTS: Allene Oxide Synthase was associated with reduced neuronal damage scores when given 45 min, but not 3 h, after HI injury (P < 0.0001) in all brain regions. AOS treatment (10 µg) improved neuronal survival in the striatum, cortex, and hippocampus (P < 0.05, P < 0.001) and reduced the microglia reaction (P < 0.05) and numbers of caspase-3-positive cells in the hippocampus (P < 0.01). CONCLUSIONS: Early blockade of oxidative stress after HI injury reduces inflammatory response, neuronal necrosis, and apoptosis. The neuroprotective effects of AOS were time of administration-dependent suggesting a relatively restricted window of opportunity for acute brain injury.

Macrophage migration inhibitory factor mediates the antidepressant actions of voluntary exercise.

Voluntary exercise is known to have an antidepressant effect. However, the underlying mechanism for this antidepressant action of exercise remains unclear, and little progress has been made in identifying genes that are directly involved. We have identified macrophage migration inhibitory factor (MIF) by analyzing existing mRNA microarray data and confirmed the augmented expression of selected genes under two experimental conditions: voluntary exercise and electroconvulsive seizure. A proinflammatory cytokine, MIF is expressed in the central nervous system and involved in innate and adaptive immune responses. A recent study reported that MIF is involved in antidepressant-induced hippocampal neurogenesis, but the mechanism remains elusive. In our data, tryptophan hydroxylase 2 (Tph2) and brain-derived neurotrophic factor (Bdnf) expression were induced after MIF treatment in vitro, as well as during both exercise and electroconvulsive seizure in vivo. This increment of Tph2 was accompanied by increases in the levels of total serotonin in vitro. Moreover, the MIF receptor CD74 and the ERK1/2 pathway mediate the MIF-induced Tph2 and Bdnf gene expression as well as serotonin content. Experiments in Mif(-/-) mice revealed depression-like behaviors and a blunted antidepressant effect of exercise, as reflected by changes in Tph2 and Bdnf expression in the forced swim test. In addition, administration of recombinant MIF protein produced antidepressant-like behavior in rats in the forced swim test. Taken together, these results suggest a role of MIF in mediating the antidepressant action of exercise, probably by enhancing serotonin neurotransmission and neurotrophic factor-induced neurogenesis in the brain.

Tumor growth control using red blood cells as the antigen delivery system and poly(I:C).

The goal of most current vaccines in tumor immunology is to induce an efficient immune response against the tumor cells. The use of red blood cells (RBCs) for the delivery of tumor-associated antigen to antigen-presenting cells is an innovative approach for cancer immunotherapy. The induction of antigen-specific immune responses after administration of antigen-loaded RBCs has been demonstrated previously in mice. In this paper, we show the utility of this delivery system for cancer immunotherapy in 2 tumor mouse models, using the E.G7-OVA and the B16F10 tumor cells. The non-self-antigen, ovalbumin, loaded in RBCs and the self-tumor antigen, tyrosinase-related protein 2, loaded in RBCs were tested in the E.G7-OVA and the B16F10 tumor models, respectively. We showed that not only protein but also peptide could be efficiently entrapped in RBCs by a controlled lysis/resealing process. In both antigen models, the administration of a small quantity of antigen loaded in RBCs combined with polyinosinic-polycytidylic acid induced an antigen-specific T-cell response and the control of tumor growth in mice, whereas the injection of the same quantity of free antigen did not. The intensity of the T-cell response was dependent on the concentrations of antigen entrapped and the treatment performed on the RBC membrane (antibody coating and heat treatment) to improve antigen delivery. In summary, these results support the use of RBCs as an antigen delivery system for a powerful cancer immunotherapy approach.

Drug delivery system for poorly water-soluble compounds using lipocalin-type prostaglandin D synthase.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a member of the lipocalin superfamily and a secretory lipid-transporter protein, which binds a wide variety of hydrophobic small molecules. Here we show the feasibility of a novel drug delivery system (DDS), utilizing L-PGDS, for poorly water-soluble compounds such as diazepam (DZP), a major benzodiazepine anxiolytic drug, and 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist and anticonvulsant. Calorimetric experiments revealed for both compounds that each L-PGDS held three molecules with high binding affinities. By mass spectrometry, the 1:3 complex of L-PGDS and NBQX was observed. L-PGDS of 500µM increased the solubility of DZP and NBQX 7- and 2-fold, respectively, compared to PBS alone. To validate the potential of L-PGDS as a drug delivery vehicle in vivo, we have proved the prospective effects of these compounds via two separate delivery strategies. First, the oral administration of a DZP/L-PGDS complex in mice revealed an increased duration of pentobarbital-induced loss of righting reflex. Second, the intravenous treatment of ischemic gerbils with NBQX/L-PGDS complex showed a protective effect on delayed neuronal cell death at the hippocampal CA1 region. We propose that our novel DDS could facilitate pharmaceutical development and clinical usage of various water-insoluble compounds.

The treatment and prevention of mouse melanoma with an oral DNA vaccine carried by attenuated Salmonella typhimurium.

Therapeutic vaccines of cancer are attractive for their capacity of breaking the immune tolerance and invoking long-term immune response targeting cancer cells without autoimmunity. An efficient antigen delivery system is the key issue of developing an effective cancer vaccine. Attenuated Salmonella typhimurium as the carrier of cancer vaccine are able to transfer DNA from the prokaryote to the eukaryote and preferentially replicate within the tumor tissue. Heat shock protein 70 delivers the tumor-associated antigens to antigen presenting cells through its polypeptide-binding domain and breaks immune tolerance of the cancer cells. Here we described a novel low-copy-number DNA vaccine based on the Hsp70-TAA complex and carried by the attenuated S. typhimurium strain SL3261. Oral administration of this vaccine elicited specific CTL-mediated lysis of the melanoma tumor cells and marked activation of the T-cells. The therapeutic vaccine effectively protected 57.1% C57BL/6J mice from lethal challenge with B16F10 melanoma tumor cells in prophylactic settings and eraicated 62.5% tumor growth in therapeutic settings. This approach may provide a new strategy for the prevention and treatment of cancer.

Immunotherapy can reject intracranial tumor cells without damaging the brain despite sharing the target antigen.

Although vaccines targeting tissue differentiation Ags represent a promising strategy for cancer immunotherapy, the risk of triggering autoimmune damage to normal tissues remains to be determined. Immunizing against a melanoma-associated Ag, dopachrome tautomerase (DCT), which normal melanocytes and glial cells also express, allowed concurrent analysis of autoimmune consequences in multiple tissues. We show that vaccination with recombinant adenovirus expressing DCT elicited a strong CTL response in C57BL/6 mice, leading to protection against intracranial challenge with B16-F10 melanoma cells. Both histological analysis and behavioral testing indicated that there was no evidence of neuropathology in vaccinated animals and long-term survivors. Although vitiligo or demyelination could be induced by additional stimuli (i.e., surgery or inflammation) in DCT-vaccinated mice, it did not extend beyond the inflammatory area, suggesting that there is self-regulatory negative feedback in normal tissues. These results demonstrate that it is possible to vaccinate against a tumor embedded in a vital organ that shares the target Ag.

Optimal epitope composition after antigen screening using a live bacterial delivery vector: application to TRP-2.

Immunotherapeutic approaches, based on the generation of tumor-specific cytotoxic T-lymphocytes (CTL), are currently emerging as promising strategies of anti-tumor therapy. The potential use of attenuated bacteria as engineered vectors for vaccine development offers several advantages, including the stimulation of innate immunity. We developed an attenuated live bacterial vector using the type III secretion system (TTSS) of Pseudomonas aeruginosa to deliver in vivo tumor antigens. Using an inducible and rapid expression plasmid, vaccination with several antigens of different length and epitope composition, including TRp-2, gp100 and MUC18, was evaluated against glioma tumor cells. We observed similar CTL immunity and T-cell receptor (TCR) repertoire diversity with the vaccines, TRP2(125-243), TRP2L(125-376) and TRP2S(291-376). However, only immunization with TRP2L(125-376) induced significant anti-tumor immunity. Taken together, our data indicate the importance of the epitopes composition and/or peptide length of these peptides for inducing cytotoxic T-lymphocyte (CTL) mediated immunity. Characteristics that consistently improved anti-tumor immunity include: long peptides with immunodominant and cryptic CD8(+) epitopes, and strong CD4(+) Th epitopes. Our bacterial vector is versatile, easy-to-use and quick to produce. This vector is suitable for rapid screening and evaluation of antigens of varying length and epitope composition.

Antigen mRNA-transfected, allogeneic fibroblasts loaded with NKT-cell ligand confer antitumor immunity.

The maturation of dendritic cells (DCs) in situ by danger signals plays a central role in linking innate and adaptive immunity. We previously demonstrated that the activation of invariant natural killer T (iNKT) cells by administration of alpha-galactosylceramide (alpha-GalCer)-loaded tumor cells can act as a cellular adjuvant through the DC maturation. In the current study, we used allogeneic fibroblasts loaded with alpha-GalCer and transfected with antigen-encoding mRNA, thus combining the adjuvant effects of iNKT-cell activation with delivery of antigen to DCs in vivo. We found that these cells produce antigen protein and activate NK and iNKT cells. When injected into major histocompatibility complex (MHC)-mismatched mice, they elicited antigen-specific T-cell responses and provided tumor protection, suggesting that these immune responses depend on host DCs. In addition, antigen-expressing fibroblasts loaded with alpha-GalCer lead to a more potent T-cell response than those expressing NK cell ligands. Thus, glycolipid-loaded, mRNA-transfected allogeneic fibroblasts act as cellular vectors to provide iNKT-cell activation, leading to DC maturation and T-cell immunity. By harnessing the innate immune system and generating an adaptive immune response to a variety of antigens, this unique tool could prove clinically beneficial in the development of immunotherapies against malignant and infectious diseases.

Novel C5a agonist-based dendritic cell vaccine in a murine model of melanoma.

A novel method to improve targeting and presentation of poorly immunogenic tumor-related antigens was investigated. This was performed with a molecular adjuvant constructed by covalently linking a response selective peptide agonist of C5a (YSFKDMP(MeL)aR) to known melanoma tumor-related antigens. C57Bl/6J mice were injected subcutaneously with bone marrow derived dendritic cells (DCs) pulsed with a melanoma epitope (TRP2-P2/Agonist), melanoma epitope tyrosinase (TYR/Agonist), a nonfunctional reverse conformation C5a agonist bound to TYR(reverse peptide) or DMSO-PBS vehicle. Mice were injected with the pulsed DCs and cytokines IL-2 and GMCSF three times prior to subcutaneous challenge with B16-F10 melanoma cells. All groups subsequently received DC vaccine boosters twice per week. Tumor growth was reduced and survival enhanced in mice immunized with the combination of TRP2-P2/Agonist and TYR/Agonist compared to mice receiving reverse peptide or vehicle.

A modified tyrosinase-related protein 2 epitope generates high-affinity tumor-specific T cells but does not mediate therapeutic efficacy in an intradermal tumor model.

The generation of tumor-specific T cells is hampered by the presentation of poorly immunogenic tumor-specific epitopes by the tumor. Here, we demonstrate that, although CD8+ T cells specific for the self/tumor Ag tyrosinase-related protein 2 (TRP2) are readily detected in tumor-bearing hosts, vaccination of either tumor-bearing or naive mice with an epitope derived from TRP2 fails to generate significant numbers of tetramer-staining TRP2-specific T cells or antitumor immunity. We identified an altered peptide epitope, called deltaV, which elicits T cell responses that are cross-reactive to the wild-type TRP2 epitope. Immunization with deltaV generates T cells with increased affinity for TRP2 compared with immunization with the wild-type TRP2 epitope, although TRP2 immunization often generates a greater number of TRP2-specific T cells based on intracellular IFN-gamma analysis. Despite generating higher affinity responses, deltaV immunization alone fails to provide any greater therapeutic efficacy against tumor growth than TRP2 immunization. This lack of tumor protection is most likely a result of both the deletion of high affinity and functional tolerance induction of lower affinity TRP2-specific T cells. Our data contribute to a growing literature demonstrating the ability of variant peptide epitopes to generate higher affinity T cell responses against tumor-specific Ags. However, consistent with most clinical data, simple generation of higher affinity T cells is insufficient to mediate tumor immunity.

Provision of granulocyte-macrophage colony-stimulating factor converts an autoimmune response to a self-antigen into an antitumor response.

Many tumor Ags recognized by T cells are self-Ags. Because high avidity, self-reactive T cells are deleted in the thymus, any residual self-reactive T cells existing in the periphery are likely to be low avidity and nonresponsive due to peripheral tolerance mechanisms. Activation of these residual T cells is critical for targeting tumors for immunotherapy. In this study, we studied immune responses against the murine B16 melanoma using a tyrosinase-related protein 2 (TRP-2) peptide as a model tumor/self-Ag. Our results showed that TRP-2 peptide vaccination alone elicited a weak T cell response and modestly decreased B16 lung tumor nodules. The combination of peptide vaccination and treatment with an Ab directed against the inhibitory receptor CTLA-4 enhanced the immune response against TRP-2 peptide, inducing autoimmune depigmentation and further decreasing lung tumor nodules. However, both vaccination methods failed to protect against orthotopic (s.c.) B16 tumor challenge. The addition of an irradiated GM-CSF-expressing, amelanotic tumor cell vaccine significantly delayed s.c. B16 tumor growth. Subsequent studies revealed that provision of GM-CSF increased dendritic cell numbers in lymph nodes and spleen. Furthermore, addition of CTLA-4 blockade increased the frequency of TRP-2-specific, IFN-secreting T cells in spleen and lymph nodes. Overall, our results indicate that combining enhancement of Ag presentation with removal of CTLA-4-mediated inhibition can convert a "weaker" autoimmune response into a more potent antitumor immune response.

Autoimmune depigmentation following sensitization to melanoma antigens.

Generating an antitumor immune response can be thought of as eliciting an immune response to cells derived from self-tissue. As such, tumor immunity may result in autoimmunity. Melanoma patients undergoing immunotherapy often develop a form of autoimmune depigmentation referred to as vitiligo, in which T cells with antigenic specificity for pigmentation antigens destroy normal melanocytes. The models described in this chapter can be used to study immunity to melanoma antigens. These models employ a well-characterized pigmentation antigen relevant to melanoma and a common transplantable murine melanoma cell line. As more sophisticated approaches to cancer therapy are developed, models such as these may be key in understanding how immunity to self-antigens can be manipulated to elicit tumor immunity.

Shift from systemic to site-specific memory by tumor-targeted IL-2.

IL-2 has been approved for treatment of patients with cancer. Moreover, it has been used as a component of vaccines against cancer. In this regard, we have recently demonstrated that dendritic cell-based peptide vaccination in mice required IL-2 to mount an effective immune response against established melanoma metastases. In this study, we confirm this observation by use of tumor-targeted IL-2. However, the development of a protective systemic memory was substantially impaired by this measure, i.e., mice, which successfully rejected s.c. tumors of B16 melanoma after vaccination with dendritic cells pulsed with tyrosinase-related protein 2-derived peptides plus a boost with targeted IL-2, failed to reject a rechallenge with experimental pulmonary metastases. Detailed analysis revealed a change in the distribution of the tumor-reactive T cell population: although targeted IL-2 expanded the local effector population, tyrosinase-related protein 2-reactive T cells were almost completely depleted from lymphatic tissues.