scholarly journals Different Profile of Interleukin-10 Production in Circulating T Cells from Atopic Asthmatics Compared with Healthy Subjects

2004 ◽  
Vol 11 (1) ◽  
pp. 33-38 ◽  
Author(s):  
K Matsumoto ◽  
S Narita ◽  
T Rerecich ◽  
DP Snider ◽  
PM O'Byrne
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Interleukin 10 ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Circulating T Cells ◽  
Normal Controls

BACKGROUND:Interleukin (IL)-10 is a pleiotropic cytokine released from various cells, including T cells. Although IL-10 is suggested to inhibit allergic responses, its role in asthma remains uncertain. The purpose of the present study was to compare the profile of IL-10 in circulating T cells from stable atopic asthmatics, atopic nonasthmatics and healthy controls.METHODS:Peripheral blood mononuclear cells were isolated, stained with anti-CD3 and CD4/CD8 antibodies, and then processed for intracellular IL-10 detection by flow cytometry.RESULTS:A kinetic study in healthy controls showed that stimulation with phorbol 12-myristate 13-acetate and ionomycin significantly increased the frequencies of IL-10-producing CD3+, CD4+and CD8+cells. Without stimulation, the frequencies of IL-10-producing CD3+, CD4+and CD8+cells were significantly higher in asthmatics than in healthy controls, while a similar trend was observed in atopic nonasthmatics. Stimulation for 24 h significantly increased IL-10-producing CD3+, CD4+and CD8+cells in healthy controls and atopic nonasthmatics, but not in asthmatics.CONCLUSIONS:The frequency of IL-10-producing T cells is increased in the circulation of stable atopic asthmatics compared with normal controls. The lack of enhancement in their frequency by phorbol 12-myristate 13-acetate and ionomycin in asthmatics suggests that the circulating T cells of asthmatic subjects are maximally stimulated with regards to IL-10 production; alternatively, IL-10 production by T cells from asthmatics may be regulated differently than T cells from other subjects.

scholarly journals Comprehensive Evaluation of the Effects of Long-term Cryopreservation on Peripheral Blood Mononuclear Cells using Flow Cytometry

2021 ◽  
Author(s):  
Bo Li ◽  
Chunmei Yang ◽  
Gui Ja ◽  
Yansheng Liu ◽  
Na Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Effective Utilization ◽  
Blood Mononuclear Cells ◽  
Important Evidence

Abstract Human peripheral blood mononuclear cells (PBMCs) originate from hematopoietic stem cells (HSCs) in the bone marrow, which mainly includes lymphocytes (T cells, B cells, and natural killer [NK] cells) and monocytes. Cryopreserved PBMCs providing biobank resources are crucial for clinical application or scientific research. Here, we used flow cytometry to explore the influence of long-term cryopreservation on the quality of PBMCs with the aim of providing important evidence for the effective utilization of biobank resources. The PBMCs were isolated from the peripheral blood, which was collected from volunteers in the hospital. After long-term cryopreservation in liquid nitrogen, we analyzed the changes in cell numbers, viability, and multiple subtypes of PBMCs and studied the apoptosis, proliferation, activation, function, and status of T cells in comparison with freshly isolated PBMCs by flow cytometry, and then further tracked the effects of long-term cryopreservation on the same sample. Although the different cell types in the PBMCs dynamically changed compared with those in the freshly isolated samples, PBMC recovery and viability remained stable after long-term cryopreservation, and the number of most innate immune cells (e.g., monocytes and B cells) was significantly reduced compared to that of the freshly isolated PBMCs or long-term cryopreserved PBMCs; more importantly, the proportion of T cell subtypes, apoptosis, proliferation, and functional T cells, except for Tregs, were not affected by long-term cryopreservation. However, the proportions of activated T, naïve T, central memory T, effector T, and effector memory T cells dynamically changed after long-term cryopreservation. This article provides important evidence for the effective utilization of biobank resources. Long-term cryopreserved PBMCs can be partly used as biological resources for clinical research or basic studies, but the effect of cryopreservation on PBMCs should be considered when selecting cell samples, especially in research relating to activating or inhibiting function.

scholarly journals Zika virus infects human blood mononuclear cells

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Carolina V. Messias ◽  
Julia P. Lemos ◽  
Daniela P. Cunha ◽  
Zilton Vasconcelos ◽  
Lidiane M. S. Raphael ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nervous System ◽  
Zika Virus ◽  
Mononuclear Cells ◽  
Health Concern ◽  
Peripheral Blood Mononuclear ◽  
Healthy Donors ◽  
Blood Mononuclear Cells ◽  
The Central Nervous System

Abstract Background Zika virus (ZIKV) infection gained public health concern after the 2015 outbreak in Brazil, when microcephaly rates increased in babies born from infected mothers. It was demonstrated that ZIKV causes a congenital Zika virus syndrome, including various alterations in the development of the central nervous system. Although the infection of cells from the nervous system has been well documented, less is known in respect of ZIKV ability to infect immune cells. Herein, we investigated if peripheral blood mononuclear cells (PBMCs), freshly-isolated from healthy donors, could be infected by ZIKV. Methods PBMCs from healthy donors were isolated and cultured in medium with ZIKV strain Rio-U1 (MOI = 0.1). Infection was analyzed by RT-qPCR and flow cytometry. Results We detected the ZIKV RNA in PBMCs from all donors by RT-qPCR analysis. The detection of viral antigens by flow cytometry revealed that PBMC from more than 50% the donors were infected by ZIKV, with CD3+CD4+ T cells, CD3−CD19+ B cells and CD3+CD8+ T cells being, respectively, the most frequently infected subpopulations, followed by CD14+ monocytes. Additionally, we observed high variability in PBMC infection rates among different donors, either by numbers or type infected cells. Conclusions These findings raise the hypothesis that PBMCs can act as a reservoir of the virus, which may facilitate viral dissemination to different organs, including immune-privileged sites.

scholarly journals Transforming Growth Factor β1 Production by CD4+ CD25+ Regulatory T Cells in Peripheral Blood Mononuclear Cells from Healthy Subjects Stimulated with Leishmania guyanensis

2005 ◽  
Vol 73 (9) ◽  
pp. 5908-5914 ◽  
Author(s):  
A. Kariminia ◽  
E. Bourreau ◽  
H. Pascalis ◽  
P. Couppié ◽  
D. Sainte-Marie ◽  
...  
Keyword(s):  
T Cells ◽  
Growth Factor ◽  
Healthy Subjects ◽  
Mononuclear Cells ◽  
Transforming Growth Factor ◽  
Suppressive Activity ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Leishmania Guyanensis ◽  
Tgf Β1

ABSTRACT Transforming growth factor β (TGF-β) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF-β production in response to Leishmania stimulation in healthy subjects. In this study, TGF-β1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-β1-producing cells were described as a distinct subpopulation of CD4+ CD25+ regulatory T cells. The suppressive properties of CD4+ CD25+ T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4+ CD25− T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-β1 did not reverse the suppressive activity of CD4+ CD25+ T cells activated by anti-CD3, it reversed the suppressive activity of CD4+ CD25+ T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-β1 is involved in the suppressive activity of L. guyanensis-stimulated CD4+ CD25+ T cells from healthy controls.

PBMC-06- Intracellular Cytokine Staining (ICS) of IFN-gamma, IL-4 and IL-17 on Human Peripheral Blood Mononuclear Cells (PBMC) v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Emanuela Rasini ◽  
Maria Giulia Albizzati ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Brefeldin A ◽  
Intracellular Cytokine ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

This present protocol is developed to analyze the frequency of IFN-γ-, IL-4- and IL-17-producing CD4+T cells, identified from ex vivo human peripheral blood mononuclear cells (PBMC). The frequencies of cytokine producing cells derived from activation of PBMC was induced trough the stimulus phorbol 12-myristate 13-acetate (PMA) and ionomycin. According onpreviously published protocols concentrations of stimulating substances were in the range from 10, to 50 ng/ml for PMA and 1 µg/ml for ionomycin (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The PMA concentrations of 10, 20 and 50 ng/ml were tested and finally the PMA concentration of 10 ng/ml was chosen since it was sufficient to obtain a frequency of cytokines comparable to that obtained with higher stimulus concentrations. PMA/ionomycin and brefeldin A are incubate together for a time of 5 h (Gupta and Maecker, 2015, Foster et al., 2007, Freer and Rindi, 2013, https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The protein secretion inhibitor brefeldin A, was used at the concentration of 10 µg/ml (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013). Cell concentrations may vary in a range from 2.5 x106 to 10 x106 cells/ml (Maecker, 2004; Freer and Rindi, 2013a; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). Concentration of 1x106 cells/ml, 4x106 cells/ml and 8x106cells/ml were tested. Cell tritation have shown a higher functional response proportional to the cell concentration when exposed to a fixed concentration of stimulants. Cell concentration of 8 milions/ml was selected in order to obtain the higher percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells. In conclusion the present protocol provides that, for a optimal optimal percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells as assessed by flow cytometry (Table 1), PBMC in a concentration 8 milions/ml were stimulated with PMA 10 ng/ml and ionomycin 1 µg/ml, and cultured for 5 h in presence of brefeldin A 10 µg/ml according to the procedure described in detail below. References Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: Comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303–313. https://doi.org/10.1128/CDLI.8.2.303-313.2001 Foster, B., Prussin, C., Liu, F., Whitmire, J.K., Whitton, J.L., 2007. Detection of intracellular cytokines by flow cytometry. Curr. Protoc. Immunol. Chapter 6. https://doi.org/10.1002/0471142735.im0624s78 Freer, G., Rindi, L., 2013. Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances. Methods 61, 30–38. https://doi.org/10.1016/j.ymeth.2013.03.035 Gupta, S., Maecker, H., 2015. Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs). BIO-PROTOCOL 5. https://doi.org/10.21769/bioprotoc.1442 Maecker, H.T., 2004. Cytokine flow cytometry. Methods Mol. Biol. 263, 95–108. https://doi.org/10.1385/1-59259-773-4:095 https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.us.560751.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using, sterile culture medium and sterile plastic disposable as well.

Evaluation of Th1/Th2 Lymphocyte Balance in Peripheral Blood Mononuclear Cells of Patients with Hashimoto's Thyroiditis

2020 ◽  
Author(s):  
Hajar VASEGHI ◽  
Fatemeh ESFAHANIAN ◽  
Zohreh JADALI
Keyword(s):  
T Cells ◽  
Transcription Factors ◽  
Peripheral Blood Mononuclear Cells ◽  
Hashimoto’S Thyroiditis ◽  
Mononuclear Cells ◽  
Hashimoto's Thyroiditis ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

Background: The role of T cells in the pathogenesis of Hashimoto’s thyroiditis is well established, whereas the precise and likely the overlapping contributions of different T-cell subpopulations to thyroid injury are less understood. The purpose of this study was to assess the expression pattern of two lineage determining transcription factors, T-bet and GATA-3 that regulate differentiation of T cells into Th1 or Th2 cell fates, respectively. Moreover, the mRNA expression and plasma concentration of Th1(IFN-γ) and Th2(IL-4) cytokines were analyzed. Methods: In this case-control study, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to determine the expression patterns of various transcripts in 20 patients (in Endocrinology Clinic, Imam Khomeini Hospital Complex, Tehran University of Medical Sciences, Tehran, Iran, in 2015) with Hashimoto’s thyroiditis (HT) and 22 healthy controls. Plasma IL-4 and IFN-γ concentrations were also measured using enzyme-linked immunosorbent assay. Results: T-bet gene expression was significantly lower in patients compared to healthy controls (P=0.014). The expression of IL-4 mRNAs was significantly increased in the peripheral blood mononuclear cells from patients as compared to normal controls (P=0.001). In addition, a marked increase in plasma IL-4 levels were observed in patient group compared to controls (P=0.043). Conclusion: Altered balance between Th1 and Th2 related transcription factors and cytokines may be implicated in the pathogenesis of Hashimoto’s thyroiditis.

scholarly journals Single-cell RNA sequencing of peripheral blood mononuclear cells from acute Kawasaki disease patients

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhen Wang ◽  
Lijian Xie ◽  
Guohui Ding ◽  
Sirui Song ◽  
Liqin Chen ◽  
...  
Keyword(s):  
T Cells ◽  
Kawasaki Disease ◽  
Immune Responses ◽  
Single Cell ◽  
Rna Sequencing ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Single Cell Rna Sequencing ◽  
Blood Mononuclear Cells

AbstractKawasaki disease (KD) is the most common cause of acquired heart disease in children in developed countries. Although functional and phenotypic changes of immune cells have been reported, a global understanding of immune responses underlying acute KD is unclear. Here, using single-cell RNA sequencing, we profile peripheral blood mononuclear cells from seven patients with acute KD before and after intravenous immunoglobulin therapy and from three age-matched healthy controls. The most differentially expressed genes are identified in monocytes, with high expression of pro-inflammatory mediators, immunoglobulin receptors and low expression of MHC class II genes in acute KD. Single-cell RNA sequencing and flow cytometry analyses, of cells from an additional 16 KD patients, show that although the percentage of total B cells is substantially decreased after therapy, the percentage of plasma cells among the B cells is significantly increased. The percentage of CD8+ T cells is decreased in acute KD, notably effector memory CD8+ T cells compared with healthy controls. Oligoclonal expansions of both B cell receptors and T cell receptors are observed after therapy. We identify biological processes potentially underlying the changes of each cell type. The single-cell landscape of both innate and adaptive immune responses provides insights into pathogenesis and therapy of KD.

Alteration in mononuclear cell subpopulations in dogs immunized with gentamicin-attenuatedLeishmania infantum

Parasitology ◽  
2012 ◽  
Vol 139 (13) ◽  
pp. 1689-1696 ◽  
Author(s):  
HAMID DANESHVAR ◽  
FARNAZ SEDGHY ◽  
SHAHRIAR DABIRI ◽  
HOSSEIN KAMIABI ◽  
MOHAMMAD M. MOLAEI ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Mononuclear Cells ◽  
Lower Percentage ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Mhc Ii ◽  
Blood Mononuclear Cells ◽  
Cell Subpopulations ◽  
The Impact

SUMMARYThe impact of immunization with gentamicin-attenuatedLeishmania infantum(H-line) on the immunophenotypic profile of popliteal lymph node (PLN) and peripheral blood mononuclear cells (PBMCs) of dogs was assessed by flow cytometry and immunohistochemistry. Compared with the dogs infected withL. infantumwild-type (Group WT), there was a significantly higher percentage of CD4+, CD44+T cells and CD14+, MHC-II+cells and a lower percentage of CD4+CD25+regulatory T cells in PLN of the immunized dogs withL. infantumH-line (Group H). The percentage of CD4+and CD8+T cells in PBMCs of immunized dogs was higher than that in dogs of Group WT. The CD4:CD8 ratio in PLN of dogs of Group H was significantly higher than that in dogs of Group WT. A significantly higher percentage of CD21+B cells and a lower percentage of CD79b+cells were found in PLN of the immunized dogs compared with dogs of Group WT. Immunohistochemical investigation showed no parasites in the PLN of immunized dogs whereas there were parasites in the PLN of 60% of dogs infected withL. infantumWT. In this study, the immunophenotypic profile of mononuclear cells of the immunized dogs correlates with cellular immunity.

scholarly journals Immunophenotyping and Function of Peripheral Blood Mononuclear Cells in Patients Undergoing Unrelated Allogeneic Transplantation with Post-Transplantation Cyclophosphamide in Combination with ATG Anti-Graft Versus Host Disease Prophylaxis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1989-1989
Author(s):  
Katia Beider ◽  
Valeria Voevoda ◽  
Noga Shem-Tov ◽  
Ronit Yerushalmi ◽  
Ivetta Danilesko ◽  
...  
Keyword(s):  
T Cells ◽  
Graft Versus Host Disease ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Healthy Controls ◽  
Peripheral Blood Mononuclear ◽  
Cd8 Cells ◽  
Graft Versus Host ◽  
Gvhd Prophylaxis ◽  
Blood Mononuclear Cells

Post-transplant cyclophosphamide (PTCy) effectively prevents alloreactive responses from unmanipulated grafts, but its effect on subsequent immune reconstitution remains largely undetermined. A total of 32 patients (pts) (ages > 18) who underwent allogeneic hematopoietic stem cell transplantation (HCT) mainly for acute leukemia and myelodysplastic syndrome were evaluated. Blood samples were collected at 15, 30, 60, 90, 180 and 270 days post-HCT. Cell phenotype was assessed on peripheral blood mononuclear cells (PBMC) using multiparametric flow cytometry. The proliferative and activation T cell capacity in response to mitogenic stimulation was determined. Pts who received graft versus host disease (GVHD) prophylaxis with PTCy/ATG combination (n=8) were compared with pts who received ATG (Grafalon®, Neovii) alone (n=13), and pts undergoing HCT with no ATG (n=11). Five healthy controls served for the functional assays. First, CD4+ and CD8+ T cell differentiation was evaluated assessing CD62L, CD45RA, and CCR7 expression. A smaller percentage of effector cells was detected in the PTCy/ATG group at days 30, 60 and 90 after HCT, compared to the no-ATG and ATG cohorts. Accordingly, the percentage of central memory and effector memory cells was significantly increased in PTCy/ATG pts at the same time points. Next, immuno-modulatory cell populations were assessed. A transient increase in the percentage of CD4+CD25+CD127- Treg cells was detected in both ATG and PTCy/ATG cohorts in the early post-HCT period, reaching maximal frequency of 21% Treg out of the CD4+ subset (range 14-42%) in the ATG group and 19% (range 12-30%) in the PTCy/ATG group on day 21, suggesting that Treg cells are relatively spared by Cy. However, a distinct expression pattern of checkpoint molecules was revealed in pts receiving PTCy/ATG. The percentage of PD1-positive CD4+ and CD8+ cells was comparable in the no-ATG and ATG cohorts (44% vs 45% and 35% vs 41%, respectively). Yet, PD1 expression was significantly increased on both CD4+ (61%, p<0.05) and CD8+ cells (60%, p<0.02) from pts receiving PTCy/ATG. Similarly, an increased frequency of LAG-3+ CD8+ cells was observed in the PTCy /ATG pts, in comparison to the no-ATG and ATG groups. Concomitantly, HLA-DR expression on CD56+ NK subset was decreased in PTCy/ATG pts versus the ATG and non-ATG groups (47% vs 61% vs 67%, respectively, p<0.05), indicating reduced activation of NK cells. Furthermore, a significant increase in the percentage of CD28-CD127- cells out of CD8+ cells was detected in PTCy/ATG pts compared to the ATG and no-ATG groups on day 60 post-HCT (82% vs 56% vs 38%, respectively, p<0.02). CD57 up-regulation combined with CD28 down-regulation on T cells reflected an exhausted/senescent phenotype; therefore, we further analyzed the frequency of CD28-CD57+ T cells. Significantly elevated expression of CD57 on CD8+CD28- cells was detected in the PTCy/ATG cohort, in comparison to the ATG and no-ATG groups (65% vs 43% vs 21%, respectively, p<0.01). Exhausted/senescent T cells showed functional impairment, which manifested as reduced potential for cytokine production and poor proliferative capacity. Notably, both CD4+ and CD8+ cells from ATG and PTCy/ATG cohorts demonstrated diminished proliferation in response to ex-vivo αCD3/αCD28 stimulation, when compared to the no-ATG cohort and healthy controls (p<0.01). Moreover, significantly reduced production of IFNγ in response to stimulation was detected in PTCy/ATG and ATG pts (p<0.01). PBMC from the no-ATG cohort displayed inducible IFNγ production levels comparable with healthy controls. Finally, PTCy/ATG-treated patients demonstrated altered phenotypic and functional recovery of the CD8+ cells. While CD4+ cells gradually downregulated PD1 expression and up-regulated co-stimulatory molecules CD28 and CD127 on days 180 and 270, notably CD8+ cells retained exhausted/tolerogenic phenotype as late as 270 days post-HCT. Our results demonstrate that the acquisition of senescent/exhausted phenotype by cytotoxic CD8+ cells is a distinctive feature of HCT with ATG conditioning. PTCy further compromises recovery of CD8+ cells and impairs NK activation, resulting in a sustained immunosuppression. Hopefully, these results will not only lead to a better understanding of the tolerance mechanisms behind PTCy/ATG anti GVHD prophylaxis but will also assist in the development of novel clinical treatments. Disclosures No relevant conflicts of interest to declare.

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