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.

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.

scholarly journals The Mycobacterium tuberculosis Early Secreted Antigenic Target of 6 kDa Inhibits T Cell Interferon-γ Production through the p38 Mitogen-activated Protein Kinase Pathway

2011 ◽  
Vol 286 (27) ◽  
pp. 24508-24518 ◽  
Author(s):  
Hui Peng ◽  
Xisheng Wang ◽  
Peter F. Barnes ◽  
Hua Tang ◽  
James C. Townsend ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
P38 Mapk ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Dependent Manner ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

We reported previously that the early secreted antigenic target of 6 kDa (ESAT-6) from Mycobacterium tuberculosis directly inhibits human T cell IFN-γ production and proliferation in response to stimulation with anti-CD3 and anti-CD28. To determine the mechanism of this effect, we treated T cells with kinase inhibitors before stimulation with ESAT-6. Only the p38 MAPK inhibitor, SB203580, abrogated ESAT-6-mediated inhibition of IFN-γ production in a dose-dependent manner. SB203580 did not reverse ESAT-6-mediated inhibition of IL-17 and IL-10 production, suggesting a specific effect of SB203580 on IFN-γ production. SB203580 did not act through inhibition of AKT (PKB) as an AKT inhibitor did not affect ESAT-6 inhibition of T cell IFN-γ production and proliferation. ESAT-6 did not reduce IFN-γ production by expanding FoxP3+ T regulatory cells. Incubation of T cells with ESAT-6 induced phosphorylation and increased functional p38 MAPK activity, but not activation of ERK or JNK. Incubation of peripheral blood mononuclear cells with ESAT-6 induced activation of p38 MAPK, and inhibition of p38 MAPK with SB203580 reversed ESAT-6 inhibition of M. tuberculosis-stimulated IFN-γ production by peripheral blood mononuclear cells from subjects with latent tuberculosis infection. Silencing of p38α MAPK with siRNA rendered T cells resistant to ESAT-6 inhibition of IFN-γ production. Taken together, our results demonstrate that ESAT-6 inhibits T cell IFN-γ production in a p38 MAPK-dependent manner.

scholarly journals Ex Vivo Stimulation and Expansion of both CD4+ and CD8+ T Cells from Peripheral Blood Mononuclear Cells of Human Cytomegalovirus-Seropositive Blood Donors by Using a Soluble Recombinant Chimeric Protein, IE1-pp65

2001 ◽  
Vol 75 (17) ◽  
pp. 7840-7847 ◽  
Author(s):  
Jocelyn Vaz-Santiago ◽  
Jacqueline Lulé ◽  
Pierre Rohrlich ◽  
Céline Jacquier ◽  
Nicolas Gibert ◽  
...  
Keyword(s):  
T Cells ◽  
Human Cytomegalovirus ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Blood Donors ◽  
Mononuclear Cells ◽  
Chimeric Protein ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

ABSTRACT The transfer of anti-human cytomegalovirus (HCMV) effector T cells to allogeneic bone marrow recipients results in protection from HCMV disease associated with transplantation, suggesting the direct control of CMV replication by T cells. IE1 and pp65 proteins, both targets of CD4+ and CD8+ T cells, are considered the best candidates for immunotherapy and vaccine design against HCMV. In this report, we describe the purification of a 165-kDa chimeric protein, IE1-pp65, and its use for in vitro stimulation and expansion of anti-HCMV CD4+ and CD8+ T cells from peripheral blood mononuclear cells (PBMC) of HCMV-seropositive donors. We demonstrate that an important proportion of anti-HCMV CD4+T cells was directed against IE1-pp65 in HCMV-seropositive donors and that the protein induced activation of HLA-DR3-restricted anti-IE1 CD4+ T-cell clones, as assessed by gamma interferon (IFN-γ) secretion and cytotoxicity. Moreover, soluble IE1-pp65 stimulated and expanded anti-pp65 CD8+ T cells from PBMC of HLA-A2, HLA-B35, and HLA-B7 HCMV-seropositive blood donors, as demonstrated by cytotoxicity, intracellular IFN-γ labeling, and quantitation of peptide-specific CD8+ cells using an HLA-A2–peptide tetramer and staining of intracellular IFN-γ. These results suggest that soluble IE1-pp65 may provide an alternative to infectious viruses used in current adoptive strategies of immunotherapy.

scholarly journals Multi-parameter immune profiling of peripheral blood mononuclear cells by multiplexed single-cell mass cytometry in patients with early multiple sclerosis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Chotima Böttcher ◽  
Camila Fernández-Zapata ◽  
Stephan Schlickeiser ◽  
Desiree Kunkel ◽  
Axel R. Schulz ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Cell Mass ◽  
Healthy Controls ◽  
Mass Cytometry ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells

AbstractMultiple sclerosis (MS) is an inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). Studies in rodent models demonstrated an association of CNS-infiltrating monocyte-derived macrophages with disease severity. However, little is known about humans. Here, we performed an exploratory analysis of peripheral blood mononuclear cells (PBMCs) isolated from healthy controls and drug-naïve patients with early MS using multiplexed single-cell mass cytometry and algorithm-based data analysis. Two antibody panels comprising a total of 64 antibodies were designed to comprehensively analyse diverse immune cell populations, with particular emphasis on monocytes. PBMC composition and marker expression were overall similar between the groups. However, an increased abundance of CCR7+ and IL-6+ T cells was detected in early MS-PBMCs, whereas NFAT1hiT-bethiCD4+ T cells were decreased. Similarly, we detected changes in the subset composition of the CCR7+ and MIPβhi HLA-DR+ lymphocyte compartment. Only mild alterations were detected in monocytes/myeloid cells of patients with early MS, namely a decreased abundance of CD141hiIRF8hiCXCR3+CD68− dendritic cells. Unlike in Crohn’s disease, no significant differences were found in the monocyte fraction of patients with early MS compared to healthy controls. This study provides a valuable resource for future studies designed to characterise and target diverse PBMC subsets in MS.

scholarly journals Single-unit dominance after double-unit umbilical cord blood transplantation coincides with a specific CD8+ T-cell response against the nonengrafted unit

Blood ◽  
2010 ◽  
Vol 115 (4) ◽  
pp. 757-765 ◽  
Author(s):  
Jonathan A. Gutman ◽  
Cameron J. Turtle ◽  
Thomas J. Manley ◽  
Shelly Heimfeld ◽  
Irwin D. Bernstein ◽  
...  
Keyword(s):  
T Cells ◽  
Cord Blood ◽  
Peripheral Blood Mononuclear Cells ◽  
Mononuclear Cells ◽  
Cord Blood Transplantation ◽  
Peripheral Blood Mononuclear ◽  
Blood Transplantation ◽  
Blood Mononuclear Cells ◽  
Cord Blood Unit ◽  
Ifn Γ

AbstractWe investigated the potential role of an immune reaction in mediating the dominant engraftment of 1 cord blood unit in 14 patients who received a double-unit cord blood transplantation (CBT). In 10 patients, dominant engraftment of a single donor unit emerged by day 28 after CBT. In 9 of these 10 patients, a significant subset of CD8+ CD45RO+/−CCR7− T cells, present in peripheral blood mononuclear cells and derived from the engrafting cord blood unit, produced interferon-γ (IFN-γ) in response to the nonengrafting unit. No significant population of IFN-γ–secreting cells was detectable when posttransplantation peripheral blood mononuclear cells were stimulated against cells from the engrafted unit (P < .001) or from a random human leukocyte antigen disparate third party (P = .003). Three patients maintained persistent mixed chimerism after CBT, and no significant IFN-γ–secreting cells were detected after similar stimulations in these patients (P < .005). Our data provide the first direct evidence in human double-unit CBT recipients that immune rejection mediated by effector CD8+ T cells developing after CBT from naive precursors is responsible for the failure of 1 unit to engraft. Future investigations based on these findings may result in strategies to predict a dominant unit and enhance graft-versus-leukemia effect.

Preventive Cancer Vaccination by P5 HER-2/neo Derived Peptide‐pulsed Peripheral Blood Mononuclear Cells in Mouse Model of Breast Cancer

2021 ◽  
Author(s):  
Mahdi Dehghan-Manshadi ◽  
Amin Reza Nikpoor ◽  
Hossein Hadinedoushan ◽  
Fateme Zare ◽  
Mojtaba Sankian ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Fas Ligand ◽  
Mononuclear Cells ◽  
Mrna Levels ◽  
Mice Model ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

This study aimed to compare the prophylactic effects of dendritic cells (DCs) and peripheral blood mononuclear cells (PBMCs) based vaccines by pulsing them in vitro with p5 peptide. The different groups of mice were injected by free peptide or peptide pulsed with DCs or PBMCs. Two weeks after the last boosting dose, immunological tests were performed on splenocyte suspensions of three mice, and the remaining mice in each group were evaluated for tumor growth and survival. The IFN-γ, granzyme B, and IL-10 were detected in T cells. Additionally, IFN-γ and perforin as well as mRNA levels of some genes associated with immune responses were assessed after challenging of splenocytes with TUBO cells. A significant increase was observed in frequency of CD4+ IFN-γ+, CD8+ IFN-γ+ and CD8+ granzymeB+ T cells, and the perforin of supernatants in DC and PBMC groups. A significant Fas ligand (FasL) and forkhead box P3 (Foxp3) expression was observed in DC and PBMC groups. These responses led to lower tumor sizes and longer survival time in tumor mice model. The efficacy of this PBMC-based vaccine in improving the protective immune response makes it a simpler and less expensive candidate vaccine compared to DCs-based vaccines.

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