Cells In Peripheral Blood Mononuclear Cells Research Articles

Role of oxidative stress in the suppression of immune responses in peripheral blood mononuclear cells exposed to combustible tobacco product preparations

Abstract Cigarette smoking is a major risk factor for several human diseases. Chronic inflammation, resulting from increased oxidative stress, has been suggested as a mechanism that contributes to the increased susceptibility of smokers to cancer and microbial infections. We have previously shown that whole smoke-conditioned medium (WS-CM) and total particulate matter (TPM) prepared from Kentucky 3R4F reference cigarettes potently suppressed agonist-stimulated cytokine secretion and target cell killing in peripheral blood mononuclear cells (PBMCs) under ex vivo conditions. Here, we sought to investigate the role of oxidative stress from cigarette smoke exposure in the compromised immunity observed in smokers. Particularly, we investigated the mechanisms of WS-CM and TPM induced suppression of select cytokine secretions in TLR agonist-stimulated cells, and target cell killing by effector cells in PBMCs. Pre-treatment with N-acetyl cysteine (NAC), a precursor of reduced glutathione and an established antioxidant, protected against DNA damage and cytotoxicity caused by exposure to WS-CM and TPM. Similarly, secretion of IFN-γ, TNF, IL-6 and IL-8 in response to TLR-4 stimulation was restored by NAC. Target cell killing, which is used as a functional measure of cytolytic cells in PBMCs, is suppressed by WS-CM. Pretreatment with NAC restored the target cell killing in WS-CM treated PBMCs. This was accompanied by higher perforin levels in the effector cell populations. Collectively, these data suggest that reducing oxidative stress caused by cigarette smoke components restores select immune responses in this ex vivo model.

High leukocyte mtDNA content contributes to poor prognosis through ROS-mediated immunosuppression in hepatocellular carcinoma patients.

Compelling epidemiological evidences indicate a significant association between leukocyte mitochondrial DNA (mtDNA) content and incidence risk of several malignancies, including hepatocellular carcinoma (HCC). However, whether leukocyte mtDNA content affect prognosis of HCC patients and underlying mechanism has never been explored. In our study, leukocyte mtDNA content was measured in 618 HCC patients and its prognostic value was analyzed. Moreover, we detected the immunophenotypes of peripheral blood mononuclear cells (PBMCs) and plasma concentrations of several cytokines in 40 HCC patients and assessed the modulating effects of mtDNA content on immunosuppression in cell models. Our results showed that HCC patients with high leukocyte mtDNA content exhibited a significantly worse recurrence-free survival (RFS) and overall survival (OS) than those with low leukocyte mtDNA content. Leukocyte mtDNA content and TNM stage exhibited a notable joint effect in prognosis prediction. Furthermore, we found that patients with high leukocyte mtDNA content exhibited a higher frequency of CD4+CD25+FOXP3+ regulatory T (Treg) cells and lower frequency of NK cells in PBMCs and had higher TGF-β1 and lower TNF-α and IFN-γ plasma concentration when compared with those with low leukocyte mtDNA content, which suggests an immunosuppressive status. High leukocyte mtDNA content significantly enhanced the ROS-mediated secretion of TGF-β1, which accounted for higher Treg and lower NK frequency in PBMCs. In a conclusion, our study for the first time demonstrates that leukocyte mtDNA content is an independent prognostic marker complementing TNM stage and associated with an ROS-mediated immunosuppressive phenotype in HCC patients.

CD19+ B cell subsets in the peripheral blood and skin lesions of psoriasis patients and their correlations with disease severity.

T lymphocytes are important in the pathogenesis of psoriasis, and increasing evidence indicates that B cells also play an important role. The mechanisms of action, however, remain unclear. We evaluated the ratios of CD19+ B cells in peripheral blood mononuclear cells (PBMCs) from 157 patients with psoriasis (65 patients with psoriasis vulgaris, 32 patients with erythrodermic psoriasis, 30 patients with arthropathic psoriasis, and 30 patients with pustular psoriasis) and 35 healthy controls (HCs). Ratios of CD19+ B cells in skin lesions were compared with non-lesions in 7 erythrodermic psoriasis patients. The Psoriasis Area Severity Index (PASI) was used to measure disease severity. CD19+ B cell ratios in PBMCs from psoriasis vulgaris (at both the active and stationary stage) and arthropathic psoriasis patients were higher compared with HCs (P<0.01), but ratios were lower in erythrodermic and pustular psoriasis patients (P<0.01). CD19+ B cell ratios in erythrodermic psoriasis skin lesions were higher than in non-lesion areas (P<0.001). Different subsets of CD19+CD40+, CD19+CD44+, CD19+CD80+, CD19+CD86+, CD19+CD11b+, and CD19+HLA-DR+ B cells in PBMCs were observed in different psoriasis clinical subtypes. PASI scores were positively correlated with CD19+ B cell ratios in psoriasis vulgaris and arthropathic psoriasis cases (r=0.871 and r=0.692, respectively, P<0.01), but were negatively correlated in pustular psoriasis (r=-0.569, P<0.01). The results indicated that similar to T cells, B cells activation may also play important roles in different pathological stages of psoriasis.

Vitamin B6 Modifies the Immune Cross-Talk between Mononuclear and Colon Carcinoma Cells

The role of vitamin B6 as a key component in a number of biological events has been well established. Based on the relationship between chronic inflammation and carcinogenesis on the one hand, and the interaction between immune and cancer cells expressed by modulated cytokine production on the other hand, the aim of the present work was to examine the possibility that vitamin B6 affects cancer development by an interference in the cross-talk between human peripheral blood mononuclear cells (PBMC) and those from two colon carcinoma cell lines. Both non-stimulated PBMC and mononuclear cells induced for cytokine production by HT-29 and RKO cells from human colon carcinoma lines were incubated without and with 4, 20 and 100 μg/ml of pyridoxal hydrochloride (vitamin B6) and secretion of TNF-α, IL-1β, IL-6, IFN-γ, IL-10, and IL-1ra was examined. Vit B6 caused a dose-dependent decrease in production of all cytokines examined, except for that of IL-1ra. The results indicate that vitamin B6 exerts an immunomodulatory effect on human PBMC. The finding that production of inflammatory cytokines is more pronounced when PBMC are in contact with malignant cells and markedly inhibited by the vitamin suggests an additional way by which vitamin B6 may exert its carcinopreventive effect.

The effect of antibiotics on cytokine production by mononuclear cells and the cross-talk with colon cancer cells

Context: Antibiotics belong to the powerful weapons applied against microbial infections. It is notable that in addition to their antimicrobial effect they express immunomodulatory and anti-cancer activities. Aims: To explore the effect of four antibiotics on the immune cross-talk between peripheral blood mononuclear cells (PBMC) and colon carcinoma cells from two human lines. Methods: Cefotaxime, meropenem, ampicillin and vancomycin were separately added to PBMC co-incubated with cells from two human colon carcinoma cell lines, i.e. HT-29 and RKO. After 24 hours, the level of the following cytokines produced by PBMC was evaluated: IL-6, IL-1ra, IL-1β, TNFα, IFNγ and IL-10. Results: All four antibiotics did not affect the generation of IL-6 and IL-1ra in both co-cultures. On the other hand all of them restrained the production of IL-1β by PBMC incubated with HT-29 cells. In the same incubation mixture cefotaxime, vancomycin and meropenem decreased IFNγ and IL-10 production, while ampicillin and vancomycin inhibited TNFα. As for PBMC incubated with RKO carcinoma cells, cefotaxime inhibited the production of IL-1β, IFNγ and mildly of IL-10, whereas vancomycin repressed that of IL-1β, TNFα and IFNγ. Notably, vancomycin increased the production of IL-1β and decreased that of TNFα and IFNγ. The results indicate that the four antibiotics examined exert a modulatory effect on the immune cross-talk between PBMC and human colon cancer cells from two lines expressed by a different impact on pro-and anti-inflammatory cytokines generation. Conclusions: These findings support the conception that antibiotics may express not only an anti-microbial effect, but also possess an anti-cancer activity that may be considered for integration to the therapeutic arsenal against cancer.

Toxicity evaluation of high-fluorescent rare-earth metal nanoparticles for bioimaging applications.

Research on nanometer-sized luminescent semiconductors and their biological applications in detectors and contrasting agents is an emergent field in nanotechnology. When new nanosize technologies are developed for human health applications, their interaction with biological systems should be studied in depth. Rare-earth elements are used in medical and industrial applications, but their toxic effects are not known. In this work, the biological interaction between terbium-doped gadolinium oxysulfide nanoparticles (GOSNPs) with human peripheral blood mononuclear cells (PBMC), human-derived macrophages (THP-1), and human cervical carcinoma cell (HeLa) were evaluated. The GOSNPs were synthetized using a hydrothermal method to obtain monodisperse nanoparticles with an average size of 91 ± 9 nm. Characterization techniques showed the hexagonal phase of the Gd2 O2 S:Tb3+ free of impurities, and a strong green emission at λemi = 544 nm produced by Tb3+ was observed. Toxic effects of GOSNPs were evaluated using cell viability, apoptosis, cell-cycle progression, and immunological response techniques. In addition, an Artemia model was used to assess the toxicity in vivo. Results indicated cell apoptosis in both types of cells with less sensitivity for PBMC cells compared to HeLa cells. In addition, no toxic effects were observed in the in vivo model of Artemia. Moreover, GOSNPs significantly reduced the activation and cell-cycle progression of PBMC and HeLa cells, respectively. Interestingly, an increase in proinflammatory cytokines was not observed. Our data suggest that fluorescence applications of GOSNPs for biolabeling are not toxic in primary immune cells and they may have an immunomodulatory effect. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 605-615, 2017.

Upregulation of toll-like receptor 4 on T cells in PBMCs is associated with disease aggravation of HBV-related acute-on-chronic liver failure

Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure (ACLF). Toll-like receptors (TLRs) have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was to characterize the TLR4 expression in peripheral blood mononuclear cells (PBMCs) of ACLF patients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected (CHB) patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 expression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was analyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expression of TLR4 on CD4(+) and CD8(+) T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4(+) and CD8(+) T cells were positively correlated with serum total bilirubin (TBIL), direct bilirubin (DBIL), international normalized ratio (INR) levels and white blood cells (WBCs), and negatively correlated with serum albumin (ALB) levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4(+) T cells, which was also positively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.

Trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules through engagement of natural killer cells and stimulation of IFNγ secretion

ABSTRACTIt is increasingly recognized that trastuzumab, an antibody approved for treating human epidermal growth factor receptor 2 (HER2)-overexpressing breast cancer, exerts some of its antitumor effects through enhanced T cell responses. Full activation of CD8+ T cells requires both expression of major histocompatibility complex class I molecules (HLA-ABC) and expression of the T cell costimulatory molecule CD80 or CD86; however, the impact of trastuzumab treatment on the expression of HLA-ABC and CD80 and CD86 has not been investigated in HER2-overexpressing breast cancer cells. In this study, we found that, while there is no direct correlation between the expression of HER2 and HLA-ABC in breast cancer, knockdown of HER2 or inhibition of HER2 kinase by lapatinib downregulated HLA-ABC expression. Trastuzumab had no meaningful effects on HLA-ABC expression in HER2-overexpressing breast cancer cells in monoculture; however, treatment of such cells with trastuzumab in co-culture with human peripheral blood mononuclear cells (PBMC) significantly upregulated not only HLA-ABC expression but also CD86 expression. We showed that this upregulation was mediated by interferon gamma (IFNγ) secreted from the natural killer (NK) cells in PBMC as a result of engagement of NK cells by trastuzumab. We further confirmed this effect of trastuzumab in vivo using a mouse mammary tumor model transduced to overexpress human HER2. Together, our data provide evidence that trastuzumab upregulates expression of HLA-ABC and T cell costimulatory molecules in HER2-overexpressing breast cancer cells in the presence of PBMC, which supports the view that T-cell-mediated immune responses are involved in trastuzumab-mediated antitumor effects.

What makes children outgrow food allergy?

What makes children outgrow food allergy?

Indoleamine 2, 3-Dioxgenase Transfected Mesenchymal Stem Cells Induce Kidney Allograft Tolerance by Increasing the Production and Function of Regulatory T Cells.

The immunoregulatory properties of mesenchymal stem cells (MSCs) have been well characterized in vitro. However, the role of MSCs in organ transplantation remains unclear. The purpose of this study was to examine the role of indoleamine 2, 3-dioxgenase (IDO)-transfected MSCs in immunoregulation both in vitro and in vivo. Wild-type (WT) MSCs, empty lentivirus-transfected MSCs (Lenti-MSCs) or IDO-lentivirus-transfected MSCs (IDO-MSCs) were cocultured with peripheral blood mononuclear cells (PBMCs) or CD4CD25 regulatory T (Treg) cells to examine the impact of IDO on the immunoregulatory properties of MSCs in vitro. WT-MSCs, Lenti-MSCs or IDO-MSCs (2 × 10/kg) were intravenously injected into rabbit renal transplant recipients immediately after surgery to examine the role of IDO-MSCs in tolerance induction in vivo. Lentivirus infection of MSCs resulted in stable expression of IDO. The IDO-MSCs inhibited the proliferation of CD4CD25 effector T cells to a greater extent than WT-MSCs. Coculture of PBMCs and IDO-MSCs induced a higher percentage of CD4CD25Foxp3 Treg cells in PBMCs. Additionally, the antigen-specific suppressive function of these CD4CD25 Treg cells was increased. The IDO-MSCs-treated Treg cells showed upregulated expression of cytotoxic T-lymphocyte-associated antigen 4 and increased secretion of IL-10 and TGF-β. Low doses of IDO-MSCs prolonged graft survival and induced tolerance by inducing antigen-specific CD4CD25 Treg cells, as evidenced by the finding that IDO-MSCs-treated kidney transplant recipients accepted donor-specific skin grafts but rejected third-party grafts. The IDO increased the direct immunoregulatory properties of MSCs. The IDO-MSCs enhanced the expression and function of CD4CD25 Foxp3 Treg cells and induced allograft tolerance.

Immune cell subsets and their gene expression profiles from human PBMC isolated by Vacutainer Cell Preparation Tube (CPT™) and standard density gradient.

BackgroundHigh quality genetic material is an essential pre-requisite when analyzing gene expression using microarray technology. Peripheral blood mononuclear cells (PBMC) are frequently used for genomic analyses, but several factors can affect the integrity of nucleic acids prior to their extraction, including the methods of PBMC collection and isolation. Due to the lack of the relevant data published, we compared the Ficoll-Paque density gradient centrifugation and BD Vacutainer cell preparation tube (CPT) protocols to determine if either method offered a distinct advantage in preparation of PBMC-derived immune cell subsets for their use in gene expression analysis. We evaluated the yield and purity of immune cell subpopulations isolated from PBMC derived by both methods, the quantity and quality of extracted nucleic acids, and compared gene expression in PBMC and individual immune cell types from Ficoll and CPT isolation protocols using Affymetrix microarrays.ResultsThe mean yield and viability of fresh PBMC acquired by the CPT method (1.16 × 106 cells/ml, 93.3 %) were compatible to those obtained with Ficoll (1.34 × 106 cells/ml, 97.2 %). No differences in the mean purity, recovery, and viability of CD19+ (B cells), CD8+ (cytotoxic T cells), CD4+ (helper T cell) and CD14+ (monocytes) positively selected from CPT- or Ficoll-isolated PBMC were found. Similar quantities of high quality RNA and DNA were extracted from PBMC and immune cells obtained by both methods. Finally, the PBMC isolation methods tested did not impact subsequent recovery and purity of individual immune cell subsets and, importantly, their gene expression profiles.ConclusionsOur findings demonstrate that the CPT and Ficoll PBMC isolation protocols do not differ in their ability to purify high quality immune cell subpopulations. Since there was no difference in the gene expression profiles between immune cells obtained by these two methods, the Ficoll isolation can be substituted by the CPT protocol without conceding phenotypic changes of immune cells and compromising the gene expression studies. Given that the CPT protocol is less elaborate, minimizes cells’ handling and processing time, this method offers a significant operating advantage, especially in large-scale clinical studies aiming at dissecting gene expression in PBMC and PBMC-derived immune cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-015-0113-0) contains supplementary material, which is available to authorized users.

Peripheral Blood Mononuclear Cells Enhance Cartilage Repair in in vivo Osteochondral Defect Model.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC’s were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

The Crohn's disease-associated polymorphism in ATG16L1 (rs2241880) reduces SHIP gene expression and activity in human subjects.

Crohn's disease (CD) is a polygenic immune-mediated disease characterized by gastrointestinal inflammation. Mice deficient in the hematopoietic-restricted SH2 domain-containing inositolpolyphosphate 5'-phosphatase (SHIP) develop spontaneous CD-like ileal inflammation. Intriguingly, SHIP mRNA is not upregulated in biopsies from patients with ileal CD despite immune cell infiltration, but SHIP's role in human CD remains unknown. We analyzed SHIP mRNA expression and activity in biopsies and peripheral blood mononuclear cells (PBMCs) from control and treatment-naive subjects with ileal CD, and demonstrated that SHIP mRNA and activity were lower in hematopoietic cells in ileal biopsies and PBMCs from subjects with CD. In all tissues from our patient cohort and in PBMCs from a second healthy control cohort, subjects homozygous for the autophagy-related 16-like protein (ATG16L1) CD-associated gene variant (rs2241880), had low SHIP mRNA expression and activity. SHIP protein expression increased during autophagy and SHIP upregulation was dependent on ATG16L1 and/or autophagy, as well as the ATG16L1 CD-associated gene variant. Finally, homozygosity for the ATG16L1 risk variant and low SHIP mRNA expression is inversely related to increased (LPS+ATP)-induced IL-1β production by PBMCs in our cohorts and was regulated by increased transcription of ILIB. These data suggest a novel mechanism by which the ATG16L1 CD-associated gene variant may predispose people to develop intestinal inflammation.

The International Society of Biological and Environmental Repositories presents Abstracts from their Annual MeetingBridging the Canyon – Connecting Biobank Communities through Innovations in Global Health, Research and Environmental PreservationMay 5–9, 2015Phoenix, Arizona

The International Society of Biological and Environmental Repositories presents Abstracts from their Annual MeetingBridging the Canyon – Connecting Biobank Communities through Innovations in Global Health, Research and Environmental PreservationMay 5–9, 2015Phoenix, Arizona

High leukocyte mitochondrial DNA content contributes to poor prognosis in glioma patients through its immunosuppressive effect.

Background:Epidemiological studies have indicated significant associations of leukocyte mitochondrial DNA (mtDNA) copy number with risk of several malignancies, including glioma. However, whether mtDNA content can predict the clinical outcome of glioma patients has not been investigated.Methods:The mtDNA content of peripheral blood leukocytes from 336 glioma patients was examined using a real-time PCR-based method. Kaplan–Meier curves and Cox proportional hazards regression model were used to examine the association of mtDNA content with overall survival (OS) and progression-free survival (PFS) of patients. To explore the potential mechanism, the immune phenotypes of peripheral blood mononuclear cells (PBMCs) and plasma concentrations of several cytokines from another 20 glioma patients were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively.Results:Patients with high mtDNA content showed both poorer OS and PFS than those with low mtDNA content. Multivariate Cox regression analysis demonstrated that mtDNA content was an independent prognostic factor for both OS and PFS. Stratified analyses showed that high mtDNA content was significantly associated with poor prognosis of patients with younger age, high-grade glioma or adjuvant radiochemotherapy. Immunological analysis indicated that patients with high mtDNA content had significantly lower frequency of natural killer cells in PBMCs and higher plasma concentrations of interleukin-2 and tumour necrosis factor-α, suggesting an immunosuppression-related mechanism involved in mtDNA-mediated prognosis.Conclusions:Our study for the first time demonstrated that leukocyte mtDNA content could serve as an independent prognostic marker and an indicator of immune functions in glioma patients.

Immunomodulatory effects of the pentapeptide YGSRS on human peripheral-blood mononuclear cells

Context: The pentapeptide YGSRS is originated from coffee bean, while its pharmacological features have little been examined.Objectives: We investigated the effects of YGSRS on proliferation, cytokine production and CD4+ CD25+ Foxp3+ regulatory T (Treg) cell frequency of human peripheral blood mononuclear cells (PBMCs) activated by T-cell mitogen.Materials and methods: The effects of YGSRS on T-cell mitogen-activated PBMCs were assessed by WST assay procedures. Concentrations of Th1/Th2/Th17 cytokines in the PBMCs culture medium were analyzed with beads-array procedures followed by analysis with flow cytometry. The CD4+ CD25+ Foxp3+ Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry.Results: YGSRS at 1–10 000 ng/ml (1.56–15 600 nM) has a tendency to promote the mitogen-activated proliferation of PBMCs, but the effects were not statistically significant. YGSRS affect the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, IL-6 and IL-10 from the activated PBMCs, and statistically significant increase in the concentrations of IL-6 and IL-10 in the medium were observed at 1–1000 ng/ml (1.56–1560 nM) (p < 0.05). YGSRS has a tendency to decrease the frequency of Treg cells in the activated PBMCs, but the difference was not statistically significant.Discussion and conclusions: The data suggest that the pentapeptide YGSRS affects the production of several types of cytokines from activated human peripheral T cells, which may modulate Th2 type immunity.

114. Integrins Are Required for Adenovirus 5 Transduction in Cells of Lymphocyte Origin

Adenovirus serotype 5 (Ad5) is the most widely used vector in cancer gene therapies. The high efficiency and broad range of host transduction, easy genome manipulation, non-integration into the host genome, and the well-characterized molecular biology of adenovirus have made them an excellent choice for cancer therapeutics. Ad5 infects cells by binding to the coxsackie-adenovirus receptor (CAR) followed by internalization mediated by binding of viral penton RGD motifs to cell surface integrins (avb3, avβ5). Low levels of Ad5 infection of both human and murine lymphocytes have been linked to the absence of CAR on these cells. We have previously reported that low levels of integrins on canine lymphoma cells are a potential obstacle to Ad5 infection. In the current report we have evaluated CAR and integrins at the molecular level by using quantitative reverse transcriptase PCR (Q-RT-PCR) in a broad range of canine tumor cells including lymphomas (OSW, 17-71), mast cells (BR, MPT1), melanocytes (CML7, CML10), histiocytoma (DH82) and mammary adenocarcinomas (CMT28). Normal canine fibroblasts, peripheral blood mononuclear cells (PBMC) and HEK293 cells were used as controls. All of the cell lines were exposed to Ad5G/L virus at multiplicities of infection (MOI) of 10, 100, and 1000 viral particles per cell. Ad5G/L is a replication incompetent virus vector that infect cells utilizing the classical CAR and integrin infection mechanism. Ad5G/L expresses both GFP and luciferase after infecting cells. Fluorescence microscopy demonstrated that Ad5G/L successfully infected CMT28, DH82, HEK293, CML7, CML10, and NCF at 48 hrs. post infection at all 3 multiplicities of infection. There was limited or no Ad5G/L infection in the lymphoma cell lines, PBMCs, and MPT1 mast cells. The mast cell line BR was the exception, showing relatively high levels of infection. Q-RT-PCR results showed low level of integrins in MPT1, 17-71, OSW, and PBMC cells in comparison to CMT28, DH82, and BR cells. Interestingly, CAR mRNA levels were comparatively higher in MPT1, 17-71, OSW, and PBMC cells. These data indicate that lack of integrins and not CAR is the primary reason for the inability of Ad5G/L infection to infect cells of lymphocyte origin. The defect in cell surface integrins must be addressed if Adenoviral vectors are to be used to transfer genes to lymphocytes or tumors in cells of lymphocyte origin.

CD49a/b-based human natural killer cell subsets in the blood and the liver: phenotypic and functional characteristics (INC6P.312)

Abstract Natural killer (NK) cells in the mouse liver sinusoid are divided into CD49a+DX5(CD49b)- and CD49a-DX5+, whereas those in the blood are mostly CD49a-DX5+. We therefore investigated whether human NK cells are also separated into distinct subsets according to CD49a/b expression. Peripheral blood mononuclear cells (PBMC) and intrasinusoidal lymphocytes (ISL) from healthy human donors were used for phenotypic and functional characterization of NK cells. Primary human NK cells expressed CD49a and CD49b and could be split into four subsets: CD49a+CD49b-, CD49a-CD49b+, CD49a+CD49b+, and CD49a-CD49b-. Frequencies of human NK cell subsets, however, differed from mouse NK cell subsets, as majority of NK cells in human PBMC are CD49a-CD49b- or CD49a+CD49b-. NK cells in ISL expressed lower CD49b but higher CD49a than those in PBMC. In addition, CD49a+ subset showed higher affinity to collagen. CD49b+ NK cells expressed more activation marker CD69 and inhibitory receptor ILT2, indicating they are activated subsets. While degranulation capacity was not affected by CD49a/b expression, CD49a-b+ subset produced higher IFN-γ and CD49a+b+ subset expressed more TRAIL upon activation. In conclusion, human NK cells were divided into four subsets based on CD49a/b expression. It appeared that NK cells in the liver have higher capacity to bind to extracelluar matrix. Small amount of CD49b+ NK cell subsets with activated phenotype were found in the blood but healthy liver had few CD49b+ NK cells.

566. Selective and Stable Transduction of Human CD4+ T Cells In Vivo Upon Systemic Administration of CD4-Targeted Lentiviral Vectors

Playing a central role in both innate and adaptive immunity, CD4+ T cells are the key target for genetic modifications in basic research and immunotherapy. Specific and stable delivery of therapeutic genes into these cells is therefore highly desirable. Here, we describe novel lentiviral vectors (CD4-LV) that have been rendered selective for human or simian CD4+ cells by surface engineering. This novel CD4-LV was highly specific and effective in genetic modification of human CD4+ T cells both in vitro and in vivo. When applied to peripheral blood mononuclear cells (PBMC), CD4-LV transduced CD4+ but not CD4− cells. Notably, also unstimulated T cells were stably genetically modified. Upon systemic or intrasplenic administration into mice reconstituted with human PBMC or hematopoietic stem cells, reporter gene expression was predominantly detected in lymphoid organs. Evaluation of GFP expression in organ-derived cells and blood by flow cytometry demonstrated exclusive gene transfer into CD4+ human lymphocytes. In bone marrow and spleen memory T cells were preferentially hit. Towards potential therapeutic applications, we show that CD4-LV can deliver genes protecting T cells from infection with HIV-1 or encoding chimeric antigen receptors thereby inducing tumor cell killing. This study demonstrates for the first time highly selective and stable gene delivery into human CD4+ T lymphocytes upon in vivo administration of lentiviral vectors, opening an avenue towards novel strategies in immunotherapy.

Mucosal-associated invariant T cell is a potential marker to distinguish fibromyalgia syndrome from arthritis.

BackgroundFibromyalgia (FM) is defined as a widely distributed pain. While many rheumatologists and pain physicians have considered it to be a pain disorder, psychiatry, psychology, and general medicine have deemed it to be a syndrome (FMS) or psychosomatic disorder. The lack of concrete structural and/or pathological evidence has made patients suffer prejudice that FMS is a medically unexplained symptom, implying inauthenticity. Furthermore, FMS often exhibits comorbidity with rheumatoid arthritis (RA) or spondyloarthritis (SpA), both of which show similar indications. In this study, disease specific biomarkers were sought in blood samples from patients to facilitate objective diagnoses of FMS, and distinguish it from RA and SpA.MethodsPeripheral blood mononuclear cells (PBMCs) from patients and healthy donors (HD) were subjected to multicolor flow cytometric analysis. The percentage of mucosal-associated invariant T (MAIT) cells in PBMCs and the mean fluorescent intensity (MFI) of cell surface antigen expression in MAIT cells were analyzed.ResultsThere was a decrease in the MAIT cell population in FMS, RA, and SpA compared with HD. Among the cell surface antigens in MAIT cells, three chemokine receptors, CCR4, CCR7, and CXCR1, a natural killer (NK) receptor, NKp80, a signaling lymphocyte associated molecule (SLAM) family, CD150, a degrunulation marker, CD107a, and a coreceptor, CD8β emerged as potential biomarkers for FMS to distinguish from HD. Additionally, a memory marker, CD44 and an inflammatory chemokine receptor, CXCR1 appeared possible markers for RA, while a homeostatic chemokine receptor, CXCR4 deserved for SpA to differentiate from FMS. Furthermore, the drug treatment interruption resulted in alternation of the expression of CCR4, CCR5, CXCR4, CD27, CD28, inducible costimulatory molecule (ICOS), CD127 (IL-7 receptor α), CD94, NKp80, an activation marker, CD69, an integrin family member, CD49d, and a dipeptidase, CD26, in FMS.ConclusionsCombined with the currently available diagnostic procedures and criteria, analysis of MAIT cells offers a more objective standard for the diagnosis of FMS, RA, and SpA, which exhibit multifaceted and confusingly similar clinical manifestations.