Quantitation of dengue virus specific CD4+ T cells by intracellular cytokine staining

2004 ◽  
Vol 284 (1-2) ◽  
pp. 89-97 ◽  
Author(s):  
Marlou M. Mangada ◽  
Francis A. Ennis ◽  
Alan L. Rothman
Keyword(s):  
T Cells ◽  
Dengue Virus ◽  
Cd4 T Cells ◽  
Intracellular Cytokine Staining ◽  
Intracellular Cytokine

scholarly journals T helper cell polarisation as a measure of the maturation of the immune response

2003 ◽  
Vol 12 (5) ◽  
pp. 285-292 ◽  
Author(s):  
Scott B. Cameron ◽  
Ellen H. Stolte ◽  
Anthony W. Chow ◽  
Huub F. J. Savelkoul
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Intracellular Cytokine Staining ◽  
T Helper Cell ◽  
Helper Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Intracellular Cytokine ◽  
T Helper

Background:T helper cell polarisation is important under chronic immune stimulatory conditions and drives the type of the evolving immune response. Mice treated with superantigensin vivodisplay strong effects on Thsubset differentiation. The aim of the study was to detect the intrinsic capacity of T cells to polarise under variousex vivoconditions.Methods:Purified CD4+T cells obtained from superantigen-treated mice were cultured under Thpolarising conditionsin vitro. By combining intracellular cytokine staining and subsequent flow cytometric analysis with quantitative cytokine measurements in culture supernatants by enzyme-linked immunosorbent assay (ELISA), the differential Thpolarising capacity of the treatment can be detected in a qualitative and quantitative manner.Results and conclusions:BALB/c mice were shown to be biased to develop strong Th2 polarised immune responses using Th0 stimulation of purified CD4+T cells from phosphate-buffered saline-treated mice. Nevertheless, our analysis methodology convincingly showed that even in these mice, Toxic Shock Syndrome Toxin-1 treatmentin vivoresulted in a significantly stronger Th1 polarising effect than control treatment. Our results indicate that populations of Thcells can be assessed individually for their differential Th1 or Th2 maturation capacityin vivoby analysing robustin vitropolarisation cultures combined with intracellular cytokine staining and ELISA.

scholarly journals Cytokine production in ex-vivo stimulated fresh and cryopreserved T-cells

2021 ◽  
Vol 67 (2) ◽  
pp. 95-101
Author(s):  
Monica Vuță ◽  
Ionela-Maria Cotoi ◽  
Ion Bogdan Mănescu ◽  
Doina Ramona Manu ◽  
Minodora Dobreanu
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Gradient Centrifugation ◽  
Intracellular Cytokine Staining ◽  
Interleukin 2 ◽  
Middle Aged

Abstract Objective: In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) is an important and reliable measure of immunocompetence. PBMC can be stimulated directly after isolation or frozen for later use. However, cryopreservation may affect cell recovery, viability and functionality. This study aims to investigate cytokine synthesis in ex-vivo stimulated fresh and cryopreserved CD4+ and CD4- T cells. Methods: PBMCs were obtained by Ficoll gradient centrifugation from heparinized peripheral blood of 6 middle-aged clinically healthy subjects. Half of these cells (labeled “Fresh”) was further processed and the other half (labeled “Cryo”) was cryopreserved at -140°C for up to 3 months. Fresh-PBMCs were activated with Phorbol-Myristate-Acetate/Ionomycin/Monensin for 5 hours immediately after isolation while Cryo-PBMCs were identically activated after thawing and cell resting. Activated cells were fixed, permeabilized and intracellular cytokine staining was performed using Phycoerythrin (PE)-conjugated antibodies for Interleukin-2 (IL-2), Tumor Necrosis Factor-alpha (TNF-a), and Interferon-gamma (IFN-g). All samples were analyzed within 24 hours by flow cytometry. Results: Both Fresh and Cryo CD3+CD4+/CD3+CD4- sub-populations partially produced each of the three cytokines. A higher percentage of CD4+ T cells produced IL-2 and TNF-a and a greater percentage of CD4- T cells were found to produce IFN-g. A significantly higher percentage of Cryo-lymphocytes was shown to produce TNF-a in both CD3+CD4+ (31.4% vs 24.9%, p=0.031) and CD3+CD4- (22.7% vs 17.9%, p=0.031) subpopulations. No notable difference was found for IL-2 and IFN-g production between Fresh and Cryo T cells. Conclusion: Cryopreservation for up to 3 months significantly increases TNF-a production of T-cells in clinically healthy middle-aged subjects.

Selective Depletion of CD11b+ Dendritic Cells from Murine Allogeneic Bone Marrow Grafts Promotes a Th1 Phenotype in Donor T Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4610-4610
Author(s):  
Kataryna A. Darlak ◽  
Jian-Ming Li ◽  
Wayne Harris ◽  
Edmund K. Waller
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Weight Change ◽  
Intracellular Cytokine Staining ◽  
Allogeneic Bone ◽  
Intracellular Cytokine ◽  
Post Transplant ◽  
Donor T Cells ◽  
Selective Depletion

Abstract Background: Allogeneic hematopoietic stem cell (HSC) transplant is a treatment used to cure patients with high risk and relapsed acute leukemia, via a graft versus leukemia (GvL) effect. In our previous studies, depletion of CD11b+ cells (containing CD11b+ dendritic cells, CD11b+ NK cells, and myeloid suppressor progenitor cells) from donor BM improved immune reconstitution and enhanced GvL effects without increased rates of GvHD in mice (Li, et al. BBMT 2004). In order to elucidate which subset of donor CD11b+ cells was responsible for this effect, we have transplanted combinations of FACS purified HSC, donor T cells, and CD11b- dendritic cells (DC) and found similar improvement in GvL activity associated with donor T cells polarized towards a Th1 phenotype (Li, et al. Blood 2007). In contrast, grafts containing FACS purified HSC, donor spleen T cells, and CD11b+ DC did not have significant GvL activity and donor T cells were polarized towards a Th2 phenotype. The objective of this study was to determine if selective depletion of CD11b+ dendritic cells by FACS would be a clinically relevant method, producing similar results as engraftment with purified populations of HSC, Cd11b- DC, and T cells. Methods: Selective CD11b+ DC depletion was achieved by FACS sorting in which cells from BM were sorted following gating of all nucleated cells with the exception of the CD11b+CD11c+Lineage- cells. The CD11b+ cells comprised ~1% of the BM. Undepleted BM was also stained and sorted using only a light scatter gate as a control for stress encountered during sorting. To study the long term effects of specific CD11b+ DC depletion, B10.BR or BA.B10 recipients were transplanted with 5×10^6 CD11b+ DC FACS-depleted or undepleted BM cells and 1×10^6 spleen T cells from C57BL/6J, Balb/C, and BA donors. Recipient mice were irradiated with 11Gy one day prior to the transplant. Recipient mice were monitored for survival, weight change, and GvHD score (based on weight change, activity, posture, fur texture, and skin condition) throughout the duration of the experiment and for chimerism of engraftment at days 30, 60, and 100 post transplant. Donor T cells were recovered from transplant recipients on days 3 and 10 post transplant and were examined for their Th1/Th2 polarization by flow cytometry after intracellular cytokine staining and ELISA of supernatants following short term culture. Results: B10.BR mice receiving undepleted BM from C57BL/6J or Balb/C mice had 100 day survivals of 60% and 40% respectively. Mice receiving CD11b+ DC depleted BM from C57BL/6J or Balb/C mice had equivalent survival at 80% and 60% survival, respectively, compared with mice receiving undepleted BM (p=ns). T cell chimerism at day 100 was over 95% donor chimerism for all mice, regardless of CD11b+ DC depletion. Differences in concentration of donor T cells in the blood were not significant between CD11b+ DC depleted and undepleted groups. The level of GvHD in recipient mice also did not differ significantly between groups. At day 3 post transplant, intracellular cytokine staining of donor T cells from mice receiving CD11b+ DC depleted BM had significantly higher levels of TNF-alpha compared with donor T cells from recipients of undepleted BM (p<0.05). Levels of IL-4, IL-10, IL-5 and IFN-gamma among donor spleen T cell population were not significantly different between undepleted and CD11b+ depleted BM recipients. Conclusions: Selective depletion of CD11b+ DC by FACS may be a clinically relevant method for increasing GvL activity without increasing GvHD. Depletion of CD11b+ DC from BM grafts polarizes donor T cells towards a Th1 phenotype, without increasing GvHD or affecting survival in recipient mice as compared with mice receiving undepleted BM grafts. The GvL effect of selective CD11b+ FACS depletion will be discussed.

scholarly journals Optimization of stimulation and staining conditions for intracellular cytokine staining (ICS) for determination of cytokine-producing T cells and monocytes

2021 ◽  
Vol 2 ◽  
pp. 184-193
Author(s):  
Wilson Mandala ◽  
Visopo Harawa ◽  
Alinane Munyenyembe ◽  
Monica Soko ◽  
Herbert Longwe
Keyword(s):  
T Cells ◽  
Intracellular Cytokine Staining ◽  
Intracellular Cytokine

scholarly journals OMIP‐056: Evaluation of Human Conventional T Cells, Donor‐Unrestricted T Cells, and NK Cells Including Memory Phenotype by Intracellular Cytokine Staining

2019 ◽  
Vol 95 (7) ◽  
pp. 722-725 ◽  
Author(s):  
One Dintwe ◽  
Shamiska Rohith ◽  
Katharine V. Schwedhelm ◽  
M. Juliana McElrath ◽  
Erica Andersen‐Nissen ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Intracellular Cytokine Staining ◽  
Intracellular Cytokine ◽  
Memory Phenotype

scholarly journals T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice

2014 ◽  
Vol 211 (4) ◽  
pp. 595-604 ◽  
Author(s):  
Benjamin D.S. Clarkson ◽  
Changying Ling ◽  
Yejie Shi ◽  
Melissa G. Harris ◽  
Aditya Rayasam ◽  
...  
Keyword(s):  
T Cells ◽  
Brain Injury ◽  
T Cell ◽  
Cerebral Ischemia ◽  
Reperfusion Injury ◽  
Cd4 T Cells ◽  
Ischemia Reperfusion Injury ◽  
Tissue Injury ◽  
Ischemia Reperfusion ◽  
Intracellular Cytokine Staining

T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion injury, but the relevant T cell–derived mediators of tissue injury remain unknown. Using a mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated in the injured mouse brain after cerebral ischemia. IL-21–deficient mice have smaller infarcts, improved neurological function, and reduced lymphocyte accumulation in the brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer experiments revealed that brain-infiltrating CD4+ T cells are the predominant IL-21 source. Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4+ T cells in the area surrounding acute stroke lesions, suggesting that IL-21–mediated brain injury may be relevant to human stroke.

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