Dual transcriptional analysis reveals adaptation of host and pathogen to intracellular survival of Pseudomonas aeruginosa associated with urinary tract infection.

Long-term survival of bacterial pathogens during persistent bacterial infections can be associated with antibiotic treatment failure and poses a serious public health problem. Infections caused by the Gram-negative pathogen Pseudomonas aeruginosa, which can cause both acute and chronic infections, are particularly challenging due to its high intrinsic resistance to antibiotics. The ineffectiveness of antibiotics is exacerbated when bacteria reside intracellularly within host cells where they can adopt a drug tolerant state. While the early steps of adherence and entry of P. aeruginosa into mammalian cells have been described, the subsequent fate of internalized bacteria, as well as host and bacterial molecular pathways facilitating bacterial long-term survival, are not well defined. In particular, long-term survival within bladder epithelial cells has not been demonstrated and this may have important implications for the understanding and treatment of UTIs caused by P. aeruginosa. Here, we demonstrate and characterize the intracellular survival of wild type (WT) P. aeruginosa inside bladder epithelial cells and a mutant with a disruption in the bacterial two-component regulator AlgR that is unable to survive intracellularly. Using simultaneous dual RNA-seq transcriptional profiling, we define the transcriptional response of intracellular bacteria and their corresponding invaded host cells. The bacterial transcriptional response demonstrates that WT bacteria rapidly adapt to the stress encountered in the intracellular environment in contrast to ΔalgR bacteria. Analysis of the host transcriptional response to invasion suggests that the NF-κB signaling pathway, previously shown to be required for extracellular bacterial clearance, is paradoxically also required for intracellular bacterial survival. Lastly, we demonstrate that intracellular survival is important for pathogenesis of P. aeruginosa in vivo using a model of murine urinary tract infection. We propose that the unappreciated ability of P. aeruginosa to survive intracellularly may play an important role in contributing to the chronicity and recurrence of P. aeruginosa in urinary tract infections.

Hybridization-based capture of pathogen mRNA enables paired host-pathogen transcriptional analysis.

Dual transcriptional profiling of host and bacteria during infection is challenging due to the low abundance of bacterial mRNA. We report Pathogen Hybrid Capture (PatH-Cap), a method to enrich for bacterial mRNA and deplete bacterial rRNA simultaneously from dual RNA-seq libraries using transcriptome-specific probes. By addressing both the differential RNA content of the host relative to the infecting bacterium and the overwhelming abundance of uninformative structural RNAs (rRNA, tRNA) of both species in a single step, this approach enables analysis of very low-input RNA samples. By sequencing libraries before (pre-PatH-Cap) and after (post-PatH-Cap) enrichment, we achieve dual transcriptional profiling of host and bacteria, respectively, from the same sample. Importantly, enrichment preserves relative transcript abundance and increases the number of unique bacterial transcripts per gene in post-PatH-Cap libraries compared to pre-PatH-Cap libraries at the same sequencing depth, thereby decreasing the sequencing depth required to fully capture the transcriptional profile of the infecting bacteria. We demonstrate that PatH-Cap enables the study of low-input samples including single eukaryotic cells infected by 1-3 Pseudomonas aeruginosa bacteria and paired host-pathogen temporal gene expression analysis of Mycobacterium tuberculosis infecting macrophages. PatH-Cap can be applied to the study of a range of pathogens and microbial species, and more generally, to lowly-abundant species in mixed populations.

Defining the core essential genome of Pseudomonas aeruginosa.

Genomics offered the promise of transforming antibiotic discovery by revealing many new essential genes as good targets, but the results fell short of the promise. While numerous factors contributed to the disappointing yield, one factor was that essential genes for a bacterial species were often defined based on a single or limited number of strains grown under a single or limited number of in vitro laboratory conditions. In fact, the essentiality of a gene can depend on both the genetic background and growth condition. We thus developed a strategy for more rigorously defining the core essential genome of a bacterial species by studying many pathogen strains and growth conditions. We assessed how many strains must be examined to converge on a set of core essential genes for a species. We used transposon insertion sequencing (Tn-Seq) to define essential genes in nine strains of Pseudomonas aeruginosa on five different media and developed a statistical model, FiTnEss, to classify genes as essential versus nonessential across all strain-medium combinations. We defined a set of 321 core essential genes, representing 6.6% of the genome. We determined that analysis of four strains was typically sufficient in P. aeruginosa to converge on a set of core essential genes likely to be essential across the species across a wide range of conditions relevant to in vivo infection, and thus to represent attractive targets for novel drug discovery.

Single-Cell RNA Sequencing to Understand Host-Pathogen Interactions.

Host-pathogen interactions, particularly in the context of bacterial infections, are dynamic exchanges where transcriptional heterogeneity from both the host and the pathogen can lead to many diverse outcomes via distinct molecular pathways. Transcriptional profiling at the single-cell level, on a genome-wide scale, has enabled a greater appreciation of the cellular diversity in complex biological organisms and the myriad of host transcriptional states during infection. Here, we highlight recent reports of single-cell RNA sequencing within the context of host-pathogen interactions, describe current limitations for detecting and profiling the transcriptome of invading pathogens at the single-cell level, and suggest exciting future prospects for this technology in the study of infection. We propose that understanding infection as an integrated process between pathogen and host with resolution at the single-cell level will ultimately inform development of vaccines with greater productive and protective host immunity, enable the development of novel therapeutics that harness host mechanisms, and yield more accurate biomarkers to guide better diagnostics.

Generation of mouse-zebrafish hematopoietic tissue chimeric embryos for hematopoiesis and host-pathogen interaction studies.

Xenografts of the hematopoietic system are extremely useful as disease models and for translational research. Zebrafish xenografts have been widely used to monitor blood cancer cell dissemination and homing due to the optical clarity of embryos and larvae, which allow unrestricted in vivo visualization of migratory events. Here, we have developed a xenotransplantation technique that transiently generates hundreds of hematopoietic tissue chimeric embryos by transplanting murine bone marrow cells into zebrafish blastulae. In contrast to previous methods, this procedure allows mammalian cell integration into the fish developmental hematopoietic program, which results in chimeric animals containing distinct phenotypes of murine blood cells in both circulation and the hematopoietic niche. Murine cells in chimeric animals express antigens related to (i) hematopoietic stem and progenitor cells, (ii) active cell proliferation and (iii) myeloid cell lineages. We verified the utility of this method by monitoring zebrafish chimeras during development using in vivo non-invasive imaging to show novel murine cell behaviors, such as homing to primitive and definitive hematopoietic tissues, dynamic hematopoietic cell and hematopoietic niche interactions, and response to bacterial infection. Overall, transplantation into the zebrafish blastula provides a useful method that simplifies the generation of numerous chimeric animals and expands the range of murine cell behaviors that can be studied in zebrafish chimeras. In addition, integration of murine cells into the host hematopoietic system during development suggests highly conserved molecular mechanisms of hematopoiesis between zebrafish and mammals.This article has an associated First Person interview with the first author of the paper.

Pathogen Cell-to-Cell Variability Drives Heterogeneity in Host Immune Responses.

Encounters between immune cells and invading bacteria ultimately determine the course of infection. These interactions are usually measured in populations of cells, masking cell-to-cell variation that may be important for infection outcome. To characterize the gene expression variation that underlies distinct infection outcomes and monitor infection phenotypes, we developed an experimental system that combines single-cell RNA-seq with fluorescent markers. Probing the responses of individual macrophages to invading Salmonella, we find that variation between individual infected host cells is determined by the heterogeneous activity of bacterial factors in individual infecting bacteria. We illustrate how variable PhoPQ activity in the population of invading bacteria drives variable host type I IFN responses by modifying LPS in a subset of bacteria. This work demonstrates a causative link between host and bacterial variability, with cell-to-cell variation between different bacteria being sufficient to drive radically different host immune responses. This co-variation has implications for host-pathogen dynamics in vivo.

Aberrant innate immune activation following tissue injury impairs pancreatic regeneration.

Normal tissue architecture is disrupted following injury, as resident tissue cells become damaged and immune cells are recruited to the site of injury. While injury and inflammation are critical to tissue remodeling, the inability to resolve this response can lead to the destructive complications of chronic inflammation. In the pancreas, acinar cells of the exocrine compartment respond to injury by transiently adopting characteristics of progenitor cells present during embryonic development. This process of de-differentiation creates a window where a mature and stable cell gains flexibility and is potentially permissive to changes in cellular fate. How de-differentiation can turn an acinar cell into another cell type (such as a pancreatic ß-cell), or a cell with cancerous potential (as in cases of deregulated Kras activity) is of interest to both the regenerative medicine and cancer communities. While it is known that inflammation and acinar de-differentiation increase following pancreatic injury, it remains unclear which immune cells are involved in this process. We used a combination of genetically modified mice, immunological blockade and cellular characterization to identify the immune cells that impact pancreatic regeneration in an in vivo model of pancreatitis. We identified the innate inflammatory response of macrophages and neutrophils as regulators of pancreatic regeneration. Under normal conditions, mild innate inflammation prompts a transient de-differentiation of acinar cells that readily dissipates to allow normal regeneration. However, non-resolving inflammation developed when elevated pancreatic levels of neutrophils producing interferon-γ increased iNOS levels and the pro-inflammatory response of macrophages. Pancreatic injury improved following in vivo macrophage depletion, iNOS inhibition as well as suppression of iNOS levels in macrophages via interferon-γ blockade, supporting the impairment in regeneration and the development of chronic inflammation arises from aberrant activation of the innate inflammatory response. Collectively these studies identify targetable inflammatory factors that can be used to influence the development of non-resolving inflammation and pancreatic regeneration following injury.

Production of α-galactosylceramide by a prominent member of the human gut microbiota.

While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer(Bf)), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer(Bf) has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.

IL-7 receptor blockade reverses autoimmune diabetes by promoting inhibition of effector/memory T cells.

To protect the organism against autoimmunity, self-reactive effector/memory T cells (T(E/M)) are controlled by cell-intrinsic and -extrinsic regulatory mechanisms. However, how some T(E/M) cells escape regulation and cause autoimmune disease is currently not understood. Here we show that blocking IL-7 receptor-α (IL-7Rα) with monoclonal antibodies in nonobese diabetic (NOD) mice prevented autoimmune diabetes and, importantly, reversed disease in new-onset diabetic mice. Surprisingly, IL-7-deprived diabetogenic T(E/M) cells remained present in the treated animals but showed increased expression of the inhibitory receptor Programmed Death 1 (PD-1) and reduced IFN-γ production. Conversely, IL-7 suppressed PD-1 expression on activated T cells in vitro. Adoptive transfer experiments revealed that T(E/M) cells from anti-IL-7Rα-treated mice had lost their pathogenic potential, indicating that absence of IL-7 signals induces cell-intrinsic tolerance. In addition to this mechanism, IL-7Rα blockade altered the balance of regulatory T cells and T(E/M) cells, hence promoting cell-extrinsic regulation and further increasing the threshold for diabetogenic T-cell activation. Our data demonstrate that IL-7 contributes to the pathogenesis of autoimmune diabetes by enabling T(E/M) cells to remain in a functionally competent state and suggest IL-7Rα blockade as a therapy for established T-cell-dependent autoimmune diseases.

Anti-CD3 therapy promotes tolerance by selectively depleting pathogenic cells while preserving regulatory T cells.

Monoclonal anti-CD3 Abs have been used clinically for two decades to reverse steroid-resistant acute graft rejection. In autoimmune diabetes, short course treatment with FcR-nonbinding (FNB) anti-CD3 mAb in mice with recent onset of diabetes induces long-term disease remission. Induction of tolerogenic regulatory T cells (Tregs) has been implicated to be one of the mechanisms of action by FNB anti-CD3 mAb in these settings. In this study, we examined the effect of FNB anti-CD3 mAb treatment on the homeostasis of naive, effector, and regulatory T cells in vivo. Anti-CD3 treatment induced a transient systemic rise in the percentage but not absolute number of CD4(+)Foxp3(+) Tregs due to selective depletion of CD4(+)Foxp3(-) conventional T cells. T cell depletion induced by FNB anti-CD3 mAb was independent of the proapoptotic proteins Fas, caspase-3, and Bim and was not inhibited by overexpression of the anti-apoptotic protein, Bcl-2. Tregs were not preferentially expanded and we found no evidence of conversion of conventional T cells into Tregs, suggesting that the pre-existing Tregs are resistant to anti-CD3-induced cell death. Interestingly, expression of the transcription factor Helios, which is expressed by thymus-derived natural Tregs, was increased in Tregs after FNB anti-CD3 mAb treatment, suggesting that the anti-CD3 treatment can alter, and potentially stabilize, Treg function. Taken together, the results suggest that FNB anti-CD3 therapy promotes tolerance by restoring the balance between pathogenic and regulatory T cells.

Intrinsic and extrinsic control of peripheral T-cell tolerance by costimulatory molecules of the CD28/ B7 family.

Positive and negative costimulation by members of the CD28 family is critical for the development of productive immune responses against foreign pathogens and their proper termination to prevent inflammation-induced tissue damage. In addition, costimulatory signals are critical for the establishment and maintenance of peripheral tolerance. This paradigm has been established in many animal models and has led to the development of immunotherapies targeting costimulation pathways for the treatment of cancer, autoimmune disease, and allograft rejection. During the last decade, the complexity of the biology of costimulatory pathways has greatly increased due to the realization that costimulation does not affect only effector T cells but also influences regulatory T cells and antigen-presenting cells. Thus, costimulation controls T-cell tolerance through both intrinsic and extrinsic pathways. In this review, we discuss the influence of costimulation on intrinsic and extrinsic pathways of peripheral tolerance, with emphasis on members of the CD28 family, CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), and programmed death-1 (PD-1), as well as the downstream cytokine interleukin-1 (IL-2).

Prevention of diabetes by FTY720-mediated stabilization of peri-islet tertiary lymphoid organs.

OBJECTIVE: The nonobese diabetic (NOD) mouse is a well-established mouse model of spontaneous type 1 diabetes, which is characterized by an autoimmune destruction of the insulin-secreting pancreatic beta-cells. In this study, we address the role of tertiary lymphoid organs (TLOs) that form in the pancreas of NOD mice during disease progression. METHODS: We developed a model designed to "lock" lymphocytes in the pancreatic lymph node (PLN) and pancreas by the use of FTY720, which blocks the exit of lymphocytes from lymph nodes. A combination of flow cytometry, immunofluorescence, and analysis of clinical scores was used to study the effects of long-term FTY720 treatment on TLO development and development of diabetes. RESULTS: Continuous treatment of NOD mice with FTY720 prevented diabetes development even at a time of significant insulitis. Treatment withdrawal led to accelerated disease independent of the PLN. Interestingly, naive T-cells trafficked to and proliferated in the TLOs. In addition, morphological changes were observed that occurred during the development of the disease. Remarkably, although the infiltrates are not organized into T/B-cell compartments in 8-week-old mice, by 20 weeks of age, and in age-matched mice undergoing FTY720 treatment, the infiltrates showed a high degree of organization. However, in naturally and FTY720-induced diabetic mice, T/B-cell compartmentalization was lost. CONCLUSION: Our data show that TLOs are established during diabetes development and suggest that islet destruction is due to a loss of TLO integrity, which may be prevented by FTY720 treatment.

Is antigen specificity of autoreactive T cells the key to islet entry?

It has been widely hypothesized that pancreatic islet infiltrates include both islet-antigen-specific and nonspecific T cells. In this issue of Immunity, Lennon et al. (2009) demonstrate that islet-antigen specificity is required for accumulation in the islets.

Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo.

Regulatory T cells (T(reg) cells) are central to the maintenance of immune homeostasis. However, little is known about the stability of T(reg) cells in vivo. In this study, we demonstrate that a substantial percentage of cells had transient or unstable expression of the transcription factor Foxp3. These 'exFoxp3' T cells had an activated-memory T cell phenotype and produced inflammatory cytokines. Moreover, exFoxp3 cell numbers were higher in inflamed tissues in autoimmune conditions. Adoptive transfer of autoreactive exFoxp3 cells led to the rapid onset of diabetes. Finally, analysis of the T cell receptor repertoire suggested that exFoxp3 cells developed from both natural and adaptive T(reg) cells. Thus, the generation of potentially autoreactive effector T cells as a consequence of Foxp3 instability has important implications for understanding autoimmune disease pathogenesis.

Central role of defective interleukin-2 production in the triggering of islet autoimmune destruction.

The dynamics of CD4(+) effector T cells (Teff cells) and CD4(+)Foxp3(+) regulatory T cells (Treg cells) during diabetes progression in nonobese diabetic mice was investigated to determine whether an imbalance of Treg cells and Teff cells contributes to the development of type 1 diabetes. Our results demonstrated a progressive decrease in the Treg cell:Teff cell ratio in inflamed islets but not in pancreatic lymph nodes. Intra-islet Treg cells expressed reduced amounts of CD25 and Bcl-2, suggesting that their decline was due to increased apoptosis. Additionally, administration of low-dose interleukin-2 (IL-2) promoted Treg cell survival and protected mice from developing diabetes. Together, these results suggest intra-islet Treg cell dysfunction secondary to defective IL-2 production is a root cause of the progressive breakdown of self-tolerance and the development of diabetes in nonobese diabetic mice.

Inhibition of IGF-1R and lipoxygenase by nordihydroguaiaretic acid (NDGA) analogs.

Herein, we pursue the hypothesis that the structure of nordihydroguaiaretic acid (NDGA) can be refined for selective potency against the insulin-like growth factor 1 receptor (IGF-1R) as a potential therapeutic target for breast cancer while diminishing its action against other cellular targets. Thus, a set of NDGA analogs (7a-7h) was prepared and examined for inhibitory potency against IGF-1R kinase and an alternative target, 15-lipoxygenase (15 LOX). The anti-cancer effects of these compounds were determined by their ability to inhibit IGF-1 mediated cell growth of MCF-7 breast cancer cells. The design of the analogs was based upon a cursory Topliss approach in which one of NDGA's aromatic rings was modified with various substituents. Structural modification of one of the two catechol rings of NDGA was found to have little effect upon the inhibitory potency against both kinase activity of the IGF-1R and IGF-1 mediated cell growth of MCF-7 cells. 15-LOX was found to be most sensitive to structural modifications of NDGA. From the limited series of NDGA analogs examined, the compound that exhibited the greatest selectivity for IGF-1 mediated growth compared to 15-LOX inhibition was a cyclic analog 7h with a framework similar to a natural product isolated from Larrea divaricata. The results for 7h are significant because while NDGA displays biological promiscuity, 7h exhibits greater specificity toward the breast cancer target IGF-1R with that added benefit of possessing a 10-fold weaker potency against 15-LOX, an enzyme which has a purported tumor suppressing role in breast cancer. With increased specificity and potency, 7h may serve as a new lead in developing novel therapeutic agents for breast cancer.

Parallel synthesis of diarylureas and their evaluation as inhibitors of insulin-like growth factor receptor.

Diarylurea (DAU) compounds, particularly species composed of a heteroaryl ring system conjugated through a urea linkage to a substituted arene, were previously identified by the screening of a diverse chemical library to be active against the insulin growth factor receptor (IGF-1R). DAU compounds 4{a,b} were synthesized in parallel by the coupling of aryl amines 2{a} with aryl isocyanates 3{b}. Preparative RP-HPLC purification was found necessary to remove an impurity 5{b}, the symmetric urea resulting from the hydrolytic degradation of aryl isocyanates. Two libraries of DAU compounds were prepared to perform preliminary optimization of the two-ring systems for inhibitory activity against IGF-1R. In the first library, we explored a series of heteroaryl ring systems and found the 4-aminoquinaldine ring system to be optimal among those evaluated. The second library fixed the 4-aminoquinaldine ring system and we evaluated a series of substituted arenes conjugated to it. Overall, eight compounds based on the 4-aminoquinaldine heteroaryl system were found to have moderate activity against IGF-1R with IC(50) values better than 40 microM.

Diarylureas are small-molecule inhibitors of insulin-like growth factor I receptor signaling and breast cancer cell growth.

In breast and certain other cancers, receptor tyrosine kinases, including the insulin-like growth factor I receptor (IGF-IR), play an important role in promoting the oncogenic process. The IGF-IR is therefore an important target for developing new anti-breast cancer therapies. An initial screening of a chemical library against the IGF-IR in breast cancer cells identified a diaryl urea compound as a potent inhibitor of IGF-IR signaling. This class of compounds has not been studied as inhibitors of the IGF-IR. We studied the effectiveness of one diaryl urea compound, PQ401, at antagonizing IGF-IR signaling and inhibiting breast cancer cell growth in culture and in vivo. PQ401 inhibited autophosphorylation of the IGF-IR in cultured human MCF-7 cells with an IC50 of 12 micromol/L and autophosphorylation of the isolated kinase domain of the IGF-IR with an IC50 <1 micromol/L. In addition, PQ401 inhibited the growth of cultured breast cancer cells in serum at 10 micromol/L. PQ401 was even more effective at inhibiting IGF-I-stimulated growth of MCF-7 cells (IC50, 6 micromol/L). Treatment of MCF-7 cells with PQ401 was associated with a decrease in IGF-I-mediated signaling through the Akt antiapoptotic pathway. Twenty-four hours of treatment with 15 micromol/L PQ401 induced caspase-mediated apoptosis. In vivo, treatment with PQ401 (i.p. injection thrice a week) reduced the growth rate of MCNeuA cells implanted into mice. These studies indicate that diaryl urea compounds are potential new agents to test in the treatment of breast and other IGF-I-sensitive cancers.

Nordihydroguaiaretic acid (NDGA) inhibits the IGF-1 and c-erbB2/HER2/neu receptors and suppresses growth in breast cancer cells.

Nordihydroguaiaretic acid (NDGA) is a phenolic compound isolated from the creosote bush Larrea divaricatta that has anti-cancer activities both in vitro and in vivo. We can now attribute certain of these anti-cancer properties in breast cancer cells to the ability of NDGA to directly inhibit the function of two receptor tyrosine kinases (RTKs), the insulin-like growth factor receptor (IGF-1R) and the c-erbB2/HER2/neu (HER2/neu) receptor. In MCF-7 human breast cancer cells, low micromolar concentrations of NDGA inhibited activation of the IGF-1R, and downstream phosphorylation of both the Akt/PKB serine kinase and the pro-apoptotic protein BAD. In mouse MCNeuA cells, NDGA also inhibited ligand independent phosphorylation of HER2/neu. To study whether this inhibitory effect in cells was due to a direct action on these receptors, we studied the IGF-1-stimulated tyrosine kinase activity of isolated IGF-1R, which was inhibited by NDGA at 10 muM or less. NDGA was also effective at inhibiting autophosphorylation of the isolated HER2/neu receptor at similar concentrations. In addition, NDGA inhibited IGF-1 specific growth of cultured breast cancer cells with an IC50 of approximately 30 muM. NDGA treatment (intraperitoneal injection 3 times per week) also decreased the activity of the IGF-1R and the HER2/neu receptor in MCNeuA cells implanted into mice. This inhibition of RTK activity was associated with decreased growth rates of MCNeuA cells in vivo. These studies indicate that the anti-breast cancer properties of NDGA are related to the inhibition of two important RTKs. Agents of this class may therefore provide new insights into potential therapies for this disease.

T-bet is required for optimal production of IFN-gamma and antigen-specific T cell activation by dendritic cells.

IFN-gamma is well known as the signature cytokine of CD4+ T helper 1, CD8+, and natural killer cells, but recent studies demonstrate that antigen-presenting cells, in particular dendritic cells (DCs), are another potent source for this proinflammatory cytokine. T-bet, a transcription factor that controls IFN-gamma expression in CD4+ T cells, was reported recently to be expressed in human monocytes and myeloid DCs. In this study we investigate the role of T-bet in this important cell type. The development, differentiation, and activation of bone marrow and splenic DCs were unimpaired in mice lacking T-bet. However, T-bet was essential for the optimal production of IFN-gamma by both CD8alpha+ and CD8alpha- DCs. T-bet-deficient DCs were significantly impaired in their capacity to secrete IFN-gamma after both stimulation with IL-12 alone or in combination with IL-18. Further, T-bet-/- DCs were impaired in their ability to activate the T helper 1 program of adoptively transferred antigen-specific T cells in vivo. The rapid up-regulation of T-bet by IFN-gamma in DCs coupled with a function for DC-derived IFN-gamma in T cell activation may constitute a positive feedback loop to maximize type 1 immunity.