The Effects of the Repeated Administration of Resveratrol Monomer and Resveratrol Dimer on Circulating Immune Cells in Healthy Individuals

Abstract Background and aims: Resveratrol is a natural occurring polyphenol that has various health promoting properties including anti-inflammatory, anti-aging and heart protective effects, and it has also shown a promising anticancer potential (reviewed in Nature Reviews Drug Discovery, 2006). We recently reported that resveratrol may promote tumor immunosurveillance by inducing NKG2D ligands in tumor cells thereby sensitizing the transformed cells to NK cells killing (Cancer Science, 2013). Notably, despite the fact that resveratrol has been extensively studied in several in vitro and preclinical models, little is still known about its in vivo effects in humans. Based on preclinical data, we therefore initiated a phase I clinical trial to explore the safety of repeated doses of resveratrol in Japanese healthy volunteers and to also evaluate its effects on circulating immune cells. Methods: In this study, we evaluated the cellular and molecular immune response in 14 healthy volunteers at baseline and after the oral administration of both resveratrol monomer at 1 Gr/day in seven individuals and resveratrol dimer, given to 7 subjects at 500 mg/day for 28 days. We obtained blood specimens at baseline, and then every 2 weeks for a total of 12 weeks. We assessed the plasma levels of 13 cytokines and chemokines by a luminex assay. In addition, we performed high-performance liquid chromatography-UV to measure resveratrol and its metabolites in both plasma and urine samples. We also evaluated the lymphocyte phenotypes by flow cytometry. Results: The consumption of resveratrol monomer and resveratrol dimer did not cause any adverse events in the individuals evaluated in this study and the plasma levels of resveratrol and its metabolites could thus be accurately measured in both plasma and urine. Consistent with their reported antioxidant properties, the administration of resveratrol monomer (1.5 fold, p=0.01) or resveratrol dimer (2 fold p=0.01) resulted in a significant increase in the antioxidant activity of the plasma compared with the corresponding antioxidant baseline activity. The administration of resveratrol monomer, but not resveratrol dimer, resulted in an increase in the number of CD3- CD56+ and NKG2D+ circulating lymphocytes (p=0.05). Conversely, the circulating numbers of monocytes, B and T lymphocytes remained essentially unchanged throughout the course of the study. Phenotypic studies revealed a significant increase in the expression level of NKG2D receptor on NK cells (p=0.01) and gamma delta T cells (p=0.05) in the individuals receiving either resveratrol dimer or monomer. The activator receptor DNAM-1 was also upregulated in the NK cells obtained from individuals receiving resveratrol. Other NK cells receptors including NKp46, NKp30 and NKG2A remained essentially unchanged. Protein profiling demonstrated significant increases in the circulating levels of immune-related cytokines including CXCL10 ( P=0.01), and MCP-1 (P=0.001), which became apparent by the second week after resveratrol administration. In contrast, the circulating levels of IL-6, IL-10, IL-15, VEGF, and ILR1a remained at baseline levels after resveratrol administration. Conclusions: This is the first study to characterize the effects of resveratrol administration on human circulating immune cells in vivo, and these findings demonstrate that resveratrol clearly has some biological effects on immune cells. Disclosures No relevant conflicts of interest to declare.

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Plant polyphenols: free radical scavengers or chain-breaking antioxidants?

Biochemical Society Symposium ◽

10.1042/bss0610103 ◽

1995 ◽

Vol 61 ◽

pp. 103-116 ◽

Cited By ~ 117

Author(s):

Catherine Rice-Evans

Keyword(s):

Biological Effects ◽

Relative Effectiveness ◽

Antioxidant Properties ◽

Peroxyl Radicals ◽

Free Radical Scavengers ◽

Dietary Polyphenols ◽

Chain Breaking ◽

Lipid Peroxyl Radicals

There is increasing interest in the biological effects of tea- and wine-derived polyphenols and many studies in vitro and in vivo are demonstrating their antioxidant properties. Tea is a major source of dietary polyphenols and an even richer source of the flavanols, the catechins and catechin/gallate esters. Although there are limited studies on the bioavailability of the polyphenols, the absorption of flavanols in humans has been shown. The studies described in this chapter discuss the relative antioxidant potentials of the polyphenolic flavonoids in vitro against radicals generated in the aqueous phase in comparison with their relative effectiveness as antioxidants against propagating lipid peroxyl radicals, and how their activity influences that of α-tocopherol in low-density lipoproteins exposed to oxidative stress.

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A Novel Method to Study the in Vivo Trafficking and Homing of Adoptively Transferred NK Cells in Rhesus Macaques and Humans

Blood ◽

10.1182/blood.v124.21.659.659 ◽

2014 ◽

Vol 124 (21) ◽

pp. 659-659 ◽

Cited By ~ 3

Author(s):

Jan Davidson-Moncada ◽

Noriko Sato ◽

Robert F Hoyt ◽

Robert N Reger ◽

Marvin Thomas ◽

...

Keyword(s):

Nk Cells ◽

Adoptive Transfer ◽

Nk Cell ◽

Conflicts Of Interest ◽

Raji Cell ◽

K562 Cells ◽

Ct Imaging ◽

Pet Ct ◽

Hematological Cancers

Abstract Adoptive transfer of allogeneic or autologous natural killer (NK) cells is now being developed for therapy of both hematological and solid malignancies. The efficacy of NK immunotherapy to mediate anti-tumor effects will ultimately be dependent on their ability to traffic and home to the tumor microenvironment. Recent data suggest expanded NK cells are ineffective at homing to the bone marrow (BM) and lymph nodes (LN) where hematological malignancies reside. A variety of techniques to maintain and/or enforce expression of homing receptors in NK cells are now being explored in preclinical models to improve their localization to the BM and LN. Historically, xenogeneic human into mouse or mouse into mouse models have been utilized for preclinical development of adoptive NK transfer. These experiments often use fluorescent dye-labeled NK cells and require repeated invasive biopsies, which can be confounded by sampling error, or the requirement for post mortem analysis. Here we present a method to track in real time and in vivo adoptively infused zirconium-89 (89Zr) labelled NK cells by PET imaging. A rhesus macaque (RM) model was used for these preclinical experiments as RM and human NK cells have similar expansion kinetics, and have greater similarity than mice in their phenotype, function, and homing receptors and ligands. PBMCs collected from the PB of 13 RMs were enriched for NK cells by CD3+ T-cell depletion and were then expanded for 14 days by culturing with irradiated human EBV-LCL cells in X-VIVO 20 media containing 10% human AB serum and 500 IU/μl of human IL-2. RM NK cells expanded a mean 145±41 fold and contained >99% pure CD3- and CD56+ cells. The phenotype and tumor cytotoxicity of RM NK cells were similar to NK cells expanded from humans (n=3) using similar expansion cultures; at a 10:1 E:T ratio, 67% and 73% of K562 cells were lysed by RM and human NK cell respectively. To label NK cells, 89Zr was conjugated to oxine, which readily permeabilized the cellular membrane and was retained in the cells. Expanded NK cells from both humans and RM showed no changes in CD16 or CD56 expression for up to 6 days following radiolabeling. Human and RM NK cell viability 0 to 24 hours following radiolabelling was 60-100% then declined to 20-30% after 6 days. 89Zr retention by both human and RM NK cells was 75-80% in the first 24 hours of culture but gradually declined with time, decreasing to 20-30% after 7 days of culture. Culturing radiolabeled human NK cells for 24-36 hours with different cellular populations including Ramos and Raji cell lines and normal human PBMCs revealed no significant transfer of radioactivity (max 2% above baseline), establishing that 89Zr was not transferred from labeled to unlabeled cells. Oxine labeling did not alter the cytotoxicity of human or RM NK cells vs K562 cells compared to unlabeled controls. 89Zr-oxine labeling of expanded RM NK cells is currently being used to quantify NK cell trafficking and survival following adoptive transfer in autologous macaques. In these experiments, RM recipients of adoptively infused 89Zr labeled NK cells receive concurrent deferoxamine to chelate and then enhance renal excretion of any free 89Zr that is released from dead cells. In the experiments shown below, 13 x 107 autologous ex vivo expanded 89Zr-labeled RM NK cells were injected IV into a 5.7 kg RM and tracked by sequential PET/CT imaging for 7 days. Up to 1-hour post infusion, most NK cell activity was restricted to the lungs. By 4 hours, NK cells began to traffic from the lungs to the liver and spleen. By 2 days, NK cells were no longer detectable in the lungs and resided largely in the liver and spleen, where they remained for the remainder of the 7 day imaging period. During the entire observation period, little to no NK cell radioactivity was detected in the LN or BM. In conclusion, 89Zr oxine labelling of NK cells followed by PET/CT imaging represents a powerful tool to track the in vivo fate of adoptively transferred NK cells. The RM model presented here provides a method to evaluate and optimize various strategies aimed at altering the phenotype of NK cells, with the goal of improving their homing to the BM and LN where hematological cancers reside. These preclinical in vitro and in vivo data suggest this technology could be safely extended to humans and could be applied to other cellular populations besides NK cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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In Vitro and In Vivo Immunomodulator Activities of Allium sativum L.

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/4984659 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 9

Author(s):

Mouna Moutia ◽

Norddine Habti ◽

Abdallah Badou

Keyword(s):

Immune Cells ◽

Allium Sativum ◽

Therapeutic Potential ◽

Inflammatory Diseases ◽

Biological Activities ◽

Antioxidant Properties ◽

Allergic Airway Inflammation ◽

Cytokine Gene Expression

Allium Sativum L. (garlic), which is a species of the onion family, Alliaceae, is one of the most used plants in traditional medicine worldwide. More than 200 chemicals with diverse properties have been found in garlic extracts. Several garlic compounds were suggested to be efficient in improving various pathologies including certain types of cancer. This paper is an overview of data about garlic biological activities in vitro and/or in vivo on immune cells, on the development of certain inflammatory diseases, and on different types of carcinomas and sarcomas. Garlic and its compounds were found to have notable antioxidant properties. Garlic therapeutic potential has also been studied in several inflammatory diseases such as allergic-airway inflammation, inflammatory bowel disease, arthritic rheumatism, and atherosclerosis. Furthermore, garlic was found to be able to maintain the immune system homeostasis and to exhibit beneficial effects on immune cells especially through regulation of proliferation and cytokine gene expression. Finally, we will show how major garlic components such as sulfur compounds and polyphenols might be responsible for the garlic biological activities revealed in different situations. If identified, specific compounds present in garlic could potentially be used in therapy.

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Granulocyte colony-stimulating factor administration to healthy volunteers: analysis of the immediate activating effects on circulating neutrophils

Blood ◽

10.1182/blood.v84.11.3885.bloodjournal84113885 ◽

1994 ◽

Vol 84 (11) ◽

pp. 3885-3894 ◽

Cited By ~ 129

Author(s):

M de Haas ◽

JM Kerst ◽

CE van der Schoot ◽

J Calafat ◽

CE Hack ◽

...

Keyword(s):

Healthy Volunteers ◽

Plasma Levels ◽

Subcutaneous Administration ◽

Secretory Vesicles ◽

Granulocyte Colony Stimulating Factor ◽

Colony Stimulating Factor ◽

Specific Granules ◽

Stimulating Factor

In four healthy volunteers, we analyzed in detail the immediate in vivo effects on circulating neutrophils of subcutaneous administration of 300 micrograms of granulocyte colony-stimulating factor (G-CSF). Neutrophil activation was assessed by measurement of degranulation. Mobilization of secretory vesicles was shown by a decrease in leukocyte alkaline phosphatase content of the circulating neutrophils. Furthermore, shortly postinjection, Fc gamma RIII was found to be upregulated from an intracellular pool that we identified by immunoelectron microscopy as secretory vesicles. Intravascular release of specific granules was shown by increased plasma levels of lactoferrin and by upregulation of the expression of CD66b and CD11b on circulating neutrophils. Moreover, measurement of fourfold elevated plasma levels of elastase, bound to its physiologic inhibitor alpha 1- antitrypsin, indicated mobilization of azurophil granules. However, no expression of CD63, a marker of azurophil granules, was observed on circulating neutrophils. G-CSF--induced mobilization of secretory vesicles and specific granules could be mimicked in whole blood cultures in vitro, in contrast to release of azurophil granules. Therefore, we postulate that the most activated neutrophils leave the circulation, as observed shortly postinjection, and undergo subsequent stimulation in the endothelial microenvironment, resulting in mobilization of azurophil granules. Our data demonstrate that G-CSF should be regarded as a potent immediate activator of neutrophils in vivo.

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Development of a Novel Pharmaceutical Formula of Nanoparticle Lipid Carriers of Gentamicin/α-Tocopherol and In Vivo Assessment of the Antioxidant Protective Effect of α-Tocopherol in Gentamicin-Induced Nephrotoxicity

Antibiotics ◽

10.3390/antibiotics8040234 ◽

2019 ◽

Vol 8 (4) ◽

pp. 234 ◽

Cited By ~ 1

Author(s):

Mahmoud A. Elfaky ◽

Abrar K. Thabit ◽

Alaa Sirwi ◽

Usama A. Fahmy ◽

Raghdah M. Bahabri ◽

...

Keyword(s):

Protective Effect ◽

Kidney Function ◽

Plasma Levels ◽

Antioxidant Properties ◽

Glycol Succinate ◽

Plasma Parameters ◽

Sod Activity ◽

Innovative Strategy ◽

Significant Difference

Gentamicin is a potent antibiotic with a nephrotoxicity drawback which limits its use. D-α-tocopherol polyethylene glycol succinate (α-tocopherol) is widely used as a surfactant and have potent antioxidant properties. This study aimed to assess the protective effect of α-tocopherol on gentamicin-induced nephrotoxicity by loading gentamicin on nanostructured lipid carriers (NLC). In vivo, the product was administered intravenously to three groups of rabbits (control, gentamicin and gentamicin/α-tocopherol NLC) for 10 consecutive days. Blood was collected on days 1, 5 and 10 to assess renal function. A significant difference in all plasma parameters related to kidney function were observed in the gentamicin group compared to the control by day 5 and 10, confirming the nephrotoxicity effect. On the other hand, the same parameter levels of the NLC group were significantly different compared to the gentamicin group, confirming the protective effect on kidney function. Gentamicin also caused significant decreases in plasma levels of glutathione sulfhydryl (GSH) and superoxide dismutase (SOD) activity. However, gentamicin-α-tocopherol NLC significantly elevates both plasma levels of GSH as well as SOD activity. The present work indicates that, loading of gentamicin on NLC by using α-tocopherol, is an innovative strategy to protect against aminoglycoside-induced nephrotoxicity due to its antioxidant activity.

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ABCG2 C.421C>A Is Associated with Higher Trough Imatinib Plasma Levels in Patients with Chronic Myeloid Leukemia.

Blood ◽

10.1182/blood.v114.22.110.110 ◽

2009 ◽

Vol 114 (22) ◽

pp. 110-110

Author(s):

Naoto Takahashi ◽

Masatomo Miura ◽

Stuart A Scott ◽

Kenichi Sawada

Keyword(s):

Chronic Myeloid Leukemia ◽

Plasma Levels ◽

Myeloid Leukemia ◽

Drug Transport ◽

Conflicts Of Interest ◽

Nucleotide Polymorphisms ◽

Wild Type ◽

Large Patient

Abstract Abstract 110 [Background] Despite the excellent efficacy of imatinib for the treatment of chronic myeloid leukemia (CML), trough imatinib plasma levels can vary widely among patients. This may be due, in part, to inter-individual variation in imatinib metabolism and drug transport efficacy. To investigate the role of genetic variation in the pharmacokinetics of imatinib, we analyzed common single nucleotide polymorphisms within important imatinib pathway genes including ABCG2 (BCRP), ABCB1 (MDR1), ABCC2 (MRP2), CYP3A5, and SLC22A1 (OCT1) in 67 CML patients treated with imatinib. In addition, trough imatinib plasma levels were determined using high-performance liquid chromatography-tandem mass spectrometry. [Results] Distinct imatinib pharmacokinetics were identified in association with ABCG2 c.421C>A (p.Q141K; rs2231142) genotype. Specifically, the presence of the variant c.421A allele was significantly (p=0.024) associated with higher imatinib concentrations [median Cmin/Dose 2.70 (range: 1.50-8.30) ng/ml/mg; n=25] compared to patients with the wild-type ABCG2 (c.421C/C) genotype [median Cmin/Dose 2.27 (range: 0.37-5.30) ng/ml/mg; n=42]. ABCG2 is an efflux transporter for many xenobiotics, including imatinib, and is expressed at high levels in the human liver. Previous studies indicate that c.421A causes a 40% reduction in imatinib transport in vitro when compared to the wild-type genotype. Our data suggest that CML patients with ABCG2 c.421A allele may have deficient ABCG2 activity in vivo, resulting in reduced hepatic excretion of imatinib. Of note, although less common among Africans and individuals of European decent, the ABCG2 c.421C>A allele occurs at a high frequency in the Japanese (0.311) and Han Chinese (0.289) populations. [Conclusion] The association of ABCG2 c.421C>A with imatinib pharmacokinetics may explain why some Japanese CML patients administered less than 400 mg/day of imatinib have clinically sufficient trough imatinib plasma levels. Prospective studies are warranted to confirm the association between ABCG2 genotype and imatinib pharmacokinetics in large patient populations. Disclosures: No relevant conflicts of interest to declare.

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Study On the Molecular Mechanisms of Cytotoxicity of NK Cells against RPMI 8226 Cells.

Blood ◽

10.1182/blood.v114.22.4745.4745 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4745-4745

Author(s):

Xiuju Wang ◽

Songmei Yin ◽

Liping Ma ◽

Danian Nie ◽

Shangfeng Xie ◽

...

Keyword(s):

Monoclonal Antibody ◽

Healthy Volunteers ◽

Nk Cells ◽

Molecular Mechanisms ◽

Nk Cell ◽

Conflicts Of Interest ◽

Control Cell ◽

Killing Activity ◽

Rpmi 8226 ◽

Hla Molecules

Abstract Abstract 4745 To observe how did the allogeneic NK cells kill human multiple myeloma cell RPMI-8266 and the immune mechanisms why RPMI-8266 cells escape from the NK cytotoxicity. Methods Detect the cytotoxicity of NK cells against RPMI 8226 cells by LDH release assay at different effect-to-target cell ratios in vitro, K562, high-sensitive with NK cells,was control cell. Test the expression of MHC-± chain-related molecules (MICA/B), human cytomegalovirus glycoprotein UL16-binding protein (ULBP1-3) and HLA -± MHC molecules on K562 and RPMI-8266 cell by flow cytometry. Detect mRNA level of MICA / B and ULBP1 ∼ 3 in K562, RPMI-266, and KIR genotyping of NK cell from 9 cases of healthy volunteers' by PCR method. As the E:T was 20:1, we used AMO-1, BMO-1, M295, M310, M551 and W6/32 mAb to block the effect of cell-surface protein MICA, MICB, ULBP1, ULBP2, ULBP3 and HLA-± in K562 and RPMI-8266 cell respectively, then observe the change of cytotoxicity of NK cells on K562 and RPMI-8266 cell. Results When the E:T was 5:1, cytotoxicity of NK cells on K562 and RPMI-8266 were (29.52 ± 0.27)% and (2.15 ± 0.32)% respectively; As the E:T was 10:1, the cytotoxicity were (36.37 ± 0.78) % and (5.26 ± 0.84)%; As the E:T was 20:1, they were (59.57 ± 1.05)% and (7.63 ± 1.05)%; As the E:T was 40:1, they were (70.64 ± 1.34)% and (10.18 ± 1.53)%. There wre significantly difference of the cytotoxicity of NK cell between on RPMI-8266 and K562 at all E:T ratio(P <0.05); As for different E:T ratio, the cytotoxicity of NK cell on 8266 cells were statistic difference among them (P <0.05). In K562 cell, we detect MICA / B and ULBP 1 ∼ 3 mRNA and protein, but no HLA -± molecules; RPMI-8266 cell expressed MICA / B and ULBP 1 ∼ 3 and HLA -± molecule, but no MICA / B and ULBP 1 ∼ 3. The genotype of KIR were mismatch between RPMI-8266 cell and NK cells from 9 cases of healthy volunteers. The E:T set as 20:1, we used monoclonal antibodies AMO-1, BMO-1, M295, M310 and M551, respectively, to block the effect of MICA, MICB, ULBP1, ULBP2 and ULBP3, killing activity of NK cell on K562 cells significantly decreased, respectively, (40.82 ± 1.47)%, (43.26 ± 2.41)%, (45.42 ± 1.58)%, (50.74 ± 2.16)%, (41.72 ± 1.66)%, compared with the previous block, they were significantly different (P <0.05); The killing activity of NK cell on RPMI-8266 did not significant change after bloking by all monoclonal antibody, they were (5.81 ± 0.72)%, (7.83 ± 0.91)%, (6.77 ± 0.82)%, (8.25 ± 1.46)%, (6.42 ± 0.87)% respectively (P> 0.05); But as we bloked HLA -± molecules with W6/32 monoclonal antibody, the cytotoxicity of NK cell on RPMI-8266 increased to (48.77 ± 4.61)%, there were significant differences between before and after bloking (P <0.05). Conclusion For 8266 cell, the mechanisms of immune escape from NK killing effect may be related to high expression of HLA -± molecule, and did not express the NKG2D ligands MICA / B and ULBP1 ∼ 3 molecules. Disclosures: No relevant conflicts of interest to declare.

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Modulation of Mesenchymal Stem Cell MHC-I Complex Increases Engraftment In Vivo.

Blood ◽

10.1182/blood.v116.21.1457.1457 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1457-1457

Author(s):

Melisa Soland ◽

Evan J Colletti ◽

Mariana Bego ◽

Chad Sanada ◽

Christopher D Porada ◽

...

Keyword(s):

Nk Cells ◽

Conflicts Of Interest ◽

Surface Expression ◽

Large Animal ◽

Specific Lysis ◽

Fetal Sheep ◽

Mhc I ◽

Npt Ii

Abstract Abstract 1457 Mesenchymal stem cells (MSC) are good candidates for cell therapies due to their immunomodulatory properties, ability to home to/engraft damaged tissues, and potential to differentiate into different cell types. However, when transplanted (Tx) in an allogeneic setting, MSC can elicit an immune response, activating the recipient's cytotoxic T lymphocytes (CTL) and Natural Killer (NK) cells, resulting in rejection of the Tx cells and reduced therapeutic efficacy. Human cytomegalovirus (HCMV, has developed several strategies to evade CTL and NK cell recognition. HCMV avoids CTL attack by producing proteins that downregulate MHC-I surface expression. These proteins are coded for by the unique short regions (US) 2, 3, 6 and 11 of HCMV's genome. We have previously shown that when MSC are transduced with retroviral vectors encoding each one of these US proteins, US6 and US11 were the most effective in reducing MSC's HLA-I surface expression and allogeneic CTL recognition and proliferation. However, HLA-I downregulation may render MSC transduced with US6 (MSC-US6) and US11 (MSC-US11) more susceptible to NK killing, undermining MSC's inherent ability to inhibit function of allogeneic NK cells. Here, we first investigated the role of US6 or US11 on MSC allorecognition by NK cells, and on MSC in vivo engraftment capability. NK killing assays demonstrated that US11 generated the most protective effect at the highest NK concentration (E:T ratio 20:1) (% specific lysis for MSC-US6: 60.4 ± 5.7 %; MSC-US11: 45.5 ± 2.4 % vs. MSC: 88.5 ± 3.4 % respectively). However, at an E:T ratio of 10:1 and 5:1 US11 produced the same degree of protection as US6 (E:T ratio of 10:1; % specific lysis for MSC-US6: 30.1 ± 5.6 %; MSC-US11: 26.3 ± 1.9 % vs. MSC: 54.7 ± 1.9 %); (E:T ratio 5:1; % specific lysis for MSC-US6: 11.9 ± 4.2; MSC-US11: 13.4 ± 2.3; vs. MSC: 25.5 ± 4 respectively). Only at an E:T ratio of 1:1 were US6 and US11 similar to untransduced MSCs (% specific lysis for MSC-US6: 4.7 ± 1.6; MSC-US11: 2.1 ± 0.5; vs. MSC: 4.9 ± 1.8; respectively) in terms of inhibition of NK killing. We also studied the role of US6 and 11 on the expression of beta-2-microglobulin (b2m) and other HLA-I molecules, and we found that US6 reduced b2m by 87± 2 % and HLA-G1 by 44±4.7 %, while US11 reduced b2m by 70± 0.6 % but increased HLA-G1 expression by 176.6±1.9 %. Therefore, the increase in HLA-G1 expression induced by US11 may explain the decrease in NK killing observed in the MSC-US11 cells. Furthermore, we investigated whether US6 or US11 could play a role in mediating complement resistance. While US6 increased the expression of CD59 in transduced cells (Mean fluorescence intensity (MFI) increased by 123.3±1), US11 increased the number of cells expressing CD59 by 121.4 ± 0.8 %, but did not modify their MFI. We next compared the in vivo engraftment potential of MSC, MSC-US6 and MSC-US11 by Tx 5.6×10^4 of each cell population into fetal sheep at 60 days of gestation (n=6). Since we have previously reported the ability of MSC to generate liver cells, we first investigated whether the expression of US6 and 11 would allow higher levels of liver engraftment and hepatocyte formation when compared to MSC (MSC-E) transduced with a retroviral vector encoding only NPT-II. Two months after Tx, liver tissues were collected and stained with NPT-II antibody. This revealed that US6 and US11 increased engraftment efficiency by 241% for MSC-US6 and 277% for MSC-US11 (MSC-E: 5.3 ± 0.4 %, MSC-US6: 12.8 ± 0.9 % and MSC-US:11 14.7 ± 0.8 %). Despite the higher level of liver engraftment seen with MSC-US6 and MSC-US11, co-expression of NPT-II and albumin (MSC-US6: 57% MSC-US1: 50% MSC-E: 75%) or NPT-II and Ov-6 was found at significantly lower levels in MSC-US11 and MSC-US6 Tx animals than in those Tx with MSC-E. Nevertheless, similar numbers of NPT-II/CD34 double-positive cells were found in the liver of MSC-US6 and MSC-US11 Tx animals when compared to MSC-E alone. In conclusion, engineering MSC to over-express US6 or US11 is an effective way to reduce CTL proliferation, NK killing and destruction of engrafted cells by the complement membrane attack complex. In agreement with the in vitro studies, transplantation of these cells into a large animal sheep model resulted in significantly higher levels of overall cell engraftment, but not differentiation towards a hepatocytic phenotype. Studies are underway to determine the mechanism by which HCMV proteins are interfering with MSC differentiation. Disclosures: No relevant conflicts of interest to declare.

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Chemokine Receptor Expression, Migration, and Survival of NK Cells Expanded with Mbil-15 or Mbil-21

Blood ◽

10.1182/blood.v120.21.4683.4683 ◽

2012 ◽

Vol 120 (21) ◽

pp. 4683-4683

Author(s):

Dean Lee ◽

Maureen Aliru ◽

Cecele J. Denman ◽

Srinivas S. Somanchi

Keyword(s):

Nk Cells ◽

Chemokine Receptors ◽

Conflicts Of Interest ◽

Specific Antigen ◽

Xenograft Model ◽

Receptor Expression ◽

Target Cells ◽

Expression Levels ◽

Tissue Migration

Abstract Abstract 4683 Natural killer (NK) cells can kill malignant or virus-infected cells through the interaction of activating and inhibitory receptors without needing specific antigen recognition of target cells, and therefor have broad therapeutic applications for treatment of human malignancies. However, due to their limited life-span in vivo and poor expansion in vitro, production of sufficient numbers of NK cells for effective adoptive immunotherapy poses an obstacle. Genetically engineered artificial antigen presenting cells (aAPCs) consisting of K562 modified 4-1BBL and membrane bound IL-15 or IL-21 have been reported for their ability to support ex vivo NK cell proliferation. aAPCs with mbIL-21 were shown to promote increased proliferation of NK cells with shorter telomeres, but differences in in vivo survival or tumor or tissue migration have not been assessed. Tumor and/or tissue migration is primarily mediated by the expression of chemokine receptors. Using aAPCs bearing mbIL15 or mbIL21, we expanded NK cells for 3 weeks and assessed their expression of chemokine receptors, organ migration, and in vivo survival in a xenograft model. Propagated NK cells showed relatively similar levels of low to modest expression of CCR2, CCR7, CXCR4 and CXCR5, and high expression levels of CXCR3. Mean CCR5 expression levels were similar on cells that were positive, but CCR5 was expressed on a higher percentage of NK cells expanded with mbIL-15 than those expanded with mbIL-21. In contrast, about 20% of mbIL-21 expanded NK cells expressed CX3CR1 expression whereas mbIL-15 NK cells showed almost no expression of this receptor. Results from ongoing migration and survival experiments will also be presented. Disclosures: No relevant conflicts of interest to declare.

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Recognition of tumor cells by Dectin-1 orchestrates innate immune cells for anti-tumor responses

eLife ◽

10.7554/elife.04177 ◽

2014 ◽

Vol 3 ◽

Cited By ~ 77

Author(s):

Shiho Chiba ◽

Hiroaki Ikushima ◽

Hiroshi Ueki ◽

Hideyuki Yanai ◽

Yoshitaka Kimura ◽

...

Keyword(s):

Immune System ◽

Nk Cells ◽

Tumor Cells ◽

Immune Cells ◽

Innate Immune ◽

Tumoricidal Activity ◽

Effector Cells ◽

Therapeutic Implications ◽

Tumor Killing

The eradication of tumor cells requires communication to and signaling by cells of the immune system. Natural killer (NK) cells are essential tumor-killing effector cells of the innate immune system; however, little is known about whether or how other immune cells recognize tumor cells to assist NK cells. Here, we show that the innate immune receptor Dectin-1 expressed on dendritic cells and macrophages is critical to NK-mediated killing of tumor cells that express N-glycan structures at high levels. Receptor recognition of these tumor cells causes the activation of the IRF5 transcription factor and downstream gene induction for the full-blown tumoricidal activity of NK cells. Consistent with this, we show exacerbated in vivo tumor growth in mice genetically deficient in either Dectin-1 or IRF5. The critical contribution of Dectin-1 in the recognition of and signaling by tumor cells may offer new insight into the anti-tumor immune system with therapeutic implications.

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