An in vitro quantitative systems pharmacology approach for deconvolving mechanisms of drug-induced, multilineage cytopenias.

Myelosuppression is one of the most common and severe adverse events associated with anti-cancer therapies and can be a source of drug attrition. Current mathematical modeling methods for assessing cytopenia risk rely on indirect measurements of drug effects and primarily focus on single lineage responses to drugs. However, anti-cancer therapies have diverse mechanisms with varying degrees of effect across hematopoietic lineages. To improve predictive understanding of drug-induced myelosuppression, we developed a quantitative systems pharmacology (QSP) model of hematopoiesis in vitro for quantifying the effects of anti-cancer agents on multiple hematopoietic cell lineages. We calibrated the system parameters of the model to cell kinetics data without treatment and then validated the model by showing that the inferred mechanisms of anti-proliferation and/or cell-killing are consistent with the published mechanisms for three classes of drugs with different mechanisms of action. Using a set of compounds as a reference set, we then analyzed novel compounds to predict their mechanisms and magnitude of myelosuppression. Further, these quantitative mechanisms are valuable for the development of translational in vivo models to predict clinical cytopenia effects.

In vitro assessment of farnesoid X receptor antagonism to predict drug-induced liver injury risk.

Drug-induced liver injury (DILI) continues to be a major cause of drug attrition and restrictive labeling. Given the importance of farnesoid X receptor (FXR) in bile acid homeostasis, drug-related FXR antagonism may be an important mechanism of DILI. However, a comprehensive assessment of this phenomenon broadly in the context of DILI is lacking. As such, we used an orthogonal approach comprising a FXR target gene assay in primary human hepatocytes and a commercially available FXR reporter assay to investigate the potential FXR antagonistic effects of an extensive test set of 159 compounds with and without association with clinical DILI. Data were omitted from analysis based on the presence of cytotoxicity to minimize false positive assay signals and other complications in data interpretation. Based on the experimental approaches employed and corresponding data, the prevalence of FXR antagonism was relatively low across this broad DILI test set, with 16-24% prevalence based on individual assay results or combined signals in both assays. Moreover, FXR antagonism was not highly predictive for identifying clinically relevant hepatotoxicants retrospectively, where FXR antagonist classification alone had minimal to moderate predictive value as represented by positive and negative likelihood ratios of 2.24-3.84 and 0.72-0.85, respectively. The predictivity did not increase significantly when considering only compounds with high clinical exposure (maximal or efficacious plasma exposures > 1.0 µM). In contrast, modest gains in predictive value of FXR antagonism were observed considering compounds that also inhibit bile salt export pump. In addition, we have identified novel FXR antagonistic effects of well-studied hepatotoxic drugs, including bosentan, tolcapone and ritonavir. In conclusion, this work represents a comprehensive evaluation of FXR antagonism in the context of DILI, including its overall predictivity and challenges associated with detecting this phenomenon in vitro.

Proof of Concept for an Automated Image Analysis Method to Quantify Rat Bone Marrow Hematopoietic Lineages on H&E Sections.

The bone marrow is an important site for assessment of the hematopoietic toxicity of new drug candidates. Here, we extended our previous work, where we developed a computer algorithm to automatically quantitate overall bone marrow cell density by analyzing digitized images of standard hematoxylin and eosin (H&E) slides of rat bone marrow and further evaluated the capability to quantify myeloid: erythroid + lymphoid (M:EL) ratio and megakaryocyte cell density. We tested the algorithm in a toxicity study, where rats were dosed with two molecules known to affect bone marrow composition, monomethyl auristatin E, and a Bcl-xL inhibitor. The image analysis method detected significant changes in M:EL and megakaryocyte number that were either not found or semiquantitatively described by manual microscopic observation of the same slides. The image analysis results were consistent with other more established but time-consuming methods that measure changes in bone marrow cell composition: smear cytology, flow cytometry, and microscopic assessment. Our work demonstrates the feasibility of a rapid and more quantitative assessment of changes in bone marrow cell lineage composition using a computer algorithm compared to microscopic examination of H&E-stained bone marrow sections.

Drug-induced allergic hepatitis develops in mice when myeloid-derived suppressor cells are depleted prior to halothane treatment.

UNLABELLED: Clinical evidence suggests that many cases of serious idiosyncratic drug-induced liver injury are mediated by the adaptive immune system in response to hepatic drug-protein adducts, also referred to as "drug-induced allergic hepatitis"; but detailed mechanistic proof has remained elusive due to the lack of animal models. We have hypothesized that drug-induced allergic hepatitis is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver. We provide evidence that immune tolerance can be overcome in a murine model of halothane-induced liver injury initiated by trifluoroacetylated protein adducts of halothane formed in the liver. Twenty-four hours after female Balb/cJ mice were initially treated with halothane, perivenous necrosis and an infiltration of CD11b(+) Gr-1(high) cells were observed in the liver. Further study revealed a subpopulation of myeloid-derived suppressor cells within the CD11b(+) Gr-1(high) cell fraction that inhibited the proliferation of both CD4(+) and CD8(+) T cells. When CD11b(+) Gr-1(high) cells were depleted from the liver with Gr-1 antibody treatment, enhanced liver injury was observed at 9 days after halothane rechallenge. Toxicity was associated with increased serum levels of interleukin-4 and immunoglobulins G1 and E directed against hepatic trifluoroacetylated protein adducts, as well as increased hepatic infiltration of eosinophils and CD4(+) T cells, all features of an allergic reaction. When hepatic CD4(+) T cells were depleted 5 days after halothane rechallenge, trifluoroacetylated protein adduct-specific serum immunoglobulin and hepatotoxicity were reduced. CONCLUSION: Our data provide a rational approach for developing animal models of drug-induced allergic hepatitis mediated by the adaptive immune system and suggest that impaired liver tolerance may predispose patients to this disease.

Safety Lead Optimization and Candidate Identification: Integrating New Technologies into Decision-Making.

Discovery toxicology focuses on the identification of the most promising drug candidates through the development and implementation of lead optimization strategies and hypothesis-driven investigation of issues that enable rational and informed decision-making. The major goals are to [a] identify and progress the drug candidate with the best overall drug safety profile for a therapeutic area, [b] remove the most toxic drugs from the portfolio prior to entry into humans to reduce clinical attrition due to toxicity, and [c] establish a well-characterized hazard and translational risk profile to enable clinical trial designs. This is accomplished through a framework that balances the multiple considerations to identify a drug candidate with the overall best drug characteristics and provides a cogent understanding of mechanisms of toxicity. The framework components include establishing a target candidate profile for each program that defines the qualities of a successful candidate based on the intended therapeutic area, including the risk tolerance for liabilities; evaluating potential liabilities that may result from engaging the therapeutic target (pharmacology-mediated or on-target) and that are chemical structure-mediated (off-target); and characterizing identified liabilities. Lead optimization and investigation relies upon the integrated use of a variety of technologies and models (in silico, in vitro, and in vivo) that have achieved a sufficient level of qualification or validation to provide confidence in their use. We describe the strategic applications of various nonclinical models (established and new) for a holistic and integrated risk assessment that is used for rational decision-making. While this review focuses on strategies for small molecules, the overall concepts, approaches, and technologies are generally applicable to biotherapeutics.

Thymic stromal lymphopoietin and interleukin-4 mediate the pathogenesis of halothane-induced liver injury in mice.

UNLABELLED: Liver eosinophilia has been associated with incidences of drug-induced liver injury (DILI) for more than 50 years, although its role in this disease has remained largely unknown. In this regard, it was recently shown that eosinophils played a pathogenic role in a mouse model of halothane-induced liver injury (HILI). However, the signaling events that drove hepatic expression of eosinophil-associated chemokines, eotaxins, eosinophil infiltration, and subsequent HILI were unclear. We now provide evidence implicating hepatic epithelial-derived cytokine thymic stromal lymphopoietin (TSLP) and type 2 immunity, in particular, interleukin-4 (IL-4) production, in mediating hepatic eosinophilia and injury during HILI. TSLP was constitutively expressed by mouse hepatocytes and increased during HILI. Moreover, the severity of HILI was reduced in mice deficient in either the TSLP receptor (TSLPR) or IL-4 and was accompanied by decreases in serum levels of eotaxins and hepatic eosinophilia. Similarly, concanavalin A-induced liver injury, where type 2 cytokines and eosinophils play a significant role in its pathogenesis, was also reduced in TSLPR-deficient mice. Studies in vitro revealed that mouse and human hepatocytes produce TSLP and eotaxins in response to treatment with combinations of IL-4 and proinflammatory cytokines IL-1ß and tumor necrosis factor alpha. CONCLUSION: This report provides the first evidence implicating roles for hepatic TSLP signaling, type 2 immunity, and eosinophilia in mediating liver injury caused by a drug.

Complexin vitromodels positioned for impact to drug testing in pharma: a review.

Recent years have seen the creation and popularization of various complexin vitromodels (CIVMs), such as organoids and organs-on-chip, as a technology with the potential to reduce animal usage in pharma while also enhancing our ability to create safe and efficacious drugs for patients. Public awareness of CIVMs has increased, in part, due to the recent passage of the FDA Modernization Act 2.0. This visibility is expected to spur deeper investment in and adoption of such models. Thus, end-users and model developers alike require a framework to both understand the readiness of current models to enter the drug development process, and to assess upcoming models for the same. This review presents such a framework for model selection based on comparative -omics data (which we term model-omics), and metrics for qualification of specific test assays that a model may support that we term context-of-use (COU) assays. We surveyed existing healthy tissue models and assays for ten drug development-critical organs of the body, and provide evaluations of readiness and suggestions for improving model-omics and COU assays for each. In whole, this review comes from a pharma perspective, and seeks to provide an evaluation of where CIVMs are poised for maximum impact in the drug development process, and a roadmap for realizing that potential.

Discovery of TRPA1 Antagonist GDC-6599: Derisking Preclinical Toxicity and Aldehyde Oxidase Metabolism with a Potential First-in-Class Therapy for Respiratory Disease.

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metabolite─generated by aldehyde oxidase (AO)─possessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.

2,3,7,8-TCDD enhances the sensitivity of mice to concanavalin A immune-mediated liver injury.

Inflammation plays a major role in immune-mediated liver injury, and exposure to environmental pollutants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been reported to alter the inflammatory response as well as affect immune cell activity. In this study, we tested the hypothesis that TCDD pretreatment exacerbates hepatotoxicity in a murine model of immune-mediated liver injury induced by concanavalin A (Con A) administration. Mice were pretreated with 30 µg/kg TCDD or vehicle control on day zero and then given either Con A or saline intravenously on day four. Mice treated with TCDD did not develop liver injury; however, TCDD pretreatment increased liver injury resulting from moderate doses of Con A (4-10 mg/kg). TCDD-pretreated mice had altered plasma concentrations of inflammatory cytokines, including interferon gamma (IFNγ), and TCDD/Con A-induced hepatotoxicity was attenuated in IFNγ knockout mice. At various times after treatment, intrahepatic immune cells were isolated, and expression of cell activation markers as well as cytolytic proteins was determined. TCDD pretreatment increased the proportion of activated natural killer T (NKT) cells and the percent of cells expressing Fas ligand (FasL) after Con A administration. In addition FasL knockout mice and mice treated with CD18 antiserum were both protected from TCDD/Con A-induced hepatotoxicity, suggesting a requirement for direct cell-cell interaction between effector immune cells and parenchymal cell targets in the development of liver injury from TCDD/Con A treatment. In summary, exposure to TCDD increased NKT cell activation and exacerbated immune-mediated liver injury induced by Con A through a mechanism involving IFNγ and FasL expression.

A BCMA/CD16A bispecific innate cell engager for the treatment of multiple myeloma.

Despite the recent progress, multiple myeloma (MM) is still essentially incurable and there is a need for additional effective treatments with good tolerability. RO7297089 is a novel bispecific BCMA/CD16A-directed innate cell engager (ICE®) designed to induce BCMA+ MM cell lysis through high affinity binding of CD16A and retargeting of NK cell cytotoxicity and macrophage phagocytosis. Unlike conventional antibodies approved in MM, RO7297089 selectively targets CD16A with no binding of other Fcγ receptors, including CD16B on neutrophils, and irrespective of 158V/F polymorphism, and its activity is less affected by competing IgG suggesting activity in the presence of M-protein. Structural analysis revealed this is due to selective interaction with a single residue (Y140) uniquely present in CD16A opposite the Fc binding site. RO7297089 induced tumor cell killing more potently than conventional antibodies (wild-type and Fc-enhanced) and induced lysis of BCMA+ cells at very low effector-to-target ratios. Preclinical toxicology data suggested a favorable safety profile as in vitro cytokine release was minimal and no RO7297089-related mortalities or adverse events were observed in cynomolgus monkeys. These data suggest good tolerability and the potential of RO7297089 to be a novel effective treatment of MM patients.

Perspectives on the evaluation and adoption of complex in vitro models in drug development: Workshop with the FDA and the pharmaceutical industry (IQ MPS Affiliate).

Complex in vitro models (CIVM) offer the potential to improve pharmaceutical clinical drug attrition due to safety and/ or efficacy concerns. For this technology to have an impact, the establishment of robust characterization and qualifi­cation plans constructed around specific contexts of use (COU) is required. This article covers the output from a workshop between the Food and Drug Administration (FDA) and Innovation and Quality Microphysiological Systems (IQ MPS) Affiliate. The intent of the workshop was to understand how CIVM technologies are currently being applied by pharma­ceutical companies during drug development and are being tested at the FDA through various case studies in order to identify hurdles (real or perceived) to the adoption of microphysiological systems (MPS) technologies, and to address evaluation/qualification pathways for these technologies. Output from the workshop includes the alignment on a working definition of MPS, a detailed description of the eleven CIVM case studies presented at the workshop, in-depth analysis, and key take aways from breakout sessions on ADME (absorption, distribution, metabolism, and excretion), pharmacology, and safety that covered topics such as qualification and performance criteria, species differences and concordance, and how industry can overcome barriers to regulatory submission of CIVM data. In conclusion, IQ MPS Affiliate and FDA scientists were able to build a general consensus on the need for animal CIVMs for preclinical species to better determine species concordance. Furthermore, there was acceptance that CIVM technologies for use in ADME, pharmacology and safety assessment will require qualification, which will vary depending on the specific COU.

Antibody-Mediated Delivery of Chimeric BRD4 Degraders. Part 2: Improvement of In Vitro Antiproliferation Activity and In Vivo Antitumor Efficacy.

Heterobifunctional compounds that direct the ubiquitination of intracellular proteins in a targeted manner via co-opted ubiquitin ligases have enormous potential to transform the field of medicinal chemistry. These chimeric molecules, often termed proteolysis-targeting chimeras (PROTACs) in the chemical literature, enable the controlled degradation of specific proteins via their direction to the cellular proteasome. In this report, we describe the second phase of our research focused on exploring antibody-drug conjugates (ADCs), which incorporate BRD4-targeting chimeric degrader entities. We employ a new BRD4-binding fragment in the construction of the chimeric ADC payloads that is significantly more potent than the corresponding entity utilized in our initial studies. The resulting BRD4-degrader antibody conjugates exhibit potent and antigen-dependent BRD4 degradation and antiproliferation activities in cell-based experiments. Multiple ADCs bearing chimeric BRD4-degrader payloads also exhibit strong, antigen-dependent antitumor efficacy in mouse xenograft assessments that employ several different tumor models.

Application of microphysiological systems in biopharmaceutical research and development.

Within the last 10 years, several tissue microphysiological systems (MPS) have been developed and characterized for retention of morphologic characteristics and specific gene/protein expression profiles from their natural in vivo state. Once developed, their utility is typically further tested by comparing responses to known toxic small-molecule pharmaceuticals in efforts to develop strategies for further toxicity testing of compounds under development. More recently, application of this technology in biopharmaceutical (large molecules) development is beginning to be more appreciated. In this review, we describe some of the advances made for tissue-specific MPS and outline the advantages and challenges of applying and further developing MPS technology in preclinical biopharmaceutical research.

General Principles for the Validation of Proarrhythmia Risk Prediction Models: An Extension of the CiPA In Silico Strategy.

This white paper presents principles for validating proarrhythmia risk prediction models for regulatory use as discussed at the In Silico Breakout Session of a Cardiac Safety Research Consortium/Health and Environmental Sciences Institute/US Food and Drug Administration-sponsored Think Tank Meeting on May 22, 2018. The meeting was convened to evaluate the progress in the development of a new cardiac safety paradigm, the Comprehensive in Vitro Proarrhythmia Assay (CiPA). The opinions regarding these principles reflect the collective views of those who participated in the discussion of this topic both at and after the breakout session. Although primarily discussed in the context of in silico models, these principles describe the interface between experimental input and model-based interpretation and are intended to be general enough to be applied to other types of nonclinical models for proarrhythmia assessment. This document was developed with the intention of providing a foundation for more consistency and harmonization in developing and validating different models for proarrhythmia risk prediction using the example of the CiPA paradigm.

The role of the hemostatic system in murine liver injury induced by coexposure to lipopolysaccharide and trovafloxacin, a drug with idiosyncratic liability.

The use of the fluoroquinolone antibiotic trovafloxacin (TVX) was severely restricted in 1999 due to its association with idiosyncratic hepatotoxicity. Previously, we reported that a nontoxic dose of TVX interacts with a nontoxic dose of lipopolysaccharide (LPS) to cause robust hepatocellular injury in mice. This interaction with LPS was not seen in mice treated with levofloxacin (LVX), a fluoroquinolone not associated with hepatotoxicity in people. TVX/LPS-coexposure caused an increase in plasma alanine aminotransferase (ALT) activity as early as 4.5 h after LPS administration which progressed through 15 h.We examined the role of the hemostatic system in TVX/LPS-induced liver injury. At the onset of liver injury, coexposure to TVX/LPS, but not exposure to TVX, LVX, LPS or LVX/LPS, caused increased plasma concentration of thrombin-antithrombin dimers and decreased plasma circulating fibrinogen. LPS treatment induced a small increase in plasma plasminogen activator inhibitor-1 (PAI-1) concentration, and TVX pretreatment enhanced this effect. TVX/LPS coexposure also resulted in hepatic fibrin deposition. Anticoagulant heparin administration reduced TVX/LPS-induced hepatic fibrin deposition and liver injury. PAI-1-/- mice treated with TVX/LPS exhibited similar fibrin deposition to wild-type mice but had significantly reduced hepatocellular injury. PAI-1-/- mice, but not heparin-treated mice, had reduced plasma concentrations of several cytokines compared to TVX/LPS-treated controls. In summary, TVX/LPS-coexposure caused an imbalance in the hemostatic system, resulting in thrombin activation increased, plasma concentration of PAI-1 and hepatic fibrin deposition. Both thrombin activation and PAI-1 play critical roles in the progression of TVX/LPS-induced liver injury, but through different modes of action.

Sequential azacitidine and lenalidomide for patients with relapsed and refractory acute myeloid leukemia: Clinical results and predictive modeling using computational analysis.

BACKGROUND: Patients with relapsed and refractory (R/R) acute myeloid leukemia (AML) have limited treatment options. Genomically-defined personalized therapies are only applicable for a minority of patients. Therapies without identifiable targets can be effective but patient selection is challenging. The sequential combination of azacitidine with high-dose lenalidomide has shown activity; we aimed to determine the efficacy of this genomically-agnostic regimen in patients with R/R AML, with the intention of applying sophisticated methods to predict responders. METHODS: Thirty-seven R/R AML/myelodysplastic syndrome patients were enrolled in a phase 2 study of azacitidine with lenalidomide. The primary endpoint was complete remission (CR) and CR with incomplete blood count recovery (CRi) rate. A computational biological modeling (CBM) approach was applied retrospectively to predict outcomes based on the understood mechanisms of azacitidine and lenalidomide in the setting of each patients' disease. FINDINGS: Four of 37 patients (11%) had a CR/CRi; the study failed to meet the alternative hypothesis. Significant toxicity was observed in some cases, with three treatment-related deaths and a 30-day mortality rate of 14%. However, the CBM method predicted responses in 83% of evaluable patients, with a positive and negative predictive value of 80% and 89%, respectively. INTERPRETATION: Sequential azacitidine and high-dose lenalidomide is effective in a minority of R/R AML patients; it may be possible to predict responders at the time of diagnosis using a CBM approach. More efforts to predict responses in non-targeted therapies should be made, to spare toxicity in patients unlikely to respond and maximize treatments for those with limited options.

Trovafloxacin-induced replication stress sensitizes HepG2 cells to tumor necrosis factor-alpha-induced cytotoxicity mediated by extracellular signal-regulated kinase and ataxia telangiectasia and Rad3-related.

Use of the fluoroquinolone antibiotic trovafloxacin (TVX) was restricted due to idiosyncratic, drug-induced liver injury (IDILI). Previous studies demonstrated that tumor necrosis factor-alpha (TNF) and TVX interact to cause death of hepatocytes in vitro that was associated with prolonged activation of c-Jun N-terminal kinase (JNK), activation of caspases 9 and 3, and DNA damage. The purpose of this study was to explore further the mechanism by which TVX interacts with TNF to cause cytotoxicity. Treatment with TVX caused cell cycle arrest, enhanced expression of p21 and impaired proliferation, but cell death only occurred after cotreatment with TVX and TNF. Cell death involved activation of extracellular signal-related kinase (ERK), which in turn activated caspase 3 and ataxia telangiectasia and Rad3-related (ATR), both of which contributed to cytotoxicity. Cotreatment of HepG2 cells with TVX and TNF caused double-strand breaks in DNA, and ERK contributed to this effect. Inhibition of caspase activity abolished the DNA strand breaks. The data suggest a complex interaction of TVX and TNF in which TVX causes replication stress, and the downstream effects are exacerbated by TNF, leading to hepatocellular death. These results raise the possibility that IDILI from TVX results from MAPK and ATR activation in hepatocytes initiated by interaction of cytokine signaling with drug-induced replication stress.

Molecular mechanisms of hepatocellular apoptosis induced by trovafloxacin-tumor necrosis factor-alpha interaction.

Idiosyncratic drug-induced liver injury (IDILI) continues to be a significant human health problem. IDILI is characterized as occurring in a minority of individuals exposed to a drug, yet it accounts for as much as 17% of all cases of acute liver failure. Despite these concerns, the mechanisms underlying IDILI remain unknown. Trovafloxacin (TVX), which causes IDILI in humans, also causes hepatocellular death in vitro when combined with tumor necrosis factor-alpha (TNF) treatment. However, the molecular mechanisms involved in this toxicity are not fully characterized. The purpose of this study was to identify mechanisms by which TVX and TNF interact to cause hepatocellular death, with a focus on a human hepatocyte cell line. TVX and TNF interacted to cause cytotoxicity in HepG2 cells at drug concentrations similar to those in people undergoing TVX therapy. TVX/TNF treatment caused apoptosis and DNA damage in HepG2 cells that depended on caspase activation. Prolonged activation of JNK occurred in TVX/TNF-induced cytotoxicity, and treatment with the JNK selective inhibitor SP600125 attenuated cytotoxicity. TVX/TNF cotreatment also caused cytotoxicity in isolated primary murine hepatocytes that was dependent on caspase activation. These results increase understanding of molecular signaling pathways involved in hepatocellular death caused by a drug with idiosyncratic liability in the presence of TNF.

Pretreatment with TCDD exacerbates liver injury from Concanavalin A: critical role for NK cells.

For many liver diseases, including viral and autoimmune hepatitis, immune cells play an important role in the development and progression of liver injury. Concanavalin A (Con A) administration to rodents has been used as a model of immune-mediated liver injury resembling human autoimmune hepatitis. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to alter the development of immune-mediated diseases. Mice pretreated with TCDD developed exacerbated liver injury in response to administration of a mild dose (6 mg/kg) of Con A. In the present study, we tested the hypothesis that TCDD pretreatment exacerbates Con A-induced liver injury by enhancing the activation and recruitment of accessory cell types including neutrophils, macrophages, and natural killer (NK) cells. Mice were treated with 0, 0.3, 3, or 30 µg/kg TCDD and 4 days later with Con A or saline. TCDD pretreatment with doses of 3 and 30 µg/kg significantly increased liver injury from Con A administration. The plasma concentrations of neutrophil chemokines were significantly increased in TCDD-pretreated mice after Con A administration. NKT cell-deficient (CD1d KO) mice were used to examine whether NKT cells were required for TCDD/Con A-induced liver injury. CD1d KO mice were completely protected from liver injury induced by treatment with Con A alone, whereas the injury from TCDD/Con A treatment was reduced but not eliminated. However, T-cell deficient (RAG1 KO) mice were protected from liver injury induced by Con A irrespective of pretreatment with TCDD. TCDD/Con A treatment increased the percentage of NK cells expressing the activation marker CD69. Depletion of NK cells prior to treatment resulted in significant reductions in plasma interferon-γ and liver injury from TCDD/Con A treatment. In summary, exposure to TCDD exacerbated the immune-mediated liver injury induced by Con A, and our findings suggest that NK cells play a critical role in this response.