KSHV infection of endothelial precursor cells with lymphatic characteristics as a novel model for translational Kaposi's sarcoma studies.

Kaposi's sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a hyperplasia consisting of enlarged malformed vasculature and spindle-shaped cells, the main proliferative component of KS. While spindle cells express markers of lymphatic and blood endothelium, the origin of spindle cells is unknown. Endothelial precursor cells have been proposed as the source of spindle cells. We previously identified two types of circulating endothelial colony forming cells (ECFCs), ones that expressed markers of blood endothelium and ones that expressed markers of lymphatic endothelium. Here we examined both blood and lymphatic ECFCs infected with KSHV. Lymphatic ECFCs are significantly more susceptible to KSHV infection than the blood ECFCs and maintain the viral episomes during passage in culture while the blood ECFCs lose the viral episome. Only the KSHV-infected lymphatic ECFCs (K-ECFCLY) grew to small multicellular colonies in soft agar whereas the infected blood ECFCs and all uninfected ECFCs failed to proliferate. The K-ECFCLYs express high levels of SOX18, which supported the maintenance of high copy number of KSHV genomes. When implanted subcutaneously into NSG mice, the K-ECFCLYs persisted in vivo and recapitulated the phenotype of KS tumor cells with high number of viral genome copies and spindling morphology. These spindle cell hallmarks were significantly reduced when mice were treated with SOX18 inhibitor, SM4. These data suggest that KSHV-infected lymphatic ECFCs can be utilized as a KSHV infection model for in vivo translational studies to test novel inhibitors representing potential treatment modalities for KS.

Development of an adenosquamous carcinoma histopathology - selective lung metastasis model.

Preclinical tumor models with native tissue microenvironments provide essential tools to understand how heterogeneous tumor phenotypes relate to drug response. Here we present syngeneic graft models of aggressive, metastasis-prone histopathology-specific NSCLC tumor types driven by KRAS mutation and loss of LKB1 (KL): adenosquamous carcinoma (ASC) and adenocarcinoma (AC). We show that subcutaneous injection of primary KL; ASC cells results in squamous cell carcinoma (SCC) tumors with high levels of stromal infiltrates, lacking the source heterogeneous histotype. Despite forming subcutaneous tumors, intravenously injected KL;AC cells were unable to form lung tumors. In contrast, intravenous injection of KL;ASC cells leads to their lung re-colonization and lesions recapitulating the mixed AC and SCC histopathology, tumor immune suppressive microenvironment and oncogenic signaling profile of source tumors, demonstrating histopathology-selective phenotypic dominance over genetic drivers. Pan-ERBB inhibition increased survival, while selective ERBB1/EGFR inhibition did not, suggesting a role of the ERBB network crosstalk in resistance to ERBB1/EGFR. This immunocompetent NSCLC lung colonization model hence phenocopies key properties of the metastasis-prone ASC histopathology, and serves as a preclinical model to dissect therapy responses and metastasis-associated processes.

Receptor Tyrosine Kinase Signaling Networks Define Sensitivity to ERBB Inhibition and Stratify Kras-Mutant Lung Cancers.

Most non-small cell lung cancers (NSCLC) contain nontargetable mutations, including KRAS, TP53, or STK11/LKB1 alterations. By coupling ex vivo drug sensitivity profiling with in vivo drug response studies, we aimed to identify drug vulnerabilities for these NSCLC subtypes. Primary adenosquamous carcinoma (ASC) or adenocarcinoma (AC) cultures were established from KrasG12D/+;Lkb1fl/fl (KL) tumors or AC cultures from KrasG12D/+;p53fl/fl (KP) tumors. Although p53-null cells readily propagated as conventional cultures, Lkb1-null cells required conditional reprograming for establishment. Drug response profiling revealed short-term response to MEK inhibition, yet long-term clonogenic assays demonstrated resistance, associated with sustained or adaptive activation of receptor tyrosine kinases (RTK): activation of ERBBs in KL cultures, or FGFR in AC cultures. Furthermore, pan-ERBB inhibition reduced the clonogenicity of KL cultures, which was exacerbated by combinatorial MEK inhibition, whereas combinatorial MEK and FGFR inhibition suppressed clonogenicity of AC cultures. Importantly, in vivo studies confirmed KL-selective sensitivity to pan-ERBB inhibition, which correlated with high ERBB ligand expression and activation of ERBB receptors, implying that ERBB network activity may serve as a predictive biomarker of drug response. Interestingly, in human NSCLCs, phosphorylation of EGFR or ERBB3 was frequently detected in ASCs and squamous cell carcinomas. We conclude that analysis of in situ ERBB signaling networks in conjunction with ex vivo drug response profiling and biochemical dissection of adaptive RTK activities may serve as a valid diagnostic approach to identify tumors sensitive to ERBB network inhibition.

Targeting ß1-integrin inhibits vascular leakage in endotoxemia.

Loss of endothelial integrity promotes capillary leakage in numerous diseases, including sepsis, but there are no effective therapies for preserving endothelial barrier function. Angiopoietin-2 (ANGPT2) is a context-dependent regulator of vascular leakage that signals via both endothelial TEK receptor tyrosine kinase (TIE2) and integrins. Here, we show that antibodies against ß1-integrin decrease LPS-induced vascular leakage in murine endotoxemia, as either a preventative or an intervention therapy. ß1-integrin inhibiting antibodies bound to the vascular endothelium in vivo improved the integrity of endothelial cell-cell junctions and protected mice from endotoxemia-associated cardiac failure, without affecting endothelial inflammation, serum proinflammatory cytokine levels, or TIE receptor signaling. Moreover, conditional deletion of a single allele of endothelial ß1-integrin protected mice from LPS-induced vascular leakage. In endothelial monolayers, the inflammatory agents thrombin, lipopolysaccharide (LPS), and IL-1ß decreased junctional vascular endothelial (VE)-cadherin and induced actin stress fibers via ß1- and α5-integrins and ANGPT2. Additionally, ß1-integrin inhibiting antibodies prevented inflammation-induced endothelial cell contractility and monolayer permeability. Mechanistically, the inflammatory agents stimulated ANGPT2-dependent translocation of α5ß1-integrin into tensin-1-positive fibrillar adhesions, which destabilized the endothelial monolayer. Thus, ß1-integrin promotes endothelial barrier disruption during inflammation, and targeting ß1-integrin signaling could serve as a novel means of blocking pathological vascular leak.

Card9-dependent IL-1ß regulates IL-22 production from group 3 innate lymphoid cells and promotes colitis-associated cancer.

Inflammatory bowel diseases (IBD) are key risk factors for the development of colorectal cancer, but the mechanisms that link intestinal inflammation with carcinogenesis are insufficiently understood. Card9 is a myeloid cell-specific signaling protein that regulates inflammatory responses downstream of various pattern recognition receptors and which cooperates with the inflammasomes for IL-1ß production. Because polymorphisms in Card9 were recurrently associated with human IBD, we investigated the function of Card9 in a colitis-associated cancer (CAC) model. Card9-/- mice develop smaller, less proliferative and less dysplastic tumors compared to their littermates and in the regenerating mucosa we detected dramatically impaired IL-1ß generation and defective IL-1ß controlled IL-22 production from group 3 innate lymphoid cells. Consistent with the key role of immune-derived IL-22 in activating STAT3 signaling during normal and pathological intestinal epithelial cell (IEC) proliferation, Card9-/- mice also exhibit impaired tumor cell intrinsic STAT3 activation. Our results imply a Card9-controlled, ILC3-mediated mechanism regulating healthy and malignant IEC proliferation and demonstrates a role of Card9-mediated innate immunity in inflammation-associated carcinogenesis.

T-bet and Gata3 in controlling type 1 and type 2 immunity mediated by innate lymphoid cells.

Innate lymphoid cells (ILCs) are an emerging group of innate lymphocytes that share functional and transcriptional attributes with the various T helper cell effector fates (e.g. Th1, Th2, Th17). ILCs are substantially represented in the intestinal mucosa but are rare in secondary lymphoid organs. They play important roles in epithelial homeostasis, tissue repair and in immunity to intestinal infections. They are also involved in immune-mediated pathology. Here, we will review the emerging roles of the transcription factors T-bet and Gata3 in the development, lineage specification and function of distinct ILC lineages. We will also highlight the requirement of these transcriptional programs for the control of infections and the pathogenesis of inflammatory diseases.

A T-bet gradient controls the fate and function of CCR6-RORγt+ innate lymphoid cells.

At mucosal surfaces, the immune system should not initiate inflammatory immune responses to the plethora of antigens constantly present in the environment, but should remain poised to unleash a potent assault on intestinal pathogens. The transcriptional programs and regulatory factors required for immune cells to switch from homeostatic (often tissue-protective) function to potent antimicrobial immunity are poorly defined. Mucosal retinoic-acid-receptor-related orphan receptor-γt-positive (RORγt(+)) innate lymphoid cells (ILCs) are emerging as an important innate lymphocyte population required for immunity to intestinal infections. Various subsets of RORγt(+) ILCs have been described but the transcriptional programs controlling their specification and fate remain largely unknown. Here we provide evidence that the transcription factor T-bet determines the fate of a distinct lineage of CCR6(-)RORγt(+) ILCs. Postnatally emerging CCR6(-)RORγt(+) ILCs upregulated T-bet and this was controlled by cues from the commensal microbiota and interleukin-23 (IL-23). In contrast, CCR6(+)RORγt(+) ILCs, which arise earlier during ontogeny, did not express T-bet. T-bet instructed the expression of T-bet target genes such as interferon-γ (IFN-γ) and of the natural cytotoxicity receptor NKp46. Mice genetically lacking T-bet showed normal development of CCR6(-)RORγt(+) ILCs, but they could not differentiate into NKp46-expressing RORγt(+) ILCs (that is, IL-22-producing natural killer (NK-22) cells) and failed to produce IFN-γ. The production of IFN-γ by T-bet-expressing CCR6(-)RORγt(+) ILCs was essential for the release of mucus-forming glycoproteins required to protect the epithelial barrier during Salmonella enterica infection. Salmonella infection also causes severe enterocolitis that is at least partly driven by IFN-γ. Mice deficient for T-bet or depleted of ILCs developed only mild enterocolitis. Thus, graded expression of T-bet in CCR6(-)RORγt(+) ILCs facilitates the differentiation of IFN-γ-producing CCR6(-)RORγt(+) ILCs required to protect the epithelial barrier against Salmonella infections. Co-expression of T-bet and RORγt, which is also found in subsets of IL-17-producing T-helper (T(H)17) cells, may be an evolutionarily conserved transcriptional program that originally developed as part of the innate defence against infections but that also confers an increased risk of immune-mediated pathology.

Unlike αß T cells, γδ T cells, LTi cells and NKT cells do not require IRF4 for the production of IL-17A and IL-22.

Apart from conventional CD4(+) Th17 cells, the cytokines IL-17A and IL-22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T-cell receptor stimulation in the presence of TGF-ß, IL-6 and IL-23. In contrast, a subset of γδ T cells ("γδT17") is committed for fast IL-17 production already in the thymus; however, γδ T cells can also produce IL-17 after prolonged in vitro stimulation via their γδ T-cell receptor plus IL-23. Here, we show that γδ T-, LTi- and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL-17A and IL-22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL-17A-producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4(-/-)) mice during experimental autoimmune encephalomyelitis. IL-17A-producing WT and Irf4(-/-) γδ T cells equally express CCR6 and lack CD27. The underlying IRF4-independent pathway partially involves STAT3 during in vitro stimulation.

Aryl hydrocarbon receptor: a molecular link between postnatal lymphoid follicle formation and diet.

Intestinal homeostasis results from a complex mutualism between gut microbiota and host cells. Defining the molecular network regulating such mutualism is currently of increasing interest, as its deregulation is reported to lead to increased susceptibility to infections, chronic inflammatory bowel diseases and cancer. Until now, the focus has been on the mechanism, by which the composition of indigenous microbiota shapes the immune system. In a recent study, we have shown that dietary compounds have also the ability to affect innate immune system. This regulation involves aryl hydrocarbon receptor (AhR), a sensor of plant-derived phytochemicals, which mediates the maintenance of Retinoic acid related orphan receptor γ t-expressing innate lymphoid cells (RORγt(+) ILC) in the gut and consequently formation of postnatal lymphoid follicles. Thus, AhR represents the first evidence of a molecular link between diet and immunity at intestinal mucosal surfaces.

Role of the Aryl Hydrocarbon Receptor in Controlling Maintenance and Functional Programs of RORγt(+) Innate Lymphoid Cells and Intraepithelial Lymphocytes.

Mucosal retinoic receptor-related orphan receptor (ROR)γt-expressing innate lymphoid cells (ILC) play an important role in the defense against intestinal pathogens and in promoting epithelial homeostasis and adaptation, thereby effectively protecting the vertebrate host against intestinal inflammatory disorders. The functional activity of RORγt(+) ILC is under the control of environmental cues. However, the molecular sensors for such environmental signals are largely unknown. Recently, the aryl hydrocarbon receptor (AhR) has emerged as a master regulator for the postnatal maintenance of intestinal RORγt(+) ILC and intraepithelial lymphocytes. AhR is a highly conserved transcription factor whose activity is regulated by environmental and dietary small molecule ligands. Here, we review the role of AhR signaling for the maintenance of intestinal immune cells and its impact on the immunological protection against intestinal infections and debilitating chronic inflammatory disorders.

Transcriptional control of innate lymphocyte fate decisions.

It has recently emerged that innate lymphocytes are more diverse than previously appreciated. In addition to natural killer cells, various subsets of innate lymphoid cells are now being characterized. It has become apparent that the transcriptional programs underlying lineage specification and cell fate decisions of innate lymphocytes strikingly resemble those of T cell subsets, suggesting that such transcriptional circuitry was already pre-formed in the evolutionary older innate immune system. Here, we will review recent advances in our understanding of the core transcriptional programs driving development and cell fate decisions of innate lymphocytes. We will also discuss whether these transcriptional programs are stable or flexible, thereby allowing for plastic adaptation of immune responses.

Natural aryl hydrocarbon receptor ligands control organogenesis of intestinal lymphoid follicles.

Innate lymphoid cells (ILC) expressing the transcription factor RORγt induce the postnatal formation of intestinal lymphoid follicles and regulate intestinal homeostasis. RORγt(+) ILC express the aryl hydrocarbon receptor (AhR), a highly conserved, ligand-inducible transcription factor believed to control adaptation of multicellular organisms to environmental challenges. We show that AhR is required for the postnatal expansion of intestinal RORγt(+) ILC and the formation of intestinal lymphoid follicles. AhR activity within RORγt(+) ILC could be induced by dietary ligands such as those contained in vegetables of the family Brassicaceae. AhR-deficient mice were highly susceptible to infection with Citrobacter rodentium, a mouse model for attaching and effacing infections. Our results establish a molecular link between nutrients and the formation of immune system components required to maintain intestinal homeostasis and resistance to infections.

Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer.

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the 68Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Regulated expression of nuclear receptor RORγt confers distinct functional fates to NK cell receptor-expressing RORγt(+) innate lymphocytes.

Whether the recently identified innate lymphocyte population coexpressing natural killer cell receptors (NKRs) and the nuclear receptor RORγt is part of the NK or lymphoid tissue inducer (LTi) cell lineage remains unclear. By using adoptive transfer of genetically tagged LTi-like cells, we demonstrate that NKR⁻RORγt(+) innate lymphocytes but not NK cells were direct progenitors to NKR(+)RORγt(+) cells in vivo. Genetic lineage tracing revealed that the differentiation of LTi-like cells was characterized by the stable upregulation of NKRs and a progressive loss of RORγt expression. Whereas interleukin-7 (IL-7) and intestinal microbiota stabilized RORγt expression within such NKR-LTi cells, IL-12 and IL-15 accelerated RORγt loss. RORγt(+) NKR-LTi cells produced IL-22, whereas RORγt⁻ NKR-LTi cells released IFN-γ and were potent inducers of colitis. Thus, the RORγt gradient in NKR-LTi cells serves as a tunable rheostat for their functional program. Our data also define a previously unappreciated role of RORγt⁻ NKR-LTi cells for the onset or maintenance of inflammatory bowel diseases.