Crohn's disease under single-cell map: from INFLARE metaplastic cells to rare immune cell subpopulations.

Hepatitis B is a noncytopathic virus, which exclusively replicates within the liver and affects 350 million people worldwide [1]. Chronic infection can lead to variable disease manifestations such as cirrhosis, decompensated liver disease and hepatocellular carcinoma causing 1 million deaths per year [2]. These important clinical outcomes are a consequence of the host immune response to HBV, which constitutes a double-edged sword responsible for both viral clearance and hepatocellular damage. <br> <br> The precipitants for natural history milestones such as HBV-related hepatic flares (HF) and hepatitis B e Antigen (HBeAg) seroconversion remain unknown. Virus-specific and non-specific cytotoxic T lymphocytes (CTLs), T regulatory (Treg) cells, Natural Killer (NK), Natural Killer T (NKT) cells and dendritic cells (DCs) have been postulated to play a role [3]. The contribution of these immune cells and the nature of their interaction in the immune pathogenesis of HBV-related liver disease require further characterization. The logistical restraints of longitudinal, peripheral and intrahepatic sampling of the human host as well as inadequate small animal and cell culture models have hampered investigation of these immune mechanisms. As a caveat to human based studies in HBV, the circulating immunological cells may not reflect the phenotype and function of equivalent cells sequestered within the human liver. Current knowledge about the immune response to HBV is extrapolated from transgenic mouse models, many of which are models of viral replication rather than liver injury. <br> <br> Natural Killer (NK) and Natural Killer T (NKT) cells are cytotoxic lymphocytes that constitute a key effector arm of the innate immune system. Efforts to characterise the immunological determinants of Hepatitis B virus (HBV) infection have focused on the adaptive immune system whilst overlooking the potential interaction between virus, hepatocyte and NK or NKT cells, which play an important role in host defense against viral pathogens through direct cytotoxicity and the production of proinflammatory and immune regulatory cytokines. There is debate as to whether NK or NKT cells are effectors of antiviral activity or mediators of hepatic injury and fibrogenesis in chronic hepatitis B infection. NK and NKT cells have been implicated in the pathogenesis of liver disease due to other hepatotropic viruses such as hepatitis C and E as well as autoimmune liver disease, as shown in animal models of liver injury [4]. Human intrahepatic lymphocytes consist of 30%-50% NK and 5-10% NKT cells. Peripheral blood lymphocytes contain 13% NK cells and 2% NKT cells [5]. The hepatic enrichment of NK and NKT cells reflects their role as regulators at the interface between the innate and adaptive immune response to liver disease. NK and NKT cells in the peripheral and intrahepatic compartments share effector functions such as direct killing of viral-infected cells and cytokine production. The latter is considered the more important effector function in CHB [4]. NK and NKT cells demonstrate reciprocal interactions (“crosstalk”) with hepatic macrophages, Kupffer cells (KC), DCs and T cells as part of an amalgamated immune response to HBV [4]. <br> <br> The role of NK and NKT cells in the initiation and orchestration of a dynamic host immune response against HBV-related liver disease is investigated in this thesis. This hepatotrophic virus has evolved direct and indirect strategies to evade or inhibit the large hepatic reservoir of NK and NKT cells. In this thesis, I will focus on the dynamic phenotype and function of NK and NKT cells throughout the different phases of HBV infection, which so far have been poorly characterized. I will also examine the effect of activated NK and NKT cells on liver injury, fibrosis, and their attenuation following <br> HBV treatment, which remains controversial. Understanding the role of NK and NKT cells in the pathogenesis of CHB may help to develop new biomarkers for disease and treatment activity and design novel immunotherapies.

Natural Killer T (NKT) cells play an important role in the immune system as demonstrated by their involvement in tumour surveillance, infection and inflammation. Unlike conventional T cells that recognises peptide antigens when presented by the Major Histocompatibility Complex (pMHC), NKT cells recognise glycolipids, presented by a MHC class I-like molecule (CD1d-α-GalCer) to invoke an immune response. Human semi-invariant NKT cells are unique as the majority of them express a T cell receptor (TCR) bearing an invariant α-chain and restricted β-chain repertoire (Vα24-Jα18;Vβ 11). The mouse orthologue also expresses an invariant α-chain (Vα14-Jα18) but a slightly more diverse β-chain repertoire (Vβ2, Vβ7 and Vβ8.2), where Vβ8.2 is most commonly expressed. The crystal structures of the human NKT TCR-CD1d-α-GalCer and mouse orthologue complexes have provided structural insights into how the NKT TCR can recognise a lipid antigen. While it is clear that the NKT TCR docks CD1d-α-GalCer in a different conformation compared to the TCR-pMHC complexes, there was no clear information on the energetic footprint of the NKT TCR's recognition of CD1d-α-GalCer and also how the NKT TCR can differentiate between closely related α-GalCer analogues to induce a biased cytokine response. In this study, an alanine scanning mutagenesis experiment carried out on the human NKT TCR (Vα24-Jα18;Vβ11) and CD1d, as well as the use of α-GalCer analogues, demonstrated that the Jα18-encoded CDR3α loop and Vβ11-encoded CDR2β loop of the NKT TCR play a crucial role in maintaining its interactions with CD1d-α-GalCer. The minimal usage of only six residues, which are also evolutionary conserved in the mouse NKT TCR (Vα14-Jα18;Vβ8.2), explains the semi-invariant nature of the NKT TCR as well as the basis of NKT cell cross-species reactivity. Furthermore, the interactions these residues made are localised directly above the F′ pocket of CD1d, distal from the galactosyl head group of α-GalCer. The use of α-GalCer analogues that contains glycosyl head group modifications, further demonstrated the lesser energetic contribution the NKT TCR CDR1α loop plays in its interactions with the sugar head group of α-GalCer. In contrast, the NKT TCR CDR3α loop, which makes interactions with α-GalCer as well as CD1d, was determined to be the key CDR loop that is energetically important in glycolipid recognition. NKT cells can differentiate between α-GalCer analogues with small modifications on their glycosyl head group to stimulate a biased T helper (Th) cytokine response. Therefore, the ability of how the mouse Vβ8.2 NKT TCR is able to distinguish between different α-GalCer analogues was further investigated through a combination of biophysical, structural and functional experiments. These data had provided further insight into how NKT cells can recognise and differentiate between structurally similar variants of α-GalCer, also referred to as Altered Glycolipid Ligands (AGLs). The crystal structures of all five NKT TCR-CD1d-AGL complexes revealed minimal structural differences. Variations in terms of affinity and kinetics of the NKT TCR engagement onto CD1d-AGLs as well as differences in cellular responses between AGLs were observed. Modifications on the glycosyl head group of the AGLs, directly impacted NKT cell activation as well as the affinity and t1/2 of the NKT TCR recognition. Furthermore, for these glycosyl head group modified AGLs, ligand potency, as determined by the amount of cytokines produced by the NKT cells, was directly affected by the t1/2 of the NKT TCR-CD1d-AGL interaction. In addition, modifications on the acyl chain of the AGLs do not affect the NKT TCR interaction but reduced NKT cell proliferation. This indicated an alternative antigen processing and presentation pathway for these AGLs by CD1d. On the other hand, truncation of the sphingosine chain resulted in a reduction of NKT TCR affinity resulting in an induced-fit mechanism by the NKT TCR. Collectively, the minimal binding requirements of CD1d restriction as well as the molecular basis of NKT fine specificity in CD1d-AGLs recognition were elucidated.

β-Glycoglycosphingolipid-Induced Alterations of the STAT Signaling Pathways Are Dependent on CD1d and the Lipid Raft Protein Flotillin-2

Research Highlights: Immunotherapy

Inflammatory bowel disease.

Natural killer T cells are important in the pathogenesis of asthma: The many pathways to asthma

ATP, adenosine triphosphate; IFN-γ, interferon-gamma; NK, natural killer cells; NKT, natural killer T cells; PH, partial hepatectomy. Liver regeneration is a highly integrated process involving a wide array of cellular interactions occurring between the different liver cell types, but also between the liver and the extrahepatic environment. Besides the main growth factors and cytokines acting in a paracrine way, diverse endocrine and neuroendocrine interactions, as well as the recruitment of blood cells, affect liver regeneration. This entire network acts in concert to achieve a fine-tuning of proliferative and hepatoprotective cascades, ultimately leading to liver mass restoration after injury or resection.1 A study published in this issue of HEPATOLOGY,2 sheds new light on the complex interactions between immune cells and liver regeneration processes, with the purinergic system as one possible regulator. Among modulators of liver regeneration, the innate immune system constitutes a complex network of interacting cells and cytokines, of which the resulting effect on liver regeneration is not entirely understood. Surprisingly, the complement system3 as well as cytotoxic cytokines, like tumor necrosis factor alpha,1 are reported as positive regulators for hepatocyte regeneration. The role of Kupffer cells, the hepatic resident macrophages, is still controversial, because these cells secrete numerous cytokines, stimulating or inhibiting hepatocyte proliferation, after liver injury.1 As to natural killer (NK) and natural killer T (NKT) cells, the main lymphoid population in human and mouse liver, although the majority of studies reported on their negative effect on liver regeneration,4, 5 their role still remains controversial.6-8 NK cells would inhibit regeneration, in particular through the secretion of interferon-gamma (IFN-γ), whereas NKT cells would play a minor role in the normal liver after partial hepatectomy (PH).5 However, NK and NKT cells may have a positive effect on oval cell-dependent regeneration after acute liver injury when hepatocytes cannot replicate.9 The study by Graubardt et al. revisited the controversy on NK cells during liver regeneration by introducing a new parameter: the purinergic system, currently recognized as a major component of the inflammatory response after injury.10 Signaling by extracellular adenosine triphosphate (ATP) acting on plasma membrane purinergic receptors, that is, P2Y1,2,4,6,11-14 (G-protein-coupled receptors) and P2X1-7 (ATP-gated channels with high Ca2+ permeability), is vital not only in excitable, but also in nonexcitable cells and tissues.11 Vascular shear stress, organ distension, or cellular injury are known to trigger ATP release from endothelial, epithelial, and other cell types.12 Once in the extracellular medium, ATP is degraded by the powerful ecto-ATPases expressed on cell surfaces to avoid excessive activation (and desensitization) of purinergic receptors. Adenosine, the breakdown product of ATP, can bind P1 purinergic receptors, with specific downstream biological responses.11 Thus, in a given tissue, the final purinergic input will be determined by the complex combination of numerous parameters, including mainly mechanisms of ATP release, purinergic receptors expression and distribution, and ecto-ATPases expression.11 In the liver, each cell type expresses its own repertoire of purinoceptors and ecto-ATPases, and evidence for a crucial effect of purinergic signaling in liver physiology is growing, including modulation of bile secretion and ischemia protection.13-15 In vitro studies suggested that extracellular ATP had a positive effect on proliferation of primary rat hepatocytes.16 Recently, we showed that an extracellular ATP release from the liver occurred immediately after PH, contributing to liver regeneration in the rat.15 We also observed an immediate ATP release from the liver after PH for living donor transplantation, suggesting that purinergic signaling may be also involved during human liver regeneration.15 However, the purinoceptors and precise mechanisms involved in ATP-mediated effect on liver regeneration still remain to be defined. The authors of the present study previously reported on a coordinating role of the ectonucleotidase CD39-mediated ATP and adenosine diphosphate degradation after PH in mice, suggesting that both hepatocytes and endothelial cells would need, in their microenvironment, a fine-tuned balance between the different nucleotide species to adequately proliferate after PH.17 The same group also showed that the lack of CD39 protected mice from liver injury in models known to be, at least in part, mediated by NK and NKT cells.14, 18 In the absence of CD39, ATP-induced NKT cell apoptosis18 and inhibition of IFN-γ secretion14 cannot be refrained, thereby limiting liver injury. In their study, Graubardt et al.2 propose that hepatic NK cell activation is required for optimal liver regeneration in a process that would depend on extracellular ATP hydrolysis. The authors reported an impaired regeneration and increased liver injury after PH in mice lacking T, B, NKT, and NK cells (Rag2/common gamma-null), whereas this was not observed in mice lacking only B, T, and NKT lymphocytes (Rag1-null). In wild-type mice, the authors also showed that PH increased cytotoxicity and immaturity of hepatic NK cells. Because ATP is released immediately after PH,15 and based on their previous studies,14, 18 the authors asked whether hepatic NK cell activity was dependent on extracellular ATP hydrolysis during liver regeneration. They observed that, when ATP was hydrolyzed by apyrase (soluble ectonucleotidase) treatment in vivo, hepatocyte entry in S phase after PH was enhanced, and that this effect was dependent on NK cells. Also, ATP clearance by apyrase treatment further increased hepatic NK cell cytotoxicity after PH. In vitro, the authors showed that ATP stimulation decreases NK cell cytotoxicity, possibly through P2X3 receptors. They propose that NK cells, which cytotoxicity is enhanced after PH, promote liver regeneration in a process inhibited by extracellular ATP and enhanced by its hydrolysis. In light of the literature, this interesting study adds complexity on top of controversial questions. First, in contradiction with a number of previous reports, in this study, NK cells appear to be beneficial for liver regeneration; the more cytotoxic they are, the best it seems to be for the liver after PH. Although this apparent paradox during liver regeneration had already been reported on for other cytotoxic components of the innate immune system,1, 3 as well as for Fas receptor engagement,19 the mechanisms by which cytotoxic NK cells would enhance hepatocyte regeneration remain to be defined. It is also important to note that, because Graubardt et al. used mice lacking not only NK, but also NKT, B, and T cells, the observed inhibition of liver regeneration may not result only from the absence of NK cells, but also from the lack of other immune cells, which interaction with NK cells may affect the final response to injury. Second, it may be important to consider that, besides ATP clearance, in vivo experiments with the soluble ectonucleotidase apyrase also provide the liver with ATP degradation products, such as adenosine, which could contribute to the liver response to PH.11 Third, it is not possible, at present, to define unequivocally a global resulting effect of ATP release during liver repair. Although, after PH, extracellular ATP can directly or indirectly promote hepatocyte proliferation,15, 16 it could also indirectly inhibit it through NK cell inactivation, as highlighted in this study. However, in the context of liver injury (toxic, ischemic, or autoimmune), extracellular ATP would be globally beneficial, whereas ectonucleotidase-mediated ATP clearance would be deleterious, although this view could be challenged by considering the differential effect of extracellular ATP on the different liver cell types and their specific purinoceptor repertoires.20 Here, there is obviously still room for further research about these apparently opposite endpoints of purinergic signaling in liver regeneration before any therapeutic strategy could be rationally anticipated. 1 Schematic overview of NK/NKT cells and purinergic system after PH or liver injury. Left panel: After PH, ATP is released inside the liver, with paracrine effects favoring hepatocyte progression in the cell cycle through P2Y and, possibly, P2X stimulation. ATP also acts on NK cells, possibly through P2X3 receptors, inhibiting cytotoxicity. When ATP is degraded by endogeneous ectonucleotidase CD39, or by apyrase treatment, NK cells, through unknown mechanisms, enhance hepatocyte entry into the cell cycle after PH. It is also possible that inhibition of IFN-γ secretion by ATP in NK cells contributes to hepatocyte cell cycle entry after PH. Blue lines result in inhibition of hepatocyte proliferation. Red lines result in stimulation of hepatocyte proliferation. Right panel: After liver injury (ischemic, toxic, or autoimmune), ATP released from stressed cells contributes to NKT cell apoptosis, possibly through P2X7, thereby reducing hepatocyte injury. ATP also inhibits IFN-γ secretion, further reducing cell damages. Blue lines result in reduction of hepatocyte injury. Red lines result in the enhancement of hepatocyte injury. Dashed lines are related to hypothetical mechanisms.

The immune system plays a major role in the elimination of tumors. CD8 T cell infiltration is known to be a good prognostic indicator. The development of therapies based on checkpoint inhibitor antibodies was an important breakthrough in increasing survival by limiting the exhaustion of cytotoxic cells and increasing tumor cell eradication. However, the immune system also contains regulatory cells that protect the organism from inappropriate activation of immune cells against self-antigens. Thus, the fact that tumor cells are autologous leads regulatory cells to inhibit the activation of anti-tumor cytotoxic cells and thus increase tumor escape. Therefore, understanding the function and the activation of regulatory cells might help to develop therapies to limit the activation of regulatory cells in order to increase tumor clearance. Natural killer T (NKT) cells are lymphocytes with features of natural killer (NK) and of T cells placing them at the interface of innate and adaptive immunity. Like NK cells, they rapidly produce cytokines after stimulation, orienting the immune response. As T cells, they express a T cell receptor (TCR) that allows the recognition of specific lipids presented by the non-classical MHC-I molecule CD1d. According to their TCR usage, two populations of NKT cells are described: type I and type II. All type I NKT cells express a semi-invariant TCR (Valpha24Jalpha18 in humans, Valpha14Jalpah18 in mice) that recognizes α-galactosylceramide (αGalCer). They can be identified with the αGalCer-loaded CD1d tetramer. In contrast, type II NKT cells express a more diverse TCR repertoire. There is no currently identified lipid antigen recognized by all type II NKT cells, making their identification more difficult. A fraction of them recognize sulfatide. By using sulfatide-loaded CD1d-tetramers, we observed for the first time that sulfatide-reactive type II NKT cells were enriched in the lung, a major site of tumor metastasis. Moreover, we previously showed that in vivo stimulation of type II NKT cells with sulfatide increased the number of tumor nodules in the lung. An in-depth phenotype analysis revealed that they were CD4 or CD8 single positive cells, like conventional T cells, whereas type I NKT cells are either CD4+CD8- or double negative. Type II NKT cells do not express PLZF, the master regulator of NKT cell development and exist in PLZF-/- mice contrary to type I NKT cells. We showed that type II NKT cells also expressed markers of myeloid cells, c-Kit, CD11b and Ly6C even though histological analysis revealed lymphocytic morphology. Interestingly, at steady state, type II NKT cells expressed granzyme A but not granzyme B or perforin. The in vivo injection of sulfatide increased the expression of the activation markers CD69 and CD44 as well as granzyme A. Since the regulatory functions of type II NKT cells have been shown to be critical in tumor immunity, the detailed characterization of these cells could help to develop a new immunotherapy for cancer. Citation Format: Lise Pasquet, Shingo Kato, Tony Adams, Susan Sharrow, Theresa Davies-Hill, Elaine Jaffe, Xia Zheng, Motoshi Suzuki, Damian Kovalovsky, Jay Berzofsky, Masaki Terabe. Characterization of sulfatide reactive type II NKT cells from mouse lung [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4622. doi:10.1158/1538-7445.AM2017-4622

Immune privilege in the eye is, in part, associated with the development of an antigen-specific systemic tolerance termed anterior chamber-associated immune deviation (ACAID). Natural killer T (NKT) cells express T-cell receptor and natural killer (NK) markers and are classified as innate immune cells partly because they produce cytokines within minutes of signals. The aim of this study was to elucidate the role of murine NKT cells in the induction of T regulatory cells in anterior chamber-associated immune deviation. Anterior chamber-associated immune deviation T regulatory cell generation ability was examined in the following NKT cell-deficient mice: SJL mice, CDld or Jalpha281 knockout (KO) mice on C57BL/6 (B6) background, and NKT cell-depleted mice. To detect T regulatory cells, splenic T cells were harvested 7 days after anterior chamber inoculation of ovalbumin (50 microg/2 microL in Hanks balanced salt solution [HBSS]), mixed with ovalbumin-primed T cells (effector) and ovalbumin-pulsed antigen-presenting cells (stimulator), and then cotransferred into the ear pinnae of a syngeneic naive mouse (local adoptive transfer assay). Ear swelling was measured 24 hours later. Anterior chamber-inoculated B6 mice developed T regulatory cells, but all natural killer T cell-deficient mice did not generate T regulatory cells unless they were reconstituted with natural killer T cells. We also found that the number of splenic natural killer T cells were increased in anterior chamber-inoculated B6 mice and those natural killer T cells produced IL-10. CD1d-reactive natural killer T cells are essential for the induction of T regulatory cells in anterior chamber-associated immune deviation through their IL-10 production and are involved in the maintenance of immune privilege of the eye.

Starting Over: The Search for Endogenous NKT Cell Ligands

Pancreatic cancer (PC) is seldom detected at early stages and most PC tumors are unresectable. The five-year survival rate for PC is &amp;lt;6%. Natural Killer (NK) and Natural Killer T (NKT) cell dysfunction is linked to aggressive tumor growth and poor PC prognosis. NKT cells are known to regulate pro-inflammatory responses of tumor-associated macrophages (TAM). We have shown that pancreatic tumors are high in TAM and overexpress microsomal prostaglandin synthase-1 (mPGES-1). Thus, we performed experiments to clarify the roles of NK, NKT, TAM, and mPGES-1 in K-rasG12D driven pancreatic tumor growth in mice. To understand the regulatory role of NKs, we initially crossed Rag1 mice, which have NK cells and lack T cells, with Kras (p48Cre/+-LSL-KrasG12D/+) mice to generate Rag1-Kras mice. At seven months of age, the Rag1-Kras mice showed reduced pancreatic intraepithelial neoplasia (PanIN) lesions without evidence of carcinoma compared with Kras mice (p&amp;lt;0.0001) which displayed high PanINs (p&amp;lt;0.001) and invasive carcinoma. Flow cytometry (FC) analysis of pancreatic tumors (PTs) showed 75% NK cells (NKp46and NK1.1-positive) in PTs of Rag1-Kras mice compared with 6.7% NK cells in PTs from K-ras mice. These results suggest an inhibitory role of NKs on PT. To further study the role of NKTs, we crossbred CD1d−/− mice with K-ras mice, and generated CD1d−/−-Kras mice deficient in both iNKT and vNKT cells. At 5 months of age, the PTs were analyzed histologically, PTs and spleens were analyzed by FC for macrophages (CD68, stabilin) and mPGES-1 expressions, along with stem-like cells (Epcam-, Dclk1-, and Lgr5- positive cells). CD1d−/−-Kras mice were observed to have significantly increased PT weights, and a 50% increase in total PanINs compared with Kras mice (PanIN1, 175 Vs 362, p&amp;lt;0.0001; PanIN2, 80 Vs 162, p&amp;lt;0.0003; PanIN 3, 17 vs 30, p&amp;lt;0.008). PTs and spleens from CD1d−/−-Kras mice showed significantly increased mPGES-1 expression in M2 type macrophages (p&amp;lt;0.005) compared with Kras mice. Tumors from CD1d−/−-Kras mice had more cancer stem-like cells that were positive for Epcam, Dclk-1, and Lgr5 (25%, p&amp;lt;0.02) compared with tumors from Kras mice. To further confirm the relationship between NKT cells, M2-derived mPGES-1, and PC development, we treated CD1d−/−-Kras mice with mPGES-1-specific inhibitor YS-121 after PanIN lesions formed at 12 weeks of age. Mice were killed after 22 weeks of age. We analyzed PanIN formation and PC. Inhibition of mPGES-1 led to a decrease in PanINs and PC development. Thus, the absence or functional loss of NKT cells leads to increased TAMs (M2) with high mPGES-1, resulting in enhanced pancreatic tumor growth and invasion. These results suggest that NKT cells play a regulatory role on macrophages during Kras-induced pancreatic tumor progression. {Supported in part by Kerley-Cade Chair Endowment and NCI-CN-53300}. Citation Format: Naveena B. Janakiram, Altaf Mohammed, Taylor Bryant, Rebekah Ritchie, Gopal Pathuri, Stan Lightfoot, Mark L. Lang, Chinthalapally V. Rao. Enhanced pancreatic tumor progression in p48Cre-KrasG12D mice is regulated by natural killer T (NKT) cells dependent on mPGES-1 in tumor-associated macrophages. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3179. doi:10.1158/1538-7445.AM2015-3179

The causes of Crohn’s disease (CD) and ulcerative colitis (UC), the two most common forms of inflammatory bowel disease (IBD), are multi-factorial and include dysregulation of immune cells in the intestine. Cannabinoids mediate protection against intestinal inflammation by binding to the G-protein coupled cannabinoid receptors 1 and 2 (CB1 and CB2). Here, we investigate the effects of the CB2 inverse agonist SMM-189 on dextran sodium sulfate (DSS)-induced experimental colitis. We observed that SMM-189 effectively attenuated the overall clinical score, reversed colitis-associated pathogenesis, and increased both body weight and colon length. Treatment with SMM-189 also increased the expression of CB2 and protein kinase A (PKA) in colon lamina propria lymphocytes (LPLs). We noticed alterations in the percentage of Th17, neutrophils, and natural killer T (NKT) cells in the spleen, mesenteric lymph nodes (MLNs), and LPLs of mice with DSS-induced colitis after treatment with SMM-189 relative to DSS alone. Further, myeloid-derived suppressor cells (MDSCs) during colitis progression increased with SMM-189 treatment as compared to DSS alone or with control cohorts. These findings suggest that SMM-189 may ameliorate experimental colitis by inducing the expression of endogenous CB2 and PKA in LPLs, increasing numbers of MDSCs in the spleen, and reducing numbers of Th17 cells and neutrophils in the spleen, MLNs, and LPLs. Taken together, these data support the idea that SMM-189 may be developed as a safe novel therapeutic target for IBD.

Aberrant immune cell infiltrates and microcircumstances represent characteristic features of liver fibrosis. In this study, we profiled the transcriptomes of intrahepatic CD45+ immune cells, from mice, using single-cell RNA sequencing (scRNA-seq) technology to understand the landscape of intrahepatic immune cells during the pathogenesis of fibrosis. Analysis of approximately 10,000 single-cell transcriptomes revealed an increase in dendritic cells (DCs), macrophages, and neutrophils and a decrease in T and natural killer T (NKT) cells. In addition, we report changes in the transcriptomes of diverse immune cell types, implying a deteriorating intrahepatic immune microcircumstance. Furthermore, we uncovered a novel fibrosis-associated CD8 T (Ccl5+, Ccl4+) and CD4 T (mt-Co1+) cell subpopulation, which infiltrates fibrotic liver and is characterized by abnormal activation or inactivation as well as a TCR decline. The results from scRNA-seq and bulk immune repertoire sequencing (IR-seq) revealed an obvious decline in T cell receptor (TCR) clonotypes combined with shrinking VJ and VDJ segment usage, as well as lower complementarity-determining region 3 (CDR3) amino acid (AA) diversity from fibrotic liver. Interestingly, a deficiency of TCR IR (TcrbKO mice) led to a deterioration of liver fibrosis, coupled with activation of hepatic stellate cells (HSCs) induced by the upregulation of macrophage and γδ T cell distribution in fibrotic TcrbKO livers. Our findings reveal the landscape and dynamics of single immune cells in liver fibrosis, and clarify the protective role of TCR IR in response to chronic liver injury.

Natural killer T (NKT) cells are αβ T cell receptor (TCR) expressing innate‐like T cells that display natural killer (NK) cell markers. Based on TCR characteristics, they are divided into two groups restricted to the MHC class I‐like molecule CD1d. Type I NKT cells, most extensively studied, are identified by a semi‐invariant Vα14‐Jα18 (mouse, Vα24‐Jα18 in humans) TCR reactive to the prototypic ligand α‐galactosylceramide presented on CD1d. In contrast, type II NKT cells display diverse TCR reacting to different CD1d‐presented ligands. There are no reagents that identify all type II NKT cells, limiting their exploration. Here, we searched for novel type II NKT cells by comparing Jα18−/−MHCII−/− mice that harbour type II but not type I NKT cells, and CD1d−/−MHCII−/− mice, lacking all NKT cells. We identified significantly larger populations of CD4+ and CD4−CD8− (double negative, DN) TCRβ+ cells expressing NKG2D or NKG2A/C/E in Jα18−/−MHCII−/− mice compared with CD1d−/−MHCII−/− mice, suggesting that 30%‐50% of these cells were type II NKT cells. They expressed CD122, NK1.1, CXCR3 and intermediate/low levels of CD45RB. Further, the CD4+ subset was CD69+, while the DN cells were CD49b+ and CD62L+. Both subsets expressed the NKT cell‐associated promyelocytic leukaemia zinc finger (PLZF) transcription factor and Tbet, while fewer cells expressed RORγt. NKG2D+ CD4+ and DN populations were producers of IFN‐γ, but rarely IL‐4 and IL‐17. Taken together, we identify a novel subset of primary CD4+ and DN type II NKT cells that expresses NKG2 receptors have typical NKT cell phenotypes and a TH1‐like cytokine production.

The pathogenesis of HIV-1 infection is a complex process in which Natural Killer (NK) and Natural Killer T (NKT) cells play an important role. NKT cells express markers for NK cells and a TCR of the conventional T cells and recognize lipid antigens presented by the non-polymorphic CD1 molecule. CD1d-restricted type I NKT cells express an invariant TCR and can recognize αGalCer, whereas a major subset of type II NKT expressing diverse TCR can recognize a self-glycolipid, sulfatide. It has been shown that CD4+ type I NKT cells are infected by HIV-1 and decreased in HIV-1-infected individuals. However, their exact role in HIV-1 infection as well as the biology and function of the type II NKT cell subset in HIV-1 infection and disease progression are not known. Our earlier studies have shown that activation of CD1d-restricted type II NKT cells by sulfatide and their interactions with plasmacytoid dendritic (pDC) and myeloid dendritic (mDC) cells result in anergy induction in type I NKT cells in several models. Here we used SCID-Hu (Thy/Liv) animals, co-implanted with human fetal liver and thymus, and found that these implants contain both type I and type II NKT cells, CD161+CD3+ NKT cells, NK cells and dendritic cells during HIV-infection. We found that the administration of sulfatide (bi-weekly, 20 μg/animal, i.p.) in SCID-Hu animals inhibits HIV-1 replication more efficiently than treatment with the nucleoside analog reverse transcriptase inhibitor, AZT. Virus replication was lowered significantly up to 4-8 weeks post infection. Furthermore sulfatide administration also resulted in significant retention of hematopoeisis that is lost during HIV-1 infection. Advantageously, sulfatide administration itself was not associated with anemia or bone marrow suppression, that are severe side effects of HAART. Since the CD1d-mediated immune pathway is highly conserved between rodents and humans, sulfatide treatment may represent a novel HLA-independent approach for intervention of HIV-1 pathogenesis.