Enhanced Tumor Rejection Research Articles

Cord blood T cells mediate enhanced antitumor effects compared with adult peripheral blood T cells

AbstractUnrelated cord blood transplantation (CBT) without in vivo T-cell depletion is increasingly used to treat high-risk hematologic malignancies. Following T-replete CBT, naïve CB T cells undergo rapid peripheral expansion with memory-effector differentiation. Emerging data suggest that unrelated CBT, particularly in the context of HLA mismatch and a T-replete graft, may reduce leukemic relapse. To study the role of CB T cells in mediating graft-versus-tumor responses and dissect the underlying immune mechanisms for this, we compared the ability of HLA-mismatched CB and adult peripheral blood (PB) T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma in a xenogeneic NOD/SCID/IL2rgnull mouse model. CB T cells mediated enhanced tumor rejection compared with equal numbers of PB T cells, leading to improved survival in the CB group (P < .0003). Comparison of CB T cells that were autologous vs allogeneic to the lymphoma demonstrated that this antitumor effect was mediated by alloreactive rather than EBV-specific T cells. Analysis of tumor-infiltrating lymphocytes demonstrated that CB T cells mediated this enhanced antitumor effect by rapid infiltration of the tumor with CCR7+CD8+ T cells and prompt induction of cytotoxic CD8+ and CD4+ T-helper (Th1) T cells in the tumor microenvironment. In contrast, in the PB group, this antilymphoma effect is impaired because of delayed tumoral infiltration of PB T cells and a relative bias toward suppressive Th2 and T-regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-replete CBT may provide superior Tc1-Th1 antitumor effects against high-risk hematologic malignancies.

Antibody opsonization of tumor cell membranes using hapten-PEG-lipid conjugates

Dendritic cell (DC) immunotherapy is a newly developed type of tumor immunotherapy. As a method for generating antigen-presenting DCs, the phagocytosis of whole tumor cells (WTCs) opsonized with antibodies by DCs (antibody-dependent cellular phagocytosis, ADCP) is especially efficient. To opsonize tumor cells, tumor-specific antibodies are usually required. Therefore conventional ADCP was limited to tumor types for which antigens have been identified and antigen-specific antibodies are available. Herein, we developed a general method to promote ADCP by DCs using an arbitrary antibody and evaluated its antitumor effects. To opsonize tumor cells with a tumor-nonspecific antibody, we modified tumor cell surfaces with PEG-lipid-hapten conjugates and a conjugated anti-hapten antibody. By co-culturing DCs with the antibody-opsonized tumor cells, the phagocytosis and DC maturation rates were enhanced by ~20% and ~10%, respectively, compared with co-cultured non-opsonized tumor cells. The administration of tumor antigen-loaded matured DCs to mice resulted in enhanced tumor rejection and long-term survival. The advantages of this method are that tumor-specific antibodies are not required and DCs can present uncharacterized antigens by phagocytosing WTCs and associated antigens. Therefore, this method might be applicable to various types of tumors for which specific antigens have not yet been identified.

Abstract B06: Imaging regulatory mechanisms that limit intratumoral CTL function in a mouse melanoma model

Abstract Introduction: Immunotherapies that harness CTLs to eradicate solid tumors, melanoma in particular, have re-emerged as potent clinical options in recent years. Improving the dispersion and cytotoxicity of intratumoral CTLs may further benefit patients. To gain insight into the regulatory processes that limit CTL activity in melanoma we investigated the influence of PD-1 signaling and Tregs on CTL behavior. Methodology: To mimic adoptive cell therapy we transferred mice bearing B16.OVA melanoma tumors with OT-I CTLs. To study Treg-mediated regulation, we used FOXP3-GFP-LuciDTR-4 host mice, in which 95% of the cells expressing Foxp3 could be depleted using diphtheria toxin. To study PD-1 mediated regulation we injected anti PD-1 to WT host mice. Tumor rejection was monitored for 2-5 weeks. Two-photon imaging of live mice was used to follow the behavior of CTLs and Tregs within tumors. Time-lapse confocal microscopy was used to follow the influence of PD-1 signaling on the behavior of CTLs and their interaction with B16-Ova cells. Results: Tumor rejection was closely regulated by Tregs as tumors were densely infiltrated by these cells and their depletion dramatically accelerated tumor rejection. Interactions between CTLs and Tregs seemed indirect, as they did not form conjugates. Following their transfer, antigen-specific OT-I CTLs, as well as polyclonal CTLs, entered murine tumors through selected ICAM-decorated intratumoral vessels. As CTLs accumulated, Treg numbers in the tumors dropped and CTLs could be observed conjugating tumor cells and killing them. Treatment with anti PD-1 increased CTL velocities and accelerated killing of B16.OVA cells in vitro, and was mirrored in vivo by enhanced tumor rejection. Whereas CTL migration and cytolysis was limited to oxygenated tumor areas within 50 µm of flowing blood vessels, Tregs exhibited higher velocities, were not affected by the proximity of blood vessels, and penetrated deeper into avascular areas. Conclusions: Our findings suggest that regulatory mechanisms influence CTL efficiency partly by affecting their migration. CTLs, more than Tregs, depend on oxygen for intratumoral locomotion and cytolysis. This disparity allows tumor cells to escape CTL lysis in deep hypoxic tumor niches where high Treg to CTL ratios favor their survival. Thus, differential motility and dependence on oxygen among CTLs and Tregs can dictate the efficiency of CTL-based therapies of melanoma. Citation Format: Tali Feferman, Natalia G. Zabolinsky, Yoav Manaster, Anat Hutzler, Guy Shakhar. Imaging regulatory mechanisms that limit intratumoral CTL function in a mouse melanoma model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B06.

Vaccine-induced but not tumor-derived Interleukin-10 dictates the efficacy of Interleukin-10 blockade in therapeutic vaccination

ABSTRACTBlocking antibodies against immunosuppressive molecules have shown promising results in cancer patients. However, there are not enough data to define those conditions dictating treatment efficacy. In this scenario, IL-10 is a cytokine with controversial effects on tumor growth. Thus, our aim was to characterize in which setting IL-10 blockade may potentiate the beneficial effects of a therapeutic vaccine In the IL-10-expressing B16-OVA and TC-1 P3 (A15) tumor models, therapeutic vaccination with tumor antigens plus the TLR7 ligand Imiquimod increased IL-10 production. Although blockade of IL-10 signal with anti-IL-10R antibodies did not inhibit tumor growth, when combined with vaccination it enhanced tumor rejection, associated with stronger innate and adaptive immune responses. Interestingly, a similar enhancement on immune responses was observed after simultaneous vaccination and IL-10 blockade in naive mice. However, when using vaccines containing as adjuvants the TLR3 ligand poly(I:C) or anti-CD40 agonistic antibodies, despite tumor IL-10 expression, anti-IL-10R antibodies did not provide any beneficial effect on tumor growth and antitumor immune responses. Of note, as opposed to Imiquimod, vaccination with this type of adjuvants did not induce IL-10 and correlated with a lack of in vitro IL-10 production by dendritic cells (DC). Finally, in B16-OVA-bearing mice, blockade of IL-10 during therapeutic vaccination with a multiple adjuvant combination (MAC) with potent immunostimulatory properties but still inducing IL-10 led to superior antitumor immunity and complete tumor rejection. These results suggest that for therapeutic antitumor vaccination, blockade of vaccine-induced IL-10 is more relevant than tumor-associated IL-10.

Dipeptidylpeptidase 4 inhibition enhances lymphocyte trafficking, improving both naturally occurring tumor immunity and immunotherapy.

The success of antitumor immune responses depends on the infiltration of solid tumors by effector T cells, a process guided by chemokines. Here we show that in vivo post-translational processing of chemokines by dipeptidylpeptidase 4 (DPP4, also known as CD26) limits lymphocyte migration to sites of inflammation and tumors. Inhibition of DPP4 enzymatic activity enhanced tumor rejection by preserving biologically active CXCL10 and increasing trafficking into the tumor by lymphocytes expressing the counter-receptor CXCR3. Furthermore, DPP4 inhibition improved adjuvant-based immunotherapy, adoptive T cell transfer and checkpoint blockade. These findings provide direct in vivo evidence for control of lymphocyte trafficking via CXCL10 cleavage and support the use of DPP4 inhibitors for stabilizing biologically active forms of chemokines as a strategy to enhance tumor immunotherapy.

Genetic depletion of cyclin dependent Kinase-5 results in enhanced tumor rejection via a CD4+ T cell dependent mechanism (TUM7P.1023)

Abstract CDK5 is a non-canonical member of the CDK family that does not require cell cycling for activity but relies on binding to its obligate activators, p35 and p39 which are mostly expressed in post mitotic neurons. CDK5 is required for normal Central Nervous System (CNS) development. Medulloblastoma (MB), derived from primitive cerebellar granule neuron precursors, is the most common pediatric CNS malignancy. Utilizing a short hairpin RNA approach to silence Cdk5 expression in a C57BL/6 (B6) syngenic MB line derived from p53+/- PTCH-/- (MM1), we found that 19 days after subcutaneous inoculation, mice with silenced Cdk5 (MM1-CDK5-KD) revealed a 78% tumor free survival (N=36) compared to 4% (N=24) of mice inoculated with wild type MB (MM1-WT). Furthermore, when injected into Nu/Nu mice, MM1-WT and MM1-CDK5-KD had similar subcutaneous growth kinetics. When CD4+ cells were depleted in B6 mice inoculated with MM1-CDK5-KD tumor free survival decreased to 28% (N=15). We sought to interrogate the role of the immune system in enhanced killing of MM1-CDK5-KD tumors. When exposed to IFN-γ stimulation in vitro, genetic profiling of MM1-CDK5-KD revealed a decrease in PD-L1 expression, a known immune checkpoint surface molecule, compared to MM1-WT. These results indicate a functional role for Cdk5 in MB immune evasion which, when circumvented via Cdk5 inhibition, leads to increased susceptibility to anti-tumor surveillance.

Immune evasion in cancer: Mechanistic basis and therapeutic strategies

Cancer immune evasion is a major stumbling block in designing effective anticancer therapeutic strategies. Although considerable progress has been made in understanding how cancers evade destructive immunity, measures to counteract tumor escape have not kept pace. There are a number of factors that contribute to tumor persistence despite having a normal host immune system. Immune editing is one of the key aspects why tumors evade surveillance causing the tumors to lie dormant in patients for years through "equilibrium" and "senescence" before re-emerging. In addition, tumors exploit several immunological processes such as targeting the regulatory T cell function or their secretions, antigen presentation, modifying the production of immune suppressive mediators, tolerance and immune deviation. Besides these, tumor heterogeneity and metastasis also play a critical role in tumor growth. A number of potential targets like promoting Th1, NK cell, γδ T cell responses, inhibiting Treg functionality, induction of IL-12, use of drugs including phytochemicals have been designed to counter tumor progression with much success. Some natural agents and phytochemicals merit further study. For example, use of certain key polysaccharide components from mushrooms and plants have shown to possess therapeutic impact on tumor-imposed genetic instability, anti-growth signaling, replicative immortality, dysregulated metabolism etc. In this review, we will discuss the advances made toward understanding the basis of cancer immune evasion and summarize the efficacy of various therapeutic measures and targets that have been developed or are being investigated to enhance tumor rejection.

Efficacy of imiquimod-based transcutaneous immunization using a nano-dispersed emulsion gel formulation.

BackgroundTranscutaneous immunization (TCI) approaches utilize skin associated lymphatic tissues to elicit specific immune responses. In this context, the imidazoquinoline derivative imiquimod formulated in Aldara applied onto intact skin together with a cytotoxic T lymphocyte (CTL) epitope induces potent CTL responses. However, the feasibility and efficacy of the commercial imiquimod formulation Aldara is limited by its physicochemical properties as well as its immunogenicity.Methodology/Principal FindingsTo overcome these obstacles, we developed an imiquimod-containing emulsion gel (IMI-Gel) and characterized it in comparison to Aldara for rheological properties and in vitro mouse skin permeation in a Franz diffusion cell system. Imiquimod was readily released from Aldara, while IMI-Gel showed markedly decreased drug release. Nevertheless, comparing vaccination potency of Aldara or IMI-Gel-based TCI in C57BL/6 mice against the model cytotoxic T-lymphocyte epitope SIINFEKL, we found that IMI-Gel was equally effective in terms of the frequency of peptide-specific T-cells and in vivo cytolytic activity. Importantly, transcutaneous delivery of IMI-Gel for vaccination was clearly superior to the subcutaneous or oral route of administration. Finally, IMI-Gel based TCI was at least equally effective compared to Aldara-based TCI in rejection of established SIINFEKL-expressing E.G7 tumors in a therapeutic setup indicated by enhanced tumor rejection and survival.Conclusion/SignificanceIn summary, we developed a novel imiquimod formulation with feasible pharmaceutical properties and immunological efficacy that fosters the rational design of a next generation transcutaneous vaccination platform suitable for the treatment of cancer or persistent virus infections.

Nanoscale artificial antigen presenting cells for T cell immunotherapy

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100nm in diameter) and avidin-coated quantum dot nanocrystals (~30nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo. From the Clinical EditorArtifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.

Human Embryonic Stem Cells - a Potential Vaccine for Ovarian Cancer

To investigate the therapeutic potential of human embryonic stem cells (hESCs) as a vaccine to induce an immune response and provide antitumor protection in a rat model. Cross-reactivity of antigens between hESCs and tumour cells was screened by immunohistochemistry. Fischer 344 rats were divided into 7 groups, with 6 rats in each, immunized with: Group 1, hESC; Group 2, pre-inactivated mitotic NuTu-19; Group 3 PBS; Group 4, hESC; Group 5, pre-inactivated mitotic NuTu-19; Group 6, PBS; Group 7, hESC only. At 1 (Groups 1-3) or 4 weeks (Groups 4-6) after the last vaccination, each rat was challenged intraperitoneally with NuTu-19. Tumor growth and animal survival were closely monitored. Rats immunized with H9 and NuTu- 19 were tested by Western blot analysis of rat orbital venous blood for cytokines produced by Th1 and Th2 cells. hESCs presented tumour antigens, markers, and genes related to tumour growth, metastasis, and signal pathway interactions. The vaccine administered to rats in Group 1 led to significant antitumor responses and enhanced tumor rejection in rats with intraperitoneal inoculation of NuTu-19 cells compared to control groups. In contrast, rats in Group 4 did not display any elevation of antitumour responses. Western blot analysis found cross-reactivity among antibodies generated between H9 and NuTu-19. However, the cytokines did not show significant differences, and no side effects were detected. hESC-based vaccination is a promising modality for immunotherapy of ovarian cancer.

Combination CTLA-4 Blockade and 4-1BB Activation Enhances Tumor Rejection by Increasing T-Cell Infiltration, Proliferation, and Cytokine Production

BackgroundThe co-inhibitory receptor Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) attenuates immune responses and prevent autoimmunity, however, tumors exploit this pathway to evade the host T-cell response. The T-cell co-stimulatory receptor 4-1BB is transiently upregulated on T-cells following activation and increases their proliferation and inflammatory cytokine production when engaged. Antibodies which block CTLA-4 or which activate 4-1BB can promote the rejection of some murine tumors, but fail to cure poorly immunogenic tumors like B16 melanoma as single agents.Methodology/Principal FindingsWe find that combining αCTLA-4 and α4-1BB antibodies in the context of a Flt3-ligand, but not a GM-CSF, based B16 melanoma vaccine promoted synergistic levels of tumor rejection. 4-1BB activation elicited strong infiltration of CD8+ T-cells into the tumor and drove the proliferation of these cells, while CTLA-4 blockade did the same for CD4+ effector T-cells. Anti-4-1BB also depressed regulatory T-cell infiltration of tumors. 4-1BB activation strongly stimulated inflammatory cytokine production in the vaccine and tumor draining lymph nodes and in the tumor itself. The addition of CTLA-4 blockade further increased IFN-γ production from CD4+ effector T-cells in the vaccine draining node and the tumor. Anti 4-1BB treatment, with or without CTLA-4 blockade, induced approximately 75% of CD8+ and 45% of CD4+ effector T-cells in the tumor to express the killer cell lectin-like receptor G1 (KLRG1). Tumors treated with combination antibody therapy showed 1.7-fold greater infiltration by these KLRG1+CD4+ effector T-cells than did those treated with α4-1BB alone.Conclusions/SignificanceThis study shows that combining T-cell co-inhibitory blockade with αCTLA-4 and active co-stimulation with α4-1BB promotes rejection of B16 melanoma in the context of a suitable vaccine. In addition, we identify KLRG1 as a useful marker for monitoring the anti-tumor immune response elicited by this therapy. These findings should aid in the design of future trials for the immunotherapy of melanoma.

Effective colon cancer prophylaxis in mice using embryonic stem cells and carbon nanotubes

IntroductionIn recent years, a new concept of an anticancer vaccine has been proposed to prevent and control the proliferation and expansion of cancer cells by eliciting an immune boost in biological systems. The recent literature supports the role of embryonic stem cells (ESC) as cellular agents that stimulate the biological systems to destroy cancer cells. However, at present, a true anticancer vaccine remains elusive. There are several lines of evidence showing that carbon nanotubes may be used to initiate and maintain immune responses.ObjectiveThe authors proposed to test the therapeutic potential of multiwalled carbon nanotubes (MWCNTs) combined with ESC as agents to induce an immune boost and provide subsequent anticancer protection in mice.MethodsC57 BL/6 mice were immunized with ESC and MWCNTs.ResultsThe proposed vaccine led to significant antitumor responses and enhanced tumor rejection in mice with subcutaneous inoculation of MC38 colon malign cells compared with groups only administered ESC, only MWCNTs, and controls.ConclusionThe application and potential of ESC combined with MWCNTs as anticancer immunization agents may represent the beginning of a new chapter in the treatment of colon cancer.

DAI (DLM-1/ZBP1) as a Genetic Adjuvant for DNA Vaccines That Promotes Effective Antitumor CTL Immunity

DNA vaccination is an attractive approach to induce antigen-specific cytotoxic CD8(+) T lymphocytes (CTLs), which can mediate protective antitumor immunity. The potency of DNA vaccines encoding weakly immunogenic tumor-associated antigens (TAAs) can be enhanced by codelivering gene-encoded adjuvants. Pattern recognition receptors (PRRs) that sense intracellular DNA could potentially be used to harness intrinsic immune-stimulating properties of plasmid DNA vaccines. Consequently, the cytosolic DNA sensor, DNA-dependent activator of interferon (IFN) regulatory factors (DAI), was used as a genetic adjuvant. In vivo electroporation (EP) of mice with a DAI-encoding plasmid (pDAI) promoted transcription of genes encoding type I IFNs, proinflammatory cytokines, and costimulatory molecules. Coimmunization with pDAI and antigen-encoding plasmids enhanced in vivo antigen-specific proliferation, and induction of effector and memory CTLs. Moreover, codelivery of pDAI effectively promoted CTL and CD4(+) Th1 responses to the TAA survivin. The DAI-enhanced CTL induction required nuclear factor κB (NF-κB) activation and type I IFN signaling, but did not involve the IFN regulatory factor 3 (IRF3). Codelivery of pDAI also increased CTL responses to the melanoma-associated antigen tyrosinase-related protein-2 (TRP2), enhanced tumor rejection and conferred long-term protection against B16 melanoma challenge. This study constitutes "proof-of-principle" validating the use of intracellular PRRs as genetic adjuvants to enhance DNA vaccine potency.

Heterologous Prime/Boost Immunization With p53-based Vaccines Combined With Toll-like Receptor Stimulation Enhances Tumor Regression

The p53 gene product is overexpressed in approximately 50% of cancers, making it an ideal target for cancer immunotherapy. We previously demonstrated that a modified vaccinia Ankara (MVA) vaccine expressing human p53 (MVA-p53) was moderately active when given as a homologous prime/boost in a human p53 knock in (Hupki) mouse model. We needed to improve upon the inefficient homologous boosting approach, because development of neutralizing immunity to the vaccine viral vector backbone suppresses its immunogenicity. To enhance specificity, we examined the combination of 2 different vaccine vectors provided in sequence as a heterologous prime/boost. Hupki mice were evaluated as a human p53 tolerant model to explore the capacity of heterologous p53 immunization to reject human p53-expressing tumors. We employed attenuated recombinant Listeria monocytogenes expressing human p53 (LmddA-LLO-p53) in addition to MVA-p53. Heterologous p53 immunization resulted in a significant increase in p53-specific CD8 and CD4 T cells compared with homologous single vector p53 immunization. Heterologous p53 immunization induced protection against tumor growth but had only a modest effect on established tumors. To enhance the immune response we used synthetic double-strand RNA (polyinsosinic:polycytidylic acid) and unmethylated CpG-containing oligodeoxynucleotide to activate the innate immune system via Toll-like receptors. Treatment of established tumor-bearing Hupki mice with polyinsosinic:polycytidylic acid and CpG-oligodeoxynucleotide in combination with heterologous p53 immunization resulted in enhanced tumor rejection relative to treatment with either agent alone. These results suggest that heterologous prime/boost immunization and Toll-like receptor stimulation increases the efficacy of a cancer vaccine, targeting a tolerized tumor antigen.

Boosting anti-tumor immunity by combining gp96-Ig vaccination and Ig-mTL1A fusion protein (131.33)

Abstract We previously developed a cell-based vaccine system that secretes heat shock protein gp96 as an Ig fusion protein that efficiently enhances the cross presentation of tumor antigens to CD8+ cytotoxic T lymphocyte (CTL), leading to a tumor antigen-specific clonal expansion. Regulatory T cells (Tregs) in the priming site impose a brake that restricts the full potential of this CTL expansion. In independent studies of TNFR25 and its ligand, TL1A, we found that TNFR25 signaling inhibits the suppressive activity of Tregs and blocks their de novo biogenesis. Combination immunotherapy studies using hamster anti-mouse TNFR25 agonist antibody, 4C12, and vaccine show enhanced tumor rejection in comparison to vaccine only. We have genetically engineered Ig-mTL1A fusion proteins that differ in retention of the cleavage site in the extracellular domain fused to the C-terminus of the mIgG Fc fragment. These fusion proteins are expected to show similar tumor rejection when used in combination therapy with vaccine and, unlike 4C12, are not expected to elicit an antibody response due to prolonged use. The in vitro potencies of the secreted fusion proteins were confirmed by cytotoxicity and binding assays and protein molecular weights were determined by fast protein liquid chromatography (FPLC). We will optimize purification methods to produce the most functional Fc-mTL1A resulting in the strongest TNFR25 signaling, which can be utilized in combination with vaccine for therapeutic benefit.

Delivery of noncarrier naked DNA vaccine into the skin by supersonic flow induces a polarized T helper type 1 immune response to cancer

DNA vaccine is a new and powerful approach to generate immunological responses against infectious disease and cancer. The T helper type (Th)1 immune response is usually required for generating effective anti-tumor responses. A microparticulate bombardment system can induce an immune response using very low amounts of DNA. Using nozzle aerodynamics, a low pressure gene gun has been developed to decrease the noise associated with high pressure gene guns. Particles are propelled by supersonic flow through this novel nozzle. To test whether this gun could inoculate a DNA vaccine that stimulates an anti-tumor Th1 immune response, we examined the effect of direct delivery of naked DNA (i.e. without any carrier) on the anti-tumor immune response of mice. The luciferase reporter plasmid DNA was delivered using a low-pressure biolistic device and expressed in C3H/HeN, BALB/c, and C57BL/6 mice. Plasmid DNA expression was mainly in the epidermis. Noncarrier naked neu DNA vaccine and gold particle-coated neu DNA vaccine (at 1 microg per mouse) had similar anti-tumor effects in C3H mice. However, cytokine profile examination showed the Th1-bias of the response induced by naked DNA vaccine and the Th2-bias of the response induced by coated DNA vaccine. A shift in the immune response to favour enhanced tumor rejection can be achieved by skin delivery of naked DNA vaccine.

OX40 signaling directly triggers the antitumor effects of NKT cells

Pathways involving the costimulatory molecule OX40 and OX40 ligand (OX40L) enhance tumor rejection. It was presumed that this effect was mediated by changes in DCs and/or T cells. In this issue of the JCI, Zaini et al. report that, in mice, intratumoral injection of DCs genetically modified to express OX40L suppressed the growth of a preexisting melanoma by directly triggering an antitumor NKT cell response (see the related article beginning on page 3330). This work suggests that the intratumoral NKT cell population may be harnessed for cancer immunotherapy and that OX40 costimulation may be used as a unique trigger of the antitumor activity of these cells.

Role for CD40L in the therapy of human cancer

CD40L-CD40 interaction is central to the control of thymusdependent humoral immunity and cell mediated immune responses. CD40, a member of the tumor necrosis factor receptor (TNFR) family, has been found on the surface of B lymphocytes, monocytes, hematopoietic progenitors, dendritic cells (DCs), endothelial cells, epithelial cells and so on. Its natural ligand (CD40 ligand, CD40L), CD154, a member of the tumor necrosis factor (TNF) family, is mainly expressed on activated CD4+ T lymphocytes. A direct growth-inhibitory effect can be found when ligated CD40 is on human breast, ovarian, cervical, bladder, non-small cell lung, and squamous epithelial carcinoma cells. This effect is related to the induction of cell cycle blockage and/or apoptosis. CD40L induces phenotypic and functional maturation of DCs, and can increase tumor immunogenicity through up-regulation of costimulatory molecular expression and cytokine production by epithelial cancer cells. As a result, CD40L can enhance tumor rejection immune responses. Furthermore, by means of a “bystander effect”, even CD40-negative tumor subsets can be eliminated by activated tumor-reactive cytotoxic T lymphocytes(CTL). This review summarizes recent findings on CD40L recombinant protein and gene therapy-based tumor treatment approaches.

IL-21 Enhances Tumor Rejection through a NKG2D-Dependent Mechanism

IL-21 is a cytokine that can promote the anti-tumor responses of the innate and adaptive immune system. Mice treated with IL-21 reject tumor cells more efficiently, and a higher percentage of mice remain tumor-free compared with untreated controls. In this study, we demonstrate that in certain tumor models IL-21-enhanced tumor rejection is NKG2D dependent. When engagement of the NKG2D receptor was prevented, either due to the lack of ligand expression on the tumor cells or due to direct blocking with anti-NKG2D mAb treatment, the protective effects of IL-21 treatment were abrogated or substantially diminished. Specifically, IL-21 only demonstrated a therapeutic effect in mice challenged with a retinoic acid early inducible-1delta-bearing lymphoma but not in mice bearing parental RMA tumors lacking NKG2D ligands. Furthermore, treatment with a blocking anti-NKG2D mAb largely prevented the therapeutic effect of IL-21 in mice challenged with the 4T1 breast carcinoma, the 3LL lung carcinoma, and RM-1 prostate carcinoma. By contrast, IL-21 did mediate beneficial effects against both the parental DA3 mammary carcinoma and DA3 tumors transfected with H60, a NKG2D ligand. We also observed that IL-21 treatment could enhance RMA-retinoic acid early inducible-1delta tumor rejection in RAG-1(-/-) deficient mice, thereby demonstrating that the IL-21-induced protective effect can be mediated by the innate immune system and that, in this case, IL-21 does not require the adaptive immune response. Collectively, these findings suggest that IL-21 therapy may work optimally against tumors that can elicit a NKG2D-mediated immune response.

A mutant B7-1/Ig fusion protein that selectively binds to CTLA-4 ameliorates anti-tumor DNA vaccination and counters regulatory T cell activity

We have shown that a plasmid encoding a B7-1/Ig fusion protein enhanced DNA vaccination against human carcinoembryonic antigen (CEA) more effectively than the plasmid encoding membrane-bound B7-1. However, it was not known if B7-1/Ig acted only by binding CD28 (amplifying a stimulatory signal) or by blocking CTLA-4 on T cells (removing inhibitory signals). Here, we aimed to determine this using a plasmid encoding mutant B7-1/Ig (B7-1wa/Ig), which binds only to CTLA-4 but not to CD28. Our results showed that both the B7-1/Ig and B7-1wa/Ig plasmids, when co-administered with a CEA plasmid, enhanced tumor rejection and the in vitro anti-CEA response. Therefore, B7-1wa/Ig ameliorates DNA vaccination, presumably by binding to CTLA-4. This could result from a number of non-exclusive mechanisms, such as a reduced threshold for T-cell activation, or blockade of CTLA-4/B7-mediated tolerogenic signals in DCs or T cells. We found that, in vitro, a significant fraction of CD3/CD28-activated T cells (in the absence of DCs) expressed CTLA-4 and B7-1. Primed T cells of CTLA-4 +B7-1 +/− phenotype acted as regulatory T cells by inhibiting IFNγ production by re-stimulated CTLA-4 −B7-1 − cells, and this was reversed by antibodies against IL-10 or TGF-β1. Both B7-1wa/Ig and CTLA-4/Ig, which bind to CTLA-4 and B7-1/B7-2 respectively, enhanced IFNγ production, but not the proliferation or IL-4 release in mixed T-cell populations containing these two cell types. In contrast, CTLA-4 −B7-1 − T cells produced IFNγ which was not affected by B7-1wa/Ig or CTLA-4/Ig. These results suggest that blocking of CTLA-4/B7-1 binding in T cell/T cell interactions blocks negative regulatory signals. This might be the mechanism, at least in part, of the enhancement of anti-tumor immunity by the B7-1wa/Ig and B7-1/Ig plasmids.