Myelin-specific CD8+ T cells exacerbate brain inflammation in CNS autoimmunity.

Multiple sclerosis (MS) is an inflammatory, demyelinating disease of the CNS. Although CD4+ T cells are implicated in MS pathogenesis and have been the main focus of MS research using the animal model experimental autoimmune encephalomyelitis (EAE), substantial evidence from patients with MS points to a role for CD8+ T cells in disease pathogenesis. We previously showed that an MHC class I-restricted epitope of myelin basic protein (MBP) is presented in the CNS during CD4+ T cell-initiated EAE. Here, we investigated whether naive MBP-specific CD8+ T cells recruited to the CNS during CD4+ T cell-initiated EAE engaged in determinant spreading and influenced disease. We found that the MBP-specific CD8+ T cells exacerbated brain but not spinal cord inflammation. We show that a higher frequency of monocytes and monocyte-derived cells presented the MHC class I-restricted MBP ligand in the brain compared with the spinal cord. Infiltration of MBP-specific CD8+ T cells enhanced ROS production in the brain only in these cell types and only when the MBP-specific CD8+ T cells expressed Fas ligand (FasL). These results suggest that myelin-specific CD8+ T cells may contribute to disease pathogenesis via a FasL-dependent mechanism that preferentially promotes lesion formation in the brain.

Durable multitransgene expression in vivo using systemic, nonviral DNA delivery.

Recombinant adeno-associated virus (AAV) vectors are transforming therapies for rare human monogenic deficiency diseases. However, adaptive immune responses to AAV and its limited DNA insert capacity, restrict their therapeutic potential. HEDGES (high-level extended duration gene expression system), a nonviral DNA- and liposome-based gene delivery platform, overcomes these limitations in immunocompetent mice. Specifically, one systemic HEDGES injection durably produces therapeutic levels of transgene-encoded human proteins, including FDA-approved cytokines and monoclonal antibodies, without detectable integration into genomic DNA. HEDGES also controls protein production duration from <3 weeks to >1.5 years, does not induce anti-vector immune responses, is reexpressed for prolonged periods following reinjection, and produces only transient minimal toxicity. HEDGES can produce extended therapeutic levels of multiple transgene-encoded therapeutic human proteins from DNA inserts >1.5-fold larger than AAV-based therapeutics, thus creating combinatorial interventions to effectively treat common polygenic diseases driven by multigenic abnormalities.

Logic-Gated ROR1 Chimeric Antigen Receptor Expression Rescues T Cell-Mediated Toxicity to Normal Tissues and Enables Selective Tumor Targeting.

Many potential targets for CAR-T cells in solid tumors are expressed in some normal tissues, raising concern for off-tumor toxicity. Following lymphodepletion, CAR-T cells targeting the tumor-associated antigen ROR1 lysed tumors in mice but induced lethal bone marrow failure due to recognition of ROR1+ stromal cells. To improve selectivity, we engineered T cells with synthetic Notch (synNotch) receptors specific for EpCAM or B7-H3, which are expressed on ROR1+ tumor cells but not ROR1+ stromal cells. SynNotch receptors induced ROR1 CAR expression selectively within the tumor, resulting in tumor regression without toxicity when tumor cells were segregated from, but not when co-localized with, normal ROR1+ cells. This strategy, thus, permits safe targeting of tumors that are sufficiently separated from normal cells.

Targeting NOTCH activation in small cell lung cancer through LSD1 inhibition.

Small cell lung cancer (SCLC) is a recalcitrant, aggressive neuroendocrine-type cancer for which little change to first-line standard-of-care treatment has occurred within the last few decades. Unlike nonsmall cell lung cancer (NSCLC), SCLC harbors few actionable mutations for therapeutic intervention. Lysine-specific histone demethylase 1A (LSD1 also known as KDM1A) inhibitors were previously shown to have selective activity in SCLC models, but the underlying mechanism was elusive. Here, we found that exposure to the selective LSD1 inhibitor ORY-1001 activated the NOTCH pathway, resulting in the suppression of the transcription factor ASCL1 and the repression of SCLC tumorigenesis. Our analyses revealed that LSD1 bound to the NOTCH1 locus, thereby suppressing NOTCH1 expression and downstream signaling. Reactivation of NOTCH signaling with the LSD1 inhibitor reduced the expression of ASCL1 and neuroendocrine cell lineage genes. Knockdown studies confirmed the pharmacological inhibitor-based results. In vivo, sensitivity to LSD1 inhibition in SCLC patient-derived xenograft (PDX) models correlated with the extent of consequential NOTCH pathway activation and repression of a neuroendocrine phenotype. Complete and durable tumor regression occurred with ORY-1001-induced NOTCH activation in a chemoresistant PDX model. Our findings reveal how LSD1 inhibitors function in this tumor and support their potential as a new and targeted therapy for SCLC.

Host Nitric Oxide Disrupts Microbial Cell-to-Cell Communication to Inhibit Staphylococcal Virulence.

Staphylococcus aureus is a commensal bacterium that can asymptomatically colonize its host but also causes invasive infections. Quorum sensing regulates S. aureus virulence and the transition from a commensal to a pathogenic organism. However, little is known about how host innate immunity affects interbacterial communication. We show that nitric oxide suppresses staphylococcal virulence by targeting the Agr quorum sensing system. Nitric oxide-mediated inhibition occurs through direct modification of cysteine residues C55, C123, and C199 of the AgrA transcription factor. Cysteine modification decreases AgrA promoter occupancy as well as transcription of the agr operon and quorum sensing-activated toxin genes. In a staphylococcal pneumonia model, mice lacking inducible nitric oxide synthase develop more severe disease with heightened mortality and proinflammatory cytokine responses. In addition, staphylococcal α-toxin production increases in the absence of nitric oxide or nitric oxide-sensitive AgrA cysteine residues. Our findings demonstrate an anti-virulence mechanism for nitric oxide in innate immunity.

Tomographic magnetic particle imaging of cancer targeted nanoparticles.

Magnetic Particle Imaging (MPI) is an emerging, whole body biomedical imaging technique, with sub-millimeter spatial resolution and high sensitivity to a biocompatible contrast agent consisting of an iron oxide nanoparticle core and a biofunctionalized shell. Successful application of MPI for imaging of cancer depends on the nanoparticles (NPs) accumulating at tumors at sufficient levels relative to other sites. NPs' physiochemical properties such as size, crystallographic structure and uniformity, surface coating, stability, blood circulation time and magnetization determine the efficacy of their tumor accumulation and MPI signal generation. Here, we address these criteria by presenting strategies for the synthesis and surface functionalization of efficient MPI tracers, that can target a typical murine brain cancer model and generate three dimensional images of these tumors with very high signal-to-noise ratios (SNR). Our results showed high contrast agent sensitivities that enabled us to detect 1.1 ng of iron (SNR ∼ 3.9) and enhance the spatial resolution to about 600 µm. The biodistribution of these NPs was also studied using near-infrared fluorescence (NIRF) and single-photon emission computed tomography (SPECT) imaging. NPs were mainly accumulated in the liver and spleen and did not show any renal clearance. This first pre-clinical study of cancer targeted NPs imaged using a tomographic MPI system in an animal model paves the way to explore new nanomedicine strategies for cancer diagnosis and therapy, using clinically safe magnetic iron oxide nanoparticles and MPI.

The A946T variant of the RNA sensor IFIH1 mediates an interferon program that limits viral infection but increases the risk for autoimmunity.

The single-nucleotide polymorphism rs1990760 in the gene encoding the cytosolic viral sensor IFIH1 results in an amino-acid change (A946T; IFIH1T946) that is associated with multiple autoimmune diseases. The effect of this polymorphism on both viral sensing and autoimmune pathogenesis remains poorly understood. Here we found that human peripheral blood mononuclear cells (PBMCs) and cell lines expressing the risk variant IFIH1T946 exhibited heightened basal and ligand-triggered production of type I interferons. Consistent with those findings, mice with a knock-in mutation encoding IFIH1T946 displayed enhanced basal expression of type I interferons, survived a lethal viral challenge and exhibited increased penetrance in autoimmune models, including a combinatorial effect with other risk variants. Furthermore, IFIH1T946 mice manifested an embryonic survival defect consistent with enhanced responsiveness to RNA self ligands. Together our data support a model wherein the production of type I interferons driven by an autoimmune risk variant and triggered by ligand functions to protect against viral challenge, which probably accounts for its selection within human populations but provides this advantage at the cost of modestly promoting the risk of autoimmunity.

Safe and Effective Gene Therapy for Murine Wiskott-Aldrich Syndrome Using an Insulated Lentiviral Vector.

Wiskott-Aldrich syndrome (WAS) is a life-threatening immunodeficiency caused by mutations within the WAS gene. Viral gene therapy to restore WAS protein (WASp) expression in hematopoietic cells of patients with WAS has the potential to improve outcomes relative to the current standard of care, allogeneic bone marrow transplantation. However, the development of viral vectors that are both safe and effective has been problematic. While use of viral transcriptional promoters may increase the risk of insertional mutagenesis, cellular promoters may not achieve WASp expression levels necessary for optimal therapeutic effect. Here we evaluate a self-inactivating (SIN) lentiviral vector combining a chromatin insulator upstream of a viral MND (MPSV LTR, NCR deleted, dl587 PBS) promoter driving WASp expression. Used as a gene therapeutic in Was-/- mice, this vector resulted in stable WASp+ cells in all hematopoietic lineages and rescue of T and B cell defects with a low number of viral integrations per cell, without evidence of insertional mutagenesis in serial bone marrow transplants. In a gene transfer experiment in non-human primates, the insulated MND promoter (driving GFP expression) demonstrated long-term polyclonal engraftment of GFP+ cells. These observations demonstrate that the insulated MND promoter safely and efficiently reconstitutes clinically effective WASp expression and should be considered for future WAS therapy.

ST-11: A New Brain-Penetrant Microtubule-Destabilizing Agent with Therapeutic Potential for Glioblastoma Multiforme.

Glioblastoma multiforme is a devastating and intractable type of cancer. Current antineoplastic drugs do not improve the median survival of patients diagnosed with glioblastoma multiforme beyond 14 to 15 months, in part because the blood-brain barrier is generally impermeable to many therapeutic agents. Drugs that target microtubules (MT) have shown remarkable efficacy in a variety of cancers, yet their use as glioblastoma multiforme treatments has also been hindered by the scarcity of brain-penetrant MT-targeting compounds. We have discovered a new alkylindole compound, ST-11, that acts directly on MTs and rapidly attenuates their rate of assembly. Accordingly, ST-11 arrests glioblastoma multiforme cells in prometaphase and triggers apoptosis. In vivo analyses reveal that unlike current antitubulin agents, ST-11 readily crosses the blood-brain barrier. Further investigation in a syngeneic orthotopic mouse model of glioblastoma multiforme shows that ST-11 activates caspase-3 in tumors to reduce tumor volume without overt toxicity. Thus, ST-11 represents the first member of a new class of brain-penetrant antitubulin therapeutic agents. Mol Cancer Ther; 15(9); 2018-29. ©2016 AACR.

In vivo delivery, pharmacokinetics, biodistribution and toxicity of iron oxide nanoparticles.

Iron oxide nanoparticles (IONPs) have been extensively used during the last two decades, either as effective bio-imaging contrast agents or as carriers of biomolecules such as drugs, nucleic acids and peptides for controlled delivery to specific organs and tissues. Most of these novel applications require elaborate tuning of the physiochemical and surface properties of the IONPs. As new IONPs designs are envisioned, synergistic consideration of the body's innate biological barriers against the administered nanoparticles and the short and long-term side effects of the IONPs become even more essential. There are several important criteria (e.g. size and size-distribution, charge, coating molecules, and plasma protein adsorption) that can be effectively tuned to control the in vivo pharmacokinetics and biodistribution of the IONPs. This paper reviews these crucial parameters, in light of biological barriers in the body, and the latest IONPs design strategies used to overcome them. A careful review of the long-term biodistribution and side effects of the IONPs in relation to nanoparticle design is also given. While the discussions presented in this review are specific to IONPs, some of the information can be readily applied to other nanoparticle systems, such as gold, silver, silica, calcium phosphates and various polymers.

A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury.

Group B streptococci (GBS) are Gram-positive bacteria that cause infections in utero and in newborns. We recently showed that the GBS pigment is hemolytic and increased pigment production promotes bacterial penetration of human placenta. However, mechanisms utilized by the hemolytic pigment to induce host cell lysis and the consequence on fetal injury are not known. Here, we show that the GBS pigment induces membrane permeability in artificial lipid bilayers and host cells. Membrane defects induced by the GBS pigment trigger K(+) efflux leading to osmotic lysis of red blood cells or pyroptosis in human macrophages. Macrophages lacking the NLRP3 inflammasome recovered from pigment-induced cell damage. In a murine model of in utero infection, hyperpigmented GBS strains induced fetal injury in both an NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent manner. These results demonstrate that the dual mechanism of action of the bacterial pigment/lipid toxin leading to hemolysis or pyroptosis exacerbates fetal injury and suggest that preventing both activities of the hemolytic lipid is likely critical to reduce GBS fetal injury and preterm birth.

Hepatocellular heme oxygenase-1: a potential mechanism of erythropoietin-mediated protection after liver ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) results in progressive injury; initiated by oxidative stress during ischemia and compounded by cytokine-mediated inflammation during reperfusion. Recovery requires strict regulation of these events. Recombinant human erythropoietin (rhEPO) is thought to mitigate hepatocellular IR injury by altering the nonparenchymal liver microenvironment. This study sought to identify additional mechanisms whereby rhEPO is protective after liver IR injury. Mice were treated with rhEPO (4 units/g s.c.) at the onset of partial liver ischemia and assessed for transaminase and histologic injury at intervals after reperfusion. Induction of cytokines, activation of signal transducers and activators of transcription (STATs), suppressors of cytokine signaling (Socs1, Socs3, Cis), caspase-3 activation, and heme oxygenase-1 (HO-1) expression were assessed in postischemic liver. Effects of rhEPO stimulation were further characterized in whole-liver lysates from mice undergoing rhEPO injection alone and in cultured AML-12 hepatocytes. Recombinant human erythropoietin treatment at the onset of severe (90 min) hepatic IR confirmed commensurate biochemical and histological protection without affecting tissue cytokine levels. Although Socs3 and STAT5 activation were induced in normal liver after in vivo rhEPO injection, this treatment did not augment expression beyond that seen with IR alone, and neither was induced in cultured hepatocytes treated with rhEPO. Recombinant human erythropoietin inhibited caspase-3 activation in nonparenchymal cells, whereas hepatocellular HO-1 was rapidly induced both in vivo and in vitro with rhEPO treatment. These data suggest HO-1 as a potent mechanism of rhEPO-mediated protection after liver IR, which involves both direct hepatocellular and nonparenchymal mechanisms.

CTCF haploinsufficiency destabilizes DNA methylation and predisposes to cancer.

Epigenetic alterations, particularly in DNA methylation, are ubiquitous in cancer, yet the molecular origins and the consequences of these alterations are poorly understood. CTCF, a DNA-binding protein that regulates higher-order chromatin organization, is frequently altered by hemizygous deletion or mutation in human cancer. To date, a causal role for CTCF in cancer has not been established. Here, we show that Ctcf hemizygous knockout mice are markedly susceptible to spontaneous, radiation-, and chemically induced cancer in a broad range of tissues. Ctcf(+/-) tumors are characterized by increased aggressiveness, including invasion, metastatic dissemination, and mixed epithelial/mesenchymal differentiation. Molecular analysis of Ctcf(+/-) tumors indicates that Ctcf is haploinsufficient for tumor suppression. Tissues with hemizygous loss of CTCF exhibit increased variability in CpG methylation genome wide. These findings establish CTCF as a prominent tumor-suppressor gene and point to CTCF-mediated epigenetic stability as a major barrier to neoplastic progression.

Inhalation of nebulized perfluorochemical enhances recombinant adenovirus and adeno-associated virus-mediated gene expression in lung epithelium.

Use of perfluorochemical liquids during intratracheal vector administration enhances recombinant adenovirus and adeno-associated virus (AAV)-mediated lung epithelial gene expression. We hypothesized that inhalation of nebulized perfluorochemical vapor would also enhance epithelial gene expression after subsequent intratracheal vector administration. Freely breathing adult C57BL/6 mice were exposed for selected times to nebulized perflubron or sterile saline in a sealed Plexiglas chamber. Recombinant adenoviral vector was administered by transtracheal puncture at selected times afterward and mice were killed 3 days after vector administration to assess transgene expression. Mice tolerated the nebulized perflubron without obvious ill effects. Vector administration 6 hr after nebulized perflubron exposure resulted in an average 540% increase in gene expression in airway and alveolar epithelium, compared with that with vector alone or saline plus vector control (p<0.05). However, vector administration 1 hr, 1 day, or 3 days after perflubron exposure was not different from either nebulized saline with vector or vector alone and a 60-min exposure to nebulized perflubron is required. In parallel pilot studies in macaques, inhalation of nebulized perflubron enhanced recombinant AAV2/5 vector expression throughout the lung. Serial chest radiographs, bronchoalveolar lavages, and results of complete blood counts and serum biochemistries demonstrated no obvious adverse effects of nebulized perflubron. Further, one macaque receiving nebulized perflubron only was monitored for 1 year with no obvious adverse effects of exposure. These results demonstrate that inhalation of nebulized perflubron, a simple, clinically more feasible technique than intratracheal administration of liquid perflubron, safely enhances lung gene expression.

WASp-deficient B cells play a critical, cell-intrinsic role in triggering autoimmunity.

Patients with the immunodeficiency Wiskott-Aldrich syndrome (WAS) frequently develop systemic autoimmunity. Here, we demonstrate that mutation of the WAS gene results in B cells that are hyperresponsive to B cell receptor and Toll-like receptor (TLR) signals in vitro, thereby promoting a B cell-intrinsic break in tolerance. Whereas this defect leads to autoantibody production in WAS protein-deficient (WASp(-/-)) mice without overt disease, chimeric mice in which only the B cell lineage lacks WASp exhibit severe autoimmunity characterized by spontaneous germinal center formation, class-switched autoantibodies, renal histopathology, and early mortality. Both T cell help and B cell-intrinsic TLR engagement play important roles in promoting disease in this model, as depletion with anti-CD4 antibodies or generation of chimeric mice with B cells deficient in both WASp and MyD88 prevented development of autoimmune disease. These data highlight the potentially harmful role for cell-intrinsic loss of B cell tolerance in the setting of normal T cell function, and may explain why WAS patients with mixed chimerism after stem cell transplantation often develop severe humoral autoimmunity.

Plasma proteome profiles associated with inflammation, angiogenesis, and cancer.

Tumor development is accompanied by a complex host systemic response, which includes inflammatory and angiogenic reactions. Both tumor-derived and systemic response proteins are detected in plasma from cancer patients. However, given their non-specific nature, systemic response proteins can confound the detection or diagnosis of neoplasia. Here, we have applied an in-depth quantitative proteomic approach to analyze plasma protein changes in mouse models of subacute irritant-driven inflammation, autoreactive inflammation, and matrix associated angiogenesis and compared results to previously described findings from mouse models of polyoma middle T-driven breast cancer and Pdx1-Cre Kras(G12D) Ink4a/Arf (lox/lox)-induced pancreatic cancer. Among the confounding models, approximately 1/3 of all quantified plasma proteins exhibited a significant change in abundance compared to control mice. Of the proteins that changed in abundance, the majority were unique to each model. Altered proteins included those involved in acute phase response, inflammation, extracellular matrix remodeling, angiogenesis, and TGFß signaling. Comparison of changes in plasma proteins between the confounder models and the two cancer models revealed proteins that were restricted to the cancer-bearing mice, reflecting the known biology of these tumors. This approach provides a basis for distinguishing between protein changes in plasma that are cancer-related and those that are part of a non-specific host response.

A phase I study of concurrent chemotherapy (paclitaxel and carboplatin) and thoracic radiotherapy with swallowed manganese superoxide dismutase plasmid liposome protection in patients with locally advanced stage III non-small-cell lung cancer.

Manganese superoxide dismutase (MnSOD) is a genetically engineered therapeutic DNA/liposome containing the human MnSOD transgene. Preclinical studies in mouse models have demonstrated that the expression of the human MnSOD transgene confers protection of normal tissues from ionizing irradiation damage. This is a phase I study of MnSOD plasmid liposome (PL) in combination with standard chemoradiation in surgically unresectable stage III non-small-cell lung cancer. Chemotherapy (carboplatin and paclitaxel) was given weekly (for 7 weeks), concurrently with radiation. MnSOD PL was swallowed twice a week (total 14 doses), at three dose levels: 0.3, 3, and 30 mg. Dose escalation followed a standard phase I design. Esophagoscopy was done at baseline, day 4, and 6 weeks after radiation with biopsies of the squamous lining cells. DNA was extracted and analyzed by PCR for the detection of the MnSOD transgene DNA. Ten patients with AJCC stage IIIA (three) and IIIB (seven) completed the course of therapy. Five had squamous histology, two adenocarcinoma, one large cell, and two not specified. Patients were treated in three cohorts at three dose levels of MnSOD PL: 0.3 (three patients), 3 (three patients), and 30 mg (four patients). The median dose of radiation was 77.7 Gy (range 63-79.10 Gy). Overall response rate for the standard chemoradiation regimen was 70% (n = 10). There were no dose-limiting toxicities reported in all three dosing tiers. It is concluded that the oral administration of MnSOD PL is feasible and safe. The phase II recommended dose is 30 mg.

Vascular endothelial platelet endothelial cell adhesion molecule 1 (PECAM-1) regulates advanced metastatic progression.

Most patients who die from cancer succumb to treatment-refractory advanced metastatic progression. Although the early stages of tumor metastasis result in the formation of clinically silent micrometastatic foci, its later stages primarily reflect the progressive, organ-destructive growth of already advanced metastases. Early-stage metastasis is regulated by multiple factors within tumor cells as well as by the tumor microenvironment (TME). In contrast, the molecular determinants that control advanced metastatic progression remain essentially uncharacterized, precluding the development of therapies targeted against it. Here we show that the TME, functioning in part through platelet endothelial cell adhesion molecule 1 (PECAM-1), drives advanced metastatic progression and is essential for progression through its preterminal end stage. PECAM-1-KO and chimeric mice revealed that its metastasis-promoting effects are mediated specifically through vascular endothelial cell (VEC) PECAM-1. Anti-PECAM-1 mAb therapy suppresses both end-stage metastatic progression and tumor-induced cachexia in tumor-bearing mice. It reduces proliferation, but not angiogenesis or apoptosis, within advanced tumor metastases. Because its antimetastatic effects are mediated by binding to VEC rather than to tumor cells, anti-PECAM-1 mAb appears to act independently of tumor type. A modified 3D coculture assay showed that anti-PECAM-1 mAb inhibits the proliferation of PECAM-1-negative tumor cells by altering the concentrations of secreted factors. Our studies indicate that a complex interplay between elements of the TME and advanced tumor metastases directs end-stage metastatic progression. They also suggest that some therapeutic interventions may target late-stage metastases specifically. mAb-based targeting of PECAM-1 represents a TME-targeted therapeutic approach that suppresses the end stages of metastatic progression, until now a refractory clinical entity.

G alpha q-containing G proteins regulate B cell selection and survival and are required to prevent B cell-dependent autoimmunity.

Survival of mature B cells is regulated by B cell receptor and BAFFR-dependent signals. We show that B cells from mice lacking the G(alphaq) subunit of trimeric G proteins (Gnaq(-/-) mice) have an intrinsic survival advantage over normal B cells, even in the absence of BAFF. Gnaq(-/-) B cells develop normally in the bone marrow but inappropriately survive peripheral tolerance checkpoints, leading to the accumulation of transitional, marginal zone, and follicular B cells, many of which are autoreactive. Gnaq(-/-) chimeric mice rapidly develop arthritis as well as other manifestations of systemic autoimmune disease. Importantly, we demonstrate that the development of the autoreactive B cell compartment is the result of an intrinsic defect in Gnaq(-/-) B cells, resulting in the aberrant activation of the prosurvival factor Akt. Together, these data show for the first time that signaling through trimeric G proteins is critically important for maintaining control of peripheral B cell tolerance induction and repressing autoimmunity.

A non-coding cationic lipid DNA complex produces lasting anti-leukemic effects.

Cationic lipid DNA complex (CLDC) is an immunostimulatory preparation that has significant anti-leukemic effects in multiple murine models of leukemia: BCR-ABL(+) myelogenous leukemia in C3H/HeJ animals and myelomonocytic leukemia in BALB/c mice. Following leukemic challenge, CLDC treatment inhibits tumor cell growth in vivo and extends survival, sometimes resulting in apparent eradication of tumor cells. CLDC induces multiple cytokines including interferon-gamma (IFNγ), and intravenous treatment results in a more rapid and robust response than subcutaneous treatment. IFNγ is induced in a dose-dependent manner, and tachyphylaxis results from repeated doses of CLDC. Tachyphylaxis of therapeutic effects is exacerbated at higher doses, thus the optimal survival benefits are seen at intermediate doses. Animals whose leukemia has been successfully treated with CLDC exhibit a survival advantage when faced with a secondary leukemic challenge, suggesting the existence of an adaptive anti-leukemic response. This work demonstrates the effectiveness of CLDC in multiple experimental leukemias and is consistent with a stimulation of a lasting TH(1) anti-leukemic immune response.