Activation of natural killer T cells contributes to Th1 bias in the murine liver after 14 d of ethinylestradiol exposure

BACKGROUND As the main component of oral contraceptives(OCs),ethinylestradiol(EE)has been widely applied as a model drug to induce murine intrahepatic cholestasis.The clinical counterpart of EE-induced cholestasis includes women who are taking OCs,sex hormone replacement therapy,and susceptible pregnant women.Taking intrahepatic cholestasis of pregnancy(ICP)as an example,ICP consumes the medical system due to its high-risk fetal burden and the impotency of ursodeoxycholic acid in reducing adverse perinatal outcomes.AIM To explore the mechanisms and therapeutic strategies of EE-induced cholestasis based on the liver immune microenvironment.METHODS Male C57BL/6J mice or invariant natural killer T(iNKT)cell deficiency(Jα18-/-mice)were administered with EE(10 mg/kg,subcutaneous)for 14 d.RESULTS Both Th1 and Th2 cytokines produced by NKT cells increased in the liver skewing toward a Th1 bias.The expression of the chemokine/chemokine receptor Cxcr6/Cxcl16,toll-like receptors,Ras/Rad,and PI3K/Bad signaling was upregulated after EE administration.EE also influenced bile acid synthase Cyp7a1,Cyp8b1,and tight junctions ZO-1 and Occludin,which might be associated with EEinduced cholestasis.iNKT cell deficiency(Jα18-/-mice)robustly alleviated cholestatic liver damage and lowered the expression of the abovementioned signaling pathways.CONCLUSION Hepatic NKT cells play a pathogenic role in EE-induced intrahepatic cholestasis.Our research improves the understanding of intrahepatic cholestasis by revealing the hepatic immune microenvironment and also provides a potential clinical treatment by regulating iNKT cells.

Human CD4- CD8- Invariant Natural Killer T Cells Promote IgG Secretion from B Cells Stimulated by Cross-Linking of Their Antigen Receptors

Immunoglobulin (Ig) M production can be induced by the interaction of thymus-independent type-2 (TI-2) antigen (Ag) with B cell Ag receptors (BCRs) without the involvement of conventional T cells;for IgG production through the same process, however, a second signal is required. Previous studies have reported that invariant natural killer T (iNKT) cells may be responsible for the second signal involved in IgG production. In the present study, we addressed whether human iNKT cells could participate in the production of Ig against TI-2 Ag in vitro. Two major distinct subsets of human iNKT cells, CD4+ CD8β- (CD4) and CD4- CD8β- [double negative (DN)] cells, were generated from peripheral blood monocytes from a healthy volunteer. BCR engagement, triggered by anti-IgM antibody stimulation, examined here as a model of BCR engagement triggered by TI-2 Ag, induced abundant IgM production by B cells. Both CD4 and DN iNKT cells reduced IgM production and conversely enhanced IgG production in a dose-dependent manner. In addition, IgG production by CD19+CD27- (naïve) and CD19+CD27+ (memory) B cells was predominantly promoted by DNiNKT cells rather than CD4 iNKT cells;nevertheless, IgM production by both B cell subsets was similarly reduced by either subset of iNKT cells. These results suggest that the DN iNKT subsets may preferentially promote Ig class switching by B cells upon stimulation with TI-2 Ag.

SRSF1 plays a critical role in invariant natural killer T cell development and function

Invariant natural killer T(iNKT)cells are highly conserved innate-like T lymphocytes that originate from CD4^(+)CD8^(+)double-positive(DP)thymocytes.Here,we report that serine/arginine splicing factor 1(SRSF1)intrinsically regulates iNKT cell development by directly targeting Myb and balancing the abundance of short and long isoforms.Conditional ablation of SRSF1 in DP cells led to a substantially diminished iNKT cell pool due to defects in proliferation,survival,and TCRαrearrangement.The transition from stage 0 to stage 1 of iNKT cells was substantially blocked,and the iNKT2 subset was notably diminished in SRSF1-deficient mice.SRSF1 deficiency resulted in aberrant expression of a series of regulators that are tightly correlated with iNKT cell development and iNKT2 differentiation,including Myb,PLZF,Gata3,ICOS,and CD5.In particular,we found that SRSF1 directly binds and regulates pre-mRNA alternative splicing of Myb and that the expression of the short isoform of Myb is substantially reduced in SRSF1-deficient DP and iNKT cells.Strikingly,ectopic expression of the Myb short isoform partially rectified the defects caused by ablation of SRSF1.Furthermore,we confirmed that the SRSF1-deficient mice exhibited resistance to acute liver injury uponα-GalCer and Con A induction.Our findings thus uncovered a previously unknown role of SRSF1 as an essential post-transcriptional regulator in iNKT cell development and functional differentiation,providing new clinical insights into iNKT-correlated disease.

The in vitro proliferation and cytokine production of Vα24＋Vβ11＋ natural killer T cells in patients with systemic lupus erythematosus

Background Activation in vitro of natural killer T （NKT） cells in systemic lupus erythematosus （SLE） with a-galactosylceramide （a-GalCer） and dendritic cells （DC） may affect the immunoregulatory role of NKT cells. This study was designed to compare the number of NKT cells in patients with SLE to the number in healthy volunteers and measure the cytokines secreted from these NKT cells in vitro. Methods Three sets of culture conditions using （i） a-GalCer, （ii） DC, or （iii） both a-GalCer and DC （a-GalCer＋DC） were adopted to expand NKT cells from peripheral blood mononuclear cells （PBMC） of patients with SLE and healthy volunteers. Flow cytometry was used to assess the levels of interleukin （IL）-4, IL-10, interferon （IFN）-y and tumor necrosis factor （TNF）-a produced by the Vα24＋Vβ11＋ NKT cells. Results After 14 days in culture, the total cell count and percentage of Vα24＋Vβ11＋ NKT cells were increased under all conditions but were highest in the a-GalCer＋DC group. The level of IL-4 and IL-10 secreted by Vα24＋Vβ11＋ NKT cells from patients with active SLE was found to be higher than that of inactive patients and the control group （P 〈0.05）, while the levels of IFN-y and TNF-a were lower than those found in the inactive and control groups （P 〈0.05）. Conclusions Va24＋V^11＋ NKT cells showed the greatest expansion in vitro with a-GalCer and DC. Th2-type cytokines from Vα24＋Vβ11＋ NKT cells are the predominant type in patients with SLE, while Th 1 cytokines predominate in the control group. This evolution of NKT cell function during the progression of the disease may have important implications in understanding the mechanism of SLE and for the development of possible therapies using NKT cell agonists.

Updating targets for natural killer/T-cell lymphoma immunotherapy

Natural killer/T-cell lymphoma(NKTCL)is a highly invasive subtype of non-Hodgkin lymphoma,typically positive for cytoplasmic CD3,CD56,cytotoxic markers,including granzyme B and TIA1,and Epstein-Barr virus(EBV).The current treatment methods for NKTCL are associated with several drawbacks.For example,chemotherapy can lead to drug resistance,while treatment with radiotherapy alone is inadequate and results in frequent relapses.Moreover,hematopoietic stem cell transplantation exhibits limited efficacy and is not well recognized by domestic and foreign experts.In recent years,immunotherapy has shown good clinical results and has become a hot spot in cancer research.Clinical activity of targeted antibodies,such as daratumumab(anti-CD38 antibody)and brentuximab vedotin(anti-CD30 antibody),have been reported in NKTCL.Additionally,dacetuzumab and Campath-1 H have demonstrated promising results.Further encouraging data have been obtained using checkpoint inhibitors.The success of these immunotherapy agents is attributed to high expression levels of programmed death-ligand 1 in NKTCL.Furthermore,anti-CCR4 monoclonal antibodies(m Abs)exert cytotoxic actions on both CCR4+tumor cells and regulatory T cells.Depletion of these cells and the long half-life of anti-CCR4 m Abs result in enhanced induction of antitumor effector T cells.The role of IL10 in NKTCL has also been investigated.It has been proposed that exploitation of this cytokine might provide potential novel therapeutic strategies.Cellular immunotherapy with engineered cytotoxic T lymphocytes targeted against LMP1 and LMP2 has shown promising results and sustained remission.Cellular immunotherapy may be used either as maintenance therapy following initial induction chemotherapy or in cases of relapsed/refractory disease.The present review outlines the known immunotherapy targets for the treatment of NKTCL.

Muscular involvement of extranodal natural killer/T cell lymphoma misdiagnosed as polymyositis: A case report and review of literature

BACKGROUND Natural killer(NK)/T cell lymphoma is a rare and highly aggressive malignant tumor,and is a special form of non-Hodgkin's lymphoma.Although extranodal involvement is frequently found in tissues such as the skin,testicular and gastrointestinal tract etc,its presence in skeletal muscle has scarcely been reported in the literature.CASE SUMMARY We report a case of extranodal NK/T cell lymphoma with muscle swelling as the first clinical manifestation.A 42-year-old man,who initially presented with localized swelling in the double lower extremities,demonstrated gradual facial and eyelid swelling,and his imaging results showed multiple sites of muscle damage throughout the body.The final pathological results suggested NK/T cell lymphoma,and immunohistochemistry showed CD20(-),CD3(+),CD30(+),CD56(-),EBER(+),Ki67(60%),TIA-1(+)and CD68(±)staining.The muscle swelling significantly improved after treatment with chemotherapy regimens.CONCLUSION This disease is difficult to diagnose and highly invasive,and should be included in the differential diagnosis of unexplained muscle swelling.

Roles of liver innate immune cells in nonalcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) has become the most common liver disease in the United States and other developed countries and is expected to increase in the next few years. Emerging data suggest that some patients with NAFLD may progress to nonalcoholic steatohepatitis (NASH), cirrhosis and even hepatocellular carcinoma. NAFLD can also promote the development and progression of disease in other organ systems, such as the cardiovascular and endocrine (i.e. diabetes) systems. Thus, understanding the pathogenesis of NAFLD is of great clinical importance and is critical for the prevention and treatment of the disease. Although the "two-hit hypothesis" is generally accepted, the exact pathogenesis of NAFLD has not been clearly established. The liver is an important innate immune organ with large numbers of innate immune cells, including Kupffer cells (KCs), natural killer T (NKT) cells and natural killer (NK) cells. Recent data show that an imbalance in liver cytokines may be implicated in the development of fatty liver disease. For example, Th1 cytokine excess may be a common pathogenic mechanism for hepatic insulin resistance and NASH. Innate immune cells in the liver play important roles in the excessive production of hepatic Th1 cytokines in NAFLD. In addition, liver innate immune cells participate in the pathogenesis of NAFLD in other ways. For example, activated KCs can generate reactive oxygen species, which induce liver injury. This review will focus primarily on the possible effect and mechanism of KCs, NKT cells and NK cells in the development of NAFLD.

Evaluatingα-galactosylceramide as an adjuvant for live attenuated infuenza vaccines in pigs

Natural killer T(NKT)cells activated with the glycolipid ligandα-galactosylceramide(α-GalCer)stimulate a wide variety of immune cells that enhance vaccine-mediated immune responses.Several studies have used this approach to adjuvant inactivated and subunit infuenza A virus(IAV)vaccines,including to enhance cross-protective infuenza immunity.However,less is known about whetherα-GalCer can enhance live attenuated infuenza virus(LAIV)vaccines,which usually induce superior heterologous and heterosubtypic immunity compared to non-replicating infuenza vaccines.The current study used the swine infuenza challenge model to assess whetherα-GalCer can enhance cross-protective immune responses elicited by a recombinant H3N2 LAIV vaccine(TX98ΔNS1)encoding a truncated NS1 protein.In one study,weaning pigs were administered the H3N2 TX98ΔNS1 LAIV vaccine with 0,10,50,and 100μg/kg doses ofα-GalCer,and subsequently challenged with a heterologous H3N2 virus.All treatment groups were protected from infection.However,the addition ofα-GalCer appeared to suppress nasal shedding of the LAIV vaccine.In another experiment,pigs vaccinated with the H3N2 LAIV,with or without 50μg/kg ofα-GalCer,were challenged with the heterosubtypic pandemic H1N1 virus.Pigs vaccinated with the LAIV alone generated cross-reactive humoral and cellular responses which blocked virus replication in the airways,and signifcantly decreased virus shedding.On the other hand,combining the vaccine withα-GalCer reduced cross-protective cellular and antibody responses,and resulted in higher virus titers in respiratory tissues.These fndings suggest that:(i)high doses ofα-GalCer impair the replication and nasal shedding of the LAIV vaccine;and(ii)α-GalCer might interfere with heterosubtypic cross-protective immune responses.This research raise concerns that should be considered before trying to use NKT cell agonists as a possible adjuvant approach for LAIV vaccines.

NKT cells in liver diseases

Natural killer T cells are innate-like and tissue-resident lymphocytes, which recognize lipid antigens and are enriched in the liver. Natural killer T cells play important roles in infections, tumors, autoimmune diseases, and metabolic diseases. In this study, we summarize recent findings on biology of natural killer T cells and their roles in hepatitis B virus and hepatitis C virus infection, autoimmune liver diseases, alcoholic liver disease, nonalcoholic fatty liver disease, and hepatocellular carcinoma. Controversial results from previous studies are discussed, and indicate the dynamic alteration in the role of natural killer T cells during the progression of liver diseases, which might be caused by changes in natural killer T subsets, factors skewing cytokine responses, and intercellular crosstalk between natural killer T cells and CDld-expressing cells or bystander cells.

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types.Although a role for cancer cell invasion programs has been well characterized,whether and how liver-intrinsic factors drive metastatic spread is incompletely understood.Here,we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase(CCRK)signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment.Using an inducible liverspecific transgenic model,we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer(CRC)metastasis to the liver,which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells(PMNMDSCs)and lacking natural killer T(NKT)cells.Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7%(metastatic tumor weights)in the melanoma and CRC models,respectively.Mechanistically,CCRK activated nuclear factor-kappa B(NF-κB)signaling to increase the PMN-MDSC trafficking chemokine C-X-C motif ligand 1(CXCL1),which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice.Accordingly,CRC liver metastasis patients exhibited hyperaaivation of hepatic CCRK/NF-κB/CXCL1 signaling,which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors.In summary,this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance.Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

MicroRNAs are key regulators controlling iNKT and regulatory T-cell development and function

MicroRNAs(miRNAs)are an abundant class of evolutionarily conserved,small,non-coding RNAs that post-transcriptionally regulate expression of their target genes.Emerging evidence indicates that miRNAs are important regulators that control the development,differentiation and function of different immune cells.Both CD4^(+)CD25^(+)Foxp3^(+) regulatory T(Treg)cells and invariant natural killer T(iNKT)cells are critical for immune homeostasis and play a pivotal role in the maintenance of self-tolerance and immunity.Here,we review the important roles of miRNAs in the development and function of iNKT and Treg cells.

自然杀伤T细胞在抗感染免疫中的作用研究进展

自然杀伤T细胞（NKT）是一类同时表达T细胞受体（TCR）dB和NK细胞表面标志的T细胞亚群，能识别由抗原提呈细胞（APC）表面CD1d分子提呈的脂类抗原。NKT细胞经抗原刺激后能迅速分泌大量的细胞因子（如IFN-1和IL-4），调节固有免疫和适应性免疫，还能直接杀伤靶细胞，具有效应细胞的功能，在机体抗感染、肿瘤和自身免疫病方面发挥重要作用。