Interactions of the anti-FcRn monoclonal antibody, rozanolixizumab, with Fcγ receptors and functional impact on immune cells in vitro.

Rozanolixizumab is a humanized anti-neonatal Fc receptor (FcRn) monoclonal antibody (mAb) of the immunoglobulin G4 (IgG4) sub-class, currently in clinical development for the treatment of IgG autoantibody-driven diseases. This format is frequently used for therapeutic mAbs due to its intrinsic lower affinity for Fc gamma receptors (FcγR) and lack of C1q engagement. However, with growing evidence suggesting that no Fc-containing agent is truly "silent" in this respect, we explored the engagement of FcγRs and potential functional consequences with rozanolixizumab. In the study presented here, rozanolixizumab was shown to bind to FcγRs in both protein-protein and cell-based assays, and the kinetic data were broadly as expected based on published data for an IgG4 mAb. Rozanolixizumab was also able to mediate antibody bipolar bridging (ABB), a phenomenon that led to a reduction of labeled FcγRI from the surface of human macrophages in an FcRn-dependent manner. However, the presence of exogenous human IgG, even at low concentrations, was able to prevent both binding and ABB events. Furthermore, data from in vitro experiments using relevant human cell types that express both FcRn and FcγRI indicated no evidence for functional sequelae in relation to cellular activation events (e.g., intracellular signaling, cytokine production) upon either FcRn or FcγR binding of rozanolixizumab. These data raise important questions about whether therapeutic antagonistic mAbs like rozanolixizumab would necessarily engage FcγRs at doses typically administered to patients in the clinic, and hence challenge the relevance and interpretation of in vitro assays performed in the absence of competing IgG.

Differential regulation of IL-17A and IL-17F via STAT5 contributes to psoriatic disease.

BACKGROUND: IL-17A plays a pivotal pathogenic role in several immune-mediated inflammatory diseases. Despite sharing 50% sequence homology with IL-17A, the role of IL-17F remains less clear. Clinical findings suggest that dual inhibition of IL-17A and IL-17F in psoriatic disease is more efficacious than IL-17A inhibition alone, positing a pathogenic role for IL-17F. OBJECTIVE: We characterized the regulation of IL-17A and IL-17F in psoriatic disease. METHODS: Using both in vitro systems and lesional skin tissue from patients, we interrogated the chromosomal, transcriptional, and protein expression landscape of IL-17A+ and IL-17F+ TH17 cells. Alongside established assays such as single-cell RNA sequencing, we developed a novel cytokine-capture technique that was combined with chromatin immunoprecipitation sequencing and RNA sequencing. RESULTS: We confirm a preferential elevation of IL-17F over IL-17A in psoriatic disease and show that expression of each isoform predominantly occurs in distinct cell populations. The expression of both IL-17A and IL-17F exhibited a high degree of plasticity, with the balance between the 2 isoforms influenced by proinflammatory signaling and by anti-inflammatory drugs such as methylprednisolone. This plasticity was reflected in a broad H3K4me3 region at the IL17A-F locus, while opposing effects of STAT5/IL-2 signaling were observed for each of the 2 genes. Functionally, higher IL17F expression was linked to greater cell proliferation. CONCLUSION: There are key differences in the regulation of IL-17A and IL-17F in psoriatic disease, leading to distinct inflammatory cell populations. As such, we propose that both IL-17A and IL-17F neutralization may be required to maximally inhibit IL-17-driven pathology.

TRYBE®: an Fc-free antibody format with three monovalent targeting arms engineered for long in vivo half-life.

TrYbe® is an Fc-free therapeutic antibody format, capable of engaging up to three targets simultaneously, with long in vivo half-life conferred by albumin binding. This format is shown by small-angle X-ray scattering to be conformationally flexible with favorable 'reach' properties. We demonstrate the format's broad functionality by co-targeting of soluble and cell surface antigens. The benefit of monovalent target binding is illustrated by the lack of formation of large immune complexes when co-targeting multivalent antigens. TrYbes® are manufactured using standard mammalian cell culture and protein A affinity capture processes. TrYbes® have been formulated at high concentrations and have favorable drug-like properties, including stability, solubility, and low viscosity. The unique functionality and inherent developability of the TrYbe® makes it a promising multi-specific antibody fragment format for antibody therapy.

Interleukin (IL)-12 and IL-18 Synergize to Promote MAIT Cell IL-17A and IL-17F Production Independently of IL-23 Signaling.

IL-23 is considered a critical regulator of IL-17 in Th17 cells; however, its requirement for inducing IL-17 production in other human immune subsets remains incompletely understood. Mucosal associated invariant T (MAIT) cells uniformly express retinoic acid receptor-related orphan receptor gamma t (RORγt) but only a minor population have been shown to produce IL-17A. Here we show that IL-17F is the dominant IL-17 isoform produced by MAIT cells, not IL-17A. For optimal MAIT cell derived IL-17A and IL-17F production, T cell receptor (TCR) triggering, IL-18 and monocyte derived IL-12 signaling is required. Unlike Th17 cells, this process is independent of IL-23 signaling. Using an in vitro skin cell activation assay, we demonstrate that dual neutralization of both IL-17A and IL-17F resulted in greater suppression of inflammatory proteins than inhibition of IL-17A alone. Finally, we extend our findings by showing that other innate-like lymphocytes such as group 3 innate lymphoid cells (ILC3) and gamma delta (γδ) T cells are also capable of IL-23 independent IL-17A and IL-17F production. These data indicate both IL-17F and IL-17A production from MAIT cells may contribute to tissue inflammation independently of IL-23, in part explaining the therapeutic disconnect between targeting IL-17 or IL-23 in certain inflammatory diseases.

Bimekizumab, a Novel Humanized IgG1 Antibody That Neutralizes Both IL-17A and IL-17F.

Interleukin (IL)-17A is a key driver of inflammation and the principal target of anti-IL-17 therapeutic monoclonal antibodies. IL-17A, and its structurally similar family member IL-17F, have been shown to be functionally dysregulated in certain human immune-mediated inflammatory diseases such as psoriasis, psoriatic arthritis, and axial spondyloarthritis. Given the overlapping biology of these two cytokines, we postulated that dual neutralization of IL-17A and IL-17F may provide a greater depth of clinical response in IL-17-mediated diseases than IL-17A inhibition alone. We identified 496.g1, a humanized antibody with strong affinity for IL-17A but poor affinity for IL-17F. Affinity maturation of 496.g1 to 496.g3 greatly enhanced the affinity of the Fab fragment for IL-17F while retaining strong binding to IL-17A. As an IgG1, the affinity for IL-17A and IL-17F was 3.2 pM and 23 pM, respectively. Comparison of 496.g3 IgG1 with the commercially available anti-IL-17A monoclonal antibodies ixekizumab and secukinumab, by surface plasmon resonance and in a human in vitro IL-17A functional assay, showed that 496.g3 and ixekizumab display equivalent affinity for IL-17A, and that both antibodies are markedly more potent than secukinumab. In contrast to ixekizumab and secukinumab, 496.g3 exhibited the unique feature of also being able to neutralize the biological activity of IL-17F. Therefore, antibody 496.g3 was selected for clinical development for its ability to neutralize the biologic function of both IL-17A and IL-17F and was renamed bimekizumab (formerly UCB4940). Early clinical data in patients with psoriasis, in those with psoriatic arthritis, and from the Phase 2 studies in psoriasis, psoriatic arthritis, and ankylosing spondylitis, are encouraging and support the targeted approach of dual neutralization of IL-17A and IL-17F. Taken together, these findings provide the rationale for the continued clinical evaluation of bimekizumab in patients with immune-mediated inflammatory diseases.

Dual neutralisation of IL-17F and IL-17A with bimekizumab blocks inflammation-driven osteogenic differentiation of human periosteal cells.

OBJECTIVES: Interleukin (IL)-17 signalling has been shown to be a key regulator of disease in ankylosing spondylitis (AS) with several IL-17 blockers currently clinically approved. Despite this, the role of IL-17 in bone pathology is poorly understood. This study aimed to investigate IL-17 signalling in the context of pathological bone formation. METHODS: A biomimetic human periosteum-derived cell (hPDC) model of osteogenic differentiation was used in combination with recombinant IL-17 cytokines, T-cell supernatants or serum from patients with AS. IL-17A, IL-17F and bimekizumab monoclonal antibodies were used to block IL-17 cytokine action. RESULTS: Recombinant IL-17A and IL-17F are pro-osteogenic with respect to hPDC differentiation. T helper 17 or γδ-T cell supernatants also potently stimulated in vitro bone formation, which was blocked deeper by dual inhibition of IL-17A and IL-17F than by neutralisation of IL-17A or IL-17F individually. Osteogenic blockade may be due to an increase in expression of the Wnt antagonist DKK1. Interestingly, osteocommitment was also induced by serum obtained from patients with AS, which was also abrogated by dual neutralisation of IL-17A and IL-17F. CONCLUSIONS: These data show for the first time that IL-17A and IL-17F enhance in vitro osteogenic differentiation and bone formation from hPDCs, inhibition of which may offer an attractive therapeutic strategy to prevent pathological bone formation.

Spatiotemporal Modeling of the Key Migratory Events During the Initiation of Adaptive Immunity.

Initiation of adaptive immunity involves distinct migratory cell populations coming together in a highly dynamic and spatially organized process. However, we lack a detailed spatiotemporal map of these events due to our inability to track the fate of cells between anatomically distinct locations or functionally identify cell populations as migratory. We used photo-convertible transgenic mice (Kaede) to spatiotemporally track the fate and composition of the cell populations that leave the site of priming and enter the draining lymph node to initiate immunity. We show that following skin priming, the lymph node migratory population is principally composed of cells recruited to the site of priming, with a minor contribution from tissue resident cells. In combination with the YAe/Eα system, we also show that the majority of cells presenting antigen are CD103+CD11b+ dendritic cells that were recruited to the site of priming during the inflammatory response. This population has previously only been described in relation to mucosal tissues. Comprehensive phenotypic profiling of the cells migrating from the skin to the draining lymph node by mass cytometry revealed that in addition to dendritic cells, the migratory population also included CD4+ and CD8+ T cells, B cells, and neutrophils. Taking our complex spatiotemporal data set, we then generated a model of cell migration that quantifies and describes the dynamics of arrival, departure, and residence times of cells at the site of priming and in the draining lymph node throughout the time-course of the initiation of adaptive immunity. In addition, we have identified the mean migration time of migratory dendritic cells as they travel from the site of priming to the draining lymph node. These findings represent an unprecedented, detailed and quantitative map of cell dynamics and phenotypes during immunization, identifying where, when and which cells to target for immunomodulation in autoimmunity and vaccination strategies.

CD4+ Recent Thymic Emigrants Are Recruited into Granulomas during Leishmania donovani Infection but Have Limited Capacity for Cytokine Production.

Recent thymic emigrants (RTEs) represent a source of antigen-naïve T cells that enter the periphery throughout life. However, whether RTEs contribute to the control of chronic parasitic infection and how their potential might be harnessed by therapeutic intervention is currently unclear. Here, we show that CD4+ recent thymic emigrants emerging into the periphery of mice with ongoing Leishmania donovani infection undergo partial activation and are recruited to sites of granulomatous inflammation. However, CD4+ RTEs displayed severely restricted differentiation either into IFNγ+ or IFNγ+TNFα+ effectors, or into IL-10-producing regulatory T cells. Effector cell differentiation in the chronically infected host was not promoted by adoptive transfer of activated dendritic cells or by allowing extended periods of post-thymic differentiation in the periphery. Nevertheless, CD4+ RTEs from infected mice retained the capacity to transfer protection into lymphopenic RAG2-/- mice. Taken together, our data indicate that RTEs emerging into a chronically inflamed environment are not recruited into the effector pool, but retain the capacity for subsequent differentiation into host protective T cells when placed in a disease-free environment.

Intranasal vaccination promotes detrimental Th17-mediated immunity against influenza infection.

Influenza disease is a global health issue that causes significant morbidity and mortality through seasonal epidemics. Currently, inactivated influenza virus vaccines given intramuscularly or live attenuated influenza virus vaccines administered intranasally are the only approved options for vaccination against influenza virus in humans. We evaluated the efficacy of a synthetic toll-like receptor 4 agonist CRX-601 as an adjuvant for enhancing vaccine-induced protection against influenza infection. Intranasal administration of CRX-601 adjuvant combined with detergent split-influenza antigen (A/Uruguay/716/2007 (H3N2)) generated strong local and systemic immunity against co-administered influenza antigens while exhibiting high efficacy against two heterotypic influenza challenges. Intranasal vaccination with CRX-601 adjuvanted vaccines promoted antigen-specific IgG and IgA antibody responses and the generation of polyfunctional antigen-specific Th17 cells (CD4(+)IL-17A(+)TNFα(+)). Following challenge with influenza virus, vaccinated mice transiently exhibited increased weight loss and morbidity during early stages of disease but eventually controlled infection. This disease exacerbation following influenza infection in vaccinated mice was dependent on both the route of vaccination and the addition of the adjuvant. Neutralization of IL-17A confirmed a detrimental role for this cytokine during influenza infection. The expansion of vaccine-primed Th17 cells during influenza infection was also accompanied by an augmented lung neutrophilic response, which was partially responsible for mediating the increased morbidity. This discovery is of significance in the field of vaccinology, as it highlights the importance of both route of vaccination and adjuvant selection in vaccine development.

A transcriptomic network identified in uninfected macrophages responding to inflammation controls intracellular pathogen survival.

Intracellular pathogens modulate host cell function to promote their survival. However, in vitro infection studies do not account for the impact of host-derived inflammatory signals. Examining the response of liver-resident macrophages (Kupffer cells) in mice infected with the parasite Leishmania donovani, we identified a transcriptomic network operating in uninfected Kupffer cells exposed to inflammation but absent from Kupffer cells from the same animal that contained intracellular Leishmania. To test the hypothesis that regulated expression of genes within this transcriptomic network might impact parasite survival, we pharmacologically perturbed the activity of retinoid X receptor alpha (RXRα), a key hub within this network, and showed that this intervention enhanced the innate resistance of Kupffer cells to Leishmania infection. Our results illustrate a broadly applicable strategy for understanding the host response to infection in vivo and identify Rxra as the hub of a gene network controlling antileishmanial resistance.

IL-10-producing Th1 cells and disease progression are regulated by distinct CD11c⁺ cell populations during visceral leishmaniasis.

IL-10 is a critical regulatory cytokine involved in the pathogenesis of visceral leishmaniasis caused by Leishmania donovani and clinical and experimental data indicate that disease progression is associated with expanded numbers of CD4⁺ IFNγ⁺ T cells committed to IL-10 production. Here, combining conditional cell-specific depletion with adoptive transfer, we demonstrate that only conventional CD11c(hi) DCs that produce both IL-10 and IL-27 are capable of inducing IL-10-producing Th1 cells in vivo. In contrast, CD11c(hi) as well as CD11c(int/lo) cells isolated from infected mice were capable of reversing the host protective effect of diphtheria toxin-mediated CD11c⁺ cell depletion. This was reflected by increased splenomegaly, inhibition of NO production and increased parasite burden. Thus during chronic infection, multiple CD11c⁺ cell populations can actively suppress host resistance and enhance immunopathology, through mechanisms that do not necessarily involve IL-10-producing Th1 cells.

B cell: T cell interactions occur within hepatic granulomas during experimental visceral leishmaniasis.

Hepatic resistance to Leishmania donovani infection in mice is associated with the development of granulomas, in which a variety of lymphoid and non-lymphoid populations accumulate. Although previous studies have identified B cells in hepatic granulomas and functional studies in B cell-deficient mice have suggested a role for B cells in the control of experimental visceral leishmaniasis, little is known about the behaviour of B cells in the granuloma microenvironment. Here, we first compared the hepatic B cell population in infected mice, where ≈60% of B cells are located within granulomas, with that of naïve mice. In infected mice, there was a small increase in mIgM(lo)mIgD(+) mature B2 cells, but no enrichment of B cells with regulatory phenotype or function compared to the naïve hepatic B cell population, as assessed by CD1d and CD5 expression and by IL-10 production. Using 2-photon microscopy to quantify the entire intra-granuloma B cell population, in conjunction with the adoptive transfer of polyclonal and HEL-specific BCR-transgenic B cells isolated from L. donovani-infected mice, we demonstrated that B cells accumulate in granulomas over time in an antigen-independent manner. Intra-vital dynamic imaging was used to demonstrate that within the polyclonal B cell population obtained from L. donovani-infected mice, the frequency of B cells that made multiple long contacts with endogenous T cells was greater than that observed using HEL-specific B cells obtained from the same inflammatory environment. These data indicate, therefore, that a subset of this polyclonal B cell population is capable of making cognate interactions with T cells within this unique environment, and provide the first insights into the dynamics of B cells within an inflammatory site.

Therapeutic vaccination with recombinant adenovirus reduces splenic parasite burden in experimental visceral leishmaniasis.

Therapeutic vaccines, when used alone or in combination therapy with antileishmanial drugs, may have an important place in the control of a variety of forms of human leishmaniasis. Here, we describe the development of an adenovirus-based vaccine (Ad5-KH) comprising a synthetic haspb gene linked to a kmp11 gene via a viral 2A sequence. In nonvaccinated Leishmania donovani-infected BALB/c mice, HASPB- and KMP11-specific CD8(+) T cell responses were undetectable, although IgG1 and IgG2a antibodies were evident. After therapeutic vaccination, antibody responses were boosted, and IFNγ(+)CD8(+) T cell responses, particularly to HASPB, became apparent. A single vaccination with Ad5-KH inhibited splenic parasite growth by ∼66%, a level of efficacy comparable to that observed in early stage testing of clinically approved antileishmanial drugs in this model. These studies indicate the usefulness of adenoviral vectors to deliver leishmanial antigens in a potent and host protective manner to animals with existing L. donovani infection.

Compartment-specific remodeling of splenic micro-architecture during experimental visceral leishmaniasis.

Progressive splenomegaly is a hallmark of visceral leishmaniasis in humans, canids, and rodents. In experimental murine visceral leishmaniasis, splenomegaly is accompanied by pronounced changes in microarchitecture, including expansion of the red pulp vascular system, neovascularization of the white pulp, and remodeling of the stromal cell populations that define the B-cell and T-cell compartments. Here, we show that Ly6C/G(+) (Gr-1(+)) cells, including neutrophils and inflammatory monocytes, accumulate in the splenic red pulp during infection. Cell depletion using monoclonal antibody against either Ly6C/G(+) (Gr-1; RB6) or Ly6G(+) (1A8) cells increased parasite burden. In contrast, depletion of Ly6C/G(+) cells, but not Ly6G(+) cells, halted the progressive remodeling of Meca-32(+) and CD31(+) red pulp vasculature. Strikingly, neither treatment affected white pulp neovascularization or the remodeling of the fibroblastic reticular cell and follicular dendritic cell networks. These findings demonstrate a previously unrecognized compartment-dependent selectivity to the process of splenic vascular remodeling during experimental murine visceral leishmaniasis, attributable to Ly6C(+) inflammatory monocytes.

Innate killing of Leishmania donovani by macrophages of the splenic marginal zone requires IRF-7.

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells.

Dynamic imaging of experimental Leishmania donovani-induced hepatic granulomas detects Kupffer cell-restricted antigen presentation to antigen-specific CD8 T cells.

Kupffer cells (KCs) represent the major phagocytic population within the liver and provide an intracellular niche for the survival of a number of important human pathogens. Although KCs have been extensively studied in vitro, little is known of their in vivo response to infection and their capacity to directly interact with antigen-specific CD8(+) T cells. Here, using a combination of approaches including whole mount and thin section confocal microscopy, adoptive cell transfer and intra-vital 2-photon microscopy, we demonstrate that KCs represent the only detectable population of mononuclear phagocytes within granulomas induced by Leishmania donovani infection that are capable of presenting parasite-derived peptide to effector CD8(+) T cells. This restriction of antigen presentation to KCs within the Leishmania granuloma has important implications for the identification of new candidate vaccine antigens and for the design of novel immuno-therapeutic interventions.

Inhibition of receptor tyrosine kinases restores immunocompetence and improves immune-dependent chemotherapy against experimental leishmaniasis in mice.

Receptor tyrosine kinases are involved in multiple cellular processes, and drugs that inhibit their action are used in the clinic to treat several types of cancer. However, the value of receptor tyrosine kinase inhibitors (RTKIs) for treating infectious disease has yet to be explored. Here, we have shown in mice that administration of the broad-spectrum RTKI sunitinib maleate (Sm) blocked the vascular remodeling and progressive splenomegaly associated with experimental visceral leishmaniasis. Furthermore, Sm treatment restored the integrity of the splenic microarchitecture. Although restoration of splenic architecture was accompanied by an increase in the frequency of IFN-gamma+CD4+ T cells, Sm treatment alone was insufficient to cause a reduction in tissue parasite burden. However, preconditioning by short-term Sm treatment proved to be successful as an adjunct therapy, increasing the frequency of IFN-gamma+ and IFN-gamma+TNF+CD4+ T cells, enhancing NO production by splenic macrophages, and providing dose-sparing effects when combined with a first-line immune-dependent anti-leishmanial drug. We propose, therefore, that RTKIs may prove clinically useful as agents to restore immune competence before the administration of chemo- or immunotherapeutic drugs in the treatment of visceral leishmaniasis or other diseases involving lymphoid tissue remodeling, including cancer.

Leishmania donovani-induced expression of signal regulatory protein alpha on Kupffer cells enhances hepatic invariant NKT-cell activation.

Signal regulatory protein alpha (SIRPalpha) and its cognate ligand CD47 have been documented to have a broad range of cellular functions in development and immunity. Here, we investigated the role of SIRPalpha-CD47 signalling in invariant NKT (iNKT) cell responses. We found that CD47 was required for the optimal production of IFN-gamma from splenic iNKT cells following exposure to the alphaGalCer analogue PBS-57 and in vivo infection of mice with Leishmania donovani. Surprisingly, although SIRPalpha was undetectable in the liver of uninfected mice, the hepatic iNKT-cell response to infection was also impaired in CD47-/- mice. However, we found that SIRPalpha was rapidly induced on Kupffer cells following L. donovani infection, via a mechanism involving G-protein-coupled receptors. Thus, we describe a novel amplification pathway affecting cytokine production by hepatic iNKT cells, which may facilitate the breakdown of hepatic tolerance after infection.

N-myristoyltransferase from Leishmania donovani: structural and functional characterisation of a potential drug target for visceral leishmaniasis.

N-Myristoyltransferase (NMT) catalyses the attachment of the 14-carbon saturated fatty acid, myristate, to the amino-terminal glycine residue of a subset of eukaryotic proteins that function in multiple cellular processes, including vesicular protein trafficking and signal transduction. In these pathways, N-myristoylation facilitates association of substrate proteins with membranes or the hydrophobic domains of other partner peptides. NMT function is essential for viability in all cell types tested to date, demonstrating that this enzyme has potential as a target for drug development. Here, we provide genetic evidence that NMT is likely to be essential for viability in insect stages of the pathogenic protozoan parasite, Leishmania donovani, causative agent of the tropical infectious disease, visceral leishmaniasis. The open reading frame of L. donovani NMT has been amplified and used to overproduce active recombinant enzyme in Escherichia coli, as demonstrated by gel mobility shift assays of ligand binding and peptide-myristoylation activity in scintillation proximity assays. The purified protein has been crystallized in complex with the non-hydrolysable substrate analogue S-(2-oxo)pentadecyl-CoA, and its structure was solved by molecular replacement at 1.4 A resolution. The structure has as its defining feature a 14-stranded twisted beta-sheet on which helices are packed so as to form an extended and curved substrate-binding groove running across two protein lobes. The fatty acyl-CoA is largely buried in the N-terminal lobe, its binding leading to the loosening of a flap, which in unliganded NMT structures, occludes the protein substrate binding site in the carboxy-terminal lobe. These studies validate L. donovani NMT as a potential target for development of new therapeutic agents against visceral leishmaniasis.