A Novel Tool to Quantify Immune Suppression after Pediatric Transplantation: Flow Cytometric Activation Assays

Penner, Robert; Ionescu, Lavinia; Mital, Seema; Foster, Bethany J.; Birk, Patricia E.; Phan, Véronique; Blydt‐Hansen, Tom; Allen, U.; Hamiwka, Lorraine; Morgan, Catherine; Urschel, Simon

doi:10.1016/j.healun.2019.01.197

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“…↓CD4+IFN+ and ↓CD8+IFN+ as indicators of relapse ↑CD4+IFN+ and ↑CD8+IFN+ as indicators of aGVHD [12] SOT Therapeutic immunodefficiency is dependant on drug dose and duration of treatment [27] CMV-CD8+ as predictors of CMV viremia, disease severity and relapse [28] ↓CMV-CD8+CD69+IFN+ as predictor of CMV infection in SOT patients (kidney and liver) [29] 1B), denying the assessment of intracellular colocalization of multiple cytokines. However, this issue can be easily avoided in future research by using customized antibodies instead.…”

Section: Nstmentioning

confidence: 99%

“…ICS proved to be a very versatile and reliable method and is now widely used in research for the study of vaccines, antigen response, autoimmune disorders, cancer, etc. (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29) The aim of this study was to investigate the variability of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) synthetic capacity of ex-vivo stimulated CD4+/CD8+ lymphocyte subpopulations in healthy subjects.…”

Section: Introductionmentioning

confidence: 99%

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Variability of ex-vivo stimulated T-cells secretory profile in healthy subjects

Manescu

Manu

Șerban

et al. 2020

Revista Romana De Medicina De Laborator

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Peripheral blood lymphocytes (PBL) are able to synthesize various cytokines that play key roles in the immune response and intercellular signaling. Since alterations in cytokine production and/or activity occur in many pathological processes, the study of cytokine synthetic capacity of PBL is a valuable tool for assessing the immune profile. In this paper, we aimed to investigate the variability of interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α) and interferon gamma (IFN-γ) synthetic capacity of CD4+/CD8+ T-cells stimulated ex-vivo in healthy subjects, by means of a commercial intracellular cytokine staining (ICS) protocol. Peripheral blood mononuclear cells were isolated from 16 healthy subjects by Ficoll gradient centrifugation and activated ex-vivo with PMA/Ionomycin/Brefeldin-A for 4 hours. Activated PBL were surface-stained for CD3/CD4/CD8, fixed and permeabilized. ICS was performed using anti-human IL-2/TNF-α/IFN-γ and samples were analyzed on a BD-FACSAria-III flow cytometer. We recorded high post-isolation and post-activation mean viabilities: 82.1% and 82.4% respectively, p=0.84. Both CD4+/CD8+ subpopulations were found to partially produce each of the three cytokines, but in different proportions. On average, a significantly greater percentage of CD4+ cells was shown to produce IL-2 and TNF-α, compared with CD8+ cells (61.5%+/-5.8 vs. 25%+/-5.6 and 26.9%+/-11 vs. 7.5%+/-3.3 respectively, p---lt---0.0001 for both). Contrarily, IFN-γ was produced by a higher proportion of CD8+ cells (8.4%+/-3.9 vs. 6.8%+/-3.2, p=0.01). These results show that the employed ICS protocol elicits a satisfactory and consistent cytokine response from PBL of healthy subjects. The collected data may be used to outline a preliminary reference range for future studies on both healthy/pathological subjects.

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