scholarly journals Comparison of Intracellular Cytokine Flow Cytometry and an Enzyme Immunoassay for Evaluation of Cellular Immune Response to Active Tuberculosis

2009 ◽  
Vol 16 (3) ◽  
pp. 344-351 ◽  
Author(s):  
Wai Lin Leung ◽  
Kai Leung Law ◽  
Veronica Sui Shan Leung ◽  
Chi Wai Yip ◽  
Chi Chiu Leung ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Active Tuberculosis ◽  
Interleukin 4 ◽  
Confirmatory Test ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intracellular Cytokine ◽  
Cell Counts ◽  
Cytokine Flow Cytometry ◽  
Ifn Γ

ABSTRACT A prospective cross-sectional blinded study of 28 patients (21 male and 7 female patients; mean age, 44 years) with suspected active tuberculosis (TB) attending a TB and chest clinic is described. Blood was taken for immune cell enumeration, a whole-blood enzyme-linked immunosorbent assay (ELISA) for the detection of gamma interferon (IFN-γ) by the QuantiFERON-TB Gold (QFT-G) assay, and intracellular cytokine flow cytometry (ICC) analysis; and sputum was simultaneously taken for bacteriological culture for Mycobacterium tuberculosis. Twelve healthy subjects were included as controls. The performance characteristics of the QFT-G and ICC assays for the detection of active TB were compared. Among the patients with active TB, we found (i) normal to slightly elevated peripheral CD4+ and CD8+ T-cell counts but a significant reduction in the number of NK cells; (ii) CD4+ T cells were the major cell type producing IFN-γ, a type 1 cytokine; (iii) small percentages of CD8+ T cells were also primed for IFN-γ production; (iv) the production of interleukin-4 (IL-4), a type 2 cytokine, was not prominent; and (v) the sensitivity and the specificity of the QFT-G assay were 88.2% and 18%, respectively, and those of the ICC assay were 94.1% and 36.4%, respectively. The specificities of the blood tests were likely underestimated due to cross-reaction to a non-M. tuberculosis mycobacterial infection and the lack of a confirmatory test that could be used to diagnose latent M. tuberculosis infection. Flow cytometry accurately locates the pool of immunological effector cells responsible for cytokine production during active TB. The ICC assay is an additional useful tool for the diagnosis of active TB.

Abstract 001: Axl Expression by CD4+ T Lymphocytes Promotes Salt-Dependent Hypertension

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Kristine M Wadosky ◽  
Sri N Batchu ◽  
Angie Hughson ◽  
Kathy Donlon ◽  
Craig N Morrell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Lymphocytes ◽  
Cd4 T Cells ◽  
White Blood Cells ◽  
Interleukin 4 ◽  
Th2 Cells ◽  
Salt Hypertension ◽  
Ifn Γ

Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10 9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

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 Dichotomy of Cytokine Profiles in Patients and High-Risk Healthy Subjects Exposed to Tuberculosis

1999 ◽  
Vol 67 (11) ◽  
pp. 5597-5603 ◽  
Author(s):  
S. Bhattacharyya ◽  
R. Singla ◽  
A. B. Dey ◽  
H. K. Prasad
Keyword(s):  
T Cells ◽  
High Risk ◽  
Healthy Subjects ◽  
Ex Vivo ◽  
Close Contact ◽  
Interleukin 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mycobacterial Antigen ◽  
Ifn Γ

ABSTRACT To better understand the role of cytokines in susceptible and resistant subjects exposed to Mycobacterium tuberculosisinfection, intracellular gamma interferon (IFN-γ) and interleukin-4 (IL-4) in ex vivo peripheral blood-derived CD4+ T cells were examined by flow cytometry. Of the 37 individuals examined, 20 had clinical evidence of pulmonary tuberculosis and showed acid-fast bacilli in the sputum. Other individuals in close contact with these patients showed no evidence of disease. Patients had a higher number of CD4+ T cells expressing IFN-γ and IL-4 in unstimulated cultures compared to healthy subjects. Despite this, the ratio of IFN-γ+ to IL-4+ CD4+ T cells was similar in both groups. The Th1 response seen in CD4+ T cells in patients was also observed in the overall pattern of IFN-γ and IL-4 detected in control culture supernatants by enzyme-linked immunosorbent assay (ELISA). However, after in vitro stimulation of PBMC with heat-killed M. tuberculosis there was a significant reduction in the percentage of IFN-γ+CD4+ T cells (P < 0.001) in patients. This trend was reflected in the IFN-γ ELISA assay with supernatants derived from stimulated cultures. However, the accumulated levels of IFN-γ were higher than those for IL-4. The reduction of IFN-γ+ CD4+ T cells resulted in the dominance of IL-4+ CD4+ T cells in 13 patients (P < 0.05). The elevated levels of IL-4+CD4+ T cells seen in patients may contribute to the downregulation of IFN-γ expression and the crucial effector function of CD4 T cells, leading to the persistence of disease and the immunopathology characteristically seen in patients. Preliminary data on the indicators of apoptosis in antigen-stimulated cultures in PBMC derived from patients are presented. Of the 17 high-risk healthy individuals examined, 11 differed in that, after mycobacterial-antigen stimulation, there was an enhancement in IFN-γ+CD4+ T cells.

scholarly journals Evaluation of the Expression of CCR5 and CX3CR1 Receptors and Correlation with the Functionality of T Cells in Women infected with ZIKV during Pregnancy

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Débora Familiar-Macedo ◽  
Iury Amancio Paiva ◽  
Jessica Badolato-Corrêa da Silva ◽  
Fabiana Rabe de Carvalho ◽  
Helver Gonçalves Dias ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Clinical Outcome ◽  
Cell Responses ◽  
Functional Assays ◽  
Asymptomatic Children ◽  
Cd107a Expression ◽  
Congenital Zika Syndrome ◽  
Ifn Γ

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child’s clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

scholarly journals Populations of Human T Lymphocytes That Traverse the Vascular Endothelium Stimulated by Borrelia burgdorferi Are Enriched with Cells That Secrete Gamma Interferon

2004 ◽  
Vol 72 (3) ◽  
pp. 1530-1536 ◽  
Author(s):  
Edna I. Gergel ◽  
Martha B. Furie
Keyword(s):  
T Cells ◽  
T Lymphocytes ◽  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Vascular Endothelium ◽  
Human Peripheral Blood ◽  
Gamma Interferon ◽  
Interleukin 4 ◽  
Ifn Γ

ABSTRACT Some diseases are characterized by prevalence in the affected tissues of type 1 T lymphocytes, which secrete gamma interferon (IFN-γ) and other proinflammatory cytokines. For example, type 1 T cells predominate in the lesions of patients with Lyme disease, which is caused by the bacterium Borrelia burgdorferi. We used an in vitro model of the blood vessel wall to test the premise that the vascular endothelium actively recruits circulating type 1 T cells to such lesions. When T lymphocytes isolated from human peripheral blood were examined, the populations that traversed monolayers of resting human umbilical vein endothelial cells (HUVEC) or HUVEC stimulated by interleukin-1β or B. burgdorferi were markedly enriched for T cells that produced IFN-γ compared to the initially added population of T cells. No enrichment was seen for cells that produced interleukin-4, a marker for type 2 T lymphocytes. Very late antigen-4 and CD11/CD18 integrins mediated passage of the T cells across both resting and stimulated HUVEC, and the endothelium-derived chemokine CCL2 (monocyte chemoattractant protein 1) was responsible for the enhanced migration of T cells across stimulated HUVEC. These results suggest that the vascular endothelium may contribute to the selective accumulation of type 1 T cells in certain pathological lesions, including those of Lyme disease.

scholarly journals Gamma Interferon Produced by Antigen-Specific CD4+ T Cells Regulates the Mucosal Immune Responses to Citrobacter rodentium Infection

2010 ◽  
Vol 78 (6) ◽  
pp. 2653-2666 ◽  
Author(s):  
Hideyuki Shiomi ◽  
Atsuhiro Masuda ◽  
Shin Nishiumi ◽  
Masayuki Nishida ◽  
Tetsuya Takagawa ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Gamma Interferon ◽  
Immune Defense ◽  
Interleukin 4 ◽  
Mucosal Inflammation ◽  
Citrobacter Rodentium ◽  
Macrophage Phagocytosis ◽  
Ifn Γ ◽  
Deficient Mice

ABSTRACT Citrobacter rodentium, a murine model pathogen for enteropathogenic Escherichia coli, colonizes the surface of intestinal epithelial cells and causes mucosal inflammation. This bacterium is an ideal model for investigating pathogen-host immune interactions in the gut. It is well known that gene transcripts for Th1 cytokines are highly induced in colonic tissue from mice infected with C. rodentium. However, it remains to be seen whether the Th1 or Th2 cytokines produced by antigen-specific CD4+ T cells provide effective regulation of the host immune defense against C. rodentium infection. To investigate the antigen-specific immune responses, C. rodentium expressing ovalbumin (OVA-C. rodentium), a model antigen, was generated and used to define antigen-specific responses under gamma interferon (IFN-γ)-deficient or interleukin-4 (IL-4)-deficient conditions in vivo. The activation of antigen-specific CD4+ T cells and macrophage phagocytosis were evaluated in the presence of IFN-γ or IL-4 in vitro. IFN-γ-deficient mice exhibited a loss of body weight and a higher bacterial concentration in feces during OVA-C. rodentium infection than C57BL/6 (wild type) or IL-4-deficient mice. This occurred through the decreased efficiency of macrophage phagocytosis and the activation of antigen-specific CD4+ T cells. Furthermore, a deficiency in antigen-specific CD4+ T-cell-expressed IFN-γ led to a higher susceptibility to mucosal and gut-derived systemic OVA-C. rodentium infection. These results show that the IFN-γ produced by antigen-specific CD4+ T cells plays an important role in the defense against C. rodentium.

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