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Immunomodulatory Therapies
Published in David E. Thurston, Ilona Pysz, Chemistry and Pharmacology of Anticancer Drugs, 2021
Peripheral Blood Mononuclear Cells (PBMCs) are first collected by leukapheresis, followed by cell washing to remove contaminants such as red blood cells and platelets which have been shown to influence the clinical efficacy of the final cell product and may lead to cell clumping. Following this, the collected T lymphocytes are either immediately cultured for transduction or cryopreserved for later use. As with the TIL approach, it is possible to enrich for (or deplete) specific cell subsets through the use of methods such as the CliniMACS system, which is based on a relevantly targeted antibody linked to paramagnetic beads. Next, the T cells are activated which, as with TIL-based therapy, may involve anti-CD3 monoclonal antibodies and IL-2.
Interleukin-1 Inhibitors and Their Significance in Rheumatoid Arthritis
Published in Thomas F. Kresina, Monoclonal Antibodies, Cytokines, and Arthritis, 2020
Gloria C. Higgins, Arnold E. Postlethwaite
In an early report, Zembala and Lemmel (176) described inhibition of PHA mitogenesis of normal human PBMCs by synovial fluids from rheumatoid arthritis patients and by SFMC supernatants. This activity has an apparent molecular weight of 50-100 kD by ultrafiltration. The suppression of mitogenesis by exogenous inhibitor depended on the presence of adherent cells in the normal PBMC population. Later, Lotz and coworkers (177) described decreased proliferative responses of rheumatoid SFMC compared to autologous PBMC, which in turn showed lower responses than PBMC from normal controls. They identified an activity in culture supernatants of rheumatoid SFMC and PBMC that decreased the response of mouse thymocytes to purified human IL-1 and PHA and of normal PBMC to anti-CD3 antibody and IL-1. Inhibition could be overcome by excess IL-1. No effect was seen on the proliferation of thymocytes in response to PHA alone or on the proliferation of the CTLL-2 cell line in response to human recombinant IL-2. Production of this inhibitory activity depended upon the presence of adherent cells in the rheumatoid mononuclear cell cultures. In a second report (178) these investigators found that supernatants from adherent rheumatoid PBMC inhibited the synthesis of IL-2 and interferon-γ by normal PBMC in response to in vitro Epstein-Barr virus infection.
Intra-cytoplasmic Cytokine Staining (ICS): Optimizing antigen stimulation for measuring M. tuberculosis-specific T cell response
Published in Ade Gafar Abdullah, Isma Widiaty, Cep Ubad Abdullah, Medical Technology and Environmental Health, 2020
A total volume of 15 ml of venous blood was collected from two healthy persons who were known to have a positive protein-purified derivative (PPD) skin test. The PBMC was isolated by standard density-gradient centrifugation technique using Ficoll-Plaque Plus (GE Healthcare, Amersham, UK). Briefly, heparinized whole blood was diluted 1:3 in phosphate-buffered saline (PBS). Diluted blood was gently layered into tubes containing Ficoll (10 ml for 3 ml diluted blood) to have the blood layered on top of the Ficoll. The tube was then centrifuged at 1800 rpm for 10 minutes at room temperature with the brake off. The PBMC layer was collected, washed three times with PBS, and resuspended in 2 ml of pre-warmed (room temperature) complete RPMI tissue culture media consisting of RPMI 1640 (Gibco, Australia), 10% foetal calf serum (FCS), penicillin and streptomycin (100 μg/ml, Gibco). The number of viable PBMC was counted in a hemocytometer. Approximately 1 million PBMC were isolated per 1 ml of whole blood.
Current approaches to evaluate the function of cytotoxic T-cells in non-human primates
Published in Journal of Immunotoxicology, 2023
Cris Kamperschroer, Brendon Frank, Caroline Genell, Hervé Lebrec, Shermaine Mitchell-Ryan, Brigitte Molinier, Courtni Newsome, Marie-Soleil Piche, Daniel Weinstock, Mark Collinge, Wendy Freebern, Daniel Rubio
This model relies on the use of an adenovirus vaccine to evaluate antigen-specific T-cell responses. Animals are immunized with antigen and whole blood is collected at least 3 weeks later, a timepoint that was selected based on preclinical studies detailing this as the optimal time period to mount a robust response to antigen vaccination. Here, PBMC are isolated from whole blood using density gradient centrifugation. The cells are then incubated overnight with mitogens and/or antigen. T-Cell activation is then generally measured by flow cytometry. Isolation of PBMC may pose logistical issues on larger monkey animal studies, given the time and technical expertise involved in this isolation. However, method development comparisons of whole blood, lysed whole blood, and isolated PBMC showed that sensitivity and dynamic range (resolution) of the measurements were greatly reduced in whole blood and lysed whole blood preparations, compared to PBMC isolates. Given the inherent variability of memory responses and the reduction in sensitivity and resolution, isolated PBMC are recommended to interpret test article effects (either suppression or enhancement of activation) using these methods on memory T-cell responses.
Mitotic index maximization with no effect on radiation-induced dicentric chromosome frequency
Published in International Journal of Radiation Biology, 2023
Kai Takebayashi, Keito Echizenya, Yuki Kameya, Daichi Nakajima, Ryo Nakayama, Yohei Fujishima, Valerie Swee Ting Goh, Yu Abe, Kosuke Kasai, Donovan A. Anderson, William F. Blakely, Tomisato Miura
To increase the proportion of lymphocytes in blood cultures, peripheral blood mononuclear cell (PBMC)-cultures can be used. As each blood cell type in peripheral blood has a different specific density, it is possible to separate blood cells by density gradient centrifugation. Hayata et al. (1992) used an iso-osmotic medium with a density of 1.077 g/ml for PBMC isolation as erythrocytes and granulocytes can be removed and good MI was obtained. IAEA recommends 0.5–2.0 × 106 PBMCs/ml with reference to the reports of Hayata et al. (1992) and McFee et al. (1997). Furthermore, PBMC cell concentration is also dependent on the exposed individual’s blood cell counts. As blood cell count information is not required to be shared between medical facilities handling exposed patient admissions and the biodosimetry laboratory, culturing blood within the recommended PBMC cell count range might be more complicated.
Mucosal-associated invariant T-Cell (MAIT) activation is altered by chlorpyrifos- and glyphosate-treated commensal gut bacteria
Published in Journal of Immunotoxicology, 2020
Anne Mendler, Florian Geier, Sven-Bastiaan Haange, Arkadiusz Pierzchalski, Jannike Lea Krause, Ivonne Nijenhuis, Jean Froment, Nico Jehmlich, Urs Berger, Grit Ackermann, Ulrike Rolle-Kampczyk, Martin von Bergen, Gunda Herberth
After in vitro stimulation, PBMC were stained with Fixable Viability Dye eFluorTM 506 (eBioscience, Frankfurt/Main, Germany) for dead cell exclusion, followed by cell surface stain-ing for 30 min at room temperature (Supplementary Table S2). Thereafter, the cells were fixed in FACSTM lysing Solution (BD) and permeabilized using FACSTM Permeabilizing Solution 2 (BD Biosciences, San Jose, CA). Finally, the cells were intracellularly stained for 30 min at room temperature (Supplementary Table S2), and then analyzed in a BD FACSCantoTM II cytometer with FACS Diva software version 8.0.1 (BD Biosciences, San Jose, CA, USA). Flow cytometry data were analyzed with FlowJo Version 10.2 (FlowJo, Ashland, OR). A minimum of 150,000 viable T-cells/sample was acquired. The lymphocytes among the human PBMC were identified by means of FSC-A and SSC-A. MAIT cells were identified (as CD3+CD8a+CD161+TCRVα7.2+) after exclusion of doublets and dead cells.