[Effect of Spatholobi Caulis extract from ethyl acetate on immune killing function of NK cells].

This study aims to investigate the regulatory effect of the Spatholobi Caulis extract from ethyl acetate(SEA) on natural killer(NK) cells under physiological conditions and elucidate the underlying mechanism. The C57BL/6 mice were randomized into NC and SEA groups, and NK-92 cells were respectively treated with 0, 25, 50, and 100 µg·mL~(-1) SEA. The body weight and immune organ index of the mice were compared between groups. The lactate dehydrogenase(LDH) assay was employed to examine the cytotoxicity of NK-92 cells treated with SEA and the killing activity of mouse NK cells against YAC-1 cells. The cell-counting kit-8(CCK-8) was used to examine the impact of SEA on the proliferation of NK-92 cells. Flow cytometry was employed to measure the number of NK cells in the peripheral blood as well as the expression levels of natural killer group 2 member A(NKG2A) and natural killer group 2 member D(NKG2D). The enzyme-linked immunosorbent assay(ELISA) was performed to determine the interferon(IFN)-γ secretion in the serum. Semi-quantitative PCR was conducted to determine the mRNA levels of NKG2A, NKG2D, and IFN-γ in spleen cells. Western blot was employed to investigate the involvement of phosphoinositide 3-kinase(PI3K)/extracellular regulated protein kinase 1(ERK1) signaling pathway. The results showed that SEA exhibited no adverse effects on the body, while significantly enhance the number of NK cells and augment the cytotoxicity of NK-92 cells against YAC-1 cells. Moreover, it suppressed the expression of NKG2A, enhanced the expression of NKG2D, promoted IFN-γ secretion, and upregulated the protein levels of PI3K and ERK. The findings suggest that SEA has the potential to enhance the immune recognition and effector function of NK cells by increasing the cell number, modulating the expression of functional receptors, and promoting IFN-γ secretion via the PI3K/ERK signaling pathway.

Group 1 innate lymphoid cell activation via recognition of NKG2D and liver resident macrophage MULT-1: Collaborated roles in triptolide induced hepatic immunotoxicity in mice.

Triptolide (TP) is the major bioactive component of traditional Chinese medicine Tripterygium wilfordii Hook. F., a traditional Chinese medicinal plant categorized within the Tripterygium genus of the Celastraceae family. It is recognized for its therapeutic potential in addressing a multitude of diseases. Nonetheless, TP is known to exhibit multi-organ toxicity, notably hepatotoxicity, which poses a significant concern for the well-being of patients undergoing treatment. The precise mechanisms responsible for TP-induced hepatotoxicity remain unresolved. In our previous investigation, it was determined that TP induces heightened hepatic responsiveness to exogenous lipopolysaccharide (LPS). Additionally, natural killer (NK) cells were identified as a crucial effector responsible for mediating hepatocellular damage in this context. However, associated activating receptors and the underlying mechanisms governing NK cell represented innate lymphoid cell (ILC) activation remained subjects of inquiry and were not yet investigated. Herein, activating receptor Killer cell lectin like receptor K1 (NKG2D) of group 1 ILCs was specifically upregulated in TP- and LPS-induced acute liver failure (ALF), and in vivo blockade of NKG2D significantly reduced group 1 ILC mediated cytotoxicity and mitigated TP- and LPS-induced ALF. NKG2D ligand UL16-binding protein-like transcript 1 (MULT-1) was found upregulated in liver resident macrophages (LRMs) after TP administration, and LRMs did exhibit NK cell activating effect. Furthermore, M1 polarization of LRMs cells was observed, along with an elevation in intracellular tumor necrosis factor (TNF)-α levels. In vivo neutralization of TNF-α significantly alleviated TP- and LPS-induced ALF. In conclusion, the collaborative role of group 1 ILCs and LRMs in mediating hepatotoxicity was confirmed in TP- and LPS-induced ALF. TP-induced MULT-1 expression in LRMs was the crucial mechanism in the activation of group 1 ILCs via MULT-1-NKG2D signal upon LPS stimulation, emphasizing the importance of infection control after TP administration.

Lack of NKG2D in MAGT1-deficient patients is caused by hypoglycosylation.

Mutations in the X-linked gene MAGT1 cause a Congenital Disorder of Glycosylation (CDG), with two distinct clinical phenotypes: a primary immunodeficiency (XMEN disorder) versus intellectual and developmental disability. It was previously established that MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (encoded by KLRK1) in lymphocytes. Here, we show that the reduced steady-state levels of NKG2D are caused by hypoglycosylation of the protein and we pinpoint the exact site that is underglycosylated in MAGT1-deficient patients. Furthermore, we challenge the possibility that supplementation with magnesium restores NKG2D levels and show that the addition of this ion does not significantly improve NKG2D steady-state expression nor does it rescue the hypoglycosylation defect in CRISPR-engineered human cell lines. Moreover, magnesium supplementation of an XMEN patient did not result in restoration of NKG2D expression on the cell surface of lymphocytes. In summary, we demonstrate that in MAGT1-deficient patients, the lack of NKG2D is caused by hypoglycosylation, further elucidating the pathophysiology of XMEN/MAGT1-CDG.

Exercise Benefits Meet Cancer Immunosurveillance: Implications for Immunotherapy.

Regular exercise reduces the risk of cancer. One potential mechanism for this efficacy is improved antitumor immunity. This is an important issue because evading immune destruction is a hallmark of cancer and immunotherapy is reshaping cancer treatment. Here we review recent developments reported by Wennerberg et al., Garritson et al., Martín-Ruiz et al., and Rundqvist et al. on the effects of exercise on anticancer immune cell effectors.

Long-chain polyunsaturated omega-3 fatty acids reduce multiple myeloma exosome-mediated suppression of NK cell cytotoxicity.

BACKGROUND: Despite the advances in the treatment of multiple myeloma (MM), complete remission is usually challenging. The interactions between tumor and host cells, in which exosomes (EXs) play critical roles, have been shown to be among the major deteriorative tumor-promoting factors herein. Therefore, any endeavor to beneficially target these EX-mediated interactions could be of high importance. OBJECTIVES: a) To investigate the effects of myeloma EXs on natural killer (NK) cell functions. b) To check whether treatment of myeloma cells with eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), two polyunsaturated omega-3 fatty acids with known anti-cancer effects, can modify myeloma EXs in terms of their effects on natural killer functions. METHODS: L363 cells were treated with either EPA or DHA or left untreated and the released EXs (designated as E-EX, D-EX and C-EX, respectively) were used to treat NK cells for functional studies. RESULTS: Myeloma EXs (C-EXs) significantly reduced NK cytotoxicity against K562 cells (P ≤ 0.05), while the cytotoxicity suppression was significantly lower (P ≤ 0.05) in the (E-EX)- and (D-EX)-treated NK cells compared to the (C-EX)-treated cells. The expression of the activating NK receptor NKG2D and NK degranulation, after treatment with the EXs, were both altered following the same pattern. However, C-EXs could increase IFN-γ production in NK cells (P < 0.01), which was not significantly affected by EPA/DHA treatment. This indicates a dual effect of myeloma EXs on NK cells functions. CONCLUSION: Our observations showed that myeloma EXs have both suppressive and stimulatory effects on different NK functions. Treatment of myeloma cells with EPA/DHA can reduce the suppressive effects of myeloma EXs while maintaining their stimulatory effects. These findings, together with the previous findings on the anti-cancer effects of EPA/DHA, provide stronger evidence for the repositioning of the currently existing EPA/DHA supplements to be used in the treatment of MM as an adjuvant treatment. EXs released from L363 (myeloma) cells in their steady state increase IFN-γ production of NK cells, while reduce their cytotoxicity against the K562 cell line (right blue trace). EXs from L363 cells pre-treated with either EPA or DHA are weaker stimulators of IFN-γ production. These EXs also increase NK cytotoxicity and NKG2D expression (left brown trace) compared to the EXs obtained from untreated L363 cells. Based on these findings, myeloma EXs have both suppressive and stimulatory effects on different NK functions depending on the properties of their cells of origin, which can be exploited in the treatment of myeloma.

Kupffer Cells Regulate Natural Killer Cells Via the NK group 2, Member D (NKG2D)/Retinoic Acid Early Inducible-1 (RAE-1) Interaction and Cytokines in a Primary Biliary Cholangitis Mouse Model.

BACKGROUND Kupffer cells and natural killer (NK) cells has been identified as contributing factors in the pathogenesis of hepatitis, but the detailed mechanism of these cell types in the pathogenesis of primary biliary cholangitis (PBC) is poorly understood. MATERIAL AND METHODS In this study, polyinosinic: polycytidylic acid (poly I: C), 2-octynoic acid-bovine serum albumin (2OA-BSA) and Freund's adjuvant (FA) were injected to establish a murine PBC model, from which NK cells and Kupffer cells were extracted and isolated. The cells were then co-cultivated in a designed culture system, and then NK group 2, member D (NKG2D), retinoic acid early inducible-1 (RAE-1), F4/80, and cytokine expression levels were detected. RESULTS The results showed close crosstalk between Kupffer cells and NK cells. PBC mice showed increased surface RAE-1 protein expression and Kupffer cell cytokine secretion, which subsequently activated NK cell-mediated target cell killing via NKG2D/RAE-1 recognition, and increased inflammation. NK cell-derived interferon-γ (IFN-γ) and Kupffer cell-derived tumor necrosis factor alpha (TNF-alpha) were found to synergistically regulate inflammation. Moreover, interleukin (IL)-12 and IL-10 improved the crosstalk between NK cells and Kupffer cells. CONCLUSIONS Our findings in mice are the first to suggest the involvement of the NKG2D/RAE-1 interaction and cytokines in the synergistic effects of NK and Kupffer cells in PBC.

Diversity of XMEN Disease: Description of 2 Novel Variants and Analysis of the Lymphocyte Phenotype.

Variants in MAGT1 have been identified as the cause of an immune deficiency termed X-linked immunodeficiency with magnesium defect, Epstein-Barr virus (EBV) infection and neoplasia (XMEN) disease. Here, we describe 2 cases of XMEN disease due to novel mutations in MAGT1, one of whom presented with classical features of XMEN disease and another who presented with a novel phenotype including probable CNS vasculitis, HHV-8 negative multicentric Castelman disease and severe molluscum contagiosum, thus highlighting the clinical diversity that may be seen in this condition. Peripheral blood immunophenotyping of these 2 patients, together with an additional 4 XMEN patients, revealed reduced NKG2D expression, impaired CD28 expression on CD8+ T cells, CD4+ T cell lymphopenia, an inverted CD4:CD8 ratio and decreased memory B cells. In addition, we showed for the first time alterations to the CD8+ T cell memory compartment, reduced CD56hi NK cells, MAIT and iNKT cells, as well as compromised differentiation of naïve CD4+ T cells into IL-21-producing Tfh-type cells in vitro. Both patients were treated with supplemental magnesium with limited benefit. However, one patient has undergone allogeneic haematopoietic stem cell transplant, with full donor chimerism and immune reconstitution. These results expand our understanding of the clinical and immunological phenotype in XMEN disease, adding to the current literature, which we further discuss here.

Selenium nanoparticles as new strategy to potentiate γδ T cell anti-tumor cytotoxicity through upregulation of tubulin-α acetylation.

Immune cell therapy presents a paradigm for the treatment of malignant tumors. Human Vγ9Vδ2 T cells, a subset of peripheral γδ T cells, have been shown to have promising anti-tumor activity. However, new methodology on how to achieve a stronger anti-tumor activity of Vγ9Vδ2 T cells is under continuous investigation. In this work, we used selenium nanoparticles (SeNPs) to strengthen the anti-tumor cytotoxicity of Vγ9Vδ2 T cells. We found SeNPs pretreated γδ T cells had significantly stronger cancer killing and tumor growth inhibition efficacy when compared with γδ T cells alone. Simultaneously, SeNPs pretreatment could significantly upregulate the expression of cytotoxicity related molecules including NKG2D, CD16, and IFN-γ, meanwhile, downregulate PD-1 expression of γδ T cells. Importantly, we observed that SeNPs promoted tubulin acetylation modification in γδ T cells through interaction between microtubule network and lysosomes since the latter is the primary resident station of SeNPs shown by confocal visualization. In conclusion, SeNPs could significantly potentiate anti-tumor cytotoxicity of Vγ9Vδ2 T cells, and both cytotoxicity related molecules and tubulin acetylation were involved in fine-tuning γδ T cell toxicity against cancer cells. Our present work demonstrated a new strategy for further enhancing anti-tumor cytotoxicity of human Vγ9Vδ2 T cells by using SeNPs-based nanotechnology, not gene modification, implicating SeNPs-based nanotechnology had a promising clinical perspective in the γδ T cell immunotherapy for malignant tumors.

Immunomodulatory and Metabolic Changes after Gnetin-C Supplementation in Humans.

Gnetin-C is a naturally occurring stilbene derived from the seeds of Gnetum gnemon L., an edible plant native to Southeast Asia that is called melinjo. Although the biological properties and safety of G. gnemon extract, which contains nearly 3% Gnetin-C, have been confirmed in various human studies, whether or not pure Gnetin-C is safe for humans is unclear at present. We conducted a randomized, double-blind, placebo-controlled trial. Healthy subjects were randomly divided into two groups. The interventional group (n = 6) was given Gnetin-C, and the control group (n = 6) was provided a placebo, for 14 days. Lipid profiles, biomarkers of oxidative stress and circulating blood cells were assessed before and after the intervention. All subjects completed the study, with no side effects reported across the study duration. Gnetin-C supplementation demonstrated a statistically significant increase in the absolute number of circulating natural killer (NK) cells expressing the activating receptors NKG2D and NKp46. NK cells derived from subjects who received Gnetin-C for two weeks showed higher cytotoxicity against K562 target cells than those before receiving Gnetin-C. In addition, Gnetin-C also resulted in a significant decrease in the absolute neutrophil count in the blood compared with the placebo. Furthermore, Gnetin-C significantly reduced the levels of uric acid, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, total adiponectin, and high-molecular-weight adiponectin. These data indicate that Gnetin-C has biological effects of enhancing the NK activity on circulating human immune cells. The immunomodulatory effects are consistent with a putative improvement in cancer immunosurveillance via the upregulation of the NKG2D receptor. The study was registered with UMIN-CTR, number 000030364, on 12 December 2017.

CD8+ T cells mediate the antitumor activity of frankincense and myrrh in hepatocellular carcinoma.

BACKGROUND: Tumor-promoting inflammation is an emerging hallmark of cancer, which participates in both cancer progression and immune escape. Hepatocellular carcinoma (HCC) is a typical inflammation-related cancer with an extremely poor prognosis. Frankincense and myrrh are anti-inflammation agents commonly used in clinic. The purpose of this study is to investigate whether extract of frankincense and myrrh (FM) downregulates inflammatory microenvironment of HCC and thereby restores antitumor immune responses. METHODS: The water-decocting FM was obtained and quantified. HCC cell lines HCCLM3 and Hepa1-6 were used to evaluate the efficacy of FM targeting NF-κB and STAT3 signaling with western blot and qRT-PCR analysis. CD8+NKG2D+ cells were derived from human peripheral blood and were used for evaluation of immune cells-mediated inflammation and oncolysis on HCCLM3 cells. The antitumor efficacy of FM was investigated both in immune compromised and immune competent mice bearing subcutaneous HCC. Mice received daily oral gavage of FM at 60 mg/kg. Immune activity within tumor microenvironment (TME) was assessed by ELISpot assay and flow cytometry, respectively. Depletion of CD8+ T cells or NK cells was achieved by intraperitoneal injection of respective neutralizing antibody. RESULTS: FM significantly inhibited the activation of NF-κB and STAT3 signaling in HCC cells induced by cytokines (TNF-α or IL-6) and in co-culture system with CD8+NKG2D+ cells. Furthermore, FM sensitized HCC cells to CD8+NKG2D+ cells-mediated oncolysis. In HCC-bearing mice, FM at a non-toxic dose failed to reduce tumor growth in immune compromised mice, whereas it significantly inhibited tumor growth and prolonged life span in immune competent mice. While the number of IFN-γ-producing cells within TME was increased in mice treated with FM, the infiltration of CD8+ T cells and NK cells was not increased. Finally, we identified that depletion of CD8+ T cells rather than NK cells abrogated the antitumor activity of FM. CONCLUSIONS: Our results show for the first time that CD8+ T cells mediate the antitumor activity of FM at a non-toxic dose. This may provide new insights to this ancient mysterious prescription in cancer therapy, which offers a novel and practical therapeutic strategy and the possibilities of combined immunotherapy for HCC as well as other inflammation-related cancers in clinic.

Differential effect of dietary vitamin D supplementation on natural killer cell activity in lean and obese mice.

Vitamin D has an immunoregulatory effect on both innate and adaptive immunity. Contradictory results regarding vitamin D and natural killer (NK) cell functions have been reported with in vitro studies, but little is known about this in vivo. We investigated whether vitamin D levels (50, 1000 or 10,000 IU/kg of diet: DD, DC or DS) affect NK cell functions in mice fed a control or high-fat diet (10% or 45% kcal fat: CD or HFD) for 12 weeks. The splenic NK cell activity was significantly higher in the CD-DS group than the HFD-DS group, and the CD-DS group showed significantly higher NK cell activity compared with the CD-DD and CD-DC groups. However, no difference in NK cell activity was observed among the HFD groups fed different levels of vitamin D. The splenic population of NK cells was significantly higher in the CD-DS group than the HFD-DS group. There was no difference in the intracellular expression of IFN-γ and the surface expression of NKG2D and CD107a in NK cells by both dietary fat and vitamin D content. The splenic mRNA expression of Ifng and Ccl5 was significantly lower in the HFD groups compared with the CD groups, but there was no difference in the mRNA levels of Vdup1 and Vdr among the groups. Taken together, these results suggest that dietary vitamin D supplementation can modulate innate immunity by increasing NK activity in control mice but not in obese mice. This effect might be mediated through alternation of the splenic NK cell population.

Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis.

Natural-killer receptor group 2, member D (NKG2D) is a well characterized natural killer (NK) cell activating receptor that recognizes several ligands poorly expressed on healthy cells but up-regulated upon stressing stimuli in the context of cancer or viral infection. Although NKG2D ligands represent danger signals that render target cells more susceptible to NK cell lysis, accumulating evidence demonstrates that persistent exposure to ligand-expressing cells causes the decrease of NKG2D surface expression leading to a functional impairment of NKG2D-dependent NK cell functions. Upon ligand binding, NKG2D is internalized from the plasma membrane and sorted to lysosomes for degradation. However, receptor endocytosis is not only a mechanism of receptor clearance from the cell surface, but is also required for the proper activation of signalling events leading to the functional program of NK cells. This review is aimed at providing a summary of current literature relevant to the molecular mechanisms leading to NKG2D down-modulation with particular emphasis given to the role of NKG2D endocytosis in both receptor degradation and signal propagation. Examples of chronic ligand-induced down-regulation of NK cell activating receptors other than NKG2D, including natural cytotoxicity receptors (NCRs), DNAX accessory molecule-1 (DNAM1) and CD16, will be also discussed.

Exploiting natural killer group 2D receptors for CAR T-cell therapy.

Chimeric antigen receptors (CARs) are genetically engineered proteins that combine an extracellular antigen-specific recognition domain with one or several intracellular T-cell signaling domains. When expressed in T cells, these CARs specifically trigger T-cell activation upon antigen recognition. While the clinical proof of principle of CAR T-cell therapy has been established in hematological cancers, CAR T cells are only at the early stages of being explored to tackle solid cancers. This special report discusses the concept of exploiting natural killer cell receptors as an approach that could broaden the specificity of CAR T cells and potentially enhance the efficacy of this therapy against solid tumors. New data demonstrating feasibility of this approach in humans and supporting the ongoing clinical trial are also presented.

Tetramethypyrazine inhibits renal cell carcinoma cells through inhibition of NKG2D signaling pathways.

Tetramethypyrazine (TMP), one of the active compounds extracted from the traditional Chinese medicinal herb (Chuanxiong), has been verified as an anticancer compound against several types of cancer. However, understanding of the molecular mechanisms have not been fully elucidated. In the present study, the anticancer efficacy of TMP was investigated in human clear cell renal cell carcinoma (ccRCC) cells. We showed that TMP significantly inhibited ccRCC cell viability, proliferation, apoptosis, invasion and migration through the methods of MTT, flow cytometry, wound healing and transwell assays. Furthermore, reverse transcription polymerase chain reaction (RT-PCR), western blotting and immunofluorescence results demonstrated TMP upregulation of the expression of NKG2D ligands (NKG2DLs) MHC class I chain-related molecules A and B (MICA/B) and epithelial cell expression marker of E-cadherin, and downregulation of mesenchymal cell expression markers of vimentin and fibronectin. Taken together, the inhibition of TMP on ccRCC cells might be mediated via inhibition of NKG2D related signaling pathway to further suppress epithelial-mesenchymal transition (EMT) progression. The binding of NKG2D to its ligands activates NK cells, giving the rationale for studies on the utilization of TMP as a potential cancer therapeutic compound to increase NK cells-mediated cytotoxicity against high MICA/B expression in cancer cells.

Immunomodulatory properties of medicinal mushrooms: differential effects of water and ethanol extracts on NK cell-mediated cytotoxicity.

Medicinal mushrooms have been used for centuries in Asian countries owing to their beneficial effects on health and longevity. Previous studies have reported that a single medicinal mushroom may produce both stimulatory and inhibitory effects on immune cells, depending on conditions, but the factors responsible for this apparent dichotomy remain obscure. We show here that water and ethanol extracts of cultured mycelium from various species (Agaricus blazei Murrill, Antrodia cinnamomea, Ganoderma lucidum and Hirsutella sinensis) produce opposite effects on NK cells. Water extracts enhance NK cell cytotoxic activity against cancer cells, whereas ethanol extracts inhibit cytotoxicity. Water extracts stimulate the expression and production of cytolytic proteins (perforin and granulysin) and NKG2D/NCR cell surface receptors, and activate intracellular signaling kinases (ERK, JNK and p38). In contrast, ethanol extracts inhibit expression of cytolytic and cell surface receptors. Our results suggest that the mode of extraction of medicinal mushrooms may determine the nature of the immunomodulatory effects produced on immune cells, presumably owing to the differential solubility of stimulatory and inhibitory mediators. These findings have important implications for the preparation of medicinal mushrooms to prevent and treat human diseases.

IL-30 (IL27p28) attenuates liver fibrosis through inducing NKG2D-rae1 interaction between NKT and activated hepatic stellate cells in mice.

UNLABELLED: Chronic hepatic diseases, such as cirrhosis, hepatocellular carcinoma, and virus-mediated immunopathogenic infections, affect billions of people worldwide. These diseases commonly initiate with fibrosis. Owing to the various side effects of antifibrotic therapy and the difficulty of diagnosing asymptomatic patients, suitable medication remains a major concern. To overcome this drawback, the use of cytokine-based sustained therapy might be a suitable alternative with minimal side effects. Here, we studied the therapeutic efficacy and potential mechanisms of interleukin (IL)-30 as antifibrosis therapy in murine liver fibrosis models. CCl4 or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) 0.1% (wt/wt) Purina 5015 Chow (LabDiet, St. Louis, MO) was fed for 3 weeks to induce liver fibrosis. Either control vector (pCtr) or pIL30 was injected hydrodynamically once per week. A significant decrease in collagen deposition and reduced expression of alpha-smooth muscle actin (α-SMA) protein indicated that IL-30-based gene therapy dramatically reduced bridging fibrosis that was induced by CCl4 or DDC. Immunophenotyping and knockout studies showed that IL-30 recruits natural-killer-like T (NKT) cells to the liver to remove activated hepatic stellate cells (HSCs) significantly and ameliorate liver fibrosis. Both flow cytometric and antibody-mediated neutralization studies showed that liver NKT cells up-regulate the natural killer group 2, member D (NKG2D) ligand and bind with the NKG2D ligand, retinoic acid early inducible 1 (Rae1), and positively activated HSCs to ameliorate liver fibrosis. Furthermore, adoptive transfer of liver NKT cells in T-cell-deficient mice showed reduction of fibrosis upon IL-30 administration. CONCLUSIONS: Highly target-specific liver NKT cells selectively remove activated HSCs through an NKG2D-Rae1 interaction to ameliorate liver fibrosis after IL-30 treatment.

The selenium metabolite methylselenol regulates the expression of ligands that trigger immune activation through the lymphocyte receptor NKG2D.

For decades, selenium research has been focused on the identification of active metabolites, which are crucial for selenium chemoprevention of cancer. In this context, the metabolite methylselenol (CH3SeH) is known for its action to selectively kill transformed cells through mechanisms that include increased formation of reactive oxygen species, induction of DNA damage, triggering of apoptosis, and inhibition of angiogenesis. Here we reveal that CH3SeH modulates the cell surface expression of NKG2D ligands. The expression of NKG2D ligands is induced by stress-associated pathways that occur early during malignant transformation and enable the recognition and elimination of tumors by activating the lymphocyte receptor NKG2D. CH3SeH regulated NKG2D ligands both on the transcriptional and the posttranscriptional levels. CH3SeH induced the transcription of MHC class I polypeptide-related sequence MICA/B and ULBP2 mRNA. However, the induction of cell surface expression was restricted to the ligands MICA/B. Remarkably, our studies showed that CH3SeH inhibited ULBP2 surface transport through inhibition of the autophagic transport pathway. Finally, we identified extracellular calcium as being essential for CH3SeH regulation of NKG2D ligands. A balanced cell surface expression of NKG2D ligands is considered to be an innate barrier against tumor development. Therefore, our work indicates that the application of selenium compounds that are metabolized to CH3SeH could improve NKG2D-based immune therapy.

α-1-Antitrypsin (AAT)-modified donor cells suppress GVHD but enhance the GVL effect: a role for mitochondrial bioenergetics.

Hematopoietic cell transplantation is curative in many patients. However, graft-versus-host disease (GVHD), triggered by alloreactive donor cells, has remained a major complication. Here, we show an inverse correlation between plasma α-1-antitrypsin (AAT) levels in human donors and the development of acute GVHD in the recipients (n = 111; P = .0006). In murine models, treatment of transplant donors with human AAT resulted in an increase in interleukin-10 messenger RNA and CD8(+)CD11c(+)CD205(+) major histocompatibility complex class II(+) dendritic cells (DCs), and the prevention or attenuation of acute GVHD in the recipients. Ablation of DCs (in AAT-treated CD11c-DTR donors) decreased CD4(+)CD25(+)FoxP3(+) regulatory T cells to one-third and abrogated the anti-GVHD effect. The graft-versus-leukemia (GVL) effect of donor cells (against A20 tumor cells) was maintained or even enhanced with AAT treatment of the donor, mediated by an expanded population of NK1.1(+), CD49B(+), CD122(+), CD335(+) NKG2D-expressing natural killer (NK) cells. Blockade of NKG2D significantly suppressed the GVL effect. Metabolic analysis showed a high glycolysis-high oxidative phosphorylation profile for NK1.1(+) cells, CD4(+)CD25(+)FoxP3(+) T cells, and CD11c(+) DCs but not for effector T cells, suggesting a cell type-specific effect of AAT. Thus, via altered metabolism, AAT exerts effective GVHD protection while enhancing GVL effects.

[Autologous CIK cell infusion promotes amplification ability of CD3⁺ CD56⁺ cells when re-preparation from patients with malignant tumors].

OBJECTIVE: To investigate the effects of the autologous cytokine-induced killer (CIK) cell infusion on the subpopulation distribution and activity of the CIK cells from the patients with malignant tumors when prepared in the same liquid culture system again. METHODS: A total of 201 patients who gave a written consent and received 2 courses of therapeutic infusion of CIK cells were divided into ≤ 90-day group in which the secondary preparation of CIK cells was performed in less than 90 days after the primary infusion and >90-day group in which the secondary preparation of CIK cells was performed in more than 90 days after the primary infusion. The proliferation and subtypes, including CD3⁺ cells, CD3⁺ CD4⁺ cells, CD3⁺ CD8⁺ cells, and CD3⁺ CD56⁺ cells, of CIK cells were analyzed by hemocytometer with trypan blue exclusion and flow cytometry, respectively. The expression of NKG2D receptor was also detected using flow cytometry. The cytotoxicity against K562 cells was analyzed using lactate dehydrogenase (LDH) release. RESULTS: The percentage of CD3⁺ CD56⁺ cell subpopulation in the secondary preparation of CIK cells in ≤ 90-day group [(16.7 ± 9.1)%] was higher than that in the primary preparation of CIK cells [(13.5 ± 8.6)%] (P<0.01). Furthermore, the percentage of NKG2D in the secondary CIK cell preparation [(84.1 ± 10.8)%] was significantly higher than that in the primary CIK cell preparation [(81.1 ± 14.8)%] in ≤ 90-day group (P<0.05). In contrast, the percentage of CD3⁺ CD4⁺ cells in the secondary CIK cell preparation [(15.2 ± 9.7)%] was significantly lower than that in the primary CIK cell preparation [(17.6 ± 12.5)%] (P<0.01). However, no significant differences in CD3⁺ CD56⁺ cell subpopulation and expression of NKG2D was detected between the primary and secondary CIK cell preparation in >90 d group, although the percentage of CD3⁺ CD4⁺ cells in the secondary CIK cell preparation [(14.5 ± 9.4)%] was significantly lower than that in the primary CIK cell preparation [(18.2 ± 12.9)%] (P<0.01). In addition, no significant differences in total cell number and cytotoxic activity against K562 cells between the primary and secondary CIK cell preparation was detected either in >90-day group or in ≤ 90-day group. CONCLUSION: CIK cell infusion can facilitate and enhance the proliferation and differentiation of the precursor cells of the CD3⁺ CD56⁺ subpopulation in the same CIK cell culture system and this effect does not last more than 90 days, suggesting that the secondary CIK cell infusion should be performed within 90 days in order to obtain the better therapeutic efficacy.

Collagen-induced arthritis: severity and immune response attenuation using multivalent N-acetyl glucosamine.

Rheumatoid arthritis is an autoimmunity leading to considerable impairment of quality of life. N-acetyl glucosamine (GlcNAc) has been described previously as a potent modulator of experimental arthritis in animal models and is used for osteoarthritis treatment in humans, praised for its lack of adverse effects. In this study we present a comprehensive immunological analysis of multivalent GlcNAc-terminated glycoconjugate (GC) application in the treatment of collagen-induced arthritis (CIA) and its clinical outcome. We used immunohistochemistry and FACS to describe conditions on the inflammation site. Systemic and clinical effects were evaluated by FACS, cytotoxicity assay, ELISA, cytometric bead array (CBA), RT-PCR and clinical scoring. We found reduced inflammatory infiltration, NKG2D expression on NK and suppression of T, B and antigen-presenting cells (APC) in the synovia. On the systemic level, GCs prevented the activation of monocyte- and B cell-derived APCs, the rise of TNF-α and IFN-γ levels, and subsequent type II collagen (CII)-specific IgG2a formation. Moreover, we detected an increase of anti-inflammatory IL-4 mRNA in the spleen. Similar to the synovia, the GCs caused a significant reduction of NKG2D-expressing NK cells in the spleen without influencing their lytic function. GCs effectively postponed the onset of arthritic symptoms, reduced their severity and in 18% (GN8P) and 31% (GN4C) of the cases completely prevented their appearance. Our data prove that GlcNAc glycoconjugates prevent the inflammatory response, involving proinflammatory cytokine rise, APC activation and NKG2D expression, leading to the attenuation of clinical symptoms. These results support the glycobiological approach to the treatment of collagen-induced arthritis/rheumatoid arthritis (CIA/RA) as a way of bringing new prospects for more effective therapeutic interventions.