scholarly journals Influence of immunomodulation on intracellular cytokine expression by spleen T-helpers of mice immunized by Yersinia pestis EV NIIEG

2021 ◽  
Author(s):  
S. N. Klyueva ◽  
A. Yu. Goncharova ◽  
A. L. Kravtsov ◽  
S. A. Bugorkova
Keyword(s):  
Flow Cytometry ◽  
Yersinia Pestis ◽  
Cytokine Expression ◽  
Intracellular Cytokine ◽  
Immunization Schedule ◽  
Spontaneous Production ◽  
Intracellular Expression ◽  
Ifn Γ ◽  
T Helpers ◽  
Expression Measurement

Aim. To characterize the intracellular expression of cytokines by spleen T-helpers and the spontaneous production of cytokines in the blood of BALB/c mice immunized with Yersinia pestis EV NIIEG against the background of immunomodulation.Materials and methods. Intracellular expression of CD4+IFN-γ+, CD4+IL-4+, CD4+IL-17+ was determined in mice spleen cell suspensions by flow cytometry, IFN-γ and IL-10 were measured in ELISA in blood supernatants on day 3 and day 21 after the immunization with Y. pestis EV against the background of immunomodulation. On day 21 after the immunization animals were infected by Y. pestis 231 at a dose of 400 LD50.Results. Differences in cytokine response to studied drugs, correlated with CD4+IFN-γ+ levels in animals, were identified. On day 3, a significant decrease in CD4+IFN-γ+ was observed in response to Y. pestis EV and to recombinant gamma interferon (Ingaron). A significant increase in CD4+IFN-γ+ was detected in response to vaccine strain administered with azoximer bromide (Polyoxidonium). Intracellular expression of IFN-γ, IL-4 and IL-17 increased on day 21by an average of 2,3 times when immunomodulators were used in the immunization schedule. In addition, on day 21 a significant (p ˂ 0.05) increase in the proportion of T-helpers expressing IFN-γ, as well as in level of spontaneous IFN-γ production in blood supernatants was observed only in animals immunized by schedules that included immunomodulators. After the challenge with Y. pestis 231 of animals previously immunized by schedules that included Polyoxidonium, the correlation analysis confirmed the association (r = 0,94; p = 0,0004) of mice survival with intensity of CD4+IFN-γ+ expression.Conclusion. The data obtained confirm the effectiveness of Polyoxidonium application in experimental animal Y. pestis EV immunization schedule and the usefulness of intracellular cytokine expression measurement for assessment of the level of protection following the immunization.

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 lncRNA028466 regulates Th1/Th2 cytokine expression and associates with Echinococcus granulosus antigen P29 immunity

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Chan Wang ◽  
Song-Hao Yang ◽  
Nan Niu ◽  
Jia Tao ◽  
Xian-Cai Du ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
Echinococcus Granulosus ◽  
Noncoding Rna ◽  
Cytokine Expression ◽  
Qrt Pcr ◽  
Th2 Cytokine ◽  
Ifn Γ ◽  
Th1 And Th2

Abstract Background Cystic echinococcosis (CE) is a parasitic disease that is caused by Echinococcus granulosus (Eg). The recombinant Echinococcus granulosus antigen P29 (rEg.P29) was shown to confer effective immunity to sheep and mice during E. granulosus secondary infection in our previous study. In this study, we sought to investigate the ability of long noncoding RNA 028466 (lncRNA028466) as a regulator for the protective immunity mediated by rEg.P29 vaccination and to study the effects of lncRNA028466 on CD4+T cell differentiation in mice spleen. Methods Female BALB/c mice were divided into two groups and were vaccinated subcutaneously with rEg.P29 antigen and PBS as a control (12 mice each group). Following prime-boost vaccination, CD4+T, CD8+T, and B cells from the spleen were isolated by flow cytometry. Quantitative real-time PCR (qRT-PCR) was performed to measure the expression of lncRNA028466 in these three kinds of cells. Then, lncRNA028466 was overexpressed and knocked down in naive CD4+T cells, and Th1 and Th2 cytokine expression was detected. qRT-PCR, western blot, and ELISA were performed to evaluate the production of IFN-γ, IL-2, IL-4, and IL-10, and flow cytometry was performed to detect the differentiation of Th1 and Th2 subgroups. Results lncRNA028466 was significantly decreased after the second week of immunization with rEg.P29 antigen. The proportion of CD4+ T cells was increased after rEg.P29 immunization. Overexpression of lncRNA028466 facilitated the production of IL-4, IL-10 and suppressed the production of IFN-γ, IL-2. Furthermore, after transfection with siRNA028466, IL-2 production was facilitated and IL-10 production was suppressed in naive CD4+ T cells. Conclusions Immunization with rEg.P29 downregulated the expression of lncRNA028466, which was related to a higher Th1 immune response and a lower Th2 immune response. Our results suggest that lncRNA028466 may be involved in rEg.P29-mediated immune response by regulating cytokine expression of Th1 and Th2.

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.

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.

Intracellular Cytokine Expression by T Cells Differs in ZAP-70-Positive and ZAP-70-Negative Chronic Lymphocytic Leukaemia Patients

2007 ◽  
Vol 118 (2) ◽  
pp. 106-110
Author(s):  
Monika Podhorecka ◽  
Agnieszka Bojarska-Junmak ◽  
Jacek Rolinski ◽  
Anna Dmoszynska
Keyword(s):  
T Cells ◽  
Chronic Lymphocytic Leukaemia ◽  
Lymphocytic Leukaemia ◽  
Cytokine Expression ◽  
Intracellular Cytokine

scholarly journals PERCENTAGE OF CD3+ T LYMPHOCYTES EXPRESSING IFN-γ AFTER CFP-10 STIMULATION (Persentase Limfosit T-CD3+ yang Mengekspresikan Interferon Gamma Setelah Stimulasi Antigen CFP-10)

2018 ◽  
Vol 23 (1) ◽  
pp. 31
Author(s):  
Yulia Nadar Indrasari ◽  
Betty Agustina Tambunan ◽  
Jusak Nugraha ◽  
Fransiska Sri Oetami
Keyword(s):  
Flow Cytometry ◽  
Mycobacterium Tuberculosis ◽  
T Lymphocytes ◽  
Interferon Gamma ◽  
Tuberculosis Antigen ◽  
Mycobacterium Tuberculosis Antigen ◽  
Ifn Γ

Tuberkulosis (TB) merupakan penyakit infeksi menular, disebabkan oleh Mycobacterium tuberculosis. Respons imun adaptif yangdiperantarai oleh limfosit T berperan sangat penting dalam menyingkirkan bakteri intraseluler. Hasilan sitokin IFN-γ merupakanmekanisme efektor utama dari limfosit T. Pengembangan vaksin yang efektif dalam melawan infeksi TB mempertimbangkan faktor yangmengatur hasilan IFN-γ. CFP-10 merupakan antigen yang disekresikan oleh Mycobacterium tuberculosis. Antigen ini dikenal sebagaikomponen vaksin potensial untuk TB. Tujuan penelitian ini adalah membandingkan respons imun seluler yaitu persentase limfosit T-CD3+yang mengekspresikan IFN-γ setelah dirangsang antigen CFP-10 di pasien TB paru kasus baru, TB laten dan orang sehat. Penelitianini menggunakan desain eksperimen murni di laboratorium secara in vitro pada kultur PBMC pasien TB paru kasus baru, TB latendan orang sehat. Subjek penelitian adalah 8 pasien TB paru kasus baru, 7 TB laten dan 7 orang sehat di RS Khusus Paru Surabaya.Pemeriksaan persentase limfosit T-CD3+ yang mengekspresikan IFN-γ dengan metode Flow cytometry (BD FACSCalibur). Hasil dianalisisdengan Kruskal-Wallis atau ANOVA satu arah. Rerata persentase limfosit T-CD3+ yang mengekspresikan IFN-γ di TB paru kasus barusetelah stimulasi antigen CFP-10 (4,36%) lebih tinggi daripada sebelum stimulasi (3,50%) (nilai P=0,015). Rerata persentase limfositT-CD3+ yang mengekspresikan IFN-γ di TB laten setelah stimulasi antigen CFP-10 (3,96%) lebih tinggi dibandingkan sebelum stimulasi(2,50%) tetapi tidak bermakna (nilai P=0,367). Rerata persentase limfosit T- CD3+ yang mengekspresikan IFN-γ di orang sehat setelahstimulasi (1,66%) lebih rendah daripada sebelum stimulasi (2,89%) tetapi tidak bermakna (nilai P=0,199). Perubahan persentaselimfosit T-CD3+ yang mengekspresikan IFN-γ setelah stimulasi antigen CFP-10 antarkelompok tidak berbeda bermakna (nilai P=0,143).Berdasarkan hasil telitian ini dapat disimpulkan bahwa terdapat peningkatan persentase limfosit T-CD3+ yang mengekspresikan IFN-γdi TB paru kasus baru setelah stimulasi antigen CFP-10. Hal ini menunjukkan limfosit T-CD3+ yang mengekspresikan IFN-γ berperandalam perlindungan terhadap infeksi TB paru.

scholarly journals CXCL12-CXCR4-Mediated Chemotaxis Supports Accumulation of Mucosal-Associated Invariant T Cells Into the Liver of Patients With PBC

2021 ◽  
Vol 12 ◽  
Author(s):  
Zhilei Chen ◽  
Suying Liu ◽  
Chengmei He ◽  
Jinlei Sun ◽  
Li Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cytokine Production ◽  
Immunofluorescence Staining ◽  
Primary Biliary Cholangitis ◽  
Hepatic Hemangioma ◽  
Cxcr4 Antagonist ◽  
Mait Cells ◽  
Ifn Γ ◽  
Invariant T Cells

Objectives: To explore the potential role of CD3+CD8+CD161high TCRVα7.2+ mucosal-associated invariant T (MAIT) cells in the pathogenesis of primary biliary cholangitis (PBC).Methods: We enrolled 55 patients with PBC, 69 healthy controls (HCs), and 8 patients with hepatic hemangioma. Circulating MAIT cells and their chemokine receptor profiles and cytokine production were quantified using flow cytometry. Liver-resident MAIT cells were examined by immunofluorescence staining. CXCL12-mediated chemotaxis of MAIT cells was measured using a transwell migration assay. Plasma interleukin (IL)-18 was measured using ELISA, and cytokine production in IL-18-stimulated MAIT cells was detected using flow cytometry.Result: Peripheral MAIT cells were found to be significantly lower in patients with PBC (3.0 ± 3.2% vs. 9.4 ± 8.0%, p < 0.01) and negatively correlated with alkaline phosphatase (ALP) levels (r = −0.3209, p < 0.05). Liver immunofluorescence staining suggested that MAIT cells might accumulate in PBC liver. MAIT cells from patients with PBC expressed higher levels of CXCR4 (84.8 ± 18.0% vs. 58.7 ± 11.4%, p < 0.01), and the expression of CXCL12 was higher in PBC liver. CXCL12 promoted MAIT cell chemotaxis (70.4 ± 6.8% vs. 52.2 ± 3.5%, p < 0.01), which was attenuated by CXCR4 antagonist. MAIT cells from PBC produced significantly more interferon-γ (IFN-γ) (88.3 ± 4.2% vs. 64.2 ± 10.1%, p < 0.01), tumor necrosis factor-α (TNF-α) (93.0 ± 1.1% vs. 80.1 ± 5.3%, p < 0.01), Granzyme B (89.3 ± 3.3% vs. 72.1 ± 7.0%, p < 0.01), and perforin (46.8 ± 6.6% vs. 34.8 ± 7.7%, p < 0.05). MAIT cells from PBC expressed higher levels of IL18-Rα (83.8 ± 10.2% vs. 58.3 ± 8.7%, p < 0.01). Plasma IL-18 was more abundant in patients with PBC (286.8 ± 75.7 pg/ml vs. 132.9 ± 78.1 pg/ml, p < 0.01). IL-18 promoted IFN-γ production in MAIT cells (74.9 ± 6.6% vs. 54.7 ± 6.7%, p < 0.01), which was partially attenuated by blocking IL-18R (68.6 ± 8.3% vs. 43.5 ± 4.2%, p < 0.01).Conclusion: Mucosal-associated invariant T cells from patients with PBC accumulated in the liver via CXCL12-CXCR4-mediated chemotaxis, produced pro-inflammatory cytokines, and contributed to portal inflammation, which was potentially mediated by elevated IL-18. Targeting MAIT cells might be a therapeutic approach for PBC.

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