Lipids regulate peripheral serotonin release via gut CD1d.

The crosstalk between the immune and neuroendocrine systems is critical for intestinal homeostasis and gut-brain communications. However, it remains unclear how immune cells participate in gut sensation of hormones and neurotransmitters release in response to environmental cues, such as self-lipids and microbial lipids. We show here that lipid-mediated engagement of invariant natural killer T (iNKT) cells with enterochromaffin (EC) cells, a subset of intestinal epithelial cells, promoted peripheral serotonin (5-HT) release via a CD1d-dependent manner, regulating gut motility and hemostasis. We also demonstrated that inhibitory sphingolipids from symbiotic microbe Bacteroides fragilis represses 5-HT release. Mechanistically, CD1d ligation on EC cells transduced a signal and restrained potassium conductance through activation of protein tyrosine kinase Pyk2, leading to calcium influx and 5-HT secretion. Together, our data reveal that by engaging with iNKT cells, gut chemosensory cells selectively perceive lipid antigens via CD1d to control 5-HT release, modulating intestinal and systemic homeostasis.

Aging unconventionally: γδ T cells, iNKT cells, and MAIT cells in aging.

Unconventional T cells include γδ T cells, invariant Natural Killer T cells (iNKT) cells and Mucosal Associated Invariant T (MAIT) cells, which are distinguished from conventional T cells by their recognition of non-peptide ligands presented by non-polymorphic antigen presenting molecules and rapid effector functions that are pre-programmed during their development. Here we review current knowledge of the effect of age on unconventional T cells, from early life to old age, in both mice and humans. We then discuss the role of unconventional T cells in age-associated diseases and infections, highlighting the similarities between members of the unconventional T cell family in the context of aging.

Calcium signals regulate the functional differentiation of thymic iNKT cells.

How natural or innate-like lymphocytes generate the capacity to produce IL-4 and other cytokines characteristic of type 2 immunity remains unknown. Invariant natural killer T (iNKT) cells differentiate in the thymus into NKT1, NKT2, and NKT17 subsets, similar to mature, peripheral CD4+ T helper cells. The mechanism for this differentiation was not fully understood. Here, we show that NKT2 cells required higher and prolonged calcium (Ca2+ ) signals and continuing activity of the calcium release-activated calcium (CRAC) channel, than their NKT1 counterparts. The sustained Ca2+ entry via CRAC pathway in NKT2 cells was apparently mediated by ORAI and controlled in part by the large mitochondrial Ca2+ uptake. Unique properties of mitochondria in NKT2 cells, including high activity of oxidative phosphorylation, may regulate mitochondrial Ca2+ buffering in NKT2 cells. In addition, the low Ca2+ extrusion rate may also contribute to the higher Ca2+ level in NKT2 cells. Altogether, we identified ORAI-dependent Ca2+ signaling connected with mitochondria and cellular metabolism, as a central regulatory pathway for the differentiation of NKT2 cells.

Deconstructing iNKT cell development at single-cell resolution.

Invariant natural killer T (iNKT) cells are increasingly regarded as disease biomarkers and immunotherapeutic targets. However, a greater understanding of their biology is necessary to effectively target these cells in the clinic. The discovery of iNKT1/2/17 cell effector subsets was a milestone in our understanding of iNKT cell development and function. Recent transcriptomic studies have uncovered an even greater heterogeneity and challenge our understanding of iNKT cell ontogeny and effector differentiation. We propose a refined model whereby iNKT cells differentiate through a dynamic and continuous instructive process that requires the accumulation and integration of various signals within the thymus or peripheral tissues. Within this framework, we question the existence of true iNKT2 cells and discuss the parallels between mouse and human iNKT cells.

Human CD4+ iNKT cell adoptive immunotherapy induces anti-tumour responses against CD1d-negative EBV-driven B lymphoma.

Invariant natural killer T (iNKT) cells are a conserved population of innate T lymphocytes that are uniquely suitable as off-the-shelf cellular immunotherapies due to their lack of alloreactivity. Two major subpopulations of human iNKT cells have been delineated, a CD4- subset that has a TH1/cytolytic profile, and a CD4+ subset that appears polyfunctional and can produce both regulatory and immunostimulatory cytokines. Whether these two subsets differ in anti-tumour effects is not known. Using live cell imaging, we found that CD4- iNKT cells limited growth of CD1d+ Epstein-Barr virus (EBV)-infected B-lymphoblastoid spheroids in vitro, whereas CD4+ iNKT cells showed little or no direct anti-tumour activity. However, the effects of the two subsets were reversed when we tested them as adoptive immunotherapies in vivo using a xenograft model of EBV-driven human B cell lymphoma. We found that EBV-infected B cells down-regulated CD1d in vivo, and administering CD4- iNKT cells had no discernable impact on tumour mass. In contrast, xenotransplanted mice bearing lymphomas showed rapid reduction in tumour mass after administering CD4+ iNKT cells. Immunotherapeutic CD4+ iNKT cells trafficked to both spleen and tumour and were associated with subsequently enhanced responses of xenotransplanted human T cells against EBV. CD4+ iNKT cells also had adjuvant-like effects on monocyte-derived DCs and promoted antigen-dependent responses of human T cells in vitro. These results show that allogeneic CD4+ iNKT cellular immunotherapy leads to marked anti-tumour activity through indirect pathways that do not require tumour cell CD1d expression and that are associated with enhanced activity of antigen-specific T cells.

Beyond CAR T cells: exploring alternative cell sources for CAR-like cellular therapies.

Chimeric antigen receptor (CAR)-T cell therapy has led to remarkable clinical outcomes in the treatment of hematological malignancies. However, challenges remain, such as limited infiltration into solid tumors, inadequate persistence, systemic toxicities, and manufacturing insufficiencies. The use of alternative cell sources for CAR-based therapies, such as natural killer cells (NK), macrophages (MΦ), invariant Natural Killer T (iNKT) cells, γδT cells, neutrophils, and induced pluripotent stem cells (iPSC), has emerged as a promising avenue. By harnessing these cells' inherent cytotoxic mechanisms and incorporating CAR technology, common CAR-T cell-related limitations can be effectively mitigated. We herein present an overview of the tumoricidal mechanisms, CAR designs, and manufacturing processes of CAR-NK cells, CAR-MΦ, CAR-iNKT cells, CAR-γδT cells, CAR-neutrophils, and iPSC-derived CAR-cells, outlining the advantages, limitations, and potential solutions of these therapeutic strategies.

Severity of critical illness correlates with CD3-low expression on iNKT-cells among septic surgical patients.

BACKGROUND: iNKT-cells are innate regulatory lymphocytes capable of directing immune and inflammatory responses to sepsis. Repeat stimulation of iNKT-cells leads to the induction of anergy with the emergence of a hyporesponsive CD3low iNKT-cell subpopulation. METHODS: iNKT-cells were isolated from critical ill surgical patients with sepsis and phenotyped for CD3 expression. This was correlated with degree of severity of illness, as denoted by APACHE-II score. RESULTS: Comparing healthy volunteers to critically ill septic patients, it was noted that increasing severity of sepsis was associated with increasing frequency of circulating CD3low-iNKT-cell populations. CONCLUSION: The emergence of CD3low -iNKT-cells may serve as a clinically translatable marker of degree of sepsis-induced immune dysfunction.

Alpha-galactosylceramide pre-treatment attenuates clinical symptoms of LPS-induced acute neuroinflammation by converting pathogenic iNKT cells to anti-inflammatory iNKT10 cells in the brain.

BACKGROUND: Invariant natural killer T (iNKT) cells play protective or pathogenic roles in a variety of immune and inflammatory diseases. However, whether iNKT cells contribute to the progression of acute neuroinflammation remains unclear. Thus, we addressed this question with a mouse model of lipopolysaccharide (LPS)-induced acute neuroinflammation. METHODS: For induction of acute neuroinflammation, wild-type (WT) C57BL/6 (B6) mice were injected intraperitoneally (i.p.) with LPS for either three or five consecutive days, and then these mice were analyzed for brain-infiltrating leukocytes or mouse behaviors, respectively. To examine the role of iNKT cell activation in LPS-induced neuroinflammation, mice were injected i.p. with the iNKT cell agonist α-galactosylceramide (α-GalCer) seven days prior to LPS treatment. Immune cells infiltrated into the brain during LPS-induced neuroinflammation were determined by flow cytometry. In addition, LPS-induced clinical behavior symptoms such as depressive-like behavior and memory impairment in mice were evaluated by the open field and Y-maze tests, respectively. RESULTS: We found that iNKT cell-deficient Jα18 mutant mice display delayed disease progression and decreased leukocyte infiltration into the brain compared with WT mice, indicating that iNKT cells contribute to the pathogenesis of LPS-induced neuroinflammation. Since it has been reported that pre-treatment with α-GalCer, an iNKT cell agonist, can convert iNKT cells towards anti-inflammatory phenotypes, we next explored whether pre-activation of iNKT cells with α-GalCer can regulate LPS-induced neuroinflammation. Strikingly, we found that α-GalCer pre-treatment significantly delays the onset of clinical symptoms, including depression-like behavior and memory impairment, while decreasing brain infiltration of pro-inflammatory natural killer cells and neutrophils, in this model of LPS-induced neuroinflammation. Such anti-inflammatory effects of α-GalCer pre-treatment closely correlated with iNKT cell polarization towards IL4- and IL10-producing phenotypes. Furthermore, α-GalCer pre-treatment restored the expression of suppressive markers on brain regulatory T cells during LPS-induced neuroinflammation. CONCLUSION: Our findings provide strong evidence that α-GalCer-induced pre-activation of iNKT cells expands iNKT10 cells, mitigating depressive-like behaviors and brain infiltration of inflammatory immune cells induced by LPS-induced acute neuroinflammation. Thus, we suggest the prophylactic potential of iNKT cells and α-GalCer against acute neuroinflammation.

The cytokine receptor DR3 identifies and promotes the activation of thymic NKT17 cells.

Invariant natural killer T (iNKT) cells correspond to a population of thymus-generated T cells with innate-like characteristics and effector functions. Among the various iNKT subsets, NKT17 is the only subset that produces the proinflammatory cytokine IL-17. But, how NKT17 cells acquire this ability and what would selectively trigger their activation remain incompletely understood. Here, we identified the cytokine receptor DR3 being specifically expressed on thymic NKT17 cells and mostly absent on other thymic iNKT subsets. Moreover, DR3 ligation promoted the in vivo activation of thymic NKT17 cells and provided costimulatory effects upon agonistic α-GalCer stimulation. Thus, we identified a specific surface marker for thymic NKT17 cells that triggers their activation and augments their effector functions both in vivo and in vitro. These findings provide new insights for deciphering the role and function of murine NKT17 cells and for understanding the development and activation mechanisms of iNKT cells in general.

Preferential and persistent impact of acute HIV-1 infection on CD4+ iNKT cells in colonic mucosa.

Acute HIV-1 infection (AHI) results in the widespread depletion of CD4+ T cells in peripheral blood and gut mucosal tissue. However, the impact on the predominantly CD4+ immunoregulatory invariant natural killer T (iNKT) cells during AHI remains unknown. Here, iNKT cells from peripheral blood and colonic mucosa were investigated during treated and untreated AHI. iNKT cells in blood were activated and rapidly depleted in untreated AHI. At the time of peak HIV-1 viral load, these cells showed the elevated expression of cell death-associated transcripts compared to preinfection. Residual peripheral iNKT cells suffered a diminished responsiveness to in vitro stimulation early into chronic infection. Additionally, HIV-1 DNA, as well as spliced and unspliced viral RNA, were detected in iNKT cells isolated from blood, indicating the active infection of these cells in vivo. The loss of iNKT cells occurred from Fiebig stage III in the colonic mucosa, and these cells were not restored to normal levels after initiation of ART during AHI. CD4+ iNKT cells were depleted faster and more profoundly than conventional CD4+ T cells, and the preferential infection of CD4+ iNKT cells over conventional CD4+ T cells was confirmed by in vitro infection experiments. In vitro data also provided evidence of latent infection in iNKT cells. Strikingly, preinfection levels of peripheral blood CD4+ iNKT cells correlated directly with the peak HIV-1 load. These findings support a model in which iNKT cells are early targets for HIV-1 infection, driving their rapid loss from circulation and colonic mucosa.

Transnuclear mice reveal Peyer's patch iNKT cells that regulate B-cell class switching to IgG1.

Tissue-resident iNKT cells maintain tissue homeostasis and peripheral surveillance against pathogens; however, studying these cells is challenging due to their low abundance and poor recovery from tissues. We here show that iNKT transnuclear mice, generated by somatic cell nuclear transfer, have increased tissue resident iNKT cells. We examined expression of PLZF, T-bet, and RORγt, as well as cytokine/chemokine profiles, and found that both monoclonal and polyclonal iNKT cells differentiated into functional subsets that faithfully replicated those seen in wild-type mice. We detected iNKT cells from tissues in which they are rare, including adipose, lung, skin-draining lymph nodes, and a previously undescribed population in Peyer's patches (PP). PP-NKT cells produce the majority of the IL-4 in Peyer's patches and provide indirect help for B-cell class switching to IgG1 in both transnuclear and wild-type mice. Oral vaccination with α-galactosylceramide shows enhanced fecal IgG1 titers in iNKT cell-sufficient mice. Transcriptional profiling reveals a unique signature of PP-NKT cells, characterized by tissue residency. We thus define PP-NKT as potentially important for surveillance for mucosal pathogens.

Hobit and Blimp-1 instruct the differentiation of iNKT cells into resident-phenotype lymphocytes after lineage commitment.

iNKT cells are CD1d-restricted T cells that play a pro-inflammatory or regulatory role in infectious and autoimmune diseases. Thymic precursors of iNKT cells eventually develop into distinct iNKT1, iNKT2, and iNKT17 lineages in the periphery. It remains unclear whether iNKT cells retain developmental potential after lineage commitment. iNKT cells acquire a similar phenotype as tissue-resident memory T cells, suggesting that they also differentiate along a trajectory that enables them to persist in peripheral tissues. Here, we addressed whether lineage commitment and memory differentiation are parallel or sequential developmental programs of iNKT cells. We defined three subsets of peripheral iNKT cells using CD62L and CD69 expression that separate central, effector, and resident memory phenotype cells. The majority of iNKT1 cells displayed a resident phenotype in contrast to iNKT2 and iNKT17 cells. The transcription factor Hobit, which is upregulated in iNKT cells, plays an essential role in their development together with its homolog Blimp-1. Hobit and Blimp-1 instructed the differentiation of central memory iNKT cells into resident memory iNKT cells, but did not impact commitment into iNKT1, iNKT2, or iNKT17 lineages. Thus, we conclude that memory differentiation and the establishment of residency occur after lineage commitment through a Hobit and Blimp-1-driven transcriptional program.

Dynamics of IL-15/IL-15R-α expression in response to HSV-1 infection reveal a novel mode of viral immune evasion counteracted by iNKT cells.

Herpes simplex virus type 1 (HSV-1) infects and persists in most of the human population. Interleukin-15 (IL-15) has an important role in the activation of cell-mediated immune responses and acts in complex with IL-15 receptor alpha (IL-15R-α) through cell surface transpresentation. Here, we have examined the IL-15/IL-15R-α complex response dynamics during HSV-1 infection in human keratinocytes. Surface expression of the IL-15/IL-15R-α complex rapidly increased in response to HSV-1, reaching a peak around 12 h after infection. This response was dependent on detection of viral replication by TLR3, and enhancement of IL15 and IL15RA gene expression. Beyond the peak of expression, levels of IL-15 and IL-15R-α gradually declined, reaching a profound loss of surface expression beyond 24 h of infection. This involved the loss of IL15 and IL15RA transcription. Interestingly, invariant natural killer T (iNKT) cells inhibited the viral interference with IL-15/IL-15R-α complex expression in an IFNγ-dependent manner. These results indicate that rapid upregulation of the IL-15/IL-15R-α complex occurs in HSV-1 infected keratinocytes, and that this response is targeted by viral interference. Shutdown of the IL-15 axis represents a novel mode of HSV-1 immune evasion, which can be inhibited by the host iNKT cell response.

Cytokine receptor γc effectuates the generation of proinflammatory innate CD8 T cells by non-classical MHC-I molecules.

Innate CD8 T cells correspond to a population of terminally differentiated effector T cells that phenotypically appear as antigen-experienced memory cells and functionally resemble proinflammatory CD8 T cells, expressing copious amounts of IFNγ. Innate CD8 T cells, however, are distinct from conventional effector-memory CD8 T cells as they acquire functional maturity during their generation in the thymus. Understanding the molecular mechanisms that drive their thymic development and differentiation is an intensely studied subject in T cell immunity, and here we identified the cytokine receptor γc as a critical mediator of innate CD8 T cell generation that promotes their selection even in the absence of classical MHC-I molecules. Consequently, overexpression of γc resulted in a dramatic increase of innate CD8 T cells in KbDb-deficient mice. We mapped its underlying mechanism to the expansion of IL-4-producing invariant NKT cells, so that it is the increased availability of intrathymic IL-4 which augments the selection of innate CD8 T cells. Collectively, these results unravel the selection of innate CD8 T cells being mediated by non-classical MHC-I molecules and being modulated by the abundance of the γc cytokine, IL-4.

Nur77 controls tolerance induction, terminal differentiation, and effector functions in semi-invariant natural killer T cells.

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels of Nr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77tg) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearranged Vα14-Jα18 TCR-α chain gene into the Nur77tg (Nur77tg;Vα14tg) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4-producing NKT2 cell subset but not IFN-γ-producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions.

Pbrm1 intrinsically controls the development and effector differentiation of iNKT cells.

Under static condition, the pool size of peripheral invariant natural killer T (iNKT) cells is determined by their homeostatic proliferation, survival and thymic input. However, the underlying mechanism is not fully understood. In the present study, we found that the percentage and number of iNKT cells were significantly reduced in the spleen, but not in the thymus of mice with deletion of polybromo-1 (Pbrm1) compared to wild type (WT) mice. Pbrm1 deletion did not affect iNKT cell proliferation and survival, instead significantly impaired their development from stage 1 to stage 2. Importantly, loss of Pbrm1 led to a dysfunction of RORγt expression and iNKT17 cell differentiation, but not iNKT1 and iNKT2 proportion. Collectively, our study reveals a novel mechanism of Pbrm1 controlling the peripheral size of iNKT cells through regulating their development and differentiation.

Early Activation of iNKT Cells Increased Survival Time of BALB/c Mice in a Murine Model of Melioidosis.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei. High interferon gamma (IFN-γ) levels in naive mice were reported to mediate protection against B. pseudomallei infection. Invariant natural killer T (iNKT) cells can produce and secrete several cytokines, including IFN-γ. When iNKT cell-knockout (KO) BALB/c mice were infected with B. pseudomallei, their survival time was significantly shorter than wild-type mice. Naive BALB/c mice pretreated intraperitoneally with α-galactosylceramide (α-GalCer), an iNKT cell activator, 24 h before infection demonstrated 62.5% survival at the early stage, with prolonged survival time compared to nonpretreated infected control mice (14 ± 1 days versus 6 ± 1 days, respectively). At 4 h after injection with α-GalCer, treated mice showed significantly higher levels of serum IFN-γ, interleukin-4 (IL-4), IL-10, and IL-12 than control mice. Interestingly, the IFN-γ levels in the α-GalCer-pretreated group were decreased at 4, 24, and 48 h after infection, while they were highly increased in the control group. At 24 h postinfection in the α-GalCer group, bacterial loads were significantly lower in blood (no growth and 1,780.00 ± 51.21, P < 0.0001), spleens (no growth and 34,300 ± 1,106.04, P < 0.0001), and livers (1,550 ± 68.72 and 13,400 ± 1,066.67, P < 0.0001) than in the control group, but not in the lungs (15,300 ± 761.10 and 1,320 ± 41.63, P < 0.0001), and almost all were negative at 48 h postinfection. This study for the first time shows that early activation of iNKT cells by α-GalCer helps clearance of B. pseudomallei and prolongs mouse survival.

MAIT cells accumulate in ovarian cancer-elicited ascites where they retain their capacity to respond to MR1 ligands and cytokine cues.

The low mutational burden of epithelial ovarian cancer (EOC) is an impediment to immunotherapies that rely on conventional MHC-restricted, neoantigen-reactive T lymphocytes. Mucosa-associated invariant T (MAIT) cells are MR1-restricted T cells with remarkable immunomodulatory properties. We sought to characterize intratumoral and ascitic MAIT cells in EOC. Single-cell RNA sequencing of six primary human tumor specimens demonstrated that MAIT cells were present at low frequencies within several tumors. When detectable, these cells highly expressed CD69 and VSIR, but otherwise exhibited a transcriptomic signature inconsistent with overt cellular activation and/or exhaustion. Unlike mainstream CD8+ T cells, CD8+ MAIT cells harbored high transcript levels of TNF, PRF1, GZMM and GNLY, suggesting their arming and cytotoxic potentials. In a congenic, MAIT cell-sufficient mouse model of EOC, MAIT and invariant natural killer T cells amassed in the peritoneal cavity where they showed robust IL-17A and IFN-γ production capacities, respectively. However, they gradually lost these functions with tumor progression. In a cohort of 23 EOC patients, MAIT cells were readily detectable in all ascitic fluids examined. In a sub-cohort in which we interrogated ascitic MAIT cells for functional impairments, several exhaustion markers, most notably VISTA, were present on the surface. However, ascitic MAIT cells were capable of producing IFN-γ, TNF-α and granzyme B, but neither IL-17A nor IL-10, in response to an MR1 ligand, bacterial lysates containing MR1 ligands, or a combination of IL-12 and IL-18. In conclusion, ascitic MAIT cells in EOC possess inducible effector functions that may be modified in future immunotherapeutic strategies.

Protein abundance of the cytokine receptor γc controls the thymic generation of innate-like T cells.

Innate-like T (iT) cells comprise a population of immunoregulatory T cells whose effector function is imposed during their development in the thymus to provide protective immunity prior to antigen encounter. The molecular mechanism that drives the generation of iT cells remains unclear. Here, we report that the cytokine receptor γc plays a previously unappreciated role for thymic iT cells by controlling their cellular abundance, lineage commitment, and subset differentiation. As such, γc overexpression on thymocytes dramatically altered iT cell generation in the thymus, as it skewed the subset composition of invariant NKT (iNKT) cells and promoted the generation of IFNγ-producing innate CD8 T cells. Mechanistically, we found that the γc-STAT6 axis drives the differentiation of IL-4-producing iNKT cells, which in turn induced the generation of innate CD8 T cells. Collectively, these results reveal a cytokine-driven circuity of thymic iT cell differentiation that is controlled by the abundance of γc proteins.

microRNA dynamic expression regulates invariant NKT cells.

Invariant natural killer T cells (iNKT) are a prevalent population of innate-like T cells in mice, but quite rare in humans that are critical for regulation of the innate and adaptive immune responses during antimicrobial immunity, tumor rejection, and inflammatory diseases. Multiple transcription factors and signaling molecules that contribute to iNKT cell selection and functional differentiation have been identified. However, the full molecular network responsible for regulating and maintaining iNKT populations remains unclear. MicroRNAs (miRNAs) are an abundant class of evolutionarily conserved, small, non-coding RNAs that regulate gene expression post-transcriptionally. Previous reports uncovered the important roles of miRNAs in iNKT cell development and function using Dicer mutant mice. In this review, we discuss the emerging roles of individual miRNAs in iNKT cells reported by our group and other groups, including miR-150, miR-155, miR-181, let-7, miR-17 ~ 92 cluster, and miR-183-96-182 cluster. It is likely that iNKT cell development, differentiation, homeostasis, and functions are orchestrated through a multilayered network comprising interactions among master transcription factors, signaling molecules, and dynamically expressed miRNAs. We provide a comprehensive view of the molecular mechanisms underlying iNKT cell differentiation and function controlled by dynamically expressed miRNAs.